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## **Meet the editors**

Deepa Bhartiya is currently head of Stem Cell Biology Department at National Institute for Research in Reproductive Health, Mumbai, India. Her group has derived two well characterized human embryonic stem cell lines: KIND1 and KIND2. Pre-clinical evaluation of safety, efficacy and feasibility of pancreatic and tripotent cardiac progenitors obtained by directed differentiation

are ongoing in animal models. Besides the group, she is also interested in a unique population of pluripotent stem cells which exist in adult body tissues termed very small embryonic-like stem cells (VSELs). VSELs have been studied in cord blood, bone marrow and mammalian gonads resulting in several publications.

Nibedita Lenka serves as a senior faculty scientist at National Centre for Cell Science, Pune, India. The prime focus of her group pertains to exploring the guiding cues underlying the cell fate decision machinery using pluripotent embryonic stem cells as a model system. Besides, her group is indulged in understanding the controversial phenomenon of transdifferentiation from

mesenchymal stem cells derived from sources ranging bone marrow, umbilical cord blood and cord tissues. The ongoing investigations aim at understanding the mechanistic basis of stem cells maintenance, the temporal action and cross-talk among factors contributing to mesodermal and neuroectodermal specifications and subsequent differentiation into functional cardiomyocytes and neurons with special focus on dopaminergic neuronal subtypes respectively, and exploring their therapeutic efficacy by using rodent models.

Contents

**Preface IX**

Ambreen Shaikh

Kerkis

and Herman S. Cheung

**Section 1 Pluripotent Stem Cells: The Genesis and Means 1**

Chapter 2 **Pluripotent Adult Stem Cells: A Potential Revolution in**

Chapter 4 **The Dark Side of Pluripotency – Cancer Stem Cell 65**

Chapter 6 **Human Testis–Derived Pluripotent Cells and Induced**

Patricia Ng and Wang Cheng-I

**Derivation and Culture 85**

**Pluripotent Stem Cells 117**

Kun-Hsiung Lee

**Somatic Cells 131**

Deepa Bhartiya, Punam Nagvenkar, Kalpana Sriraman and

**Regenerative Medicine and Tissue Engineering 25**

Chapter 3 **De-Differentiation of Somatic Cells to a Pluripotent State 39**

Chapter 5 **Conditions and Techniques for Mouse Embryonic Stem Cell**

Hideyuki Kobayashi, Koichi Nagao and Koichi Nakajima

Chapter 7 **Generation of Induced Pluripotent Stem Cells from Dental Pulp**

Nelson F. Lizier, Irina Kerkis and Cristiane V. Wenceslau

Tsz Kin Ng, Daniel Pelaez, Veronica R. Fortino, Jordan Greenberg

Cristiane V. Wenceslau, Irina Kerkis, Nelson F. Lizier and Alexandre

Chapter 1 **An Overview of Pluripotent Stem Cells 3**

## Contents



Nelson F. Lizier, Irina Kerkis and Cristiane V. Wenceslau

#### X Contents


Chapter 19 **Disease Models for the Genetic Cardiac Diseases 415**

Calvin C. Sheng and Charles C. Hong

Chapter 23 **Induced Pluripotent Stem Cells as a Source of**

**Bioethical Considerations 529**

**Diseases 477**

**Hepatocytes 517**

**Formation 555**

Günter Virt

Chapter 26 **Stem Cells in Tissue Engineering 567**

Chapter 21 **Pluripotent Stem Cells for Cardiac Cell Therapy: The Application of Cell Sheet Technology 459** Hidetoshi Masumoto and Jun K. Yamashita

Chapter 20 **Pluripotent Stem Cells to Model Human Cardiac Diseases 439**

Chapter 22 **Human Pluripotent Stem Cells Modeling Neurodegenerative**

Roxana Nat, Andreas Eigentler and Georg Dechant

Chapter 24 **Induced Pluripotent Stem Cells: Therapeutic Applications in**

Antonio Liras, Cristina Segovia and Aline S. Gabán

Chapter 25 **Safety Assessment of Reprogrammed Cells Prior to Clinical**

Juan Carlos Polanco and Andrew L. Laslett

Chapter 27 **Ethical Considerations on Stem Cell Research 603**

Minoru Tomizawa, Fuminobu Shinozaki, Takao Sugiyama, Shigenori Yamamoto, Makoto Sueishi and Takanobu Yoshida

**Monogenic and Metabolic Diseases, and Regulatory and**

**Applications: Potential Approaches to Eliminate Teratoma**

Shohreh Mashayekhan, Maryam Hajiabbas and Ali Fallah

Andreas M. Weiss, Michael Breitenbach, Mark Rinnerthaler and

Mari Pekkanen-Mattila, Kristiina Rajala and Katriina Aalto-Setälä

Contents **VII**


**Section 2 Mechanistic Underpinning 151**

**VI** Contents

Chapter 8 **Molecular Mechanisms Underlying Pluripotency 153** Erhard Bieberich and Guanghu Wang

Perla Cota, Mehdi Shafa and Derrick E. Rancourt

Richard L. Eckert, Yasin Kizilyer and Candace L. Kerr

Antonio Lo Nigro and Catherine M. Verfaillie

Chapter 12 **Induced Pluripotent Stem Cells: Current and Emerging**

Guofeng Han, Hongtao Wang and Jijun Hao

Chapter 16 **β1,4-Galactosyltransferases, Potential Modifiers of Stem Cell**

Chapter 14 **Epigenetic Instability in Embryonic Stem Cells 301**

**Pluripotency and Differentiation 345**

**Section 3 Therapeutic Implications and Ethical Concerns 373**

Chapter 18 **Embryonic Stem Cell Therapy – From Bench to Bed 397**

Takuro Horii and Izuho Hatada

Chapter 15 **Function of KLF4 in Stem Cell Biology 317**

Ying Shi and Walden Ai

Chapter 17 **Advances in Stem Cell Therapies 375** Joel Sng and Thomas Lufkin

Michael Wassler

Laura E. Sperling

Jacob Kimmel and Kiminobu Sugaya

Chapter 13 **Molecular Mechanisms of Embryonic Stem Cell**

Chapter 9 **Stem Cells and Epigenetic Reprogramming 179**

Chapter 10 **Epigenetic Reprogramming in Stem Cells 205**

Chapter 11 **Multiple Paths to Reprogramming 227**

**Technologies 263**

**Pluripotency 283**


Antonio Liras, Cristina Segovia and Aline S. Gabán


Preface

Pluripotency is basically defined as the potential to give rise to all three germ layer deriva‐ tives reflecting to 200 odd cell types present in the body, and except the extra-embryonic cell types. It could be either (i) inherent as seen in case of embryonic stem (ES) cells derived from the inner cell mass of blastocyst stage embryo, embryonic/primordial germ (EG/PG) cells derived from developing gonads, embronal carcinoma (EC) cells or (ii) acquired. The latter category includes the cells from adult tissues reprogrammed to attain the pluripotent state and functioning similar to ES cells. This could be achieved either by somatic cell nucle‐ ar transfer (SCNT) or by incorporating a set of transcription factors specific to ES cells into somatic cells and their transient activation leading to induction of pluripotency in them, the latter being designated as induced pluruipotent stem cells (iPSCs) pioneered by Dr. Shinya Yamanaka (the recipient of Nobel Prize in Physiology / Medicine for the year 2012 along

Undoubtedly the recent progress in Stem Cells research field has opened up a wider horizon with various interesting avenues for explorations in basic Biology and Development and on successful broad spectral implication of stem cells in cell replacement therapy, gene therapy, live stock improvement and tissue engineering as well as in pharmaceutical industries. However, a priori requirement is to have the mechanistic understanding and address the safety vs. the efficacy issue including the ethical concerns. In an effort in this line, the book, "Pluripotent Stem Cells" is a compendium addressing the aforesaid aspects. Indeed wide gamut of topics has been covered in the book under various sections with contributions from experts in the stated field concerning diverse aspects of pluripotent stem cells. We

hope that the book would add up to the existing knowledge on pluripotent stem cells.

National Institute for Research in Reproductive Health (NIRRH),

**Dr. Deepa Bhartiya**

**Dr. Nibedita Lenka**

National Centre for Cell Science,

Pune University Campus, Ganeshkhind, Pune, India

India

with Sir Dr. John Gordon, the father of reprogramming phenomenon).

## Preface

Pluripotency is basically defined as the potential to give rise to all three germ layer deriva‐ tives reflecting to 200 odd cell types present in the body, and except the extra-embryonic cell types. It could be either (i) inherent as seen in case of embryonic stem (ES) cells derived from the inner cell mass of blastocyst stage embryo, embryonic/primordial germ (EG/PG) cells derived from developing gonads, embronal carcinoma (EC) cells or (ii) acquired. The latter category includes the cells from adult tissues reprogrammed to attain the pluripotent state and functioning similar to ES cells. This could be achieved either by somatic cell nucle‐ ar transfer (SCNT) or by incorporating a set of transcription factors specific to ES cells into somatic cells and their transient activation leading to induction of pluripotency in them, the latter being designated as induced pluruipotent stem cells (iPSCs) pioneered by Dr. Shinya Yamanaka (the recipient of Nobel Prize in Physiology / Medicine for the year 2012 along with Sir Dr. John Gordon, the father of reprogramming phenomenon).

Undoubtedly the recent progress in Stem Cells research field has opened up a wider horizon with various interesting avenues for explorations in basic Biology and Development and on successful broad spectral implication of stem cells in cell replacement therapy, gene therapy, live stock improvement and tissue engineering as well as in pharmaceutical industries. However, a priori requirement is to have the mechanistic understanding and address the safety vs. the efficacy issue including the ethical concerns. In an effort in this line, the book, "Pluripotent Stem Cells" is a compendium addressing the aforesaid aspects. Indeed wide gamut of topics has been covered in the book under various sections with contributions from experts in the stated field concerning diverse aspects of pluripotent stem cells. We hope that the book would add up to the existing knowledge on pluripotent stem cells.

> **Dr. Deepa Bhartiya** National Institute for Research in Reproductive Health (NIRRH), India

> > **Dr. Nibedita Lenka** National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune, India

**Section 1**

**Pluripotent Stem Cells: The Genesis and Means**

**Pluripotent Stem Cells: The Genesis and Means**

**Chapter 1**

**An Overview of Pluripotent Stem Cells**

This book is entitled **Pluripotent Stem Cells** (PSCs) and various contributors have written on different aspects of the PSCs. But I will fail as an editor of this book if I do not bring to the

Professor Thomson and Prof Gearhart published landmark papers in 1998 wherein they published derivation of PSCs from inner cell mass of spare human blastocyst [1] and from early fetal germ cells [2] respectively. Recently Professor Yamanaka was awarded the Nobel prize for medicine for establishing protocols to reprogram somatic cells to embryonic state with the help of 4 factors [3, 4]. Besides this there are several papers which have reported derivation of ES-like colonies from adult testicular biopsies in both mice [5, 6] and men [7-10]. Similarly Gong et al [11] reported ES-like culture using ovarian tissue. There is a huge body

> © 2013 Bhartiya et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Bhartiya et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Deepa Bhartiya, Punam Nagvenkar,

http://dx.doi.org/10.5772/55130

**1. Introduction**

Kalpana Sriraman and Ambreen Shaikh

Additional information is available at the end of the chapter

reader's attention the all the sources of PSCs (Figure 1).

**Figure 1.** Potential sources for pluripotent stem cells

**Chapter 1**

## **An Overview of Pluripotent Stem Cells**

Deepa Bhartiya, Punam Nagvenkar, Kalpana Sriraman and Ambreen Shaikh

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55130

## **1. Introduction**

This book is entitled **Pluripotent Stem Cells** (PSCs) and various contributors have written on different aspects of the PSCs. But I will fail as an editor of this book if I do not bring to the reader's attention the all the sources of PSCs (Figure 1).

**Figure 1.** Potential sources for pluripotent stem cells

Professor Thomson and Prof Gearhart published landmark papers in 1998 wherein they published derivation of PSCs from inner cell mass of spare human blastocyst [1] and from early fetal germ cells [2] respectively. Recently Professor Yamanaka was awarded the Nobel prize for medicine for establishing protocols to reprogram somatic cells to embryonic state with the help of 4 factors [3, 4]. Besides this there are several papers which have reported derivation of ES-like colonies from adult testicular biopsies in both mice [5, 6] and men [7-10]. Similarly Gong et al [11] reported ES-like culture using ovarian tissue. There is a huge body

© 2013 Bhartiya et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Bhartiya et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of literature suggesting that mesenchymal stem cells (MSCs) have pluripotent characteristics and can transdifferentiate [12]. We have recently published that adult gonads [13, 14] umbilical cord blood/tissue, bone marrow [15] etc. harbor a sub-population of similar kind of pluripotent stem cells termed very small embryonic-like stem cells (VSELs). We also developed a case for VSELs which may be resulting in ES-like colonies rather than de-differentiation of spermato‐ gonial stem cells into pluripotent state [16]. Moreover, the VSELs have confused the field of MSCs, since they are always present as a sub-population amongst MSCs but have remained unnoticed and the pluripotent properties were conferred incorrectly on to the MSCs. VSELs are not widely accepted at present, but have been shown to have promising application towards regenerative medicine.

induces expression of *Id* (Inhibitor of differentiation) genes via the Smad pathway. Overex‐ pression of *Id* indeed allows proliferation of mES cells in the presence of LIF and without need

An Overview of Pluripotent Stem Cells http://dx.doi.org/10.5772/55130 5

A breakthrough occurred with the derivation of human ES (hES) cells in 1998 [1]. Since the first report on derivation of hES cell lines at least 1071 hES cell lines have been derived worldwide [26]. Besides spare human blastocysts, hES cell lines have also been derived from morula stage embryos [27], abnormally developing and arrested embryos [28], single blasto‐ meres of 8-cell stage embryos [29] and 4-cell stage embryos [30, 31]. Mitotically inactivated feeder cells and serum containing medium along with basic fibroblast growth factor (bFGF) are generally used to maintain hES cells. LIF and its related cytokines fail to support hES cells in serum-containing media that supports mES cells despite the existence of a functional LIF/ STAT3 signaling pathway in hES cells [1, 32, 33]. In contrast to mES cells, FGF and TGF/Activin/ Nodal signaling are essential for the self-renewal of hES cells [34]. Although, elements of the BMP pathway exist in hES cells [35], but unlike mES cells, BMPs added to hES cells in conditions that would otherwise support self-renewal, cause rapid differentiation [36]. Recent studies have revealed multiple interactions between the FGF, TGFβ, and BMP pathways in hES cells. Activin induces bFGF expression [37], and bFGF induces Tgfβ1/TGFβ1 and Grem1/ GREM1 (a BMP antagonist) expression and inhibits Bmp4/BMP4 expression in both fibroblast

Although similar in their characteristics such as expression of Oct-4, Nanog, alkaline phos‐ phatase activity, formation of embryoid bodies, teratoma formation, some potential differences exist between mES cells and hES cells. In contrast to mES cells which show expression of SSEA-1, hES cells express SSEA-3/4, TRA-1-60/81. Further, the average population doubling

Although hES cell lines were first derived on MEF feeder layers, continuous efforts towards developing xeno-free culture system has resulted in establishment of human feeders derived from fallopian tube epithelium [39], fetal foreskin, muscle [40, 41], or amniotic epithelium [42]. Attempts have been made to derive new hES cell lines in more defined conditions including serum-free or feeder-free conditions in the presence of extracellular matrices such as matrigel and fibronectin [43-45]. Crook et al [46] derived six clinical-grade hES cell lines using GMPgrade human feeder grown in a medium with GMP-quality FBS and propagated the cell lines using a GMP formulation of Knockout Serum Replacement (KO-SR). Although not xeno-free, the cell lines meet clinical quality. Sidhu et al [47] reported the derivation of hES cell line in culture using human-derived collagen coated plates and KO-SR to maintain human feeder

time for hES cells is longer compared to mES cells (30-35 hr vs. 12-15 hr).

*5. In vitro* **culture and differentiation of hES cells**

of BMP4 or serum.

**4. Human ES cells**

feeders and in hES cells [38].

Thus the aim of the present chapter is to update the readers with the recent advances with embryonic stem cells, induced pluripotent stem cells and VSELs which have been implicated with maximum potential for use in cell-based therapies.

## **2. Embryonic stem cells**

Embryonic stem (ES) cells, as the name suggests, are derived from embryos, more specifically from the inner cell mass (ICM) of the blastocyst. ES cells are characterized by two hallmark properties viz., self-renewal - ability to proliferate indefinitely and pluripotency - capacity to give rise to cells of all the three embryonic germ lineages such as ectoderm, mesoderm and endoderm. They possess a high nucleo-cytoplasmic ratio and telomerase activity. ES cells display high activity of endogenous alkaline phosphatase and express several nuclear and cellsurface markers of pluripotency. They tend to cluster together when cultured in suspension on a non-adherent surface to form 3D aggregates known as embryoid bodies that may be simple or cystic. Moreover, they produce teratomas on injection in immune deficient (SCID) mice, are clonogenic and are capable of producing chimeras when injected into blastocysts in the mouse model.

## **3. Mouse ES cells**

ES cells were first derived from ICM of mouse blastocyst stage embryos [17, 18]. Besides ICM of blastocyst mouse ES (mES) cells have also been derived from cleavage stage embryos and even from biopsied individual blastomeres of two- to eight-cell stage embryos [19- 21]. In general, mES cells can be cultured on a layer of mitotically inactive mouse embryonic fibro‐ blasts (MEF) in the presence of serum and leukaemia inhibitory factor (LIF). The cytokine LIF sustains the self-renewing and pluripotency features of mES cells. LIF, a soluble glycoprotein of interleukin (IL)-6 family of cytokines acts via binding to heterodimers of the LIF-receptor and the signal transducer gp130 resulting in activation of STAT3 signaling [22-24]. In absence of serum, LIF is incapable of maintaining pluripotency of mES cells; however, in combination with bone morphogenetic protein-4 (BMP4) prevents differentiation of mES cells [25]. BMP4

induces expression of *Id* (Inhibitor of differentiation) genes via the Smad pathway. Overex‐ pression of *Id* indeed allows proliferation of mES cells in the presence of LIF and without need of BMP4 or serum.

## **4. Human ES cells**

of literature suggesting that mesenchymal stem cells (MSCs) have pluripotent characteristics and can transdifferentiate [12]. We have recently published that adult gonads [13, 14] umbilical cord blood/tissue, bone marrow [15] etc. harbor a sub-population of similar kind of pluripotent stem cells termed very small embryonic-like stem cells (VSELs). We also developed a case for VSELs which may be resulting in ES-like colonies rather than de-differentiation of spermato‐ gonial stem cells into pluripotent state [16]. Moreover, the VSELs have confused the field of MSCs, since they are always present as a sub-population amongst MSCs but have remained unnoticed and the pluripotent properties were conferred incorrectly on to the MSCs. VSELs are not widely accepted at present, but have been shown to have promising application

Thus the aim of the present chapter is to update the readers with the recent advances with embryonic stem cells, induced pluripotent stem cells and VSELs which have been implicated

Embryonic stem (ES) cells, as the name suggests, are derived from embryos, more specifically from the inner cell mass (ICM) of the blastocyst. ES cells are characterized by two hallmark properties viz., self-renewal - ability to proliferate indefinitely and pluripotency - capacity to give rise to cells of all the three embryonic germ lineages such as ectoderm, mesoderm and endoderm. They possess a high nucleo-cytoplasmic ratio and telomerase activity. ES cells display high activity of endogenous alkaline phosphatase and express several nuclear and cellsurface markers of pluripotency. They tend to cluster together when cultured in suspension on a non-adherent surface to form 3D aggregates known as embryoid bodies that may be simple or cystic. Moreover, they produce teratomas on injection in immune deficient (SCID) mice, are clonogenic and are capable of producing chimeras when injected into blastocysts in

ES cells were first derived from ICM of mouse blastocyst stage embryos [17, 18]. Besides ICM of blastocyst mouse ES (mES) cells have also been derived from cleavage stage embryos and even from biopsied individual blastomeres of two- to eight-cell stage embryos [19- 21]. In general, mES cells can be cultured on a layer of mitotically inactive mouse embryonic fibro‐ blasts (MEF) in the presence of serum and leukaemia inhibitory factor (LIF). The cytokine LIF sustains the self-renewing and pluripotency features of mES cells. LIF, a soluble glycoprotein of interleukin (IL)-6 family of cytokines acts via binding to heterodimers of the LIF-receptor and the signal transducer gp130 resulting in activation of STAT3 signaling [22-24]. In absence of serum, LIF is incapable of maintaining pluripotency of mES cells; however, in combination with bone morphogenetic protein-4 (BMP4) prevents differentiation of mES cells [25]. BMP4

towards regenerative medicine.

4 Pluripotent Stem Cells

**2. Embryonic stem cells**

the mouse model.

**3. Mouse ES cells**

with maximum potential for use in cell-based therapies.

A breakthrough occurred with the derivation of human ES (hES) cells in 1998 [1]. Since the first report on derivation of hES cell lines at least 1071 hES cell lines have been derived worldwide [26]. Besides spare human blastocysts, hES cell lines have also been derived from morula stage embryos [27], abnormally developing and arrested embryos [28], single blasto‐ meres of 8-cell stage embryos [29] and 4-cell stage embryos [30, 31]. Mitotically inactivated feeder cells and serum containing medium along with basic fibroblast growth factor (bFGF) are generally used to maintain hES cells. LIF and its related cytokines fail to support hES cells in serum-containing media that supports mES cells despite the existence of a functional LIF/ STAT3 signaling pathway in hES cells [1, 32, 33]. In contrast to mES cells, FGF and TGF/Activin/ Nodal signaling are essential for the self-renewal of hES cells [34]. Although, elements of the BMP pathway exist in hES cells [35], but unlike mES cells, BMPs added to hES cells in conditions that would otherwise support self-renewal, cause rapid differentiation [36]. Recent studies have revealed multiple interactions between the FGF, TGFβ, and BMP pathways in hES cells. Activin induces bFGF expression [37], and bFGF induces Tgfβ1/TGFβ1 and Grem1/ GREM1 (a BMP antagonist) expression and inhibits Bmp4/BMP4 expression in both fibroblast feeders and in hES cells [38].

Although similar in their characteristics such as expression of Oct-4, Nanog, alkaline phos‐ phatase activity, formation of embryoid bodies, teratoma formation, some potential differences exist between mES cells and hES cells. In contrast to mES cells which show expression of SSEA-1, hES cells express SSEA-3/4, TRA-1-60/81. Further, the average population doubling time for hES cells is longer compared to mES cells (30-35 hr vs. 12-15 hr).

## *5. In vitro* **culture and differentiation of hES cells**

Although hES cell lines were first derived on MEF feeder layers, continuous efforts towards developing xeno-free culture system has resulted in establishment of human feeders derived from fallopian tube epithelium [39], fetal foreskin, muscle [40, 41], or amniotic epithelium [42]. Attempts have been made to derive new hES cell lines in more defined conditions including serum-free or feeder-free conditions in the presence of extracellular matrices such as matrigel and fibronectin [43-45]. Crook et al [46] derived six clinical-grade hES cell lines using GMPgrade human feeder grown in a medium with GMP-quality FBS and propagated the cell lines using a GMP formulation of Knockout Serum Replacement (KO-SR). Although not xeno-free, the cell lines meet clinical quality. Sidhu et al [47] reported the derivation of hES cell line in culture using human-derived collagen coated plates and KO-SR to maintain human feeder fibroblasts. A fully defined xeno-free medium (RegES), capable of supporting the expansion of hES cell lines, induced pluripotent stem (iPS) cells and adipose stem cells has been described [48]. Recently, Wang et al [49] have developed a xeno-free and feeder-cell-free culture system for propagating hES cells and hiPS cells using human plasma and human placenta extracts.

they would continue to monitor existing patients, and were attempting to find a partner that could continue their research. The recent success of a prospective clinical study of Advanced Cell Technology (CA and MA, USA) to establish the safety and tolerability of subretinal transplantation of hES cell-derived retinal pigment epithelium (RPE) in patients with Star‐ gardt's Macular Dystrophy (SMD) and Dry age-related Macular Degeneration (Dry AMD) represents an important step towards therapeutic use of hES cells [68]. Although long-term follow up is essential and eye is an immune-privileged site; it is still encouraging to note that there are no associated signs of hyperproliferation, tumorigenicity, ectopic tissue formation,

An Overview of Pluripotent Stem Cells http://dx.doi.org/10.5772/55130 7

A major progress in the stem cell field was generation of induced pluripotent stem (iPS) cells by the reprogramming of somatic cells to an embryonic stem cell state using a cocktail of transcription factors. In 2006, Takahashi and Yamanaka reprogrammed mouse fibroblasts through retroviral transduction with 24 candidate genes [3]. The pool of genes was gradually reduced to four transcription factors, Oct4, Sox2, c-Myc, and Klf4. The results were rapidly confirmed by various researchers [69-71]. Soon the technology was successfully applied to generate iPS cells from human fibroblasts [4, 72, 73]. Concurrently, another group identified Oct4, Sox2, Nanog, and Lin28 to be sufficient to reprogram human cells, with Oct4 and Sox2 appearing essential and the other two factors either strongly (Nanog) or modestly (Lin28)

The different ways for generation of mouse and human iPS cells using various reprogramming factors has been well summarized by Maherali and Hochedlinger [75] and Kiskinis and Eggan [76]. The choice of a gene delivery system is a key aspect for generation of iPS cells and has been very well reviewed by Oh et al [77]. Many researchers have reported use of integrating viral vectors such as retroviral [4, 73, 78] and lentiviral vectors [74, 79], non-integrating viral vectors such as adenoviral [80] and Sendaiviral vectors [81], nonviral methods such as plasmid DNA [82], piggyBac transposons [83, 84], recombinant proteins [85, 86], mRNAs [87] and small molecules such as valproic acid [88]. Moreover, derivation of iPS cells from patients suffering from the neurodegenerative disease amyotrophic lateral sclerosis (ALS) [89] as well as patients with other diseases, including juvenile onset type 1 diabetes mellitus, Parkinson disease (PD)

As a potential application in cell based therapy, one of the major advantages of iPS cells is the avoidance of immune rejection, since they are derived from a patient's own cells, as well as ethical issues associated with the use of human embryos. Furthermore, iPS cells are similar to ES cells in many aspects, including cell morphology, expression of pluripotency markers, long

or immune- rejection after 4 months of transplantation.

**7. Induced pluripotent stem cells**

influencing the efficiency of reprogramming [74].

[90], and spinal muscular atrophy (SMA) [91] has been reported.

**8. Advantages and disadvantages of iPS cells**

Human ES cells have the ability to form 200 odd cell types in our body. Essentially, ES cells can be differentiated spontaneously by embryoid body formation or by directed differentiation using a cocktail of growth factors. Several growth factors have been shown to direct differen‐ tiation of ES cells namely activin-A and transforming growth factor (TGF-β1) mainly induce mesodermal cells; retinoic acid (RA), epidermal growth factor (EGF), BMP-4, and bFGF activate ectodermal and mesodermal cells; β nerve growth factor (NGF) and hepatocyte growth factor (HGF) differentiate all three embryonic germ layers [50-53]. Directed differen‐ tiation is a more controlled process involving stage specific sequential addition of growth inducers and inhibitors which are known to effect key pathways. For e.g. activin A and BMP4 are two such growth factors which have been used widely for cardiogenic differentiation. Various studies have shown that hES cells can be differentiated into neuronal [54], hemato‐ poietic [55], endothelial [56], muscle [57], cardiac [58, 59] pancreatic [60, 61], hepatic [62] lineages. Although hES stem cell lines are similar with respect to self-renewal and expression of pluripotency markers, published literature however suggests that they exhibit differences in their differentiation ability under identical culture conditions [63, 64].

## **6. Potential use of ES cells**

The remarkable features of hES cells has served as an important breakthrough for basic research and has great potential for regenerative medicine. ES cells may act as key research tools for understanding the complex events that occur during embryonic development which may explain the causes of birth defects. They are ideal candidates for studying apoptosis in early stage of embryo, mechanism of differentiation, mutagenesis, immune rejection and aging. Human ES cells and their derivatives may be used for testing therapeutic drug efficacy and toxicity. They also have wide applications in tissue engineering. Following their culture on polymer scaffold, it has been reported to coax stem cells to form tissues with characteristics of developing human cartilage, liver, neurons and blood vessels.

Despite being associated with the risk of inducing teratomas and immune rejection, the vital potential application of hES cells is the generation of cells and tissues that could be used for cell-based therapies. Human ES cells directed to differentiate into specific cell types offer the possibility of a renewable source of replacement cells and tissues to treat a myriad of diseases and disabilities including Parkinson's and Alzheimer's diseases, spinal cord injury, burns, heart failure and diabetes etc. The first FDA-approved phase-1 clinical trial for safety began with Geron's (Menlo Park, CA, USA) GRNOPC1 derived oligodendrocyte progenitor cells to treat complete thoracic-level spinal cord injury [65]. The trial was initially stalled for occurrence of microscopic cysts in animal transplants but was later approved [66, 67]. However, in November 2011 Geron dropped out of stem cell research for financial reasons and said that they would continue to monitor existing patients, and were attempting to find a partner that could continue their research. The recent success of a prospective clinical study of Advanced Cell Technology (CA and MA, USA) to establish the safety and tolerability of subretinal transplantation of hES cell-derived retinal pigment epithelium (RPE) in patients with Star‐ gardt's Macular Dystrophy (SMD) and Dry age-related Macular Degeneration (Dry AMD) represents an important step towards therapeutic use of hES cells [68]. Although long-term follow up is essential and eye is an immune-privileged site; it is still encouraging to note that there are no associated signs of hyperproliferation, tumorigenicity, ectopic tissue formation, or immune- rejection after 4 months of transplantation.

## **7. Induced pluripotent stem cells**

fibroblasts. A fully defined xeno-free medium (RegES), capable of supporting the expansion of hES cell lines, induced pluripotent stem (iPS) cells and adipose stem cells has been described [48]. Recently, Wang et al [49] have developed a xeno-free and feeder-cell-free culture system for propagating hES cells and hiPS cells using human plasma and human placenta extracts.

Human ES cells have the ability to form 200 odd cell types in our body. Essentially, ES cells can be differentiated spontaneously by embryoid body formation or by directed differentiation using a cocktail of growth factors. Several growth factors have been shown to direct differen‐ tiation of ES cells namely activin-A and transforming growth factor (TGF-β1) mainly induce mesodermal cells; retinoic acid (RA), epidermal growth factor (EGF), BMP-4, and bFGF activate ectodermal and mesodermal cells; β nerve growth factor (NGF) and hepatocyte growth factor (HGF) differentiate all three embryonic germ layers [50-53]. Directed differen‐ tiation is a more controlled process involving stage specific sequential addition of growth inducers and inhibitors which are known to effect key pathways. For e.g. activin A and BMP4 are two such growth factors which have been used widely for cardiogenic differentiation. Various studies have shown that hES cells can be differentiated into neuronal [54], hemato‐ poietic [55], endothelial [56], muscle [57], cardiac [58, 59] pancreatic [60, 61], hepatic [62] lineages. Although hES stem cell lines are similar with respect to self-renewal and expression of pluripotency markers, published literature however suggests that they exhibit differences

The remarkable features of hES cells has served as an important breakthrough for basic research and has great potential for regenerative medicine. ES cells may act as key research tools for understanding the complex events that occur during embryonic development which may explain the causes of birth defects. They are ideal candidates for studying apoptosis in early stage of embryo, mechanism of differentiation, mutagenesis, immune rejection and aging. Human ES cells and their derivatives may be used for testing therapeutic drug efficacy and toxicity. They also have wide applications in tissue engineering. Following their culture on polymer scaffold, it has been reported to coax stem cells to form tissues with characteristics

Despite being associated with the risk of inducing teratomas and immune rejection, the vital potential application of hES cells is the generation of cells and tissues that could be used for cell-based therapies. Human ES cells directed to differentiate into specific cell types offer the possibility of a renewable source of replacement cells and tissues to treat a myriad of diseases and disabilities including Parkinson's and Alzheimer's diseases, spinal cord injury, burns, heart failure and diabetes etc. The first FDA-approved phase-1 clinical trial for safety began with Geron's (Menlo Park, CA, USA) GRNOPC1 derived oligodendrocyte progenitor cells to treat complete thoracic-level spinal cord injury [65]. The trial was initially stalled for occurrence of microscopic cysts in animal transplants but was later approved [66, 67]. However, in November 2011 Geron dropped out of stem cell research for financial reasons and said that

in their differentiation ability under identical culture conditions [63, 64].

of developing human cartilage, liver, neurons and blood vessels.

**6. Potential use of ES cells**

6 Pluripotent Stem Cells

A major progress in the stem cell field was generation of induced pluripotent stem (iPS) cells by the reprogramming of somatic cells to an embryonic stem cell state using a cocktail of transcription factors. In 2006, Takahashi and Yamanaka reprogrammed mouse fibroblasts through retroviral transduction with 24 candidate genes [3]. The pool of genes was gradually reduced to four transcription factors, Oct4, Sox2, c-Myc, and Klf4. The results were rapidly confirmed by various researchers [69-71]. Soon the technology was successfully applied to generate iPS cells from human fibroblasts [4, 72, 73]. Concurrently, another group identified Oct4, Sox2, Nanog, and Lin28 to be sufficient to reprogram human cells, with Oct4 and Sox2 appearing essential and the other two factors either strongly (Nanog) or modestly (Lin28) influencing the efficiency of reprogramming [74].

The different ways for generation of mouse and human iPS cells using various reprogramming factors has been well summarized by Maherali and Hochedlinger [75] and Kiskinis and Eggan [76]. The choice of a gene delivery system is a key aspect for generation of iPS cells and has been very well reviewed by Oh et al [77]. Many researchers have reported use of integrating viral vectors such as retroviral [4, 73, 78] and lentiviral vectors [74, 79], non-integrating viral vectors such as adenoviral [80] and Sendaiviral vectors [81], nonviral methods such as plasmid DNA [82], piggyBac transposons [83, 84], recombinant proteins [85, 86], mRNAs [87] and small molecules such as valproic acid [88]. Moreover, derivation of iPS cells from patients suffering from the neurodegenerative disease amyotrophic lateral sclerosis (ALS) [89] as well as patients with other diseases, including juvenile onset type 1 diabetes mellitus, Parkinson disease (PD) [90], and spinal muscular atrophy (SMA) [91] has been reported.

#### **8. Advantages and disadvantages of iPS cells**

As a potential application in cell based therapy, one of the major advantages of iPS cells is the avoidance of immune rejection, since they are derived from a patient's own cells, as well as ethical issues associated with the use of human embryos. Furthermore, iPS cells are similar to ES cells in many aspects, including cell morphology, expression of pluripotency markers, long telomeres and capability to form embryoid bodies, teratoma, and viable chimeras [92, 93]. Apart from use in cell-based therapy, iPS cells derived from patients with disease can serve as an effective model to understand the mechanisms of diseases.

multipotent adult stem cells (MASCs) [103], very small embryonic like stem cells (VSELs) [104]. Although these cells may represent an overlapping type of stem cells, the most characterized among these cells to the single cell level is VSELs and they have been isolated and identified

VSELs are defined as epiblast derived stem cells, which are deposited early during organo‐ genesis and may serve as source of tissue commited stem cells. Pluripotent VSELs (Oct4+

tissues [105]; highest numbers being in brain, kidneys, muscles, pancreas and bone marrow [106]. These are diploid cells with high telomerase activity, express other pluripotent (Rex-1, Nanog, SSEA and Klf-4) and germ cell (Mvh, Stella, Fragilis, Nobox and Hdac-6) markers and decrease in numbers with age [107]. An important evidence for pluripotency of VSELs is hypomethylated status of OCT-4 promoter and its association with transcription promoting histones [108] as well as presence of bivalent domains [109]. Like embryonic stem cells they do not express MHC class I and HLA-DR antigens and are also negative for mesenchymal stem cell markers like CD90, CD105, CD29. They are very small in size (3-5 um in mice), have a large nucleo-cytoplasmic ratio, and open chromatin structure for OCT-4 and Nanog promoter [107]. OCT-4 expression at mRNA and protein level in VSELs has been confirmed using sequence specific primers. VSELs have the ability to differentiate into three germ layers *in vitro*, however unlike ES cells, VSELs neither complement during blastocyst development nor form teratomas in immuno-deficient mice [110]. Attempts have been made to propagate them on feeder layers, but they do not self-renew as easily as the established embryonic stem cell lines possibly because of altered methylation status of some developmentally crucial genes. Similar VSELs have also been isolated from human umbilical cord blood, mobilized peripheral blood, and

, lin-

VSELs are descendants of epiblast stage pluripotent stem cells. They get deposited in various body organs including the gonads in early stages of development, as a quiescent stem cell population which possibly serves as a back up to the tissue committed stem cells (TCSCs) [112]. These two populations of stem cells (VSELs and TCSCs) together are responsible in bringing about tissue renewal, homeostasis and regeneration after injury throughout life and decrease in number with age. The co-existence of two stem cell populations (the more primitive being quiescent and the progenitor being more rapidly dividing) has been recently proposed [113, 114]. VSELs are the DNA label-retaining (e.g. BrdU), quiescent stem cells with lower metabolic state whereas the tissue committed stem cells actively divide and do not retain DNA label over time. They are highly mobile, respond to the SDF-1 gradient and enter into circulation in case of any injury to bring about regeneration and homeostasis. They are also considered as a

missing link to support the germ-line hypothesis of cancer development [115, 116].

*VSELs in Umbilical Cord Blood (UCB):* A population of human cells similar to murine bone marrow derived VSELs was first reported by Kucia et al in umbilical cord blood [117]. These UCB derived VSELs (Lin-/CD45-/CD133+) ranged between 6-8 um in size, possess large nuclei and express nuclear embryonic transcription factors OCT-4, Nanog and cell surface SSEA-4. The strategy of isolation of VSELs from cord blood is hampered by their small size as they get

, CD45- [104] and also by differential

) were first reported by Ratajczak and group in various adult mice

,

9

An Overview of Pluripotent Stem Cells http://dx.doi.org/10.5772/55130

in several adult body organs.

, Lin-

, CD45-

adult bone marrow by flow cytometry as CD133+

centrifugation method [15, 111].

, Sca1+

SSEA1+

However, use of iPS cells have several drawbacks and are mostly related to current reprog‐ ramming methods. Viral vectors employed for gene delivery has led to the integration of multiple viruses into iPS cell genomes, resulting in tumorigenesis due to genetic abnormalities in the cells. Moreover, the efficiency of reprogramming of human iPS cells from fibroblasts is very low, approximately less than 0.02% [94]. The use of Myc gene as a reprogramming factor and/or the reactivation of a silenced Myc gene might cause iPS cells to become cancer cells [95].

Recently, three studies published in Nature showed that the reprogramming process and the subsequent culture of iPS cells *in vitro* can induce genetic and epigenetic abnormalities in these cells. Gore et al [96] found on an average of five point mutations in each of the iPS cell line analyzed, with the majority of the mutations being non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. Hussein and colleagues [97] showed that copy number variations (CNVs) occurred at a high rate during the process of reprogramming leading to genetic mosaicism in early-passage iPSCs. Analysis of the CG methylation patterns by Lister et al [98] identified numerous differentially methy‐ lated CG regions (CG-DMRs) between iPS cells and ES cells. The presence of a core set of CG-DMRs in every iPS cell line suggests hotspots of failed epigenomic reprogramming. These studies raise concerns over the implications of such aberrations for future applications of iPS cells. A much more in-depth research is necessary to understand about the reprogramming process and the biological consequences of these genomic and epigenomic changes needs to be investigated.

## **9. Very small embryonic like stem cells**

The ethical and other technical issues concerning the use of ES cells in regenerative medicine have led to search for alternative stem cells with therapeutic potential. In this regard adult stem cells can potentially provide a therapeutic alternative to ES or iPS cells. Though adult stem cells are known to be tissue specific and can only differentiate into cells of their tissues of origin, nevertheless several studies have reported that adult stem cells can differentiate in to cells of completely different lineage. The process is termed as adult stem cell plasticity. Wagers and Weissman proposed few potential mechanisms and explanations for the observed adult stem cell plasticity [99]. The potential mechanisms include trans-differentiation or dedifferentiation of stem cells, presence of multiple different stem cells in a tissue, presence of pluripotent stem cells in addition to adult stem cells and cell fusion of stem cell with cell of different lineage. However, several lines of evidence support existence of pluripotent stem cells in adult tissues that can differentiate into all three lineages explaining adult plasticity the best. Many investigators have reported presence of pluripotent stem cells in adult tissues and were defined either as mesenchymal stem cells (MSCs) [100], multipotent adult progenitor cells (MAPCs) [101], marrow isolated adult multilineage inducible cells (MIAMI) [102], multipotent adult stem cells (MASCs) [103], very small embryonic like stem cells (VSELs) [104]. Although these cells may represent an overlapping type of stem cells, the most characterized among these cells to the single cell level is VSELs and they have been isolated and identified in several adult body organs.

telomeres and capability to form embryoid bodies, teratoma, and viable chimeras [92, 93]. Apart from use in cell-based therapy, iPS cells derived from patients with disease can serve as

However, use of iPS cells have several drawbacks and are mostly related to current reprog‐ ramming methods. Viral vectors employed for gene delivery has led to the integration of multiple viruses into iPS cell genomes, resulting in tumorigenesis due to genetic abnormalities in the cells. Moreover, the efficiency of reprogramming of human iPS cells from fibroblasts is very low, approximately less than 0.02% [94]. The use of Myc gene as a reprogramming factor and/or the reactivation of a silenced Myc gene might cause iPS cells to become cancer cells [95].

Recently, three studies published in Nature showed that the reprogramming process and the subsequent culture of iPS cells *in vitro* can induce genetic and epigenetic abnormalities in these cells. Gore et al [96] found on an average of five point mutations in each of the iPS cell line analyzed, with the majority of the mutations being non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. Hussein and colleagues [97] showed that copy number variations (CNVs) occurred at a high rate during the process of reprogramming leading to genetic mosaicism in early-passage iPSCs. Analysis of the CG methylation patterns by Lister et al [98] identified numerous differentially methy‐ lated CG regions (CG-DMRs) between iPS cells and ES cells. The presence of a core set of CG-DMRs in every iPS cell line suggests hotspots of failed epigenomic reprogramming. These studies raise concerns over the implications of such aberrations for future applications of iPS cells. A much more in-depth research is necessary to understand about the reprogramming process and the biological consequences of these genomic and epigenomic changes needs to

The ethical and other technical issues concerning the use of ES cells in regenerative medicine have led to search for alternative stem cells with therapeutic potential. In this regard adult stem cells can potentially provide a therapeutic alternative to ES or iPS cells. Though adult stem cells are known to be tissue specific and can only differentiate into cells of their tissues of origin, nevertheless several studies have reported that adult stem cells can differentiate in to cells of completely different lineage. The process is termed as adult stem cell plasticity. Wagers and Weissman proposed few potential mechanisms and explanations for the observed adult stem cell plasticity [99]. The potential mechanisms include trans-differentiation or dedifferentiation of stem cells, presence of multiple different stem cells in a tissue, presence of pluripotent stem cells in addition to adult stem cells and cell fusion of stem cell with cell of different lineage. However, several lines of evidence support existence of pluripotent stem cells in adult tissues that can differentiate into all three lineages explaining adult plasticity the best. Many investigators have reported presence of pluripotent stem cells in adult tissues and were defined either as mesenchymal stem cells (MSCs) [100], multipotent adult progenitor cells (MAPCs) [101], marrow isolated adult multilineage inducible cells (MIAMI) [102],

an effective model to understand the mechanisms of diseases.

be investigated.

8 Pluripotent Stem Cells

**9. Very small embryonic like stem cells**

VSELs are defined as epiblast derived stem cells, which are deposited early during organo‐ genesis and may serve as source of tissue commited stem cells. Pluripotent VSELs (Oct4+ , SSEA1+ , Sca1+ , Lin- , CD45- ) were first reported by Ratajczak and group in various adult mice tissues [105]; highest numbers being in brain, kidneys, muscles, pancreas and bone marrow [106]. These are diploid cells with high telomerase activity, express other pluripotent (Rex-1, Nanog, SSEA and Klf-4) and germ cell (Mvh, Stella, Fragilis, Nobox and Hdac-6) markers and decrease in numbers with age [107]. An important evidence for pluripotency of VSELs is hypomethylated status of OCT-4 promoter and its association with transcription promoting histones [108] as well as presence of bivalent domains [109]. Like embryonic stem cells they do not express MHC class I and HLA-DR antigens and are also negative for mesenchymal stem cell markers like CD90, CD105, CD29. They are very small in size (3-5 um in mice), have a large nucleo-cytoplasmic ratio, and open chromatin structure for OCT-4 and Nanog promoter [107]. OCT-4 expression at mRNA and protein level in VSELs has been confirmed using sequence specific primers. VSELs have the ability to differentiate into three germ layers *in vitro*, however unlike ES cells, VSELs neither complement during blastocyst development nor form teratomas in immuno-deficient mice [110]. Attempts have been made to propagate them on feeder layers, but they do not self-renew as easily as the established embryonic stem cell lines possibly because of altered methylation status of some developmentally crucial genes. Similar VSELs have also been isolated from human umbilical cord blood, mobilized peripheral blood, and adult bone marrow by flow cytometry as CD133+ , lin- , CD45- [104] and also by differential centrifugation method [15, 111].

VSELs are descendants of epiblast stage pluripotent stem cells. They get deposited in various body organs including the gonads in early stages of development, as a quiescent stem cell population which possibly serves as a back up to the tissue committed stem cells (TCSCs) [112]. These two populations of stem cells (VSELs and TCSCs) together are responsible in bringing about tissue renewal, homeostasis and regeneration after injury throughout life and decrease in number with age. The co-existence of two stem cell populations (the more primitive being quiescent and the progenitor being more rapidly dividing) has been recently proposed [113, 114]. VSELs are the DNA label-retaining (e.g. BrdU), quiescent stem cells with lower metabolic state whereas the tissue committed stem cells actively divide and do not retain DNA label over time. They are highly mobile, respond to the SDF-1 gradient and enter into circulation in case of any injury to bring about regeneration and homeostasis. They are also considered as a missing link to support the germ-line hypothesis of cancer development [115, 116].

*VSELs in Umbilical Cord Blood (UCB):* A population of human cells similar to murine bone marrow derived VSELs was first reported by Kucia et al in umbilical cord blood [117]. These UCB derived VSELs (Lin-/CD45-/CD133+) ranged between 6-8 um in size, possess large nuclei and express nuclear embryonic transcription factors OCT-4, Nanog and cell surface SSEA-4. The strategy of isolation of VSELs from cord blood is hampered by their small size as they get discarded along with debris. Recently our studies reported that VSELs settle along with RBCs and are not enriched in interphase layer of MNCs obtained after ficoll separation of cord blood [15].These VSELs expressed pluripotent markers OCT-4,primitive marker CD133 along with primordial germ cell marker stella and fragilis indicating their epiblast origin. Our studies have also shown the presence of VSELs in the discarded fractions of bone marrow and cord blood obtained after processing [15].

testis as mentioned earlier. We have recently reviewed various publications on ovarian stem cells and explained the results in the context of VSEL biology [122]. Readers are encouraged

An Overview of Pluripotent Stem Cells http://dx.doi.org/10.5772/55130 11

Based on our studies in ovarian stem cells and other literature, we have proposed a model for oogenesis and follicular assembly in adult mammalian ovaries [122]. According to the model, VSELs with nuclear Oct-4 that are located in the OSE undergo asymmetric cell division and give rise to cells with cytoplasmic Oct-4 (OGSCs, which intensely stain with Haematoxylin). The OGSCs undergo further proliferation, meiosis and differentiation to assemble into primordial follicles in the OSE. The granulosa cells are formed by the epithelial cells through epithelial mesenchymal transition. As the follicles grow and further mature they shift into the

The clinical potential of VSELs, isolated from cord blood or bone marrow by flow cytometry, is justbeginningtoemerge.Invariousdiseasemodelslikemyocardialinfarct[125,126],stroke[127], skin burn injury [128], neural regeneration [129] etc. these cells get mobilized into circulation within24hours.Formyocardialregeneration,theVSELsareveryefficienttoimproveLVejection fraction and attenuation of myocardial hypertrophy [126]. As they become scarce with age,

The identification of VSELs in gonads has far reaching implications in reproductive health issues. Understanding the biology of VSELs in gonads may help explain the mechanisms of different pathologies of gonads and may pave for new treatments for infertility. However, application of VSELs to improve reproductive health needs to be researched and established. We have recently studied the differential effect of busulphan on the relatively quiescent VSELs versus rapidly dividing germ cells in adult mice gonads (unpublished results). The VSELs were found to be resistant to the treatment, however were unable to differentiate probably due to the altered niche of VSELs due to treatment. Ratajczak group recently reported that VSELs in mouse bone marrow are resistant to total body irradiation [130]. They observed that there was increase in proliferation of VSELs post treatment, although were unable to reconstitute the bone marrow. These studies open up newer and exciting avenues for fertility preservation

VSELs can be derived easily from autologus source and do not form teratoma easily [131]. Thus both the major concerns associated with ES cells of immune-rejection and risk of teratoma

regeneration becomes inefficient resulting in age-related disease manifestations.

in cancer survivors who are rendered infertile by various cancer treatments.

**11. Advantages of VSELs over ES cells**

formation is taken care of.

to read the review for more details.

**10. Clinical potential of VSELs**

ovarian medulla.

*VSELs in adult mammalian gonads:* Initial studies by Ratajczak group have shown that mouse testis harbor VSELs [106]. Our group has identified presence of VSELs in testis of human and mice as well as in ovaries of human, sheep, monkey, rabbit and mice [13]. These VSELs are localized in the basal layer of cells adjacent to the basement membrane in seminiferous tubules [13] and were found interspersed with the ovarian surface epithelial cells [14]. The main approach in identifying the VSELs in adult mammalian gonads involves studying differential expression of a pluripotent marker OCT-4. OCT-4 is an octamer binding transcription factor required for maintaining pluripotency of cell. Published literature on OCT-4 in somatic stem cells has confused stem cell researchers [118-120] because of the presence of several pseudo‐ genes and alternatively spliced transcripts [118, 121]. Thus a careful designing of primers for RT-PCR analysis and proper selection of antibodies becomes essential to detect specific transcripts. Also a careful selection of OCT-4 antibodies is essential to detect pluripotent stem cells [119]. We used a polyclonal OCT-4 antibody that enabled the simultaneous identification of VSELs with nuclear OCT-4 and tissue committed stem cells with cytoplasmic OCT-4. In addition, careful selection of primers for OCT-4A and total OCT-4 for Q-PCR studies has helped us generate interesting results [13-15, 122].

*VSELs in Testis:* We have documented that adult human testis harbors a population of pluripotent VSELs (with nuclear OCT-4A) which are more primitive to Adark Spermatogonial Stem Cell (SSC) (with cytoplasmic OCT-4B). The VSELs possibly give rise to Adark SSCs, which in turn undergo clonal expansion as evident by the presence of cytoplasmic bridges between the rapidly dividing cells [13]. OCT-4 is not immuno-localized in more differentiated male germ cells. Based on this study a new hierarchy of testicular cells was proposed with all testicular cells originating from VSELs and not from SSCs as generally believed. Similarly presence of VSELs distinct from SSCs was also identified in mouse testicular tissue.

*VSELs in Ovaries:* The long- held dogma in female biology is that women and other mammalian females are born with fixed and non-renewing pool of germ cells, which are enclosed in structures called follicles. Their number decrease with age due to ovulation or atresia and their exhaustion lead to menopause. However in last 8 years several investigators with access to modern molecular techniques have convincingly demonstrated that adult mammalian ovaries harbor stem cells and undergo postnatal oogeneisis and thus have challenged the central dogma. Presence of PSCs in adult ovary has been demonstrated by many groups [11, 14, 123, 124]. Our group has identified two distinct types of stem cells in ovarian surface epithelium (OSE) of human and other mammalian species [14, 122]. The two stem cells are VSELs that express OCT-4 in nucleus, which are pluripotent and slightly larger progenitor committed cells (termed Ovarian Germ Stem Cells-OGSCs) that express OCT-4 cytoplasmically. This is very similar to reported presence of VSELs and Spermatogonial stem cells in adult mammalian testis as mentioned earlier. We have recently reviewed various publications on ovarian stem cells and explained the results in the context of VSEL biology [122]. Readers are encouraged to read the review for more details.

Based on our studies in ovarian stem cells and other literature, we have proposed a model for oogenesis and follicular assembly in adult mammalian ovaries [122]. According to the model, VSELs with nuclear Oct-4 that are located in the OSE undergo asymmetric cell division and give rise to cells with cytoplasmic Oct-4 (OGSCs, which intensely stain with Haematoxylin). The OGSCs undergo further proliferation, meiosis and differentiation to assemble into primordial follicles in the OSE. The granulosa cells are formed by the epithelial cells through epithelial mesenchymal transition. As the follicles grow and further mature they shift into the ovarian medulla.

## **10. Clinical potential of VSELs**

discarded along with debris. Recently our studies reported that VSELs settle along with RBCs and are not enriched in interphase layer of MNCs obtained after ficoll separation of cord blood [15].These VSELs expressed pluripotent markers OCT-4,primitive marker CD133 along with primordial germ cell marker stella and fragilis indicating their epiblast origin. Our studies have also shown the presence of VSELs in the discarded fractions of bone marrow and cord

*VSELs in adult mammalian gonads:* Initial studies by Ratajczak group have shown that mouse testis harbor VSELs [106]. Our group has identified presence of VSELs in testis of human and mice as well as in ovaries of human, sheep, monkey, rabbit and mice [13]. These VSELs are localized in the basal layer of cells adjacent to the basement membrane in seminiferous tubules [13] and were found interspersed with the ovarian surface epithelial cells [14]. The main approach in identifying the VSELs in adult mammalian gonads involves studying differential expression of a pluripotent marker OCT-4. OCT-4 is an octamer binding transcription factor required for maintaining pluripotency of cell. Published literature on OCT-4 in somatic stem cells has confused stem cell researchers [118-120] because of the presence of several pseudo‐ genes and alternatively spliced transcripts [118, 121]. Thus a careful designing of primers for RT-PCR analysis and proper selection of antibodies becomes essential to detect specific transcripts. Also a careful selection of OCT-4 antibodies is essential to detect pluripotent stem cells [119]. We used a polyclonal OCT-4 antibody that enabled the simultaneous identification of VSELs with nuclear OCT-4 and tissue committed stem cells with cytoplasmic OCT-4. In addition, careful selection of primers for OCT-4A and total OCT-4 for Q-PCR studies has

*VSELs in Testis:* We have documented that adult human testis harbors a population of pluripotent VSELs (with nuclear OCT-4A) which are more primitive to Adark Spermatogonial Stem Cell (SSC) (with cytoplasmic OCT-4B). The VSELs possibly give rise to Adark SSCs, which in turn undergo clonal expansion as evident by the presence of cytoplasmic bridges between the rapidly dividing cells [13]. OCT-4 is not immuno-localized in more differentiated male germ cells. Based on this study a new hierarchy of testicular cells was proposed with all testicular cells originating from VSELs and not from SSCs as generally believed. Similarly

*VSELs in Ovaries:* The long- held dogma in female biology is that women and other mammalian females are born with fixed and non-renewing pool of germ cells, which are enclosed in structures called follicles. Their number decrease with age due to ovulation or atresia and their exhaustion lead to menopause. However in last 8 years several investigators with access to modern molecular techniques have convincingly demonstrated that adult mammalian ovaries harbor stem cells and undergo postnatal oogeneisis and thus have challenged the central dogma. Presence of PSCs in adult ovary has been demonstrated by many groups [11, 14, 123, 124]. Our group has identified two distinct types of stem cells in ovarian surface epithelium (OSE) of human and other mammalian species [14, 122]. The two stem cells are VSELs that express OCT-4 in nucleus, which are pluripotent and slightly larger progenitor committed cells (termed Ovarian Germ Stem Cells-OGSCs) that express OCT-4 cytoplasmically. This is very similar to reported presence of VSELs and Spermatogonial stem cells in adult mammalian

presence of VSELs distinct from SSCs was also identified in mouse testicular tissue.

blood obtained after processing [15].

10 Pluripotent Stem Cells

helped us generate interesting results [13-15, 122].

The clinical potential of VSELs, isolated from cord blood or bone marrow by flow cytometry, is justbeginningtoemerge.Invariousdiseasemodelslikemyocardialinfarct[125,126],stroke[127], skin burn injury [128], neural regeneration [129] etc. these cells get mobilized into circulation within24hours.Formyocardialregeneration,theVSELsareveryefficienttoimproveLVejection fraction and attenuation of myocardial hypertrophy [126]. As they become scarce with age, regeneration becomes inefficient resulting in age-related disease manifestations.

The identification of VSELs in gonads has far reaching implications in reproductive health issues. Understanding the biology of VSELs in gonads may help explain the mechanisms of different pathologies of gonads and may pave for new treatments for infertility. However, application of VSELs to improve reproductive health needs to be researched and established. We have recently studied the differential effect of busulphan on the relatively quiescent VSELs versus rapidly dividing germ cells in adult mice gonads (unpublished results). The VSELs were found to be resistant to the treatment, however were unable to differentiate probably due to the altered niche of VSELs due to treatment. Ratajczak group recently reported that VSELs in mouse bone marrow are resistant to total body irradiation [130]. They observed that there was increase in proliferation of VSELs post treatment, although were unable to reconstitute the bone marrow. These studies open up newer and exciting avenues for fertility preservation in cancer survivors who are rendered infertile by various cancer treatments.

## **11. Advantages of VSELs over ES cells**

VSELs can be derived easily from autologus source and do not form teratoma easily [131]. Thus both the major concerns associated with ES cells of immune-rejection and risk of teratoma formation is taken care of.

## **12. Advantages of VSELs over iPS cells**

There is no need for reprogramming somatic cells (which may harbor mutations) to embryonic state when pluripotent ES-like stem cells can be harvested from adult tissues. They may also be superior to iPS cells since they are derived from a very quiescent stem cell population and are thus 'young' cells with long telomeres that could be isolated from an aged body, in contrast to iPS cells which are derived from terminally differentiated somatic skin fibroblasts (with shortened telomeres) that tend to accumulate DNA mutations over time. In addition, VSELs do not have epigenetic issues associated with iPS cells. Unlike iPS cells, there is no requirement of viral vectors and hence risk of transformation of VSELs into cancer cells is avoided.

[2] Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW, Donovan PJ, Blumenthal PD, Huggins GR, Gearhart JD (1998) Derivation of Pluripotent Stem Cells from Cul‐ tured Human Primordial Germ Cells. Proc Natl Acad Sci U S A. 95:13726-13731.

An Overview of Pluripotent Stem Cells http://dx.doi.org/10.5772/55130 13

[3] Takahashi K, Yamanaka S (2006) Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell 126: 663–676.

[4] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by De‐

[5] Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, Baba S, Ka‐ to T, Kazuki Y, Toyokuni S, Toyoshima M, Niwa O, Oshimura M, Heike T, Nakahata T, Ishino F, Ogura A, Shinohara T (2004) Generation of pluripotent stem cells from

[6] Guan K, Nayernia K, Maier LS, Wagner S, Dressel R, Lee JH, Nolte J, Wolf F, Li M, Engel W, Hasenfuss G (2006) Pluripotency of spermatogonial stem cells from adult

[7] Conrad S, Renninger M, Hennenlotter J, Wiesner T, Just L, Bonin M, Aicher W, Buhr‐ ing HJ, Mattheus U, Mack A, Wagner HJ, Minger S, Matzkies M, Reppel M, Heschel‐ er J, Sievert KD, Stenzl A, Skutella T (2008) Generation of pluripotent stem cells from

[8] Golestaneh N, Kokkinaki M, Pant D, Jiang J, DeStefano D, Fernandez-Bueno C, Rone JD, Haddad BR, Gallicano GI, Dym M (2009) Pluripotent stem cells derived from

[9] Kossack N, Meneses J, Shefi S, Nguyen HN, Chavez S, Nicholas C, Gromoll J, Turek PJ, Reijo-Pera RA (2009) Isolation and characterization of pluripotent human sperma‐

[10] Mizrak SC, Chikhovskaya JV, Sadri-Ardekani H, van Daalen S, Korver CM, Hovingh SE, Roepers-Gajadien HL, Raya A, Fluiter K, de Reijke TM, de la Rosette JJ, Knegt AC, Belmonte JC, van der Veen F, de Rooij DG, Repping S, van Pelt AM (2009) Em‐ bryonic stem cell-like cells derived from adult human testis. Human Reprod 25:

[11] Gong SP, Lee ST, Lee EJ, Kim DY, Lee G, Chi SG, Ryu BK, Lee CH, Yum KE, Lee HJ, Han JY, Tilly JL, Lim JM (2010) Embryonic Stem Cell-Like Cells Established by Cul‐

[12] Phinney DG, Prockop DJ (2007) Concise Review: Mesenchymal Stem/Multipotent Stromal Cells: The State of Transdifferentiation and Modes of Tissue Repair--Current

[13] Bhartiya D, Kasiviswanathan S, Unni SK, Pethe P, Dhabalia JV, Patwardhan S, Ton‐ gaonkar HB (2010) Newer Insights into Premeiotic Development of Germ Cells in

fined Factors. Cell 131: 861–872.

neonatal mouse testis. Cell 119: 1001-1012.

mouse testis. Nature 440: 1199-1203.

adult human testis. Nature 456: 344-349.

158-167.

Views. Stem Cells 25:2896-902.

adult human testes. Stem Cells Dev 18: 1115-1126.

togonial stem cell-derived cells. Stem Cells 27: 138-149.

ture of Adult Ovarian Cells in Mice. Fertil Steril 93:2594-2601.

## **13. Future perspectives**

Embryonic stem cells are considered to be 'magic bullets' having a great potential for cell-based therapy, however future clinical use of ES cells are still plagued by ethical issues. Hence there is urgent need to expand research in derivation and culture of pluripotent stem cells from alternate sources. Induced pluripotent stem cells though believed to be ideal candidates need to be exploited further to realize their clinical potential. Considering the potential advantages of VSELs over ES and iPS cells, the need for research to harness potentials of VSELs is high. Currently the availability of large number of VSELs for effective use in clinical applications is limited. Research is progressing towards expansion of VSELs in culture and is still in nascent stages. Also, many key questions have to be answered before realizing the full potential of stem cells.

### **Author details**

Deepa Bhartiya\* , Punam Nagvenkar, Kalpana Sriraman and Ambreen Shaikh

\*Address all correspondence to: deepa.bhartiya@yahoo.in; bhartiyad@nirrh.res.in

Stem Cell Biology Department, National Institute for Research in Reproductive Health, Par‐ el, Mumbai, India

## **References**

[1] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic Stem Cell Lines Derived from Human Blastocysts. Sci‐ ence 282:1145-1147.

[2] Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW, Donovan PJ, Blumenthal PD, Huggins GR, Gearhart JD (1998) Derivation of Pluripotent Stem Cells from Cul‐ tured Human Primordial Germ Cells. Proc Natl Acad Sci U S A. 95:13726-13731.

**12. Advantages of VSELs over iPS cells**

**13. Future perspectives**

12 Pluripotent Stem Cells

stem cells.

**Author details**

Deepa Bhartiya\*

el, Mumbai, India

**References**

ence 282:1145-1147.

There is no need for reprogramming somatic cells (which may harbor mutations) to embryonic state when pluripotent ES-like stem cells can be harvested from adult tissues. They may also be superior to iPS cells since they are derived from a very quiescent stem cell population and are thus 'young' cells with long telomeres that could be isolated from an aged body, in contrast to iPS cells which are derived from terminally differentiated somatic skin fibroblasts (with shortened telomeres) that tend to accumulate DNA mutations over time. In addition, VSELs do not have epigenetic issues associated with iPS cells. Unlike iPS cells, there is no requirement of viral vectors and hence risk of transformation of VSELs into cancer cells is avoided.

Embryonic stem cells are considered to be 'magic bullets' having a great potential for cell-based therapy, however future clinical use of ES cells are still plagued by ethical issues. Hence there is urgent need to expand research in derivation and culture of pluripotent stem cells from alternate sources. Induced pluripotent stem cells though believed to be ideal candidates need to be exploited further to realize their clinical potential. Considering the potential advantages of VSELs over ES and iPS cells, the need for research to harness potentials of VSELs is high. Currently the availability of large number of VSELs for effective use in clinical applications is limited. Research is progressing towards expansion of VSELs in culture and is still in nascent stages. Also, many key questions have to be answered before realizing the full potential of

, Punam Nagvenkar, Kalpana Sriraman and Ambreen Shaikh

Stem Cell Biology Department, National Institute for Research in Reproductive Health, Par‐

[1] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic Stem Cell Lines Derived from Human Blastocysts. Sci‐

\*Address all correspondence to: deepa.bhartiya@yahoo.in; bhartiyad@nirrh.res.in


Adult Human Testis using Oct-4 as a Stem Cell Marker. J Histochem Cytochem 58:1093-1106.

[25] Ying QL, Nichols J, Chambers I, Smith A (2003) BMP Induction of Id Proteins Sup‐ presses Differentiation and Sustains Embryonic Stem Cell Self-Renewal in Collabora‐

An Overview of Pluripotent Stem Cells http://dx.doi.org/10.5772/55130 15

[26] Loser P, Schirm J, Guhr A, Wobus AM, Kurtz A (2010) Human Embryonic Stem Cell

[27] Strelchenko N, Verlinsky O, Kukharenko V, Verlisnky Y (2004) Morula-derived Em‐

[28] Zhang X, Stojkovic P, Przyborski S, Cooke M, Armstrong L, Lako M, Stojkovic M (2006) Derivation of Human Embryonic Stem Cells From Developing And Arrested

[29] Klimanskaya I, Chung Y, Becker S, Lu SJ, Lanza R (2007) Derivation of Human Em‐

[30] Feki A, Hovatta O, Jaconi M (2008) Derivation of Human Embryonic Stem Cell Lines from Single Cells of 4-Cell Stage Embryos: Be Aware of the Risks. Hum Reprod 23:

[31] Geens M, Mateizel I, Sermon K, De Rycke M, Spits C, Cauffman G, Devroey P, Tour‐ naye H, Liebaers I, Van de Velde H (2009) Human Embryonic Stem Cell Lines De‐ rived from Single Blastomeres of Two 4-Cell Stage Embryos. Hum Reprod 24:

[32] Daheron L, Opitz SL, Zaehres H, Lensch MW, Andrews PW, Itskovitz-Eldor J, Daley GQ (2004) LIF/STAT3 Signaling Fails to Maintain Self-Renewal of Human Embryonic

[33] Humphrey RK, Beattie GM, Lopez AD, Bucay N, King CC, Firpo MT, Rose-John S, Hayek A (2004) Maintenance of Pluripotency in Human Embryonic Stem Cells is

[34] Vallier L, Alexander M, Pedersen RA (2005) Activin/Nodal and FGF Pathways Coop‐ erate to Maintain Pluripotency of Human Embryonic Stem Cells. J Cell Sci 118:4495–

[35] Rho JY, Yu K, Han, JS, Chae JI, Koo DB, Yoon HS, Moon SY, Lee KK, Han YM (2006) Transcriptional Profiling of the Developmentally Important Signalling Pathways in

[36] Xu RH, Chen X, Li DS, Li R, Addicks GC, Glennon C, Zwaka TP Thomson JA (2002) BMP4 Initiates Human Embryonic Stem Cell Differentiation to Trophoblast. Nat Bio‐

[37] Xiao L, Yuan X, Sharkis SJ (2006) Activin A Maintains Self-Renewal and Regulates Fi‐ broblast Growth Factor, Wnt, and Bone Morphogenic Protein Pathways in Human

Lines And Their Use In International Research. Stem Cells 28:240-246.

bryonic Stem Cells from Single Blastomeres. Nat Protoc 2: 1963-1972.

bryonic Stem Cells. Reprod. Biomed. Online 9: 623-629.

tion With STAT3. Cell 115:281–292.

Embryos. Stem Cells 24:2669-2676.

Stem Cells. Stem Cells 22: 770–778.

STAT3 Independent. Stem Cells 22:522–530.

Human Embryonic Stem Cells. Hum Reprod 21:405–412.

Embryonic Stem Cells. Stem Cells 24: 1476–1486.

2874.

2709-2717.

4509.

technol 20: 1261–1264.


[25] Ying QL, Nichols J, Chambers I, Smith A (2003) BMP Induction of Id Proteins Sup‐ presses Differentiation and Sustains Embryonic Stem Cell Self-Renewal in Collabora‐ tion With STAT3. Cell 115:281–292.

Adult Human Testis using Oct-4 as a Stem Cell Marker. J Histochem Cytochem

[14] Parte S, Bhartiya D, Telang J, Daithankar V, Salvi V, Zaveri K, Hinduja I (2011) Detec‐ tion, Characterization, and Spontaneous Differentiation *In Vitro* of Very Small Em‐ bryonic-Like Putative Stem Cells in Adult Mammalian Ovary. Stem Cells Dev

[15] Bhartiya D, Shaikh A, Nagvenkar P, Kasiviswanathan S, Pethe P, Pawani H, Mohan‐ ty S, Rao SG, Zaveri K, Hinduja I (2012) Very Small Embryonic-Like Stem Cells with Maximum Regenerative Potential get Discarded during Cord Blood Banking and Bone Marrow Processing for Autologous Stem Cell Therapy. Stem Cells Dev 21:1-6.

[16] Bhartiya D, Kasiviswanathan S, Shaikh A (2012) Cellular Origin of Testis derived

[17] Evans M, Kaufman M (1981) Establishment in Culture of Pluripotent Cells from

[18] Martin GR (1981) Isolation of a Pluripotent Cell Line from Early Mouse Embryos Cultured in Medium Conditioned by Teratocarcinoma Stem Cells. Proc Natl Acad

[19] Chung Y, Klimanskaya I, Becker S, Marh J, Lu SJ, Johnson J, Meisner L, Lanza R (2006) Embryonic and Extraembryonic Stem Cell Lines Derived from Single Mouse

[20] Wakayama S, Hikichi T, Suetsugu R, Sakaide Y, Bui HT, Mizutani E, Wakayama T (2007) Efficient Establishment of Mouse Embryonic Stem Cell Lines from Single Blas‐

[21] Lorthongpanich C, Yang SH, Piotrowska-Nitsche K, Parnpai R, Chan AW (2008) De‐ velopment of Single Mouse Blastomeres into Blastocysts, Outgrowths and the Estab‐

[22] Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl M, Rogers D (1988) Inhibition of Pluripotential Embryonic Stem Cell Differentiation by Purified Polypep‐

[23] Williams RL, Hilton DJ, Pease S, Willson TA, Stewart CL, Gearing DP, Wagner EF, Metcalf D, Nicola NA, Gough NM (1988) Myeloid Leukaemia Inhibitory Factor Maintains the Developmental Potential of Embryonic Stem Cells. Nature 336:684–

[24] Yoshida K, Chambers I, Nichols J, Smith A, Saito M, Yasukawa K, Shoyab M, Taga T, Kishimoto T (1994) Maintenance of the Pluripotential Phenotype of Embryonic Stem Cells Through Direct Activation of gp130 Signalling Pathways. Mech Dev 45:163–

Pluripotent Stem Cells: A Case for VSELs. Stem Cells Dev 21:670-674.

Mouse Embryos. Nature 292:154–156.

Blastomeres. Nature 439: 216–219.

tides. Nature 336:688–690.

687.

171.

tomeres and Polar Bodies. Stem Cells 25: 986–993.

lishment of Embryonic Stem Cells. Reproduction 135:805–813.

Sci USA 78:7634–7638.

58:1093-1106.

14 Pluripotent Stem Cells

20:1451-64.


[38] Greber B, Lehrach H, Adjaye J (2007) Fibroblast Growth Factor 2 Modulates Trans‐ forming Growth Factor β signaling in Mouse Embryonic Fibroblasts and Human ESCs (hESCs) to Support hESC Self-renewal. Stem Cells 25: 455–464.

[50] Slager HG, Van Inzen W, Freund E, Van der Eijnden-Van Raaij AJ, Mummery CL (1993) Transforming Growth Factor-beta in the Early Mouse Embryo: Implications for the Regulation of Muscle Formation and Implantation. Dev Genet 14: 212-224. [51] Rohwedel J, Maltsev V, Bober E, Arnold HH, Hescheler J, Wobus AM (1994) Muscle Cell Differentiation of Embryonic Stem Cells Reflects Myogenesis in vivo: Develop‐ mentally Regulated Expression of Myogenic Determination Genes and Functional

An Overview of Pluripotent Stem Cells http://dx.doi.org/10.5772/55130 17

[52] Bain G, Kitchens D, Yao M, Huettner JE, Gottlieb DI (1995) Embryonic Stem Cells Ex‐

[53] Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton DA, Benvenisty N (2000). Effects of Eight Growth Factors on the Differentiation of Cells Derived from Human Embry‐

[54] Kirkeby A, Grealish S, Wolf DA, Nelander J, Wood J, Lundblad M, Lindvall O, Par‐ mar M (2012) Generation of Regionally Specified Neural Progenitors and Functional Neurons from Human Embryonic Stem Cells Under Defined Conditions. Cell Rep

[55] Chang KH, Bonig H, Papayannopoulou T (2011) Generation and Characterization of Erythroid Cells from Human Embryonic Stem Cells and Induced Pluripotent Stem

[56] Li Z, Hu S, Ghosh Z, Han Z, Wu JC (2011) Functional Characterization and Expres‐ sion Profiling of Human Induced Pluripotent Stem Cell- and Embryonic Stem Cell-

[57] Barberi T, Bradbury M, Dincer Z, Panagiotakos G, Socci ND, Studer L (2007) Deriva‐ tion of Engraftable Skeletal Myoblasts from Human Embryonic Stem Cells. Nat Med

[58] Laflamme MA, Chen KY, Naumova AV, Muskheli V, Fugate JA, Dupras SK, Rein‐ ecke H, Xu C, Hassanipour M, Police S, O'Sullivan C, Collins L, Chen Y, Minami E, Gill EA, Ueno S, Yuan C, Gold J, Murry CE (2007) Cardiomyocytes Derived from Hu‐ man Embryonic Stem Cells in Pro-Survival Factors Enhance Function of Infarcted

[59] Yang L, Soonpaa MH, Adler ED, Roepke TK, Kattman SJ, Kennedy M, Henckaerts E, Bonham K, Abbott GW, Linden RM, Field LJ., Keller GM (2008) Human Cardiovas‐ cular Progenitor Cells Develop from a KDR+ Embryonic-Stem-Cell-Derived Popula‐

[60] Jiang J, Au M, Lu K, Eshpeter A, Korbutt G, Fisk G, Majumdar AS (2007) Generation of Insulin Producing Islet-Like from Human Embryonic Stem Cells. Stem Cells

[61] Kroon E, Martinson LA, Kadoya K, Bang AG, Kelly OG, Eliazer S, Young H, Richard‐ son M, Smart NG, Cunningham J, Agulnick AD, D'Amour KA, Carpenter MK,

Expression of Ionic Currents. Dev Biol 164: 87-101.

onic Stem Cells. PNAS 97: 11307-11312.

Cells: An Overview. Stem Cells Int 2011:791604.

Rat Hearts. Nat Biotechnol 25:1015–1024.

tion. Nature 453: 524-528.

25:1940-1953.

Derived Endothelial Cells. Stem Cells Dev 20:1701-1710.

1:703-714.

13: 642-648.

press Neuronal Properties in vitro. Dev Biol 168: 342-357.


[50] Slager HG, Van Inzen W, Freund E, Van der Eijnden-Van Raaij AJ, Mummery CL (1993) Transforming Growth Factor-beta in the Early Mouse Embryo: Implications for the Regulation of Muscle Formation and Implantation. Dev Genet 14: 212-224.

[38] Greber B, Lehrach H, Adjaye J (2007) Fibroblast Growth Factor 2 Modulates Trans‐ forming Growth Factor β signaling in Mouse Embryonic Fibroblasts and Human

[39] Bongso A, Fong CY, Ng SC, Ratnam SS (1994) Isolation and culture of inner cell mass

[40] Amit M, Margulets V, Segev H, Shariki K, Laevsky I, Coleman R, Itskovitz-Eldor J (2003) Human Feeder Layers for Human Embryonic Stem Cells. Biol Reprod

[41] Richards M, Fong CY, Chan WK, Wong PC, Bongso A (2002) Human Feeders Sup‐ port Prolonged Undifferentiated Growth of Human Inner Cell Masses and Embryon‐

[42] Miyamoto K, Hayashi K, Suzuki T, Ichihara S, Yamada T, Kano Y, Yamabe T, Ito Y (2004) Human placenta feeder layers support undifferentiated growth of primate em‐

[43] Genbacev O, Krtolica A, Zdravkovic T, Brunette E, Powell S, Nath A, Caceres E, McMaster M, McDonagh S, Li Y, Mandalam R, Lebkowski J, Fisher SJ (2005) Serumfree derivation of human embryonic stem cell lines on human placental fibroblast

[44] Klimanskaya I, Chung Y, Meisner L, Johnson J, West MD, Lanza R (2005) Human Embryonic Stem Cells Derived Without Feeder Cells. Lancet 365:1636-1641.

[45] Ludwig TE, Levenstein ME, Jones JM, Berggren WT, Mitchen ER, Frane JL, Crandall LJ, Daigh CA, Conard KR, Piekarczyk MS, Llanas RA, Thomson JA (2006) Derivation of Human Embryonic Stem Cells in Defined Conditions. Nat Biotechnol 24:185-187.

[46] Crook JM, Peura TT, Kravets L, Bosman AG, Buzzard JJ, Horne R, Hentze H, Dunn NR, Zweigerdt R, Chua F, Upshall A, Colman A (2007) The Generation of Six Clini‐

[47] Sidhu KS, Ryan JP, Tuch BE (2008). Derivation of a New hESC Line, Endeavour-1

[48] Rajala K, Lindroos B, Hussein SM, Lappalainen RS, Pekkanen-Mattila M, Inzunza J, Rozell B, Miettinen S, Narkilahti S, Kerkelä E, Aalto-Setälä K, Otonkoski T, Suuronen R, Hovatta O, Skottman H (2010) A Defined and Xeno-Free Culture Method Enabling the Establishment of Clinical Grade Human Embryonic, Induced Pluripotent and

[49] Wang Q, Mou X, Cao H, Meng Q, Ma Y, Han P, Jiang J, Zhang H, Ma Y (2012) A Novel Xeno-Free and Feeder-Cell-Free System for Human Pluripotent Stem Cell Cul‐

cal-Grade Human Embryonic Stem Cell Lines. Cell Stem Cell 1: 490–494.

and its Clonal Propagation. Stem Cells Dev 17:41-52.

Adipose Stem Cells. PLoS One 5:e10246.

ture. Protein Cell 3:51-59.

ESCs (hESCs) to Support hESC Self-renewal. Stem Cells 25: 455–464.

cells from human blastocysts. Hum Reprod 9:2110–2117.

ic Stem Cells. Nat Biotechnol 20:933–936.

bryonic stem cells. Stem Cells 22:433–440.

feeders. Fertil Steril 83:1517-1529.

68:2150–2156.

16 Pluripotent Stem Cells


Baetge EE (2008) Pancreatic Endoderm Derived from Human Embryonic Stem Cells Generates Glucose-Responsive Insulin-Secreting Cells in vivo. Nat Biotechnol 26:443–452.

[74] Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA (2007) Induced Pluripo‐ tent Stem Cell Lines Derived from Human Somatic Cells. Science 318: 1917–1920.

An Overview of Pluripotent Stem Cells http://dx.doi.org/10.5772/55130 19

[75] Maherali N, Hochedlinger K (2008) Guidelines and Techniques for the Generation of

[76] Kiskinis E, Eggan K (2010) Progress toward the clinical application of patient-specific

[77] Oh SI, Lee CK, Cho KJ, Lee KO, Cho SG, Hong S (2012) Technological Progress in Generation of Induced Pluripotent Stem Cells for Clinical Applications. Scientific

[78] Aasen T, Raya A, Barrero MJ, Garreta E, Consiglio A, Gonzalez F, Vassena R, Bilic J, Pekarik V, Tiscornia G, Edel M, Boue S, Izpisua Belmonte JC (2008) Efficient and Rapid Generation of Induced Pluripotent Stem Cells from Human Keratinocytes. *Nat*

[79] Sommer CA, Stadtfeld M, Murphy GJ, Hochedlinger K, Kotton DN, Mostoslavsky G (2009) Induced Pluripotent Stem Cell Generation Using a Single Lentiviral Stem Cell

[80] Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K (2008) Induced Pluripotent

[81] Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M (2009) Efficient Induction of Transgene-Free Human Pluripotent Stem Cells Using a Vector Based on Sendai Vi‐ rus, an RNA Virus that Does Not Integrate Into the Host Genome. Proceedings of the

[82] Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S (2008) Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors. Science 322:949–953.

[83] Woltjen K, Michael IP, Mohseni P, Desai R, Mileikovsky M, Hämäläinen R, Cowling R, Wang W, Liu P, Gertsenstein M, Kaji K, Sung HK, Nagy A (2009) piggyBac Trans‐ position Reprograms Fibroblasts To Induced Pluripotent Stem Cells. Nature 458:766–

[84] Yusa K, Rad R, Takeda J, Bradley A (2009) Generation of Transgene-Free Induced Pluripotent Mouse Stem Cells by the piggyBac Transposon. Nature Methods 6: 363–

[85] Kim D, Kim CH, Moon JI, Chung YG, Chang MY, Han BS, Ko S, Yang E, Cha KY, Lanza R, Kim KS (2009) Generation of Human Induced Pluripotent Stem Cells by Di‐

rect Delivery of Reprogramming Proteins. Cell Stem Cell 4:472–476.

Stem Cells Generated Without Viral Integration. Science 322:945–949.

Induced Pluripotent Stem Cells. Cell Stem Cell 3:595–605.

pluripotent stem cells. J Clin Invest 120:51-59.

World Journal 2012:417809.

Biotechnol 26:1276–1284.

Cassette. Stem Cells 27:543-9.

Japan Academy Series B 85: 348–362.

770.


[74] Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA (2007) Induced Pluripo‐ tent Stem Cell Lines Derived from Human Somatic Cells. Science 318: 1917–1920.

Baetge EE (2008) Pancreatic Endoderm Derived from Human Embryonic Stem Cells Generates Glucose-Responsive Insulin-Secreting Cells in vivo. Nat Biotechnol

[62] Touboul T, Hannan NR, Corbineau S, Martinez A, Martinet C, Branchereau S, Main‐ ot S, Strick-Marchand H, Pedersen R, Di Santo J, Weber A, Vallier L (2010) Genera‐ tion of Functional Hepatocytes from Human Embryonic Stem Cells Under Chemically Defined Conditions that Recapitulate Liver Development. Hepatology

[63] Osafune K, Caron L, Borowiak M, Martinez RJ, Fitz-Gerald CS, Sato Y, Cowan CA, Chien KR, Melton DA (2008) Marked Differences in Differentiation Propensity

[64] Tavakoli T, Xu X, Derby E, Serebryakova Y, Reid Y, Rao MS, Mattson MP, Ma W (2009) Self-renewal and Differentiation Capabilities are Variable Between Human

[65] Mayor S (2010) First Patient Enters Trial to Test Stem Cells in Spinal Injury. BMJ

[66] Alper J (2009) Geron Gets Green Light for Human Trial of ES-cell Derived Product.

[68] Schwartz SD, Hubschman JP, Heilwell G, Franco-Cardenas V, Pan CK, Ostrick RM, Mickunas E, Gay R, Klimanskaya I, Lanza R (2012) Embryonic Stem Cell Trials for

[69] Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, Arnold K, Stadtfeld M, Yachech‐ ko R, Tchieu J, Jaenisch R, Plath K, Hochedlinger K (2007) Directly Reprogrammed Fibroblasts Show Global Epigenetic Remodeling and Widespread Tissue Contribu‐

[70] Okita K, Ichisaka T, Yamanaka S (2007) Generation of Germline-Competent Induced

[71] Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, Bernstein BE, Jaenisch R (2007). In vitro Reprogramming of Fibroblasts into a Pluripotent ES-

[72] Lowry WE, Richter L, Yachechko R, Pyle AD, Tchieu J, Sridharan R, Clark AT, Plath K (2008) Generation of Human Induced Pluripotent Stem Cells from Dermal Fibro‐

[73] Park IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA, Lerou PH, Lensch MW, Da‐ ley GQ (2008) Reprogramming of Human Somatic Cells to Pluripotency with De‐

among Human Embryonic Stem Cell Lines. Nat Biotechnol 26:313-315.

Embryonic Stem Cell Lines I3, I6 and BG01V. BMC Cell Biol 10: 44.

[67] De Francesco L (2009) Fits and start for Geron. Nat Biotechnol 27:877.

Macular Degeneration: A Preliminary Report. Lancet 379:713-20.

26:443–452.

18 Pluripotent Stem Cells

51:1754-1765.

341:c5724.

Nat Biotechnol 27:213-4.

tion. Cell Stem Cell 1:55–70.

Pluripotent Stem Cells. Nature 448: 313–317.

Cell Like State. Nature 448: 318–324.

fined Factors. Nature 451: 141–146.

blasts. Proc Natl Acad Sci 105: 2883–2888.


[86] Zhou H, Wu S, Joo JY, Zhu S, Han DW, Lin T, Trauger S, Bien G, Yao S, Zhu Y, Siuz‐ dak G, Scholer HR, Duan L, Ding S (2009) Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins. Cell Stem Cell 4: 381–384.

zett-Jones DP, Alitalo K, Lahesmaa R, Nagy A, Otonkoski T (2011) Copy Number Variation and Selection During Reprogramming to Pluripotency. Nature 471:58-62.

An Overview of Pluripotent Stem Cells http://dx.doi.org/10.5772/55130 21

[98] Lister R, Pelizzola M, Kida YS, Hawkins RD, Nery JR, Hon G, Antosiewicz-Bourget J, O'Malley R, Castanon R, Klugman S, Downes M, Yu R, Stewart R, Ren B, Thomson JA, Evans RM, Ecker JR. Hotspots of Aberrant Epigenomic Reprogramming in Hu‐

[99] Wagers AJ, Weissman IL (2004) Plasticity of Adult Stem Cells. Cell 116:639–648.

[100] Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, D. J., Horwitz, E. (2006). Minimal criteria for defin‐ ing multipotent mesenchymal stromal cells. The International Society for Cellular

[101] Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Lar‐ gaespada DA, Verfaillie CM (2002) Pluripotency of Mesenchymal Stem Cells Derived

[102] D'Ippolito G, Diabira S, Howard GA, Menei P, Roos BA, Schiller PC (2004) Marrow-Isolated Adult Multilineage Inducible (MIAMI) Cells, A Unique Population of Post‐ natal Young and Old Human Cells with Extensive Expansion and Differentiation

[103] Beltrami AP, Cesselli, D, Bergamin N, Marcon P, Rigo S, Puppato E, D'Aurizio F, Ve‐ rardo R, Piazza S, Pignatelli A, Poz A, Baccarani U, Damiani D, Fanin R, Mariuzzi L, Finato N, Masolini P, Burelli S, Belluzzi O, Schneider C, Beltrami CA (2007) Multipo‐ tent Cells Can Be Generated In Vitro from Several Adult Human Organs (Heart, Liv‐

[104] Kucia M, Reca R, Campbell FR, Zuba-Surma E, Majka M, Ratajczak J, Ratajczak MZ (2006) A Population of Very Small Embryonic-Like (VSEL) CXCR4(+)SSEA-1(+)Oct-4+ Stem Cells Identified in Adult Bone Marrow. Leukemia

[105] Ratajczak MZ, Kucia M, Majka M, Reca R, Ratajczak J (2004) Heterogeneous Popula‐ tions of Bone Marrow Stem Cells--Are We Spotting on the Same Cells from the Dif‐

[106] Zuba-Surma EK, Kucia M, Wu W, Klich I, Lillard JW Jr, Ratajczak J, Ratajczak MZ. (2008). Very Small Embryonic-Like Stem Cells Are Present in Adult Murine Organs: Imagestream-based Morphological Analysis and Distribution Studies. Cytometry A

[107] Zuba-Surma EK, Wu W, Ratajczak J, Kucia M, Ratajczak MZ (2009) Very Small Em‐ bryonic-Like Stem Cells in Adult Tissues-Potential Implications for Aging. Mech

man Induced Pluripotent Stem Cells. Nature 471:68-73.

Therapy position statement. Cytotherapy 8:315–317.

from Adult Marrow. Nature 418:41–49.

Potential. J Cell Sci 117:2971–2981.

20:857-869.

73A:1116-1127.

Ageing Dev 130:58-66.

er And Bone Marrow). Blood 110:3438–3446.

ferent Angles? Folia Histochem Cytobio 42:139-146.


zett-Jones DP, Alitalo K, Lahesmaa R, Nagy A, Otonkoski T (2011) Copy Number Variation and Selection During Reprogramming to Pluripotency. Nature 471:58-62.


[86] Zhou H, Wu S, Joo JY, Zhu S, Han DW, Lin T, Trauger S, Bien G, Yao S, Zhu Y, Siuz‐ dak G, Scholer HR, Duan L, Ding S (2009) Generation of Induced Pluripotent Stem

[87] Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD, Meissner A, Daley GQ, Brack AS, Collins JJ, Cowan C, Schlaeger TM, Rossi DJ (2010) Highly Efficient Reprogramming to Pluripotency and Directed Differentiation

[88] Huangfu D, Maehr R, Guo W, Eijkelenboom A, Snitow M, Chen AE, Melton DA (2008) Induction of Pluripotent Stem Cells by Defined Factors is Greatly Improved by

[89] Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF, Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K (2008) In‐ duced Pluripotent Stem Cells Generated from Patients with ALS can be Differentiat‐

[90] Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ (2008) Disease-specific induced pluripotent stem cells.

[91] Ebert AD, Yu J, Rose FF Jr, Mattis VB, Lorson CL, Thomson JA, Svendsen CN (2009). Induced Pluripotent Stem Cells from a Spinal Muscular Atrophy Patient Nature

[92] Amabile G, Meissner A (2009) Induced pluripotent stem cells: current progress and

[93] Marion RM, Strati K, Li H, Tejera A, Schoeftner S, Ortega S, Serrano M, Blasco MA (2009) Telomeres Acquire Embryonic Stem Cell Characteristics in Induced Pluripo‐

[94] Takahashi K, Okita K, Nakagawa M, Yamanaka S (2007) Induction of Pluripotent

[95] Takahashi K, Ichisaka T, Yamanaka S (2006) Identification of Genes Involved in Tu‐ mor-Like Properties of Embryonic Stem Cells. Methods in Molecular Biology 329:

[96] Gore A, Li Z, Fung HL, Young JE, Agarwal S, Antosiewicz-Bourget J, Canto I, Gior‐ getti A, Israel MA, Kiskinis E, Lee JH, Loh YH, Manos PD, Montserrat N, Panopoulos AD, Ruiz S, Wilbert ML, Yu J, Kirkness EF, Izpisua Belmonte JC, Rossi DJ, Thomson JA, Eggan K, Daley GQ, Goldstein LS, Zhang K (2011) Somatic Coding Mutations in

[97] Hussein SM, Batada NN, Vuoristo S, Ching RW, Autio R, Narva E, Ng S, Sourour M, Hamalainen R, Olsson C, Lundin K, Mikkola M, Trokovic R, Peitz M, Brustle O, Ba‐

potential for regenerative medicine. Trends Mol Med 15:59–68.

Stem Cells from Fibroblast Cultures Nature Protocols 2: 3081–3089.

Human Induced Pluripotent Stem Cells. Nature 471:63-37.

of Human Cells with Synthetic Modified mRNA. Cell Stem Cell 7: 618–630.

Cells Using Recombinant Proteins. Cell Stem Cell 4: 381–384.

Small-Molecule Compounds. Nature Biotechnol 26: 795–797.

ed into Motor Neurons Science 321:1218–1221.

tent Stem Cells. Cell Stem Cell 4:141–154.

Cell 134:877–886.

457:277–280.

20 Pluripotent Stem Cells

449–458.


[108] Shin DM, Zuba-Surma EK, Wu W, Ratajczak J, Wysoczynski M, Ratajczak MZ, Kucia M (2009) Novel Epigenetic Mechanisms that Control Pluripotency and Quiescence of Adult Bone Marrow Derived Oct4(+) Very Small Embryonic-Like Stem Cells. Leuke‐ mia 23:2042-2051.

[121] Takeda J, Seino S, Bell GI (1992) Human Oct3 Gene Family: cDNA Sequences, Alter‐ native Splicing, Gene Organization, Chromosomal Location, and Expression at Low

An Overview of Pluripotent Stem Cells http://dx.doi.org/10.5772/55130 23

[122] Bhartiya D, Sriraman K, Parte S (2012) Stem Cell Interaction with Somatic Niche may hold the Key to Fertility Restoration in Cancer Patients. Obstet and Gynec Intl 2012:

[123] Virant-Klun I, Rozman P, Cvjeticanin B, Vrtacnik-Bokal E, Novakovic S, Rülicke T, Dovc P, Meden-Vrtovec H (2009) Parthenogenetic Embryo-Like Structures in The Human Ovarian Surface Epithelium Cell Culture in Postmenopausal Women with

[124] Bukovsky A, Svetlikova M, Caudle MR (2005) Oogenesis in Cultures Derived from

[125] Zuba-Surma EK, Wojakowski W, Ratajczak MZ, Dawn B (2011) Very Small Embry‐ onic-Like Stem Cells: Biology and Therapeutic Potential for Heart Repair. Antiox Re‐

[126] Wojakowski W, Kucia M, Zuba-Surma E, Jadczyk T, Książek B, Ratajczak MZ, Ten‐ dera M (2010) Very Small Embryonic-Like Stem Cells in Cardiovascular Repair.

[127] Paczkowska E, Kucia M, Koziarska D, Halasa M, Safranow K, Masiuk M, Karbicka A, Nowik M, Nowacki P, Ratajczak MZ, Machalinski B (2009) Clinical Evidence that Very Small Embryonic-Like Stem Cells are Mobilized into Peripheral Blood in Pa‐

[128] Drukała J, Paczkowska E, Kucia M, Młyńska E, Krajewski A, Machaliński B, Madeja Z, Ratajczak MZ (2012) Stem Cells, Including A Population of Very Small Embryon‐ ic-Like Stem Cells, Are Mobilized into Peripheral Blood in Patients After Skin Burn

[129] Ratajczak J, Zuba-Surma E, Paczkowska E, Kucia M, Nowacki P, Ratajczak MZ (2011) Stem Cells for Neural Regeneration--A Potential Application of Very Small Embry‐

[130] Ratajczak J, Wysoczynski M, Zuba-Surma E, Wan W, Kucia M, Yoder MC, Ratajczak MZ (2011). Adult Murine Bone Marrow-Derived Very Small Embryonic-Like Stem Cells Differentiate into the Hematopoietic Lineage after Coculture over OP9 Stromal

[131] Ratajczak MZ, Liu R, Marlicz W, Blogowski W, Starzynska T, Wojakowski W, Zuba-Surma E (2011) Identification of Very Small Embryonic/Epiblast-Like Stem Cells (Vsels) Circulating in Peripheral Blood During Organ/Tissue Injuries. Methods Cell

onic-Like Stem Cells. Journal of Physiology and Pharmacology 62:3-12.

No Naturally Present Follicles and Oocytes. Stem Cells Dev 18:137-149.

Adult Human Ovaries. Reprod Biol Endocrinol. 3:17.

Levels in Adult Tissues. Nucleic Acids Research 20:4613-4620.

921082.

dox Signal 15:1821-1834.

Pharmacology & Therapeutics 129:21-28.

tients after Stroke. Stroke. 40(4):1237-1244.

Injury. Stem Cell Review 8:184-194.

Cells. Exp Hematol 39:225-237.

Biol 103:31- 54.


[121] Takeda J, Seino S, Bell GI (1992) Human Oct3 Gene Family: cDNA Sequences, Alter‐ native Splicing, Gene Organization, Chromosomal Location, and Expression at Low Levels in Adult Tissues. Nucleic Acids Research 20:4613-4620.

[108] Shin DM, Zuba-Surma EK, Wu W, Ratajczak J, Wysoczynski M, Ratajczak MZ, Kucia M (2009) Novel Epigenetic Mechanisms that Control Pluripotency and Quiescence of Adult Bone Marrow Derived Oct4(+) Very Small Embryonic-Like Stem Cells. Leuke‐

[109] Shin DM, Liu R, Wu W, Waigel SJ, Zacharias W, Ratajczak MZ, Kucia M (2012) Glob‐ al Gene Expression Analysis of Very Small Embryonic-Like Stem Cells Reveals That the Ezh2-Dependent Bivalent Domain Mechanism Contributes To Their Pluripotent

[110] Ratajczak MZ, Machalinski B, Wojakowski W, Ratajczak J, Kucia M (2007) A Hypoth‐ esis for An Embryonic Origin of Pluripotent Oct-4(+) Stem Cells in Adult Bone Mar‐

[111] Bhartiya D (2012) Pluripotent Very Small Embryonic-Like Stem Cells Get Discarded During Cord Blood and Bone Marrow Processing. Stem Cells Dev 21:2563-2564.

[112] Ratajczak MZ, Zuba-Surma EK, Shin DM, Ratajczak J, Kucia M (2008) Very Small Embryonic-Like (VSEL) Stem Cells in Adult Organs and Their Potential Role in Reju‐

[113] Li L, Clevers H (2010) Coexistence of Quiescent and Active Adult Stem Cells in

[114] De Rosa L, De Luca M (2012) Cell Biology Dormant and Skin Stem Cells. Nature

[115] Ratajczak MZ, Shin DM, Kucia M (2009) Very Small Embryonic/Epiblast-Like Stem Cells: A Missing Link to Support the Germ Line Hypothesis of Cancer Development?

[116] Ratajczak MZ, Shin DM, Liu R, Marlicz W, Tarnowski M, Ratajczak J, Kucia M. (2010) Epiblast/Germ Line Hypothesis of Cancer Development Revisited: Lesson

from the Presence of Oct-4(1) Cells In Adult Tissues. Stem Cell Rev 6:307-316.

[117] Kucia M, Halasa M, Wysoczynski M, Baskiewicz-Masiuk M, Moldenhawer S, Zuba-Surma E, Czajka R, Wojakowski W, Machalinski B and Ratajczak MZ (2007) Morpho‐ logical and Molecular Characterization of Novel Population of cxcr4+ SSEA-4+ Oct-4+ Very Small Embryonic-Like Cells Purified From Human Cord Blood – Pre‐

[118] Liedtke S, Enczmann J, Waclawczyk S, Wernet P, Kögler G (2007) Oct4 and Its Pseu‐

[119] Liedtke S, Stephan M, Kogler G (2008) Oct4 Expression Revisited: Potential Pitfalls for Data Misinterpretation in Stem Cell Research. J Biol Chem 389:845-850.

[120] Wang X, Dai J (2010) Isoforms Of OCT4 Contribute to the Confusing Diversity in

dogenes Confuse Stem Cell Research. Cell Stem Cell 1:364-366.

venation of Tissues and Longevity. ExpGerontol 43:1009-1017.

mia 23:2042-2051.

22 Pluripotent Stem Cells

State. Stem Cells Dev 21:1639-52.

Mammals. Science 327:542-5.

Am J Path 174:1985-1992.

liminary Report. Leukemia 21:297–303

Stem Cell Biology. Stem Cells 28:885-893.

489:215-217.

row and Other Tissues. Leukemia 21:860-867.


**Chapter 2**

**Pluripotent Adult Stem Cells: A Potential Revolution in**

Stem cells are undifferentiated cells defined by their abilities to self-renew and differentiate into mature cells. Stem cells found in fully developed tissues are defined as adult stem cells. The function of adult stem cells is the maintenance of adult tissue specificity by homeostatic cell replacement and tissue regeneration (Wagers and Weissman, 2004). Adult stem cells are presumed quiescent within adult tissues, but divide infrequently to generate a stem cell clone and a transiently-amplifying cell. The transiently-amplifying cells will undergo a lim‐ ited number of cell divisions before terminal differentiation into mature functional tissue cells. The existence of adult stem cells has been reported in multiple organs; these include: brain, heart, skin, intestine, testis, muscle and blood, among others. This chapter focuses on four adult stem cell populations: hematopoietic, mesenchymal, periodontal ligament-de‐

Hematopoietic stem cells are the most characterized adult stem cell population. They func‐ tion to generate all cell lineages found in mature blood (erythroid, myeloid and lymphoid) and to sustain blood production during the entire life of an animal (Kondo et al., 2003). Adult bone marrow, umbilical cord blood and mobilized peripheral blood are sources of hematopoietic stem cells for transplantation in many blood-related diseases. Hematopoietic stem cells can be characterized by positive selection of CD34, CD45, and CD133 markers and

Mesenchymal stem cells, also called marrow stromal cells, are another well-studied adult stem cell population. Mesenchymal stem cells were originally identified in the bone mar‐ row, but have since been found in other systems such as adipose tissue, umbilical cord and

> © 2013 Ng et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Ng et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

negative selection of CD31, CD105 and CD146 markers (Tárnok et al., 2010).

**Regenerative Medicine and Tissue Engineering**

Tsz Kin Ng, Daniel Pelaez, Veronica R. Fortino,

Jordan Greenberg and Herman S. Cheung

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54366

rived, and spermatogonial (Table 1).

**1. Introduction**

## **Pluripotent Adult Stem Cells: A Potential Revolution in Regenerative Medicine and Tissue Engineering**

Tsz Kin Ng, Daniel Pelaez, Veronica R. Fortino, Jordan Greenberg and Herman S. Cheung

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54366

## **1. Introduction**

Stem cells are undifferentiated cells defined by their abilities to self-renew and differentiate into mature cells. Stem cells found in fully developed tissues are defined as adult stem cells. The function of adult stem cells is the maintenance of adult tissue specificity by homeostatic cell replacement and tissue regeneration (Wagers and Weissman, 2004). Adult stem cells are presumed quiescent within adult tissues, but divide infrequently to generate a stem cell clone and a transiently-amplifying cell. The transiently-amplifying cells will undergo a lim‐ ited number of cell divisions before terminal differentiation into mature functional tissue cells. The existence of adult stem cells has been reported in multiple organs; these include: brain, heart, skin, intestine, testis, muscle and blood, among others. This chapter focuses on four adult stem cell populations: hematopoietic, mesenchymal, periodontal ligament-de‐ rived, and spermatogonial (Table 1).

Hematopoietic stem cells are the most characterized adult stem cell population. They func‐ tion to generate all cell lineages found in mature blood (erythroid, myeloid and lymphoid) and to sustain blood production during the entire life of an animal (Kondo et al., 2003). Adult bone marrow, umbilical cord blood and mobilized peripheral blood are sources of hematopoietic stem cells for transplantation in many blood-related diseases. Hematopoietic stem cells can be characterized by positive selection of CD34, CD45, and CD133 markers and negative selection of CD31, CD105 and CD146 markers (Tárnok et al., 2010).

Mesenchymal stem cells, also called marrow stromal cells, are another well-studied adult stem cell population. Mesenchymal stem cells were originally identified in the bone mar‐ row, but have since been found in other systems such as adipose tissue, umbilical cord and

menstrual blood (Ding et al., 2011). Mesenchymal stem cells differentiate into osteocytes, chondrocytes and adipocytes (Arita et al., 2011; Pittenger et al., 1999). Human mesenchymal stem cells can be characterized by the positive expression of CD29, CD44, CD73, CD90, CD105, CD146 and STRO-1, and the negative expression of CD31, CD34, CD45, CD49f and CD133 (Mödder et al., 2012; Tárnok et al., 2010).

somatic and germ-line cells in the developing embryo. The first pluripotent cells were derived in 1976 from a type of germ-line tumor known as a teratocarcinoma (Hogan, 1976). Embryonic stem cells, derived from the inner cell mass of a blastocyst prior to gastrulation, are still considered the gold standard for pluripotent stem cells. Even though adult cells are terminally differentiated, pluripotency has also been conferred to these cells in past studies, by the technique of somatic cell nuclear transfer (Perry, 2005), parthenogenesis of unfertilized eggs (Brevini et al., 2008), and reprogramming by cell fu‐ sion (Pralong et al., 2006). Research into adult cell pluripotency was slow to progress un‐ til a major breakthrough in 2006 brought with it the technique of "induced pluripotent stem cells". In this process adult skin fibroblasts were induced into a pluripotent state by the forced expression of key transcription factors (OCT4, SOX2, KLF4 and c-MYC; Taka‐ hashi et al., 2007) or (OCT4, SOX2, NANOG and LIN28; Yu et al., 2007). Despite the low reprogramming efficiency, this has become a convenient method for generating new plu‐

Pluripotent Adult Stem Cells: A Potential Revolution in Regenerative Medicine and Tissue Engineering

http://dx.doi.org/10.5772/54366

27

Adult stem cells are thought to be tissue-specific and only able to differentiate into progeny cells of their tissues of origin. An increasing number of studies, however, re‐ port that adult stem cells are capable of giving rise to cells of an entirely distinct line‐ age. The concept of adult stem cell plasticity might be explained by 5 potential mechanisms: cell fusion, trans-differentiation, de-differentiation, heterogeneous stem cell populations, or pluripotency (Wagers and Weissman, 2004). Cell-cell fusion occurs at a low frequency, but is implicated in the transplantation of bone marrow cells to liver hepatocytes, cardiomyocytes and Purkinje neurons (Alvarez-Dolado et al., 2003). In cell fusion events, the stem cells acquire the mature phenotype of the tissue they are em‐ bedded within and can be easily mistaken for correct differentiation of the transplant‐ ed cells. Trans-differentiation is a direct lineage conversion by the activation of a dormant differentiation program to alter the lineage specificity of the cell. De-differen‐ tiation is another lineage conversion phenomenon in which a tissue-specific cell sponta‐ neously de-differentiates into a more basal multipotent cell and re-differentiates to a new lineage. While the heterogeneity of the stem cell population employed can ac‐ count for some of the apparent trans-differentiation and de-differentiation events ob‐ served *in vivo*, it is worth discussing as a separate factor in the resulting multi-lineage tissues, which are often seen after transplantation. The characterization of homogene‐ ous stem cell populations that contribute to the regeneration of one cell type remains an active field of study for most cellular therapy applications. Lastly, pluripotent stem cells are present in adult tissues as minute sub-populations in certain stem cell niches. Such a population has already been identified and reported in bone marrow derived mesenchymal stem cells (Jiang et al., 2002). In addition, pluripotent stem cells in adult tissues can also arise from remnants of the migrating neural crest. The neural crest is a transient embryonic structure that affords various organs with cells which could un‐ dergo a more stochastic type of differentiation than other embryonic progenitor cells (Slack, 2008). Neural crest cells are pluripotent and may retain some of their character‐

ripotent stem cell lines for research from differentiated adult cells.

istics after their migration and engraftment into their terminal sites.


**Table 1.** Feasible sources and characterization of adult stem cells

Periodontal ligament, derived from the cranial neural crest, is a soft connective tissue embedded between the tooth root and the alveolar bone socket, supporting the teeth *in situ* and preserving tissue homeostasis. The periodontal ligament contains stem cell pop‐ ulations that can differentiate into cementum-forming cells or bone-forming cells (Seo et al., 2004). Periodontal ligament-derived stem cells are heterogeneous, composed of mes‐ enchymal stem cells and putative neural crest cells. Therefore, human periodontal liga‐ ment-derived stem cell populations have been characterized not only by mesenchymal stem cell markers, but also by neural crest cell markers, such as p75, nestin, Slug and SOX10 (Huang et al., 2009; Mrozik et al., 2010).

Testicular spermatogonial stem cells are the germ-line cells for spermatogenesis, an ongoing process throughout the lifespan of the male animals. They are unipotent in nature and con‐ tinuously generate differentiating daughter cells for subsequent production of spermatozoa (Fagoonee et al., 2011). Human spermatogonial stem cells can be purified by antibodies against cell surface markers CD9, CD49f and GPR125 (Conrad et al., 2008).

### **2. Pluripotent stem cells**

Pluripotency refers to the ability of cells to self-renew and differentiate into all 3 germ layers (ectoderm, endoderm and mesoderm). Pluripotent stem cells are the origin of all somatic and germ-line cells in the developing embryo. The first pluripotent cells were derived in 1976 from a type of germ-line tumor known as a teratocarcinoma (Hogan, 1976). Embryonic stem cells, derived from the inner cell mass of a blastocyst prior to gastrulation, are still considered the gold standard for pluripotent stem cells. Even though adult cells are terminally differentiated, pluripotency has also been conferred to these cells in past studies, by the technique of somatic cell nuclear transfer (Perry, 2005), parthenogenesis of unfertilized eggs (Brevini et al., 2008), and reprogramming by cell fu‐ sion (Pralong et al., 2006). Research into adult cell pluripotency was slow to progress un‐ til a major breakthrough in 2006 brought with it the technique of "induced pluripotent stem cells". In this process adult skin fibroblasts were induced into a pluripotent state by the forced expression of key transcription factors (OCT4, SOX2, KLF4 and c-MYC; Taka‐ hashi et al., 2007) or (OCT4, SOX2, NANOG and LIN28; Yu et al., 2007). Despite the low reprogramming efficiency, this has become a convenient method for generating new plu‐ ripotent stem cell lines for research from differentiated adult cells.

menstrual blood (Ding et al., 2011). Mesenchymal stem cells differentiate into osteocytes, chondrocytes and adipocytes (Arita et al., 2011; Pittenger et al., 1999). Human mesenchymal stem cells can be characterized by the positive expression of CD29, CD44, CD73, CD90, CD105, CD146 and STRO-1, and the negative expression of CD31, CD34, CD45, CD49f and

**Adult stem cells Feasible sources Characterization**

Spermatogonial stem cells Testis (+): CD9, CD49f and GPR125

Periodontal ligament, derived from the cranial neural crest, is a soft connective tissue embedded between the tooth root and the alveolar bone socket, supporting the teeth *in situ* and preserving tissue homeostasis. The periodontal ligament contains stem cell pop‐ ulations that can differentiate into cementum-forming cells or bone-forming cells (Seo et al., 2004). Periodontal ligament-derived stem cells are heterogeneous, composed of mes‐ enchymal stem cells and putative neural crest cells. Therefore, human periodontal liga‐ ment-derived stem cell populations have been characterized not only by mesenchymal stem cell markers, but also by neural crest cell markers, such as p75, nestin, Slug and

Testicular spermatogonial stem cells are the germ-line cells for spermatogenesis, an ongoing process throughout the lifespan of the male animals. They are unipotent in nature and con‐ tinuously generate differentiating daughter cells for subsequent production of spermatozoa (Fagoonee et al., 2011). Human spermatogonial stem cells can be purified by antibodies

Pluripotency refers to the ability of cells to self-renew and differentiate into all 3 germ layers (ectoderm, endoderm and mesoderm). Pluripotent stem cells are the origin of all

against cell surface markers CD9, CD49f and GPR125 (Conrad et al., 2008).

(+): CD34, CD45, CD133

(-): CD31, CD105, CD146

(+): CD29, CD44, CD73, CD90, CD105, CD146, STRO-1 (-): CD31, CD34, CD45, CD49f, CD133

Mesenchymal stem cell markers: CD29, CD44, CD73, CD90, CD105, CD146, STRO-1 Neural crest cell markers: p75, nestin, Slug, SOX10

Bone marrow, umbilical cord blood, mobilized peripheral blood

umbilical cord, menstrual blood

CD133 (Mödder et al., 2012; Tárnok et al., 2010).

Mesenchymal stem cells Bone marrow, adipose tissue,

stem cells Periodontal ligament

**Table 1.** Feasible sources and characterization of adult stem cells

SOX10 (Huang et al., 2009; Mrozik et al., 2010).

**2. Pluripotent stem cells**

Hematopoietic stem cells

26 Pluripotent Stem Cells

Periodontal ligament-derived

Adult stem cells are thought to be tissue-specific and only able to differentiate into progeny cells of their tissues of origin. An increasing number of studies, however, re‐ port that adult stem cells are capable of giving rise to cells of an entirely distinct line‐ age. The concept of adult stem cell plasticity might be explained by 5 potential mechanisms: cell fusion, trans-differentiation, de-differentiation, heterogeneous stem cell populations, or pluripotency (Wagers and Weissman, 2004). Cell-cell fusion occurs at a low frequency, but is implicated in the transplantation of bone marrow cells to liver hepatocytes, cardiomyocytes and Purkinje neurons (Alvarez-Dolado et al., 2003). In cell fusion events, the stem cells acquire the mature phenotype of the tissue they are em‐ bedded within and can be easily mistaken for correct differentiation of the transplant‐ ed cells. Trans-differentiation is a direct lineage conversion by the activation of a dormant differentiation program to alter the lineage specificity of the cell. De-differen‐ tiation is another lineage conversion phenomenon in which a tissue-specific cell sponta‐ neously de-differentiates into a more basal multipotent cell and re-differentiates to a new lineage. While the heterogeneity of the stem cell population employed can ac‐ count for some of the apparent trans-differentiation and de-differentiation events ob‐ served *in vivo*, it is worth discussing as a separate factor in the resulting multi-lineage tissues, which are often seen after transplantation. The characterization of homogene‐ ous stem cell populations that contribute to the regeneration of one cell type remains an active field of study for most cellular therapy applications. Lastly, pluripotent stem cells are present in adult tissues as minute sub-populations in certain stem cell niches. Such a population has already been identified and reported in bone marrow derived mesenchymal stem cells (Jiang et al., 2002). In addition, pluripotent stem cells in adult tissues can also arise from remnants of the migrating neural crest. The neural crest is a transient embryonic structure that affords various organs with cells which could un‐ dergo a more stochastic type of differentiation than other embryonic progenitor cells (Slack, 2008). Neural crest cells are pluripotent and may retain some of their character‐ istics after their migration and engraftment into their terminal sites.

## **3. Isolation of pluripotent adult stem cells**

The expression of embryonic stem cell markers in some adult stem cells suggest a sub-popu‐ lation of pluripotent cells in these niches (Table 2). The common embryonic stem cell mak‐ ers, such as OCT4, SOX2, NANOG, KLF4, LIN28, SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, are all expressed in hematopoietic stem cells (Wang et al., 2010; Zhao et al., 2006; Zulli et al., 2008) and mesenchymal stem cells (Anjos-Afonso and Bonnet, 2007; Jaramillo-Ferrada et al., 2012; Riekstina et al., 2009; Sung et al., 2010). Similarly, expressions of most of these markers, except for LIN28, have been reported in periodontal ligament-derived stem cells, a tissue arising from the migrating cranial neural crest (Huang et al., 2009; Kawanabe et al., 2010). Previous studies show that spermatogonial stem cells also express most of the embryonic stem cell markers, except SSEA-3 and TRA-1-60 (Izadyar et al., 2008; Izadyar et al., 2011; Kanatsu-Shinohara et al., 2008; Panda et al., 2011; Zheng et al., 2009). These find‐ ings suggest that pluripotent stem cells exist as sub-populations in adult stem cell reservoirs. cells, sharing cellular and molecular similarities with human embryonic stem cells, can be purified by α6 integrin (CD49f) antibody (Conrad et al., 2008). Moreover, a human hematopoietic stem cell subpopulation, highly efficient in generating long-term multi-lin‐ eage grafts, can also be isolated by the same α6 integrin expression (Notta et al., 2011). In addition, stem cells from granulocyte colony-stimulating factor-mobilized human periph‐ eral blood can divide indefinitely without reaching replicative senescence and differenti‐

Pluripotent Adult Stem Cells: A Potential Revolution in Regenerative Medicine and Tissue Engineering

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29

Recently, a cell surfaceome map of mouse embryonic stem cells and induced pluripotent stem cells was reported (Gundry et al., 2012). Previously unidentified cellular surface mark‐ ers, such as CD31, CD49f, CD123 and CD326, indicated a purified population of pluripotent stem cells. Further analyses should be performed to determine the expression of these mark‐ ers in different adult stem cell populations. Their presence in adult stem cell populations could facilitate the purification of homogeneous pluripotent stem cells within an otherwise

The standard tests for pluripotency are teratoma and chimera formation assays. Terato‐ mas can be formed when pluripotent stem cells are injected into immunodeficient ani‐ mals; they consist of foci with derivatives of ectodermal, mesodermal and endodermal embryonic germs layers (Wobus et al., 1984). Chimeras can be generated when pluripo‐ tent stem cells are microinjected into mouse blastocysts and are induced to differentiate into multiple cell types during normal developmental processes (Becker et al., 1984). Ter‐ atoma formation assays can be used to test for the pluripotency of human stem cells, whereas both teratoma and chimera formation can test for the pluripotency of mouse stem cells. Spermatogonial stem cells isolated from human testis by positive expression of CD49f are able to form teratomas when injected into immunodeficient mice (Conrad et al., 2008). Mesenchymal stem cells isolated from murine bone marrow contribute to most of the somatic cell types (chimerism ranged between 0.1% and 45%) when they are singly injected into an early mouse blastocyst (Jiang et al., 2002). Moreover, human hem‐ atopoietic stem cells isolated by CD49f cell surface marker display multi-lineage chimer‐ ism when transplanted into the NOD-*scid*-*IL2Rgc*-/- mice (Notta et al., 2011). However, human bone marrow-derived mesenchymal stem cells purified by the SSEA-3 cell sur‐ face marker do not form teratomas in immunodeficient mouse testes even though cells positive for human ectodermal, endodermal and mesodermal lineage markers were de‐ tected within the injected mouse testes (Kuroda et al., 2010). Conversely, pluripotency as‐ says of human periodontal ligament-derived stem cells isolated by SSEA-4 cell surface

ate into multiple lineages (Cesselli et al., 2009).

heterogeneous pool of regenerative adult cells.

**4. Characterization of pluripotent adult stem cells**

marker expression have not yet been reported (Kawanabe et al., 2010).

Although most of the adult stem cells are unable to form teratomas in immunodeficient mice, can they still be defined as pluripotent stem cells? Considering this apparent inability as well as the variability in teratoma formation efficiency even when using a known pluripo‐


HSC: hematopoietic stem cells; MSC: mesenchymal stem cells; PDLSC; periodontal ligament-derived stem cells; SSC: spermatogonial stem cells;

**Table 2.** Embryonic stem cell marker expression in different adult stem cell populations

The existence of cells with a defined pluripotency-associated phenotypic expression with‐ in adult tissues enables researchers to isolate and purify a homogeneous subpopulation of adult pluripotent stem cells. In fact, with the use of magnetic affinity cell sorting, adult human mesenchymal stem cells, shown to differentiate into endodermal, ectoder‐ mal and mesodermal cells, were isolated by antibody against SSEA-3 (Kuroda et al., 2010). Similarly, stem cells exhibiting the potential to generate specialized cells of the three embryonic germ layers can be isolated by positive SSEA-4 expression from human periodontal ligament (Kawanabe et al., 2010). Furthermore, human spermatogonial stem cells, sharing cellular and molecular similarities with human embryonic stem cells, can be purified by α6 integrin (CD49f) antibody (Conrad et al., 2008). Moreover, a human hematopoietic stem cell subpopulation, highly efficient in generating long-term multi-lin‐ eage grafts, can also be isolated by the same α6 integrin expression (Notta et al., 2011). In addition, stem cells from granulocyte colony-stimulating factor-mobilized human periph‐ eral blood can divide indefinitely without reaching replicative senescence and differenti‐ ate into multiple lineages (Cesselli et al., 2009).

Recently, a cell surfaceome map of mouse embryonic stem cells and induced pluripotent stem cells was reported (Gundry et al., 2012). Previously unidentified cellular surface mark‐ ers, such as CD31, CD49f, CD123 and CD326, indicated a purified population of pluripotent stem cells. Further analyses should be performed to determine the expression of these mark‐ ers in different adult stem cell populations. Their presence in adult stem cell populations could facilitate the purification of homogeneous pluripotent stem cells within an otherwise heterogeneous pool of regenerative adult cells.

## **4. Characterization of pluripotent adult stem cells**

**3. Isolation of pluripotent adult stem cells**

28 Pluripotent Stem Cells

The expression of embryonic stem cell markers in some adult stem cells suggest a sub-popu‐ lation of pluripotent cells in these niches (Table 2). The common embryonic stem cell mak‐ ers, such as OCT4, SOX2, NANOG, KLF4, LIN28, SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, are all expressed in hematopoietic stem cells (Wang et al., 2010; Zhao et al., 2006; Zulli et al., 2008) and mesenchymal stem cells (Anjos-Afonso and Bonnet, 2007; Jaramillo-Ferrada et al., 2012; Riekstina et al., 2009; Sung et al., 2010). Similarly, expressions of most of these markers, except for LIN28, have been reported in periodontal ligament-derived stem cells, a tissue arising from the migrating cranial neural crest (Huang et al., 2009; Kawanabe et al., 2010). Previous studies show that spermatogonial stem cells also express most of the embryonic stem cell markers, except SSEA-3 and TRA-1-60 (Izadyar et al., 2008; Izadyar et al., 2011; Kanatsu-Shinohara et al., 2008; Panda et al., 2011; Zheng et al., 2009). These find‐ ings suggest that pluripotent stem cells exist as sub-populations in adult stem cell reservoirs.

**Embryonic stem cell marker HSC MSC PDLSC SSC** SOX2 + + + + OCT4 + + + + NANOG + + + + KLF4 + + + + LIN28 + + + SSEA-1 + + + +

SSEA-4 + + + +

TRA-1-81 + + + +

HSC: hematopoietic stem cells; MSC: mesenchymal stem cells; PDLSC; periodontal ligament-derived stem cells; SSC:

The existence of cells with a defined pluripotency-associated phenotypic expression with‐ in adult tissues enables researchers to isolate and purify a homogeneous subpopulation of adult pluripotent stem cells. In fact, with the use of magnetic affinity cell sorting, adult human mesenchymal stem cells, shown to differentiate into endodermal, ectoder‐ mal and mesodermal cells, were isolated by antibody against SSEA-3 (Kuroda et al., 2010). Similarly, stem cells exhibiting the potential to generate specialized cells of the three embryonic germ layers can be isolated by positive SSEA-4 expression from human periodontal ligament (Kawanabe et al., 2010). Furthermore, human spermatogonial stem

SSEA-3 + + +

TRA-1-60 + + +

**Table 2.** Embryonic stem cell marker expression in different adult stem cell populations

spermatogonial stem cells;

The standard tests for pluripotency are teratoma and chimera formation assays. Terato‐ mas can be formed when pluripotent stem cells are injected into immunodeficient ani‐ mals; they consist of foci with derivatives of ectodermal, mesodermal and endodermal embryonic germs layers (Wobus et al., 1984). Chimeras can be generated when pluripo‐ tent stem cells are microinjected into mouse blastocysts and are induced to differentiate into multiple cell types during normal developmental processes (Becker et al., 1984). Ter‐ atoma formation assays can be used to test for the pluripotency of human stem cells, whereas both teratoma and chimera formation can test for the pluripotency of mouse stem cells. Spermatogonial stem cells isolated from human testis by positive expression of CD49f are able to form teratomas when injected into immunodeficient mice (Conrad et al., 2008). Mesenchymal stem cells isolated from murine bone marrow contribute to most of the somatic cell types (chimerism ranged between 0.1% and 45%) when they are singly injected into an early mouse blastocyst (Jiang et al., 2002). Moreover, human hem‐ atopoietic stem cells isolated by CD49f cell surface marker display multi-lineage chimer‐ ism when transplanted into the NOD-*scid*-*IL2Rgc*-/- mice (Notta et al., 2011). However, human bone marrow-derived mesenchymal stem cells purified by the SSEA-3 cell sur‐ face marker do not form teratomas in immunodeficient mouse testes even though cells positive for human ectodermal, endodermal and mesodermal lineage markers were de‐ tected within the injected mouse testes (Kuroda et al., 2010). Conversely, pluripotency as‐ says of human periodontal ligament-derived stem cells isolated by SSEA-4 cell surface marker expression have not yet been reported (Kawanabe et al., 2010).

Although most of the adult stem cells are unable to form teratomas in immunodeficient mice, can they still be defined as pluripotent stem cells? Considering this apparent inability as well as the variability in teratoma formation efficiency even when using a known pluripo‐ tent stem cell line, a teratoma assay might not be a suitable assay for pluripotency of adult stem cells. Instead, *in vitro* and *in vivo* differentiation into cells of the 3 embryonic germ lay‐ ers along with chimera formation in xeno-transplanted mice can be applied for testing adult stem cell potency. The conventional concept of development involves a hierarchical struc‐ ture of cellular commitment extending outward from embryonic and pluripotent, to adult terminally differentiated tissues. However, recent ideas propose that all or most tissues in the postnatal body are continuously turning over and contain a pluripotent stem cell reser‐ voir (Slack, 2008). These pluripotent stem cell populations are able to differentiate into mul‐ tiple cell types depending on their microenvironmental cues. Therefore, the stem cell status should be defined by plasticity (Zipori, 2005). Pluripotency refers to the ability of cells to dif‐ ferentiate into any cell type of the 3 germ layers (ectoderm, endoderm and mesoderm), whereas multipotency refers to the ability of cells to differentiate only into a closely related family of cells (Ilic and Polak, 2011). All of the previously described adult stem cells (hema‐ topoietic, mesenchymal, periodontal ligament-derived, and spermatogonial) could differen‐ tiate into specialized cells of the three germ layers: neurons (ectodermal lineage), adipocytes, cardiomyocytes, osteoblasts, and chondrocytes (mesoderm lineage), and hepatocytes and in‐ sulin-producing cells (endodermal lineage) (Conrad et al., 2008; Jiang et al., 2002; Kuroda et al., 2010; Kawanabe et al., 2010; Notta et al., 2011). Therefore, these adult stem cells could also be defined as pluripotent stem cells.

er, concerning generation of induced pluripotent stem cells (Hayden, 2011). Firstly, the de‐ livery of reprogramming factors (OCT4, SOX2, NANOG, LIN28, KLF4 and c-MYC) relies on the use of viral vectors for delivery (Takahashi et al., 2007). Retroviral sequences could inte‐ grate into the DNA of the host cells, potentially disrupting the gene structure as well as re‐ sulting in an aberrant phenotypic expression. Ultimately this could result in pathological mutations and cancer formation. Alternative methods such as direct protein or small mole‐ cule delivery have been adopted, although the reprogramming efficiency of these techni‐ ques is lower than with viral vectors (Kim et al., 2009; Shi et al., 2008). Secondly, two of the reprogramming factors, *c-MYC* and *KLF4*, are proto-oncogenes, which raise the concern of cancer formation further. Omitting *c-MYC* would lower the reprogramming efficiency, whereas silencing *c-MYC* could lead to its reactivation. Moreover, reprogramming can in‐ duce other genomic changes, such as DNA mutations (Gore et al., 2011), copy number varia‐ tions (Hussein et al., 2011) and chromosomal aberrations (Mayshar et al., 2010). Genomic instability could have unpredictable and undesirable effects on the reprogrammed cells. Furthermore, induced pluripotent stem cells carry their epigenetic signatures from the origi‐ nal differentiated adult cells (Lister et al., 2011). The reprogrammed cells, therefore, unlike embryonic stem cells, may not develop into some cell types. In addition, induced pluripo‐

Pluripotent Adult Stem Cells: A Potential Revolution in Regenerative Medicine and Tissue Engineering

http://dx.doi.org/10.5772/54366

31

tent stem cells can still cause immune reactions when transplanted allogeneically.

The sources of adult stem cells are multiple and feasibly obtained from various adult tissues, such as bone marrow, blood, adipose tissue, teeth and testes (Table 1). These adult stem cells can be collected from the human body at anytime throughout life. This makes them readily available and does not raise the moral and ethical issues involved with the attainment of embryonic stem cells. Moreover, pluripotent adult stem cells can easily be isolated and puri‐ fied by cell surface markers, such as CD49f, SSEA-3 and SSEA4 (Conrad et al., 2008; Kuroda et al., 2010; Kawanabe et al., 2010; Notta et al., 2011). The pluripotent status of these adult stem cells is naturally acquired and does not require reprogramming by the introduction of pluripotent transcriptional factors, thus eliminating the use of viral vectors and the chance of aberrant chromosomal changes. Furthermore, transplantation of mesenchymal stem cells and periodontal ligament-derived stem cells can be autogenic or allogeneic. Immuno-sup‐ pression is not necessary since mesenchymal stem cells have strong immunomodulatory properties against alloreactivity of T lymphocytes and dendritic cells (Chen et al., 2011). Similarly, mesenchymal stem cells and periodontal ligament-derived stem cells inhibit the proliferation of peripheral blood mononuclear cells (Wada et al., 2009). Spermatogonial stem cells, however, are killed by cytotoxic T lymphocytes after transplantation (Dressel et al., 2009), whereas allogeneic hematopoietic stem cell transplantation induces graft-vs-host dis‐ ease (Strober et al., 2011). Therefore, transplantation of spermatogonial stem cells and hema‐ topoietic stem cells should only be autogenic, without the application of immunosuppressive drugs. Similar to embryonic stem cells and induced pluripotent stem cells, pluripotent adult stem cells can differentiate into specialized cells of the three germ layers. Except for spermatogonial stem cells (Conrad et al., 2008), teratoma formation was not found in pluripotent hematopoietic stem cells, mesenchymal stem cells and periodontal ligament-derived stem cells (Kuroda et al., 2010; Kawanabe et al., 2010; Notta et al., 2011). This suggests a reduction in the probabilities of tumor formation post-transplantation, and

## **5. Advantages of pluripotent adult stem cells over embryonic stem cells and induced pluripotent stem cells**

Human embryonic stem cells come from the inner cell mass of human blastocysts. There‐ fore, embryonic stem cells used for cell therapy are allogenic; the transplanted donor cells do not originate from the recipient. This raises a concern about the immunogenic response of the host, and the need for immune-suppressive therapy concurrent with em‐ bryonic stem cell transplantation (Charron et al., 2009). Moreover, embryonic stem cellbased therapy has been hampered by the moral, legal and ethical dilemma surrounding the use of human embryos for derivation of the stem cell lines (Zarzeczny and Caulfield, 2009). Furthermore, as the gold standard of pluripotent stem cells, embryonic stem cells have the potential to form teratomas in the host. Tumorigenic potential can be reduced by differentiating the embryonic stem cells into lineage-specific progenitor cells or ma‐ ture tissue cells prior to transplantation (Schwartz et al., 2012). In order to better control standards of good manufacturing practices and reduce variability as much as possible, the *in vitro* manipulation of embryonic stem cells should be minimized as recommend by the Food and Drug Administration (Lysaght and Campbell, 2011). Furthermore, tumori‐ genic potential remains a concern if the entirety of the embryonic stem cell population does not completely differentiate into fully mature cells.

Differentiated adult cells used for the generation of the induced pluripotent stem cells can be collected from the recipient body, avoiding the contentious need for a human embryo. This also circumvents the problem of immune rejection. There are technical hurdles, howev‐ er, concerning generation of induced pluripotent stem cells (Hayden, 2011). Firstly, the de‐ livery of reprogramming factors (OCT4, SOX2, NANOG, LIN28, KLF4 and c-MYC) relies on the use of viral vectors for delivery (Takahashi et al., 2007). Retroviral sequences could inte‐ grate into the DNA of the host cells, potentially disrupting the gene structure as well as re‐ sulting in an aberrant phenotypic expression. Ultimately this could result in pathological mutations and cancer formation. Alternative methods such as direct protein or small mole‐ cule delivery have been adopted, although the reprogramming efficiency of these techni‐ ques is lower than with viral vectors (Kim et al., 2009; Shi et al., 2008). Secondly, two of the reprogramming factors, *c-MYC* and *KLF4*, are proto-oncogenes, which raise the concern of cancer formation further. Omitting *c-MYC* would lower the reprogramming efficiency, whereas silencing *c-MYC* could lead to its reactivation. Moreover, reprogramming can in‐ duce other genomic changes, such as DNA mutations (Gore et al., 2011), copy number varia‐ tions (Hussein et al., 2011) and chromosomal aberrations (Mayshar et al., 2010). Genomic instability could have unpredictable and undesirable effects on the reprogrammed cells. Furthermore, induced pluripotent stem cells carry their epigenetic signatures from the origi‐ nal differentiated adult cells (Lister et al., 2011). The reprogrammed cells, therefore, unlike embryonic stem cells, may not develop into some cell types. In addition, induced pluripo‐ tent stem cells can still cause immune reactions when transplanted allogeneically.

tent stem cell line, a teratoma assay might not be a suitable assay for pluripotency of adult stem cells. Instead, *in vitro* and *in vivo* differentiation into cells of the 3 embryonic germ lay‐ ers along with chimera formation in xeno-transplanted mice can be applied for testing adult stem cell potency. The conventional concept of development involves a hierarchical struc‐ ture of cellular commitment extending outward from embryonic and pluripotent, to adult terminally differentiated tissues. However, recent ideas propose that all or most tissues in the postnatal body are continuously turning over and contain a pluripotent stem cell reser‐ voir (Slack, 2008). These pluripotent stem cell populations are able to differentiate into mul‐ tiple cell types depending on their microenvironmental cues. Therefore, the stem cell status should be defined by plasticity (Zipori, 2005). Pluripotency refers to the ability of cells to dif‐ ferentiate into any cell type of the 3 germ layers (ectoderm, endoderm and mesoderm), whereas multipotency refers to the ability of cells to differentiate only into a closely related family of cells (Ilic and Polak, 2011). All of the previously described adult stem cells (hema‐ topoietic, mesenchymal, periodontal ligament-derived, and spermatogonial) could differen‐ tiate into specialized cells of the three germ layers: neurons (ectodermal lineage), adipocytes, cardiomyocytes, osteoblasts, and chondrocytes (mesoderm lineage), and hepatocytes and in‐ sulin-producing cells (endodermal lineage) (Conrad et al., 2008; Jiang et al., 2002; Kuroda et al., 2010; Kawanabe et al., 2010; Notta et al., 2011). Therefore, these adult stem cells could

**5. Advantages of pluripotent adult stem cells over embryonic stem cells**

Human embryonic stem cells come from the inner cell mass of human blastocysts. There‐ fore, embryonic stem cells used for cell therapy are allogenic; the transplanted donor cells do not originate from the recipient. This raises a concern about the immunogenic response of the host, and the need for immune-suppressive therapy concurrent with em‐ bryonic stem cell transplantation (Charron et al., 2009). Moreover, embryonic stem cellbased therapy has been hampered by the moral, legal and ethical dilemma surrounding the use of human embryos for derivation of the stem cell lines (Zarzeczny and Caulfield, 2009). Furthermore, as the gold standard of pluripotent stem cells, embryonic stem cells have the potential to form teratomas in the host. Tumorigenic potential can be reduced by differentiating the embryonic stem cells into lineage-specific progenitor cells or ma‐ ture tissue cells prior to transplantation (Schwartz et al., 2012). In order to better control standards of good manufacturing practices and reduce variability as much as possible, the *in vitro* manipulation of embryonic stem cells should be minimized as recommend by the Food and Drug Administration (Lysaght and Campbell, 2011). Furthermore, tumori‐ genic potential remains a concern if the entirety of the embryonic stem cell population

Differentiated adult cells used for the generation of the induced pluripotent stem cells can be collected from the recipient body, avoiding the contentious need for a human embryo. This also circumvents the problem of immune rejection. There are technical hurdles, howev‐

also be defined as pluripotent stem cells.

30 Pluripotent Stem Cells

**and induced pluripotent stem cells**

does not completely differentiate into fully mature cells.

The sources of adult stem cells are multiple and feasibly obtained from various adult tissues, such as bone marrow, blood, adipose tissue, teeth and testes (Table 1). These adult stem cells can be collected from the human body at anytime throughout life. This makes them readily available and does not raise the moral and ethical issues involved with the attainment of embryonic stem cells. Moreover, pluripotent adult stem cells can easily be isolated and puri‐ fied by cell surface markers, such as CD49f, SSEA-3 and SSEA4 (Conrad et al., 2008; Kuroda et al., 2010; Kawanabe et al., 2010; Notta et al., 2011). The pluripotent status of these adult stem cells is naturally acquired and does not require reprogramming by the introduction of pluripotent transcriptional factors, thus eliminating the use of viral vectors and the chance of aberrant chromosomal changes. Furthermore, transplantation of mesenchymal stem cells and periodontal ligament-derived stem cells can be autogenic or allogeneic. Immuno-sup‐ pression is not necessary since mesenchymal stem cells have strong immunomodulatory properties against alloreactivity of T lymphocytes and dendritic cells (Chen et al., 2011). Similarly, mesenchymal stem cells and periodontal ligament-derived stem cells inhibit the proliferation of peripheral blood mononuclear cells (Wada et al., 2009). Spermatogonial stem cells, however, are killed by cytotoxic T lymphocytes after transplantation (Dressel et al., 2009), whereas allogeneic hematopoietic stem cell transplantation induces graft-vs-host dis‐ ease (Strober et al., 2011). Therefore, transplantation of spermatogonial stem cells and hema‐ topoietic stem cells should only be autogenic, without the application of immunosuppressive drugs. Similar to embryonic stem cells and induced pluripotent stem cells, pluripotent adult stem cells can differentiate into specialized cells of the three germ layers. Except for spermatogonial stem cells (Conrad et al., 2008), teratoma formation was not found in pluripotent hematopoietic stem cells, mesenchymal stem cells and periodontal ligament-derived stem cells (Kuroda et al., 2010; Kawanabe et al., 2010; Notta et al., 2011). This suggests a reduction in the probabilities of tumor formation post-transplantation, and the elimination of the need to manipulate the cells into mature tissue prior to transplanta‐ tion. In addition, transplanted stem cell-induced regeneration may not be due to stem cell differentiation per se (Johnson et al., 2010; Williams and Hare, 2011). Instead, a paracrine ef‐ fect has been hypothesized in which the adult stem cells secrete cytokines, chemokines, or protective proteins (Bai et al., 2012; Bráz et al., 2012) that nourish the host tissue cells and facilitate the healing process. This special feature has not yet been reported with the use of embryonic stem cells or induced pluripotent stem cells in a clinical setting.

ble over embryonic stem cells and induced pluripotent stem cells. The secretion of cyto‐ kines, chemokines, and/or protective proteins from the adult stem cells could nourish the

Pluripotent Adult Stem Cells: A Potential Revolution in Regenerative Medicine and Tissue Engineering

http://dx.doi.org/10.5772/54366

33

Adult stem cells are found all over the body. They can be conveniently obtained from different accessible tissues: bone marrow, blood, adipose tissue, teeth and testes. Pluripo‐ tent adult stem cells, which reside as a subpopulation within adult stem cells, can be easily isolated by pluripotent cell surface markers, such as SSEA-3, SSEA-4 and CD49f. Moreover, pluripotent adult stem cells can be characterized by their ability to differenti‐ ate into cells of 3 germ layers (ectoderm, mesoderm and endoderm) as well as by the chimera formation in xeno-transplanted mice. Pluripotent adult stem cells are better than embryonic stem cells and induced pluripotent stem cells as they are an autologous source, require minimal manipulation and do not have the ability to form teratomas. In addition, they are more appropriate to be used as a clinical product for therapeutic treat‐ ments, as a cellular replacement or secretory protein reservoir. However, there are uncer‐ tainties that still remain unanswered. Which stem cell types are optimal for regenerative medicine? What is the optimal cell number for transplantation? Should the cells be pre‐ emptively differentiated or used as is? Further research is needed to understand the

mechanisms of stem cells in regenerating damaged tissues after transplantation.

, Veronica R. Fortino2

1 Geriatric Research, Education and Clinical Center, Miami Veterans Affairs Medical Center,

2 Department of Biomedical Engineering, College of Engineering, University of Miami, Coral

[1] Alvarez-dolado, M. R, Pardal, J. M, Garcia-verdugo, J. R, Fike, H. O, Lee, K, Pfeffer, C, Lois, S. J, & Morrison, A. Alvarez-Buylla. (2003). Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature, 425, 968-973.

\*Address all correspondence to: hcheung@med.maimi.edu

, Jordan Greenberg2

and

host tissue and facilitate the healing process (Bai et al., 2012; Bráz et al., 2012).

**7. Summary**

**Author details**

Herman S. Cheung1,2\*

Miami, FL, USA

Gables, FL, USA

**References**

, Daniel Pelaez1

Tsz Kin Ng1

### **6. Potential applications of pluripotent adult stem cells**

Stem cell clinical trials have advanced rapidly for a broad spectrum of diseases, such as dia‐ betes, neurodegeneration, immune diseases, heart disease, and bone disease. In 2011, there were 123 clinical trials using mesenchymal stem cells (Trounson et al., 2011). It is predicted that stem cell therapy will eventually become the treatment of choice in regenerative medi‐ cine, especially the use of adult stem cells. As stem cell products become more wide-spread and maintained under various conditions, the need for global standardization and regula‐ tion of processes will become necessary for the viable application of these products in a clin‐ ical setting. The Food and Drug Administration regulates interstate commerce in human cells and tissue-based products under the Public Health Service Act and the Code of Federal Regulations for Food and Drugs (Lysaght and Campbell, 2011). Human cells and tissuebased products are defined as "articles containing or consisting of human cells or tissues that are intended for implantation, transplantation, infusion, or transfer into a human recipi‐ ent" (Lysaght and Campbell, 2011). Human cells and tissue-based products must be: (1) minimally manipulated, (2) intended only for homologous use, (3) not combined with an‐ other article (except for water, or sterilization, preservation, or storage agents), and (4) ei‐ ther: (a) have no systemic or metabolic effect, or (b) be for autologous use, allogeneic use in first- or second-degree blood relative, or reproductive use.

Pluripotent adult stem cells fall under the criteria for human cells and tissue-based prod‐ ucts as stated by the Food and Drug Administration. Unlike induced pluripotent stem cells, pluripotent adult stem cells can be minimally manipulated as their pluripotent state occurs naturally. Unlike embryonic stem cells, pluripotent adult stem cells are suited for autologous use. Similar to embryonic stem cells and induced pluripotent stem cells, plu‐ ripotent adult stem cells are able to differentiate into specialized cells of the three germ layers. In addition, embryonic stem cells and induced pluripotent stem cells have the po‐ tential to form teratomas (an unfavorable side-effect in clinical applications) although a recent study suggests that the teratoma-forming cells could be removed by the antibody against SSEA-5 (Tang et al., 2011). In contrast, most pluripotent adult stem cells do not form teratomas *in vivo*, eliminating the need for preemptive differentiation of pluripotent adult stem cells into mature specialized cells.

If stem cell-aided regeneration is not due to stem cell differentiation to replace damaged cells (Johnson et al., 2010; Williams and Hare, 2011), pluripotent adult stem cells are favora‐ ble over embryonic stem cells and induced pluripotent stem cells. The secretion of cyto‐ kines, chemokines, and/or protective proteins from the adult stem cells could nourish the host tissue and facilitate the healing process (Bai et al., 2012; Bráz et al., 2012).

## **7. Summary**

the elimination of the need to manipulate the cells into mature tissue prior to transplanta‐ tion. In addition, transplanted stem cell-induced regeneration may not be due to stem cell differentiation per se (Johnson et al., 2010; Williams and Hare, 2011). Instead, a paracrine ef‐ fect has been hypothesized in which the adult stem cells secrete cytokines, chemokines, or protective proteins (Bai et al., 2012; Bráz et al., 2012) that nourish the host tissue cells and facilitate the healing process. This special feature has not yet been reported with the use of

Stem cell clinical trials have advanced rapidly for a broad spectrum of diseases, such as dia‐ betes, neurodegeneration, immune diseases, heart disease, and bone disease. In 2011, there were 123 clinical trials using mesenchymal stem cells (Trounson et al., 2011). It is predicted that stem cell therapy will eventually become the treatment of choice in regenerative medi‐ cine, especially the use of adult stem cells. As stem cell products become more wide-spread and maintained under various conditions, the need for global standardization and regula‐ tion of processes will become necessary for the viable application of these products in a clin‐ ical setting. The Food and Drug Administration regulates interstate commerce in human cells and tissue-based products under the Public Health Service Act and the Code of Federal Regulations for Food and Drugs (Lysaght and Campbell, 2011). Human cells and tissuebased products are defined as "articles containing or consisting of human cells or tissues that are intended for implantation, transplantation, infusion, or transfer into a human recipi‐ ent" (Lysaght and Campbell, 2011). Human cells and tissue-based products must be: (1) minimally manipulated, (2) intended only for homologous use, (3) not combined with an‐ other article (except for water, or sterilization, preservation, or storage agents), and (4) ei‐ ther: (a) have no systemic or metabolic effect, or (b) be for autologous use, allogeneic use in

Pluripotent adult stem cells fall under the criteria for human cells and tissue-based prod‐ ucts as stated by the Food and Drug Administration. Unlike induced pluripotent stem cells, pluripotent adult stem cells can be minimally manipulated as their pluripotent state occurs naturally. Unlike embryonic stem cells, pluripotent adult stem cells are suited for autologous use. Similar to embryonic stem cells and induced pluripotent stem cells, plu‐ ripotent adult stem cells are able to differentiate into specialized cells of the three germ layers. In addition, embryonic stem cells and induced pluripotent stem cells have the po‐ tential to form teratomas (an unfavorable side-effect in clinical applications) although a recent study suggests that the teratoma-forming cells could be removed by the antibody against SSEA-5 (Tang et al., 2011). In contrast, most pluripotent adult stem cells do not form teratomas *in vivo*, eliminating the need for preemptive differentiation of pluripotent

If stem cell-aided regeneration is not due to stem cell differentiation to replace damaged cells (Johnson et al., 2010; Williams and Hare, 2011), pluripotent adult stem cells are favora‐

embryonic stem cells or induced pluripotent stem cells in a clinical setting.

**6. Potential applications of pluripotent adult stem cells**

32 Pluripotent Stem Cells

first- or second-degree blood relative, or reproductive use.

adult stem cells into mature specialized cells.

Adult stem cells are found all over the body. They can be conveniently obtained from different accessible tissues: bone marrow, blood, adipose tissue, teeth and testes. Pluripo‐ tent adult stem cells, which reside as a subpopulation within adult stem cells, can be easily isolated by pluripotent cell surface markers, such as SSEA-3, SSEA-4 and CD49f. Moreover, pluripotent adult stem cells can be characterized by their ability to differenti‐ ate into cells of 3 germ layers (ectoderm, mesoderm and endoderm) as well as by the chimera formation in xeno-transplanted mice. Pluripotent adult stem cells are better than embryonic stem cells and induced pluripotent stem cells as they are an autologous source, require minimal manipulation and do not have the ability to form teratomas. In addition, they are more appropriate to be used as a clinical product for therapeutic treat‐ ments, as a cellular replacement or secretory protein reservoir. However, there are uncer‐ tainties that still remain unanswered. Which stem cell types are optimal for regenerative medicine? What is the optimal cell number for transplantation? Should the cells be pre‐ emptively differentiated or used as is? Further research is needed to understand the mechanisms of stem cells in regenerating damaged tissues after transplantation.

## **Author details**

Tsz Kin Ng1 , Daniel Pelaez1 , Veronica R. Fortino2 , Jordan Greenberg2 and Herman S. Cheung1,2\*

\*Address all correspondence to: hcheung@med.maimi.edu

1 Geriatric Research, Education and Clinical Center, Miami Veterans Affairs Medical Center, Miami, FL, USA

2 Department of Biomedical Engineering, College of Engineering, University of Miami, Coral Gables, FL, USA

### **References**

[1] Alvarez-dolado, M. R, Pardal, J. M, Garcia-verdugo, J. R, Fike, H. O, Lee, K, Pfeffer, C, Lois, S. J, & Morrison, A. Alvarez-Buylla. (2003). Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature, 425, 968-973.

[2] Anjos-afonso, F, & Bonnet, D. (2007). Nonhematopoietic/endothelial SSEA-1+ cells define the most primitive progenitors in the adult murine bone marrow mesenchy‐ mal compartment. Blood. , 109, 1298-1306.

cells, can be killed by cytotoxic T lymphocytes despite low expression of major histo‐

Pluripotent Adult Stem Cells: A Potential Revolution in Regenerative Medicine and Tissue Engineering

http://dx.doi.org/10.5772/54366

35

[14] Fagoonee, S, Pellicano, R, Silengo, L, & Altruda, F. (2011). Potential applications of germline cell-derived pluripotent stem cells in organ regeneration. *Organogenesis*, 7,

[15] Gore, A, Li, Z, Fung, H. L, Young, J. E, Agarwal, S, Antosiewicz-bourget, J, Canto, I, Gior‐ getti, A, Israel, M. A, Kiskinis, E, Lee, J. H, Loh, Y. H, Manos, P. D, Montserrat, N, Pano‐ poulos, A. D, Ruiz, S, Wilbert, M. L, Yu, J, Kirkness, E. F, Izpisua, J. C, Belmonte, D. J, Rossi, J. A, Thomson, K, Eggan, G. Q, & Daley, L. S. Goldstein, and K. Zhang. (2011). So‐ matic coding mutations in human induced pluripotent stem cells. *Nature*, 471, 63-67. [16] Gundry, R. L, Riordon, D. R, Tarasova, Y, Chuppa, S, Bhattacharya, S, Juhasz, O, Wiedemeier, O, Milanovich, S, Noto, F. K, Tchernyshyov, I, Raginski, K, Bauschfluck, D, Tae, H. J, Marshall, S, Duncan, S. A, Wollscheid, B, Wersto, R. P, Rao, S, Van Eyk, J. E, & Boheler, K. R. (2012). A Cell Surfaceome Map for Immunophenotyping

[17] Hayden, E. C. (2011). Stem cells: The growing pains of pluripotency. *Nature*, 473, 272-274. [18] Hogan, B. L. (1976). Changes in the behaviour of teratocarcinoma cells cultivated in

[19] Hussein, S. M, Batada, N. N, Vuoristo, S, Ching, R. W, Autio, R, Närvä, E, Ng, S, Sourour, M, Hämäläinen, R, Olsson, C, Lundin, K, Mikkola, M, Trokovic, R, Peitz, M, Brüstle, O, Bazett-jones, D. P, Alitalo, K, Lahesmaa, R, Nagy, A, & Otonkoski, T. (2011). Copy number variation and selection during reprogramming to pluripotency.

[20] Ilic, D, & Polak, J. M. (2011). Stem cells in regenerative medicine: introduction. *Br*

[21] Izadyar, F, Pau, F, Marh, J, Slepko, N, Wang, T, Gonzalez, R, Ramos, T, Howerton, K, Sayre, C, & Silva, F. (2008). Generation of multipotent cell lines from a distinct popu‐

[22] Izadyar, F, Wong, J, Maki, C, Pacchiarotti, J, Ramos, T, Howerton, K, Yuen, C, Grei‐ lach, S, Zhao, H. H, Chow, M, Chow, Y. C, Rao, J, Barritt, J, Bar-chama, N, & Copper‐ man, A. (2011). Identification and characterization of repopulating spermatogonial

[23] Jaramillo-ferrada, P. A, Wolvetang, E. J, & Cooper-white, J. J. (2012). Differential mes‐ engenic potential and expression of stem cell-fate modulators in mesenchymal stro‐ mal cells from human-term placenta and bone marrow. *J Cell Physiol*. , 227, 3234-3242.

[24] Jiang, Y, Jahagirdar, B. N, Reinhardt, R. L, Schwartz, R. E, Keene, C. D, Ortiz-gonza‐ lez, X. R, Reyes, M, Lenvik, T, Lund, T, Blackstad, M, Du, J, Aldrich, S, Lisberg, A, Low, W. C, Largaespada, D. A, & Verfaillie, C. M. (2002). Pluripotency of mesenchy‐

lation of male germ line stem cells. *Reproduction*. , 135, 771-784.

stem cells from the adult human testis. *Hum Reprod*. , 26, 1296-1306.

mal stem cells derived from adult marrow. *Nature*, 418, 41-49.

compatibility complex class I molecules. *Biol Direct*. 4:31.

and Sorting Pluripotent Stem Cells. *Mol Cell Proteomics*. In press.

116-122.

vitro. *Nature*, 263, 136-137.

*Nature*, 471, 58-62.

*Med Bull*. , 98, 117-126.


cells, can be killed by cytotoxic T lymphocytes despite low expression of major histo‐ compatibility complex class I molecules. *Biol Direct*. 4:31.

[14] Fagoonee, S, Pellicano, R, Silengo, L, & Altruda, F. (2011). Potential applications of germline cell-derived pluripotent stem cells in organ regeneration. *Organogenesis*, 7, 116-122.

[2] Anjos-afonso, F, & Bonnet, D. (2007). Nonhematopoietic/endothelial SSEA-1+ cells define the most primitive progenitors in the adult murine bone marrow mesenchy‐

[3] Arita, N. A, Pelaez, D, & Cheung, H. S. (2011). Activation of the extracellular signalregulated kinases 1 and 2 (ERK1/2) is needed for the TGFβ-induced chondrogenic and osteogenic differentiation of mesenchymal stem cells. *Biochem Biophys Res Com‐*

[4] Bai, L, Lennon, D. P, Caplan, A. I, Dechant, A, Hecker, J, Kranso, J, Zaremba, A, & Miller, R. H. (2012). Hepatocyte growth factor mediates mesenchymal stem cell-in‐

[5] Becker, K, Wobus, A. M, Conrad, U, & Schöneich, J. (1984). Injection of murine em‐ bryonal carcinoma cells and embryo-derived pluripotential cells into mouse blasto‐

[6] Bráz, J. M, Sharif-naeini, R, Vogt, D, Kriegstein, A, Alvarez-buylla, A, Rubenstein, J. L, & Basbaum, A. I. (2012). Forebrain GABAergic neuron precursors integrate into adult spinal cord and reduce injury-induced neuropathic pain. *Bráz, J.M., R. Sharif-Naeini, D. Vogt, A. Kriegstein, A. Alvarez-Buylla, J.L. Rubenstein, and A.I. Basbaum. 2012. Forebrain GABAergic neuron precursors integrate into adult spinal cord and reduce injury-*

[7] Brevini, T. A, Pennarossa, G, Antonini, S, & Gandolfi, F. (2008). Parthenogenesis as an approach to pluripotency: advantages and limitations involved. *Stem Cell Rev*. ,

[8] Cesselli, D, Beltrami, A. P, Rigo, S, Bergamin, N, Aurizio, F. D, Verardo, R, Piazza, S, Klaric, E, Fanin, R, Toffoletto, B, Marzinotto, S, Mariuzzi, L, Finato, N, Pandolfi, M, Leri, A, Schneider, C, Beltrami, C. A, & Anversa, P. (2009). Multipotent progenitor

[9] Charron, D, Suberbielle-boissel, C, & Al-daccak, R. (2009). Immunogenicity and allo‐ genicity: a challenge of stem cell therapy. *J Cardiovasc Transl Res*. , 2, 130-138.

[10] Chen, P. M, Yen, M. L, Liu, K. J, Sytwu, H. K, & Yen, B. L. (2011). Immunomodulato‐ ry properties of human adult and fetal multipotent mesenchymal stem cells. *J Biomed*

[11] Conrad, S, Renninger, M, Hennenlotter, J, Wiesner, T, Just, L, Bonin, M, Aicher, W, Bühring, H. J, Mattheus, U, Mack, A, Wagner, H. J, Minger, S, Matzkies, M, Reppel, M, Hescheler, J, Sievert, K. D, Stenzl, A, & Skutella, T. (2008). Generation of pluripo‐

[12] Ding, D. C, Shyu, W. C, & Lin, S. Z. (2011). Mesenchymal stem cells. *Cell Transplant*. ,

[13] Dressel, R, Guan, K, Nolte, J, Elsner, L, Monecke, S, Nayernia, K, Hasenfuss, G, & En‐ gel, W. (2009). Multipotent adult germ-line stem cells, like other pluripotent stem

cells are present in human peripheral blood. *Circ Res*. , 104, 1225-1234.

tent stem cells from adult human testis. *Nature*, 456, 344-349.

duced recovery in multiple sclerosis models. Nat Neurosci. *In press*.

mal compartment. Blood. , 109, 1298-1306.

*mun*. , 405, 564-569.

34 Pluripotent Stem Cells

4127-135.

*Sci*. 18:49.

20, 5-14.

cysts. *Cell Differ*. , 15, 195-202.

*induced neuropathic pain. Neuron. .*, 74, 663-675.


[25] Johnson, T. V, Bull, N. D, Hunt, D. P, Marina, N, Tomarev, S. I, & Martin, K. R. (2010). Neuroprotective effects of intravitreal mesenchymal stem cell transplantation in experimental glaucoma. *Invest Ophthalmol Vis Sci*. , 51, 2051-2059.

[36] Notta, F, Doulatov, S, Laurenti, E, Poeppl, A, Jurisica, I, & Dick, J. E. (2011). Isolation of single human hematopoietic stem cells capable of long-term multilineage engraft‐

Pluripotent Adult Stem Cells: A Potential Revolution in Regenerative Medicine and Tissue Engineering

http://dx.doi.org/10.5772/54366

37

[37] Panda, R. P, Barman, H. K, & Mohapatra, C. (2011). Isolation of enriched carp sper‐ matogonial stem cells from Labeo rohita testis for in vitro propagation. *Theriogenolo‐*

[38] Perry, A. C. (2005). Progress in human somatic-cell nuclear transfer. *N Engl J Med*. ,

[39] Pittenger, M. F, Mackay, A. M, Beck, S. C, Jaiswal, R. K, Douglas, R, Mosca, J. D, Moorman, M. A, Simonetti, D. W, Craig, S, & Marshak, D. R. (1999). Multilineage po‐

[40] Pralong, D, Trounson, A. O, & Verma, P. J. (2006). Cell fusion for reprogramming pluripotency: toward elimination of the pluripotent genome. *Stem Cell Rev*. , 2,

[41] Riekstina, U, Cakstina, I, Parfejevs, V, Hoogduijn, M, Jankovskis, G, Muiznieks, I, Muceniece, R, & Ancans, J. (2009). Embryonic stem cell marker expression pattern in human mesenchymal stem cells derived from bone marrow, adipose tissue, heart

[42] Schwartz, S. D, Hubschman, J. P, Heilwell, G, Franco-cardenas, V, Pan, C. K, Ostrick, R. M, Mickunas, E, Gay, R, Klimanskaya, I, & Lanza, R. (2012). Embryonic stem cell

[43] Seo, B. M, Miura, M, Gronthos, S, Bartold, P. M, Batouli, S, Brahim, J, Young, M, Ro‐ bey, P. G, Wang, C. Y, & Shi, S. (2004). Investigation of multipotent postnatal stem

[44] Shi, Y, Desponts, C, Do, J. T, Hahm, H. S, Schöler, H. R, & Ding, S. (2008). Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with

[46] Strober, S, Spitzer, T. R, Lowsky, R, & Sykes, M. (2011). Translational studies in hem‐ atopoietic cell transplantation: treatment of hematologic malignancies as a stepping

[47] Sung, H. J, Hong, S. C, Yoo, J. H, Oh, J. H, Shin, H. J, Choi, I. Y, Ahn, K. H, Kim, S. H, Park, Y, & Kim, B. S. (2010). Stemness evaluation of mesenchymal stem cells from placentas according to developmental stage: comparison to those from adult bone

[48] Takahashi, K, Tanabe, K, Ohnuki, M, Narita, M, Ichisaka, T, Tomoda, K, & Yamana‐ ka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by de‐

[45] Slack, J. M. (2008). Origin of stem cells in organogenesis. *Science*. , 322, 1498-1501.

trials for macular degeneration: a preliminary report. *Lancet*. , 379, 713-720.

cells from human periodontal ligament. *Lancet*. , 364, 149-155.

small-molecule compounds. *Cell Stem Cell*. , 3, 568-574.

stone to tolerance induction. *Semin Immunol*. , 23, 273-281.

marrow. *J Korean Med Sci*. , 25, 1418-1426.

fined factors. *Cell*. , 131, 861-872.

tential of adult human mesenchymal stem cells. *Science*. , 284, 143-147.

ment. *Science*, 333, 218-221.

and dermis. *Stem Cell Rev*. , 5, 378-386.

*gy*, 76, 241-251.

353, 87-88.

331-340.


[36] Notta, F, Doulatov, S, Laurenti, E, Poeppl, A, Jurisica, I, & Dick, J. E. (2011). Isolation of single human hematopoietic stem cells capable of long-term multilineage engraft‐ ment. *Science*, 333, 218-221.

[25] Johnson, T. V, Bull, N. D, Hunt, D. P, Marina, N, Tomarev, S. I, & Martin, K. R. (2010). Neuroprotective effects of intravitreal mesenchymal stem cell transplantation

[26] Kanatsu-shinohara, M, Lee, J, Inoue, K, Ogonuki, N, Miki, H, Toyokuni, S, Ikawa, M, Nakamura, T, Ogura, A, & Shinohara, T. (2008). Pluripotency of a single spermatogo‐

[27] Kawanabe, N, Murata, S, Murakami, K, Ishihara, Y, Hayano, S, Kurosaka, H, Kamio‐ ka, H, Takano-yamamoto, T, & Yamashiro, T. (2010). Isolation of multipotent stem cells in human periodontal ligament using stage-specific embryonic antigen-4. *Differ‐*

[28] Kim, D, Kim, C. H, Moon, J. I, Chung, Y. G, Chang, M. Y, Han, B. S, Ko, S, Yang, E, Cha, K. Y, Lanza, R, & Kim, K. S. (2009). Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. *Cell Stem Cell*, 4, 472-476. [29] Kondo, M, Wagers, A. J, Manz, M. G, Prohaska, S. S, Scherer, D. C, Beilhack, G. F, Shizuru, J. A, & Weissman, I. L. (2003). Biology of hematopoietic stem cells and pro‐

genitors: implications for clinical application. *Annu Rev Immunol*. , 21, 759-806.

[30] Kuroda, Y, Kitada, M, Wakao, S, Nishikawa, K, Tanimura, Y, Makinoshima, H, Goda, M, Akashi, H, Inutsuka, A, Niwa, A, Shigemoto, T, Nabeshima, Y, Nakahata, T, Nabeshima, Y, Fujiyoshi, Y, & Dezawa, M. (2010). Unique multipotent cells in adult human mesenchymal cell populations. *Proc Natl Acad Sci U S A*. , 107,

[31] Lister, R, Pelizzola, M, Kida, Y. S, Hawkins, R. D, Nery, J. R, Hon, G, Antosiewiczbourget, J, Malley, R. O, Castanon, R, Klugman, S, Downes, M, Yu, R, Stewart, R, Ren, B, Thomson, J. A, Evans, R. M, & Ecker, J. R. (2011). Hotspots of aberrant epige‐ nomic reprogramming in human induced pluripotent stem cells. *Nature*, 471, 68-73.

[32] Lysaght, T, & Campbell, A. V. (2011). Regulating autologous adult stem cells: the

[33] Mayshar, Y, Ben-david, U, Lavon, N, Biancotti, J. C, Yakir, B, Clark, A. T, Plath, K, Lowry, W. E, & Benvenisty, N. (2010). Identification and classification of chromoso‐ mal aberrations in human induced pluripotent stem cells. *Cell Stem Cell*, 7, 521-531.

[34] Mödder, U. I, Roforth, M. M, Nicks, K. M, Peterson, J. M, Mccready, L. K, Monroe, D. G, & Khosla, S. (2012). Characterization of mesenchymal progenitor cells isolated from human bone marrow by negative selection. *Mödder, U.I., M.M. Roforth, K.M. Nicks, J.M. Peterson, L.K. McCready, D.G. Monroe, and S. Khosla. 2012. Characterization of mesenchymal progenitor cells isolated from human bone marrow by negative selection.*

[35] Mrozik, K, Gronthos, S, Shi, S, & Bartold, P. M. (2010). A method to isolate, purify, and characterize human periodontal ligament stem cells. *Methods Mol Biol*. , 666,

in experimental glaucoma. *Invest Ophthalmol Vis Sci*. , 51, 2051-2059.

nial stem cell in mice. *Biol Reprod*. ;, 78, 681-687.

*entiation*, 79, 74-83.

36 Pluripotent Stem Cells

8639-8643.

*Bone. .*, 50, 804-810.

269-284.

FDA steps up. *Cell Stem Cell*, 9, 393-396.


[49] Tang, C, Lee, A. S, Volkmer, J. P, Sahoo, D, Nag, D, Mosley, A. R, Inlay, M. A, Arde‐ hali, R, Chavez, S. L, Pera, R. R, Behr, B, Wu, J. C, Weissman, I. L, & Drukker, M. (2011). An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells. *Nat Biotechnol*. , 29, 829-834.

**Chapter 3**

**De-Differentiation of Somatic**

**Cells to a Pluripotent State**

Cristiane V. Wenceslau, Irina Kerkis, Nelson F. Lizier and Alexandre Kerkis

http://dx.doi.org/10.5772/54372

**1. Introduction**

Additional information is available at the end of the chapter

The gametes are highly specialized haploid cells that harbor genetic background of each in‐ dividual. During fertilization, the fusion of female and male gametes occurs in order to pro‐ duce zygote. These zygotes are diploid cells and have genetic material of both individuals. Zygotes start to divide and undergo further pre-implantation development through the for‐ mation of morula, blastocysts and finally fetus (Fig. 1). When intrauterine fetal development is finished, the organism is already formed and ready to birth. The zygotes can be consid‐ ered primordial stem cells, which originate the whole organism through unequal divisions to produce blastomeres, the cells resulting by cleavage of a zygote. Sixteen blastomeres con‐ stitute a morula, the spherical embryonic mass surrounded by the zona pellucid, which fur‐ ther became a blastocyst. Blastocyst is a thin-walled hollow structure surrounded by trophoblasts layer that contains a cluster of cells called the inner cell mass (ICM) from which the embryo arises and the scientists isolate embryonic stem (ES) cells for *in vitro* cultivation and for study the process of differentiation. However, ES cells are pluripotent cells able to produce any cell type raise ethical concerns about the destruction of human embryo to pro‐ duce stem cell lines. To get the better concept of pluripotent cells for stem cell based thera‐ pies the reprogramming of patient specific adult cells to embryonic stage was suggested (Takashi & Yamanaka,2006). Cell reprogramming is a process of de-differentiation of somat‐ ic cells into pluripotent state whereby they adopt features of ES cells. De-differentiation of adult cells can be achieved through i. somatic cell nuclear transfer; ii. cell fusion - somatic cell hybrids and; iii. production of induced pluripotent stem (iPS) cells through the activa‐ tion of essential stemness genes (reprogramming factor), over-expression in fibroblasts and/or other adult cells. Small molecules and other technologies are also exploring to repro‐

> © 2013 Wenceslau et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Wenceslau et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


## **Chapter 3**

## **De-Differentiation of Somatic Cells to a Pluripotent State**

Cristiane V. Wenceslau, Irina Kerkis, Nelson F. Lizier and Alexandre Kerkis

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54372

#### **1. Introduction**

[49] Tang, C, Lee, A. S, Volkmer, J. P, Sahoo, D, Nag, D, Mosley, A. R, Inlay, M. A, Arde‐ hali, R, Chavez, S. L, Pera, R. R, Behr, B, Wu, J. C, Weissman, I. L, & Drukker, M. (2011). An antibody against SSEA-5 glycan on human pluripotent stem cells enables

[50] Tárnok, A, Ulrich, H, & Bocsi, J. (2010). Phenotypes of stem cells from diverse origin.

[51] Trounson, A, Thakar, R. G, Lomax, G, & Gibbons, D. (2011). Clinical trials for stem

[52] Wada, N, Menicanin, D, Shi, S, Bartold, P. M, & Gronthos, S. (2009). Immunomodulatory properties of human periodontal ligament stem cells. *J Cell Physiol*. , 219, 667-676.

[54] Wang, J, Zhou, X, Cui, L, Yan, L, Liang, J, Cheng, X, Qiao, L, Shi, Y, Han, Z, Cao, Y, Han, Y, & Fan, D. (2010). The significance of CD14+ monocytes in peripheral blood

[55] Williams, A. R, & Hare, J. M. (2011). Mesenchymal stem cells: biology, pathophysiol‐ ogy, translational findings, and therapeutic implications for cardiac disease. *Circ*

[56] Wobus, A. M, Holzhausen, H, Jäkel, P, & Schöneich, J. (1984). Characterization of a pluripotent stem cell line derived from a mouse embryo. *Exp Cell Res*. , 152, 212-219.

[57] Yu, J, Vodyanik, M. A, Smuga-otto, K, Antosiewicz-bourget, J, Frane, J. L, Tian, S, Nie, J, Jonsdottir, G. A, Ruotti, V, Stewart, R, Slukvin, I. I, & Thomson, J. A. (2007). Induced pluripotent stem cell lines derived from human somatic cells. *Science*. , 318,

[58] Zarzeczny, A, & Caulfield, T. (2009). Emerging ethical, legal and social issues associ‐ ated with stem cell research & and the current role of the moral status of the embryo.

[59] Zhao, Y, Wang, H, & Mazzone, T. (2006). Identification of stem cells from human umbilical cord blood with embryonic and hematopoietic characteristics. *Exp Cell*

[60] Zheng, K, Wu, X, Kaestner, K. H, & Wang, P. J. (2009). The pluripotency factor LIN28

[61] Zipori, D. (2005). The stem state: plasticity is essential, whereas self-renewal and hi‐

[62] Zulli, A, Buxton, B. F, Merrilees, M, & Hare, D. L. (2008). Human diseased arteries contain cells expressing leukocytic and embryonic stem cell markers. *Hum Pathol*. ,

marks undifferentiated spermatogonia in mouse. *BMC Dev Biol*. 9:38.

erarchy are optional. *Stem Cells*. , 23, 719-726.

[53] Wagers, A. J, & Weissman, I. L. (2004). Plasticity of adult stem cells. *Cell*. , 116, 639-648.

stem cells for the treatment of rat liver cirrhosis. *Cytotherapy*. , 12, 1022-1034.

removal of teratoma-forming cells. *Nat Biotechnol*. , 29, 829-834.

*Cytometry A*. , 77, 6-10.

38 Pluripotent Stem Cells

*Res*. , 109, 923-940.

1917-1920.

*Stem Cell Rev*. , 5, 96-101.

*Res*. , 312, 2454-2464.

39, 657-665.

cell therapies. *BMC Med*. 9:52.

The gametes are highly specialized haploid cells that harbor genetic background of each in‐ dividual. During fertilization, the fusion of female and male gametes occurs in order to pro‐ duce zygote. These zygotes are diploid cells and have genetic material of both individuals. Zygotes start to divide and undergo further pre-implantation development through the for‐ mation of morula, blastocysts and finally fetus (Fig. 1). When intrauterine fetal development is finished, the organism is already formed and ready to birth. The zygotes can be consid‐ ered primordial stem cells, which originate the whole organism through unequal divisions to produce blastomeres, the cells resulting by cleavage of a zygote. Sixteen blastomeres con‐ stitute a morula, the spherical embryonic mass surrounded by the zona pellucid, which fur‐ ther became a blastocyst. Blastocyst is a thin-walled hollow structure surrounded by trophoblasts layer that contains a cluster of cells called the inner cell mass (ICM) from which the embryo arises and the scientists isolate embryonic stem (ES) cells for *in vitro* cultivation and for study the process of differentiation. However, ES cells are pluripotent cells able to produce any cell type raise ethical concerns about the destruction of human embryo to pro‐ duce stem cell lines. To get the better concept of pluripotent cells for stem cell based thera‐ pies the reprogramming of patient specific adult cells to embryonic stage was suggested (Takashi & Yamanaka,2006). Cell reprogramming is a process of de-differentiation of somat‐ ic cells into pluripotent state whereby they adopt features of ES cells. De-differentiation of adult cells can be achieved through i. somatic cell nuclear transfer; ii. cell fusion - somatic cell hybrids and; iii. production of induced pluripotent stem (iPS) cells through the activa‐ tion of essential stemness genes (reprogramming factor), over-expression in fibroblasts and/or other adult cells. Small molecules and other technologies are also exploring to repro‐

© 2013 Wenceslau et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Wenceslau et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

gram cells without the use of viral vectors primarily by Yamanaka. The methods used for cell de-differentiation induce the expression of genes that are not normally expressed in adult cell but are expressed in pluripotent stem cells, leading to the activation of pluripotent cell transcriptional networks. A cascade of transcriptional activity switch on the changes in gene expression profile in the adult cells, which begins to express a repertoire of genes that are commonly, identified in pluripotent ES cells. Following reprograming the adult cells un‐ dergo morphological changes and begin to grow as a tightly packed cluster of cells known as a colony, which mirrors how undifferentiated ES cells grow in culture. Both processes of reprograming and resulting pluripotency of reprogrammed cells vary significantly and elu‐ cidation of different approaches can clarify the reprograming process. In this chapter we will describe different methods of reprogramming of differentiated cells to pluripotent cells and the knowledge gain from each. Additionally, we try to provide a functional vision on reprogramming process and to analyze different types of stem cell niches produced by natu‐ ral and reprogrammed cells. The better comprehension of stem cell niches will allow us to improve the reprogramming technology and to put more close in production of natural plu‐ ripotent stem cells using molecular biology approaches.

**2. Natural** *in vivo* **pluripotent stem cell niches**

Here we proposed that starting from morula, when zona pellucid arises, a first specific com‐ partment called stem cell niche is formed. This niche can be defined as a microenvironment in which stem cells are found. Stem cell niche provides the both interaction between the cells and their interaction with local microenvironment, by which their fate regulates and occurs. In morula, stem cell niche consists of pluripotent stem cells that provide expression of spe‐ cific transcription factor, such as POU domain transcription factor (Oct3/4) responsible for self-renewal capacity and pluripotency of these cells. In mammals morula first cell fate deci‐ sions is governed by key transcriptional factor: Oct3/4 (Palmieri et al., 1994). Oct3/4 is unique because it requires maintaining the pluripotency in both conditions *in vivo* and *in vi‐ tro* (Nichols et al., 1998) and it is essential for epigenetic reprogramming (Niwa et al., 2000). The depletion of transcription factors leads to increased expression of genes that are in‐

De-Differentiation of Somatic Cells to a Pluripotent State

http://dx.doi.org/10.5772/54372

41

volved in the processes of development and cell differentiation (Niwa et al., 2000).

**2.3. Epiblast stem cell niche: Naive and primed pluripotent stem cells**

The first lineage segregation is resulted in the formation of trophectoderm and ICM (Wobus, et al., 2005). Upon silencing of Oct3/4, a part of morula cells spontaneously inactivates the self-renewal process and start to differentiate into trophoblast cells, thus forming pluripo‐ tent stem cell niche in blastocyst. This blastocyst niche is a dynamic structure which follows developmental program of an organism in parallel with Oct3/4, expression of other tran‐ scriptional factor such as Nanog occurs in ICM. in early blastocyst (Nichols et al., 1998; Avi‐

In mice in late blastocyst transcription factor Sox2 starts to express in the cells of ICM in ad‐ dition to GATA6 and Nanog, which lead to formation of two distinct populations: epiblast and hypoblast (Mitsui et al., 2003). These cell populations are considered the precursors of the primitive endoderm and the pluripotent epiblast (Morrissey et al., 1998). Recent studies suggest that stem cells in rodent epiblast have two distinct stable states of pluripotency: na‐ ïve and primed, thus establishing epiblast stem cell niche (Tesar et al., 2007; Nichols, 2009). According to these classification both of states exhibit features of bona fide pluripotent stem cells, such as have indefinite self-renewal, tri-germ layer potential and depend on expres‐ sion of all three transcription factors, such as Oct3/4, Sox2 and Nanog (Tesar et al., 2009; Nichols, 2009; de Los Angeles et al., 2012). Naïve (more immature) pluripotent stem cells can be obtained from pre-implanted stage of embryo in rodents (Okamoto et al., 2003). These cells have both sex X chromosomes activated and are able to produce high-grade chi‐ meras after their reintroduction into the host blastocyst. In contrast, in humans primed plu‐ ripotent ES cells are isolated from human pre-implantation blastocysts stage of development. In these cells one of female X chromosome is inactivated, albeit human ES

**2.1. Morula stem cell niche**

**2.2. Blastocyst stem cell niche**

lon et al., 2003).

**Figure 1.** Early development of stem cells niches. According to current knowledge there are natural stem cells niches during development: morula, blastocyst, epiblast and fetus, and artificial stem cells niches: stem cell culture *in vitro*.

## **2. Natural** *in vivo* **pluripotent stem cell niches**

#### **2.1. Morula stem cell niche**

gram cells without the use of viral vectors primarily by Yamanaka. The methods used for cell de-differentiation induce the expression of genes that are not normally expressed in adult cell but are expressed in pluripotent stem cells, leading to the activation of pluripotent cell transcriptional networks. A cascade of transcriptional activity switch on the changes in gene expression profile in the adult cells, which begins to express a repertoire of genes that are commonly, identified in pluripotent ES cells. Following reprograming the adult cells un‐ dergo morphological changes and begin to grow as a tightly packed cluster of cells known as a colony, which mirrors how undifferentiated ES cells grow in culture. Both processes of reprograming and resulting pluripotency of reprogrammed cells vary significantly and elu‐ cidation of different approaches can clarify the reprograming process. In this chapter we will describe different methods of reprogramming of differentiated cells to pluripotent cells and the knowledge gain from each. Additionally, we try to provide a functional vision on reprogramming process and to analyze different types of stem cell niches produced by natu‐ ral and reprogrammed cells. The better comprehension of stem cell niches will allow us to improve the reprogramming technology and to put more close in production of natural plu‐

**Figure 1.** Early development of stem cells niches. According to current knowledge there are natural stem cells niches during development: morula, blastocyst, epiblast and fetus, and artificial stem cells niches: stem cell culture *in vitro*.

ripotent stem cells using molecular biology approaches.

40 Pluripotent Stem Cells

Here we proposed that starting from morula, when zona pellucid arises, a first specific com‐ partment called stem cell niche is formed. This niche can be defined as a microenvironment in which stem cells are found. Stem cell niche provides the both interaction between the cells and their interaction with local microenvironment, by which their fate regulates and occurs. In morula, stem cell niche consists of pluripotent stem cells that provide expression of spe‐ cific transcription factor, such as POU domain transcription factor (Oct3/4) responsible for self-renewal capacity and pluripotency of these cells. In mammals morula first cell fate deci‐ sions is governed by key transcriptional factor: Oct3/4 (Palmieri et al., 1994). Oct3/4 is unique because it requires maintaining the pluripotency in both conditions *in vivo* and *in vi‐ tro* (Nichols et al., 1998) and it is essential for epigenetic reprogramming (Niwa et al., 2000). The depletion of transcription factors leads to increased expression of genes that are in‐ volved in the processes of development and cell differentiation (Niwa et al., 2000).

#### **2.2. Blastocyst stem cell niche**

The first lineage segregation is resulted in the formation of trophectoderm and ICM (Wobus, et al., 2005). Upon silencing of Oct3/4, a part of morula cells spontaneously inactivates the self-renewal process and start to differentiate into trophoblast cells, thus forming pluripo‐ tent stem cell niche in blastocyst. This blastocyst niche is a dynamic structure which follows developmental program of an organism in parallel with Oct3/4, expression of other tran‐ scriptional factor such as Nanog occurs in ICM. in early blastocyst (Nichols et al., 1998; Avi‐ lon et al., 2003).

#### **2.3. Epiblast stem cell niche: Naive and primed pluripotent stem cells**

In mice in late blastocyst transcription factor Sox2 starts to express in the cells of ICM in ad‐ dition to GATA6 and Nanog, which lead to formation of two distinct populations: epiblast and hypoblast (Mitsui et al., 2003). These cell populations are considered the precursors of the primitive endoderm and the pluripotent epiblast (Morrissey et al., 1998). Recent studies suggest that stem cells in rodent epiblast have two distinct stable states of pluripotency: na‐ ïve and primed, thus establishing epiblast stem cell niche (Tesar et al., 2007; Nichols, 2009). According to these classification both of states exhibit features of bona fide pluripotent stem cells, such as have indefinite self-renewal, tri-germ layer potential and depend on expres‐ sion of all three transcription factors, such as Oct3/4, Sox2 and Nanog (Tesar et al., 2009; Nichols, 2009; de Los Angeles et al., 2012). Naïve (more immature) pluripotent stem cells can be obtained from pre-implanted stage of embryo in rodents (Okamoto et al., 2003). These cells have both sex X chromosomes activated and are able to produce high-grade chi‐ meras after their reintroduction into the host blastocyst. In contrast, in humans primed plu‐ ripotent ES cells are isolated from human pre-implantation blastocysts stage of development. In these cells one of female X chromosome is inactivated, albeit human ES cells are self-renewing and express key transcription factors and are able to form teratoma (Okamoto et al., 2003; Brons et al., 2007; Tesar et al., 2005). Studies of X chromosome inacti‐ vation in pre-implantation human embryos reported that *XIST* transcript accumulation on this chromosome occurs in the eight-cell stage embryo, however the identity of the cells, which show *XIST* accumulation is not clear. *In vitro* studies of *XIST* accumulation in human ES cells lines revealed three different patterns of X chromosome inactivation. The naïve state - with both active X chromosomes, intermediate state - with both *XIST* accumulation and last - state, when the cells never undergo X chromosome inactivation even under differentia‐ tion and *XIST* accumulation does not occur (Dvash and Fan, 2009). Therefore, using current technologies "true" pluripotent stem cells can be exclusively isolated from mouse and may be from some other rodents, which present similar pattern of early embryonic development with mice. It is not obvious if it is possible to obtain "true" human ES cells, once we cannot test their contribution into developing human embryo (due to ethic consideration). Howev‐ er, the lack of X chromosome reactivation indicates that probably these cells will never be able to reintegrate into early development events *in vivo* similar to rodent.

ized enucleated oocyte and oocyte-NT involves the transplantation of multiple somatic cell nuclei into immature oocyte of amphibian. Nevertheless are important differences between the two types of nuclear transfer experiment. In oocyte-NT experiments extensive cell divi‐ sion take places and new functional cell types appear as soon as the nuclear transplant em‐ bryo start to develop. In this experiment somatic cell chromatin is directly reprogrammed to express pluripotency genes within a day. In contrast to oocyte-NT experiments, in egg-NT no new cell types are formed, and neither oocyte nor nuclei divide, however direct transi‐ tion of reprogrammed nuclei that transcribe genes of pluripotency into differentiated cells occurs. Analysis of the mechanism of reprogramming in egg-NT experiments, which in‐ volves transcription pluripotency genes and others, is complicated owing to rapid DNA rep‐

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43

lication and numerous cell divisions (Halley–Scott et al., 2010; Julien et al., 2010).

(Kishigami et al., 2007).

donor cell (somatic cell).

The NT process leads to direct reprogramming of pluripotent stem cell and expression of such markers as Oct3/4, Nanog, and Sox2 that are silent in differentiated somatic cell nu‐ cleus. In general, the reactivation of silent pluripotency genes starts around 24 and 48 hours after NT (Halley–Scott et al., 2010; Julien et al., 2010; Byrne et al., 2003). Upon NT occurs the series of events when oocyte cytoplasm induces changes in the structure of donor chromatin toward pluripotent state, which became more appropriate for embryonic development. However, synchronization process which should happen between genomic DNA of donor cell and cytoplasm of recipient cell is complex and may affect significantly pluripotency of reprogrammed cells. Attempts to facilitate this reprogramming process have been made us‐ ing chemicals that alter the methylation status of the chromatin, such as TSA (trichostatin A), azacytidine, scriptaid, either before or after NT. In the mouse, the use of TSA (a histone deacetylase inhibitor, HDACi) significantly increased the success rate of mouse cloning

**Figure 2.** Stages of nuclear transfer. The nucleus is removed from an egg (or oocyte) and replaced by a nucleus from a

## **3.** *In vitro* **pluripotent stem cell niches**

After isolation, pluripotent stem cells start to organize *in vitro* stem cell niche, which up to a cer‐ tain degree simulates experience of these cells *in vivo*. *In vitro* these cells showed similar mor‐ phology with ICM, forming islands of juxtaposed cells and expressing pluripotent stem cell markers such as Oct3/4, Nanog, Sox2 (Tesar et al., 2005; Tesar et al., 2007). However, to distin‐ guish *in vitro* naïve and primed pluripotent cells are difficult or even impossible task due to high heterogenety of pluripotent cell lines established *in vitro* (Brons et al., 2007; Tesar et al., 2007). Different factors may contribute to this heterogeneity, such as natural polymorphism of the cells, selection of colonies in vitro, which can be pluripotent at different degrees, cell cul‐ ture conditions adopted in each work as well as pluripotent cells, itself, may produce an "im‐ perfect" *in vitro* microenvironment again due to their natural heterogeneity.

## **4. Generation of artificial pluripotent stem cell – Reprogramming strategies**

#### **4.1. Reprogramming by means of differentiated cells nuclear transfer**

Several strategies can be provided in order to reprogramming differentiated or committed somatic cell genome. One of these strategies is a nuclear transfer (NT) of differentiated cell nucleus to oocyte whose maternal DNA was removed (Campbell et al., 1996). This type of reprogramming uses the natural components without any previous genetic or molecular modification of nucleus–donor and oocyte-recipient. NT is relatively efficient and frequently depends on technical experience of researcher (Galli et al., 2012). There are two kinds of nu‐ clear transfer trial: egg-NT involves the transfer of a single somatic nucleus to an unfertil‐ ized enucleated oocyte and oocyte-NT involves the transplantation of multiple somatic cell nuclei into immature oocyte of amphibian. Nevertheless are important differences between the two types of nuclear transfer experiment. In oocyte-NT experiments extensive cell divi‐ sion take places and new functional cell types appear as soon as the nuclear transplant em‐ bryo start to develop. In this experiment somatic cell chromatin is directly reprogrammed to express pluripotency genes within a day. In contrast to oocyte-NT experiments, in egg-NT no new cell types are formed, and neither oocyte nor nuclei divide, however direct transi‐ tion of reprogrammed nuclei that transcribe genes of pluripotency into differentiated cells occurs. Analysis of the mechanism of reprogramming in egg-NT experiments, which in‐ volves transcription pluripotency genes and others, is complicated owing to rapid DNA rep‐ lication and numerous cell divisions (Halley–Scott et al., 2010; Julien et al., 2010).

cells are self-renewing and express key transcription factors and are able to form teratoma (Okamoto et al., 2003; Brons et al., 2007; Tesar et al., 2005). Studies of X chromosome inacti‐ vation in pre-implantation human embryos reported that *XIST* transcript accumulation on this chromosome occurs in the eight-cell stage embryo, however the identity of the cells, which show *XIST* accumulation is not clear. *In vitro* studies of *XIST* accumulation in human ES cells lines revealed three different patterns of X chromosome inactivation. The naïve state - with both active X chromosomes, intermediate state - with both *XIST* accumulation and last - state, when the cells never undergo X chromosome inactivation even under differentia‐ tion and *XIST* accumulation does not occur (Dvash and Fan, 2009). Therefore, using current technologies "true" pluripotent stem cells can be exclusively isolated from mouse and may be from some other rodents, which present similar pattern of early embryonic development with mice. It is not obvious if it is possible to obtain "true" human ES cells, once we cannot test their contribution into developing human embryo (due to ethic consideration). Howev‐ er, the lack of X chromosome reactivation indicates that probably these cells will never be

After isolation, pluripotent stem cells start to organize *in vitro* stem cell niche, which up to a cer‐ tain degree simulates experience of these cells *in vivo*. *In vitro* these cells showed similar mor‐ phology with ICM, forming islands of juxtaposed cells and expressing pluripotent stem cell markers such as Oct3/4, Nanog, Sox2 (Tesar et al., 2005; Tesar et al., 2007). However, to distin‐ guish *in vitro* naïve and primed pluripotent cells are difficult or even impossible task due to high heterogenety of pluripotent cell lines established *in vitro* (Brons et al., 2007; Tesar et al., 2007). Different factors may contribute to this heterogeneity, such as natural polymorphism of the cells, selection of colonies in vitro, which can be pluripotent at different degrees, cell cul‐ ture conditions adopted in each work as well as pluripotent cells, itself, may produce an "im‐

able to reintegrate into early development events *in vivo* similar to rodent.

perfect" *in vitro* microenvironment again due to their natural heterogeneity.

**4.1. Reprogramming by means of differentiated cells nuclear transfer**

**4. Generation of artificial pluripotent stem cell – Reprogramming**

Several strategies can be provided in order to reprogramming differentiated or committed somatic cell genome. One of these strategies is a nuclear transfer (NT) of differentiated cell nucleus to oocyte whose maternal DNA was removed (Campbell et al., 1996). This type of reprogramming uses the natural components without any previous genetic or molecular modification of nucleus–donor and oocyte-recipient. NT is relatively efficient and frequently depends on technical experience of researcher (Galli et al., 2012). There are two kinds of nu‐ clear transfer trial: egg-NT involves the transfer of a single somatic nucleus to an unfertil‐

**3.** *In vitro* **pluripotent stem cell niches**

**strategies**

42 Pluripotent Stem Cells

The NT process leads to direct reprogramming of pluripotent stem cell and expression of such markers as Oct3/4, Nanog, and Sox2 that are silent in differentiated somatic cell nu‐ cleus. In general, the reactivation of silent pluripotency genes starts around 24 and 48 hours after NT (Halley–Scott et al., 2010; Julien et al., 2010; Byrne et al., 2003). Upon NT occurs the series of events when oocyte cytoplasm induces changes in the structure of donor chromatin toward pluripotent state, which became more appropriate for embryonic development. However, synchronization process which should happen between genomic DNA of donor cell and cytoplasm of recipient cell is complex and may affect significantly pluripotency of reprogrammed cells. Attempts to facilitate this reprogramming process have been made us‐ ing chemicals that alter the methylation status of the chromatin, such as TSA (trichostatin A), azacytidine, scriptaid, either before or after NT. In the mouse, the use of TSA (a histone deacetylase inhibitor, HDACi) significantly increased the success rate of mouse cloning (Kishigami et al., 2007).

**Figure 2.** Stages of nuclear transfer. The nucleus is removed from an egg (or oocyte) and replaced by a nucleus from a donor cell (somatic cell).

In mammals, embryo obtained by NT and transferred into foster mother can result (or not) in full term development. The clones, obtained by NT method, are genetically identical to donor organism, which provide a nucleus. The sheep Dolly was the first successfully cloned farm animal. Dolly was obtained from NT of terminally differentiated mammary epithelial cell (Campbell et al., 1996). However the generation of animals by NT is not very efficient, once many clones are dying soon after implantation, and only few clones survive and born (Galli et al. 1999; Ritchie 2006). These clones frequently affected with severe abnormalities, they die prematurely and often obese. The survival rate of clones depends on species, on do‐ nor cell type, method of NT and varied significantly between different laboratories (Oback & Wells, 2002; Wilmut et al., 2002). However, pre-implantation development does not seem to be a problem (Ono et al., 2001; Ono et al., 2001a) the majority of the term losses occurs during the post implantation period and/or after birth. It has been reported in some experi‐ mental studies, that only 2-3% of the transferred embryos develop to term in mice (Ono et al., 2001a; Sakai et al., 2005). Over time the methods were improved and other species have been cloned with success from differentiated donor cells, such as cattle (Galli et al., 1999); mouse (Wakayama & Yanagimachi, 1999); pig (Polejaeva et al., 2000a); cat (Shin et al., 2002); goat (Keefer et al., 2002); mule (Woods et al., 2003); horse (Galli et al., 1999); rabbit (Challah-Jacques et al., 2003); rat (Zhou et al., 2003) and dog (Lee et al., 2005). In humans, the attempt to NT has been achieved using animal oocytes as recipients for human genetic material. The reprogramming of human somatic cell nuclei did not occur after NT into bovine and rabbit oocytes. These oocytes with human genome were not able to follow early embryonic devel‐ opment. The up-regulation of human pluripotency-associated genes did not occur. These data raised a question about the potential use of animal embryonic environment to generate patient-specific stem cells using NT technology. Ethical implications also should be taken in consideration (Chung et al., 2009).

tion of karyotype, microsatellite and single nucleotide polymorphisms (SNP) analyses con‐ firmed that both ES cell lines were originated from SCNT embryos and were not from parthenotes. These ES cell lines demonstrated typical pluripotent cells morphology, self-re‐ newal capacity and expression of stem cell markers. They were also transcriptionally similar to ES cells derived from fertilized blastocysts, and pluripotent, as demonstrated by the gen‐ eration of several tissues from three germ layers after *in vivo* teratoma formation (Byrne 2007). Additionally, the experiments using mouse pluripotent primordial germ (PG) and ES cells as nuclei donors have also been performed using single-cell NT method. The results showed that embryos obtained from PG or ES cells NT method cannot develop and com‐ plete pre-implantation stage (Kato and Tsunoda, 1995). Possibly that long term *in vitro* cul‐ ture can affect the karyotype of these cells accumulating chromosomal abnormalities, thus

De-Differentiation of Somatic Cells to a Pluripotent State

http://dx.doi.org/10.5772/54372

45

The main goal of NT technology was to multiply the genotypes of high genetic value in farm animals and species, which are under the risk of extinction. Further, this technology was used as a tool for genome reprogramming of somatic differentiated cells into pluripotent state. The principles of cloning, which were developed by Willadsen (1986), are also impor‐ tant today. All the cloning studies provided the first experimental evidence for reprogram‐ ming (Kono et al., 1997; Gurdon, 2008). Currently, NT technologies can be applied in two different ways, to produce animal clones and to reprogram the nuclei of differentiated so‐ matic cell, which can be used for basic research to analyze X chromosome inactivation or to study the dynamics of imprinting process during reprogramming and in some cases for preclinical evaluation of these cells in animal models (Hochedlinger and Jaenisch 2006). This technology yet holds medical interest to produce patient-specific stem cells, which can be

The pluripotency, characteristic feature of ES cells, can be evaluated by their capacity to dif‐ ferentiate into cells of the three germ layers. More precisely, ES cells pluripotency can be evaluated by generation of chimaeras, organisms composed of cells from two or more indi‐ viduals from the same or different species (Kaufman, 1981; Keller, 1995; Wobus, 2005). Pro‐ duction of human/animal chimaeras is a method currently in use to analyze developmental potency of mammalian ES in biomedical research (Behringer, 2007; Lensch et al., 2007). James et al (2006) showed for the first time that a nonhuman embryo surrogate environment could be used to study developmental potential of human ES cells as well as biological com‐ patibility between human ES cells and the mouse ICM. Adult stem cells (ASC) are now seen as an alternative to ES cells, which can raise a number of ethical objections due requires de‐ struction of human embryo. Populations of multipotent ASC that express ES cell markers, such as Oct3/4, Nanog and Sox2, presenting a differentiation capacity similar to that of ES cells *in vitro*, can be isolated from different fetal and adult animal and human tissues (Wen‐ ceslau et al., 2011). For example, we have reported the isolation of human immature dental pulp stem cells (hIDPSC) from deciduous (baby) teeth, which express the aforementioned pluripotent markers and can differentiate into several cell types *in vitro*, such as bone, carti‐

resulting in formation of abnormal embryos (Balbach et al., 2007).

**4.3. Reprogramming by means of early embryonic environment**

used in cell therapy and regenerative medicine.

#### **4.2. Reprogramming by means of stem cells nuclear transfer**

In 1998, Cibelli performed stem cells nuclear transfer (SCNT) using nucleus of bovine fibro‐ blasts and enucleated bovine oocytes. They obtained 330 reconstructed oocytes, generated 37 cloned blastocysts, which served for isolation of 22 ES-like cell lines. These ES-like cells were injected into bovine oocytes, cultured cultured to produce embryos that further which were transferred into recipient females. In six out of seven calves at least one tissue originat‐ ed from ES cell has been found. Other authors demonstrated the ability of karyoplast of ES cells induce Oct4 expression in the somatic genome (Tada, 2001).

In humans (Hall et al., 2007) and non-human primate (Mitalipov et al., 2002) the SCNT effi‐ ciency of blastocyst formation has typically been very low, thus suggesting a lack in or com‐ plete nuclear reprogramming. In order to overcome these difficulties modified SCNT approach was used to produce rhesus macaque blastocysts from adult skin fibroblasts and to isolate from this blastocyst two ES cell lines. This was achieved thought non-invasive ap‐ proaches for meiotic spindle detection in oocytes and their removal using high-performance imaging. Spindle imaging system supports rapid and highly efficient real-time enucleation of primate oocytes. In this experiment spindle removal efficiency was 100%. The investiga‐ tion of karyotype, microsatellite and single nucleotide polymorphisms (SNP) analyses con‐ firmed that both ES cell lines were originated from SCNT embryos and were not from parthenotes. These ES cell lines demonstrated typical pluripotent cells morphology, self-re‐ newal capacity and expression of stem cell markers. They were also transcriptionally similar to ES cells derived from fertilized blastocysts, and pluripotent, as demonstrated by the gen‐ eration of several tissues from three germ layers after *in vivo* teratoma formation (Byrne 2007). Additionally, the experiments using mouse pluripotent primordial germ (PG) and ES cells as nuclei donors have also been performed using single-cell NT method. The results showed that embryos obtained from PG or ES cells NT method cannot develop and com‐ plete pre-implantation stage (Kato and Tsunoda, 1995). Possibly that long term *in vitro* cul‐ ture can affect the karyotype of these cells accumulating chromosomal abnormalities, thus resulting in formation of abnormal embryos (Balbach et al., 2007).

The main goal of NT technology was to multiply the genotypes of high genetic value in farm animals and species, which are under the risk of extinction. Further, this technology was used as a tool for genome reprogramming of somatic differentiated cells into pluripotent state. The principles of cloning, which were developed by Willadsen (1986), are also impor‐ tant today. All the cloning studies provided the first experimental evidence for reprogram‐ ming (Kono et al., 1997; Gurdon, 2008). Currently, NT technologies can be applied in two different ways, to produce animal clones and to reprogram the nuclei of differentiated so‐ matic cell, which can be used for basic research to analyze X chromosome inactivation or to study the dynamics of imprinting process during reprogramming and in some cases for preclinical evaluation of these cells in animal models (Hochedlinger and Jaenisch 2006). This technology yet holds medical interest to produce patient-specific stem cells, which can be used in cell therapy and regenerative medicine.

#### **4.3. Reprogramming by means of early embryonic environment**

In mammals, embryo obtained by NT and transferred into foster mother can result (or not) in full term development. The clones, obtained by NT method, are genetically identical to donor organism, which provide a nucleus. The sheep Dolly was the first successfully cloned farm animal. Dolly was obtained from NT of terminally differentiated mammary epithelial cell (Campbell et al., 1996). However the generation of animals by NT is not very efficient, once many clones are dying soon after implantation, and only few clones survive and born (Galli et al. 1999; Ritchie 2006). These clones frequently affected with severe abnormalities, they die prematurely and often obese. The survival rate of clones depends on species, on do‐ nor cell type, method of NT and varied significantly between different laboratories (Oback & Wells, 2002; Wilmut et al., 2002). However, pre-implantation development does not seem to be a problem (Ono et al., 2001; Ono et al., 2001a) the majority of the term losses occurs during the post implantation period and/or after birth. It has been reported in some experi‐ mental studies, that only 2-3% of the transferred embryos develop to term in mice (Ono et al., 2001a; Sakai et al., 2005). Over time the methods were improved and other species have been cloned with success from differentiated donor cells, such as cattle (Galli et al., 1999); mouse (Wakayama & Yanagimachi, 1999); pig (Polejaeva et al., 2000a); cat (Shin et al., 2002); goat (Keefer et al., 2002); mule (Woods et al., 2003); horse (Galli et al., 1999); rabbit (Challah-Jacques et al., 2003); rat (Zhou et al., 2003) and dog (Lee et al., 2005). In humans, the attempt to NT has been achieved using animal oocytes as recipients for human genetic material. The reprogramming of human somatic cell nuclei did not occur after NT into bovine and rabbit oocytes. These oocytes with human genome were not able to follow early embryonic devel‐ opment. The up-regulation of human pluripotency-associated genes did not occur. These data raised a question about the potential use of animal embryonic environment to generate patient-specific stem cells using NT technology. Ethical implications also should be taken in

consideration (Chung et al., 2009).

44 Pluripotent Stem Cells

**4.2. Reprogramming by means of stem cells nuclear transfer**

cells induce Oct4 expression in the somatic genome (Tada, 2001).

In 1998, Cibelli performed stem cells nuclear transfer (SCNT) using nucleus of bovine fibro‐ blasts and enucleated bovine oocytes. They obtained 330 reconstructed oocytes, generated 37 cloned blastocysts, which served for isolation of 22 ES-like cell lines. These ES-like cells were injected into bovine oocytes, cultured cultured to produce embryos that further which were transferred into recipient females. In six out of seven calves at least one tissue originat‐ ed from ES cell has been found. Other authors demonstrated the ability of karyoplast of ES

In humans (Hall et al., 2007) and non-human primate (Mitalipov et al., 2002) the SCNT effi‐ ciency of blastocyst formation has typically been very low, thus suggesting a lack in or com‐ plete nuclear reprogramming. In order to overcome these difficulties modified SCNT approach was used to produce rhesus macaque blastocysts from adult skin fibroblasts and to isolate from this blastocyst two ES cell lines. This was achieved thought non-invasive ap‐ proaches for meiotic spindle detection in oocytes and their removal using high-performance imaging. Spindle imaging system supports rapid and highly efficient real-time enucleation of primate oocytes. In this experiment spindle removal efficiency was 100%. The investiga‐

The pluripotency, characteristic feature of ES cells, can be evaluated by their capacity to dif‐ ferentiate into cells of the three germ layers. More precisely, ES cells pluripotency can be evaluated by generation of chimaeras, organisms composed of cells from two or more indi‐ viduals from the same or different species (Kaufman, 1981; Keller, 1995; Wobus, 2005). Pro‐ duction of human/animal chimaeras is a method currently in use to analyze developmental potency of mammalian ES in biomedical research (Behringer, 2007; Lensch et al., 2007). James et al (2006) showed for the first time that a nonhuman embryo surrogate environment could be used to study developmental potential of human ES cells as well as biological com‐ patibility between human ES cells and the mouse ICM. Adult stem cells (ASC) are now seen as an alternative to ES cells, which can raise a number of ethical objections due requires de‐ struction of human embryo. Populations of multipotent ASC that express ES cell markers, such as Oct3/4, Nanog and Sox2, presenting a differentiation capacity similar to that of ES cells *in vitro*, can be isolated from different fetal and adult animal and human tissues (Wen‐ ceslau et al., 2011). For example, we have reported the isolation of human immature dental pulp stem cells (hIDPSC) from deciduous (baby) teeth, which express the aforementioned pluripotent markers and can differentiate into several cell types *in vitro*, such as bone, carti‐ lage, skeletal, smooth muscles and neurons (Kerkis et al., 2006; Lizier et al., 2012). We found that after their transplantation into adult mice, they engrafted within different mouse or‐ gans, such as the liver, heart, spleen, kidney and the brain. Although hIDPSC express pluri‐ potent cell markers they present fibroblast-like morphology and were isolated from adult tissues (Kerkis et al., 2006; Lizier et al., 2012). We demonstrated that hIDPSC are truly multi‐ potent cells, which were able to undergo further development similar to mouse ES cells in nonhuman embryo surrogate environment. These cells were able to contribute *in vitro* into ICM of mouse blastocyst, thus undergoing cell divisions, and *in vivo* into fetus development thus generating pretermed human/mouse chimaera, which a prerequisite to characterizing pluripotency similar for ES cells. In this study in order to analyze the ability of hIDPSC (46, XY) to contribute to ICM and trophectoderm of mouse early embryos, 6–8 cells stained with vital Vibrant fluorescent dye (Fig. 3A) were injected into the perivitelline space and/or the blastocell of 8 compacted morulae and 20 early blastocysts (Fig. 3B). After injection these cells have adopted similar size to those of the recipient mouse embryo. They proliferated in the recipient mouse embryonic environment and showed a contribution to the ICM and also to the trophoblast cell layer (Fig. 4A). To determine the developmental and pluripotent ca‐ pacity of hIDPSC, six to eight stained cells were injected into the blastocele of 57 early blas‐ tocysts (Fig. 4B) and were immediately transferred to the uterus of five foster mothers. Three mice achieved pregnancy and, according to ethical recommendations, human/mouse chi‐ maeras were collected before birth. The 18 d.p.c. mouse foetuses seemed to be well formed based on their morphological appearance (Fig. 4C).

Additionally to Vibrant fluorescent dye the anti-hIDPSC antibody, (this identifies exclusive‐ ly hIDPSC), was used to detect the presence of these cells in 18 d.p.c. mouse foetuses). Strong fluorescent signals were observed in different organs of the chimaeras, such as the brain, liver, intestine and muscles (Fig. 4D and Fig. 5B). Using a variety of methods we dem‐ onstrated hIDPSC contribution to mouse embryos, which did not present any type of mor‐ phological deficiency (Fig. 5A). We were able to produce evidence, that these cells accomplished differentiation within local tissues, by the presence of human-specific tissue proteins, such as myosin and cytokeratin. Moreover, we used a specific antibody against hu‐ man nuclei to confirm, again, that the cells were indeed of human origin (Siqueira da Fonse‐ ca et al., 2009). Little is known about the initial reprogramming events that occur after transference of ASC into mouse blastocysts (Yokoo et al., 2005). In our experiment, hIDPSC were capable of engrafting and proliferating inside mouse morulae and blastocysts and forming pretermed chimaeras. These cells contributed not only to ICM, as do human ES

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47

**Figure 4.** Developmental and pluripotent capacity of hIDPSC to generating pretermed human/mouse chimaera. Early chimera blastocyst (A) were transferred to the uterus of foster mother (B). Human/mouse chimaeras (C) were collect‐

ed before birth and fluorescent signals were observed in different organs of the chimaeras (D).

cells, but also to the trophoblast cell layer – without any embryo damage.

**Figure 3.** The hIDPSC injection in early embryonic environment. (A) hIDPSC stained with vital Vibrant fluorescent dye were injected into the perivitelline space and/or compacted morulae (B) hIDPSC showed a contribution to the ICM and also to the trophoblast cell layer.

Additionally to Vibrant fluorescent dye the anti-hIDPSC antibody, (this identifies exclusive‐ ly hIDPSC), was used to detect the presence of these cells in 18 d.p.c. mouse foetuses). Strong fluorescent signals were observed in different organs of the chimaeras, such as the brain, liver, intestine and muscles (Fig. 4D and Fig. 5B). Using a variety of methods we dem‐ onstrated hIDPSC contribution to mouse embryos, which did not present any type of mor‐ phological deficiency (Fig. 5A). We were able to produce evidence, that these cells accomplished differentiation within local tissues, by the presence of human-specific tissue proteins, such as myosin and cytokeratin. Moreover, we used a specific antibody against hu‐ man nuclei to confirm, again, that the cells were indeed of human origin (Siqueira da Fonse‐ ca et al., 2009). Little is known about the initial reprogramming events that occur after transference of ASC into mouse blastocysts (Yokoo et al., 2005). In our experiment, hIDPSC were capable of engrafting and proliferating inside mouse morulae and blastocysts and forming pretermed chimaeras. These cells contributed not only to ICM, as do human ES cells, but also to the trophoblast cell layer – without any embryo damage.

lage, skeletal, smooth muscles and neurons (Kerkis et al., 2006; Lizier et al., 2012). We found that after their transplantation into adult mice, they engrafted within different mouse or‐ gans, such as the liver, heart, spleen, kidney and the brain. Although hIDPSC express pluri‐ potent cell markers they present fibroblast-like morphology and were isolated from adult tissues (Kerkis et al., 2006; Lizier et al., 2012). We demonstrated that hIDPSC are truly multi‐ potent cells, which were able to undergo further development similar to mouse ES cells in nonhuman embryo surrogate environment. These cells were able to contribute *in vitro* into ICM of mouse blastocyst, thus undergoing cell divisions, and *in vivo* into fetus development thus generating pretermed human/mouse chimaera, which a prerequisite to characterizing pluripotency similar for ES cells. In this study in order to analyze the ability of hIDPSC (46, XY) to contribute to ICM and trophectoderm of mouse early embryos, 6–8 cells stained with vital Vibrant fluorescent dye (Fig. 3A) were injected into the perivitelline space and/or the blastocell of 8 compacted morulae and 20 early blastocysts (Fig. 3B). After injection these cells have adopted similar size to those of the recipient mouse embryo. They proliferated in the recipient mouse embryonic environment and showed a contribution to the ICM and also to the trophoblast cell layer (Fig. 4A). To determine the developmental and pluripotent ca‐ pacity of hIDPSC, six to eight stained cells were injected into the blastocele of 57 early blas‐ tocysts (Fig. 4B) and were immediately transferred to the uterus of five foster mothers. Three mice achieved pregnancy and, according to ethical recommendations, human/mouse chi‐ maeras were collected before birth. The 18 d.p.c. mouse foetuses seemed to be well formed

**Figure 3.** The hIDPSC injection in early embryonic environment. (A) hIDPSC stained with vital Vibrant fluorescent dye were injected into the perivitelline space and/or compacted morulae (B) hIDPSC showed a contribution to the ICM

based on their morphological appearance (Fig. 4C).

46 Pluripotent Stem Cells

and also to the trophoblast cell layer.

**Figure 4.** Developmental and pluripotent capacity of hIDPSC to generating pretermed human/mouse chimaera. Early chimera blastocyst (A) were transferred to the uterus of foster mother (B). Human/mouse chimaeras (C) were collect‐ ed before birth and fluorescent signals were observed in different organs of the chimaeras (D).

Furthermore, hIDPSC integrated into host embryos and developed foetuses, undergoing the process of differentiation. Obviously that due to the difference in cell cycle dynamics be‐ tween mouse and human cells, the number of human cells during mouse pre-natal develop‐ ment is decreased in comparison with hIDPSC contribution in ICM of blastocyst. However, it is not clear if hIDPSC can really undergo reprogramming into ES-like cells within nonhu‐ man embryo surrogate environment.

ogy and gene expression pattern, thus maintaining variable levels of pluripotency, how‐ ever not all TC cells able to generate chimaeras and to contribute to germ line (Papaioannou and Rossant 1983). These cells frequently have abnormal karyotype, such as loss of the Y chromosome, trisomy, deletions or translocations (Takagi et al. 1983, Rousset et al. 1983, Modlinski et al. 1990). The hybrid cells obtained from pluripotent TC cells and somatic cell partner, which express embryonic antigens, were able to produce teratomas containing derivatives of all three embryonic germ layers (Andrews and Good‐ fellow 1980, Atsumi et al., 1982; Rousset et al. 1983; Forejt et al., 1984; Takagi, 1983) and to form embryoid bodies (EBs) in suspension culture (Takagi, 1983). These hybrid cells showed also reactivation of particular genes after reprogramming (Miller and Ruddle, 1976, 1977; Andrews and Goodfellow, 1980; Rousset et al., 1983) and reactivation of inac‐ tive X chromosome originated from the somatic partner (McBurney and Adamson, 1976; McBurney and Strutt, 1980; Takagi et al., 1983, Takagi, 1988; Mise et al., 1996). However, pluripotent hybrids were obtained when lymphocytes or thymocytes, not fibroblasts, were used as the somatic parents in fusion (Rousset et al., 1979). These studies indicate that hybrid cells generated by ES cells and differentiated cells, which have less cyto‐

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49

plasm, seem to be more adequate systems to undergo reprogramming.

al., 1984; Doetschman et al., 1985; Pease et al., 1990).

Matveeva et al. (1996) has obtained cultures of intraspecific embryonic hybrid cells by fusion of mouse ES cells, denominated HM-1 cells, which were derived from HPRT-deficient strain 129 mice (Magin et al., 1992) and characterized as highly pluripotent (Magin et al., 1992; Selfridge et al.1992) with splenocytes derived from an adult DD/c female. These hybrids were denominated as hybrid embryonic stem and somatic (HESS) cells and characterized as pluripotent and HPRT positive (Matveeva et al., 1996; 1998). Our group used three mouse hybrid clones HESS-1, HESS-2 and HESS-3 in order to study their karyotypes and investi‐ gate the influence of the karyotypes on the differentiation of these cells through the forma‐ tion of embryonic bodies (Mittmann et al., 2002). The hybrid cells used in our study were near diploid (HESS-2 and HESS-3) and near tetraploid (HESS-1) and chromosome analysis showed different trisomies. The trisomies of chromosomes 1 and 11 were found in near dip‐ loid hybrids. These trisomies are probably typical of these pluripotent cells, and have previ‐ ously been described in the mouse ES cells line (Crolla et al., 1990) and in TC cells (McBurney and Rogers, 1982). We found that the sex chromosome constitution in the HESS-2 line was predominantly XY, while in the HESS-3 line it was XO. Interesting that in HESS-2 and HESS-3 lines the segregated X chromosome was of embryonic origin. Indeed, it has been demonstrated by Ringertz and Savage (1976) that hybrids lose the chromosomes originating from differentiated, more slowly dividing cells. In our experiments, hybrids showed the capacity to form EBs *in vitro*, even at late passages (Fig. 6). The EBs formed by the hybrid cells could be considered as complex as those derived from the HM-1 line and the cystic-type EBs formed by pluripotent cells (Martin and Evans, 1975; Van der Kamp et

In the EBs derived from hybrids we observed haematopoietic-like cells, cells resembling skeletal and smooth muscle and others (Fig. 7). Cells of ectodermal origin (e.g. nerve cells) were not identified in EBs derived from hybrids. Our data shows that the 'embry‐

Our finding suggests that expression of such pluripotent markers, as nanog and oct4 by hIDPSC is enough condition for these cells to contribute into different mouse tissues in early embryo-fetal development, to differentiate properly and to express human proteins within mouse fetal an immune privileged environment (Siqueira da Fonseca et al., 2009).

**Figure 5.** The hIDPSC contribution in pretermed human/mouse chimaera. (A) 18 d.p.c. mouse fetus. (B) Strong fluores‐ cent signals were observed in different organs of the chimaeras, such as the brain, liver, intestine, muscles and others.

#### **4.4. Reprogramming by means of cell fusion**

First pluripotent hybrid cells have been isolated by fusion of pluripotent teratocarcinoma (TC) cells with differentiated somatic cells, which served as a tool for investigating the interaction between different genomes. These TC cells are similar to ES cells in morphol‐ ogy and gene expression pattern, thus maintaining variable levels of pluripotency, how‐ ever not all TC cells able to generate chimaeras and to contribute to germ line (Papaioannou and Rossant 1983). These cells frequently have abnormal karyotype, such as loss of the Y chromosome, trisomy, deletions or translocations (Takagi et al. 1983, Rousset et al. 1983, Modlinski et al. 1990). The hybrid cells obtained from pluripotent TC cells and somatic cell partner, which express embryonic antigens, were able to produce teratomas containing derivatives of all three embryonic germ layers (Andrews and Good‐ fellow 1980, Atsumi et al., 1982; Rousset et al. 1983; Forejt et al., 1984; Takagi, 1983) and to form embryoid bodies (EBs) in suspension culture (Takagi, 1983). These hybrid cells showed also reactivation of particular genes after reprogramming (Miller and Ruddle, 1976, 1977; Andrews and Goodfellow, 1980; Rousset et al., 1983) and reactivation of inac‐ tive X chromosome originated from the somatic partner (McBurney and Adamson, 1976; McBurney and Strutt, 1980; Takagi et al., 1983, Takagi, 1988; Mise et al., 1996). However, pluripotent hybrids were obtained when lymphocytes or thymocytes, not fibroblasts, were used as the somatic parents in fusion (Rousset et al., 1979). These studies indicate that hybrid cells generated by ES cells and differentiated cells, which have less cyto‐ plasm, seem to be more adequate systems to undergo reprogramming.

Furthermore, hIDPSC integrated into host embryos and developed foetuses, undergoing the process of differentiation. Obviously that due to the difference in cell cycle dynamics be‐ tween mouse and human cells, the number of human cells during mouse pre-natal develop‐ ment is decreased in comparison with hIDPSC contribution in ICM of blastocyst. However, it is not clear if hIDPSC can really undergo reprogramming into ES-like cells within nonhu‐

Our finding suggests that expression of such pluripotent markers, as nanog and oct4 by hIDPSC is enough condition for these cells to contribute into different mouse tissues in early embryo-fetal development, to differentiate properly and to express human proteins within

**Figure 5.** The hIDPSC contribution in pretermed human/mouse chimaera. (A) 18 d.p.c. mouse fetus. (B) Strong fluores‐ cent signals were observed in different organs of the chimaeras, such as the brain, liver, intestine, muscles and others.

First pluripotent hybrid cells have been isolated by fusion of pluripotent teratocarcinoma (TC) cells with differentiated somatic cells, which served as a tool for investigating the interaction between different genomes. These TC cells are similar to ES cells in morphol‐

**4.4. Reprogramming by means of cell fusion**

mouse fetal an immune privileged environment (Siqueira da Fonseca et al., 2009).

man embryo surrogate environment.

48 Pluripotent Stem Cells

Matveeva et al. (1996) has obtained cultures of intraspecific embryonic hybrid cells by fusion of mouse ES cells, denominated HM-1 cells, which were derived from HPRT-deficient strain 129 mice (Magin et al., 1992) and characterized as highly pluripotent (Magin et al., 1992; Selfridge et al.1992) with splenocytes derived from an adult DD/c female. These hybrids were denominated as hybrid embryonic stem and somatic (HESS) cells and characterized as pluripotent and HPRT positive (Matveeva et al., 1996; 1998). Our group used three mouse hybrid clones HESS-1, HESS-2 and HESS-3 in order to study their karyotypes and investi‐ gate the influence of the karyotypes on the differentiation of these cells through the forma‐ tion of embryonic bodies (Mittmann et al., 2002). The hybrid cells used in our study were near diploid (HESS-2 and HESS-3) and near tetraploid (HESS-1) and chromosome analysis showed different trisomies. The trisomies of chromosomes 1 and 11 were found in near dip‐ loid hybrids. These trisomies are probably typical of these pluripotent cells, and have previ‐ ously been described in the mouse ES cells line (Crolla et al., 1990) and in TC cells (McBurney and Rogers, 1982). We found that the sex chromosome constitution in the HESS-2 line was predominantly XY, while in the HESS-3 line it was XO. Interesting that in HESS-2 and HESS-3 lines the segregated X chromosome was of embryonic origin. Indeed, it has been demonstrated by Ringertz and Savage (1976) that hybrids lose the chromosomes originating from differentiated, more slowly dividing cells. In our experiments, hybrids showed the capacity to form EBs *in vitro*, even at late passages (Fig. 6). The EBs formed by the hybrid cells could be considered as complex as those derived from the HM-1 line and the cystic-type EBs formed by pluripotent cells (Martin and Evans, 1975; Van der Kamp et al., 1984; Doetschman et al., 1985; Pease et al., 1990).

In the EBs derived from hybrids we observed haematopoietic-like cells, cells resembling skeletal and smooth muscle and others (Fig. 7). Cells of ectodermal origin (e.g. nerve cells) were not identified in EBs derived from hybrids. Our data shows that the 'embry‐ onic' X chromosome may be lost in pluripotent hybrids, but reprogramming of the 'so‐ matic' X chromosome may still occur, thus allowing restricted pluripotency. The normal karyotype may be a prerequisite for the efficient contribution of these cells to the germ line in transgenic and chimeric animals and for their ability to differentiate *in vitro* into a wide spectrum of cell types (Papaioannou et al., 1978; McBurney Rogers, 1982; Pease et al., 1990; Bronson et al., 1995; Liu et al., 1997; Suzuki et al., 1997). Therefore, we further tested the capacity of near diploid HESS-2 to differentiate *in vitro* in putative germ cells (GC) (Fig. 8). We demonstrated that two days after induction of differentiation by retino‐ ic acid, the HESS-2 derived GC-like cells presented expression patterns of a gene set, in‐ volved in the progression of early stages of gametogenesis (Vasa, Stella, Dazl, Piwil 2, Tex14, Bmp8b, Tdrd1 and Rnf17). This finding is similar to previous descriptions of GC obtained *in vitro* from mouse ES cells (Hübner et al., 2003; Geijsen et al., 2004; Kerkis et al., 2007). HESS-2 generates GC *in vitro*, which were able to differentiation into spermand oocyte-like cells. These structures resembling the formation of presumptive oocytes appeared floating in the culture medium. FISH analyses indicate that several GC derived from HESS-2 hybrid cells were able to undergo sex chromosome reduction. The expres‐ sion of ZP2 and ZP3, oocyte-specific markers, was also detected supporting our morpho‐ logical observation. Hence our observations indicate that HESS-2 cells can progress into both female- and male- GC differentiation, however, the female developmental program could be achieved only in early stages (Lavaginolli et al., 2009).

cells has been reported (Takahashi and Yamanaka, 2006). These iPS-somatic cell hybrids demonstrated the expression of markers of pluripotent cells, such as Oct4, SSEA-1, and alka‐ line phosphatase and were able to differentiate into multiple cell types similar to ES cells, thus confirming the reprogramming ability of iPS cells (Takahashi and Yamanaka, 2006).

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**Figure 7.** Electron microscopy demonstrates differentiation within cystic embryoid bodies derived from somatic cell

**Figure 8.** Germ cell derived in vitro from HESS-2. (A) Protocol of differentiation. (B) Expression of genes involved in the progression of early stages of gametogenesis during HESS-2 differentiation. (C) Sperm-like structure. (D) Oocyte-like structure. (E and F) Haploid cells with X or Y chromosomes. (G) Expression of oocyte-specific markers in oocyte-like

hybrids (HESS-2).

structures obtained from HESS-2.

**Figure 6.** Cystic embryoid body - a globular cell cluster cultured from mouse ES cells.

Therefore, we demonstrated that near diploid somatic cell hybrids obtained by the fusion of ES cells with differentiated cell can be fully reprogrammed and able to produce *in vitro* even GCs. It is not likely that these cells will be able to generate live offspring after fertilization of normal oocyte due to abnormal karyotype. However, they represent an interesting model to study the influence of karyotype on the process of GC *in vitro* formation. More recently the reprograming of somatic cell nucleus after the fusion with induced pluripotent stem (iPS) cells has been reported (Takahashi and Yamanaka, 2006). These iPS-somatic cell hybrids demonstrated the expression of markers of pluripotent cells, such as Oct4, SSEA-1, and alka‐ line phosphatase and were able to differentiate into multiple cell types similar to ES cells, thus confirming the reprogramming ability of iPS cells (Takahashi and Yamanaka, 2006).

onic' X chromosome may be lost in pluripotent hybrids, but reprogramming of the 'so‐ matic' X chromosome may still occur, thus allowing restricted pluripotency. The normal karyotype may be a prerequisite for the efficient contribution of these cells to the germ line in transgenic and chimeric animals and for their ability to differentiate *in vitro* into a wide spectrum of cell types (Papaioannou et al., 1978; McBurney Rogers, 1982; Pease et al., 1990; Bronson et al., 1995; Liu et al., 1997; Suzuki et al., 1997). Therefore, we further tested the capacity of near diploid HESS-2 to differentiate *in vitro* in putative germ cells (GC) (Fig. 8). We demonstrated that two days after induction of differentiation by retino‐ ic acid, the HESS-2 derived GC-like cells presented expression patterns of a gene set, in‐ volved in the progression of early stages of gametogenesis (Vasa, Stella, Dazl, Piwil 2, Tex14, Bmp8b, Tdrd1 and Rnf17). This finding is similar to previous descriptions of GC obtained *in vitro* from mouse ES cells (Hübner et al., 2003; Geijsen et al., 2004; Kerkis et al., 2007). HESS-2 generates GC *in vitro*, which were able to differentiation into spermand oocyte-like cells. These structures resembling the formation of presumptive oocytes appeared floating in the culture medium. FISH analyses indicate that several GC derived from HESS-2 hybrid cells were able to undergo sex chromosome reduction. The expres‐ sion of ZP2 and ZP3, oocyte-specific markers, was also detected supporting our morpho‐ logical observation. Hence our observations indicate that HESS-2 cells can progress into both female- and male- GC differentiation, however, the female developmental program

could be achieved only in early stages (Lavaginolli et al., 2009).

50 Pluripotent Stem Cells

**Figure 6.** Cystic embryoid body - a globular cell cluster cultured from mouse ES cells.

Therefore, we demonstrated that near diploid somatic cell hybrids obtained by the fusion of ES cells with differentiated cell can be fully reprogrammed and able to produce *in vitro* even GCs. It is not likely that these cells will be able to generate live offspring after fertilization of normal oocyte due to abnormal karyotype. However, they represent an interesting model to study the influence of karyotype on the process of GC *in vitro* formation. More recently the reprograming of somatic cell nucleus after the fusion with induced pluripotent stem (iPS)

**Figure 7.** Electron microscopy demonstrates differentiation within cystic embryoid bodies derived from somatic cell hybrids (HESS-2).

**Figure 8.** Germ cell derived in vitro from HESS-2. (A) Protocol of differentiation. (B) Expression of genes involved in the progression of early stages of gametogenesis during HESS-2 differentiation. (C) Sperm-like structure. (D) Oocyte-like structure. (E and F) Haploid cells with X or Y chromosomes. (G) Expression of oocyte-specific markers in oocyte-like structures obtained from HESS-2.

#### **4.5. Reprogramming by means of Yamanaka's factors**

The pluripotency manifests during short time of early mammalian development (Choen et al., 2011; Dejosez et al., 2012). Such powerful, pluripotent cells can be obtained *in vitro* from early embryo and they are very promising for the future of regenerative medicine and even for organ generation. However, in humans isolation of these cells implicates with ethical problem of embryo destruction. Thus the idea to obtain such pluripotent cells artificially took the minds of the researchers. The technologies to obtain such alternative pluripotent cells are growing continuously. Yamanaka 's group using the combination of different fac‐ tors performed the first reprograming of mouse embryonic fibroblasts. The resulted iPS cells showed gene-protein expression of ES-cell markers, teratoma formation, differentiation into the tissues of three germ layers, beside chimaeras generation. This reprogramming strategy, using defined factors (i.e. Klf4, Oct4, Sox2, and c-Myc, termed "KOSM"), is conceptually and technically simple (Takahashi & Yamanaka, 2006). However, it is a low efficient and repro‐ ducibility process, which is influenced by several variables and also could affect the quality, such as completely or non-completely reprogrammed iPS cells. These variables are the age donor, cell type, different delivery systems and reprogramming cocktail choice, factors used for reprogramming (Daley et al., 2009). Currently, several strategies, based on genes, pro‐ teins, iRNA, as well as on different chemicals, are available for the reprogramming of somat‐ ic cells (Nakagawa et al., 2008; Yu et al., 2007). In the original method of iPS generation developed by Yamanaka's group used the moloney murine leukemia virus (MMLV) retrovi‐ rus for transgene expression (Takahashi and Yamanaka 2006). This vector has cloning ca‐ pacity of around 8 kb allows delivery of genes into the genome of cells and expected to be silenced after reprogramming and induction of endogenous genes activation. The efficiency of iPS cells generated using MMLV retroviruses Expressing the KOSM set genes is around 0.1% in mouse embryonic fibroblasts and approximately 0.01% in human fibroblasts (Jahner et al., 1982; Stewart et al., 1982; Hotta et al., 2008). Lentiviral vector is also used in reprog‐ raming experiments thus exhibiting slightly higher (8–10 kb) cloning capacity and usually have higher infection efficiency than MMLV retroviruses (Blelloch et al., 2007). However, carcinogenesis may be caused by genomic integration of retro- or lentiviral fragments into host DNA (Varas et al., 2009) and use of c-Myc oncogene, which after reactivation might cause malignant tumor formation (Okita et al., 2007; Brambrink et al., 2008). Thus viral sys‐ tems are still unsafe for therapeutic application. Therefore, a number of reports demon‐ strates that iPS cells can be generated by reducing the use of viral constructs and/or minimize viral integration through substitution of key reprogramming factors by chemical compounds or employing less differentiated cells, which already express endogenously one or more of the key pluripotency factors (Hota et al., 2008). Our group reprogrammed hIDPSC-fibroblast-like cells isolated from deciduous (baby) teeth, which express endoge‐ nous Oct3/4 and Nanog, using retroviral vector and four Yamanaka' s factors (Fig. 9).

derived iPS cells were generated to in shorter time and presented higher efficiency of colo‐ nies formation And were able to form under iPS colonies feeder –free conditions conditions. For example, the time of fibroblasts reprogramming using retrovirus vectors takes 20–25 days (Aesen et al., 2008), while reprogramming of hIDPSC occurs only in eleven days after infection (Beltrão-Braga, 2011). These results suggest that age of donor and differentiation status of cell type used for reprograming may also affect reprogramming efficiency. Accord‐ ingly, Maherali and Hochedlinger et al., (2007) compared skin fibroblasts reprograming effi‐ ciency from two-month-old and two years-old mice. Older cells produced half as many iPS cell colonies as young skin fibroblasts. It has been shown that iPS cells have so-called epige‐ netic memory, which means that after reprograming their differentiation potential can re‐ flect on their lineage commitment before reprograming. Therefore, hIDPSC showed strong neural commitment, which is due to their ectomesodermal origin. After reprograming strong neural commitment was evidenced within teratomas as well as spontaneous *in vitro* differentiation into neurons hIDPS-iPSC was also detected. It was expected that ordinary human adult cells reprogrammed as iPSC may revolutionize medicine by creating new therapies unique to individual patients. However, important questions have persisted about the safety of these cells, such as it is not clear the degree to which these cells are homologous to ES cells in respect of the genes expression pattern, differentiation capacities, epigenetics and in particular interest is the question whether iPSCs genetic material is altered during the reprogramming process. The researchers, which examined 22 different human iPSC lines obtained from seven research groups showed that these cells present 10 times more muta‐ tions than they expected to find. While some of the mutations appeared to be silent, the ma‐ jority did change specific protein functions, including those in genes associated with causative effects in cancers. Anyway, the studies of iPSC provide an important new tool in the fight against human disease, but to use these cells directly in the clinic, we must ensure

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**Figure 9.** hIDPSC-derived iPS cell. (A) Representative figure of morphological characteristics of hIDPSC *in vitro* cultur‐ ing. (B) iPS cell derivation were shown to be obtained under feeder-free condition on matrigel-coated dishes. (C) A

that they are safe.

typical hIDPSC-derived iPS cell colony. Light microscopy.

Reprogrammed hIDPSC presented all key characteristics of pluripotent cells: formed juxta‐ posed colonies of ES-like morphology and produce teratoma with derivates of all three germ layers. These cells did not integrate retroviral vector in their genome and express lower lev‐ els of Oct4, Nanog and Sox 2. In contrast to iPS cells derived to fibroblast cell, the hIDPSC derived iPS cells were generated to in shorter time and presented higher efficiency of colo‐ nies formation And were able to form under iPS colonies feeder –free conditions conditions. For example, the time of fibroblasts reprogramming using retrovirus vectors takes 20–25 days (Aesen et al., 2008), while reprogramming of hIDPSC occurs only in eleven days after infection (Beltrão-Braga, 2011). These results suggest that age of donor and differentiation status of cell type used for reprograming may also affect reprogramming efficiency. Accord‐ ingly, Maherali and Hochedlinger et al., (2007) compared skin fibroblasts reprograming effi‐ ciency from two-month-old and two years-old mice. Older cells produced half as many iPS cell colonies as young skin fibroblasts. It has been shown that iPS cells have so-called epige‐ netic memory, which means that after reprograming their differentiation potential can re‐ flect on their lineage commitment before reprograming. Therefore, hIDPSC showed strong neural commitment, which is due to their ectomesodermal origin. After reprograming strong neural commitment was evidenced within teratomas as well as spontaneous *in vitro* differentiation into neurons hIDPS-iPSC was also detected. It was expected that ordinary human adult cells reprogrammed as iPSC may revolutionize medicine by creating new therapies unique to individual patients. However, important questions have persisted about the safety of these cells, such as it is not clear the degree to which these cells are homologous to ES cells in respect of the genes expression pattern, differentiation capacities, epigenetics and in particular interest is the question whether iPSCs genetic material is altered during the reprogramming process. The researchers, which examined 22 different human iPSC lines obtained from seven research groups showed that these cells present 10 times more muta‐ tions than they expected to find. While some of the mutations appeared to be silent, the ma‐ jority did change specific protein functions, including those in genes associated with causative effects in cancers. Anyway, the studies of iPSC provide an important new tool in the fight against human disease, but to use these cells directly in the clinic, we must ensure that they are safe.

**4.5. Reprogramming by means of Yamanaka's factors**

52 Pluripotent Stem Cells

The pluripotency manifests during short time of early mammalian development (Choen et al., 2011; Dejosez et al., 2012). Such powerful, pluripotent cells can be obtained *in vitro* from early embryo and they are very promising for the future of regenerative medicine and even for organ generation. However, in humans isolation of these cells implicates with ethical problem of embryo destruction. Thus the idea to obtain such pluripotent cells artificially took the minds of the researchers. The technologies to obtain such alternative pluripotent cells are growing continuously. Yamanaka 's group using the combination of different fac‐ tors performed the first reprograming of mouse embryonic fibroblasts. The resulted iPS cells showed gene-protein expression of ES-cell markers, teratoma formation, differentiation into the tissues of three germ layers, beside chimaeras generation. This reprogramming strategy, using defined factors (i.e. Klf4, Oct4, Sox2, and c-Myc, termed "KOSM"), is conceptually and technically simple (Takahashi & Yamanaka, 2006). However, it is a low efficient and repro‐ ducibility process, which is influenced by several variables and also could affect the quality, such as completely or non-completely reprogrammed iPS cells. These variables are the age donor, cell type, different delivery systems and reprogramming cocktail choice, factors used for reprogramming (Daley et al., 2009). Currently, several strategies, based on genes, pro‐ teins, iRNA, as well as on different chemicals, are available for the reprogramming of somat‐ ic cells (Nakagawa et al., 2008; Yu et al., 2007). In the original method of iPS generation developed by Yamanaka's group used the moloney murine leukemia virus (MMLV) retrovi‐ rus for transgene expression (Takahashi and Yamanaka 2006). This vector has cloning ca‐ pacity of around 8 kb allows delivery of genes into the genome of cells and expected to be silenced after reprogramming and induction of endogenous genes activation. The efficiency of iPS cells generated using MMLV retroviruses Expressing the KOSM set genes is around 0.1% in mouse embryonic fibroblasts and approximately 0.01% in human fibroblasts (Jahner et al., 1982; Stewart et al., 1982; Hotta et al., 2008). Lentiviral vector is also used in reprog‐ raming experiments thus exhibiting slightly higher (8–10 kb) cloning capacity and usually have higher infection efficiency than MMLV retroviruses (Blelloch et al., 2007). However, carcinogenesis may be caused by genomic integration of retro- or lentiviral fragments into host DNA (Varas et al., 2009) and use of c-Myc oncogene, which after reactivation might cause malignant tumor formation (Okita et al., 2007; Brambrink et al., 2008). Thus viral sys‐ tems are still unsafe for therapeutic application. Therefore, a number of reports demon‐ strates that iPS cells can be generated by reducing the use of viral constructs and/or minimize viral integration through substitution of key reprogramming factors by chemical compounds or employing less differentiated cells, which already express endogenously one or more of the key pluripotency factors (Hota et al., 2008). Our group reprogrammed hIDPSC-fibroblast-like cells isolated from deciduous (baby) teeth, which express endoge‐

nous Oct3/4 and Nanog, using retroviral vector and four Yamanaka' s factors (Fig. 9).

Reprogrammed hIDPSC presented all key characteristics of pluripotent cells: formed juxta‐ posed colonies of ES-like morphology and produce teratoma with derivates of all three germ layers. These cells did not integrate retroviral vector in their genome and express lower lev‐ els of Oct4, Nanog and Sox 2. In contrast to iPS cells derived to fibroblast cell, the hIDPSC

**Figure 9.** hIDPSC-derived iPS cell. (A) Representative figure of morphological characteristics of hIDPSC *in vitro* cultur‐ ing. (B) iPS cell derivation were shown to be obtained under feeder-free condition on matrigel-coated dishes. (C) A typical hIDPSC-derived iPS cell colony. Light microscopy.

## **5. Final considerations**

All reprogramming strategies are aimed at genomic reprogramming, which is a key biologi‐ cal process. It is still unknown, how many and what reprogramming factors, which initiate a cascade of reprogramming events, are involved in NT, SCNT, in cell fusion and even in iPS cell production. Yamanaka's study suggests that these factors may be mainly proteins of the nucleus; however the cytoplasm factors also should be taken in consideration. NT techni‐ que, which was used for Dolly the Sheep and many other species, has been abandon by many researches due to the low efficiency. Some researches try to use SCNT in stem cells research in order to obtain stem cells that are genetically matched to the donor organism. However, up to data no human ES cells were obtained using SCNT. Another limitation of this method is that resulting cells retain mitochondrial structures, which originally belonged to the egg. The great limitation of cell fusion technology is chromosome set composed by different genomes. Currently many scientists, which used all these methods moved to iPS cell production.

**Author details**

**References**

271-284.

Cristiane V. Wenceslau1,2, Irina Kerkis1,2, Nelson F. Lizier1,2 and Alexandre Kerkis1

1 Laboratory of Genetics, Butantan Institute, Federal University of Sao Paulo, Brazil

2 Department of Morphology and Genetics, Federal University of Sao Paulo, Brazil

from human keratinocytes. Nature Biotech. 26, 1276–1284.

nol. Nov 27;29(12):1132-44.

ferentiation 23: 83-86.

Dev; 17:126 – 140.

15; 308(2):309-21.

1, 259–262.

[1] Aasen, T. et al (2008) Efficient and rapid generation of induced pluripotent stem cells

De-Differentiation of Somatic Cells to a Pluripotent State

http://dx.doi.org/10.5772/54372

55

[2] Amps K et al. (2011) Screening ethnically diverse human embryonic stem cells identi‐ fies a chromosome 20 minimal amplicon conferring growth advantage. Nat Biotech‐

[3] Andrews PW, Goodfellow PN (1980) Antigen expression by somatic cell hybrids of a murine embryonal carcinoma cell with thymocytes and L cells. Somat Cell Genet 6:

[4] Atsumi T, Shirayoshi Y, Takeichi M and Okada TS (1982) Nullipotent teratocarcino‐ ma cells acquire the pluripotency for differentiation by fusion with somatic cells. Dif‐

[5] Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R (2003) Multi‐ potent cell lineages in early mouse development depend on SOX2 function Genes

[6] Balbach ST, Jauch A, Böhm-Steuer B, Cavaleri FM, Han YM, Boiani M (2007) Chro‐ mosome stability differs in cloned mouse embryos and derivative ES cells. Dev Biol.

[7] Behringer RR (2007) Human-animal chimaeras in biomedical research. Cell Stem Cell

[8] Beltrão-Braga PCB, Pignatari GC, Maiorka PC, Lizier NF, Wenceslau CV, Miglino MA, Muotri AR, Kerkis I (2011) Feeder free derivation of induced pluripotent stem

[9] Brambrink, T. et al (2008) Sequential expression of pluripotency markers during di‐

[10] Brons, L.E. Smithers, M.W. Trotter, P. Rugg-Gunn, P. Sun, S.M. Chuva de Sousa Lps, S.K. Howlett, A. Clarkson, L. Ahrlund-Richter, R.A. Pedersen, L. Vallier (2007) Deri‐

cells from human immature dental pulp stem cells. Cell Transplatation.

rect reprogramming of mouse somatic cells. Cell Stem Cell 2, 151–159.

We started this chapter with simplified description of the concept of stem cell niches for‐ mation during early development. This conception lead to comprehension that such nich‐ es are very complex and composed by heterogeneous population of different somatic and stem cells. We know, that at least two different populations of pluripotent stem cells na‐ ïve and prime can be identified *in vivo* and isolated *in vitro* in rodents. In humans these two populations are difficult to identified and isolate. Additionally, the data on the pat‐ tern of X chromosome activation of *in vitro* cultured human ES cells suggest the existence of may be three such populations. In adult organism the number of stem cell niches in‐ creased dramatically, the examples are neuronal, hematopoietic, hair follicle, skeletal muscle, dental pulp and many other stem cells niches. In order to obtain stem cells of the most excellent quality the scientist try to re-create stem cell niche *in vitro*, which ena‐ bles ad of control of culture conditions, including oxygen tension and hydrostatic pres‐ sure and various factors believed to be involved in self-renewing, division, migration, recruitment and lineage commitment of stem cells. Any strategies of reprogramming are closely related with the conception of stem cell niche, because in all strategies of reprog‐ ramming the nucleus or the cell with different developmental histories and from differ‐ ent cells niches are used. In order to translate the potential of reprogrammed cells into to the clinical reality our knowledge about reprogrammed stem cells microenvironment should be significantly improved.

### **Acknowledgements**

The authors thank Dr. Thais M. C. Lavagnolli from Imperial College London and Dr. Si‐ mone A. S. da Fonseca from University of Sao Paulo for their contribution in our research.

## **Author details**

**5. Final considerations**

54 Pluripotent Stem Cells

cell production.

should be significantly improved.

**Acknowledgements**

All reprogramming strategies are aimed at genomic reprogramming, which is a key biologi‐ cal process. It is still unknown, how many and what reprogramming factors, which initiate a cascade of reprogramming events, are involved in NT, SCNT, in cell fusion and even in iPS cell production. Yamanaka's study suggests that these factors may be mainly proteins of the nucleus; however the cytoplasm factors also should be taken in consideration. NT techni‐ que, which was used for Dolly the Sheep and many other species, has been abandon by many researches due to the low efficiency. Some researches try to use SCNT in stem cells research in order to obtain stem cells that are genetically matched to the donor organism. However, up to data no human ES cells were obtained using SCNT. Another limitation of this method is that resulting cells retain mitochondrial structures, which originally belonged to the egg. The great limitation of cell fusion technology is chromosome set composed by different genomes. Currently many scientists, which used all these methods moved to iPS

We started this chapter with simplified description of the concept of stem cell niches for‐ mation during early development. This conception lead to comprehension that such nich‐ es are very complex and composed by heterogeneous population of different somatic and stem cells. We know, that at least two different populations of pluripotent stem cells na‐ ïve and prime can be identified *in vivo* and isolated *in vitro* in rodents. In humans these two populations are difficult to identified and isolate. Additionally, the data on the pat‐ tern of X chromosome activation of *in vitro* cultured human ES cells suggest the existence of may be three such populations. In adult organism the number of stem cell niches in‐ creased dramatically, the examples are neuronal, hematopoietic, hair follicle, skeletal muscle, dental pulp and many other stem cells niches. In order to obtain stem cells of the most excellent quality the scientist try to re-create stem cell niche *in vitro*, which ena‐ bles ad of control of culture conditions, including oxygen tension and hydrostatic pres‐ sure and various factors believed to be involved in self-renewing, division, migration, recruitment and lineage commitment of stem cells. Any strategies of reprogramming are closely related with the conception of stem cell niche, because in all strategies of reprog‐ ramming the nucleus or the cell with different developmental histories and from differ‐ ent cells niches are used. In order to translate the potential of reprogrammed cells into to the clinical reality our knowledge about reprogrammed stem cells microenvironment

The authors thank Dr. Thais M. C. Lavagnolli from Imperial College London and Dr. Si‐ mone A. S. da Fonseca from University of Sao Paulo for their contribution in our research.

Cristiane V. Wenceslau1,2, Irina Kerkis1,2, Nelson F. Lizier1,2 and Alexandre Kerkis1

1 Laboratory of Genetics, Butantan Institute, Federal University of Sao Paulo, Brazil

2 Department of Morphology and Genetics, Federal University of Sao Paulo, Brazil

## **References**


vation of pluripotent epiblast stem cells from mammalian embryos,Nature, 448 pp. 191–195.

[24] Dvash T, Fan G. (2009).Epigenetic regulation of X-inactivation in human embryonic

De-Differentiation of Somatic Cells to a Pluripotent State

http://dx.doi.org/10.5772/54372

57

[25] Evans, M& Kaufman, M (1981) Establishment in culture of pluripotent cells from

[26] Forejt J, Gregorova S, Dohnal K , Nosek J (1984) Gene expression of differentiated pa‐ rent in teratocarcinoma cell hybrids. Repression or reprogramming?Cell Diff 15:

[27] Galli C, Duchi R, Moor RM, Lazzari G (1999) Mammalian leukocytes contain all the genetic information necessary for the development of a new individual. Cloning 1,

[28] Galli C, Lagutina I, Perota A, Colleoni S, Duchi R, Lucchini F, Lazzari G (2012) So‐ matic Cell Nuclear Transfer and Transgenesis in Large Animals: Current and Future

[29] Geijsen N, Horoschak M, Kim K., et al (2004) Derivation of embryonic germ cells and

[30] Hall, V. J. et al (2007) Developmental competence of human in vitro aged oocytes as

[31] Halley-Stott, R. P. et al (2010) Mammalian nuclear transplantation to germinal vesicle stage Xenopus oocytes—a method for quantitative transcriptional reprogramming.

[32] Hotta A, Ellis J (2008) Retroviral vector silencing during iPS cell induction: an epige‐ netic beacon that signals distinct pluripotent states. J. Cell. Biochem. 105, 940–948.

[33] Hubner, K., Fuhrmann, G., Christenson, L.K., Kehler, J., De La Reinbold, R., Fuente,R., Wood, J., Strauss, J.F., Boiani, M., Scholer, H.R (2000) Derivation of oocytes

[34] Jahner D. et al (1982) De novo methylation and expression of retroviral genomes dur‐

[35] James D, Noggle SA, Swigut T, Brivanlou AH (2006) Contribution of human embry‐

[36] Jullien J, Astrand C, Halley-Stott R P, Garrett N, Gurdon JB (2010) Characterization of somatic cell nuclear reprogramming by oocytes in which a linker histone is re‐ quired for pluripotency gene reactivation. Proc. Natl Acad. Sci. USA 107, 5483–5488.

[37] Kato Y, Tsunoda Y (1995) Germ cell nuclei of male fetal mice can support develop‐ ment of chimeras to midgestation following serial transplantation. Development.

stem cells. Epigenetics. 4(1):19-22.

229-234.

161–170.

Methods 51, 56–65.

121(3):779-8.

mouse embryos. Natures, 292: 154-156.

Insights. Reprod Dom Anim 47, (Suppl. 3), 2–11.

male gametes from embryonic stem cells. Nature 427, 148–154.

host cells for nuclear transfer. Hum. Reprod. 22, 52–62.

from mouse embryonicstemcells.Science300,1251–1256.

onic stem cells to mouse blastocysts. Dev. Biol. 295, 90–102.

ing mouse embryogenesis. Nature 298, 623–628.


[24] Dvash T, Fan G. (2009).Epigenetic regulation of X-inactivation in human embryonic stem cells. Epigenetics. 4(1):19-22.

vation of pluripotent epiblast stem cells from mammalian embryos,Nature, 448 pp.

[11] Bronson SK, Smithies O, Mascarello JT. High incidence of XXY and XYY males among the offspring of female chimeras from embryonic stem cells (1995) Proc Natl

[12] Byrne A,.Pedersen DA, Clepper LL, Nelson M, Sanger WG, Gokhale S, D. P. Wolf1 DP, Mitalipov SM (2007) Producing primate embryonic stem cells by somatic cell nu‐

[13] Byrne J A, Simonsson S, Western P S, Gurdon JB (2003) Nuclei of adult mammalian somatic cells are directly reprogrammed to oct‐4 stem cell gene expression by am‐

[14] Campbell, K. H., McWhir, J., Ritchie, W. A. & Wilmut, I (1996) Sheep cloned by nu‐

[15] Challah-Jacques M, Chesne P, Renard J P (2003) "Production of cloned rabbits by so‐

[16] Chung Y, Bishop CE, Treff NR, Walker SJ, Sandler VM, Becker S, Klimanskaya I, Wun WS, Dunn R, Hall RM, Su J, Lu SJ, Maserati M,Choi YH, Scott R, Atala A, Ditt‐ man R, Lanza R (2009) Reprogramming of human somatic cells using human and an‐

[17] Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce dLn, Robl JM Ž ( 1998) Cloned transgenic calves produced from non-quiescent fetal fibroblasts. Sci‐

[18] Cohen DE, Melton D (2011) Turning straw into gold: directing cell fate for regenera‐

[19] Crolla JA, Lakeman SK, Seller MJ (1990) The induction of tail malformations in triso‐ my 16 mouse fetuses heterozygous for the curly tail recessive gene. Genet Res. 55(1):

[20] Daley G Q. et al (2009) Broader implications of defining standards for the pluripoten‐

[21] De Los Angeles A, Loh YH, Tesar PJ, Daley G Q (2012) Accessing naïve human pluri‐

[22] Dejosez M, ZwakaTP (2012) Pluripotency and nuclear reprogramming. Annu Rev Bi‐

[23] Doetschaman T, Eistetter H, Katz M, Schmidt W & Kemler R (1985) The in vitro de‐ velopment of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. Journal of embryology and Experimental Mor‐

potency. Current Opinion in Genetics & Development. 22, 3, 272–282.

191–195.

56 Pluripotent Stem Cells

Acad Sci U S A. 11; 92(8):3120-3.

phibian oocytes. Curr. Biol. 13, 1206–1213.

imal. Cloning Stem Cells. Jun; 11(2):213-23.

tive medicine. Nat Rev Genet. Apr; 12(4):243-52.

cy of iPSCs. Cell Stem Cell 4, 200–201.

ochem. 2012; 81:737-65.

phology, 87:27-45.

ence 280:1256-1258.

27-32.

clear transfer from a cultured cell line. Nature 380, 64–66.

matic nuclear transfer." Cloning Stem Cells; 5(4): 295-9.

clear transfer. Nature, 450.


[38] Keefer CL, Keyston R, Lazaris A, Bhatia B, Begin I, Bilodeau AS, Zhou FJ, Kafidi N, Wang B, Baldassarre H, Karatzas CN (2002) "Production of cloned goats after nuclear transfer using adult somatic cells". Biol Reprod., 66(1).

show global epigenetic remodeling and widespread tissue contribution. Cell Stem

De-Differentiation of Somatic Cells to a Pluripotent State

http://dx.doi.org/10.5772/54372

59

[50] Martin GR, Evans M.J (1975) Differentiration of clonallines of teratocarcinoma cells: formation of embryoids in vitro. Proceedings of national academy of Sciences. USA,

[51] Matveeva NM, Shilov XG, Bayborodin SI, Philimonenko VV, Rolinskaya IV and Se‐ rov OL (1996) Hybrid between mouse embryonic stem and somatic cells retain pluri‐

[52] Matveeva NM, Shilov AG, Kaftanovskaya HM, Bayborodin SI, Fokina MM, Maxi‐ movsky LP, Zhelezova AI, Golubitsa AN and Serov OL (1998) In vitro and in vivo study of pluripotency in hybrid cells obtained by fusion of mouse embryonic stem

[53] McBurney MW, Adamson ED (1976) Studies on the activity of the X chromosomes in

[54] McBurneyMW, Strutt BJ (1980) Genetic activity of X chromosomes in pluripotent fe‐ male teratocarcinoma cells and their differentiated progeny. Cell 21: 357-364.

[55] McBurney MW, Rogers BJ (1982) Isolation of male embryonal carcinoma cells and

[56] Miller RA, Ruddle FH (1976) Pluripotent teratocarcinoma-thymus somatic cell hy‐

[57] Mise N, Sado T, Tada S and Takagi N (1996) Activation of the inactive X chromo‐ some induced by cell fusion between a murine EC and female somatic cell accompa‐ nies reproducible changes in the methylation pattern of the Xist gene. Exp Cell Res

[58] Mitalipov S M, Yeoman RR Nusser KD, Wolf DP (2002) Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells. Biol. Re‐

[59] Mittmann J, Kerkis I, Kawashima C, Sukoyan M, Santos E, Kerkis A (2002) Differen‐ tiation of mouse embryonic stem cells and their hybrids during embryoid body for‐

[60] Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M,Maeda M, Yamanaka S (2003) The homeoprotein Nanog is required for mainte‐

[61] Modlinski JA, Gerhauser D, Lioli B,Winking H and Illmensee K (1990) Nuclear trans‐ fer from teratocarcinoma cells into mouse oocytes and eggs. Development 108:

nance of pluripotency in mouse epiblast and ES cells. Cell; 113:631–642.

Cell 1, 55–70.

72: 1441-1445.

brids. Cell 9: 45-55.

prod. 66, 1367–1373.

mation. Gen. Mol. Biol. 25 (1), 103–11.

223: 193-202.

337-348.

potency. Proc Acad Sci Russia 349: 129-132.

cells with splenocytes. Mol Reprod Dev 50: 128-138.

female teratocarcinoma cells in culture. Cell. 9(1):57-70.

their chromosome replication patterns. Dev Biol. 89(2):503-8.


show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1, 55–70.

[50] Martin GR, Evans M.J (1975) Differentiration of clonallines of teratocarcinoma cells: formation of embryoids in vitro. Proceedings of national academy of Sciences. USA, 72: 1441-1445.

[38] Keefer CL, Keyston R, Lazaris A, Bhatia B, Begin I, Bilodeau AS, Zhou FJ, Kafidi N, Wang B, Baldassarre H, Karatzas CN (2002) "Production of cloned goats after nuclear

[39] Keller G.M (1995) In vitro differentiation of embryonic stem cells. Current Opnion in

[40] Kerkis I, Kerkis A, Dozortsev D, Stukart-Parsons GC, Gomes Massironi SM, Pereira LV, Caplan AI, Cerruti HF (2006) Isolation and characterization of a population of immature dental pulp stem cells expressing OCT-4 and other embryonic stem cell

[41] Kerkis A, Fonseca SAS, Serafim RC , Lavagnolli TMC, , Pereira VS, Abdelmassih S, Abdelmassih R, Kerkis I. (2007) In Vitro Differentiation of Male Mouse Embryonic Stem Cells into Both Presumptive Sperm Cells and Oocytes. Cloning and Stem Cells.

[42] Kishigami S, Bui HT, Wakayama S, Tokunaga K, Van Thuan N, Hikichi T, Mizutani E, Ohta H, Suetsugu R, Sata T, Wakayama T (2007) Successful mouse cloning of an outbred strain by trichostatin A treatment after somatic nuclear transfer. J Reprod

[43] Lavagnolli TMC, Fonseca SAS, Serafim RC, Pereira VS, Santos EJC, Abdelmassih S, Kerkis A, and Kerkis I (2009) Presumptive germ cells derived from mouse pluripo‐

[44] Lee BC, Kim MK, Jang G, Oh HJ, Yuda, F, Kim HJ, Shamim MH, Kim JJ, Kang SK, Schatten G, Hwang WS (2005) "Dogs cloned from adult somatic cells". Nature, 436:

[45] Lensch MW, Schlaeger TM, Zon LI, Daley GQ (2007) Teratoma formation assays with human embryonic stem cells: a rationale for one type of human-animal chimaera.

[46] Liu X, Wu H, Loring J, Hormuzdi S, Disteche CM, Bornstein P, Jaenisch R. (1997) Trisomy eight in ES cells is a common potential problem in gene targeting and inter‐

[47] Lizier NF, Kerkis A, Gomes CM, Hebling J, Oliveira CF, Caplan AI, Kerkis I (2012) Scaling-up of dental pulp stem cells isolated from multiple niches. PLoS One.;

[48] Magin TM, McWhir J and Melton DW (1992) A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency. Nucleic Acid Res 20:

[49] Maherali, N., Sridharan, R., Xie, W., Utikal, J., Eminli, S., Arnold, K., Stadtfeld, M., Yachechko, R., Tchieu, J., Jaenisch, R., et al. (2007) Directly reprogrammed fibroblasts

transfer using adult somatic cells". Biol Reprod., 66(1).

markers. Cells Tissues Organs. 184(3-4):105-16.

tent somatic cell hybrids. Differentiation. 78; 124–130.

feres with germ line transmission. Dev Dyn. 209(1):85-91.

cell Biology, 7-862-829.

9, 4.

58 Pluripotent Stem Cells

641.

Dev. 53(1):165-70.

Cell Stem Cell, 253–258.

7(6):e39885.

3795-3796.


[62] Nakagawa M. et al (2008) Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nature Biotech. 26, 101–106.

[77] Rousset J-P, Bucchini D, Jami J (1983) Hybrids between F9 nulli-potent teratocarcino‐ ma and thymus cells produce multi-differentiated tumors in mice. Dev Biol 96:

De-Differentiation of Somatic Cells to a Pluripotent State

http://dx.doi.org/10.5772/54372

61

[79] Martin GR, Evans M.J (1975) Differentiration of clonallines of teratocarcinoma cells: formation of embryoids in vitro. Proceedings of national academy of Sciences. USA,

[80] Rousset J-P, Dubois P, Lasserre C, Aviles D, Fellous M, Jami J (1979) Phenotype and surface antigenes of mouse teratocarcinoma x fibroblast cell hybrids. Somat Cell Gen‐

[81] Sakai, R.R.; Tamashino, K.L.K.; Yamazaki, Y.; Yanagimachi, R (2005). "Cloning and assisted reproductive techniques: influence on early development and adult pheno‐

[82] Selfridge J, Pow AM, McWhir J, Magin TM, Melton DW (1992) Gene targeting using a mouse HPRT mini/HPRT-deficient embryonic stem cell system: Inactivation of the

[83] Siqueira da Fonseca SA, Abdelmassih S, Serafim RC , Lavagnolli TMC Clemente San‐ tos ES, Mendes CM, de Souza Pereira V, Ambrosio CE., Miglino MA., Visintin JR, Abdelmassih, Kerkis A, Kerkis I (2009) Human immature dental pulp stem cells' con‐ tribution to developing mouse embryos: production of human/mouse preterm chi‐

[84] Shin T, Kraemer D, Pryor J, Liu L, Rugila J, Howe L, Buck S, Murphy K, Lyons L, Westhusin, M (2002) "A cat cloned by nuclear transplantation". Nature, 415(6874):

[85] Stewart CL, Stuhlmann H, Jahner D & Jaenisch R (1982) De novo methylation, ex‐ pression, and infectivity of retroviral genomes introduced into embryonal carcinoma

[86] Suzuki H, Kamada N, Ueda O, Jishage K, Kurihara H, Terauchi Y, Azuma S, Kado‐ waki T, Kodama T, Yazaki Y & Toyoda Y (1997) Germ-line contribution of embryonic stem cells in chimeric mice: influenceof karyotype and in vitro differentiation ability.

[87] Tada M, Takahama Y, Abe§ K, Nakatsuji N ,Tada T (2001) Nuclear reprogramming of somatic cells by in vitro hybridization with ES cell, Current Biology. 11: 9.

[88] Takagi N (1988) Requirement of mitoses for the reversal of X-inactivation in cell hy‐ brids between murine embryonal carcinoma and normal thymocytes. Expl Cell Res

[78] Ringers N.R, Savage R.E (1976) Cell Hybrids. Acad. Press, New York, 417, ppl.

331-336.

72: 1441-1445.

et 5: 739-752.

859.

175: 363-375.

type". Birth Defects Research, 75: 151-162.

maeras Cell Prolif. 42, 132–140.

Experimental Animals, 46: 17- 23.

cells. Proc. Natl Acad. Sci. USA 79, 4098–4102.

mouse ERCC-1 gene. Somat Cell Mol Genet 18:325–336.


[62] Nakagawa M. et al (2008) Generation of induced pluripotent stem cells without Myc

[63] Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Schöler H, Smith A (1998) Formation of pluripotent stem cells in the mammalian em‐

[64] Nichols J, A. Smith (2009) Naïve and primed pluripotent states. Cell Stem Cell, 4, pp.

[65] Niwa H, Miyazaki J, and Smith AG (2000) Quantitative expression of oct-3/4 defines differentiation dedifferentiation or self-renewal of ES cell. Nat Genet. 24: 372-3726.

[66] Oback B, Wells D (2002) "Donor cells for nuclear cloning: many are called, but few

[67] Okamoto, A.P. Otte, C.D. Allis, D. Reinberg, E. Heard (2003) Epigenetic dynamics of imprinted X inactivation during early mouse development Science. 303; 644–649.

[68] Okita K, Ichisaka T, & Yamanaka S (2007) Generation of germline-competent induced

[69] Ono, Y; Shimozawa, N.; Ito, M.; Kono, T. (2001a). "Cloned mice from fetal fibroblasts cells arrested at metaphase by serial nuclear transfer". Biol. Reprod., 64: 44-50.

[70] Ono, Y.; Shimozawa, N.; Muguruma, K.; Kimoto, S.; Hioki, K.; Tachibana, M.; Shin‐ kai, Y.; Ito, M.; Kono, T. (2001b). "Production of cloned mice from embryonic stem

[71] Palmieri SL, Peter W, Hess H, Scholer HR (1994) Oct-4 transcription factor is differ‐ entially expressed in the mouse embryo during establishment of the first two extra‐

[72] Papaioannou VE, Rossant J (1983) Effects of the embryonic environment on prolifera‐ tion and differentiation of embryonal carcinoma cells. Cancer Surveys 2: 165-183.

[73] Papaioannou VE, Gardner RL, McBurney MW, Babinet C, Evans MJ (1978) Participa‐ tion of cultured teratocarcinoma cells in mouse embryogenesis. J Embryol Exp Mor‐

[74] Pease S, WilliamsRL (1990) Formation of germ line chimeras from embryonic stem cells maintained with recombinant leukemia inhibitory factor. Exp Cell Res. 190(2):

[75] Polejaeva I A, Chen S-H, Vaught TD, Page R L, Mullins J, Ball S, Dai Y, Boone J, Walker S, Ayares DL, Colman A, Campbell' KHS (2000) "Cloned pigs produced by

nuclear transfer from adult somatic cells". Nature, 407: 86-90.174

[76] Ritchie WA. (2006).Nuclear transfer in sheep. Methods Mol Biol. ;325:11-23

embryonic cell lineages involved in implantation. Dev Biol; 166:259 – 267.

from mouse and human fibroblasts. Nature Biotech. 26, 101–106.

bryo depend on the POU transcription factor oct4. Cell 95:379:391

are chosen". Cloning and Stem Cells, 4 (2): 147-168.

cells arrested at metaphase". Reproduction, 122: 731-736.

pluripotent stem cells. Nature 448, 313–317.

487–492.

60 Pluripotent Stem Cells

phol. 44:93-104.

209-11.


[89] Takagi N, Yoshida MA, Sugawara O and Sasaki M (1983) Reversal of X inactivation in female mouse somatic cells hybridized with murine teratocarcinoma stem cells in vitro. Cell 34: 1053-1062.

[103] Zhou Q, Renard JP, Le Friec G, Brochard V, Beaujean N, Cherifi Y, Fraichard A, Coz‐ zi J (2003) "Generation of fertile cloned rats by regulating oocyte activation". Science,

De-Differentiation of Somatic Cells to a Pluripotent State

http://dx.doi.org/10.5772/54372

63

302(5648): 1179.


[103] Zhou Q, Renard JP, Le Friec G, Brochard V, Beaujean N, Cherifi Y, Fraichard A, Coz‐ zi J (2003) "Generation of fertile cloned rats by regulating oocyte activation". Science, 302(5648): 1179.

[89] Takagi N, Yoshida MA, Sugawara O and Sasaki M (1983) Reversal of X inactivation in female mouse somatic cells hybridized with murine teratocarcinoma stem cells in

[90] Takahashi, K. & Yamanaka, S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676.

[91] Tesar PJ, Chenoweth JG, Brook FA, Davies TJ, Evans EP, Mack DL, Gardner, RL, McKay RDG (2007) New cell lines from mouse epiblast share defining features with

[92] Tesar PJ (2005) Derivation of germ-line-competent embryonic stem cell lines from preblastocyst mouse embryos. Proc Natl Acad Sci U S A. 7; 102(23):8239-44.

[93] Van der Kamp AW, Roza-de Jongh EJ, Houwen RH, Magrane GG, van Dongen JM, EvansMJ (1984) Developmental characteristics of somatic cell hybrids between toti‐ potent mouse teratocarcinoma and rat intestinal villus cells. Exp Cell Res. 154(1):

[94] Varas, F. et al (2009) Fibroblast-derived induced pluripotent stem cells show no com‐

[95] Wakayama T, Perry AC, Zuccotti M, Johnson KR, Yanagimachi R (1998) Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Na‐

[96] Wenceslau CV, Miglino MA, Martins DS, Ambrósio CE, Lizier NF, Pignatari GC,Ker‐ kis I (2011) Mesenchymal progenitor cells from canine fetal tissues: yolk sac, liver

[97] Willadsen SM (1986) Nuclear transplantation in sheep embryos. Nature 320, 63–65.

[98] Wilmut I, Beaujeau N, de Sousa PA, Dinnyes A, King T J, Paterson LA, Wells DN,

[99] Woods G L, White K L, Vanderwall DK, Li G-P, Aston KI, Bunch T D, Meerdo LM, Pate B J (2003) "A mule cloned from fetal cells by nuclear transfer". Science,

[100] Wobus, A.M; Boheler K (2005) Embryonic stem cells: prospects for developmental bi‐

[101] Yu J. et al (2007) Induced pluripotent stem cell lines derived from human somatic

[102] Yokoo T, Ohashi T, Shen JS, Sakurai K, Miyazaki Y, Utsunomiya Y, Takahashi M, Terada Y, Eto Y, Kawamura T, Osumi N, Hosoya T (2005) Human mesenchymal stem cells in rodent whole-embryo culture are reprogrammed to contribute to kidney

Young L E (2002) "Somatic cell nuclear transfer". Nature, 149: 583-586.

human embryonic stem cells . Nature. 488, 196–199.

mon retroviral vector insertions. Stem Cells 27, 300–306.

vitro. Cell 34: 1053-1062.

62 Pluripotent Stem Cells

53-64, 1984

ture 394, 369–374.

441, 1061–1067.

301(5636): 1063.

cells. Science 318, 1917–1920.

and bone marrow. Tissue Eng Part A.

ology and cell therapy. Physiol rRev. 85: 635-678.

tissues. Proc. Natl. Acad. Sci. USA 102, 3296–3300.

**Chapter 4**

**The Dark Side of Pluripotency – Cancer Stem Cell**

Cancer stem cells are defined as cancer cells that show the two properties of stemness: un‐ limited self-renewal and, pluripotency or multipotency. These properties make cancer stem

The definition of cancer stem cell has been a topic of debate and has changed with time. Cancer stem cells were proposed in 1994 by John Dick and coworkers as the cells that initiat‐ ed leukemia [1]. It was thought that this leukemic cell was derived from the mutation of a hematopoietic stem cell. Importantly, the term was used to distinguish a small subpopula‐ tion of leukemic cells that could initiate and maintain cancer from the rest of the leukemic cells that could not. Subsequently, it was also observed in other types of cancer that only a very small subpopulation of cancer cells had the ability to initiate cancer when transplanted into a new host [2–12]. This subpopulation of cancer cells was considered as cancer stem cells. The rest of the cancer cells, which ranged from progenitor to fully differentiated cancer cells, that formed the bulk of the cancer had limited proliferative capacity and hence could not initiate cancer when transplanted. Since cancer comprise a heterogeneous collection of cells, a unique set of cell surface markers that were expressed on cancer stem cells were used

The definition underwent revision when new experimental methods showed that turmori‐ genicity had been underassigned to a small group of cells due to limitations of the detection technique used. When different experimental approaches were undertaken, tumorigenicity was found to be widespread amongst phenotypically diverse cancer cells, resulting in a paradigm shift in the definition of cancer stem cells. Hence in 2006, the American Associa‐ tion of Cancer Research (AACR) defined a cancer stem cell as any cancer cell that possessed stem cell-like properties of unlimited self-renewal and multi/pluripotency. AACR specifical‐ ly highlighted that the definition of a cancer stem cell does not imply that such cells are de‐

> © 2013 Ng and Cheng-I; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Ng and Cheng-I; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Patricia Ng and Wang Cheng-I

http://dx.doi.org/10.5772/54369

**1. Introduction**

to define them.

Additional information is available at the end of the chapter

cell tumorigenic i.e. the ability to induce and sustain cancer.

## **The Dark Side of Pluripotency – Cancer Stem Cell**

Patricia Ng and Wang Cheng-I

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54369

## **1. Introduction**

Cancer stem cells are defined as cancer cells that show the two properties of stemness: un‐ limited self-renewal and, pluripotency or multipotency. These properties make cancer stem cell tumorigenic i.e. the ability to induce and sustain cancer.

The definition of cancer stem cell has been a topic of debate and has changed with time. Cancer stem cells were proposed in 1994 by John Dick and coworkers as the cells that initiat‐ ed leukemia [1]. It was thought that this leukemic cell was derived from the mutation of a hematopoietic stem cell. Importantly, the term was used to distinguish a small subpopula‐ tion of leukemic cells that could initiate and maintain cancer from the rest of the leukemic cells that could not. Subsequently, it was also observed in other types of cancer that only a very small subpopulation of cancer cells had the ability to initiate cancer when transplanted into a new host [2–12]. This subpopulation of cancer cells was considered as cancer stem cells. The rest of the cancer cells, which ranged from progenitor to fully differentiated cancer cells, that formed the bulk of the cancer had limited proliferative capacity and hence could not initiate cancer when transplanted. Since cancer comprise a heterogeneous collection of cells, a unique set of cell surface markers that were expressed on cancer stem cells were used to define them.

The definition underwent revision when new experimental methods showed that turmori‐ genicity had been underassigned to a small group of cells due to limitations of the detection technique used. When different experimental approaches were undertaken, tumorigenicity was found to be widespread amongst phenotypically diverse cancer cells, resulting in a paradigm shift in the definition of cancer stem cells. Hence in 2006, the American Associa‐ tion of Cancer Research (AACR) defined a cancer stem cell as any cancer cell that possessed stem cell-like properties of unlimited self-renewal and multi/pluripotency. AACR specifical‐ ly highlighted that the definition of a cancer stem cell does not imply that such cells are de‐

© 2013 Ng and Cheng-I; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Ng and Cheng-I; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

rived from the stem cells of the corresponding tissue. Also, a cancer stem cell does not have to be that initial cell in the body that caused cancer. For example, a differentiated cell that reacquires immortality through genetic mutations is considered a cancer stem cell. Thus any cancer cell that possesses or acquires stemness which results in unlimited tumorigenic po‐ tential is considered a cancer stem cell.

cells within a population of 20 million leukemia cells in the bone marrow of these mice. This indicated that SCID-leukemia initiating cells in the mice had multiplied from three to a 100

The Dark Side of Pluripotency – Cancer Stem Cell

http://dx.doi.org/10.5772/54369

67

Evidence that cancer stem cells could differentiate into the rest of the cancer cell population was provided by characterizing the CD34+CD38- cells after transplantation into NOD/SCID mice [15]. Flow cytometry analysis of human cells isolated from the bone marrow of mice showed that transplanting CD34+CD38- cells resulted in an increase in cancer cell popula‐ tion, of which 98% were positive for both CD34 and CD38. This differentiative capacity, to‐ gether with self-renewal ability, led to the conclusions that cancer stem cells existed and

Following the identification of cancer stem cells in leukemia, a series of in vivo studies docu‐ menting the presence of cancer stem cells in other cancers came to light. These studies, sum‐ marized in Table 1, characterized human cancer-initiating cells by their surface markers and were based mostly on the NOD/SCID mouse xenotransplantation assay. The table highlights information regarding the frequency of expression of cancer-stem cell-associated markers in the cancer cells, and the estimated frequency of the cancer stem cells residing in the cell pop‐ ulation that bears the cancer stem cell-associated markers. Based on these studies, it was es‐ timated that cancer stem cell existed in, at most, one in ten thousand cancer cells. Importantly, it was shown that this small population of cancer stem cells had a distinct CDphenotype, which when fully defined would serve as the address for accurate delivery of

In addition to the use of NOD/SCID mice, in vitro techniques that were previously used for the isolation of normal stem cells were also used to isolate cancer stem cells. These techni‐ ques included the formation of non-adherent spheroids in tissue culture method [10] and the exclusion of the fluorescent Hoechst dye method [16]. Both methods led to the identifica‐ tion of a subpopulation of cells that, when transplanted into mice, resulted in tumorigenici‐ ty. Hence both in vivo and in vitro studies suggested that tumorigenic cancer cells were

After a decade of using human cancer cells with NOD/SCID mouse as a model for can‐ cer stem cell detection, there were concerns of incompatibility issues between the two species with regards to the cytokines and receptors involved in cancer stem cell research. Cytokines and receptors from different species could prevent critical interactions that were required for cancer cells to survive. Furthermore, the NOD/SCID mouse immune system, even though rendered compromised, could still mount some level of response to reject the human cells, thereby potentially resulting in erroneously lower count estima‐

**1.2. Cancer stem cells are not ALWAYS a distinct subpopulation of cancer cells**

and therefore behaved like stem cells, possessing unlimited self-renewal ability.

formed a distinct subpopulation of cancer cells.

cytotoxic drugs.

stem-like in phenotype.

tion of cancer stem cell population.

More recently, interesting data has emerged demonstrating that partially differentiated can‐ cer cells, when exposed to a specific set of microenvironmental factors, can reacquire stem‐ ness [13]. Induction of stemness through this mechanism is reversible and could also result in epigenetic modifications, which then becomes heritable. This finding would again modify our understanding of the nature of cancer stem cell, suggesting that the cancer stem cell can be a dynamic and reversible entity.

In this section, experimental data shaping the identification and definition of cancer stem cells are presented in three parts. It begins with a description of early studies demonstrating that cancer stem cells were found to be a small and distinct subpopulation of cancer. This is followed by evidence suggesting that cancer stem cells can also be a highly common and heterogeneous population of cancer cells. Finally, evidence that cancer stem cell is a dynam‐ ic and reversible entity in cancer is discussed.

#### **1.1. Cancer stem cells: A distinct subpopulation of cancer cells**

The concept of cancer stem cell is not new. The first experimental evidence for the existence of cancer stem cell was in 1937 when Furth and Kahn injected a single leukemic cell from a mouse into an inbred mouse and transmitted leukemia [14]. At that time, it was unclear if every cancer cell or only a subpopulation of cancer cells possessed this ability to transmit. In 1994, a landmark experiment showed that only a subpopulation of cancer cells could trans‐ mit cancer [1]. John Dick and his group isolated cancer cells from patients with acute mye‐ loid leukemia (AML) and separated these cells based on their expression of CD34 and CD38. In this study, transplanting half a million of CD34+CD38- cells into severe combined immu‐ nodeficiency (SCID) mice induced AML in mice within thirty days, while the same number of CD34+CD38+ cells did not induce any AML in mice. The subpopulation of cancer cells that could transmit cancer was termed SCID-leukemia initiating cells and was thought to be amongst CD34+CD38- cells.

To determine the amount of the SCID-leukemia initiating cells within the CD34+CD38- cell population, a quantitative transplantation approach was used [15]. The cancer cells were se‐ rially diluted and transplanted into NOD/SCID (non-obese diabetic/severe combined immu‐ nodeficiency) mice. The minimum dose required to cause leukemia was then determined. Based on this experiment, it was found that there was about one cancer stem cell per 5,000 CD34+CD38- cells. This would mean that within a population of a million cancer cells, there was about one cancer stem cell. The ability of cancer stem cell to self-renew was provided by experiments that used the same transplantation approach described above, i.e. using human leukemic cells and NOD/SCID mice [15]. In one of these experiments, the number of human cancer stem cells in mice was initially found to be about three in 16 million leukemia cells. However, after six weeks, human cancer stem cells had increased to about 100 cancer stem cells within a population of 20 million leukemia cells in the bone marrow of these mice. This indicated that SCID-leukemia initiating cells in the mice had multiplied from three to a 100 and therefore behaved like stem cells, possessing unlimited self-renewal ability.

rived from the stem cells of the corresponding tissue. Also, a cancer stem cell does not have to be that initial cell in the body that caused cancer. For example, a differentiated cell that reacquires immortality through genetic mutations is considered a cancer stem cell. Thus any cancer cell that possesses or acquires stemness which results in unlimited tumorigenic po‐

More recently, interesting data has emerged demonstrating that partially differentiated can‐ cer cells, when exposed to a specific set of microenvironmental factors, can reacquire stem‐ ness [13]. Induction of stemness through this mechanism is reversible and could also result in epigenetic modifications, which then becomes heritable. This finding would again modify our understanding of the nature of cancer stem cell, suggesting that the cancer stem cell can

In this section, experimental data shaping the identification and definition of cancer stem cells are presented in three parts. It begins with a description of early studies demonstrating that cancer stem cells were found to be a small and distinct subpopulation of cancer. This is followed by evidence suggesting that cancer stem cells can also be a highly common and heterogeneous population of cancer cells. Finally, evidence that cancer stem cell is a dynam‐

The concept of cancer stem cell is not new. The first experimental evidence for the existence of cancer stem cell was in 1937 when Furth and Kahn injected a single leukemic cell from a mouse into an inbred mouse and transmitted leukemia [14]. At that time, it was unclear if every cancer cell or only a subpopulation of cancer cells possessed this ability to transmit. In 1994, a landmark experiment showed that only a subpopulation of cancer cells could trans‐ mit cancer [1]. John Dick and his group isolated cancer cells from patients with acute mye‐ loid leukemia (AML) and separated these cells based on their expression of CD34 and CD38. In this study, transplanting half a million of CD34+CD38- cells into severe combined immu‐ nodeficiency (SCID) mice induced AML in mice within thirty days, while the same number of CD34+CD38+ cells did not induce any AML in mice. The subpopulation of cancer cells that could transmit cancer was termed SCID-leukemia initiating cells and was thought to be

To determine the amount of the SCID-leukemia initiating cells within the CD34+CD38- cell population, a quantitative transplantation approach was used [15]. The cancer cells were se‐ rially diluted and transplanted into NOD/SCID (non-obese diabetic/severe combined immu‐ nodeficiency) mice. The minimum dose required to cause leukemia was then determined. Based on this experiment, it was found that there was about one cancer stem cell per 5,000 CD34+CD38- cells. This would mean that within a population of a million cancer cells, there was about one cancer stem cell. The ability of cancer stem cell to self-renew was provided by experiments that used the same transplantation approach described above, i.e. using human leukemic cells and NOD/SCID mice [15]. In one of these experiments, the number of human cancer stem cells in mice was initially found to be about three in 16 million leukemia cells. However, after six weeks, human cancer stem cells had increased to about 100 cancer stem

tential is considered a cancer stem cell.

66 Pluripotent Stem Cells

be a dynamic and reversible entity.

amongst CD34+CD38- cells.

ic and reversible entity in cancer is discussed.

**1.1. Cancer stem cells: A distinct subpopulation of cancer cells**

Evidence that cancer stem cells could differentiate into the rest of the cancer cell population was provided by characterizing the CD34+CD38- cells after transplantation into NOD/SCID mice [15]. Flow cytometry analysis of human cells isolated from the bone marrow of mice showed that transplanting CD34+CD38- cells resulted in an increase in cancer cell popula‐ tion, of which 98% were positive for both CD34 and CD38. This differentiative capacity, to‐ gether with self-renewal ability, led to the conclusions that cancer stem cells existed and formed a distinct subpopulation of cancer cells.

Following the identification of cancer stem cells in leukemia, a series of in vivo studies docu‐ menting the presence of cancer stem cells in other cancers came to light. These studies, sum‐ marized in Table 1, characterized human cancer-initiating cells by their surface markers and were based mostly on the NOD/SCID mouse xenotransplantation assay. The table highlights information regarding the frequency of expression of cancer-stem cell-associated markers in the cancer cells, and the estimated frequency of the cancer stem cells residing in the cell pop‐ ulation that bears the cancer stem cell-associated markers. Based on these studies, it was es‐ timated that cancer stem cell existed in, at most, one in ten thousand cancer cells. Importantly, it was shown that this small population of cancer stem cells had a distinct CDphenotype, which when fully defined would serve as the address for accurate delivery of cytotoxic drugs.

In addition to the use of NOD/SCID mice, in vitro techniques that were previously used for the isolation of normal stem cells were also used to isolate cancer stem cells. These techni‐ ques included the formation of non-adherent spheroids in tissue culture method [10] and the exclusion of the fluorescent Hoechst dye method [16]. Both methods led to the identifica‐ tion of a subpopulation of cells that, when transplanted into mice, resulted in tumorigenici‐ ty. Hence both in vivo and in vitro studies suggested that tumorigenic cancer cells were stem-like in phenotype.

#### **1.2. Cancer stem cells are not ALWAYS a distinct subpopulation of cancer cells**

After a decade of using human cancer cells with NOD/SCID mouse as a model for can‐ cer stem cell detection, there were concerns of incompatibility issues between the two species with regards to the cytokines and receptors involved in cancer stem cell research. Cytokines and receptors from different species could prevent critical interactions that were required for cancer cells to survive. Furthermore, the NOD/SCID mouse immune system, even though rendered compromised, could still mount some level of response to reject the human cells, thereby potentially resulting in erroneously lower count estima‐ tion of cancer stem cell population.


genic mice bearing the oncogene Myc with the immunoglobulin heavy chain enhancer. Just ten mouse leukemic cells were sufficient to induce cancer. Indeed, this experiment recapitu‐ lates the very first experiment in 1937 in which a single cancer cell from a chemically-in‐ duced cancer mouse was able to cause cancer in an inbred mouse [14,17]. This suggested that cancer stem cells were not necessarily a small population of cancer cells but rather

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69

In the second model, human cancer cells continued to be used. However, they were trans‐ planted into mice that were rendered even more severely immunocompromised than NOD/ SCID mice [18]. In one such study where NOD/SCID ILR2γnull mice were used, 27% (and hence more than a quarter) of single cell transplantation of human melanoma cells into the mice resulted in cancer [18,19]. Importantly, this experiment showed that these cancer stem cells were not associated with any of the surface markers that were previously characterized (See Table 1). A total of 85 cell surface markers from these cancer stem cells were studied. Of these, 22 cell surface markers showed heterogeneous expression within the cancer cell popu‐ lation of which none had an association with the capacity for tumor initiation. For example, both CD133+ and CD133- cells were able to induce cancer [19]. In addition, the cancers that resulted from both CD133+ and CD133- cancer cells created a population of cancer cells that was heterogeneous in their expression of CD133. These findings implied that cancer stem cells were both common and heterogeneous. Hence, these 2 models showed that cancer stem cells were not a small, distinct subpopulation of cancer cells but rather a common and heter‐

Amidst the new findings that challenged the concept of cancer stem cells as a small, distinct subpopulation of cancer cells, there were experiments which still showed that the cancer stem cell subpopulation was indeed low, and not common and heterogeneous, even when syngenic mice were used [23]. Interestingly, human experiments (which could not have been conducted currently due to ethical reasons) provided evidence that when human cancer cells were transplanted back into the human subject from whom the cancer cells originated,

Taking all the evidence together, cancer stem cells are indeed a small and distinct subpopu‐ lation of cancer cells in some cancers, whereas in other cancers, cancer stem cells are com‐ mon and heterogeneous. An alternative explanation that could account for these varied observations about cancer stem cells is that a cancer stem cell is not a static entity but rather

Even more recently, it has been shown that a partially differentiated cancer cell, under the "right" microenvironmental influence, can reacquire stemness [13]. This finding is crucial in furthering our understanding of the cancer stem cell as a dynamic and reversible entity,

In a study on colorectal cancer, differentiated colorectal cancer cells were able to dedifferen‐ tiate back into cancer cells with cancer stem cell phenotype after being exposed to hepato‐

the likelihood of cancer-initiation in the autologous human host is rare [24–26].

could be more common than previously thought.

ogeneous population of cancer cells in cancers such as melanoma.

a state that cancer cells can transform into.

**1.3. Cancer stem cells are dynamic**

rather than a static one.

**Table 1.** Experiments using markers for the enrichment of human cancer stem cells and xenotransplation assay for the detection and quantification of human caner stem cells. An estimation of the population of cancer stem cell in a tumor is given based. Epithelial-specific antigen (ESA) and ATP-binding cassette B5 (ABCB5) are surface markers. Aldehyde dehydrogenase 1 (ALDH1) is an enzyme inside the cell. Lin refers to a collection of lineage markers CD2, CD3, CD10, CD16, CD18, CD31, CD64 and CD140b. Mice were condition by irradiation prior to receiving the transplantation. a Percentages of tumor cells expressing the selected markers. bMinimum number of surface-marker expressing cells required to induce cancer in at least 50% of the mice.

To address these concerns, alternative experimental models were used. The first model used mouse cancer cells instead of human cancer cells to circumvent the issue of cross species barrier. One of these experiments involved transplanting mouse leukemic cells from trans‐ genic mice bearing the oncogene Myc with the immunoglobulin heavy chain enhancer. Just ten mouse leukemic cells were sufficient to induce cancer. Indeed, this experiment recapitu‐ lates the very first experiment in 1937 in which a single cancer cell from a chemically-in‐ duced cancer mouse was able to cause cancer in an inbred mouse [14,17]. This suggested that cancer stem cells were not necessarily a small population of cancer cells but rather could be more common than previously thought.

In the second model, human cancer cells continued to be used. However, they were trans‐ planted into mice that were rendered even more severely immunocompromised than NOD/ SCID mice [18]. In one such study where NOD/SCID ILR2γnull mice were used, 27% (and hence more than a quarter) of single cell transplantation of human melanoma cells into the mice resulted in cancer [18,19]. Importantly, this experiment showed that these cancer stem cells were not associated with any of the surface markers that were previously characterized (See Table 1). A total of 85 cell surface markers from these cancer stem cells were studied. Of these, 22 cell surface markers showed heterogeneous expression within the cancer cell popu‐ lation of which none had an association with the capacity for tumor initiation. For example, both CD133+ and CD133- cells were able to induce cancer [19]. In addition, the cancers that resulted from both CD133+ and CD133- cancer cells created a population of cancer cells that was heterogeneous in their expression of CD133. These findings implied that cancer stem cells were both common and heterogeneous. Hence, these 2 models showed that cancer stem cells were not a small, distinct subpopulation of cancer cells but rather a common and heter‐ ogeneous population of cancer cells in cancers such as melanoma.

Amidst the new findings that challenged the concept of cancer stem cells as a small, distinct subpopulation of cancer cells, there were experiments which still showed that the cancer stem cell subpopulation was indeed low, and not common and heterogeneous, even when syngenic mice were used [23]. Interestingly, human experiments (which could not have been conducted currently due to ethical reasons) provided evidence that when human cancer cells were transplanted back into the human subject from whom the cancer cells originated, the likelihood of cancer-initiation in the autologous human host is rare [24–26].

Taking all the evidence together, cancer stem cells are indeed a small and distinct subpopu‐ lation of cancer cells in some cancers, whereas in other cancers, cancer stem cells are com‐ mon and heterogeneous. An alternative explanation that could account for these varied observations about cancer stem cells is that a cancer stem cell is not a static entity but rather a state that cancer cells can transform into.

#### **1.3. Cancer stem cells are dynamic**

**Markers for enrichment of cancer stem cells**

68 Pluripotent Stem Cells

CD44+ Lin-

CD44+ CD24-

transplantation. a

CD34+ CD38- Leukemia

CD44+ ESA+ CD24- or low Lin- Breast cancer

ALDH1+ Lin- Breast cancer

CD44+ ESA+ CD24+ Pancreatic cancer

CD44+ CD117+ Ovarian cancer

CD133+ Brain cancer

ABCB5+ Melanoma

expressing cells required to induce cancer in at least 50% of the mice.

CD44+ ESAhigh CD166+ Colon cancer

(0.02-2% of mononuclear cells)a

Head and Neck cancer (0.1-42%)

(2%)

(0.1-1.2%)

(0.2-0.8%)

(1.2-16%)

(0.2%)

(6-29%)

Colon cancer (1.8-24.5%)

Lung cancer (0.32-22%)

Pancreatic cancer (1.1-3.2%)

(1.6-20.4%)

**Table 1.** Experiments using markers for the enrichment of human cancer stem cells and xenotransplation assay for the detection and quantification of human caner stem cells. An estimation of the population of cancer stem cell in a tumor is given based. Epithelial-specific antigen (ESA) and ATP-binding cassette B5 (ABCB5) are surface markers. Aldehyde dehydrogenase 1 (ALDH1) is an enzyme inside the cell. Lin refers to a collection of lineage markers CD2, CD3, CD10, CD16, CD18, CD31, CD64 and CD140b. Mice were condition by irradiation prior to receiving the

To address these concerns, alternative experimental models were used. The first model used mouse cancer cells instead of human cancer cells to circumvent the issue of cross species barrier. One of these experiments involved transplanting mouse leukemic cells from trans‐

Percentages of tumor cells expressing the selected markers. bMinimum number of surface-marker

**Cancer type Cancer stem cell detection assay Ref.**

2 of 2 NOD/SCID mice (5,000 cells)b

5 of 7 NOD/SCID mice (5,000 cells)

4 of 4 NOD/SCID mice (200 cells)

> NOD/SCID mice (20 cells)

6 of 12 NOD/SCID mice (100 cells)

1 of 2 NOD/SCID mice (150 cells)

9 of 10 nude mice (100 cells)

4 of 4 NOD/SCID mice (100 cells)

5 of 6 NOD/SCID mice (500 cells) 15 of 30 NOD/SCID mice (3,000 cells)

> 4 of 4 SCID mice (10,000 cells)

> > Nude mice (500 cells)

11 of 11 NOD/SCID mice (1,000,000 cells)

[1,15]

[20]

[2,4]

[21]

[12]

[3]

[11]

[10]

[6] [8]

[5]

[22]

[9]

Even more recently, it has been shown that a partially differentiated cancer cell, under the "right" microenvironmental influence, can reacquire stemness [13]. This finding is crucial in furthering our understanding of the cancer stem cell as a dynamic and reversible entity, rather than a static one.

In a study on colorectal cancer, differentiated colorectal cancer cells were able to dedifferen‐ tiate back into cancer cells with cancer stem cell phenotype after being exposed to hepato‐ cyte growth factor (HGF) [27]. Upon exposure, these cells showed increased colony-forming ability (clonogenicity) and increased tumorigenicity. Biochemically, the cells exhibited an in‐ crease in the Wnt signaling pathway leading to the expression of β-catenin dependent genes. This finding is important as HGF is present within the natural microenvironment of colorec‐ tal cancer as it is normally produced by myofibroblasts which are prominent in the colorec‐ tal stroma. Hence, given the right microenvironment, non-tumorigenic cancer cells can become cancer stem cells.

**2.1. Drug resistance**

*2.1.1. Cell quiescence*

*2.1.2. DNA-repair*

*2.1.3. Drug transporters*

many cancers.

to genetic mutations, to evade anti-cancer drugs.

nisms for drug resistance as discussed below.

Normal stem cells have traits that confer high survival capacity under harsh environments. These include (1) cell quiescent, (2) active DNA-repair system, (3) expression of transporters that keeps toxic substances out, (4) high metabolism in detoxification, and (5) resistance to apoptosis. These mechanisms are thought to be employed by cancer stem cells, in addition

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71

Chemotherapeutic cancer drugs such as vincristine, vinblastine, paclitaxel and docitaxel works by arresting cancer cells in mitosis, thus leading to apoptosis [28]. One hypothesis to explain resistance of cancer stem cells to these drugs is that cancer stem cells are in a quies‐ cent state. Indeed, quiescent cancer stem cells have been shown to exist in some cancers [29]. Moreover, drug resistance in slow-cycling cancer stem cell population has also been report‐ ed [30,31]. In addition to cell quiescence, cancer stem cells are likely to have other mecha‐

Ionizing radiation and anti-cancer drugs that disrupt the genome kill cancer cells by target‐ ing their DNA. Cancer stem cells have efficient DNA-repair systems that confer resistance to these anti-cancer agents. A study on glioblastoma demonstrated that cancer stem cells, iden‐ tified by their expression of CD133, showed preferential activation of the DNA damage check point response resulting in an increase in their DNA repair capacity [32]. The study also shows that both in vitro as well as mouse brain samples of cancer cells have increased the proportion of CD133-positive cells to CD133-negative cells following radiotherapy. This suggested that the subpopulation of CD133-positive, i.e. cancer stem cells, had developed

A third mechanism of drug resistance is the expression of transporters of the ATP-binding cassette (ABC) family. ABC transporters are efflux pumps that can actively expel a wide range of chemotherapeutic drugs from the cell. ABC transporters are expressed in both nor‐ mal stem cells and cancer stem cells. Three members of this family of ABC transporters, ABCB1, ABCC1 and ABCG2 have been identified as the culprits of multidrug resistance in

A study on neuroblastoma patients illustrates how cancer stem cells use the efflux transport‐ er, ABCG2 to protect themselves from anti-cancer drugs. In this study, cells expressing ABCG2 were identified by the fluorescent Hoechst dye 33342, in flow cytometry, as a "side population" (SP) of cells that did not take up this dye. A previous study had shown that cancer stem cells reside in the SP fraction of neuroblastoma [10]. SP cells from neuroblasto‐ ma patients showed increased efflux of mitoxantrone when compared to non-SP cells. Also, treatment of neuroblastoma cell lines with mitoxantrone led to an increase in the proportion

resistance to radiotherapy and were the cause of cancer relapse in the mouse.

Similarly, in a separate study using PDGF-induced glioma in mice, exposure to nitric oxide caused differentiated glioma cancer cells to transform into glioma cancer stem cells [25]. Again, nitric oxide is normally present in the natural microenvironment of gliomas as nitric oxide is produced by blood vessels. Hence glioma cancer cells in close proximity to brain blood vessels were able to re-acquire stem-like properties. These two recent studies present‐ ed reiterate the concept that cancer stem cells are dynamic - cancer cells are able to trans‐ form back into cancer stem cells given the right micorenvironmental conditions.

#### **1.4. Conclusions**

Cancer stem cells are cancer cells that have self-renewable and multi or pluripotent abilities. Our current understanding is that cancer stem cells can be a distinct subpopulation of cancer cells in certain cancers while in other cancers, they can be relatively common and heteroge‐ neous. They are also dynamic in nature.

Understanding the defining characteristics of cancer stem cells is important as these have important therapeutic implications. In cancers in which the cancer stem cells form a distinct subpopulation, eliminating this subpopulatuion of cancer stem cells can potentially lead to a cure. In contrast, targeting one specific group of cancer stem cells in cancers in which the cancer stem cells are common and heterogeneous would be futile. In addition, learning more about the microenvironmental factors that promote the cancer stem cell state provides another interesting approach in finding a cure for cancer.

## **2. Cancer stem cell: The survivor**

Chemotherapeutic agents against cancer are able to reduce tumor mass significantly but of‐ ten a cure may not be achievable. In such cases, a cure is not possible due to a subpopulation of cells that are resistant to cancer drugs. The cancer stem cells amongst this resistant popu‐ lation then self-renew, proliferate and metastasize to cause relapse after treatment. In addi‐ tion to understanding the defining characteristics of cancer stem cells for therapeutic purposes, a working knowledge of the molecular mechanisms of drug-resistance in cancer stem cells will empower researchers to better design new therapeutic agents that can over‐ come drug resistance. We will also explore the mechanism for metastasis in cancer stem cells, which serves as another potential therapeutic target.

#### **2.1. Drug resistance**

cyte growth factor (HGF) [27]. Upon exposure, these cells showed increased colony-forming ability (clonogenicity) and increased tumorigenicity. Biochemically, the cells exhibited an in‐ crease in the Wnt signaling pathway leading to the expression of β-catenin dependent genes. This finding is important as HGF is present within the natural microenvironment of colorec‐ tal cancer as it is normally produced by myofibroblasts which are prominent in the colorec‐ tal stroma. Hence, given the right microenvironment, non-tumorigenic cancer cells can

Similarly, in a separate study using PDGF-induced glioma in mice, exposure to nitric oxide caused differentiated glioma cancer cells to transform into glioma cancer stem cells [25]. Again, nitric oxide is normally present in the natural microenvironment of gliomas as nitric oxide is produced by blood vessels. Hence glioma cancer cells in close proximity to brain blood vessels were able to re-acquire stem-like properties. These two recent studies present‐ ed reiterate the concept that cancer stem cells are dynamic - cancer cells are able to trans‐

Cancer stem cells are cancer cells that have self-renewable and multi or pluripotent abilities. Our current understanding is that cancer stem cells can be a distinct subpopulation of cancer cells in certain cancers while in other cancers, they can be relatively common and heteroge‐

Understanding the defining characteristics of cancer stem cells is important as these have important therapeutic implications. In cancers in which the cancer stem cells form a distinct subpopulation, eliminating this subpopulatuion of cancer stem cells can potentially lead to a cure. In contrast, targeting one specific group of cancer stem cells in cancers in which the cancer stem cells are common and heterogeneous would be futile. In addition, learning more about the microenvironmental factors that promote the cancer stem cell state provides

Chemotherapeutic agents against cancer are able to reduce tumor mass significantly but of‐ ten a cure may not be achievable. In such cases, a cure is not possible due to a subpopulation of cells that are resistant to cancer drugs. The cancer stem cells amongst this resistant popu‐ lation then self-renew, proliferate and metastasize to cause relapse after treatment. In addi‐ tion to understanding the defining characteristics of cancer stem cells for therapeutic purposes, a working knowledge of the molecular mechanisms of drug-resistance in cancer stem cells will empower researchers to better design new therapeutic agents that can over‐ come drug resistance. We will also explore the mechanism for metastasis in cancer stem

form back into cancer stem cells given the right micorenvironmental conditions.

become cancer stem cells.

70 Pluripotent Stem Cells

**1.4. Conclusions**

neous. They are also dynamic in nature.

**2. Cancer stem cell: The survivor**

another interesting approach in finding a cure for cancer.

cells, which serves as another potential therapeutic target.

Normal stem cells have traits that confer high survival capacity under harsh environments. These include (1) cell quiescent, (2) active DNA-repair system, (3) expression of transporters that keeps toxic substances out, (4) high metabolism in detoxification, and (5) resistance to apoptosis. These mechanisms are thought to be employed by cancer stem cells, in addition to genetic mutations, to evade anti-cancer drugs.

#### *2.1.1. Cell quiescence*

Chemotherapeutic cancer drugs such as vincristine, vinblastine, paclitaxel and docitaxel works by arresting cancer cells in mitosis, thus leading to apoptosis [28]. One hypothesis to explain resistance of cancer stem cells to these drugs is that cancer stem cells are in a quies‐ cent state. Indeed, quiescent cancer stem cells have been shown to exist in some cancers [29]. Moreover, drug resistance in slow-cycling cancer stem cell population has also been report‐ ed [30,31]. In addition to cell quiescence, cancer stem cells are likely to have other mecha‐ nisms for drug resistance as discussed below.

#### *2.1.2. DNA-repair*

Ionizing radiation and anti-cancer drugs that disrupt the genome kill cancer cells by target‐ ing their DNA. Cancer stem cells have efficient DNA-repair systems that confer resistance to these anti-cancer agents. A study on glioblastoma demonstrated that cancer stem cells, iden‐ tified by their expression of CD133, showed preferential activation of the DNA damage check point response resulting in an increase in their DNA repair capacity [32]. The study also shows that both in vitro as well as mouse brain samples of cancer cells have increased the proportion of CD133-positive cells to CD133-negative cells following radiotherapy. This suggested that the subpopulation of CD133-positive, i.e. cancer stem cells, had developed resistance to radiotherapy and were the cause of cancer relapse in the mouse.

#### *2.1.3. Drug transporters*

A third mechanism of drug resistance is the expression of transporters of the ATP-binding cassette (ABC) family. ABC transporters are efflux pumps that can actively expel a wide range of chemotherapeutic drugs from the cell. ABC transporters are expressed in both nor‐ mal stem cells and cancer stem cells. Three members of this family of ABC transporters, ABCB1, ABCC1 and ABCG2 have been identified as the culprits of multidrug resistance in many cancers.

A study on neuroblastoma patients illustrates how cancer stem cells use the efflux transport‐ er, ABCG2 to protect themselves from anti-cancer drugs. In this study, cells expressing ABCG2 were identified by the fluorescent Hoechst dye 33342, in flow cytometry, as a "side population" (SP) of cells that did not take up this dye. A previous study had shown that cancer stem cells reside in the SP fraction of neuroblastoma [10]. SP cells from neuroblasto‐ ma patients showed increased efflux of mitoxantrone when compared to non-SP cells. Also, treatment of neuroblastoma cell lines with mitoxantrone led to an increase in the proportion of SP cells to non-SP cells, suggesting that ABCG2 conferred a survival advantage to cancer stem cells [33]. Similarly, in acute myeloid leukemia, SP cells derived from mononuclear cells in bone marrow of patients showed an increase efflux of daunorubicin and mitoxan‐ trone when compared to non-SP cells [34]. Taken together, these findings suggest that can‐ cer stem cell uses efflux transporters to guard against anti-cancer drugs.

ated with a hypoxic microenvironment. Expression of hypoxia-inducible factors (HIF) are important as these factors regulate stem cell self-renewal and pluripotency [39]. Although not well studied in cancer stem cells per se, the role of HIF in regulating apoptosis has been shown in cancer cell cultures. HIF directly regulates the transcription of anti-apoptotic genes such as myeloid cell leukemia 1 (MCL-1) and B-cell lymphoma extra-large (BCL-XL) [40,41]. Hypoxia also results in a lower level of reactive oxygen species (ROS) in the cell. A lower level of ROS leads to a decrease in activation of caspase-8, a decrease in expression of proapoptotic receptor TRAIL-R2 and an increase expression of pro-survival proteins like cFLIP

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73

Besides the extrinsic chemical factors described above, another type of extrinsic factor that blocks apoptosis is the stimulating ligands produced by neighboring cells. The hematopoiet‐ ic niche has been found to confer resistance to leukemic cells via adhesion molecules such as integrins and soluble molecules of the Wnt pathway [44]. Wnt, Notch and Hedgehog are de‐ velopmental regulatory molecules that are increasingly shown to be involved in cancer stem cell self-renewal, growth and differentiation [27,45–50]. Therapeutics targeting factors of these pathways are currently undergoing clinical trials and have shown promising results in

Tumorigenicity is an essential characteristic that metastatic cancer cells must possess in or‐ der to initiate tumor formation after metastasizing to a distant site. Hence, it can be assumed that cancer stem cells are responsible for metastasis as they have tumorigenic properties by definition. Recent findings suggest that not all cancer stem cells have the capacity to meta‐ stasize. Rather, this capacity is confined to a subset of cancer stem cells. Cancer stem cells (identified by their CD 133 marker) isolated from pancreatic cancer patients were found to contain a subset of cells that expressed CXCR4, the receptor for stromal-cell derived factor 1 (SDF-1). These cancer stem cells that expressed CXCR4 were able to induce tumors in mice, spread via the blood circulation and cause liver metastasis. On the other hand, cancer stem cells that were CXCR4-negative were only able to induce tumors in mice, failing to spread and cause metastasis [22]. In light of this finding, therapies targeting this subset of cancer

The multitude of research in cancer stem cell has deepened our understanding in this field. We present a schema (figure 1) that summarizes the literature in cancer stem cell research from a therapeutic perspective. This schema illustrates that targeting cancer cells with tu‐ morigenic abilities, i.e. cancer stem cells, is not enough. It is the problematic subset of resist‐ ant cancer stem cells (outlined black in figure 1) that accounts for failure of current cancer therapies. Overcoming resistance in cancer stem cells is crucial and we have described sev‐ eral mechanisms that cancer stem cells use to stem our efforts for a cure. One innovative ap‐ proach to eliminate resistant cancer stem cells is differentiation therapy, where cancer stem

cells are made to differentiate, thereby losing their resistant capabilities [51].

eliminating cancer stem cells that are resistant to existing therapies.

and BCL-2 [42,43].

**2.2. Metastasis**

**2.3. Conclusions**

stem cells could prevent metastasis.

#### *2.1.4. High detoxification activity*

Aldehyde dehydrogenase I (ALDH1) is a detoxifying enzyme that oxidizes intracellular ald‐ hydes and is a marker of normal stem cell. Cancer stem cells from acute myeloid leukemia and breast carcinoma are known to have high levels of ALDH1. [21]. In breast cancer pa‐ tients undergoing chemotherapy with paclitaxel and epirubicin, it was found that the pro‐ portion of ALDH1-positive cancer cells increased significantly post treatment, resulting in treatment failure[35]. A high ALDH1 level is thus associated with poor clinical outcomes. This finding indicates that new therapeutic agents must be able to overcome the detoxifica‐ tion prowess of cancer stem cells in order to be effective.

#### *2.1.5. Blockage of apoptosis*

Blockage of apoptosis is a major mechanism for drug resistance as it offers protection against any therapy that results in cell destruction. This ability to prevent apoptosis from oc‐ curring in cancer stem cell is mediated by both inherent cellular factors and extrinsic micro‐ environmental factors.

Inherent cellular factors are important in blocking the apoptotic process. In a study on can‐ cer stem cells (isolated via CD133) from glioblastoma, exposure of cancer cells to conven‐ tional chemotherapeutics such as temozolomide, carboplatin, paclitaxel and etoposide, showed that CD133-positive cells had higher viability compared to CD133-negative cells [36]. In contrast to CD133-negative cells, CD133-positive cells had higher mRNA levels of the anti-apoptotic proteins, such as B-cell lymphoma (Bcl) -2 and -XL proteins, inhibitors of apoptosis proteins (IAPs), FLICE-like inhibitory protein (FLIP) and Sirtuin 1 (SIRT1). In ad‐ dition, CD133-positive cells had lower mRNA level of the pro-apoptotic protein, including Bcl-2 associated X protein (BAX). In a separate study on colon cancer, autocrine production of interleukin-4 (IL4) by CD133-positive colon cancer cells was found to prevent apoptosis of cancer stem cells from occurring when conventional chemotherapeutics and a recombi‐ nant protein called TNF-related apoptosis-inducing ligand (TRAIL) were used [37]. These findings show that cancer stem cells have an armament of proteins to protect themselves from undergoing apoptosis.

In some cancers, resistance to apoptosis is highly dependent on extrinsic microenvironmen‐ tal factors. For example, culturing ovarian cancer cells under stem cell culture conditions led to formation of spheroid cultures of cells that were self-renewing and resistant to cisplatin and paclitaxel [38]. However, this resistance was lost once the cells were cultured under a different set of conditions. One extrinsic factor that has been recognized as the main cause for resistance to cancer therapeutics is hypoxia. The normal stem cell niche has been associ‐ ated with a hypoxic microenvironment. Expression of hypoxia-inducible factors (HIF) are important as these factors regulate stem cell self-renewal and pluripotency [39]. Although not well studied in cancer stem cells per se, the role of HIF in regulating apoptosis has been shown in cancer cell cultures. HIF directly regulates the transcription of anti-apoptotic genes such as myeloid cell leukemia 1 (MCL-1) and B-cell lymphoma extra-large (BCL-XL) [40,41]. Hypoxia also results in a lower level of reactive oxygen species (ROS) in the cell. A lower level of ROS leads to a decrease in activation of caspase-8, a decrease in expression of proapoptotic receptor TRAIL-R2 and an increase expression of pro-survival proteins like cFLIP and BCL-2 [42,43].

Besides the extrinsic chemical factors described above, another type of extrinsic factor that blocks apoptosis is the stimulating ligands produced by neighboring cells. The hematopoiet‐ ic niche has been found to confer resistance to leukemic cells via adhesion molecules such as integrins and soluble molecules of the Wnt pathway [44]. Wnt, Notch and Hedgehog are de‐ velopmental regulatory molecules that are increasingly shown to be involved in cancer stem cell self-renewal, growth and differentiation [27,45–50]. Therapeutics targeting factors of these pathways are currently undergoing clinical trials and have shown promising results in eliminating cancer stem cells that are resistant to existing therapies.

#### **2.2. Metastasis**

of SP cells to non-SP cells, suggesting that ABCG2 conferred a survival advantage to cancer stem cells [33]. Similarly, in acute myeloid leukemia, SP cells derived from mononuclear cells in bone marrow of patients showed an increase efflux of daunorubicin and mitoxan‐ trone when compared to non-SP cells [34]. Taken together, these findings suggest that can‐

Aldehyde dehydrogenase I (ALDH1) is a detoxifying enzyme that oxidizes intracellular ald‐ hydes and is a marker of normal stem cell. Cancer stem cells from acute myeloid leukemia and breast carcinoma are known to have high levels of ALDH1. [21]. In breast cancer pa‐ tients undergoing chemotherapy with paclitaxel and epirubicin, it was found that the pro‐ portion of ALDH1-positive cancer cells increased significantly post treatment, resulting in treatment failure[35]. A high ALDH1 level is thus associated with poor clinical outcomes. This finding indicates that new therapeutic agents must be able to overcome the detoxifica‐

Blockage of apoptosis is a major mechanism for drug resistance as it offers protection against any therapy that results in cell destruction. This ability to prevent apoptosis from oc‐ curring in cancer stem cell is mediated by both inherent cellular factors and extrinsic micro‐

Inherent cellular factors are important in blocking the apoptotic process. In a study on can‐ cer stem cells (isolated via CD133) from glioblastoma, exposure of cancer cells to conven‐ tional chemotherapeutics such as temozolomide, carboplatin, paclitaxel and etoposide, showed that CD133-positive cells had higher viability compared to CD133-negative cells [36]. In contrast to CD133-negative cells, CD133-positive cells had higher mRNA levels of the anti-apoptotic proteins, such as B-cell lymphoma (Bcl) -2 and -XL proteins, inhibitors of apoptosis proteins (IAPs), FLICE-like inhibitory protein (FLIP) and Sirtuin 1 (SIRT1). In ad‐ dition, CD133-positive cells had lower mRNA level of the pro-apoptotic protein, including Bcl-2 associated X protein (BAX). In a separate study on colon cancer, autocrine production of interleukin-4 (IL4) by CD133-positive colon cancer cells was found to prevent apoptosis of cancer stem cells from occurring when conventional chemotherapeutics and a recombi‐ nant protein called TNF-related apoptosis-inducing ligand (TRAIL) were used [37]. These findings show that cancer stem cells have an armament of proteins to protect themselves

In some cancers, resistance to apoptosis is highly dependent on extrinsic microenvironmen‐ tal factors. For example, culturing ovarian cancer cells under stem cell culture conditions led to formation of spheroid cultures of cells that were self-renewing and resistant to cisplatin and paclitaxel [38]. However, this resistance was lost once the cells were cultured under a different set of conditions. One extrinsic factor that has been recognized as the main cause for resistance to cancer therapeutics is hypoxia. The normal stem cell niche has been associ‐

cer stem cell uses efflux transporters to guard against anti-cancer drugs.

tion prowess of cancer stem cells in order to be effective.

*2.1.4. High detoxification activity*

72 Pluripotent Stem Cells

*2.1.5. Blockage of apoptosis*

environmental factors.

from undergoing apoptosis.

Tumorigenicity is an essential characteristic that metastatic cancer cells must possess in or‐ der to initiate tumor formation after metastasizing to a distant site. Hence, it can be assumed that cancer stem cells are responsible for metastasis as they have tumorigenic properties by definition. Recent findings suggest that not all cancer stem cells have the capacity to meta‐ stasize. Rather, this capacity is confined to a subset of cancer stem cells. Cancer stem cells (identified by their CD 133 marker) isolated from pancreatic cancer patients were found to contain a subset of cells that expressed CXCR4, the receptor for stromal-cell derived factor 1 (SDF-1). These cancer stem cells that expressed CXCR4 were able to induce tumors in mice, spread via the blood circulation and cause liver metastasis. On the other hand, cancer stem cells that were CXCR4-negative were only able to induce tumors in mice, failing to spread and cause metastasis [22]. In light of this finding, therapies targeting this subset of cancer stem cells could prevent metastasis.

#### **2.3. Conclusions**

The multitude of research in cancer stem cell has deepened our understanding in this field. We present a schema (figure 1) that summarizes the literature in cancer stem cell research from a therapeutic perspective. This schema illustrates that targeting cancer cells with tu‐ morigenic abilities, i.e. cancer stem cells, is not enough. It is the problematic subset of resist‐ ant cancer stem cells (outlined black in figure 1) that accounts for failure of current cancer therapies. Overcoming resistance in cancer stem cells is crucial and we have described sev‐ eral mechanisms that cancer stem cells use to stem our efforts for a cure. One innovative ap‐ proach to eliminate resistant cancer stem cells is differentiation therapy, where cancer stem cells are made to differentiate, thereby losing their resistant capabilities [51].

stem cell antigens were injected directly into patients to prime the immune system against cancer stem cells. The peptides used in these vaccinations are found in both cancer stem cells and non-stem cancer cells. One of these antigens is Wilms' Tumor 1 (WT1). WT1 is a transcription factor that is expressed in leukemia. Although a direct association between WT1 and the leukemic stem cell has never been shown, WT1 however, has been associated with the CD34+CD38- cell population which is thought to harbor the hematopoietic stem

**Drug target Drug name Cancer Stage Company**

Telomerase (inhibitor) Imetelstat Broad range Phase II Geron Corporation

Notch pathway Anti-DLL4 (demcizumab) Solid Tumors Phase I OncoMed Anti-Notch2/3 (OPM-59R5) Solid tumors Phase I

tumors

Leukemia and mesothelioma

Advance solid tumor cancers

Advance leukemia and advance brain cancer

A third approach is the targeting of the cancer stem cell and its microenvironment. Anti-EphA3 antibody is a clinical candidate against cancer stem cell that has been developed by KaloBios. This antibody treatment is now in phase I trial. EphA3 expression is found in preB leukemia cell line and in a subset of samples from leukemia patients [56]. There is no direct evidence that links EphA3 expression to the leukemic stem cells, however, in an in-house study by KaloBios, incubating anti-EphA3 with cancer cells leads to the cancer cells losing their ability to form colonies in vitro, suggesting that the antibody was active against cancer

Solid Tumors Phase I

VS6063 Advance solid

Colorectal cancer Entering Phase III Boston Biomedicals, Inc

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75

Glioblastoma Entering phase I ImmunoCellular

Phase I completed

Leukemia Phase I KaloBios

Phase I

Phase I/II completed Therapeutics Ltd.

Verastem and Pfeizer

Pharmaceuticals

Pharmaceuticals, Inc.

Stemline Therapeutics

Phase II Formula

cell [55] and also the leukemia stem cell [15].

Undisclosed Cancer stem cell inhibitor

CD-133 Dendritic cell-based vaccine

Wilms Tumor 1 Peptides from Wilms Tumor 1

EphA3 Human monoclonal antibody

Wnt pathway Anti-Fzd7 (OMP-18R5, binds 5

**Table 2.** Current clinical drug trials in cancer stem cell therapy

Focal adhesion kinase (inhibitor)

Undisclosed cancer stem cell antigen

BBI608

ICT-121

(FPI-01)

(KB004) binds EphA3

Frizzled receptors)

Truncated Frizzled 8-Fc fusion protein (OMP-54F28)

Peptides vaccine (SL401 and SL701)

**Figure 1.** Properties of cancer cells. The subset of tumorigenic cells that are also drug-resistant should be targeted to ensure elimination of cancer.

## **3. Molecular targets for cancer therapy**

#### **3.1. Current clinical drug trials targeting cancer stem cells**

Translating research from bench to bedside is perhaps the most challenging and rewarding part of science. The development of drugs against cancer stem cell is an exciting field with many different, innovative approaches. In this section, a review of the drugs that have al‐ ready reached clinical trials is presented (Table 2).

One approach is the targeting of the cancer stem cell machinery. An example of this ap‐ proach is the telomerase inhibitors. Telomeric inhibitors block replication and a clinical can‐ didate Imetelstat have shown efficacy in cancer stem cells [52]. As a bonus, telomerase inhibitors are expected to also target the bulk of the tumor. Importantly, unlike normal stem cells, cancer stem cells express higher levels of telomerase [53]. Hence this could potentially be a drug that targets cancer stem cells without hurting normal stem cells.

In a second approach the targeting of the cancer stem cell phenotype, the immunogenic-re‐ sponse that ironically had been a problem to researchers in the detection of cancer stem cells using the mouse model, has become a solution against cancer stem cells. In a study in which cancer stem cells were injected into immunocompetent syngenic mice, cancer stem cells in‐ duced antitumor response more effectively than unselected cancer cells [54]. This finding is important and has led to the development of various clinical candidates by three different pharmaceutical companies. These candidates, all of which are currently in clinical trials, were developed based on cancer stem cell-associated proteins. These proteins serve as anti‐ gens to evoke an immune response against cancer stem cells. In essence, these proteins act as vaccines against cancer stem cells. Immunocellular Therapeutics has developed a dendriticbased vaccine comprising dendritic cells that were obtained from patients and primed in vi‐ tro by two CD133-peptides. This vaccine has just recently been approved for phase I clinical trials. Using the same approach, other clinical candidates were developed by two pharma‐ ceutical companies. Instead of obtaining dendritic cells from patients, peptides of cancer stem cell antigens were injected directly into patients to prime the immune system against cancer stem cells. The peptides used in these vaccinations are found in both cancer stem cells and non-stem cancer cells. One of these antigens is Wilms' Tumor 1 (WT1). WT1 is a transcription factor that is expressed in leukemia. Although a direct association between WT1 and the leukemic stem cell has never been shown, WT1 however, has been associated with the CD34+CD38- cell population which is thought to harbor the hematopoietic stem cell [55] and also the leukemia stem cell [15].



**Figure 1.** Properties of cancer cells. The subset of tumorigenic cells that are also drug-resistant should be targeted to

Translating research from bench to bedside is perhaps the most challenging and rewarding part of science. The development of drugs against cancer stem cell is an exciting field with many different, innovative approaches. In this section, a review of the drugs that have al‐

One approach is the targeting of the cancer stem cell machinery. An example of this ap‐ proach is the telomerase inhibitors. Telomeric inhibitors block replication and a clinical can‐ didate Imetelstat have shown efficacy in cancer stem cells [52]. As a bonus, telomerase inhibitors are expected to also target the bulk of the tumor. Importantly, unlike normal stem cells, cancer stem cells express higher levels of telomerase [53]. Hence this could potentially

In a second approach the targeting of the cancer stem cell phenotype, the immunogenic-re‐ sponse that ironically had been a problem to researchers in the detection of cancer stem cells using the mouse model, has become a solution against cancer stem cells. In a study in which cancer stem cells were injected into immunocompetent syngenic mice, cancer stem cells in‐ duced antitumor response more effectively than unselected cancer cells [54]. This finding is important and has led to the development of various clinical candidates by three different pharmaceutical companies. These candidates, all of which are currently in clinical trials, were developed based on cancer stem cell-associated proteins. These proteins serve as anti‐ gens to evoke an immune response against cancer stem cells. In essence, these proteins act as vaccines against cancer stem cells. Immunocellular Therapeutics has developed a dendriticbased vaccine comprising dendritic cells that were obtained from patients and primed in vi‐ tro by two CD133-peptides. This vaccine has just recently been approved for phase I clinical trials. Using the same approach, other clinical candidates were developed by two pharma‐ ceutical companies. Instead of obtaining dendritic cells from patients, peptides of cancer

be a drug that targets cancer stem cells without hurting normal stem cells.

ensure elimination of cancer.

74 Pluripotent Stem Cells

**3. Molecular targets for cancer therapy**

ready reached clinical trials is presented (Table 2).

**3.1. Current clinical drug trials targeting cancer stem cells**

A third approach is the targeting of the cancer stem cell and its microenvironment. Anti-EphA3 antibody is a clinical candidate against cancer stem cell that has been developed by KaloBios. This antibody treatment is now in phase I trial. EphA3 expression is found in preB leukemia cell line and in a subset of samples from leukemia patients [56]. There is no direct evidence that links EphA3 expression to the leukemic stem cells, however, in an in-house study by KaloBios, incubating anti-EphA3 with cancer cells leads to the cancer cells losing their ability to form colonies in vitro, suggesting that the antibody was active against cancer stem cells. In addition, the antibody was also found to bind to EphA3 that was expressed on cancer vasculature cells as well as cancer stromal cells. The binding was reported to cause cell-cell repulsion, resulting in the destruction of new vessels and failure to establish a can‐ cer stromal environment [57]. This strategy, which targets a protein that is found in cancer stem cell, cancer stromal cells and cancer vasculature cells, would be "killing-three-birdswith-one-stone". Other clinical candidates that are based on a similar approach have also been developed via targeting the Wnt pathway and the Notch Pathway instead [47–50].

cultured under hypoxic conditions, embryonic stem cell-association gene expression and formation of neuroshpere were seen in both fractions [65]. Thus, low levels of oxygen pro‐ motes the transformation of cancer cells into cancer stem cells by activating the pluripotency machinery in cancer cells with expression and repression of modules that are similar in pro‐ file to embryonic cells. This should be taken into consideration when targeting cancer cells. Studies on HIF have shown that both HIF-1α and HIF-2α h are associated with cancer. HIF-1α has been shown to play a role in angiogenesis [66] and anti-angiogenesis therapies targeting HIF-1α have been undertaken [67,68]. In contrast, recent findings suggest that HIF-2α is involved in the triggering of stemness in cancer which in turn promotes cancer growth and aggressiveness [69]. Hence a potential pathway to target cancer stem cell will be

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77

**Figure 2.** The three sets of genes that are activated or repressed by distinct sets of transcriptional regulators. Induce pluripotent stem (iPS) cells show similar gene expression profile as embryonic stem (ES) cells. Mouse embryonic fibro‐ blast (MEF) represents a set of differentiated cells and shows a profile that is opposite to that of ES cells. Analysis of the profile in cancer highlights a pluripotency machinery in which the core module has been suppressed and the Myc module overactivated. Figure is a reprint of the graphical abstract provided by Kim et al [60] (Reprinted with permis‐

Knowledge garnered from studies on pluripotent stem cell provides a rich resource for can‐ cer stem cell research and paves the way in identifying novel key targets for cancer therapy. Targeting molecules or pathways specific to embryonic stem cells gives us the opportunity

to kill cancer cells without harming innocent bystander cells.

the HIF-2α-mediated pathway.

sion from Cell Press)

#### **3.2. Potential new targets – Insights from Pluripotent Stem cells**

The discovery that transcription factors, namely, Oct4, Sox2, Klf4 and c-Myc, can induce pluripotency in a differentiated adult cell [58], accelerated the understanding of the molecu‐ lar machinery driving pluripotent stem cells. Systems biology approaches based on these transcription factors generated genome-wide regulatory networks that are thought to be the supporting framework for an embryonic stem cell state. These data serves as a rich resource in furthering our understanding of the cancer stem cell.

In a recent study of regulatory networks in embryonic stem cell, analysis of the protein-pro‐ tein interactions of key transcription factors and the downstream targeted genes revealed that the embryonic stem cell regulatory network can be divided into three independent modules (Figure 2). The three modules are the core module, the c-Myc module and the Poly‐ comb Repressive Complex (PRC) module. The core module comprises genes that are regu‐ lated by the embryonic stem cell-specific transcription factor Oct4 and Oct4-associated proteins while the PRC module comprises genes that are repressed by the PRC. The Mycmodule comprises genes that are regulated by c-Myc and its associated proteins. Proper functioning of all three modules are essential for having a normal pluripotent stem cell [59].

Using the 3-module model to study the genes expressed in bladder cancer and breast cancer samples, it was found that the Myc module was more active in cancers while that of the core module was more repressed, when compared to normal urothelium obtained from a distant site of the cancer [61,62]. This suggested that in cancer cells, the Myc module is re-activated but is not balanced by a core module. It should be noted that this comparison was done with the heterogeneous cancer cell population and not the cancer stem cell population. Repeating the same characterization analysis on cancer stem cell samples will likely highlight the key differences between the regulatory network of cancer stem cells and that of normal pluripo‐ tent stem cells. These differences could become potential targets for anti-cancer therapy.

Factors that are crucial for the maintenance of pluripotent stem cell have been found to be involved in cancer. Hypoxia-inducible factors (HIFs) have been found to be important in pluripotent stem cell [39,63]. Studies now show that HIFs could be the key factor in switch‐ ing on the pluripotency machinery in cancer cells to form cancer stem cells [64]. In an experi‐ ment where glioma cells and cervical cells were exposed to HIFs, activation of the embryonic stem cell marker, Oct4, was observed [65]. Subjecting glioma cells to hypoxia re‐ sulted in an increase in the level of CD133 mRNA [65]. In samples from glioma patients, subjecting the CD133-positive fraction to hypoxia resulted in increased mRNA levels of OCT4, NANOG and cMYC. Interestingly, when CD133-positive and negative fractions were cultured under hypoxic conditions, embryonic stem cell-association gene expression and formation of neuroshpere were seen in both fractions [65]. Thus, low levels of oxygen pro‐ motes the transformation of cancer cells into cancer stem cells by activating the pluripotency machinery in cancer cells with expression and repression of modules that are similar in pro‐ file to embryonic cells. This should be taken into consideration when targeting cancer cells. Studies on HIF have shown that both HIF-1α and HIF-2α h are associated with cancer. HIF-1α has been shown to play a role in angiogenesis [66] and anti-angiogenesis therapies targeting HIF-1α have been undertaken [67,68]. In contrast, recent findings suggest that HIF-2α is involved in the triggering of stemness in cancer which in turn promotes cancer growth and aggressiveness [69]. Hence a potential pathway to target cancer stem cell will be the HIF-2α-mediated pathway.

stem cells. In addition, the antibody was also found to bind to EphA3 that was expressed on cancer vasculature cells as well as cancer stromal cells. The binding was reported to cause cell-cell repulsion, resulting in the destruction of new vessels and failure to establish a can‐ cer stromal environment [57]. This strategy, which targets a protein that is found in cancer stem cell, cancer stromal cells and cancer vasculature cells, would be "killing-three-birdswith-one-stone". Other clinical candidates that are based on a similar approach have also been developed via targeting the Wnt pathway and the Notch Pathway instead [47–50].

The discovery that transcription factors, namely, Oct4, Sox2, Klf4 and c-Myc, can induce pluripotency in a differentiated adult cell [58], accelerated the understanding of the molecu‐ lar machinery driving pluripotent stem cells. Systems biology approaches based on these transcription factors generated genome-wide regulatory networks that are thought to be the supporting framework for an embryonic stem cell state. These data serves as a rich resource

In a recent study of regulatory networks in embryonic stem cell, analysis of the protein-pro‐ tein interactions of key transcription factors and the downstream targeted genes revealed that the embryonic stem cell regulatory network can be divided into three independent modules (Figure 2). The three modules are the core module, the c-Myc module and the Poly‐ comb Repressive Complex (PRC) module. The core module comprises genes that are regu‐ lated by the embryonic stem cell-specific transcription factor Oct4 and Oct4-associated proteins while the PRC module comprises genes that are repressed by the PRC. The Mycmodule comprises genes that are regulated by c-Myc and its associated proteins. Proper functioning of all three modules are essential for having a normal pluripotent stem cell [59]. Using the 3-module model to study the genes expressed in bladder cancer and breast cancer samples, it was found that the Myc module was more active in cancers while that of the core module was more repressed, when compared to normal urothelium obtained from a distant site of the cancer [61,62]. This suggested that in cancer cells, the Myc module is re-activated but is not balanced by a core module. It should be noted that this comparison was done with the heterogeneous cancer cell population and not the cancer stem cell population. Repeating the same characterization analysis on cancer stem cell samples will likely highlight the key differences between the regulatory network of cancer stem cells and that of normal pluripo‐ tent stem cells. These differences could become potential targets for anti-cancer therapy.

Factors that are crucial for the maintenance of pluripotent stem cell have been found to be involved in cancer. Hypoxia-inducible factors (HIFs) have been found to be important in pluripotent stem cell [39,63]. Studies now show that HIFs could be the key factor in switch‐ ing on the pluripotency machinery in cancer cells to form cancer stem cells [64]. In an experi‐ ment where glioma cells and cervical cells were exposed to HIFs, activation of the embryonic stem cell marker, Oct4, was observed [65]. Subjecting glioma cells to hypoxia re‐ sulted in an increase in the level of CD133 mRNA [65]. In samples from glioma patients, subjecting the CD133-positive fraction to hypoxia resulted in increased mRNA levels of OCT4, NANOG and cMYC. Interestingly, when CD133-positive and negative fractions were

**3.2. Potential new targets – Insights from Pluripotent Stem cells**

in furthering our understanding of the cancer stem cell.

76 Pluripotent Stem Cells

**Figure 2.** The three sets of genes that are activated or repressed by distinct sets of transcriptional regulators. Induce pluripotent stem (iPS) cells show similar gene expression profile as embryonic stem (ES) cells. Mouse embryonic fibro‐ blast (MEF) represents a set of differentiated cells and shows a profile that is opposite to that of ES cells. Analysis of the profile in cancer highlights a pluripotency machinery in which the core module has been suppressed and the Myc module overactivated. Figure is a reprint of the graphical abstract provided by Kim et al [60] (Reprinted with permis‐ sion from Cell Press)

Knowledge garnered from studies on pluripotent stem cell provides a rich resource for can‐ cer stem cell research and paves the way in identifying novel key targets for cancer therapy. Targeting molecules or pathways specific to embryonic stem cells gives us the opportunity to kill cancer cells without harming innocent bystander cells.

### **4. Summary**

Research in cancer is immense and complex as cancer is a diverse disease with a myriad of genetic mutations. A pressing practical concern in cancer therapeutics is the development of resistance of cancer cells to current treatment, resulting in failure of therapy and eventual death. In the last two decades, cancer stem cell hypothesis has emerged as the likely reason for this resistance in cancer. We now understand that cancer stem cells are present in differ‐ ent cancers. They can be a small, distinct population characterized by certain phenotypes in some cancers while heterogeneous and with no defining phenotypes in others. Cancer stem cells can also result from cancer cells under the influence of environmental factors such as hypoxia. They are also highly resistant to cancer drugs with several mechanisms employed for enhanced survival. Research into stem cells and pluripotency regulatory networks will provide further characterization and understanding of cancer stem cells. The information on cancer stem cells has pieced together a therapeutic framework to address cancer resistance with several potential therapies in clinical trials currently. So much more needs to be done in this field in our quest to conquer cancer totally.

[3] Dalerba P, Dylla SJ, Park I-K, Liu R, Wang X, Cho RW, et al. Phenotypic characteriza‐ tion of human colorectal cancer stem cells. Proceedings of the National Academy of

The Dark Side of Pluripotency – Cancer Stem Cell

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79

[4] Dick JE. Breast cancer stem cells revealed. Proceedings of the National Academy of

[5] Eramo a, Lotti F, Sette G, Pilozzi E, Biffoni M, Di Virgilio a, et al. Identification and expansion of the tumorigenic lung cancer stem cell population. Cell death and differ‐

[6] O'Brien C a, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of ini‐ tiating tumour growth in immunodeficient mice. Nature 2007;445(7123):106–10.

[7] Prince ME, Sivanandan R, Kaczorowski a, Wolf GT, Kaplan MJ, Dalerba P, et al. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proceedings of the National Academy of Sciences of

[8] Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, et al. Identi‐ fication and expansion of human colon-cancer-initiating cells. Nature 2007;445(7123):

[9] Schatton T, Murphy GF, Frank NY, Yamaura K, Waaga-Gasser AM, Gasser M, et al. Identification of cells initiating human melanomas. Nature 2008;451(7176):345–9. [10] Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al. Identification of human brain tumour initiating cells. Nature 2004;432(November):396–401.

[11] Zhang S, Balch C, Chan MW, Lai H-C, Matei D, Schilder JM, et al. Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Can‐

[12] Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, et al. Identification of pan‐

[13] Li Y, Laterra J. Cancer stem cells: distinct entities or dynamically regulated pheno‐

[14] Furth J, Kahn MC. The Transmission of Leukemia of Mice with a Single Cell. Ameri‐

[15] Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nature medicine 1997;3(7):730–7. [16] Ho MM, Ng AV, Lam S, Hung JY. Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer research 2007;67(10):4827–

[17] Prince ME, Sivanandan R, Kaczorowski a, Wolf GT, Kaplan MJ, Dalerba P, et al. Identification of a subpopulation of cells with cancer stem cell properties in head and

creatic cancer stem cells. Cancer research 2007;67(3):1030–7.

Sciences of the United States of America 2007;104(24):10158–63.

Sciences of the United States of America 2003;100(7):3547–9.

the United States of America 2007;104(3):973–8.

entiation 2008;15(3):504–14.

cer research 2008;68(11):4311–20.

types? Cancer research 2012;72(3):576–80.

can journal of cancer 1937;31:276–82.

111–5.

33.

## **Acknowledgements**

The authors acknowledge Dr Jansen Koh for active discussion and review of the manuscript. Patricia Ng is supported by the Agency for Science, Technology and Research (A\*STAR) Re‐ search Fellowship and the UNESCO-L'Oréal International Fellowship.

## **Author details**

Patricia Ng and Wang Cheng-I

Singapore Immunology Network, Agency for Science, Technology and Research, Singapore

### **References**


[3] Dalerba P, Dylla SJ, Park I-K, Liu R, Wang X, Cho RW, et al. Phenotypic characteriza‐ tion of human colorectal cancer stem cells. Proceedings of the National Academy of Sciences of the United States of America 2007;104(24):10158–63.

**4. Summary**

78 Pluripotent Stem Cells

in this field in our quest to conquer cancer totally.

**Acknowledgements**

**Author details**

**References**

Patricia Ng and Wang Cheng-I

1994;367:645–8.

Research in cancer is immense and complex as cancer is a diverse disease with a myriad of genetic mutations. A pressing practical concern in cancer therapeutics is the development of resistance of cancer cells to current treatment, resulting in failure of therapy and eventual death. In the last two decades, cancer stem cell hypothesis has emerged as the likely reason for this resistance in cancer. We now understand that cancer stem cells are present in differ‐ ent cancers. They can be a small, distinct population characterized by certain phenotypes in some cancers while heterogeneous and with no defining phenotypes in others. Cancer stem cells can also result from cancer cells under the influence of environmental factors such as hypoxia. They are also highly resistant to cancer drugs with several mechanisms employed for enhanced survival. Research into stem cells and pluripotency regulatory networks will provide further characterization and understanding of cancer stem cells. The information on cancer stem cells has pieced together a therapeutic framework to address cancer resistance with several potential therapies in clinical trials currently. So much more needs to be done

The authors acknowledge Dr Jansen Koh for active discussion and review of the manuscript. Patricia Ng is supported by the Agency for Science, Technology and Research (A\*STAR) Re‐

Singapore Immunology Network, Agency for Science, Technology and Research, Singapore

[1] Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, et al. A cell initiating human acute myeloi leukaemia after transplation into SCID mice. Nature

[2] Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Acade‐

my of Sciences of the United States of America 2003;100(7):3983–8.

search Fellowship and the UNESCO-L'Oréal International Fellowship.


neck squamous cell carcinoma. Proceedings of the National Academy of Sciences of the United States of America 2007;104(3):973–8.

[32] Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Na‐

The Dark Side of Pluripotency – Cancer Stem Cell

http://dx.doi.org/10.5772/54369

81

[33] Hirschmann-Jax C, Foster a E, Wulf GG, Nuchtern JG, Jax TW, Gobel U, et al. A dis‐ tinct "side population" of cells with high drug efflux capacity in human tumor cells. Proceedings of the National Academy of Sciences of the United States of America

[34] Wulf GG, Wang R-Y, Kuehnle I, Weidner D, Marini F, Brenner MK, et al. A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia. Blood

[35] Tanei T, Morimoto K, Shimazu K, Kim SJ, Tanji Y, Taguchi T, et al. Association of breast cancer stem cells identified by aldehyde dehydrogenase 1 expression with re‐ sistance to sequential Paclitaxel and epirubicin-based chemotherapy for breast can‐ cers. Clinical cancer research : an official journal of the American Association for

[36] Liu G, Yuan X, Zeng Z, Tunici P, Ng H, Abdulkadir IR, et al. Analysis of gene expres‐ sion and chemoresistance of CD133+ cancer stem cells in glioblastoma. Molecular

[37] Todaro M, Alea MP, Di Stefano AB, Cammareri P, Vermeulen L, Iovino F, et al. Co‐ lon cancer stem cells dictate tumor growth and resist cell death by production of in‐

[38] Zhang S, Balch C, Chan MW, Lai H-C, Matei D, Schilder JM, et al. Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Can‐

[39] Ji L, Liu Y, Yang C, Yue W, Shi S, Bai C, et al. Self-renewal and pluripotency is main‐ tained in human embryonic stem cells by co-culture with human fetal liver stromal cells expressing hypoxia inducible factor 1alpha. Journal of cellular physiology

[40] Shroff EH, Snyder C, Chandel NS. Role of Bcl-2 Family Members in Anoxia Induced

[41] Chen N, Chen X, Huang R, Zeng H, Gong J, Meng W, et al. BCL-xL is a target gene regulated by hypoxia-inducible factor-1{alpha}. The Journal of biological chemistry

[42] Jie W, Yi J. Cancer cell killing via ROS. Cancer Biology & Therapy 2008;7(December):

[43] Reinehr R, Becker S, Eberle A, Grether-beck S, Häussinger D. Involvement of NADPH oxidase isoforms and SRC family kinases in CD95-dependent hepatocyte

apoptosis. Journal of biological chemistry 2005;280:27179–94.

ture 2006;444(7120):756–60.

2004;101(39):14228–33.

2001;98(4):1166–73.

cancer 2006;5:67.

2009;221(1):54–66.

2009;284(15):10004–12.

1875–84.

Cancer Research 2009;15(12):4234–41.

terleukin-4. Cell stem cell 2007;1(4):389–402.

Cell Death. Cell Cycle 2007;6(April):807–9.

cer research 2008;68(11):4311–20.


[32] Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Na‐ ture 2006;444(7120):756–60.

neck squamous cell carcinoma. Proceedings of the National Academy of Sciences of

[18] Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a pre‐

[19] Hermann PC, Huber SL, Herrler T, Aicher A, Ellwart JW, Guba M, et al. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in

[21] Quintana E, Shackleton M, Sabel MS, Fullen DR, Johnson TM, Morrison SJ. Efficient tumour formation by single human melanoma cells. Nature 2008;456(7222):593–8.

[22] Quintana E, Shackleton M, Foster HR, Fullen DR, Sabel MS, Johnson TM, et al. Phe‐ notypic heterogeneity among tumorigenic melanoma cells from patients that is re‐

[23] Yilmaz OH, Valdez R, Theisen BK, Guo W, Ferguson DO, Wu H, et al. Pten depend‐ ence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature

[24] Southam CM, Brunschwig A. Quantitative studies of autotransplantation of human

[25] Koike a, Moore GE, Mendoza CB, Watne a L. Heterologous, Homologous, and Autol‐

[26] Nadler SH, Moore GE. Autotransplantation of Human Cancer. JAMA : the journal of

[27] Vermeulen L, De Sousa E Melo F, van der Heijden M, Cameron K, de Jong JH, Borov‐ ski T, et al. Wnt activity defines colon cancer stem cells and is regulated by the micro‐

[28] Ribatti D. Cancer stem cells and tumor angiogenesis. Cancer letters 2012;321(1):13–7.

[29] Moore N, Lyle S. Quiescent, slow-cycling stem cell populations in cancer: a review of the evidence and discussion of significance. Journal of oncology 2011;2011.

[30] Naumov GN, Townson JL, MacDonald IC, Wilson SM, Bramwell VH, Groom AC, et al. Ineffectiveness of Doxorubicin Treatment on Solitary Dormant Mammary Carci‐ noma Cells or Late-developing Metastases. Breast Cancer Research and Treatment

[31] Dembinski JL, Krauss S. Characterization and functional analysis of a slow cycling stem cell-like subpopulation in pancreas adenocarcinoma. Clinical & experimental

ogous Transplantation of Human Tumors. Cancer 1963;16:1065–71.

the American Medical Association 1965;191:105–6.

environment. Nature cell biology 2010;12(5):468–76.

[20] Barnes WA, Furth J. A transmissible leukemia i n m i c e w i t h atypical cells.

versible and not hierarchically organized. Cancer cell 2010;18(5):510–23.

the United States of America 2007;104(3):973–8.

2006;441(7092):475–82.

80 Pluripotent Stem Cells

2003;82:199–206.

metastasis 2009;26(7):611–23.

cancer. Cancer 1961;14:971–8.

dictor of poor clinical outcome. Cell stem cell 2007;1(5):555–67.

human pancreatic cancer. Cell stem cell 2007;1(3):313–23.


[44] De Toni F, Racaud-Sultan C, Chicanne G, Mas VM-D, Cariven C, Mesange F, et al. A crosstalk between the Wnt and the adhesion-dependent signaling pathways governs the chemosensitivity of acute myeloid leukemia. Oncogene 2006;25(22):3113–22.

[57] Garber K. Of Ephs and Ephrins : Companies Target Guidance Molecules in Cancer.

The Dark Side of Pluripotency – Cancer Stem Cell

http://dx.doi.org/10.5772/54369

83

[58] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell

[59] Fazzio TG, Huff JT, Panning B. An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity. Cell 2008;134(1):162–74.

[60] Kim J, Woo AJ, Chu J, Snow JW, Fujiwara Y, Kim CG, et al. A Myc Network Ac‐ counts for Similarities between Embryonic Stem and Cancer Cell Transcription Pro‐

[61] Sanchez-Carbayo M, Socci ND, Lozano J, Saint F, Cordon-Cardo C. Defining molecu‐ lar profiles of poor outcome in patients with invasive bladder cancer using oligonu‐ cleotide microarrays. Journal of clinical oncology : official journal of the American

[62] Kim J, Orkin SH. Embryonic stem cell-specific signatures in cancer: insights into ge‐ nomic regulatory networks and implications for medicine. Genome medicine

[63] Forristal CE, Wright KL, Hanley N a, Oreffo ROC, Houghton FD. Hypoxia inducible factors regulate pluripotency and proliferation in human embryonic stem cells cul‐

[64] Keith B, Simon MC. Hypoxia-inducible factors, stem cells, and cancer. Cell

[65] Mathieu J, Zhang Z, Zhou W, Wang AJ, Heddleston JM, Pinna CM a, et al. HIF indu‐ ces human embryonic stem cell markers in cancer cells. Cancer research 2011;71(13):

[66] Semenza GL. HIF-1 and tumor progression: pathophysiology and therapeutics.

[67] Ferrara N, Hillan KJ, Gerber H-P, Novotny W. Discovery and development of bevaci‐ zumab, an anti-VEGF antibody for treating cancer. Nature reviews. Drug discovery

[68] Tan C, Noronha RGD, Roecker AJ, Pyrzynska B, Khwaja F, Zhang Z, et al. Identifica‐ tion of a Novel Small-Molecule Inhibitor of the Hypoxia-Inducible Factor 1 Pathway

[69] Koh MY, Lemos R, Liu X, Powis G. The hypoxia-associated factor switches cells from HIF-1α- to HIF-2α-dependent signaling promoting stem cell characteristics, aggres‐

sive tumor growth and invasion. Cancer research 2011;71(11):4015–27.

tured at reduced oxygen tensions. Reproduction Research 2010;139(1):85–97.

Journal of the national cancer institute 2010;102(22):1692–4.

Society of Clinical Oncology 2006;24(5):778–89.

Trends in molecular medicine 2002;8(4 Suppl):S62–7.

Hypoxia-Inducible Factor 1 Pathway. 2005;:605–12.

2007;131(5):861–72.

grams. 2010;:313–24.

2011;3(11):75.

2007;129(3):465–72.

2004;3(5):391–400.

4640–52.


[57] Garber K. Of Ephs and Ephrins : Companies Target Guidance Molecules in Cancer. Journal of the national cancer institute 2010;102(22):1692–4.

[44] De Toni F, Racaud-Sultan C, Chicanne G, Mas VM-D, Cariven C, Mesange F, et al. A crosstalk between the Wnt and the adhesion-dependent signaling pathways governs the chemosensitivity of acute myeloid leukemia. Oncogene 2006;25(22):3113–22.

[45] Xu Q, Yuan X, Liu G, Black KL, Yu JS. Hedgehog signaling regulates brain tumorinitiating cell proliferation and portends shorter survival for patients with PTEN-co‐

[46] Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem

[47] Gurney A, Axelrod F, Bond CJ, Cain J, Chartier C, Donigan L, et al. Wnt pathway in‐ hibition via the targeting of Frizzled receptors results in decreased growth and tu‐ morigenicity of human tumors. Proceedings of the National Academy of Sciences of

[48] Gurney A, Hoey T. Anti-DLL4, a cancer therapeutic with multiple mechanisms of ac‐

[49] Fischer M, Yen W-C, Kapoun AM, Wang M, O'Young G, Lewicki J, et al. Anti-DLL4 inhibits growth and reduces tumor-initiating cell frequency in colorectal tumors with

[50] Hoey T, Yen W-C, Axelrod F, Basi J, Donigian L, Dylla S, et al. DLL4 blockade inhib‐ its tumor growth and reduces tumor-initiating cell frequency. Cell stem cell

[51] Leszczyniecka M, Roberts T, Dent P, Grant S, Fisher PB. Differentiation therapy of human cancer: basic science and clinical applications. Pharmacology & therapeutics

[52] Marian CO, Cho SK, McEllin BM, Maher E a, Hatanpaa KJ, Madden CJ, et al. The te‐ lomerase antagonist, imetelstat, efficiently targets glioblastoma tumor-initiating cells leading to decreased proliferation and tumor growth. Clinical cancer research : an official journal of the American Association for Cancer Research 2010;16(1):154–63.

[53] Shay JW, Wright WE. Telomeres and telomerase in normal and cancer stem cells.

[54] Ning N, Pan Q, Zheng F, Teitz-Tennenbaum S, Egenti M, Yet J, et al. Cancer stem cell vaccination confers significant antitumor immunity. Cancer research 2012;72(7):1853–

[55] Baird PN, Simmons PJ. Expression of the Wilms' tumor gene (WT1) in normal hemo‐

[56] Dottori M, Down M, Hüttmann A, Fitzpatrick DR, Boyd AW, Dottori BM, et al. Methylation Regulates Expression in Hematopoietic Tumor Cells. 2012;3:2477–86.

expressing glioblastomas. Stem cells (Dayton, Ohio) 2008;26(12):3018–26.

cells. Nature 2001;414(6859):105–11.

tion. Vascular cell 2011;3(1):18.

2009;5(2):168–77.

82 Pluripotent Stem Cells

2001;90(2-3):105–56.

64.

FEBS letters 2012;584(17):3819–25.

poiesis. Experimental hematology 1997;25(4):312–20.

the United States of America 2012;109(29):11717–22.

oncogenic KRAS mutations. Cancer research 2011;71(5):1520–5.


**Chapter 5**

**Conditions and Techniques for**

Additional information is available at the end of the chapter

Kun-Hsiung Lee

**1. Introduction**

http://dx.doi.org/10.5772/55105

cells, as they can only form sperm [22].

cheap is presented.

**Mouse Embryonic Stem Cell Derivation and Culture**

Stem cells, characterized by their ability for self-renewal and differentiation, have been derived from the embryo and from various postnatal animal sources. They are usually classified according to their developmental potential. Totipotency is defined as the ability of a single cell to replicate and produce all differentiated cells in an entire organism, including extraem‐ bryonic tissues that will develop and differentiate into the fetal placenta and fetal membranes [1,2]. In plants, spores are totipotent cells. In some cases, cells can de-differentiate and regain their totipotency. For instance, a plant cutting or callus can be utilized to grow an entire functional plant [3]. In mammals, only the zygote and early blastomeres are totipotent cells [4-7]. In other words, an individual cell is capable to generate a functionally normal animal with fertile ability [8-10]. Mouse embryonic stem (ES) cells, typically derived from inner cell masses (ICMs) or corresponding earlier blastomeres or later epiblasts (develop to embryo proper), are an example of pluripotent cells that can self-renew and generate all types of body cells *in vivo* and *in vitro*, but cannot generate the extraembryonic trophoblast lineage [11-14]. Under some particular conditions, an ES cell-derived mouse with germline transmission can be generated routinely [15-21]. Multipotent cells, such as hematopoietic stem cells, can give rise all cell types within a particular lineage. Spermatogonial stem cells are unipotent stem

In recent years, major improvements in deriving mouse ES cell (ESC) lines have dramatically increased success rates. Therefore, this chapter reviews and discusses the conditions and techniques for derivation and cultivation of mouse ES cell (mESC) lines. Thereafter, a proposed novel and user-friendly protocol that is efficient, reproducible, easy to carry out and relatively

> © 2013 Lee; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Lee; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **Conditions and Techniques for Mouse Embryonic Stem Cell Derivation and Culture**

Kun-Hsiung Lee

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55105

## **1. Introduction**

Stem cells, characterized by their ability for self-renewal and differentiation, have been derived from the embryo and from various postnatal animal sources. They are usually classified according to their developmental potential. Totipotency is defined as the ability of a single cell to replicate and produce all differentiated cells in an entire organism, including extraem‐ bryonic tissues that will develop and differentiate into the fetal placenta and fetal membranes [1,2]. In plants, spores are totipotent cells. In some cases, cells can de-differentiate and regain their totipotency. For instance, a plant cutting or callus can be utilized to grow an entire functional plant [3]. In mammals, only the zygote and early blastomeres are totipotent cells [4-7]. In other words, an individual cell is capable to generate a functionally normal animal with fertile ability [8-10]. Mouse embryonic stem (ES) cells, typically derived from inner cell masses (ICMs) or corresponding earlier blastomeres or later epiblasts (develop to embryo proper), are an example of pluripotent cells that can self-renew and generate all types of body cells *in vivo* and *in vitro*, but cannot generate the extraembryonic trophoblast lineage [11-14]. Under some particular conditions, an ES cell-derived mouse with germline transmission can be generated routinely [15-21]. Multipotent cells, such as hematopoietic stem cells, can give rise all cell types within a particular lineage. Spermatogonial stem cells are unipotent stem cells, as they can only form sperm [22].

In recent years, major improvements in deriving mouse ES cell (ESC) lines have dramatically increased success rates. Therefore, this chapter reviews and discusses the conditions and techniques for derivation and cultivation of mouse ES cell (mESC) lines. Thereafter, a proposed novel and user-friendly protocol that is efficient, reproducible, easy to carry out and relatively cheap is presented.

© 2013 Lee; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Lee; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. Conditions for derivation of mouse embryonic stem cells (mESCs)**

To date, the success rate in establishing mESCs via whole embryos is usually more than 50%, regardless of the mouse strains used [31,52-58]. Further progress in the derivation of mESCs from whole early-stage embryos seems limited. Conversely, the success rate in establishing mESCs via isolated single blastomeres is relatively low and highly variable [31,59]. Although the success rates in some reports are approximately 30% [59,60], it cannot be the routine yet

Conditions and Techniques for Mouse Embryonic Stem Cell Derivation and Culture

http://dx.doi.org/10.5772/55105

87

To date, FBS, with its excellent nutrient mixture, remains the most important and universal component for propagating cells. Additionally, FBS contains growth factors that support ESCs. However, FBS also contains potential differentiation factors for ESCs [61-64]. Therefore, testing and then selecting batches of FBS to support the growth of undifferentiated ESCs is necessary. Otherwise, qualified ESC-grade FBS, which is more costly than conventional FBS, can be used. To support mESCs, the biopotency of FBS (a biological product) varies from batch to batch. Additionally, its supply worldwide is sometimes limited and it is expensive. Furthermore, animal-originated materials risk introducing adventitious agents into a cell culture system. Therefore, to circumvent interference from differentiation factors and other disadvantages of

Although KSR is a commercial product, its formula remains unknown. With its well-defined chemical formula, N2B27, can replace KSR and achieve almost the same deriving and main‐ taining ESCs results [48,65]. Originally, N2B27 is an empirically mixed formulation that

Conventional ESC media usually contain 15–20% FBS or 10% FBS plus 10% newborn serum. For a chemically defined ESC medium, FBS can be replaced completely by KSR or N2B27; otherwise, 5–15% FBS can be replaced by SR. When changing FBS to a new batch, the ESCs sometimes have to adapt gradually to the new batch. For example, one can mix 50% old FBS with 50% new FBS and allow the ESCs to acclimatize to the new medium. Generally, ESCs can be changed easily from serum replacement (SR) to FBS ESC media by sequential adaptation with approximately three passages. Reversely, acclimatizing ES cells from the FBS to the SR ESC medium is sometimes difficult and can fail. When one is switching to a serum-free cell

Notably, FBS, a good buffer, is a complex solution that contains many chemicals and proteins with different molecular weights. Therefore, mESCs in FBS-free medium are more sensitive to extremes of pH, osmolarity, enzyme treatment, and mechanical forces. Furthermore, a 5- to 10-fold lower antibiotics concentration is used in an FBS-free medium because serum proteins typically bind a certain amount of the antibiotic; without these serum proteins, the antibiotic

After passing mESCs in a SR ESC medium, centrifugation is necessary to remove trypsin from the cell suspension to avoid further digestion of cells due to the lack of trypsin inhibitors in the medium. For convenience, enzymes that can stop digestion due to the decreased concen‐ tration will be a good alternative to trypsin. TrypLE™, a recombinant enzyme derived from

due to the variable results.

**2.1. Fetal bovine serum (FBS)** *vs.* **serum replacement (SR)**

FBS, chemically defined KSR was developed for use in place of FBS [28].

provides optimum cell viability and efficient neural differentiation [29,30].

culture, sequential adaptation for approximately 4–5 passages is required.

concentration may be toxic to mESCs.

Since the first mouse ES cell lines were described [23,24], various empirical combinations of conditions and techniques for derivation and cultivation of mESCs from blastocysts and isolated ICMs have been developed [25-27]. Of which, selected batches of fetal bovine serum (FBS), inactivated STO (a SIM mouse embryonic fibroblast line resistant to 6-thioguanine and ouabain) or murine embryonic fibroblast (mEF) feeder cells, conditioned media, mouse strains, embryo status, and different small growth areas of wells to initiate cultivation are the principal concerns when deriving mESCs [27].

To support fetal growth and development, FBS contains mixed combinations of cell replication stimulators and cell differentiation inducers. Notably, FBS is a biological product, such that its biopotency to support mESCs varies from batch to batch. Therefore, to circumvent interference from differentiation factors and other disadvantages associated with FBS, chemically defined KnockOut™ serum replacement (KSR) [28] and N2B27 [29,30] were developed to replace FBS. That is, when culturing established mESCs, KSR and N2B27 are usually as effective as FBS. Unfortunately, embryos in the KSR ESC medium do not result in effective derivation of ESCs [28,31]. However, a chemically defined ESC medium containing differentiation inhibitors has much better efficiency than the FBS ESC medium when deriving mESCs [31].

Zygotes to hatched embryos and blastomeres, ICMs, or epiblasts of early-stage embryos can be used to establish mESCs [31,32]. These cells have extremely high capability for cell division and differentiation. Theoretically, inhibiting endogenous differentiation and maintaining or enhancing proliferation of pre-implantation embryos can be helpful for the establishment of ES cell lines. In 1988, researchers have found that leukemia inhibitory factor (LIF) assists in the derivation and maintenance of mESCs pluripotency [33,34]. However, when protocols and media containing LIF for mESCs derivation are applied to mouse strains other than 129s, efficiency declines from about 20% to less than 5% [35-38]. Furthermore, ESC media supple‐ mented with LIF are not good for deriving ESCs other than mESCs [39,40]. Since then, regulatory mechanisms, pathways, and signal transduction of self-renewal, differentiation, proliferation, and apoptosis have been investigated [41,42]. Additionally, the corresponding synthesized inhibitors and/or stimulators/inducers/enhancers of stem cells [43-47] have been investigated intensively.

In the early 1990s, as the specificity of developed inhibitors was not sufficiently strong, their effectiveness in inhibiting differentiation and then helping to establish mESCs were not satisfactory and culturing results were inconsistent. More recent year, a breakthrough result was reported. Following the progresses, using the novel mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD0325901 or SU5402 to eliminate differentiation-inducing signaling from MAPK and using the glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021 to enhance ESC growth capacity and viability helped dramatically in establishing mESC lines [48]. Accordingly, ES cell lines of the second mammalian species (rat) with germline transmission have been reported [49,50]. The combi‐ nation of MEK inhibitor PD0325901 and GSK3 inhibitor CHIR99021 (2i) also appears to improve the generation efficiency of induced pluripotent stem cells (iPSCs) [51].

To date, the success rate in establishing mESCs via whole embryos is usually more than 50%, regardless of the mouse strains used [31,52-58]. Further progress in the derivation of mESCs from whole early-stage embryos seems limited. Conversely, the success rate in establishing mESCs via isolated single blastomeres is relatively low and highly variable [31,59]. Although the success rates in some reports are approximately 30% [59,60], it cannot be the routine yet due to the variable results.

#### **2.1. Fetal bovine serum (FBS)** *vs.* **serum replacement (SR)**

**2. Conditions for derivation of mouse embryonic stem cells (mESCs)**

concerns when deriving mESCs [27].

86 Pluripotent Stem Cells

investigated intensively.

Since the first mouse ES cell lines were described [23,24], various empirical combinations of conditions and techniques for derivation and cultivation of mESCs from blastocysts and isolated ICMs have been developed [25-27]. Of which, selected batches of fetal bovine serum (FBS), inactivated STO (a SIM mouse embryonic fibroblast line resistant to 6-thioguanine and ouabain) or murine embryonic fibroblast (mEF) feeder cells, conditioned media, mouse strains, embryo status, and different small growth areas of wells to initiate cultivation are the principal

To support fetal growth and development, FBS contains mixed combinations of cell replication stimulators and cell differentiation inducers. Notably, FBS is a biological product, such that its biopotency to support mESCs varies from batch to batch. Therefore, to circumvent interference from differentiation factors and other disadvantages associated with FBS, chemically defined KnockOut™ serum replacement (KSR) [28] and N2B27 [29,30] were developed to replace FBS. That is, when culturing established mESCs, KSR and N2B27 are usually as effective as FBS. Unfortunately, embryos in the KSR ESC medium do not result in effective derivation of ESCs [28,31]. However, a chemically defined ESC medium containing differentiation inhibitors has

Zygotes to hatched embryos and blastomeres, ICMs, or epiblasts of early-stage embryos can be used to establish mESCs [31,32]. These cells have extremely high capability for cell division and differentiation. Theoretically, inhibiting endogenous differentiation and maintaining or enhancing proliferation of pre-implantation embryos can be helpful for the establishment of ES cell lines. In 1988, researchers have found that leukemia inhibitory factor (LIF) assists in the derivation and maintenance of mESCs pluripotency [33,34]. However, when protocols and media containing LIF for mESCs derivation are applied to mouse strains other than 129s, efficiency declines from about 20% to less than 5% [35-38]. Furthermore, ESC media supple‐ mented with LIF are not good for deriving ESCs other than mESCs [39,40]. Since then, regulatory mechanisms, pathways, and signal transduction of self-renewal, differentiation, proliferation, and apoptosis have been investigated [41,42]. Additionally, the corresponding synthesized inhibitors and/or stimulators/inducers/enhancers of stem cells [43-47] have been

In the early 1990s, as the specificity of developed inhibitors was not sufficiently strong, their effectiveness in inhibiting differentiation and then helping to establish mESCs were not satisfactory and culturing results were inconsistent. More recent year, a breakthrough result was reported. Following the progresses, using the novel mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD0325901 or SU5402 to eliminate differentiation-inducing signaling from MAPK and using the glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021 to enhance ESC growth capacity and viability helped dramatically in establishing mESC lines [48]. Accordingly, ES cell lines of the second mammalian species (rat) with germline transmission have been reported [49,50]. The combi‐ nation of MEK inhibitor PD0325901 and GSK3 inhibitor CHIR99021 (2i) also appears to

improve the generation efficiency of induced pluripotent stem cells (iPSCs) [51].

much better efficiency than the FBS ESC medium when deriving mESCs [31].

To date, FBS, with its excellent nutrient mixture, remains the most important and universal component for propagating cells. Additionally, FBS contains growth factors that support ESCs. However, FBS also contains potential differentiation factors for ESCs [61-64]. Therefore, testing and then selecting batches of FBS to support the growth of undifferentiated ESCs is necessary. Otherwise, qualified ESC-grade FBS, which is more costly than conventional FBS, can be used.

To support mESCs, the biopotency of FBS (a biological product) varies from batch to batch. Additionally, its supply worldwide is sometimes limited and it is expensive. Furthermore, animal-originated materials risk introducing adventitious agents into a cell culture system. Therefore, to circumvent interference from differentiation factors and other disadvantages of FBS, chemically defined KSR was developed for use in place of FBS [28].

Although KSR is a commercial product, its formula remains unknown. With its well-defined chemical formula, N2B27, can replace KSR and achieve almost the same deriving and main‐ taining ESCs results [48,65]. Originally, N2B27 is an empirically mixed formulation that provides optimum cell viability and efficient neural differentiation [29,30].

Conventional ESC media usually contain 15–20% FBS or 10% FBS plus 10% newborn serum. For a chemically defined ESC medium, FBS can be replaced completely by KSR or N2B27; otherwise, 5–15% FBS can be replaced by SR. When changing FBS to a new batch, the ESCs sometimes have to adapt gradually to the new batch. For example, one can mix 50% old FBS with 50% new FBS and allow the ESCs to acclimatize to the new medium. Generally, ESCs can be changed easily from serum replacement (SR) to FBS ESC media by sequential adaptation with approximately three passages. Reversely, acclimatizing ES cells from the FBS to the SR ESC medium is sometimes difficult and can fail. When one is switching to a serum-free cell culture, sequential adaptation for approximately 4–5 passages is required.

Notably, FBS, a good buffer, is a complex solution that contains many chemicals and proteins with different molecular weights. Therefore, mESCs in FBS-free medium are more sensitive to extremes of pH, osmolarity, enzyme treatment, and mechanical forces. Furthermore, a 5- to 10-fold lower antibiotics concentration is used in an FBS-free medium because serum proteins typically bind a certain amount of the antibiotic; without these serum proteins, the antibiotic concentration may be toxic to mESCs.

After passing mESCs in a SR ESC medium, centrifugation is necessary to remove trypsin from the cell suspension to avoid further digestion of cells due to the lack of trypsin inhibitors in the medium. For convenience, enzymes that can stop digestion due to the decreased concen‐ tration will be a good alternative to trypsin. TrypLE™, a recombinant enzyme derived from microbial fermentation, can stay at room temperature (RT) before expiration. My laboratory routinely uses TrypLE™ Express to pass mESCs during derivation and maintenance with good outcomes [31].

primary cells harvested from fetuses approximately 12.5–13.5 days post coitum (dpc). How‐ ever, mEF can be passed and propagated only for a short period [83-85]. Therefore, they must be prepared continuously. Additionally, the traits of mEFs differ from batch to batch, and quality control of mEFs, especially for mycoplasma contamination, may be the problems [86,87]. Moreover, hFFs are more durable than mEFs in that they remain in healthy condition more than 2 weeks after inactivation by radiation. In contrast, mEF deteriorates within 1 week

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To derive and maintain mESCs, feeder cells and ESC medium supplemented with LIF (typical concentration is 1,000 unit/mL) are usually chosen. The reason is due to the LIF produced by mEFs and STO is not enough to maintain ESC properties most of the time. However, recombi‐ nant murine LIF is expensive. Therefore, STO that expresses a high level of LIF (SNL76/7, ATCC No. SCRC-1049; SNLP 76/7-4, ATCC No. SCRC-1050) has been developed [88]. This cell line also can be used as a feeder layer supporting the derivation and growth of mESCs and iPSCs [89]. The disadvantage of the SNL76/7 as a feeder cell is the highly variable level of LIF

Both teratocarcinoma stem cells and EC cells were established in the 1970s [66,94-96]. Both pluripotent cells usually undergo extensive differentiation *in vivo* and *in vitro* to generate a wide variety of cell types [97]. Mouse teratocarcinoma stem cells can condition themselves. Therefore, medium conditioned with teratocarcinoma stem cells, which is equivalent to a 5 fold concentration of LIF [92], was used to help establish mESCs [24]. Thereafter, the propa‐

Feeder cells secrete many different factors, including growth factors, to support ESCs [76,77,79-81]. The recovered conditioned medium is a complex solution containing many unidentified chemicals. However, as conditioned media are exhausted media prepared in a batch-by-batch manner, their biopotency might vary. The question is whether conditioned medium is still needed to derive ES cells when chemically defined ESC media supplemented

A few different conditioned media have been used to establish mESCs. Of which, an FBS ESC medium conditioned by a rabbit fibroblast cell line transduced with genomic rabbit LIF (10 ng/mL) allows efficient derivation and maintenance of mESC lines from all 10 inbred mouse strains tested, including some that were presumed nonpermissive for mESC derivation [56]. This commercialized conditioned medium, RESGROTM Culture Medium (Millipore), can establish and rescue established mESCs that have started drifting, and either generate low-

Teratomas (benign) and teratocarcinomas (malignant) are tumors composed of an ectoderm, endoderm, and mesoderm mixtures of adult tissues [99]. Most are found in gonads and rare in mammals, including experimental animals. In the 1950s, mouse strain 129 had found an

in culturing medium, that might have different effects on mESCs [24,56,57,90-93].

gation of mESCs in high densities reduces possible differentiation [26,98].

percentage chimeras or lose their germline transmission capability [53,92].

with differentiation inhibitors and growth factors are available.

**2.4. Mouse strain and embryo status**

after the inactivation [76].

**2.3. Conditioned media**

#### **2.2. Feeder cells**

The ESCs are extremely sensitive to culture conditions, including properties of culture media and dishes, when maintaining pluripotency without differentiation. Unfortunately, commer‐ cially available plates and dishes are not adequate for ESCs.

In the 1970s, pluripotent teratocarcinoma (stem) cell lines were established after introduction of a cell feeder layer [66-68]. Feeder (helper) cells have since been used to help study terato‐ carcinoma stem cells and embryonal carcinoma (EC) cells. The STO feeder cells then demon‐ strated to help derive the first mESCs [23] and maintain mESCs at the undifferentiated state.

Feeder cells are usually inactivated via mitomycin C or γ-irradiation treatment. While γirradiation leads to breaks in DNA strands, mitomycin C has the extraordinary ability to crosslink DNA with high efficiency and is specific for the CpG sequence [69,70]. Although feeder cells are alive, they do not replicate but gene transcription and protein synthesis are not affected.

It has been speculated that feeder cells support embryos and ESCs attachment through the physical matrix [71]. Furthermore, feeder cells may release embryo trophic factors, reduce inhibitory or toxic factors in FBS, or may be beneficial by lowering concentrations of ions and/ or glucose in medium, and thereby overcoming the developmental blockage of embryos mediated by the release of growth factors essential for activation of the embryonic genome and for normal embryonic development. Additionally, feeder cells may protect embryos from oxygen toxicity [63].

Various feeder cells, which differ in their ability to support ESCs, have been utilized to establish, propagate, and maintain the pluripotency of ESCs [35,72]. Conventionally, STO and mEF are the most popular feeder cells for deriving and maintaining ESCs. However, human foreskin fibroblast (hFF) feeder cells also support propagation and self-renewal of human [73-76] and mouse ESCs [31,77].

As feeder cells at earlier passages are used, their ability to support ESCs is increased [78]. Reports have demonstrated that mEF, STO, and hFF feeder cells secrete different growth factors to support ESCs [76,77,79-81].

To help establish mESCs, the STO, mEF, and hFF feeder cells might have roughly equal efficiency [31,82]. Recently, a study reported that hFF supported mESC self-renewal superiorly to mEFs due to the convenience. Using the hFF system, multiple lines of mESCs have been successfully derived without addition of exogenous LIF and any inhibitors. These mESCs have capacities to self-renew for a long period of time and to differentiate into various cell types of the three germ layers both *in vitro* and *in vivo* [76]. The STO is a cell line (ATCC No. CRL-1503) for unlimited propagation. The hFF may be a cell line (CCD-1112Sk, ATCC No. CRL-2429; Hs68, ATCC No. CRL-1635; HFF-1, ATCC No. SCRC-1041) or primary cells. The mEF is primary cells harvested from fetuses approximately 12.5–13.5 days post coitum (dpc). How‐ ever, mEF can be passed and propagated only for a short period [83-85]. Therefore, they must be prepared continuously. Additionally, the traits of mEFs differ from batch to batch, and quality control of mEFs, especially for mycoplasma contamination, may be the problems [86,87]. Moreover, hFFs are more durable than mEFs in that they remain in healthy condition more than 2 weeks after inactivation by radiation. In contrast, mEF deteriorates within 1 week after the inactivation [76].

To derive and maintain mESCs, feeder cells and ESC medium supplemented with LIF (typical concentration is 1,000 unit/mL) are usually chosen. The reason is due to the LIF produced by mEFs and STO is not enough to maintain ESC properties most of the time. However, recombi‐ nant murine LIF is expensive. Therefore, STO that expresses a high level of LIF (SNL76/7, ATCC No. SCRC-1049; SNLP 76/7-4, ATCC No. SCRC-1050) has been developed [88]. This cell line also can be used as a feeder layer supporting the derivation and growth of mESCs and iPSCs [89]. The disadvantage of the SNL76/7 as a feeder cell is the highly variable level of LIF in culturing medium, that might have different effects on mESCs [24,56,57,90-93].

#### **2.3. Conditioned media**

microbial fermentation, can stay at room temperature (RT) before expiration. My laboratory routinely uses TrypLE™ Express to pass mESCs during derivation and maintenance with good

The ESCs are extremely sensitive to culture conditions, including properties of culture media and dishes, when maintaining pluripotency without differentiation. Unfortunately, commer‐

In the 1970s, pluripotent teratocarcinoma (stem) cell lines were established after introduction of a cell feeder layer [66-68]. Feeder (helper) cells have since been used to help study terato‐ carcinoma stem cells and embryonal carcinoma (EC) cells. The STO feeder cells then demon‐ strated to help derive the first mESCs [23] and maintain mESCs at the undifferentiated state.

Feeder cells are usually inactivated via mitomycin C or γ-irradiation treatment. While γirradiation leads to breaks in DNA strands, mitomycin C has the extraordinary ability to crosslink DNA with high efficiency and is specific for the CpG sequence [69,70]. Although feeder cells are alive, they do not replicate but gene transcription and protein synthesis are not

It has been speculated that feeder cells support embryos and ESCs attachment through the physical matrix [71]. Furthermore, feeder cells may release embryo trophic factors, reduce inhibitory or toxic factors in FBS, or may be beneficial by lowering concentrations of ions and/ or glucose in medium, and thereby overcoming the developmental blockage of embryos mediated by the release of growth factors essential for activation of the embryonic genome and for normal embryonic development. Additionally, feeder cells may protect embryos from

Various feeder cells, which differ in their ability to support ESCs, have been utilized to establish, propagate, and maintain the pluripotency of ESCs [35,72]. Conventionally, STO and mEF are the most popular feeder cells for deriving and maintaining ESCs. However, human foreskin fibroblast (hFF) feeder cells also support propagation and self-renewal of human

As feeder cells at earlier passages are used, their ability to support ESCs is increased [78]. Reports have demonstrated that mEF, STO, and hFF feeder cells secrete different growth

To help establish mESCs, the STO, mEF, and hFF feeder cells might have roughly equal efficiency [31,82]. Recently, a study reported that hFF supported mESC self-renewal superiorly to mEFs due to the convenience. Using the hFF system, multiple lines of mESCs have been successfully derived without addition of exogenous LIF and any inhibitors. These mESCs have capacities to self-renew for a long period of time and to differentiate into various cell types of the three germ layers both *in vitro* and *in vivo* [76]. The STO is a cell line (ATCC No. CRL-1503) for unlimited propagation. The hFF may be a cell line (CCD-1112Sk, ATCC No. CRL-2429; Hs68, ATCC No. CRL-1635; HFF-1, ATCC No. SCRC-1041) or primary cells. The mEF is

cially available plates and dishes are not adequate for ESCs.

outcomes [31].

88 Pluripotent Stem Cells

**2.2. Feeder cells**

affected.

oxygen toxicity [63].

[73-76] and mouse ESCs [31,77].

factors to support ESCs [76,77,79-81].

Both teratocarcinoma stem cells and EC cells were established in the 1970s [66,94-96]. Both pluripotent cells usually undergo extensive differentiation *in vivo* and *in vitro* to generate a wide variety of cell types [97]. Mouse teratocarcinoma stem cells can condition themselves. Therefore, medium conditioned with teratocarcinoma stem cells, which is equivalent to a 5 fold concentration of LIF [92], was used to help establish mESCs [24]. Thereafter, the propa‐ gation of mESCs in high densities reduces possible differentiation [26,98].

Feeder cells secrete many different factors, including growth factors, to support ESCs [76,77,79-81]. The recovered conditioned medium is a complex solution containing many unidentified chemicals. However, as conditioned media are exhausted media prepared in a batch-by-batch manner, their biopotency might vary. The question is whether conditioned medium is still needed to derive ES cells when chemically defined ESC media supplemented with differentiation inhibitors and growth factors are available.

A few different conditioned media have been used to establish mESCs. Of which, an FBS ESC medium conditioned by a rabbit fibroblast cell line transduced with genomic rabbit LIF (10 ng/mL) allows efficient derivation and maintenance of mESC lines from all 10 inbred mouse strains tested, including some that were presumed nonpermissive for mESC derivation [56]. This commercialized conditioned medium, RESGROTM Culture Medium (Millipore), can establish and rescue established mESCs that have started drifting, and either generate lowpercentage chimeras or lose their germline transmission capability [53,92].

#### **2.4. Mouse strain and embryo status**

Teratomas (benign) and teratocarcinomas (malignant) are tumors composed of an ectoderm, endoderm, and mesoderm mixtures of adult tissues [99]. Most are found in gonads and rare in mammals, including experimental animals. In the 1950s, mouse strain 129 had found an incidence of spontaneous testicular teratoma of about 1% [100]. After progress and refinement of cell culture techniques, most notably the introduction of the cell feeder layer, allowed the reliable cultures of pluripotent teratocarcinoma (stem) cell lines [66-68].

Typically, diploid male mESCs capable of a high percentage generation of chimeras with germline transmission are selected for further utilization. However, some unusual mESCs have been reported including the androgenetic [111], germline transmitted female [112], adult somatic cell nuclear transferred [113], XO [114], parthenogenetic [115], haploid [54], and

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To date, most available ES cell lines were derived from the outgrowth of ICMs of blastocysts. However, due to ethical concerns over the derivation of human ES (hES) cells for regenerative medicine, a single blastomere (usually from 8-cell embryos) has been utilized to derive ESCs. Unfortunately, conventional methods used to establish mESCs directly from an isolated single blastomere, which is extremely sensitive to culture conditions, are unsuccessful. Actually, most of the isolated blastomeres divide to form small sheets of cells with a trophoblastic-like morphology or small blastocysts with or without visible ICM-like cells [6,31,117]. In 1996, mESlike cells were first reported from single blastomeres [118]. Since then, no ESCs were estab‐

The mESCs can condition themselves to inhibit or prevent differentiation. Therefore, blasto‐ meres aggregated with established mESCs for initial co-culturing to help in the derivation of mESCs is a logically reasonable alternative. In this manner, the single blastomere-derived mESCs have been established but overall success rates are less than 5% when using the conventional FBS ESC medium [117-119]. In spite of that, a possibility may exist for deriving

On 2004, the study showed that the KSR ESC medium do not support mES single cell culturing. Contrary, single mESCs were propagated without loss of pluripotency when the adrenocor‐ ticotropic hormone (ACTH) was added to KSR ESC medium [62]. The authors of that study hypothesized that ACTH may be integrated via a weak cross interaction with an unknown, non-physiological inhibitory G protein coupled receptor. A signaling system other than the cAMP-PKA pathway or PKA pathway may play an important role in propagation of mESCs [62]. Later, simple and efficient establishment of mES cell lines from a single blastomere of 2 to 8-cell embryos with KSR ESC medium containing ACTH fragments 1–24 (ACTH 1–24) on

Wakayama *et al.* (2007) developed a novel protocol and established mESCs via blastomeres and polar bodies. In their experiment, isolated B6D2F2 blastomeres were cultured on KSR (20%) ESC medium containing ACTH 1–24 in 96-well plates coated with mEFs. The mESC establishment rates were 33%, 8%, and 8% for blastomeres derived from 2-cell (1/2), late 4-cell (1/4), and 8-cell embryos (1/8), respectively. However, they did not aggregate blastomeres with other mESCs, suggesting that success was likely attributable to the KSR ESC medium con‐ taining ACTH 1–24 [59]. Other studies then demonstrated that mESCs can be established from 1/2, 1/4, 2/4, 1/8, 2/8, 3/8, and 4/8 blastomeres in medium containing ACTH 1–24 [31,120-122]. For blastomeres, as the developmental stage of embryos decreases and the number of isolated blastomeres used increases, the derivation efficiency of mESCs increases. Moreover, whole embryos always have better derivation efficiency than corresponding blastomeres. Further‐

androgenetic haploid [116] mESCs.

lished from blastomeres until 2006 [117].

mEF feeders was reported [59].

personalized hES cells without destruction of 8-cell embryos.

**2.5. Single blastomere**

The mESCs were first derived from 129SvE [23] and then (ICR × SWR/J) F1 hybrid embryos [24]. However, due to the demand for animal models of human diseases, strains other than 129s have been used to establish new mESC lines. Unfortunately, the following mESCs were derived mainly from 129 strains due to the permissive nature of the genetic background [27,32,35,38]. The derivation of mESCs from blastocysts is a process that is often very ineffi‐ cient, and even in the most favorable 129 strains, a success rate of 30% is regarded as high [26]. Derivation of mESCs is strongly mouse strain–dependent [32], and in practice the efficiency of derivation in strains other than 129 strains does not usually exceed 10% [36].

Strains other than 129s, such as FVB, CBA/Ca, and the non-obese diabetic (NOD) mouse [101], have the extremely low derivation rates of mESCs using conventional conditions. They are traditionally regarded as highly refractory (nonpermissive) for derivation of mESCs. More‐ over, they also produce chimeras either incapable of germline transmission [102] or restricted in their germline competence [37].

Mouse strain C57BL/6 (B6) is not usually considered as a permissive strain for mESC deriva‐ tion. Although the B6 mES cell lines have been available since the early 1990s [35,103], the efficiency in establishing B6 mESCs via FBS or KSR ESC medium containing LIF varies and is typically less than 10% [32,38,56,61,82,103-109]. When establishing C57BL/6J mESCs using FBS (15%) ESC medium containing LIF, the highest success rate was 40% (36/89) [56]. Notably, this study used mEF feeder cells coated on 96-well cell plates for initiation of blastocysts and thereafter digested ICM outgrowths cultured at 39°C under 5% CO2 in an incubator. In establishing B6 mESCs using KSR (20%) ESC medium containing LIF, the derivation rate of (C57BL/6N × C57BL/6J) F1 mES-like colony can be as high as 40% (10 of 25 blastocysts) [109]. Other studies also demonstrated that the efficiency in establishing mESCs is significantly higher for the C57BL/6N strain (53%) than for the C57BL/6J strain (20%) [109]. Recently, my study showed that C57BL/6J blastocysts in KSR ESC medium did not lead to effective deriva‐ tion of mESCs; however, the success rate in 2i medium was as high as 75%. Furthermore, the efficacy of 2i medium was also demonstrated when using morulae (60%) and 8-cell embryos (50%) for mESCs derivation [31].

Although previous results suggested that true ES cells can be derived from embryos explanted at any stage of preimplantation development in the mouse [110]. My laboratory was the first to report success in deriving mESCs from mouse zygotes. These zygote-derived mESCs are morphologically indistinguishable from mESCs derived from fertilized embryos and blasto‐ meres. Moreover, the generation of germline transmitted chimeras confirmed that the estab‐ lished mES-like cells are pluripotent mESCs [31].

In recent years, the mESC lines of nonpermissive strains have been established routinely from 3.5 dpc blastocysts when SR ESC medium containing differentiation inhibitors and/or prolif‐ eration enhancers was used.

Typically, diploid male mESCs capable of a high percentage generation of chimeras with germline transmission are selected for further utilization. However, some unusual mESCs have been reported including the androgenetic [111], germline transmitted female [112], adult somatic cell nuclear transferred [113], XO [114], parthenogenetic [115], haploid [54], and androgenetic haploid [116] mESCs.

#### **2.5. Single blastomere**

incidence of spontaneous testicular teratoma of about 1% [100]. After progress and refinement of cell culture techniques, most notably the introduction of the cell feeder layer, allowed the

The mESCs were first derived from 129SvE [23] and then (ICR × SWR/J) F1 hybrid embryos [24]. However, due to the demand for animal models of human diseases, strains other than 129s have been used to establish new mESC lines. Unfortunately, the following mESCs were derived mainly from 129 strains due to the permissive nature of the genetic background [27,32,35,38]. The derivation of mESCs from blastocysts is a process that is often very ineffi‐ cient, and even in the most favorable 129 strains, a success rate of 30% is regarded as high [26]. Derivation of mESCs is strongly mouse strain–dependent [32], and in practice the efficiency

Strains other than 129s, such as FVB, CBA/Ca, and the non-obese diabetic (NOD) mouse [101], have the extremely low derivation rates of mESCs using conventional conditions. They are traditionally regarded as highly refractory (nonpermissive) for derivation of mESCs. More‐ over, they also produce chimeras either incapable of germline transmission [102] or restricted

Mouse strain C57BL/6 (B6) is not usually considered as a permissive strain for mESC deriva‐ tion. Although the B6 mES cell lines have been available since the early 1990s [35,103], the efficiency in establishing B6 mESCs via FBS or KSR ESC medium containing LIF varies and is typically less than 10% [32,38,56,61,82,103-109]. When establishing C57BL/6J mESCs using FBS (15%) ESC medium containing LIF, the highest success rate was 40% (36/89) [56]. Notably, this study used mEF feeder cells coated on 96-well cell plates for initiation of blastocysts and thereafter digested ICM outgrowths cultured at 39°C under 5% CO2 in an incubator. In establishing B6 mESCs using KSR (20%) ESC medium containing LIF, the derivation rate of (C57BL/6N × C57BL/6J) F1 mES-like colony can be as high as 40% (10 of 25 blastocysts) [109]. Other studies also demonstrated that the efficiency in establishing mESCs is significantly higher for the C57BL/6N strain (53%) than for the C57BL/6J strain (20%) [109]. Recently, my study showed that C57BL/6J blastocysts in KSR ESC medium did not lead to effective deriva‐ tion of mESCs; however, the success rate in 2i medium was as high as 75%. Furthermore, the efficacy of 2i medium was also demonstrated when using morulae (60%) and 8-cell embryos

Although previous results suggested that true ES cells can be derived from embryos explanted at any stage of preimplantation development in the mouse [110]. My laboratory was the first to report success in deriving mESCs from mouse zygotes. These zygote-derived mESCs are morphologically indistinguishable from mESCs derived from fertilized embryos and blasto‐ meres. Moreover, the generation of germline transmitted chimeras confirmed that the estab‐

In recent years, the mESC lines of nonpermissive strains have been established routinely from 3.5 dpc blastocysts when SR ESC medium containing differentiation inhibitors and/or prolif‐

reliable cultures of pluripotent teratocarcinoma (stem) cell lines [66-68].

of derivation in strains other than 129 strains does not usually exceed 10% [36].

in their germline competence [37].

90 Pluripotent Stem Cells

(50%) for mESCs derivation [31].

eration enhancers was used.

lished mES-like cells are pluripotent mESCs [31].

To date, most available ES cell lines were derived from the outgrowth of ICMs of blastocysts. However, due to ethical concerns over the derivation of human ES (hES) cells for regenerative medicine, a single blastomere (usually from 8-cell embryos) has been utilized to derive ESCs. Unfortunately, conventional methods used to establish mESCs directly from an isolated single blastomere, which is extremely sensitive to culture conditions, are unsuccessful. Actually, most of the isolated blastomeres divide to form small sheets of cells with a trophoblastic-like morphology or small blastocysts with or without visible ICM-like cells [6,31,117]. In 1996, mESlike cells were first reported from single blastomeres [118]. Since then, no ESCs were estab‐ lished from blastomeres until 2006 [117].

The mESCs can condition themselves to inhibit or prevent differentiation. Therefore, blasto‐ meres aggregated with established mESCs for initial co-culturing to help in the derivation of mESCs is a logically reasonable alternative. In this manner, the single blastomere-derived mESCs have been established but overall success rates are less than 5% when using the conventional FBS ESC medium [117-119]. In spite of that, a possibility may exist for deriving personalized hES cells without destruction of 8-cell embryos.

On 2004, the study showed that the KSR ESC medium do not support mES single cell culturing. Contrary, single mESCs were propagated without loss of pluripotency when the adrenocor‐ ticotropic hormone (ACTH) was added to KSR ESC medium [62]. The authors of that study hypothesized that ACTH may be integrated via a weak cross interaction with an unknown, non-physiological inhibitory G protein coupled receptor. A signaling system other than the cAMP-PKA pathway or PKA pathway may play an important role in propagation of mESCs [62]. Later, simple and efficient establishment of mES cell lines from a single blastomere of 2 to 8-cell embryos with KSR ESC medium containing ACTH fragments 1–24 (ACTH 1–24) on mEF feeders was reported [59].

Wakayama *et al.* (2007) developed a novel protocol and established mESCs via blastomeres and polar bodies. In their experiment, isolated B6D2F2 blastomeres were cultured on KSR (20%) ESC medium containing ACTH 1–24 in 96-well plates coated with mEFs. The mESC establishment rates were 33%, 8%, and 8% for blastomeres derived from 2-cell (1/2), late 4-cell (1/4), and 8-cell embryos (1/8), respectively. However, they did not aggregate blastomeres with other mESCs, suggesting that success was likely attributable to the KSR ESC medium con‐ taining ACTH 1–24 [59]. Other studies then demonstrated that mESCs can be established from 1/2, 1/4, 2/4, 1/8, 2/8, 3/8, and 4/8 blastomeres in medium containing ACTH 1–24 [31,120-122]. For blastomeres, as the developmental stage of embryos decreases and the number of isolated blastomeres used increases, the derivation efficiency of mESCs increases. Moreover, whole embryos always have better derivation efficiency than corresponding blastomeres. Further‐ more, KSR ESC medium containing differentiation inhibitors and/or proliferation stimulators is better than FBS ESC medium in establishing mESCs.

LIF receptor and the transmembrane signaling molecule gp130, resulting in activation of transcription factor STAT3, which is essential and sufficient to promote self-renewal and inhibit mesoderm and endoderm differentiation of mESCs [125,130-132]. Additionally, Wnt signaling inhibits GSK3β and results in the protein stabilization of cytoplasmic β-catenin (β-Ctnn). The GSK3, a constitutively acting multi-functional serine threonine kinase, derives its name from its substrate glycogen synthase, a key enzyme involved in conversion of glucose to glycogen. Although GSK3 is kept inactive by phosphorylation, activated GSK3 enhances synthesis of glycogen and inhibits cell proliferation. The name GSK does not adequately describe the multitude of diverse substrates and functions attributed to GSK3. For instance, it is involved in various cellular processes, ranging from glycogen metabolism, insulin signaling, cell proliferation, neuronal function, and oncogenesis to embryonic development [133]. Additionally, GSK3 is one of the crucial molecules involved in regulation of the Wnt/β-catenin, Hedgehog, and Notch signaling pathways. The undifferentiated pluripotency of both mouse and human ESCs can be maintained by GSK3-specific inhibitor 6-bromoindirubin-3'-oxime (BIO), which prevents phosphorylation of β-Ctnn by GSK3β and activates Wnt [134,135]. The Wnt signaling is endogenously activated in mESCs and is down regulated upon differentiation [135]. The target genes of the Wnt signaling pathway, such as c-Myc and CyclinD1, promote cell proliferation and self-renewal. The LIF/STAT3 pathway combines with the Wnt/GSK3β/βcatenin pathway to enhance self-renewal by activation of pluripotency genes, including transcription factors Nanog, Oct3/4, and Klf4. Nevertheless, LIF also activates the MAPK/ERK

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**Figure 1.** The use of signalling pathway inhibitors and chromatin modifiers for enhancing pluripotency. (Reproduced

pathway, which induces mESC differentiation [45].

with permission from Sumer *et al*., 2010. Theriogenology 74:525–533.)

For ethical concerns, such as maintaining the developmental potential of embryos and establishing corresponding hES cells, a 1/8 blastomere is one of the best candidates. Unfortu‐ nately, derivation efficiency of mESCs via a 1/8 blastomere is usually approximately 5–10%, such that it cannot be a standard protocol for the routine operation. Obviously, reliable and efficient protocols for ESCs derivation should be developed.

Previous studies have indicated poor derivation efficiency of mESCs from a 1/8 blastomere, partly due to the low division rate of single blastomeres when compared to that of their counterparts with a higher number of blastomeres (2/8, 3/8, and 4/8). Communication and adhesion between blastomeres, from which the derivation process begins, are likely important aspects to efficiently deriving mESC lines. Therefore, an approach consisting of chimeric Ecadherin (E-cad-Fc) adhesion to the blastomere surface has been devised to recreate the signaling produced by native E-cadherin between neighboring blastomeres inside an embryo. Via this approach, the 1/8 blastomere incubated with E-cad-Fc for only 24 h can significantly improve the mESC derivation efficiency from 2.2% to 33.6% [60]. To date, this novel method via 1/8 blastomere has the best derivation efficiency for mESCs. However, its reproducibility must be confirmed by other laboratories.

#### **2.6. Pluripotent signaling pathways**

In 1988, two studies demonstrated that LIF could assist in derivation and maintenance of the pluripotency of mESCs [33,34]. Unfortunately, following studies revealed that LIF is not as effective or good for mammals other than mice in establishing ESCs. These experimental results implied that different mammals might have different regulatory mechanisms for ESCs. Actually, distinct signaling pathways have been shown to regulate the pluripotency of mouse and human ESCs [41,42,123].

The regulatory mechanisms and signal transductions of self-renewal, differentiation, prolif‐ eration, and apoptosis [41,42,124], as well as the corresponding inhibitors of stem cells [43-47, 125] have been investigated (Figure 1).

To date, self-renewal, pluripotency, and the propagation signaling transduction pathways of ESCs includes the LIF/signal transducer and activation of transcription 3 (STAT3), Wnt/βcatenin, phosphatidylinositol 3-kinase (PI3K), bone morphogenic protein 4 (BMP4)/Smad1/5, and basic fibroblast growth factor (bFGF) [42,45]. Conversely, the Ras/Raf/MEK/ERK pathway is central to the signaling networks that govern proliferation, differentiation, and cell survival [126]. The active Ras/Raf/MEK/ERK pathways induce differentiation of mESCs [42,124,126]. The mESCs have high ERK activity when they undergo differentiation. Suppression of the ERK pathway promotes self-renewal of mESCs. Moreover, BMP4 activation inhibits differentiation of mESCs in medium containing LIF due to inhibitor of differentiation (Id) genes expression and ERK inactivation [127-129].

Of the many pathways, self-renewal of mESCs largely depends on LIF/interleukin 6 (IL-6) family members [130] and BMP4 [65,127]. LIF binds to a cell surface complex composed of the LIF receptor and the transmembrane signaling molecule gp130, resulting in activation of transcription factor STAT3, which is essential and sufficient to promote self-renewal and inhibit mesoderm and endoderm differentiation of mESCs [125,130-132]. Additionally, Wnt signaling inhibits GSK3β and results in the protein stabilization of cytoplasmic β-catenin (β-Ctnn). The GSK3, a constitutively acting multi-functional serine threonine kinase, derives its name from its substrate glycogen synthase, a key enzyme involved in conversion of glucose to glycogen. Although GSK3 is kept inactive by phosphorylation, activated GSK3 enhances synthesis of glycogen and inhibits cell proliferation. The name GSK does not adequately describe the multitude of diverse substrates and functions attributed to GSK3. For instance, it is involved in various cellular processes, ranging from glycogen metabolism, insulin signaling, cell proliferation, neuronal function, and oncogenesis to embryonic development [133]. Additionally, GSK3 is one of the crucial molecules involved in regulation of the Wnt/β-catenin, Hedgehog, and Notch signaling pathways. The undifferentiated pluripotency of both mouse and human ESCs can be maintained by GSK3-specific inhibitor 6-bromoindirubin-3'-oxime (BIO), which prevents phosphorylation of β-Ctnn by GSK3β and activates Wnt [134,135]. The Wnt signaling is endogenously activated in mESCs and is down regulated upon differentiation [135]. The target genes of the Wnt signaling pathway, such as c-Myc and CyclinD1, promote cell proliferation and self-renewal. The LIF/STAT3 pathway combines with the Wnt/GSK3β/βcatenin pathway to enhance self-renewal by activation of pluripotency genes, including transcription factors Nanog, Oct3/4, and Klf4. Nevertheless, LIF also activates the MAPK/ERK pathway, which induces mESC differentiation [45].

more, KSR ESC medium containing differentiation inhibitors and/or proliferation stimulators

For ethical concerns, such as maintaining the developmental potential of embryos and establishing corresponding hES cells, a 1/8 blastomere is one of the best candidates. Unfortu‐ nately, derivation efficiency of mESCs via a 1/8 blastomere is usually approximately 5–10%, such that it cannot be a standard protocol for the routine operation. Obviously, reliable and

Previous studies have indicated poor derivation efficiency of mESCs from a 1/8 blastomere, partly due to the low division rate of single blastomeres when compared to that of their counterparts with a higher number of blastomeres (2/8, 3/8, and 4/8). Communication and adhesion between blastomeres, from which the derivation process begins, are likely important aspects to efficiently deriving mESC lines. Therefore, an approach consisting of chimeric Ecadherin (E-cad-Fc) adhesion to the blastomere surface has been devised to recreate the signaling produced by native E-cadherin between neighboring blastomeres inside an embryo. Via this approach, the 1/8 blastomere incubated with E-cad-Fc for only 24 h can significantly improve the mESC derivation efficiency from 2.2% to 33.6% [60]. To date, this novel method via 1/8 blastomere has the best derivation efficiency for mESCs. However, its reproducibility

In 1988, two studies demonstrated that LIF could assist in derivation and maintenance of the pluripotency of mESCs [33,34]. Unfortunately, following studies revealed that LIF is not as effective or good for mammals other than mice in establishing ESCs. These experimental results implied that different mammals might have different regulatory mechanisms for ESCs. Actually, distinct signaling pathways have been shown to regulate the pluripotency of mouse

The regulatory mechanisms and signal transductions of self-renewal, differentiation, prolif‐ eration, and apoptosis [41,42,124], as well as the corresponding inhibitors of stem cells [43-47,

To date, self-renewal, pluripotency, and the propagation signaling transduction pathways of ESCs includes the LIF/signal transducer and activation of transcription 3 (STAT3), Wnt/βcatenin, phosphatidylinositol 3-kinase (PI3K), bone morphogenic protein 4 (BMP4)/Smad1/5, and basic fibroblast growth factor (bFGF) [42,45]. Conversely, the Ras/Raf/MEK/ERK pathway is central to the signaling networks that govern proliferation, differentiation, and cell survival [126]. The active Ras/Raf/MEK/ERK pathways induce differentiation of mESCs [42,124,126]. The mESCs have high ERK activity when they undergo differentiation. Suppression of the ERK pathway promotes self-renewal of mESCs. Moreover, BMP4 activation inhibits differentiation of mESCs in medium containing LIF due to inhibitor of differentiation (Id) genes expression

Of the many pathways, self-renewal of mESCs largely depends on LIF/interleukin 6 (IL-6) family members [130] and BMP4 [65,127]. LIF binds to a cell surface complex composed of the

is better than FBS ESC medium in establishing mESCs.

92 Pluripotent Stem Cells

efficient protocols for ESCs derivation should be developed.

must be confirmed by other laboratories.

**2.6. Pluripotent signaling pathways**

and human ESCs [41,42,123].

125] have been investigated (Figure 1).

and ERK inactivation [127-129].

**Figure 1.** The use of signalling pathway inhibitors and chromatin modifiers for enhancing pluripotency. (Reproduced with permission from Sumer *et al*., 2010. Theriogenology 74:525–533.)

As a protein, transforming growth factor beta (TGFβ) controls proliferation, cellular differen‐ tiation, and other functions in most cells. This protein is a secreted protein of cytokines. The TGFβ family is part of the large TGFβ superfamily, which has more than 40 members, including TGFβ, activin, nodal, BMPs, inhibins, anti-müllerian hormone, decapentaplegic, and Vg-1. All of these ligands are associated with ESCs. Although BMP4 is a member of the BMP family, it functions via a different signaling pathway with TGFβ. Notably, BMP4 induces expression of Id genes and inhibits MAPK signaling as well as neuroectoderm differentiation [65,127]. Although activation of STAT3 is sufficient for self-renewal of mESCs, a study showed that LIF-STAT3 do not maintain mESCs in serum-free ESC medium. The combination of LIF and BMP4 maintained the self-renewal of mESCs in the absence of both feeder cells and FBS [65,127]. Thus, BMP4 and LIF have synergistic effects on the self-renewal of mESCs [127]. In contrast to mESCs, BMP4 does not maintain the self-renewal of hES cells; rather, it induces trophoblast or primitive endoderm differentiation of hES cells [136].

activated protein kinase phosphatase 3 (MKP-3) [41] enhanced the self-renewal of mESCs and

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It was the first report showed that the combination of PD98059 (MEK 1 inhibitor) with LIF enhances the establishment of mESCs from the refractory CBA strain [148]. A combination of the MEK1/2 inhibitor, U0126, with LIF further promoted the efficiency of mESC derivation from CBA [129]. These small molecules, PD98059 and U0126, play positive roles in the selfrenewal of mESCs, but they are incapable of maintaining the pluripotency of mESCs in long-

Notably, BMP4 inhibits both ERK and p38 MAPK pathways in mESCs. The inhibitors of the ERK and p38 MAPKs mimic the effect of BMP4 on mESCs. Inhibition of the p38 and MAPKs by SB203580 overcomes the roadblock in deriving mESCs from blastocysts lacking a functional

The self-renewal of mESCs is generally dependent on multifactorial stimulation. To support the growth and development of fetuses, FBS contains cell replication stimulators and cell differentiation inducers. Serum and serum substitutes contain various inductive stimuli that may activate commitment and differentiation programs [49]. However, simple withdrawal of serum or other exogenous stimuli cannot prevent differentiation of ESCs due to endogenous autoinductive differentiation of fibroblast growth factor 4 (FGF4) via the MEK/ERK pathway [48,149]. Therefore, to suppress endogenous autoinductive differentiation and to maintain high viability and growth rates, one must inhibit the MEK/ERK pathway and/or provide LIF, or restrict the activity of GSK3, which acts mainly via the Wnt/β-catenin signaling pathway to suppress cellular biosynthetic capacity, and subsequent cell growth and viability [134,150]. Those studies have demonstrated that inhibition of GSK3 via BIO dramatically augment mESC

derivation from isolated ICMs of both C57BL/6 (76%) [135] and BALB/c (31%) [134].

on a BALB/c background) on refractory backgrounds [57].

Through dual inhibition of the Ras GTPase-activating protein (RasGAP) and ERK1, a function-oriented and novel small molecular pluripotin, also called SC1, was developed. By using this novel pluripotin, one can propagate mESCs in an undifferentiated and pluripotent state under chemically defined conditions in the absence of feeder cells, FBS, and LIF. Moreover, long-term pluripotin-expanded mESCs can generate germline-transmit‐ ted chimeric mice [43]. By combining pluripotin and LIF for the derivation of mESCs, the successfully isolated mESCs from five strains of mice; efficiency was 57% for NOD-scid, 63% for SCID beige, 80% for CD-1, and 100% for two F1 strains from C57BL/6 x CD-1. Pluripotin combined with LIF improved the efficiency of mESC isolation by selectively maintaining Oct4-positive cells in outgrowths. This is the first work to efficiently derive mESCs from immunodeficient mice (NOD-scid on an NOD background and SCID beige

Recently, a novel protocol involves an unusually long initial incubation of 12 days for blasto‐ cysts seeded in 12-well plates coated with mEFs and containing LIF and pluripotin-supple‐ mented KSR ESC medium (15% KSR), which results in the formation of large spherical outgrowths. These outgrowths are morphologically distinct from classical ICM outgrowths and can be picked easily and trypsinized. Importantly, pluripotin needs to be omitted after the

inhibited their differentiation.

term culture without LIF [48,128,135].

Alk3, the BMP4 type-IA receptor [127].

The PI3K pathway is also important for proliferation, survival, and maintenance of pluripo‐ tency, as well as inhibiting apoptosis in ESCs. The ESC-expressed Ras (ERas) is specifically expressed in ESCs, stimulating PI3K. This PI3K activation promotes ESC proliferation [137] and self-renewal [138]. Inhibition of PI3K and Akt induces differentiation of mESCs in the presence of LIF and feeder cells [139], suggesting that PI3K/Akt signaling is necessary for maintenance of the pluripotency of ESCs.

The ESC can maintain its pluripotency with feeder cells. Exogenous supplementation of LIF is sufficient to sustain mESCs at undifferentiated state in a feeder cells free condition. However, LIF is insufficient for maintaining the pluripotency of hES cells. Human ESCs have been most commonly cultured in the presence of bFGF either on fibroblast feeder layers [140] or in fibroblast-conditioned medium. The bFGF signaling pathway appears to be important to the self-renewal of hES cells [141,142]. Reports have shown that bFGF (40 ng/mL) combined with noggin (inhibitor of BMP4) supports the undifferentiated proliferation of hES cells in the absence of feeder cells [143-145]. Furthermore, a high bFGF concentration (100 ng/ml) alone is sufficient to maintain human ESCs [145]. The post-implantation epiblast-derived stem cell (EpiSC) lines [14] express transcription factors that regulate pluripotency and robustly differentiate into the major somatic cell types as well as primordial germ cells [12]. Further‐ more, the EpiSCs and hES cells share patterns of gene expression and signaling responses in the epiblasts [12]. In fact, the similarities (FGF and activin growth factors for self-renewal; *in vitro* pluripotency; chimera formation; and spontaneous trophoblast differentiation) between hES cells and mouse EpiSCs have led to the suggestion that hES cells are equivalent to early post-implantation epiblasts, rather than their ICM progenitor [146,147].

#### **2.7. Differentiation inhibitors help to derive mouse ES cells**

In the early 1990s, as the specificity of developed inhibitors was insufficient, the effectiveness of these inhibitors to inhibit differentiation and then help to establish mESCs was not satis‐ factory and outcomes are varied. Since the late 1990s, using MAPK/ERK kinases (MEKs) inhibitors PD098059 [128] and UO126 [41], or by dephosphorylating ERKs by mitogenactivated protein kinase phosphatase 3 (MKP-3) [41] enhanced the self-renewal of mESCs and inhibited their differentiation.

As a protein, transforming growth factor beta (TGFβ) controls proliferation, cellular differen‐ tiation, and other functions in most cells. This protein is a secreted protein of cytokines. The TGFβ family is part of the large TGFβ superfamily, which has more than 40 members, including TGFβ, activin, nodal, BMPs, inhibins, anti-müllerian hormone, decapentaplegic, and Vg-1. All of these ligands are associated with ESCs. Although BMP4 is a member of the BMP family, it functions via a different signaling pathway with TGFβ. Notably, BMP4 induces expression of Id genes and inhibits MAPK signaling as well as neuroectoderm differentiation [65,127]. Although activation of STAT3 is sufficient for self-renewal of mESCs, a study showed that LIF-STAT3 do not maintain mESCs in serum-free ESC medium. The combination of LIF and BMP4 maintained the self-renewal of mESCs in the absence of both feeder cells and FBS [65,127]. Thus, BMP4 and LIF have synergistic effects on the self-renewal of mESCs [127]. In contrast to mESCs, BMP4 does not maintain the self-renewal of hES cells; rather, it induces trophoblast

The PI3K pathway is also important for proliferation, survival, and maintenance of pluripo‐ tency, as well as inhibiting apoptosis in ESCs. The ESC-expressed Ras (ERas) is specifically expressed in ESCs, stimulating PI3K. This PI3K activation promotes ESC proliferation [137] and self-renewal [138]. Inhibition of PI3K and Akt induces differentiation of mESCs in the presence of LIF and feeder cells [139], suggesting that PI3K/Akt signaling is necessary for

The ESC can maintain its pluripotency with feeder cells. Exogenous supplementation of LIF is sufficient to sustain mESCs at undifferentiated state in a feeder cells free condition. However, LIF is insufficient for maintaining the pluripotency of hES cells. Human ESCs have been most commonly cultured in the presence of bFGF either on fibroblast feeder layers [140] or in fibroblast-conditioned medium. The bFGF signaling pathway appears to be important to the self-renewal of hES cells [141,142]. Reports have shown that bFGF (40 ng/mL) combined with noggin (inhibitor of BMP4) supports the undifferentiated proliferation of hES cells in the absence of feeder cells [143-145]. Furthermore, a high bFGF concentration (100 ng/ml) alone is sufficient to maintain human ESCs [145]. The post-implantation epiblast-derived stem cell (EpiSC) lines [14] express transcription factors that regulate pluripotency and robustly differentiate into the major somatic cell types as well as primordial germ cells [12]. Further‐ more, the EpiSCs and hES cells share patterns of gene expression and signaling responses in the epiblasts [12]. In fact, the similarities (FGF and activin growth factors for self-renewal; *in vitro* pluripotency; chimera formation; and spontaneous trophoblast differentiation) between hES cells and mouse EpiSCs have led to the suggestion that hES cells are equivalent to early

In the early 1990s, as the specificity of developed inhibitors was insufficient, the effectiveness of these inhibitors to inhibit differentiation and then help to establish mESCs was not satis‐ factory and outcomes are varied. Since the late 1990s, using MAPK/ERK kinases (MEKs) inhibitors PD098059 [128] and UO126 [41], or by dephosphorylating ERKs by mitogen-

post-implantation epiblasts, rather than their ICM progenitor [146,147].

**2.7. Differentiation inhibitors help to derive mouse ES cells**

or primitive endoderm differentiation of hES cells [136].

maintenance of the pluripotency of ESCs.

94 Pluripotent Stem Cells

It was the first report showed that the combination of PD98059 (MEK 1 inhibitor) with LIF enhances the establishment of mESCs from the refractory CBA strain [148]. A combination of the MEK1/2 inhibitor, U0126, with LIF further promoted the efficiency of mESC derivation from CBA [129]. These small molecules, PD98059 and U0126, play positive roles in the selfrenewal of mESCs, but they are incapable of maintaining the pluripotency of mESCs in longterm culture without LIF [48,128,135].

Notably, BMP4 inhibits both ERK and p38 MAPK pathways in mESCs. The inhibitors of the ERK and p38 MAPKs mimic the effect of BMP4 on mESCs. Inhibition of the p38 and MAPKs by SB203580 overcomes the roadblock in deriving mESCs from blastocysts lacking a functional Alk3, the BMP4 type-IA receptor [127].

The self-renewal of mESCs is generally dependent on multifactorial stimulation. To support the growth and development of fetuses, FBS contains cell replication stimulators and cell differentiation inducers. Serum and serum substitutes contain various inductive stimuli that may activate commitment and differentiation programs [49]. However, simple withdrawal of serum or other exogenous stimuli cannot prevent differentiation of ESCs due to endogenous autoinductive differentiation of fibroblast growth factor 4 (FGF4) via the MEK/ERK pathway [48,149]. Therefore, to suppress endogenous autoinductive differentiation and to maintain high viability and growth rates, one must inhibit the MEK/ERK pathway and/or provide LIF, or restrict the activity of GSK3, which acts mainly via the Wnt/β-catenin signaling pathway to suppress cellular biosynthetic capacity, and subsequent cell growth and viability [134,150]. Those studies have demonstrated that inhibition of GSK3 via BIO dramatically augment mESC derivation from isolated ICMs of both C57BL/6 (76%) [135] and BALB/c (31%) [134].

Through dual inhibition of the Ras GTPase-activating protein (RasGAP) and ERK1, a function-oriented and novel small molecular pluripotin, also called SC1, was developed. By using this novel pluripotin, one can propagate mESCs in an undifferentiated and pluripotent state under chemically defined conditions in the absence of feeder cells, FBS, and LIF. Moreover, long-term pluripotin-expanded mESCs can generate germline-transmit‐ ted chimeric mice [43]. By combining pluripotin and LIF for the derivation of mESCs, the successfully isolated mESCs from five strains of mice; efficiency was 57% for NOD-scid, 63% for SCID beige, 80% for CD-1, and 100% for two F1 strains from C57BL/6 x CD-1. Pluripotin combined with LIF improved the efficiency of mESC isolation by selectively maintaining Oct4-positive cells in outgrowths. This is the first work to efficiently derive mESCs from immunodeficient mice (NOD-scid on an NOD background and SCID beige on a BALB/c background) on refractory backgrounds [57].

Recently, a novel protocol involves an unusually long initial incubation of 12 days for blasto‐ cysts seeded in 12-well plates coated with mEFs and containing LIF and pluripotin-supple‐ mented KSR ESC medium (15% KSR), which results in the formation of large spherical outgrowths. These outgrowths are morphologically distinct from classical ICM outgrowths and can be picked easily and trypsinized. Importantly, pluripotin needs to be omitted after the first trypsinization because it blocks the attachment of mES-like cells to the mEF feeder layer; its removal facilitates the formation of mESC colonies. In addition, pluripotin is harmful to the mEFs and it is not unusual that half of the mEFs die during the first week. However, this massive death of mEFs does not affect the mES cell derivation efficiency [55]. I also found that STO and Hs68 feeder cells were dying when culturing in KSR ESC medium supplemented with 3 μM pluripotin (unpublished observation). The modified protocol gives rise to mESCs (more then five passages) in a robust and reliable manner with an extremely high success rate of 94% (78/83) [55]. Surprisingly, 10 mESCs derived with 4 μM pluripotin showed the chro‐ mosomal instability. All of these cells generated weak chimeras. Thus, these lines are only suitable for *in vitro* analysis. In contrast, mES-cell lines derived with 2 μM pluripotin during the blastocyst outgrowth phase were generated with unusual high efficiency (100%) and these lines had a normal karyotype. In addition, strong chimeras could be derived from these mESCs [55]. Yang and colleagues (2009) derived mESCs with 3–5 μM pluripotin. Their mESCs showed some chromosomal abnormalities and were not proven to be germline competent [57].

mESC lines from permissive 129SvE delayed blastocysts, solely by culturing them on STO feeder cells. Notably, Martin (1981) was unable to establish mESC lines from immunosurgically isolated ICMs of (ICR x SWR/J) F1 and (C3H x C57BL/6) F1 fully expanded blastocysts unless the teratocarcinoma stem cell-conditioned medium, equivalent to a 5-fold LIF concentration [92], was added. A previous report revealed that a mESC medium containing 10 ng/mL rabbit LIF or conditioned by a rabbit fibroblast cell line transduced with genomic rabbit LIF facilitates efficient derivation and maintenance of mESC lines (≥ 10 passages) from all 10 inbred mouse strains tested, including some that were presumed nonpermissive for ESC derivation [56]. A more recent study demonstrated that SR ESC medium containing high concentrations of LIF (2,500 and 5,000 unit/mL) could establish mES cell lines from C57BL/6, Balb/K, nonpermissive

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Additionally, another report shows that KSR ESC medium does not support single mESC culture, likely because this medium lacked some important growth factors or such undefined factors, such as "stem-cell autocrine factors," are secreted by mESCs themselves [62]. However, when KSR ESC medium is supplemented with ACTH, single mESCs are propagated and their pluripotency is maintained. Accordingly, mESC lines have been established efficiently using single blastomeres from 2- to 8-cell embryos in KSR ESC medium containing ACTH 1–24 [59].

The CHIR99021 pathway and proposed ACTH pathway likely differ. Therefore, synergetic effects of 2i (PD0325901 + CHIR99021) and ACTH 1–24 may exist in deriving mESCs. My experimental results reveal that neither STO nor Hs68 feeder cells coated on 10-μL droplets and cultured in KSR ESC medium supports effective derivation of mESCs from embryos or blastomeres of ICR or C57BL/6J. However, by supplementing KSR ESC medium with 2i or ACTH 1–24, efficiency in establishing mESCs increased dramatically. Experimental results also demonstrate that inhibiting cell differentiation and increasing cell growth/viability (in the presence of 2i) simultaneously is better than increasing only cell survival and/or proliferation (with ACTH 1–24) when deriving mESCs [31]. Additionally, experimental results also suggest that GSK3 inhibitor CHIR99021 and ACTH 1–24 likely have different pathways in synergisti‐ cally enhancing the establishment of mESCs because 2i with ACTH 1–24 is much more effective

A previous study has demonstrated that blastomeres from 2- to 8-cell embryos developed into blastocysts within 3–5 days in KSR ESC medium containing ACTH 1–24 [59]. Another report indicates that 2- to 4-cell embryos and blastomeres, which developed into blastocysts in medium containing ACTH 1–24, PD98059 (MAP2K1 inhibitor) or SB203580 (MAPK14 inhibitor), yields developmental rates comparable to those of the control embryos [151]. My previous study showed that approximately 60% of 1.5–2.5 dpc denuded whole embryos developed into typical blastocysts or small blastocysts and approximately 80% of the corre‐ sponding blastomeres developed into small blastocysts within 2–4 days in KSR ESC medium containing 2i or ACTH 1–24. These experimental results imply that 2i and ACTH 1–24 might have little or no adverse effect on cell proliferation or the development of embryos and blastomeres. Furthermore, these observations indicate that 2i, MAP2K1, and MAPK14

CBA/Ca, and NOD mice [92].

than 2i or ACTH 1–24 alone [31].

**2.9. Developing more powerful differentiation inhibitors**

Although the derivation of novel mESCs have been improved significantly via differentiation inhibitors and/or proliferation stimulators, no germline-transmitted ESCs other than the mESCs have been reported. In recent years, the MEK inhibitor PD0325901 or SU5402 has been used to eliminate differentiation-inducing signaling from MAPK and the GSK3 inhibitor CHIR99021 has been used to enhance mESC growth, enabling derivation and propagation of germline-competent mESCs from CBA and 129 strains in an N2B27 chemically defined ESC medium. These findings reveal that self-renewal is enabled by the elimination of differentia‐ tion-inducing signaling from MAPK. Additional inhibition of GSK3 consolidates biosynthetic capacity and suppresses residual differentiation. Complete bypass of cytokine signaling is confirmed by isolating *stat3*–/– mESCs [48]. Accordingly, ES cell lines of the second mammalian species (rat) with germline transmission have been established [49,50].

Recently, my report showed that two C57BL/6J mESCs were derived from two morulae in KSR ESC medium supplemented with 2i but without LIF [31]. These experimental results confirmed previous reports indicating that inhibitors that block the MEK/ERK differentiation pathway can support self-renewal of mESCs, even without LIF signaling [43,48].

The efficiency of establishing mESCs with 2i was not reported [48]. Recently, my experimental results showed that the efficiency in establishing C57BL/6J ES cells in KSR ESC medium supplemented with 2i and LIF could be high as 65% for morulae and 70% for blastocysts [31]. Thus, the efficacy of 2i in helping establish mESCs is confirmed.

#### **2.8. Self-renewal and proliferation stimulator**

Although a previous study showed that extrinsic stimuli are dispensable for derivation, propagation, and pluripotency of mESCs [48], the self-renewal and derivation of mESCs largely depends on LIF [130] and BMP4 [65,127]. When LIF is present at concentrations higher than those used in routine cultivation (500 unit/mL for the mEF feeder and 1,000 unit/mL for the STO feeder), the derivation efficiencies of mESCs from refractory strains are improved significantly [57,91,93]. Indeed, strain difference in response to soluble growth factors is evident from two original reports of mESC derivation. Evans and Kaufman (1981) established mESC lines from permissive 129SvE delayed blastocysts, solely by culturing them on STO feeder cells. Notably, Martin (1981) was unable to establish mESC lines from immunosurgically isolated ICMs of (ICR x SWR/J) F1 and (C3H x C57BL/6) F1 fully expanded blastocysts unless the teratocarcinoma stem cell-conditioned medium, equivalent to a 5-fold LIF concentration [92], was added. A previous report revealed that a mESC medium containing 10 ng/mL rabbit LIF or conditioned by a rabbit fibroblast cell line transduced with genomic rabbit LIF facilitates efficient derivation and maintenance of mESC lines (≥ 10 passages) from all 10 inbred mouse strains tested, including some that were presumed nonpermissive for ESC derivation [56]. A more recent study demonstrated that SR ESC medium containing high concentrations of LIF (2,500 and 5,000 unit/mL) could establish mES cell lines from C57BL/6, Balb/K, nonpermissive CBA/Ca, and NOD mice [92].

Additionally, another report shows that KSR ESC medium does not support single mESC culture, likely because this medium lacked some important growth factors or such undefined factors, such as "stem-cell autocrine factors," are secreted by mESCs themselves [62]. However, when KSR ESC medium is supplemented with ACTH, single mESCs are propagated and their pluripotency is maintained. Accordingly, mESC lines have been established efficiently using single blastomeres from 2- to 8-cell embryos in KSR ESC medium containing ACTH 1–24 [59].

The CHIR99021 pathway and proposed ACTH pathway likely differ. Therefore, synergetic effects of 2i (PD0325901 + CHIR99021) and ACTH 1–24 may exist in deriving mESCs. My experimental results reveal that neither STO nor Hs68 feeder cells coated on 10-μL droplets and cultured in KSR ESC medium supports effective derivation of mESCs from embryos or blastomeres of ICR or C57BL/6J. However, by supplementing KSR ESC medium with 2i or ACTH 1–24, efficiency in establishing mESCs increased dramatically. Experimental results also demonstrate that inhibiting cell differentiation and increasing cell growth/viability (in the presence of 2i) simultaneously is better than increasing only cell survival and/or proliferation (with ACTH 1–24) when deriving mESCs [31]. Additionally, experimental results also suggest that GSK3 inhibitor CHIR99021 and ACTH 1–24 likely have different pathways in synergisti‐ cally enhancing the establishment of mESCs because 2i with ACTH 1–24 is much more effective than 2i or ACTH 1–24 alone [31].

#### **2.9. Developing more powerful differentiation inhibitors**

first trypsinization because it blocks the attachment of mES-like cells to the mEF feeder layer; its removal facilitates the formation of mESC colonies. In addition, pluripotin is harmful to the mEFs and it is not unusual that half of the mEFs die during the first week. However, this massive death of mEFs does not affect the mES cell derivation efficiency [55]. I also found that STO and Hs68 feeder cells were dying when culturing in KSR ESC medium supplemented with 3 μM pluripotin (unpublished observation). The modified protocol gives rise to mESCs (more then five passages) in a robust and reliable manner with an extremely high success rate of 94% (78/83) [55]. Surprisingly, 10 mESCs derived with 4 μM pluripotin showed the chro‐ mosomal instability. All of these cells generated weak chimeras. Thus, these lines are only suitable for *in vitro* analysis. In contrast, mES-cell lines derived with 2 μM pluripotin during the blastocyst outgrowth phase were generated with unusual high efficiency (100%) and these lines had a normal karyotype. In addition, strong chimeras could be derived from these mESCs [55]. Yang and colleagues (2009) derived mESCs with 3–5 μM pluripotin. Their mESCs showed some chromosomal abnormalities and were not proven to be germline competent [57].

Although the derivation of novel mESCs have been improved significantly via differentiation inhibitors and/or proliferation stimulators, no germline-transmitted ESCs other than the mESCs have been reported. In recent years, the MEK inhibitor PD0325901 or SU5402 has been used to eliminate differentiation-inducing signaling from MAPK and the GSK3 inhibitor CHIR99021 has been used to enhance mESC growth, enabling derivation and propagation of germline-competent mESCs from CBA and 129 strains in an N2B27 chemically defined ESC medium. These findings reveal that self-renewal is enabled by the elimination of differentia‐ tion-inducing signaling from MAPK. Additional inhibition of GSK3 consolidates biosynthetic capacity and suppresses residual differentiation. Complete bypass of cytokine signaling is confirmed by isolating *stat3*–/– mESCs [48]. Accordingly, ES cell lines of the second mammalian

Recently, my report showed that two C57BL/6J mESCs were derived from two morulae in KSR ESC medium supplemented with 2i but without LIF [31]. These experimental results confirmed previous reports indicating that inhibitors that block the MEK/ERK differentiation pathway

The efficiency of establishing mESCs with 2i was not reported [48]. Recently, my experimental results showed that the efficiency in establishing C57BL/6J ES cells in KSR ESC medium supplemented with 2i and LIF could be high as 65% for morulae and 70% for blastocysts [31].

Although a previous study showed that extrinsic stimuli are dispensable for derivation, propagation, and pluripotency of mESCs [48], the self-renewal and derivation of mESCs largely depends on LIF [130] and BMP4 [65,127]. When LIF is present at concentrations higher than those used in routine cultivation (500 unit/mL for the mEF feeder and 1,000 unit/mL for the STO feeder), the derivation efficiencies of mESCs from refractory strains are improved significantly [57,91,93]. Indeed, strain difference in response to soluble growth factors is evident from two original reports of mESC derivation. Evans and Kaufman (1981) established

species (rat) with germline transmission have been established [49,50].

can support self-renewal of mESCs, even without LIF signaling [43,48].

Thus, the efficacy of 2i in helping establish mESCs is confirmed.

**2.8. Self-renewal and proliferation stimulator**

96 Pluripotent Stem Cells

A previous study has demonstrated that blastomeres from 2- to 8-cell embryos developed into blastocysts within 3–5 days in KSR ESC medium containing ACTH 1–24 [59]. Another report indicates that 2- to 4-cell embryos and blastomeres, which developed into blastocysts in medium containing ACTH 1–24, PD98059 (MAP2K1 inhibitor) or SB203580 (MAPK14 inhibitor), yields developmental rates comparable to those of the control embryos [151]. My previous study showed that approximately 60% of 1.5–2.5 dpc denuded whole embryos developed into typical blastocysts or small blastocysts and approximately 80% of the corre‐ sponding blastomeres developed into small blastocysts within 2–4 days in KSR ESC medium containing 2i or ACTH 1–24. These experimental results imply that 2i and ACTH 1–24 might have little or no adverse effect on cell proliferation or the development of embryos and blastomeres. Furthermore, these observations indicate that 2i, MAP2K1, and MAPK14 inhibitors are not strong enough to inhibit differentiation of embryos and blastomeres entirely [31]. I also note that 0.3–3 μM pluripotin did not inhibit differentiation of ICR 2.5 dpc embryos and blastomeres completely (unpublished observation).

These experimental findings suggest that other more powerful inhibitors, via the Ras-MEK-ERK signaling pathway or other signaling pathways, may be worth developing to further enhance the success rate of deriving ESCs. Theoretically, chemical cocktails that completely inhibit endogenous differentiation, increase cell division, and decrease apoptosis of pluripo‐ tent cells should maximize the derivation efficiency of ESCs. Furthermore, is it possible to develop novel differentiation inhibitors that maintain the totipotency of very-early-stage blastomeres?

## **3. Novel efficient, reproducible, and user-friendly protocol for deriving mouse ES cells**

An excellent protocol for deriving mESCs must be efficient, reproducible, easy to perform, and relatively cheap.

The following protocol, adopted and minor modified from my previous study [31], has been used in my laboratory for more than 3 years with reproducible high derivation efficiency (always more than 50%, occasionally reaching 100%) for 2.5 dpc whole embryos, regardless of which strains (ICR, B6, ICRB6F1, and B6CBF1) are tested. Moreover, all other pre-implantation embryos can be used. Although the same protocol can be used to derive mESCs via single blastomeres, derivation of mESC varies in efficiency, which depends on the origin of blasto‐ meres (approximately 10–30%).

> **Figure 2.** The 10-μL droplets are excellent for initial cultivation to derive mESCs. Embryos and blastomeres seeded in 10-μL droplets coated with Hs68 feeder cells in KnockOut™ serum replacement (KSR) ESC medium supplemented with 0.5 µM PD0325901 (MEK inhibitor), and 3 µM CHIR99021 (GSK3 inhibitor) (2i), 10 µM ACTH–24 (adenylyl cyclase inhibitor), as well as 1,000 unit/mL LIF support the growth of 2.5 dpc embryos for at least 7 days and 1/8 blastomeres for at least 10 days. a1, early blastocyst; b1, morula; c1, 2/16—isolated single blastomere that originated from late 8 cell embryos were divided; d1, 1/8—isolated single blastomere that originated from 8-cell embryos. a2–d2, cultured for 3 days after initiation of cultivation; a3–d3, cultured for 7 days; and a4–d4, cultured for 10 days; a5–d5, cultured

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The previous study reported that the culturing blastocysts allow to hatch and expand for approximately 6 days; however, the earlier trypsinization (at day 4 or 5) of outgrowths do not seem to affect efficiency but prolonged the time needed for mESCs derivation [58]. Very interestingly, blastocyst outgrowths can be cultured in 2 μM pluripotin-containing KSR ESC medium for 12 days (or up to 18 days) without losing their ability to form mESCs. The main advantage of a long initial cultivation is to greatly increase the number of cells, which including undifferentiated cells, before the first trypsinization. The derivation efficiency could be as high

Further, my experimental results demonstrated that mESCs are established efficiently from C57BL/6J denuded whole embryos culturing in a 10-μL droplet coated with Hs68 feeder cells

of KSR ESC medium supplemented with 2i, ACTH-24, and LIF (Figure 3) [31].

for 12 days. The bar represents 100 μm.

as 94% (78/83) [55].

#### **3.1. Growth area for initial cultivation**

For initial cultivation of embryos, ICMs, and blastomeres to establish mESCs, different growth surface areas in multi-well cell plates are used. Of which, the 24-well and 96-well plates are used mainly. In practice, the amount of medium needed to half fill one well in a 24-well plate is approximately 0.5 mL and 150 μL for a 96-well plate. Both volumes are enormous to the mouse embryo (diameter, 85 μm; volume, 320 pL = 0.00032 μL) or 1/8 blastomere (diameter, 20 μm; volume, 4 pL = 0.000004 μL). Furthermore, the height of wells is not user-friendly when taking photographs or picking growing three-dimensional outgrowths. Therefore, to reduce the amount of expensive ESC media that is usually exchanged every other day, ease handling, and increase the disaggregation efficiency of growing three-dimensional outgrowths, different volumes of micro-droplets were tested. I hypothesize that homemade micro-droplets on cell culture dishes coated with feeder cells are as effective as cell plate wells for initial cultivation to establish mESCs. Finally, 10-μL and 20-μL droplets are chosen for initial and for disaggre‐ gated outgrowths cultivation, respectively. Up to 32 10-μL or 24 20-μL feeder droplets on a 60-mm cell culture dish covered with heavy weight paraffin oil can be prepared and used routinely. My culturing results show that 10-μL droplets support the growth of 2.5 dpc embryos for at least 7 days and 1/8 blastomeres for at least 10 days (Figure 2).

inhibitors are not strong enough to inhibit differentiation of embryos and blastomeres entirely [31]. I also note that 0.3–3 μM pluripotin did not inhibit differentiation of ICR 2.5 dpc embryos

These experimental findings suggest that other more powerful inhibitors, via the Ras-MEK-ERK signaling pathway or other signaling pathways, may be worth developing to further enhance the success rate of deriving ESCs. Theoretically, chemical cocktails that completely inhibit endogenous differentiation, increase cell division, and decrease apoptosis of pluripo‐ tent cells should maximize the derivation efficiency of ESCs. Furthermore, is it possible to develop novel differentiation inhibitors that maintain the totipotency of very-early-stage

**3. Novel efficient, reproducible, and user-friendly protocol for deriving**

An excellent protocol for deriving mESCs must be efficient, reproducible, easy to perform, and

The following protocol, adopted and minor modified from my previous study [31], has been used in my laboratory for more than 3 years with reproducible high derivation efficiency (always more than 50%, occasionally reaching 100%) for 2.5 dpc whole embryos, regardless of which strains (ICR, B6, ICRB6F1, and B6CBF1) are tested. Moreover, all other pre-implantation embryos can be used. Although the same protocol can be used to derive mESCs via single blastomeres, derivation of mESC varies in efficiency, which depends on the origin of blasto‐

For initial cultivation of embryos, ICMs, and blastomeres to establish mESCs, different growth surface areas in multi-well cell plates are used. Of which, the 24-well and 96-well plates are used mainly. In practice, the amount of medium needed to half fill one well in a 24-well plate is approximately 0.5 mL and 150 μL for a 96-well plate. Both volumes are enormous to the mouse embryo (diameter, 85 μm; volume, 320 pL = 0.00032 μL) or 1/8 blastomere (diameter, 20 μm; volume, 4 pL = 0.000004 μL). Furthermore, the height of wells is not user-friendly when taking photographs or picking growing three-dimensional outgrowths. Therefore, to reduce the amount of expensive ESC media that is usually exchanged every other day, ease handling, and increase the disaggregation efficiency of growing three-dimensional outgrowths, different volumes of micro-droplets were tested. I hypothesize that homemade micro-droplets on cell culture dishes coated with feeder cells are as effective as cell plate wells for initial cultivation to establish mESCs. Finally, 10-μL and 20-μL droplets are chosen for initial and for disaggre‐ gated outgrowths cultivation, respectively. Up to 32 10-μL or 24 20-μL feeder droplets on a 60-mm cell culture dish covered with heavy weight paraffin oil can be prepared and used routinely. My culturing results show that 10-μL droplets support the growth of 2.5 dpc

embryos for at least 7 days and 1/8 blastomeres for at least 10 days (Figure 2).

and blastomeres completely (unpublished observation).

blastomeres?

98 Pluripotent Stem Cells

**mouse ES cells**

relatively cheap.

meres (approximately 10–30%).

**3.1. Growth area for initial cultivation**

**Figure 2.** The 10-μL droplets are excellent for initial cultivation to derive mESCs. Embryos and blastomeres seeded in 10-μL droplets coated with Hs68 feeder cells in KnockOut™ serum replacement (KSR) ESC medium supplemented with 0.5 µM PD0325901 (MEK inhibitor), and 3 µM CHIR99021 (GSK3 inhibitor) (2i), 10 µM ACTH–24 (adenylyl cyclase inhibitor), as well as 1,000 unit/mL LIF support the growth of 2.5 dpc embryos for at least 7 days and 1/8 blastomeres for at least 10 days. a1, early blastocyst; b1, morula; c1, 2/16—isolated single blastomere that originated from late 8 cell embryos were divided; d1, 1/8—isolated single blastomere that originated from 8-cell embryos. a2–d2, cultured for 3 days after initiation of cultivation; a3–d3, cultured for 7 days; and a4–d4, cultured for 10 days; a5–d5, cultured for 12 days. The bar represents 100 μm.

The previous study reported that the culturing blastocysts allow to hatch and expand for approximately 6 days; however, the earlier trypsinization (at day 4 or 5) of outgrowths do not seem to affect efficiency but prolonged the time needed for mESCs derivation [58]. Very interestingly, blastocyst outgrowths can be cultured in 2 μM pluripotin-containing KSR ESC medium for 12 days (or up to 18 days) without losing their ability to form mESCs. The main advantage of a long initial cultivation is to greatly increase the number of cells, which including undifferentiated cells, before the first trypsinization. The derivation efficiency could be as high as 94% (78/83) [55].

Further, my experimental results demonstrated that mESCs are established efficiently from C57BL/6J denuded whole embryos culturing in a 10-μL droplet coated with Hs68 feeder cells of KSR ESC medium supplemented with 2i, ACTH-24, and LIF (Figure 3) [31].

compared to that of STO. Therefore, STO feeder cells are adopted for the large growing areas

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The basal KSR ESC medium contained KnockOut™ DMEM (glucose 4.5 g/L) supplemented with 20% KSR, 0.1 mM non-essential amino acids, 1.75 mM GlutaMAX™-I supplement, 0.1 mM β-mercaptoethanol, penicillin (15.62 unit/mL), streptomycin (15.62 μg/mL), and LIF (1,000 unit/mL). The KnockOut™ DMEM can be replaced by conventional DMEM or (KnockOut™)

Although media prepared from powder are far cheaper than using liquid directly, experiments always use embryo- or cell culture-tested grades liquid media or solutions, as they are more consistent and efficient for derivation of mESCs, especially when KSR ESC media are used in cultivations. The variable quality of homemade ultrapure ddH2O might prove problematic. However, once the mESCs established, the media prepared from powder could be used for

To enhance the successful derivation of mESCs, the basal KSR ESC medium is supplemented with 0.5 μM PD0325901 (MEK inhibitor), 3 μM CHIR99021 (GSK3 inhibitor) (2i) [48], and 10

Natural or superovulated 3.5–4.5 dpc morulae and blastocysts are used. Recovered embryos are washed and placed in the KSOM medium supplemented with 20.85 mM HEPES (HK) at RT until the next treatment. The zona pellucida of embryos is removed (denuded) in seconds using acidic Tyrode's solution. Blastomeres of 2- to 8-cell embryos are separated by incubating denuded embryos in 0.25% trypsin-EDTA for approximately 3–4 min in a 37°C incubator with humidified atmosphere of 5% CO2 in air, followed by gentle pipetting using a mouth pipette. The blastomeres and denuded embryos or hatched (naked) blastocysts are washed and then plated into 10-μL feeder droplets (P0) in KSR ESC medium supplemented with 2i and ACTH 1–24, which is exchanged 1–2 h earlier. Embryos and blastomeres are cultured in a 37°C incubator with 5% CO2 in air. Following attachment of embryos or blastomeres to the feeder cells, the media are exchanged at the second or third day. Thereafter, the media are exchanged

After culturing for 6 ± 2 d (for whole embryos) or 9 ± 2 d (for blastomeres), individual threedimensional outgrowth is identified visually. The feeder cells and flat growing cells are removed mechanically by a mouth pipette. The outgrowth in the same droplet is washed with TrypLE™ Express once and then incubated with TrypLE™ Express in a 37°C incubator for approximately 13 ± 3 min. The outgrowth is washed with KSR ESC medium once and then disaggregated into clumps and single cells, which are reseeded onto fresh 20-μL droplets (P1). After 4 ± 1 d, only morphologically mES-like colonies are then passed (via TrypLE Express) to fresh 1 or 2 wells of 4-well cell plates (P2), and thereafter to either 4-well plates or 35-mm cell culture dishes (P3) depending on the number of cells. Once mES-like cells are growing in 4-

in commercially available cell plates and dishes.

μM ACTH 1–24 (adenylyl cyclase inhibitor) [59].

**3.4. Derivation of mouse ES cells**

DMEM/F12.

routine cultivation.

every 1 to 2 days.

**3.3. KSR ESC medium supplemented with 2i and ACTH 1–24**

**Figure 3.** The C57BL/6J ES cells derived from 2.5-day post-coitum (dpc) denuded 8-cell embryos or single blastomeres cultured in 10-µL droplets. a1: An 8-cell embryo cultured in KSR ESC medium containing 0.5 µM PD0325901 (MEK inhibitor) and 3 µM CHIR99021 (GSK3 inhibitor) (2i) with STO feeders. Images show 2 (a2), 4 (a3), 11 (a4; passage #1, P1) and 14 (a5; ESC98B01, P2) days after initiation of cultivation. b1: An 8-cell embryo cultured in KSR ESC medium with Hs68 feeders. Images show 3 (b2), 4 (b3), 9 (b4; P1), and 18 (b5; ESC98B05, P4) days after initiation of cultivation. c1: A single blastomere isolated from an 8-cell embryo cultured in KSR ESC medium containing 2i with Hs68 feeders. Images show 3 (c2), 6 (c3), 9 (c4; P1), and 14 (c5; ESC98B04, P3) days after initiation of cultivation. The bar represents 100 μm. (Reproduced from Lee *et al*., 2012. Stem Cells and Development 21:373–383.)

#### **3.2. Feeder cells and medium**

The cell lines of STO and Hs68 (Caucasian human newborn foreskin fibroblast; ATCC No. CRL-1635) as well as primary mEF cells can be used as feeder cells. These cells share the same growth medium, which comprises DMEM (glucose, 4.5 g/L) containing 10% FBS, penicillin (50 unit/mL), and streptomycin (50 μg/mL).

After mitotic inactivation by 10 μg/mL mitomycin C for 2–3 h, single trypsinized cells via 0.25% trypsin-EDTA are used to prepare feeder layers directly or are frozen until thawed for feeder preparation. Up to 32 10-μL feeder droplets on a 60-mm cell culture dish, covered with heavy weight paraffin oil, is prepared 1–2 days before denuded embryos or isolated blastomeres are seeded.

No difference existed between STO and Hs68 feeders in the derivation of mESCs [31]. How‐ ever, as STO feeders occasionally detached and curled up during cultivation in 10 μL and 20 μL droplets, Hs68 is usually chosen as the feeder cells. However, propagation of Hs68 is slow compared to that of STO. Therefore, STO feeder cells are adopted for the large growing areas in commercially available cell plates and dishes.

#### **3.3. KSR ESC medium supplemented with 2i and ACTH 1–24**

The basal KSR ESC medium contained KnockOut™ DMEM (glucose 4.5 g/L) supplemented with 20% KSR, 0.1 mM non-essential amino acids, 1.75 mM GlutaMAX™-I supplement, 0.1 mM β-mercaptoethanol, penicillin (15.62 unit/mL), streptomycin (15.62 μg/mL), and LIF (1,000 unit/mL). The KnockOut™ DMEM can be replaced by conventional DMEM or (KnockOut™) DMEM/F12.

Although media prepared from powder are far cheaper than using liquid directly, experiments always use embryo- or cell culture-tested grades liquid media or solutions, as they are more consistent and efficient for derivation of mESCs, especially when KSR ESC media are used in cultivations. The variable quality of homemade ultrapure ddH2O might prove problematic. However, once the mESCs established, the media prepared from powder could be used for routine cultivation.

To enhance the successful derivation of mESCs, the basal KSR ESC medium is supplemented with 0.5 μM PD0325901 (MEK inhibitor), 3 μM CHIR99021 (GSK3 inhibitor) (2i) [48], and 10 μM ACTH 1–24 (adenylyl cyclase inhibitor) [59].

#### **3.4. Derivation of mouse ES cells**

**Figure 3.** The C57BL/6J ES cells derived from 2.5-day post-coitum (dpc) denuded 8-cell embryos or single blastomeres cultured in 10-µL droplets. a1: An 8-cell embryo cultured in KSR ESC medium containing 0.5 µM PD0325901 (MEK inhibitor) and 3 µM CHIR99021 (GSK3 inhibitor) (2i) with STO feeders. Images show 2 (a2), 4 (a3), 11 (a4; passage #1, P1) and 14 (a5; ESC98B01, P2) days after initiation of cultivation. b1: An 8-cell embryo cultured in KSR ESC medium with Hs68 feeders. Images show 3 (b2), 4 (b3), 9 (b4; P1), and 18 (b5; ESC98B05, P4) days after initiation of cultivation. c1: A single blastomere isolated from an 8-cell embryo cultured in KSR ESC medium containing 2i with Hs68 feeders. Images show 3 (c2), 6 (c3), 9 (c4; P1), and 14 (c5; ESC98B04, P3) days after initiation of cultivation. The bar represents

The cell lines of STO and Hs68 (Caucasian human newborn foreskin fibroblast; ATCC No. CRL-1635) as well as primary mEF cells can be used as feeder cells. These cells share the same growth medium, which comprises DMEM (glucose, 4.5 g/L) containing 10% FBS, penicillin (50

After mitotic inactivation by 10 μg/mL mitomycin C for 2–3 h, single trypsinized cells via 0.25% trypsin-EDTA are used to prepare feeder layers directly or are frozen until thawed for feeder preparation. Up to 32 10-μL feeder droplets on a 60-mm cell culture dish, covered with heavy weight paraffin oil, is prepared 1–2 days before denuded embryos or isolated blastomeres are

No difference existed between STO and Hs68 feeders in the derivation of mESCs [31]. How‐ ever, as STO feeders occasionally detached and curled up during cultivation in 10 μL and 20 μL droplets, Hs68 is usually chosen as the feeder cells. However, propagation of Hs68 is slow

100 μm. (Reproduced from Lee *et al*., 2012. Stem Cells and Development 21:373–383.)

**3.2. Feeder cells and medium**

100 Pluripotent Stem Cells

seeded.

unit/mL), and streptomycin (50 μg/mL).

Natural or superovulated 3.5–4.5 dpc morulae and blastocysts are used. Recovered embryos are washed and placed in the KSOM medium supplemented with 20.85 mM HEPES (HK) at RT until the next treatment. The zona pellucida of embryos is removed (denuded) in seconds using acidic Tyrode's solution. Blastomeres of 2- to 8-cell embryos are separated by incubating denuded embryos in 0.25% trypsin-EDTA for approximately 3–4 min in a 37°C incubator with humidified atmosphere of 5% CO2 in air, followed by gentle pipetting using a mouth pipette. The blastomeres and denuded embryos or hatched (naked) blastocysts are washed and then plated into 10-μL feeder droplets (P0) in KSR ESC medium supplemented with 2i and ACTH 1–24, which is exchanged 1–2 h earlier. Embryos and blastomeres are cultured in a 37°C incubator with 5% CO2 in air. Following attachment of embryos or blastomeres to the feeder cells, the media are exchanged at the second or third day. Thereafter, the media are exchanged every 1 to 2 days.

After culturing for 6 ± 2 d (for whole embryos) or 9 ± 2 d (for blastomeres), individual threedimensional outgrowth is identified visually. The feeder cells and flat growing cells are removed mechanically by a mouth pipette. The outgrowth in the same droplet is washed with TrypLE™ Express once and then incubated with TrypLE™ Express in a 37°C incubator for approximately 13 ± 3 min. The outgrowth is washed with KSR ESC medium once and then disaggregated into clumps and single cells, which are reseeded onto fresh 20-μL droplets (P1). After 4 ± 1 d, only morphologically mES-like colonies are then passed (via TrypLE Express) to fresh 1 or 2 wells of 4-well cell plates (P2), and thereafter to either 4-well plates or 35-mm cell culture dishes (P3) depending on the number of cells. Once mES-like cells are growing in 4well plates and larger dishes, KSR ESC medium (2i + ACTH 1–24 is option) is used for subsequent propagation. At passages 5 ± 2, mES-like cells are frozen in FBS supplemented with 10% (v/v) dimethyl sulfoxide (DMSO). The typical duration of the above-described process of mESC derivation (from embryos to freezing of subconfluent 35 mm-dishes) is ranging from 14 to 20 days.

NSC99-2324-B-059-001, and NSC97-2317-B-059-004. Drs. C.H. Chen, C.k. Juang, C.F. Tu, and S.F. Guo as well as Ms. H.R. Chang and T.L. Hsu at the Animal Technology Institute Taiwan (ATIT) are thanked for their critical comments, technical assistance, and routine maintenance

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Division of Biotechnology, Animal Technology Institute Taiwan, Chunan, Miaoli, Taiwan

[1] Nagy A, Gertsenstein M, Vintersten K, Behringer R, Nagy A, Gertsenstein M, Vin‐ tersten K, Behringer R. Summary of mouse development, Manipulating the Mouse Embryo: A Laboratory Manual, 3 edition. New York: Cold Spring Harbor Lab. Press;

[2] Rossant J, Tam PP. Blastocyst lineage formation, early embryonic asymmetries and

[3] Mitalipov S, Wolf D. Totipotency, pluripotency and nuclear reprogramming. Advan‐

[4] Papaioannou VE, Ebert KM. Comparative aspects of embryo manipulation in mam‐ mals. In: Rossant J, Pedersen RA (eds), Experimental Approach to Mammalian Em‐

[5] Suwinska A, Czolowska R, Ozdzenski W, Tarkowski AK. Blastomeres of the mouse embryo lose totipotency after the fifth cleavage division: Expression of Cdx2 and Oct4 and developmental potential of inner and outer blastomeres of 16- and 32-cell

[6] Tarkowski AK, Wroblewska J. Development of blastomeres of mouse eggs isolated at the 4- and 8-cell stage. Journal of Embryology and Experimental Morphology

[7] Ziomek CA, Johnson MH, Handyside AH. The developmental potential of mouse 16-

[8] Willadsen SM, Polge C. Attempts to produce monozygotic quadruplets in cattle by

cell blastomeres. Journal of Experimental Zoology 1982;221: 345-355.

blastomere separation. Veterinary Record 1981;108: 211-213.

bryonic Development. Cambridge, UK: Cambridge Univ. Press; 1986. p67-96.

axis patterning in the mouse. Development 2009;136: 701-713.

ces in Biochemical Engineering Biotechnology 2009;114: 185-199.

embryos. Developmental Biology 2008;322: 133-144.

in the laboratory and mouse facility.

Address all correspondence to: khlee@mail.atit.org.tw

**Author details**

Kun-Hsiung Lee\*

**References**

2003. p31-139.

1967;18: 155-180.

Practically, the first 2–3 passages are critical for successful derivation of mESCs. This protocol suggests that one use KSR ESC medium containing 2i + ACTH 1–24 only for the first 10-μL droplets for embryo outgrowth (P0) and second (for single cells and clumps of digested outgrowths, P1) and/or third 20-μL (for mES-like cells, P2) droplets. Once mES-like cells are growing on 4-well plates (P2–3), KSR ESC medium is used thereafter for all following culti‐ vations.

The success rates in establishing B6 mESCs by this simple protocol are always greater than 50% for 2.5–4.5 dpc embryos. This efficiency is comparable to that achieved in two other studies [56,109] and much better than those in many other studies [32,38,61,82,103-108]. This proposed protocol has a simple layout, is easy to operate, is highly efficient, is reproducible, and can be an alternative method for establishing mES cell lines routinely.

## **4. Conclusions**

Zygotes to hatched embryos and blastomeres, ICMs, and the epiblasts of early-stage preimplantation embryos can be used to establish mESCs. Both embryos and blastomeres have an extremely high capability for cell division and differentiation. Theoretically, chemical cocktails that can completely inhibit endogenous differentiation, increase cell division, and decrease apoptosis of pre-implantation embryos can be helpful to maximize the derivation efficiency of ESCs.

Culturing pre-implantation embryos, no matter what strains, on a very small surface area coated with feeder cells in a chemically defined medium supplemented with differentiation inhibitors (*e.g*., 2i) and/or proliferation enhancers/stimulators (*e.g*., LIF and ACTH 1–24) can be used as a routine protocol to establish mESCs efficiently and reproducibly (always more than 50%, occasionally reaching 100%). Unfortunately, the same protocol when using 1/8 blastomeres to derive mESCs is merely acceptable (10–25%). Obviously, the possibility to increase the efficiency of deriving mESCs from whole embryos is limited and unexciting unless novel differentiation inhibitors or proliferation enhancers/stimulators reveal the effectiveness of using 1/8 blastomeres and embryos from species other than rodents.

## **Acknowledgements**

I would like to thank the National Science Council of the Republic of China, Taiwan, for financially supporting my research under Contract Nos. NSC100-2321-B-059-001-MY3, NSC99-2324-B-059-001, and NSC97-2317-B-059-004. Drs. C.H. Chen, C.k. Juang, C.F. Tu, and S.F. Guo as well as Ms. H.R. Chang and T.L. Hsu at the Animal Technology Institute Taiwan (ATIT) are thanked for their critical comments, technical assistance, and routine maintenance in the laboratory and mouse facility.

## **Author details**

well plates and larger dishes, KSR ESC medium (2i + ACTH 1–24 is option) is used for subsequent propagation. At passages 5 ± 2, mES-like cells are frozen in FBS supplemented with 10% (v/v) dimethyl sulfoxide (DMSO). The typical duration of the above-described process of mESC derivation (from embryos to freezing of subconfluent 35 mm-dishes) is ranging from

Practically, the first 2–3 passages are critical for successful derivation of mESCs. This protocol suggests that one use KSR ESC medium containing 2i + ACTH 1–24 only for the first 10-μL droplets for embryo outgrowth (P0) and second (for single cells and clumps of digested outgrowths, P1) and/or third 20-μL (for mES-like cells, P2) droplets. Once mES-like cells are growing on 4-well plates (P2–3), KSR ESC medium is used thereafter for all following culti‐

The success rates in establishing B6 mESCs by this simple protocol are always greater than 50% for 2.5–4.5 dpc embryos. This efficiency is comparable to that achieved in two other studies [56,109] and much better than those in many other studies [32,38,61,82,103-108]. This proposed protocol has a simple layout, is easy to operate, is highly efficient, is reproducible, and can be

Zygotes to hatched embryos and blastomeres, ICMs, and the epiblasts of early-stage preimplantation embryos can be used to establish mESCs. Both embryos and blastomeres have an extremely high capability for cell division and differentiation. Theoretically, chemical cocktails that can completely inhibit endogenous differentiation, increase cell division, and decrease apoptosis of pre-implantation embryos can be helpful to maximize the derivation

Culturing pre-implantation embryos, no matter what strains, on a very small surface area coated with feeder cells in a chemically defined medium supplemented with differentiation inhibitors (*e.g*., 2i) and/or proliferation enhancers/stimulators (*e.g*., LIF and ACTH 1–24) can be used as a routine protocol to establish mESCs efficiently and reproducibly (always more than 50%, occasionally reaching 100%). Unfortunately, the same protocol when using 1/8 blastomeres to derive mESCs is merely acceptable (10–25%). Obviously, the possibility to increase the efficiency of deriving mESCs from whole embryos is limited and unexciting unless novel differentiation inhibitors or proliferation enhancers/stimulators reveal the effectiveness

I would like to thank the National Science Council of the Republic of China, Taiwan, for financially supporting my research under Contract Nos. NSC100-2321-B-059-001-MY3,

of using 1/8 blastomeres and embryos from species other than rodents.

an alternative method for establishing mES cell lines routinely.

14 to 20 days.

102 Pluripotent Stem Cells

vations.

**4. Conclusions**

efficiency of ESCs.

**Acknowledgements**

Kun-Hsiung Lee\*

Address all correspondence to: khlee@mail.atit.org.tw

Division of Biotechnology, Animal Technology Institute Taiwan, Chunan, Miaoli, Taiwan

### **References**


[9] Fehilly CB, Willadsen SM. Embryo manipulation in farm animals. Oxford Reviews of Reproductive Biology 1986;8: 379-413.

[21] Iwasaki S, Kimura H. Further studies on thermal treatment of two-cell stage embryos to produce complete embryonic stem-cell-derived mice by cell-aggregation methods.

Conditions and Techniques for Mouse Embryonic Stem Cell Derivation and Culture

http://dx.doi.org/10.5772/55105

105

[22] Jaenisch R, Young R. Stem cells, the molecular circuitry of pluripotency and nuclear

[23] Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse

[24] Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proceedings of the National

[25] Nagy A, Gertsenstein M, Vintersten K, Behringer R, Nagy A, Gertsenstein M, Vin‐ tersten K, Behringer R. Isolation and culture of blastocyst-derived stem cell line, Ma‐ nipulating the Mouse Embryo: A Laboratory Manual, 3 edition. New York: Cold

[26] Robertson EJ. Embryo-derived stem cell lines. In: Robertson EJ (ed), Teratocarcino‐ mas and Embryonic Stem Cells: A Practical Approach. Oxford UK: IRL Press; 1987.

[27] Hogan B, Costantini F, Beddington R, Lacy E. Isolation, culture, and manipulation of embryonic stem cells, manipulating the Mouse Embryo: A Laboratory Manual, 2 edi‐

[28] Goldsborough MD, Tilkins ML, Price PJ, Lobo-Alfonso J, Morrison JR, Stevens ME, Meneses J, Pederson R, Koller R, Koller B, Latour A. Serum-free culture of murine

[29] Ying QL, Smith AG. Defined conditions for neural commitment and differentiation.

[30] Nichols J, Ying QL. Derivation and propagation of embryonic stem cells in serum-

[31] Lee KH, Chuang CK, Guo SF, Tu CF. Simple and efficient derivation of mouse em‐ bryonic stem cell lines using differentiation inhibitors or proliferation stimulators.

[32] Brook FA, Gardner RL. The origin and efficient derivation of embryonic stem cells in the mouse. Proceedings of the National Academy of Sciences of the United States of

[33] Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl M, Rogers D. Inhibi‐ tion of pluripotential embryonic stem cell differentiation by purified polypeptides.

and feeder-free culture. Methods in Molecular Biology 2006;329: 91-98.

tion. Plainview: Cold Spring Harbor Lab. Press; 1994. p253-290.

embryonic stem (ES) cells. FOCUS (Gibco) 1998;20: 8-12.

Methods in Enzymology 2003;365:327-41.: 327-341.

Stem Cells and Development 2012;21: 373-383.

America 1997;94: 5709-5712.

Nature 1988;336: 688-690.

Academy of Sciences of the United States of America 1981;78: 7634-7638.

Development, Growth & Differentiation 2003;45: 427-434.

reprogramming. Cell 2008;132: 567-582.

Spring Harbor Lab. Press; 2003. p359-397.

p71-112.

embryos. Nature 1981;292: 154-156.


[21] Iwasaki S, Kimura H. Further studies on thermal treatment of two-cell stage embryos to produce complete embryonic stem-cell-derived mice by cell-aggregation methods. Development, Growth & Differentiation 2003;45: 427-434.

[9] Fehilly CB, Willadsen SM. Embryo manipulation in farm animals. Oxford Reviews of

[10] Willadsen SM. A method for culture of micromanipulated sheep embryos and its use

[11] Beddington RS, Robertson EJ. An assessment of the developmental potential of em‐ bryonic stem cells in the midgestation mouse embryo. Development 1989;105:

[12] Tesar PJ, Chenoweth JG, Brook FA, Davies TJ, Evans EP, Mack DL, Gardner RL, McKay RDG. New cell lines from mouse epiblast share defining features with human

[13] Tanaka S, Kunath T, Hadjantonakis AK, Nagy A, Rossant J. Promotion of trophoblast

[14] Brons, I. G, Smithers, L. E, Trotter M. W.B, Rugg-Gunn P, Sun B, Chuva de Sousa Lopes S, Howlett S. K, Clarkson A, hrlund-Richter L, Pedersen R. A, & Vallier, L. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature

[15] Nagy A, Gocza E, Diaz EM, Prideaux VR, Ivanyi E, Markkula M, Rossant J. Embry‐ onic stem cells alone are able to support fetal development in the mouse. Develop‐

[16] Nagy A, Rossant J, Nagy R, bramow-Newerly W, Roder JC. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America 1993;90: 8424-8428.

[17] Poueymirou WT, Auerbach W, Frendewey D, Hickey JF, Escaravage JM, Esau L, Dore AT, Stevens S, Adams NC, Dominguez MG, Gale NW, Yancopoulos GD, De‐ Chiara TM, Valenzuela DM. F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nature Biotechnolo‐

[18] Lee, K-H. Methods to generate chimeric mice from embryonic stem cells. In: Kallos MS (ed), Embryonic Stem Cells: Basic Biology to Bioengineering. Rijeka: InTech;

[19] Modlinski JA, Reed MA, Wagner TE, Karasiewicz J. Embryonic stem cells: Develop‐ mental capabilities and their possible use in mammalian embryo cloning. Animal Re‐

[20] Amano T, Kato Y, Tsunoda Y. Comparison of heat-treated and tetraploid blastocysts for the production of completely ES-cell-derived mice. Zygote 2001;9: 153-157.

Reproductive Biology 1986;8: 379-413.

733-737.

104 Pluripotent Stem Cells

2007;448: 191-195.

gy 2007;25: 91-99.

2011. p193-212.

production Science 1996;42: 437-446.

ment 1990;110: 815-821.

to produce monozygotic twins. Nature 1979;277: 298-300.

stem cell proliferation by FGF4. Science 1998;282: 2072-2075.

embryonic stem cells. Nature 2007;448: 196-199.


[34] Williams RL, Hilton DJ, Pease S, Willson TA, Stewart CL, Gearing DP, Wagner EF, Metcalf D, Nicola NA, Gough NM. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 1988;336: 684-687.

[47] Efe JA, Ding S. The evolving biology of small molecules: Controlling cell fate and identity. Philosophical Transactions of the Royal Society of London Series B: Biologi‐

Conditions and Techniques for Mouse Embryonic Stem Cell Derivation and Culture

http://dx.doi.org/10.5772/55105

107

[48] Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A. The ground state of embryonic stem cell self-renewal. Nature 2008;453: 519-523.

[49] Buehr M, Meek S, Blair K, Yang J, Ure J, Silva J, McLay R, Hall J, Ying QL, Smith A. Capture of authentic embryonic stem cells from rat blastocysts. Cell 2008;135:

[50] Li P, Tong C, Mehrian-Shai R, Jia L, Wu N, Yan Y, Maxson RE, Schulze EN, Song H, Hsieh CL, Pera MF, Ying QL. Germline competent embryonic stem cells derived

[51] Silva J, Barrandon O, Nichols J, Kawaguchi J, Theunissen TW, Smith A. Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biology

[52] Hassani S-N, Totonchi M, Farrokhi A, Taei A, Larijani MR, Gourabi H, Baharvand H. Simultaneous suppression of TGF-β and ERK signaling contributes to the highly effi‐ cient and reproducible Generation of mouse embryonic stem cells from previously considered refractory and non-permissive strains. Stem Cell Reviews and Reports

[53] Gertsenstein M, Nutter LM, Reid T, Pereira M, Stanford WL, Rossant J, Nagy A. Effi‐ cient generation of germ line transmitting chimeras from C57BL/6N ES cells by ag‐

[54] Leeb M, Wutz A. Derivation of haploid embryonic stem cells from mouse embryos.

[55] Pieters T, Haenebalcke L, Hochepied T, D'Hont, J, Haigh, J. J, Van Roy, F, & Van Hengel, J. Efficient and user-friendly pluripotin-based derivation of mouse embryon‐

[56] Schoonjans L, Kreemers V, Danloy S, Moreadith RW, Laroche Y, Collen D. Improved generation of germline-competent embryonic stem cell lines from inbred mouse

[57] Yang W, Wei W, Shi C, Zhu J, Ying W, Shen Y, Ye X, Fang L, Duo S, Che J, Shen H, Ding S, Deng H. Pluripotin combined with leukemia inhibitory factor greatly pro‐ motes the derivation of embryonic stem cell lines from refractory strains. Stem Cells

[58] Bryja V, Bonilla S, Cajanek L, Parish CL, Schwartz CM, Luo Y, Rao MS, Arenas E. An efficient method for the derivation of mouse embryonic stem cells. Stem Cells

gregation with outbred host embryos. PLoS ONE 2010;5: e11260.

ic stem cells. Stem Cell Reviews and Reports 2012;8: 768-778.

cal Sciences 2011;366: 2208-2221.

from rat blastocysts. Cell 2008;135: 1299-1310.

1287-1298.

2008;6: 2237-2247.

2012;8: 472-481.

Nature 2011;479: 131-134.

strains. Stem Cells 2003;21: 90-97.

2009;27: 383-389.

2006;24: 844-849.


[47] Efe JA, Ding S. The evolving biology of small molecules: Controlling cell fate and identity. Philosophical Transactions of the Royal Society of London Series B: Biologi‐ cal Sciences 2011;366: 2208-2221.

[34] Williams RL, Hilton DJ, Pease S, Willson TA, Stewart CL, Gearing DP, Wagner EF, Metcalf D, Nicola NA, Gough NM. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 1988;336: 684-687.

[35] Kawase E, Suemori H, Takahashi N, Okazaki K, Hashimoto K, Nakatsuji N. Strain difference in establishment of mouse embryonic stem (ES) cell lines. International

[36] McWhir J, Schnieke AE, Ansell R, Wallace H, Colman A, Scott AR, Kind AJ. Selective ablation of differentiated cells permits isolation of embryonic stem cell lines from murine embryos with a non-permissive genetic background. Nature Genetics

[37] Nagafuchi S, Katsuta H, Kogawa K, Akashi T, Kondo S, Sakai Y, Tsukiyama T, Kita‐ mura D, Niho Y, Watanabe T. Establishment of an embryonic stem (ES) cell line de‐ rived from a non-obese diabetic (NOD) mouse: In vivo differentiation into lymphocytes and potential for germ line transmission. FEBS Letters 1999;455:

[38] Suzuki O, Matsuda J, Takano K, Yamamoto Y, Asano T, Naiki M, Kusanagi M. Effect of genetic background on establishment of mouse embryonic stem cells. Experimen‐

[39] Brevini, T. A. L, Antonini, S, Cillo, F, Crestan, M, & Gandolfi, F. Porcine embryonic stem cells: Facts, challenges and hopes. Theriogenology 2007;68: S1, 206-213.

[40] Keefer CL, Pant D, Blomberg L, Talbot NC. Challenges and prospects for the estab‐ lishment of embryonic stem cell lines of domesticated ungulates. Animal Reproduc‐

[41] Burdon T, Smith A, Savatier P. Signalling, cell cycle and pluripotency in embryonic

[42] Liu N, Lu M, Tian X, Han Z. Molecular mechanisms involved in self-renewal and pluripotency of embryonic stem cells. Journal of Cellular Physiology 2007;211:

[43] Chen S, Do JT, Zhang Q, Yao S, Yan F, Peters EC, Scholer HR, Schultz PG, Ding S. Self-renewal of embryonic stem cells by a small molecule. Proceedings of the Nation‐

al Academy of Sciences of the United States of America 2006;103: 17266-17271.

[44] Emre N, Coleman R, Ding S. A chemical approach to stem cell biology. Current

[45] Li W, Ding S. Small molecules that modulate embryonic stem cell fate and somatic

[46] Sumer H, Liu J, Verma PJ. The use of signalling pathway inhibitors and chromatin

cell reprogramming. Trends in Pharmacological Sciences 2010;31: 36-45.

modifiers for enhancing pluripotency. Theriogenology 2010;74: 525-533.

Journal of Developmental Biology 1994;38: 385-390.

1996;14: 223-226.

tal Animals 1999;48: 213-216.

tion Science 2007;98: 147-168.

stem cells. Trends in Cell Biology 2002;12: 432-438.

Opinion in Chemical Biology 2007;11: 252-258.

101-104.

106 Pluripotent Stem Cells

279-286.


[59] Wakayama S, Hikichi T, Suetsugu R, Sakaide Y, Bui HT, Mizutani E, Wakayama T. Efficient establishment of mouse embryonic stem cell lines from single blastomeres and polar bodies. Stem Cells 2007;25: 986-993.

[71] Li M, Ma W, Hou Y, Sun XF, Sun QY, Wang WH. Improved isolation and culture of embryonic stem cells from Chinese miniature pig. Journal of Reproduction and De‐

Conditions and Techniques for Mouse Embryonic Stem Cell Derivation and Culture

http://dx.doi.org/10.5772/55105

109

[72] Richards M, Tan S, Fong CY, Biswas A, Chan WK, Bongso A. Comparative evalua‐ tion of various human feeders for prolonged undifferentiated growth of human em‐

[73] Amit M, Margulets V, Segev H, Shariki K, Laevsky I, Coleman R, Itskovitz-Eldor J. Human feeder layers for human embryonic stem cells. Biology of Reproduction

[74] Ellerstrom C, Strehl R, Noaksson K, Hyllner J, Semb H. Facilitated expansion of hu‐ man embryonic stem cells by single-cell enzymatic dissociation. Stem Cells 2007;25:

[75] Hovatta O, Mikkola M, Gertow K, Stromberg AM, Inzunza J, Hreinsson J, Rozell B, Blennow E, Andang M, hrlund-Richter L. A culture system using human foreskin fi‐ broblasts as feeder cells allows production of human embryonic stem cells. Human

[76] Ma Y, Gu J, Li C, Wei X, Tang F, Shi G, Jiang J, Kuang Y, Li J, Wang Z, Xie X, Jin Y. Human foreskin fibroblast produces interleukin-6 to support derivation and self-re‐ newal of mouse embryonic stem cells. Stem Cell Research & Therapy 2012;3: 29.

[77] Meng, G. L, Zur Nieden, N. I, Liu, S. Y, Cormier, J. T, Kallos, M. S, & Rancourt, D. E. Properties of murine embryonic stem cells maintained on human foreskin fibroblasts

[78] Bryja V, Bonilla S, Arenas E. Derivation of mouse embryonic stem cells. Nature Pro‐

[79] Eiselleova L, Peterkova I, Neradil J, Slaninova I, Hampl A, Dvorak P. Comparative study of mouse and human feeder cells for human embryonic stem cells. Internation‐

[80] Villa-Diaz, LG, Pacut C, Slawny NA, Ding, J, O'Shea, K. S, & Smith, G. D. Analysis of the factors that limit the ability of feeder cells to maintain the undifferentiated state of human embryonic stem cells. Stem Cells and Development 2009;18: 641-651.

[81] Lim JW, Bodnar A. Proteome analysis of conditioned medium from mouse embryon‐ ic fibroblast feeder layers which support the growth of human embryonic stem cells.

[82] Suemori H, Nakatsuji N. Establishment of the embryo-derived stem (ES) cell lines from mouse blastocysts: Effects of the feeder cell layer. Development, Growth & Dif‐

without LIF. Molecular Reproduction and Development 2008;75: 614-622.

al Journal of Developmental Biology 2008;52: 353-363.

velopment 2004;50: 237-244.

Reproduction 2003;18: 1404-1409.

tocols 2006;1: 2082-2087.

Proteomics 2002;2: 1187-1203.

ferentiation 1987;29: 133-139.

2003;68: 2150-2156.

1690-1696.

bryonic stem cells. Stem Cells 2003;21: 546-556.


[71] Li M, Ma W, Hou Y, Sun XF, Sun QY, Wang WH. Improved isolation and culture of embryonic stem cells from Chinese miniature pig. Journal of Reproduction and De‐ velopment 2004;50: 237-244.

[59] Wakayama S, Hikichi T, Suetsugu R, Sakaide Y, Bui HT, Mizutani E, Wakayama T. Efficient establishment of mouse embryonic stem cell lines from single blastomeres

[60] Gonzalez S, Ibanez E, Santalo J. Influence of E-cadherin-mediated cell adhesion on mouse embryonic stem cells derivation from isolated blastomeres. Stem Cell Reviews

[61] Cheng J, Dutra A, Takesono A, Garrett-Beal L, Schwartzberg PL. Improved genera‐ tion of C57BL/6J mouse embryonic stem cells in a defined serum-free media. Genesis

[62] Ogawa K, Matsui H, Ohtsuka S, Niwa H. A novel mechanism for regulating clonal

[63] Vanroose, G, Soom, A. V, Kruif, A. De. From co-culture to defined medium: State of the art and practical considerations. Reproduction in Domestic Animals 2001;36:

[64] Mannello F, Tonti GA. No breakthroughs for human mesenchymal and embryonic stem cell culture: Conditioned medium, feeder layer, or feeder-free; medium with fe‐ tal calf serum, human serum, or enriched plasma; serum-free, serum replacement nonconditioned medium, or ad hoc formula? All that glitters is not gold! Stem Cells

[65] Ying QL, Nichols J, Chambers I, Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with

[66] Martin GR, Evans MJ. The morphology and growth of a pluripotent teratocarcinoma

[67] Martin GR, Evans MJ. Differentiation of clonal lines of teratocarcinoma cells: forma‐ tion of embryoid bodies in vitro. Proceedings of the National Academy of Sciences of

[68] Martin GR, Evans MJ. Multiple differentiation of clonal teratocarcinoma stem sells

[69] Park JH, Kim SJ, Oh EJ, Moon SY, Roh SI, Kim CG, Yoon HS. Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell

[70] Anderson RE, Pogue L, Troup GM, Standefer JC. A comparison of irradiation and mitomycin as blocking agents in the mixed lymphocyte reaction. Archives of Pathol‐

cell line and its derivatives in tissue culture. Cell 1974;2: 163-172.

following embryoid body formation in vitro. Cell 1975;6: 467–474.

the United States of America 1975;72: 1441-1445.

line. Biology of Reproduction 2003;69: 2007-2014.

ogy & Laboratory Medicine 1984;108: 363-367.

propagation of mouse ES cells. Genes to Cells 2004;9: 471-477.

and polar bodies. Stem Cells 2007;25: 986-993.

and Reports 2011;7: 494-505.

2004;39: 100-104.

2007;25: 1603-1609.

STAT3. Cell 2003;115: 281-292.

25-28.

108 Pluripotent Stem Cells


[83] Richards, M, Fong, C. Y, Chan, W. K, Wong, P. C, & Bongso A. Human feeders sup‐ port prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nature Biotechnology 2002;20: 933-936.

[95] Kahan BW, Ephrussi B. Developmental potentialities of clonal in vitro cultures of mouse testicular teratoma. Journal of the National Cancer Institute 1970;44:

Conditions and Techniques for Mouse Embryonic Stem Cell Derivation and Culture

http://dx.doi.org/10.5772/55105

111

[96] Rosenthal MD, Wishnow RM, Sato GH. In vitro growth and differetiation of clonal populations of multipotential mouse cells derived from a transplantable testicular

[97] Strickland S, Mahdavi V. The induction of differentiation in teratocarcinoma stem

[98] Hogan B, Costantini F, Beddington R, Lacy E, Hogan B, Costantini F, Beddington R, Lacy E. In vitro manipulation of preimplantation embryos, Manipulating the Mouse Embryo: A Laboratory Manual, 2 edition. Plainview: Cold Spring Harbor Lab. Press;

[99] Solter D. From teratocarcinomas to embryonic stem cells and beyond: A history of

[100] Stevens LC, Little CC. Spontaneous testicular teratomas in an inbred strain of mice. Proceedings of the National Academy of Sciences of the United States of America

[101] Ohta H, Ohinata Y, Ikawa M, Morioka Y, Sakaide Y, Saitou M, Kanagawa O, Wa‐ kayama T. Male germline and embryonic stem cell lines from NOD mice: Efficient derivation of GS cells from a nonpermissive strain for ES cell derivation. Biology of

[102] Roach ML, Stock JL, Byrum R, Koller BH, McNeish JD. A new embryonic stem cell line from DBA/1lacJ mice allows genetic modification in a murine model of human

[103] Ledermann B, Burki K. Establishment of a germ-line competent C57BL/6 embryonic

[104] Auerbach W, Dunmore JH, Fairchild-Huntress V, Fang Q, Auerbach AB, Huszar D, Joyner AL. Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived

[105] Eistetter HR. Pluripotent embryonal stem cell lines can be established from disaggre‐ gated mouse morulae. Development, Growth & Differentiation 1989;31: 275-282.

[106] Keskintepe L, Norris K, Pacholczyk G, Dederscheck SM, Eroglu A. Derivation and comparison of C57BL/6 embryonic stem cells to a widely used 129 embryonic stem

[107] Lemckert FA, Sedgwick JD, Korner H. Gene targeting in C57BL/6 ES cells. Successful germ line transmission using recipient BALB/c blastocysts developmentally matured

inflammation. Experimental Cell Research 1995;221: 520-525.

stem cell line. Experimental Cell Research 1991;197: 254-258.

cell line. Transgenic Research 2007;16: 751-758.

in vitro. Nucleic Acids Research 1997;25: 917-918.

mouse embryonic stem cell lines. Biotechniques 2000;29: 1024-1032.

embryonic stem cell research. Nature Reviews Genetics 2006;7: 319-327.

teratocarcinoma. Journal of the National Cancer Institute 1970;44: 1001-1014.

cells by retinoic acid. Cell 1978;15: 393-403.

1015-1036.

1994. p191-206.

1954;40: 1080-1087.

Reproduction 2009;81: 1147-1153.


[95] Kahan BW, Ephrussi B. Developmental potentialities of clonal in vitro cultures of mouse testicular teratoma. Journal of the National Cancer Institute 1970;44: 1015-1036.

[83] Richards, M, Fong, C. Y, Chan, W. K, Wong, P. C, & Bongso A. Human feeders sup‐ port prolonged undifferentiated growth of human inner cell masses and embryonic

[84] Kim H, You S, Farris J, Kong BW, Christman SA, Foster LK, Foster DN. Expression profiles of p53-, p16INK4a-, and telomere-regulating genes in replicative senescent pri‐ mary human, mouse, and chicken fibroblast cells. Experimental Cell Research

[85] Suda Y, Suzuki M, Ikawa Y, Aizawa S. Mouse embryonic stem cells exhibit indefinite

[86] Cobo F, Cortes JL, Cabrera C, Nieto A, Concha A. Microbiological contamination in

[87] Markoullis K, Bulian D, Holzlwimmer G, Quintanilla-Martinez L, Heiliger KJ, Zit‐ zelsberger H, Scherb H, Mysliwietz J, Uphoff CC, Drexler HG, Adler T, Busch DH, Schmidt J, Mahabir E. Mycoplasma contamination of murine embryonic stem cells affects cell parameters, germline transmission and chimeric progeny. Transgenic Re‐

[88] McMahon AP, Bradley A. The Wnt-1 (int-1) proto-oncogene is required for develop‐

[89] Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluri‐

[90] Zandstra PW, Le HV, Daley GQ, Griffith LG, Lauffenburger DA. Leukemia inhibito‐ ry factor (LIF) concentration modulates embryonic stem cell self-renewal and differ‐ entiation independently of proliferation. Biotechnology and Bioengineering 2000;69:

[91] Iijima S, Tanimoto Y, Mizuno S, Daitoku Y, Kunita S, Sugiyama F, Yagami K. Effect of different culture conditions on establishment of embryonic stem cells from

[92] Davies TJ, Fairchild PJ. Optimization of protocols for derivation of mouse embryonic stem cell lines from refractory strains, including the non obese diabetic mouse. Stem

[93] Baharvand H, Matthaei KI. Culture condition difference for establishment of new embryonic stem cell lines from the C57BL/6 and BALB/c mouse strains. In Vitro Cel‐

[94] Evans MJ. The isolation and properties of a clonal tissue culture strain of pluripotent mouse teratoma cells. Journal of Embryology and Experimental Morphology 1972;28:

BALB/cAJ and NZB/BINJ mice. Cellular Reprogramming 2010;12: 679-688.

proliferative potential. Journal of Cellular Physiology 1987;133: 197-201.

stem cell cultures. Cell Biology International 2007;31: 991-995.

ment of a large region of the mouse brain. Cell 1990;62: 1073-1085.

potent stem cells. Nature 2007;448: 313-317.

Cells and Development 2012;21: 1688-1700.

lular & Developmental Biology: Animal 2004;40: 76-81.

stem cells. Nature Biotechnology 2002;20: 933-936.

2002;272: 199-208.

110 Pluripotent Stem Cells

search 2009;18: 71-87.

607-617.

163-176.


[108] Shimizukawa R, Sakata A, Hirose M, Takahashi A, Iseki H, Liu Y, Kunita S, Sugiya‐ ma F, Yagami K. Establishment of a new embryonic stem cell line derived from C57BL/6 mouse expressing EGFP ubiquitously. Genesis 2005;42: 47-52.

[120] Lorthongpanich C, Yang SH, Piotrowska-Nitsche, K, Parnpai, R. & Chan, A. W. S. Development of single mouse blastomeres into blastocysts, outgrowths and the es‐

Conditions and Techniques for Mouse Embryonic Stem Cell Derivation and Culture

http://dx.doi.org/10.5772/55105

113

[121] Gonzalez S, Ibanez E, Santalo J. Establishment of mouse embryonic stem cells from isolated blastomeres and whole embryos using three derivation methods. Journal of

[122] Gonzalez S, Ibanez E, Santalo J. Influence of early fate decisions at the two-cell stage on the derivation of mouse embryonic stem cell lines. Stem Cell Research 2011;7:

[123] Zhang H, Wang ZZ. Mechanisms that mediate stem cell self-renewal and differentia‐

[124] Boiani M, Scholer HR. Regulatory networks in embryo-derived pluripotent stem

[125] Hirai H, Karian P, Kikyo N. Regulation of embryonic stem cell self-renewal and plu‐ ripotency by leukaemia inhibitory factor. Biochemical Journal 2011;438: 11-23.

[126] Kolch W. Meaningful relationships: The regulation of the Ras/Raf/MEK/ERK path‐

[127] Qi X, Li TG, Hao J, Hu J, Wang J, Simmons H, Miura S, Mishina Y, Zhao GQ. BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated pro‐ tein kinase pathways. Proceedings of the National Academy of Sciences of the Unit‐

[128] Burdon T, Stracey C, Chambers I, Nichols J, Smith A. Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells. Developmental Bi‐

[129] Lodge P, McWhir J, Gallagher E, Sang H. Increased gp130 signaling in combination with inhibition of the MEK/ERK pathway facilitates embryonic stem cell isolation from normally refractory murine CBA blastocysts. Cloning Stem Cells 2005;7: 2-7.

[130] Niwa H, Burdon T, Chambers I, Smith A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes & Development 1998;12:

[131] Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl M, Rogers D. Inhibi‐ tion of pluripotential embryonic stem cell differentiation by purified polypeptides.

[132] Matsuda T, Nakamura T, Nakao K, Arai T, Katsuki M, Heike T, Yokota T. STAT3 ac‐ tivation is sufficient to maintain an undifferentiated state of mouse embryonic stem

tablishment of embryonic stem cells. Reproduction 2008;135: 805-813.

Assisted Reproduction and Genetics 2010;27: 671-682.

tion. Journal of Cellular Biochemistry 2008;103: 709-718.

ed States of America 2004;101: 6027-6032.

ology 1999;210: 30-43.

Nature 1988;336: 688-690.

cells. EMBO Journal 1999;18: 4261-4269.

2048-2060.

cells. Nature Reviews Molecular Cell Biology 2005;6: 872-881.

way by protein interactions. Biochemical Journal 2000;351: 289-305.

54-65.


[120] Lorthongpanich C, Yang SH, Piotrowska-Nitsche, K, Parnpai, R. & Chan, A. W. S. Development of single mouse blastomeres into blastocysts, outgrowths and the es‐ tablishment of embryonic stem cells. Reproduction 2008;135: 805-813.

[108] Shimizukawa R, Sakata A, Hirose M, Takahashi A, Iseki H, Liu Y, Kunita S, Sugiya‐ ma F, Yagami K. Establishment of a new embryonic stem cell line derived from

[109] Tanimoto Y, Iijima S, Hasegawa Y, Suzuki Y, Daitoku Y, Mizuno S, Ishige T, Kudo T, Takahashi S, Kunita S, Sugiyama F, Yagami K. Embryonic stem cells derived from

[110] Tesar PJ. Derivation of germ-line-competent embryonic stem cell lines from preblas‐ tocyst mouse embryos. Proceedings of the National Academy of Sciences of the Unit‐

[111] Mann JR, Gadi I, Harbison ML, Abbondanzo SJ, Stewart CL. Androgenetic mouse embryonic stem cells are pluripotent and cause skeletal defects in chimeras: Implica‐

[112] Voss AK, Thomas T, Gruss P. Germ line chimeras from female ES cells. Experimental

[113] Wakayama, T, Tabar, V, Rodriguez, I, Perry, A. C. F, Studer, L, & Mombaerts, P. Dif‐ ferentiation of embryonic stem cell lines generated from adult somatic cells by nucle‐

[114] Eggan K, Rode A, Jentsch I, Samuel C, Hennek T, Tintrup H, Zevnik B, Erwin J, Lor‐ ing J, Jackson-Grusby L, Speicher MR, Kuehn R, Jaenisch R. Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complemen‐

[115] Chen Z, Liu Z, Huang J, Amano T, Li C, Cao S, Wu C, Liu B, Zhou L, Carter MG, Keefe DL, Yang X, Liu L. Birth of parthenote mice directly from parthenogenetic em‐

[116] Li W, Shuai L, Wan H, Dong M, Wang M, Sang L, Feng C, Luo G-Z, Li T, Li X, Wang L, Zheng, Q.-Y, Sheng, C, Wu, H.-J, Liu, Z, Liu, L, Wang, L, Wang, X.-J, Zhao, X.-Y, Zhou Q. Androgenetic haploid embryonic stem cells produce live transgenic mice.

[117] Chung Y, Klimanskaya I, Becker S, Marh J, Lu SJ, Johnson J, Meisner L, Lanza R. Em‐ bryonic and extraembryonic stem cell lines derived from single mouse blastomeres.

[118] Delhaise F, Bralion V, Schuurbiers N, Dessy F. Establishment of an embryonic stem cell line from 8-cell stage mouse embryos. European Journal of morphology 1996;34:

[119] Klimanskaya I, Chung Y, Becker S, Lu SJ, Lanza R. Human embryonic stem cell lines

derived from single blastomeres. Nature 2006;444: 481-485.

C57BL/6 mouse expressing EGFP ubiquitously. Genesis 2005;42: 47-52.

C57BL/6J and C57BL/6N mice. Comparative Medicine 2008;58: 347-352.

ed States of America 2005;102: 8239-8244.

Cell Research 1997;230: 45-49.

112 Pluripotent Stem Cells

Nature (2012)., 490, 407-411.

Nature 2006;439: 216-219.

237-243.

ar transfer. Science 2001;292: 740-743.

tation. Nature Biotechnology 2002;20: 455-459.

bryonic stem cells. Stem Cells 2009;27: 2136-2145.

tions for genetic imprinting. Cell 1990;62: 251-260.


[133] Rayasam GV, Tulasi VK, Sodhi R, Davis JA, Ray A. Glycogen synthase kinase 3: More than a namesake. British Journal of Pharmacology 2009;156: 885-898.

[146] Stadtfeld M, Hochedlinger K. Induced pluripotency: History, mechanisms, and ap‐

Conditions and Techniques for Mouse Embryonic Stem Cell Derivation and Culture

http://dx.doi.org/10.5772/55105

115

[148] Buehr M, Smith A. Genesis of embryonic stem cells. Philosophical Transactions of the Royal Society of London Series B: Biological Sciences 2003;358: 1397-1402.

[149] Kunath T, Saba-El-Leil MK, Almousailleakh M, Wray J, Meloche S, Smith A. FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embry‐ onic stem cells from self-renewal to lineage commitment. Development 2007;134:

[150] Frame S, Cohen P. GSK3 takes centre stage more than 20 years after its discovery. Bi‐

[151] Lorthongpanich C, Yang SH, Piotrowska-Nitsche K, Parnpai R, Chan AW. Chemical enhancement in embryo development and stem cell derivation from single blasto‐

[147] Rossant J. Stem cells and early lineage development. Cell 2008;132: 527-531.

plications. Genes & Development 2010;24: 2239-2263.

2895-2902.

ochemical Journal 2001;359: 1-16.

meres. Cloning Stem Cells 2008;10: 503-512.


[133] Rayasam GV, Tulasi VK, Sodhi R, Davis JA, Ray A. Glycogen synthase kinase 3: More than a namesake. British Journal of Pharmacology 2009;156: 885-898.

[134] Umehara H, Kimura T, Ohtsuka S, Nakamura T, Kitajima K, Ikawa M, Okabe M, Ni‐ wa H, Nakano T. Efficient derivation of embryonic stem cells by inhibition of glyco‐

[135] Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH. Maintenance of pluripo‐ tency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nature Medicine 2004;10: 55-63.

[136] Xu RH, Chen X, Li DS, Li R, Addicks GC, Glennon C, Zwaka TP, Thomson JA. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nature Biotech‐

[137] Takahashi K, Mitsui K, Yamanaka S. Role of ERas in promoting tumour-like proper‐

[138] Takahashi K, Murakami M, Yamanaka S. Role of the phosphoinositide 3-kinase path‐ way in mouse embryonic stem (ES) cells. Biochemical Society Transactions 2005;33:

[139] Paling NR, Wheadon H, Bone HK, Welham MJ. Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling. Journal of Biological

[140] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science

[141] Xu C. Feeder-free growth of undifferentiated human embryonic stem cells. Nature

[142] Amit M. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Developmental Biology

[143] Dvorak P, Dvorakova D, Koskova S, Vodinska M, Najvirtova M, Krekac D, Hampl A. Expression and potential role of fibroblast growth factor 2 and its receptors in human

[144] Wang G, Zhang H, Zhao Y, Li J, Cai J, Wang P, Meng S, Feng J, Miao C, Ding M, Li D, Deng H. Noggin and bFGF cooperate to maintain the pluripotency of human em‐ bryonic stem cells in the absence of feeder layers. Biochemical and Biophysical Re‐

[145] Xu RH, Peck RM, Li DS, Feng X, Ludwig T, Thomson JA. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nature

embryonic stem cells. Stem Cells 2005;23: 1200-1211.

search Communications 2005;330: 934-942.

ties in mouse embryonic stem cells. Nature 2003;423: 541-545.

gen synthase kinase-3. Stem Cells 2007;25: 2705-2711.

nology 2002;20: 1261-1264.

Chemistry 2004;279: 48063-48070.

Biotechnology 2001;19: 971-974.

1998;282: 1145-1147.

2000;227: 271-278.

Methods 2005;2: 185-190.

1522-1525.

114 Pluripotent Stem Cells


**Chapter 6**

**Human Testis–Derived Pluripotent Cells and Induced**

Pregnancy rates achieved by intercourse in normal human couples are 20-25% per month, 75% by six months, and 90% by one year [1]. However, 15% of couples of unknown fertility status are unable to conceive a baby after one year of intercourse without contraception. For 30% of these couples, their infertility can be attributed to a male factor alone; in an additional 20%, failure to conceive is explained by the presence of both male and female factors [2,3,4]. Among couples known to be infertile, a male factor is involved in 50% of the cases. The most common causes of male infertility include abnormal sperm production or function, impaired delivery of sperm, and overexposure to certain gonadotoxins in the environment. The pathogenesis of male infertility can be attributed to a disorder of germ-cell proliferation and differentiation or

The induction of spermatogenesis depends on the complementary actions of FSH and testosterone. FSH establishes the requisite Sertoli cell population. In the prepubertal primate, FSH alone can induce proliferation of Sertoli cells and spermatogonia, but this does not result in qualitatively and quantitatively normal spermatogenesis unless testosterone is simultane‐ ously present [6] [7]. Testosterone affects the functional completion of meiosis and post-meiotic sperm differentiation and maturation. LH stimulates Leydig cells to produce testosterone. Although FSH appears to play a more dominant role in the maintenance of primate sperma‐ togenesis than in its initiation, normal spermatogenesis is best maintained by the combined

The most severe form of male infertility is nonobstructive azoospermia, which is typically characterized by small-volume testes and elevated FSH. Patients with this disorder cannot

> © 2013 Kobayashi et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Kobayashi et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Pluripotent Stem Cells**

Koichi Nakajima

**1. Introduction**

http://dx.doi.org/10.5772/55570

to somatic cell dysfunction [5].

effects of FSH and LH [6].

Hideyuki Kobayashi, Koichi Nagao and

Additional information is available at the end of the chapter

## **Human Testis–Derived Pluripotent Cells and Induced Pluripotent Stem Cells**

Hideyuki Kobayashi, Koichi Nagao and Koichi Nakajima

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55570

## **1. Introduction**

Pregnancy rates achieved by intercourse in normal human couples are 20-25% per month, 75% by six months, and 90% by one year [1]. However, 15% of couples of unknown fertility status are unable to conceive a baby after one year of intercourse without contraception. For 30% of these couples, their infertility can be attributed to a male factor alone; in an additional 20%, failure to conceive is explained by the presence of both male and female factors [2,3,4]. Among couples known to be infertile, a male factor is involved in 50% of the cases. The most common causes of male infertility include abnormal sperm production or function, impaired delivery of sperm, and overexposure to certain gonadotoxins in the environment. The pathogenesis of male infertility can be attributed to a disorder of germ-cell proliferation and differentiation or to somatic cell dysfunction [5].

The induction of spermatogenesis depends on the complementary actions of FSH and testosterone. FSH establishes the requisite Sertoli cell population. In the prepubertal primate, FSH alone can induce proliferation of Sertoli cells and spermatogonia, but this does not result in qualitatively and quantitatively normal spermatogenesis unless testosterone is simultane‐ ously present [6] [7]. Testosterone affects the functional completion of meiosis and post-meiotic sperm differentiation and maturation. LH stimulates Leydig cells to produce testosterone. Although FSH appears to play a more dominant role in the maintenance of primate sperma‐ togenesis than in its initiation, normal spermatogenesis is best maintained by the combined effects of FSH and LH [6].

The most severe form of male infertility is nonobstructive azoospermia, which is typically characterized by small-volume testes and elevated FSH. Patients with this disorder cannot

© 2013 Kobayashi et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Kobayashi et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

produce biological children. Although microdissection testicular sperm extraction (micro-TESE) is used to treat patients with nonobstructive azoospermia [8], this technique does not have a good success rate. Therefore, new approaches are needed to develop treatments for male infertility.

Stem cells have the potential to differentiate into a variety of functional cell types in the body, and their discovery has given rise to the fields of regenerative medicine and cloning. Stem cells are regulated by the particular microenvironment in which they reside; these microenviron‐ ments are referred to as niches. Male germline stem cells can continuously produce sperm throughout adulthood, and investigators have sought to develop methods using stem cells to improve or restore fertility.

Embryonic stem cells (ESCs) have the potential to differentiate into nearly every cell type in the body. As the cells differentiate, they lose the ability to develop into different tissues. In contrast, specific tissues (gastrointestinal, integumentary, spermatogenic, and hematopoietic systems) maintain their regenerative capacity *in vivo*, and in fact, stem cells have been functionally identified in a wide range of adult tissues. These adult stem cells are believed to hold great promise for tissue generation in clinical settings. Here, we provide a summary of the therapeutic potential of stem cells for the rejuvenation of fertility in infertile males. Our hope is that future research will provide a range of options for the preservation of male fertility or the reversal of infertility.

the stem cell pool, while their differentiation generates a large number of germ cells. Therefore, a balance between SSC self-renewal and differentiation in the adult testis is essential to maintain normal spermatogenesis and fertility throughout life. SSCs need to reside in a unique environment, or niche, that provides the factors necessary for their survival and potency. In mice, Sertoli cells in the testis are a crucial component of the spermatogonial stem cell niche. They produce glial cell line-derived neurotrophic factor (GDNF), a distant member of the TGFβ family, which controls SSC self-renewal [16]. Several groups have reported that adding GDNF to freshly isolated germ cells in culture results in the proliferation of SSCs [17,18]. Other factors within the niche influence the fate of SSCs. One example is colony-stimulating factor 1 (CSF1), which is produced by Leydig cells and some peritubular myoid cells [19], and plays a

Human Testis–Derived Pluripotent Cells and Induced Pluripotent Stem Cells

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119

The existence of SSCs was postulated almost 40 years ago on the basis of morphological studies [20] [21] [22] and observations of toxin-induced spermatogenic damage. The early studies of Clermont [23] [24] on human spermatogenesis revealed two types of spermatogonia, the Adark and Apale spermatogonia, which were differentiated by the staining pattern of their nucleus. Both cell types are generally considered stem cells [24,25]. Adark spermatogonia function as reverse stem cells that rarely divide, but can be triggered to self-renew in the case of injury or disease, while Apale spermatogonia are self-renewing stem cells [23,24,25,26]; they

In the last decade, molecular markers that can be used to identify and characterize human SSCs have been sought. A recent study reported that the expression of surface marker G protein coupled receptor 125 (GPR125) can be used in the isolation, characterization, and culture of putative human SSCs [27]. GPR125-positive spermatogonia are very rare, possibly limited to Adark spermatogonia or a sub-population of Apale spermatogonia. Human SSCs are also positive

also divide into B spermatogonia, which further divide into spermatocytes [24].

role in SSC self-renewal (Figure 1).

**Table 1.** Markers of human pluripotent stem cells and germ cells.

## **2. Differentiation and characterization of human primordial germ cells**

Human primordial germ cells (PGCs) can be isolated from tissues and their identity confirmed by observing their migratory activity in vitro [9]. Cultured human PGCs become human embryonic germ cells (hEGCs) in vitro, in the presence of feeder cells, leukemia inhibitory factor (LIF), and basic fibroblast growth factor (bFGF) [10]. hEGCs express alkaline phospha‐ tase (AP), OCT4, SOX2, NANOG, stage specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, and TRA-1-81, which are pluripotent stem cell markers. In vivo, human PGCs do not express FGF4, SOX2 [11] [12], TRA-1-60, or TRA-1-81 [13] [14], which are expressed by hESCs or hEGCs in vitro. The molecular signature of human PGCs in vivo can be characterized as C-KIT+ , SOX2- , TRA1-60- , TRA1-81- , and FGF4- , in contrast with human pluripotent stem cell lines in vitro. (This information is summarized in Table 1.) However, the full complement of genes that are expressed specifically in human PGCs and their functions remain unclear.

## **3. Spermatogonial stem cells**

Spermatogenesis is a complex and tightly regulated process in which a small pool of germline stem cells ultimately gives rise to spermatozoa [15]. These stem cells, called spermatogo‐ nial stem cells (SSCs) are found in the basal compartment of the seminiferous epithelium, where they adhere to the basement membrane. SSC self-renewal ensures the maintenance of


**Table 1.** Markers of human pluripotent stem cells and germ cells.

produce biological children. Although microdissection testicular sperm extraction (micro-TESE) is used to treat patients with nonobstructive azoospermia [8], this technique does not have a good success rate. Therefore, new approaches are needed to develop treatments for

Stem cells have the potential to differentiate into a variety of functional cell types in the body, and their discovery has given rise to the fields of regenerative medicine and cloning. Stem cells are regulated by the particular microenvironment in which they reside; these microenviron‐ ments are referred to as niches. Male germline stem cells can continuously produce sperm throughout adulthood, and investigators have sought to develop methods using stem cells to

Embryonic stem cells (ESCs) have the potential to differentiate into nearly every cell type in the body. As the cells differentiate, they lose the ability to develop into different tissues. In contrast, specific tissues (gastrointestinal, integumentary, spermatogenic, and hematopoietic systems) maintain their regenerative capacity *in vivo*, and in fact, stem cells have been functionally identified in a wide range of adult tissues. These adult stem cells are believed to hold great promise for tissue generation in clinical settings. Here, we provide a summary of the therapeutic potential of stem cells for the rejuvenation of fertility in infertile males. Our hope is that future research will provide a range of options for the preservation of male fertility

**2. Differentiation and characterization of human primordial germ cells**

, and FGF4-

cell lines in vitro. (This information is summarized in Table 1.) However, the full complement of genes that are expressed specifically in human PGCs and their functions remain unclear.

Spermatogenesis is a complex and tightly regulated process in which a small pool of germline stem cells ultimately gives rise to spermatozoa [15]. These stem cells, called spermatogo‐ nial stem cells (SSCs) are found in the basal compartment of the seminiferous epithelium, where they adhere to the basement membrane. SSC self-renewal ensures the maintenance of

, in contrast with human pluripotent stem

Human primordial germ cells (PGCs) can be isolated from tissues and their identity confirmed by observing their migratory activity in vitro [9]. Cultured human PGCs become human embryonic germ cells (hEGCs) in vitro, in the presence of feeder cells, leukemia inhibitory factor (LIF), and basic fibroblast growth factor (bFGF) [10]. hEGCs express alkaline phospha‐ tase (AP), OCT4, SOX2, NANOG, stage specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, and TRA-1-81, which are pluripotent stem cell markers. In vivo, human PGCs do not express FGF4, SOX2 [11] [12], TRA-1-60, or TRA-1-81 [13] [14], which are expressed by hESCs or hEGCs in vitro. The molecular signature of human PGCs in vivo can be characterized

male infertility.

118 Pluripotent Stem Cells

improve or restore fertility.

or the reversal of infertility.

as C-KIT+

, SOX2-

, TRA1-60-

**3. Spermatogonial stem cells**

, TRA1-81-

the stem cell pool, while their differentiation generates a large number of germ cells. Therefore, a balance between SSC self-renewal and differentiation in the adult testis is essential to maintain normal spermatogenesis and fertility throughout life. SSCs need to reside in a unique environment, or niche, that provides the factors necessary for their survival and potency. In mice, Sertoli cells in the testis are a crucial component of the spermatogonial stem cell niche. They produce glial cell line-derived neurotrophic factor (GDNF), a distant member of the TGFβ family, which controls SSC self-renewal [16]. Several groups have reported that adding GDNF to freshly isolated germ cells in culture results in the proliferation of SSCs [17,18]. Other factors within the niche influence the fate of SSCs. One example is colony-stimulating factor 1 (CSF1), which is produced by Leydig cells and some peritubular myoid cells [19], and plays a role in SSC self-renewal (Figure 1).

The existence of SSCs was postulated almost 40 years ago on the basis of morphological studies [20] [21] [22] and observations of toxin-induced spermatogenic damage. The early studies of Clermont [23] [24] on human spermatogenesis revealed two types of spermatogonia, the Adark and Apale spermatogonia, which were differentiated by the staining pattern of their nucleus. Both cell types are generally considered stem cells [24,25]. Adark spermatogonia function as reverse stem cells that rarely divide, but can be triggered to self-renew in the case of injury or disease, while Apale spermatogonia are self-renewing stem cells [23,24,25,26]; they also divide into B spermatogonia, which further divide into spermatocytes [24].

In the last decade, molecular markers that can be used to identify and characterize human SSCs have been sought. A recent study reported that the expression of surface marker G protein coupled receptor 125 (GPR125) can be used in the isolation, characterization, and culture of putative human SSCs [27]. GPR125-positive spermatogonia are very rare, possibly limited to Adark spermatogonia or a sub-population of Apale spermatogonia. Human SSCs are also positive

servation. However, most couples would prefer a naturally conceived child. Work has progressed in many laboratories to partially enrich the spermatogonial stem cells of species ranging from mice to primates. Today, many urologists bank a testicular biopsy from patients about to undergo chemotherapy, with the expectation that technology will advance rapidly

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121

Previous studies have demonstrated that neonatal and adult germline stem cells (GSCs) can be self-reprogrammed into ESC-like cells, called germline-derived pluripotent stem cells [32,33,34,35]. In addition, Conrad et al. [36] reported that pluripotent cells can be derived from human testis, which those authors called human adult GSCs (haGSCs). Other research groups subsequently claimed that ESC-like cells could be obtained from cultures of human testicular cells [37,38,39]. Conrad and colleagues compared the global gene expression profile of hESCs and haGSCs, and concluded that the populations presented a similar gene expression profile, and thus, that the haGSCs were pluripotent. However, Ko et al. claimed that the gene expres‐ sion profile of haGSCs differed substantially from the pluripotent profile of hESCs, determined by a number of laboratories [40]. For example, the haGSCs did not express NANOG, and had low OCT4 and SOX2 levels, but showed high levels of the fibroblast markers SNA12 and ACTA2 [40]. Ko and colleagues therefore suggested that the haGSCs originated from fibroblast cells, rather than from pluripotent tissue. They concluded that haGSCs were very similar to a human testicular fibroblast cell line (hTFCs) [40]. Conrad and colleagues argued that micro‐ array data sets cannot be compared unless they are processed in parallel in the same experi‐ ment, suggesting that the similarity between haGSCs and hTFCs was inconclusive. However, studies on microarray results generated by different laboratories [41,42,43] have shown that findings from microarray analyses are comparable across multiple laboratories [44], particu‐ larly when a common platform and set of procedures are used. These findings justify the utility of microarray repositories, such as the GEO database [45], not only as data warehouses but also as resources for comparative and combinatory analyses of microarray data from different laboratories. In conclusion, the global gene expression analysis of haGSCs demonstrated that

over the next 10 years and allow transplantation in the future.

**4. Pluripotency of human testis–derived ESC–like cells**

these cells resembled fibroblast hTFCs more than pluripotent hESCs.

The year 2006 saw the first description of mouse induced pluripotent stem cells (miPSCs), which were generated by the retrovirus-mediated transduction of four transcription factors (OCT3/4, SOX2, KLF4, and C-MYC) into mouse fibroblasts [46]. Human somatic cells can be reprogrammed to become human iPSCs via the introduction of a small set of genes, either those encoding OCT3/4, SOX2 and KLF4, with or without the addition of C-MYC, or an alternate combination of OCT3/4, SOX2, LIN28, and NANOG [47,48,49,50,51,52,53,54,55]. Human iPSCs (hiPSCs) have remarkable similarity to hESCs in terms of their morphology, in

**5. Induced pluripotent stem (iPS) cells**

**Figure 1.** Diagram of the spermatogonial stem cell (SSC) niche showing that extrinsic factors drive SSC maintenance and self-renewal. SSCs and Sertoli cells are attached to the basement membrane. Sertoli cells produce glial cell linederived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF). Leydig cells and peritubular cells pro‐ duce colony-stimulating factor-1 (CSF-1).

for some markers identified in mouse SSCs and other undifferentiated spermatogonia, including GFRA1, UCHL1 (PGP9.5), ZBTB16 (PLZF), and THY1 (CD90) [27,28]. We also have obtained evidence that THY1 is a potential surface marker for human SSCs [29].

Brinster and colleagues proved the existence of mouse SSCs by using unique approaches [30, 31]. These investigators transplanted cells obtained as testicular homogenates expressing the *LacZ* gene into the seminiferous tubules of otherwise sterile mice with a Sertoli-cell-only pathology. After three months, the transplanted spermatogonial stem cells had engrafted and colonized the seminiferous tubules. Spermatogenesis was restored.

The clinical implications of this work are enormous. The findings suggest that the isolation, enrichment, and cryopreservation of spermatogonial stem cells prior to chemotherapy or radiation therapy, with later autologous transplantation, may offer the potential for the subsequent restoration of fertility. The development of this technique will be especially important for survivors of childhood cancer. Adult patients can also bank sperm for cryopre‐ servation. However, most couples would prefer a naturally conceived child. Work has progressed in many laboratories to partially enrich the spermatogonial stem cells of species ranging from mice to primates. Today, many urologists bank a testicular biopsy from patients about to undergo chemotherapy, with the expectation that technology will advance rapidly over the next 10 years and allow transplantation in the future.

## **4. Pluripotency of human testis–derived ESC–like cells**

Previous studies have demonstrated that neonatal and adult germline stem cells (GSCs) can be self-reprogrammed into ESC-like cells, called germline-derived pluripotent stem cells [32,33,34,35]. In addition, Conrad et al. [36] reported that pluripotent cells can be derived from human testis, which those authors called human adult GSCs (haGSCs). Other research groups subsequently claimed that ESC-like cells could be obtained from cultures of human testicular cells [37,38,39]. Conrad and colleagues compared the global gene expression profile of hESCs and haGSCs, and concluded that the populations presented a similar gene expression profile, and thus, that the haGSCs were pluripotent. However, Ko et al. claimed that the gene expres‐ sion profile of haGSCs differed substantially from the pluripotent profile of hESCs, determined by a number of laboratories [40]. For example, the haGSCs did not express NANOG, and had low OCT4 and SOX2 levels, but showed high levels of the fibroblast markers SNA12 and ACTA2 [40]. Ko and colleagues therefore suggested that the haGSCs originated from fibroblast cells, rather than from pluripotent tissue. They concluded that haGSCs were very similar to a human testicular fibroblast cell line (hTFCs) [40]. Conrad and colleagues argued that micro‐ array data sets cannot be compared unless they are processed in parallel in the same experi‐ ment, suggesting that the similarity between haGSCs and hTFCs was inconclusive. However, studies on microarray results generated by different laboratories [41,42,43] have shown that findings from microarray analyses are comparable across multiple laboratories [44], particu‐ larly when a common platform and set of procedures are used. These findings justify the utility of microarray repositories, such as the GEO database [45], not only as data warehouses but also as resources for comparative and combinatory analyses of microarray data from different laboratories. In conclusion, the global gene expression analysis of haGSCs demonstrated that these cells resembled fibroblast hTFCs more than pluripotent hESCs.

### **5. Induced pluripotent stem (iPS) cells**

for some markers identified in mouse SSCs and other undifferentiated spermatogonia, including GFRA1, UCHL1 (PGP9.5), ZBTB16 (PLZF), and THY1 (CD90) [27,28]. We also have

**Figure 1.** Diagram of the spermatogonial stem cell (SSC) niche showing that extrinsic factors drive SSC maintenance and self-renewal. SSCs and Sertoli cells are attached to the basement membrane. Sertoli cells produce glial cell linederived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF). Leydig cells and peritubular cells pro‐

Brinster and colleagues proved the existence of mouse SSCs by using unique approaches [30, 31]. These investigators transplanted cells obtained as testicular homogenates expressing the *LacZ* gene into the seminiferous tubules of otherwise sterile mice with a Sertoli-cell-only pathology. After three months, the transplanted spermatogonial stem cells had engrafted and

The clinical implications of this work are enormous. The findings suggest that the isolation, enrichment, and cryopreservation of spermatogonial stem cells prior to chemotherapy or radiation therapy, with later autologous transplantation, may offer the potential for the subsequent restoration of fertility. The development of this technique will be especially important for survivors of childhood cancer. Adult patients can also bank sperm for cryopre‐

obtained evidence that THY1 is a potential surface marker for human SSCs [29].

colonized the seminiferous tubules. Spermatogenesis was restored.

duce colony-stimulating factor-1 (CSF-1).

120 Pluripotent Stem Cells

The year 2006 saw the first description of mouse induced pluripotent stem cells (miPSCs), which were generated by the retrovirus-mediated transduction of four transcription factors (OCT3/4, SOX2, KLF4, and C-MYC) into mouse fibroblasts [46]. Human somatic cells can be reprogrammed to become human iPSCs via the introduction of a small set of genes, either those encoding OCT3/4, SOX2 and KLF4, with or without the addition of C-MYC, or an alternate combination of OCT3/4, SOX2, LIN28, and NANOG [47,48,49,50,51,52,53,54,55]. Human iPSCs (hiPSCs) have remarkable similarity to hESCs in terms of their morphology, in vitro characteristics, proliferation rate, gene expression, and ability to differentiate into mesoderm, endoderm, and ectoderm, both in vitro and in vivo, in teratoma assays [56,57].

surface markers are available. To purify PGC-like cells from differentiating human ESCs and iPSCs, cell sorting with specific antibodies for SSEA1 [68], SSEA1 and C-KIT [50] [79], or CXCR4 [69] has been effective. In particular, Eguizabal et al. (2011) published a straightforward protocol for germline cell purification that requires only three steps. First, human iPSCs and hESCs are allowed to differentiate for 3 weeks in a monolayer, in the absence of growth cytokines. Second, the cells are cultured for 3 weeks in the presence of retinoic acid. Finally, after these 6 weeks of differentiation, the cells are sorted for a specific combination of surface markers (CD49f++, CD9+, CD90-, and SSEA4-), and the isolated fraction is cultured in the

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presence of LIF, bFGF, Forskolin, and CYP26 inhibitor for 4 more weeks [76].

germline development from piPSCs have been published to date.

**8. Conclusions**

**Acknowledgements**

Science and Technology of Japan, 2011-2015.

**7. Germline differentiation from porcine iPSCs, non–human iPSCs**

Despite their undoubted promise as sources of cells for tissue transplants, many roadblocks remain against using human ESCs clinically. Particularly troubling is the lack of tests for the efficacy of such therapies and the safety of transferring these cells in animals whose anatomy and physiology resemble those of humans better than mouse models do [80] [81] [82] [83] [84]. The pig is a potentially useful model in this regard, because of its similarities to humans in organ size, immunology, and whole animal physiology [85] [86] [87]. It was reported that porcine somatic cells can be reprogrammed to form piPSCs [88]. However, no reports on

Research on stem cells has shown remarkable progress over the past 5 years. In particular, the development of human iPSCs has opened new avenues into the generation of an *in vitro* disease model of male infertility. However, improvements are still needed before stem cells can be used clinically. For the treatment and diagnosis of male infertility, future advances may enable spermatids to be differentiated from germline stem cells or iPS cells. In addition, by examining patient-specific iPSCs that are defective in their ability to generate germ cells and comparing their differentiation capacity with that of normal human ESCs and iPSCs, researchers can hope

This study was supported in part by a Grant-in-Aid for Young Scientists (B) of the Japan Society for the Promotion of Science (JSPS) and a Grant of the Strategic Research Foundation Grantaided Project for Private schools at Heisei 23th from Ministry of Education, Culture, Sports,

to uncover the nature of male infertility and to design new methods to reverse it.

In our laboratory, we induced iPS cells from adult human testicular tissue by introducing four transcription factors, OCT4, SOX2, KLF4, and C-MYC, using lentiviral vectors [58]. We also generated ES-like cells from 293FT cells by using OCT4, SOX2, NANOG, and LIN28 [59]. Finally, we generated iPS cells derived from the human testicular tissue of individuals with Klinefelter syndrome (KS, also called 47, XXY) [60].

## **6. Germline differentiation from ESCs and iPSCs in humans**

Recent studies indicate that mouse [61,62,63,64,65] and human [66,67] [50,68,69,70,71] ESCs can differentiate in vitro into oocyte- or sperm-like cells. In particular, Clark et al. first reported the spontaneous differentiation of germ cells in embryoid bodies derived from human ESCs [66]. Male germline cells express specific RNA and protein markers, such as VASA. In 2009, Park et al. demonstrated that PGC-like cells can be differentiated from human iPSCs [50]. Subsequent reports on male germline differentiation from stem cells have used one of three approaches: (1) specific culture conditions, (2) manipulation of gene expression, and (3) purification of germ cells.

**Culture conditions supporting differentiation into germline cells.** Bucay et al. observed that as hESCs differentiate into putative germline cells, they also produce Sertoli-like support cells [69]. In addition, co-cultures of hESCs and hiPSCs with human fetal gonadal stromal cells [50], mouse Sertoli cells [72], or mouse embryonic fibroblasts [67] resulted in the increased efficiency of germ cell-like differentiation. Co-culture systems are used to mimic a suitable microenvir‐ onment for the growing germ cells. For the differentiation of germline-like cells from hESCs and hiPSCs, cytokines and other cell-signaling molecules are often added to the cultures. For example, BMP4 and other BMPs are added to promote PGC-like differentiation from hESCs and hiPSCs [73,74,75]. In addition, retinoic acid has been used to stimulate meiosis [75] [76]. Panula and colleagues reported the differentiation of fetal- and adult-derived iPSCs into germ cells, and showed that ~5% of human iPSCs differentiated into PGCs following induction with BMPs [77].

**Manipulation of gene expression.** By manipulating gene expression, researchers can regulate the cell lineage decisions of differentiating pluripotent stem cells. Overexpression of DAZL and VASA promotes PGC formation in differentiating human ESCs and iPSCs [78]. In addition, Kee and colleagues (2009) reported that hESCs differentiate into germline cells that initiate meiosis and progress to form haploid germ cells. These authors indicated that the overexpres‐ sion of members of the DAZ gene family, DAZ, DAZL, and BOULE, promoted the progression of PGCs to meiosis and the production of haploid cells, a process that is unique to germ cell development [71].

**Purification of germline cells.** The isolation and purification of germline cells from stem cell cultures (ESCs and iPSCs) can be performed efficiently when specific antibodies for germ cell surface markers are available. To purify PGC-like cells from differentiating human ESCs and iPSCs, cell sorting with specific antibodies for SSEA1 [68], SSEA1 and C-KIT [50] [79], or CXCR4 [69] has been effective. In particular, Eguizabal et al. (2011) published a straightforward protocol for germline cell purification that requires only three steps. First, human iPSCs and hESCs are allowed to differentiate for 3 weeks in a monolayer, in the absence of growth cytokines. Second, the cells are cultured for 3 weeks in the presence of retinoic acid. Finally, after these 6 weeks of differentiation, the cells are sorted for a specific combination of surface markers (CD49f++, CD9+, CD90-, and SSEA4-), and the isolated fraction is cultured in the presence of LIF, bFGF, Forskolin, and CYP26 inhibitor for 4 more weeks [76].

## **7. Germline differentiation from porcine iPSCs, non–human iPSCs**

Despite their undoubted promise as sources of cells for tissue transplants, many roadblocks remain against using human ESCs clinically. Particularly troubling is the lack of tests for the efficacy of such therapies and the safety of transferring these cells in animals whose anatomy and physiology resemble those of humans better than mouse models do [80] [81] [82] [83] [84]. The pig is a potentially useful model in this regard, because of its similarities to humans in organ size, immunology, and whole animal physiology [85] [86] [87]. It was reported that porcine somatic cells can be reprogrammed to form piPSCs [88]. However, no reports on germline development from piPSCs have been published to date.

## **8. Conclusions**

vitro characteristics, proliferation rate, gene expression, and ability to differentiate into mesoderm, endoderm, and ectoderm, both in vitro and in vivo, in teratoma assays [56,57].

In our laboratory, we induced iPS cells from adult human testicular tissue by introducing four transcription factors, OCT4, SOX2, KLF4, and C-MYC, using lentiviral vectors [58]. We also generated ES-like cells from 293FT cells by using OCT4, SOX2, NANOG, and LIN28 [59]. Finally, we generated iPS cells derived from the human testicular tissue of individuals with

Recent studies indicate that mouse [61,62,63,64,65] and human [66,67] [50,68,69,70,71] ESCs can differentiate in vitro into oocyte- or sperm-like cells. In particular, Clark et al. first reported the spontaneous differentiation of germ cells in embryoid bodies derived from human ESCs [66]. Male germline cells express specific RNA and protein markers, such as VASA. In 2009, Park et al. demonstrated that PGC-like cells can be differentiated from human iPSCs [50]. Subsequent reports on male germline differentiation from stem cells have used one of three approaches: (1) specific culture conditions, (2) manipulation of gene expression, and (3)

**Culture conditions supporting differentiation into germline cells.** Bucay et al. observed that as hESCs differentiate into putative germline cells, they also produce Sertoli-like support cells [69]. In addition, co-cultures of hESCs and hiPSCs with human fetal gonadal stromal cells [50], mouse Sertoli cells [72], or mouse embryonic fibroblasts [67] resulted in the increased efficiency of germ cell-like differentiation. Co-culture systems are used to mimic a suitable microenvir‐ onment for the growing germ cells. For the differentiation of germline-like cells from hESCs and hiPSCs, cytokines and other cell-signaling molecules are often added to the cultures. For example, BMP4 and other BMPs are added to promote PGC-like differentiation from hESCs and hiPSCs [73,74,75]. In addition, retinoic acid has been used to stimulate meiosis [75] [76]. Panula and colleagues reported the differentiation of fetal- and adult-derived iPSCs into germ cells, and showed that ~5% of human iPSCs differentiated into PGCs following induction with

**Manipulation of gene expression.** By manipulating gene expression, researchers can regulate the cell lineage decisions of differentiating pluripotent stem cells. Overexpression of DAZL and VASA promotes PGC formation in differentiating human ESCs and iPSCs [78]. In addition, Kee and colleagues (2009) reported that hESCs differentiate into germline cells that initiate meiosis and progress to form haploid germ cells. These authors indicated that the overexpres‐ sion of members of the DAZ gene family, DAZ, DAZL, and BOULE, promoted the progression of PGCs to meiosis and the production of haploid cells, a process that is unique to germ cell

**Purification of germline cells.** The isolation and purification of germline cells from stem cell cultures (ESCs and iPSCs) can be performed efficiently when specific antibodies for germ cell

**6. Germline differentiation from ESCs and iPSCs in humans**

Klinefelter syndrome (KS, also called 47, XXY) [60].

purification of germ cells.

122 Pluripotent Stem Cells

BMPs [77].

development [71].

Research on stem cells has shown remarkable progress over the past 5 years. In particular, the development of human iPSCs has opened new avenues into the generation of an *in vitro* disease model of male infertility. However, improvements are still needed before stem cells can be used clinically. For the treatment and diagnosis of male infertility, future advances may enable spermatids to be differentiated from germline stem cells or iPS cells. In addition, by examining patient-specific iPSCs that are defective in their ability to generate germ cells and comparing their differentiation capacity with that of normal human ESCs and iPSCs, researchers can hope to uncover the nature of male infertility and to design new methods to reverse it.

## **Acknowledgements**

This study was supported in part by a Grant-in-Aid for Young Scientists (B) of the Japan Society for the Promotion of Science (JSPS) and a Grant of the Strategic Research Foundation Grantaided Project for Private schools at Heisei 23th from Ministry of Education, Culture, Sports, Science and Technology of Japan, 2011-2015.

## **Author details**

Hideyuki Kobayashi\* , Koichi Nagao and Koichi Nakajima

\*Address all correspondence to: hideyukk@med.toho-u.ac.jp

Department of Urology, Toho University School of Medicine, Tokyo, Japan

#### **References**


[12] De Jong, J, Stoop, H, Gillis, A. J, & Van Gurp, R. J. van de Geijn GJ, Boer M et al. Dif‐ ferential expression of SOX17 and SOX2 in germ cells and stem cells has biological

Human Testis–Derived Pluripotent Cells and Induced Pluripotent Stem Cells

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125

[13] Kerr, C. L, Hill, C. M, Blumenthal, P. D, & Gearhart, J. D. Expression of pluripotent

[14] Kerr, C. L, Hill, C. M, Blumenthal, P. D, & Gearhart, J. D. Expression of pluripotent stem cell markers in the human fetal ovary. Hum Reprod (2008). , 23, 589-99.

[15] De Rooij, D. G, & Russell, L. D. All you wanted to know about spermatogonia but

[16] Meng, X, Lindahl, M, Hyvonen, M. E, Parvinen, M, De Rooij, D. G, Hess, M. W, et al. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science

[17] Kanatsu-shinohara, M, Ogonuki, N, Inoue, K, Miki, H, Ogura, A, Toyokuni, S, et al. Long-term proliferation in culture and germline transmission of mouse male germ‐

[18] Kubota, H, Avarbock, M. R, & Brinster, R. L. Growth factors essential for self-renew‐ al and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci U S A

[19] Oatley, J. M, Oatley, M. J, Avarbock, M. R, Tobias, J. W, & Brinster, R. L. Colony stim‐ ulating factor 1 is an extrinsic stimulator of mouse spermatogonial stem cell self-re‐

[20] Huckins, C. The spermatogonial stem cell population in adult rats. 3. Evidence for a

[21] Huckins, C. The spermatogonial stem cell population in adult rats. II. A radioauto‐ graphic analysis of their cell cycle properties. Cell Tissue Kinet (1971). , 4, 313-34.

[22] Huckins, C. The spermatogonial stem cell population in adult rats. I. Their morphol‐

[23] Clermont, Y. The cycle of the seminiferous epithelium in man. Am J Anat (1963). ,

[25] Clermont, Y. Spermatogenesis in man. A study of the spermatogonial population.

[26] Clermont, Y. Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle

[27] He, Z, Kokkinaki, M, Jiang, J, Dobrinski, I, & Dym, M. Isolation, characterization, and

[24] Clermont, Y. Renewal of spermatogonia in man. Am J Anat (1966). , 118, 509-24.

long-cycling population. Cell Tissue Kinet (1971). , 4, 335-49.

ogy, proliferation and maturation. Anat Rec (1971). , 169, 533-57.

and spermatogonial renewal. Physiol Rev (1972). , 52, 198-236.

culture of human spermatogonia. Biol Reprod (2010). , 82, 363-72.

stem cell markers in the human fetal testis. Stem Cells (2008). , 26, 412-21.

and clinical implications. J Pathol (2008). , 215, 21-30.

were afraid to ask. J Androl (2000). , 21, 776-98.

line stem cells. Biol Reprod (2003). , 69, 612-6.

newal. Development (2009). , 136, 1191-9.

(2000). , 287, 1489-93.

(2004). , 101, 16489-94.

112, 35-51.

Fertil Steril (1966). , 17, 705-21.


[12] De Jong, J, Stoop, H, Gillis, A. J, & Van Gurp, R. J. van de Geijn GJ, Boer M et al. Dif‐ ferential expression of SOX17 and SOX2 in germ cells and stem cells has biological and clinical implications. J Pathol (2008). , 215, 21-30.

**Author details**

124 Pluripotent Stem Cells

Hideyuki Kobayashi\*

**References**

phy (1985). , 22, 415-30.

failure. Am J Pathol (2007). , 170, 899-908.

cretion. Endocrinology (2010). , 151, 2800-10.

results. Semin Reprod Med (2009). , 27, 165-70.

Proc Natl Acad Sci U S A (1998). , 95, 13726-31.

ment or upon in vitro culture. Biol Reprod (2008). , 78, 852-8.

tro. Anat Rec (1983). , 205, 21-6.

, Koichi Nagao and Koichi Nakajima

Department of Urology, Toho University School of Medicine, Tokyo, Japan

[1] Spira, A. Epidemiology of human reproduction. Hum Reprod (1986). , 1, 111-5.

[3] Mosher, W. D. Reproductive impairments in the United States, 1965-1982. Demogra‐

[5] Lue, Y, Erkkila, K, Liu, P. Y, Ma, K, Wang, C, Hikim, A. S, et al. Fate of bone marrow stem cells transplanted into the testis: potential implication for men with testicular

[6] Nieschlag, E, Simoni, M, Gromoll, J, & Weinbauer, G. F. Role of FSH in the regulation of spermatogenesis: clinical aspects. Clin Endocrinol (Oxf) (1999). , 51, 139-46.

[7] Allan, C. M, Couse, J. F, Simanainen, U, Spaliviero, J, Jimenez, M, Rodriguez, K, et al. Estradiol induction of spermatogenesis is mediated via an estrogen receptor-{alpha} mechanism involving neuroendocrine activation of follicle-stimulating hormone se‐

[8] Schlegel, P. N. Nonobstructive azoospermia: a revolutionary surgical approach and

[9] Kuwana, T, & Fujimoto, T. Active locomotion of human primordial germ cells in vi‐

[10] Shamblott, M. J, Axelman, J, Wang, S, Bugg, E. M, Littlefield, J. W, Donovan, P. J, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells.

[11] Perrett, R. M, Turnpenny, L, Eckert, J. J, Shea, O, Sonne, M, & Cameron, S. B. IT et al. The early human germ cell lineage does not express SOX2 during in vivo develop‐

[2] MacLeod JHuman male infertility. Obstet Gynecol Surv (1971). , 26, 335-51.

[4] Simmons, F. A. Human infertility. N Engl J Med (1956). contd., 255, 1140-6.

\*Address all correspondence to: hideyukk@med.toho-u.ac.jp


[28] Dym, M, Kokkinaki, M, & He, Z. Spermatogonial stem cells: mouse and human com‐ parisons. Birth Defects Res C Embryo Today (2009). , 87, 27-34.

[42] Larkin, J. E, Frank, B. C, Gavras, H, Sultana, R, & Quackenbush, J. Independence and reproducibility across microarray platforms. Nat Methods (2005). , 2, 337-44.

Human Testis–Derived Pluripotent Cells and Induced Pluripotent Stem Cells

http://dx.doi.org/10.5772/55570

127

[43] Irizarry, R. A, Warren, D, Spencer, F, Kim, I. F, Biswal, S, Frank, B. C, et al. Multiplelaboratory comparison of microarray platforms. Nat Methods (2005). , 2, 345-50.

[45] Edgar, R, Domrachev, M, & Lash, A. E. Gene Expression Omnibus: NCBI gene ex‐ pression and hybridization array data repository. Nucleic Acids Res (2002). , 30,

[46] Takahashi, K, & Yamanaka, S. Induction of pluripotent stem cells from mouse em‐ bryonic and adult fibroblast cultures by defined factors. Cell (2006). , 126, 663-76.

[47] Okita, K, Ichisaka, T, & Yamanaka, S. Generation of germline-competent induced

[48] Yu, J, Vodyanik, M. A, Smuga-otto, K, Antosiewicz-bourget, J, Frane, J. L, Tian, S, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science

[49] Woltjen, K, Michael, I. P, Mohseni, P, Desai, R, Mileikovsky, M, Hamalainen, R, et al. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Na‐

[50] Park, T. S, Galic, Z, Conway, A. E, Lindgren, A, Van Handel, B. J, Magnusson, M, et al. Derivation of primordial germ cells from human embryonic and induced pluripo‐ tent stem cells is significantly improved by coculture with human fetal gonadal cells.

[51] Zou, J, Maeder, M. L, Mali, P, Pruett-miller, S. M, Thibodeau-beganny, S, Chou, B. K, et al. Gene targeting of a disease-related gene in human induced pluripotent stem

[52] Nakagawa, M, Koyanagi, M, Tanabe, K, Takahashi, K, Ichisaka, T, Aoi, T, et al. Gen‐ eration of induced pluripotent stem cells without Myc from mouse and human fibro‐

[53] Takahashi, K, Okita, K, Nakagawa, M, & Yamanaka, S. Induction of pluripotent stem

[54] Liu, H, Zhu, F, Yong, J, Zhang, P, Hou, P, Li, H, et al. Generation of induced pluripo‐ tent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell (2008). , 3,

[55] Takahashi, K, Tanabe, K, Ohnuki, M, Narita, M, Ichisaka, T, Tomoda, K, et al. Induc‐ tion of pluripotent stem cells from adult human fibroblasts by defined factors. Cell

and embryonic stem cells. Cell Stem Cell (2009). , 5, 97-110.

cells from fibroblast cultures. Nat Protoc (2007). , 2, 3081-9.

[44] Sherlock, G. Of fish and chips. Nat Methods (2005). , 2, 329-30.

pluripotent stem cells. Nature (2007). , 448, 313-7.

207-10.

587-90.

(2007). , 131, 861-72.

(2007). , 318, 1917-20.

ture (2009). , 458, 766-70.

Stem Cells (2009). , 27, 783-95.

blasts. Nat Biotechnol (2008). , 26, 101-6.


[28] Dym, M, Kokkinaki, M, & He, Z. Spermatogonial stem cells: mouse and human com‐

*NANOG* and may include spermatogonial stem cells. Reprod Med Biol (2009).

[30] Brinster, R. L, & Avarbock, M. R. Germline transmission of donor haplotype follow‐ ing spermatogonial transplantation. Proc Natl Acad Sci U S A (1994). , 91, 11303-7.

[31] Brinster, R. L, & Zimmermann, J. W. Spermatogenesis following male germ-cell

[32] Ko, K, Tapia, N, Wu, G, Kim, J. B, Bravo, M. J, Sasse, P, et al. Induction of pluripoten‐

[33] Kanatsu-shinohara, M, Inoue, K, Lee, J, Yoshimoto, M, Ogonuki, N, Miki, H, et al. Generation of pluripotent stem cells from neonatal mouse testis. Cell (2004). , 119,

[34] Seandel, M, James, D, Shmelkov, S. V, Falciatori, I, Kim, J, Chavala, S, et al. Genera‐ tion of functional multipotent adult stem cells from GPR125+ germline progenitors.

[35] Kanatsu-shinohara, M, Lee, J, Inoue, K, Ogonuki, N, Miki, H, Toyokuni, S, et al. Plu‐ ripotency of a single spermatogonial stem cell in mice. Biol Reprod (2008). , 78, 681-7.

[36] Conrad, S, Renninger, M, Hennenlotter, J, Wiesner, T, Just, L, Bonin, M, et al. Genera‐ tion of pluripotent stem cells from adult human testis. Nature (2008). , 456, 344-9.

[37] Golestaneh, N, Kokkinaki, M, Pant, D, Jiang, J, Destefano, D, Fernandez-bueno, C, et al. Pluripotent stem cells derived from adult human testes. Stem Cells Dev (2009). ,

[38] Kossack, N, Meneses, J, Shefi, S, Nguyen, H. N, Chavez, S, Nicholas, C, et al. Isola‐ tion and characterization of pluripotent human spermatogonial stem cell-derived

[39] Mizrak, S. C, Chikhovskaya, J. V, Sadri-ardekani, H, Van Daalen, S, Korver, C. M, Hovingh, S. E, et al. Embryonic stem cell-like cells derived from adult human testis.

[40] Ko, K, Arauzo-bravo, M. J, Tapia, N, Kim, J, Lin, Q, Bernemann, C, et al. Human

[41] Bammler, T, Beyer, R. P, Bhattacharya, S, Boorman, G. A, Boyles, A, Bradford, B. U, et al. Standardizing global gene expression analysis between laboratories and across

adult germline stem cells in question. Nature (2010). E1; discussion E3.

cy in adult unipotent germline stem cells. Cell Stem Cell (2009). , 5, 87-96.

adult human testicular tissues express human embryonic stem cell genes *OCT3/4* and

cells isolated from

parisons. Birth Defects Res C Embryo Today (2009). , 87, 27-34.

[29] Kobayashi, H, Nagao, K, Nakajima, K, Miura, K, & Ishii, N. Thy-1+

transplantation. Proc Natl Acad Sci U S A (1994). , 91, 11298-302.

1001-12.

126 Pluripotent Stem Cells

18, 1115-26.

Nature (2007). , 449, 346-50.

cells. Stem Cells (2009). , 27, 138-49.

Hum Reprod (2010). , 25, 158-67.

platforms. Nat Methods (2005). , 2, 351-6.


[56] Yamanaka, S. Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell (2007). , 1, 39-49.

[69] Bucay, N, Yebra, M, Cirulli, V, Afrikanova, I, Kaido, T, Hayek, A, et al. A novel ap‐ proach for the derivation of putative primordial germ cells and sertoli cells from hu‐

Human Testis–Derived Pluripotent Cells and Induced Pluripotent Stem Cells

http://dx.doi.org/10.5772/55570

129

[70] Tilgner, K, Atkinson, S. P, Yung, S, Golebiewska, A, Stojkovic, M, Moreno, R, et al. Expression of GFP under the control of the RNA helicase VASA permits fluores‐ cence-activated cell sorting isolation of human primordial germ cells. Stem Cells

[71] Kee, K, Angeles, V. T, Flores, M, & Nguyen, H. N. Reijo Pera RA. Human DAZL, DAZ and BOULE genes modulate primordial germ-cell and haploid gamete forma‐

[72] Geens, M, & Sermon, K. D. Van de Velde H, Tournaye H. Sertoli cell-conditioned me‐ dium induces germ cell differentiation in human embryonic stem cells. J Assist Re‐

[73] West, F. D, Roche-rios, M. I, Abraham, S, Rao, R. R, Natrajan, M. S, Bacanamwo, M, et al. KIT ligand and bone morphogenetic protein signaling enhances human embry‐ onic stem cell to germ-like cell differentiation. Hum Reprod (2010). , 25, 168-78.

[74] Kee, K, Gonsalves, J. M, Clark, A. T, & Pera, R. A. Bone morphogenetic proteins in‐ duce germ cell differentiation from human embryonic stem cells. Stem Cells Dev

[75] Richards, M, Fong, C. Y, & Bongso, A. Comparative evaluation of different in vitro systems that stimulate germ cell differentiation in human embryonic stem cells. Fertil

[76] Eguizabal, C, Montserrat, N, Vassena, R, Barragan, M, Garreta, E, Garcia-quevedo, L, et al. Complete meiosis from human induced pluripotent stem cells. Stem Cells

[77] Panula, S, Medrano, J. V, Kee, K, Bergstrom, R, Nguyen, H. N, Byers, B, et al. Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem

[78] Medrano, J. V, Ramathal, C, Nguyen, H. N, & Simon, C. Reijo Pera RA. Divergent RNA-binding proteins, DAZL and VASA, induce meiotic progression in human

[79] Aflatoonian, B, Ruban, L, Jones, M, Aflatoonian, R, Fazeli, A, & Moore, H. D. In vitro post-meiotic germ cell development from human embryonic stem cells. Hum Reprod

[80] Vackova, I, Ungrova, A, & Lopes, F. Putative embryonic stem cell lines from pig em‐

man embryonic stem cells. Stem Cells (2009). , 27, 68-77.

(2010). , 28, 84-92.

(2006). , 15, 831-7.

Steril (2010). , 93, 986-94.

cells. Hum Mol Genet (2011). , 20, 752-62.

bryos. J Reprod Dev (2007). , 53, 1137-49.

germ cells derived in vitro. Stem Cells (2012). , 30, 441-51.

(2011). , 29, 1186-95.

(2009). , 24, 3150-9.

tion. Nature (2009). , 462, 222-5.

prod Genet (2011). , 28, 471-80.


[69] Bucay, N, Yebra, M, Cirulli, V, Afrikanova, I, Kaido, T, Hayek, A, et al. A novel ap‐ proach for the derivation of putative primordial germ cells and sertoli cells from hu‐ man embryonic stem cells. Stem Cells (2009). , 27, 68-77.

[56] Yamanaka, S. Strategies and new developments in the generation of patient-specific

[57] Yamanaka, S. Pluripotency and nuclear reprogramming. Philos Trans R Soc Lond B

[58] Kobayashi, H, Nakajima, K, Oka, Y, Tai, T, Nagao, K, & Ishii, N. Reprogramming of adult human testicular cells by our transcription factors (OCT4, SOX2, KLF4, and C-

[59] Oka, Y, Nakajima, K, Nagao, K, Miura, K, Ishii, N, & Kobayashi, H. FT cells trans‐ duced with four transcription actors (OCT4, SOX2, NANOG, and LIN28) generate aberrant ES-like cells. J. Stem cell and Regenerative Medicine (2010). , 3, 149-156.

[60] Kobayashi, H. Pluripotent stem cells induced from testicular tissue with Klinefelter syndrome (47, XXY) by four ranscription factors (OCT4, SOX2, KLF4, and C-MYC). Methodological Advances in the Culture, Manipulation and Utilization of Embryonic

[61] Hubner, K, Fuhrmann, G, Christenson, L. K, Kehler, J, Reinbold, R, De La Fuente, R, et al. Derivation of oocytes from mouse embryonic stem cells. Science (2003). , 300,

[62] Toyooka, Y, Tsunekawa, N, Akasu, R, & Noce, T. Embryonic stem cells can form

[63] Geijsen, N, Horoschak, M, Kim, K, Gribnau, J, Eggan, K, & Daley, G. Q. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature (2004). ,

[64] Qing, T, Shi, Y, Qin, H, Ye, X, Wei, W, Liu, H, et al. Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells. Differ‐

[65] Nagano, M. C. In vitro gamete derivation from pluripotent stem cells: progress and

[66] Clark, A. T, Bodnar, M. S, Fox, M, Rodriquez, R. T, Abeyta, M. J, Firpo, M. T, et al. Spontaneous differentiation of germ cells from human embryonic stem cells in vitro.

[67] West, F. D, Machacek, D. W, Boyd, N. L, Pandiyan, K, Robbins, K. R, & Stice, S. L. Enrichment and differentiation of human germ-like cells mediated by feeder cells

[68] Tilgner, K, Atkinson, S. P, Golebiewska, A, Stojkovic, M, Lako, M, & Armstrong, L. Isolation of primordial germ cells from differentiating human embryonic stem cells.

and basic fibroblast growth factor signaling. Stem Cells (2008). , 26, 2768-76.

Stem Cells for Basic and Practical Applications (2011). , 295-306.

germ cells in vitro. Proc Natl Acad Sci U S A (2003). , 100, 11457-62.

pluripotent stem cells. Cell Stem Cell (2007). , 1, 39-49.

MYC). Reprod Med Biol (2011). , 10, 105-112.

Biol Sci (2008). , 363, 2079-87.

1251-6.

128 Pluripotent Stem Cells

427, 148-54.

entiation (2007). , 75, 902-11.

perspective. Biol Reprod (2007). , 76, 546-51.

Hum Mol Genet (2004). , 13, 727-39.

Stem Cells (2008). , 26, 3075-85.


[81] Keefer, C. L, Pant, D, Blomberg, L, & Talbot, N. C. Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates. Anim Reprod Sci (2007). , 98, 147-68.

**Chapter 7**

**Generation of Induced Pluripotent Stem Cells from**

During early development, human dental pulp is originated from neural crest, which is a transient embryonic structure (Fig. 1). According to current knowledge, neural crest stem cells (NCSCs) have the capacity to self-renewal and display a developmental potential almost the same as embryonic stem (ES) cells (Kerkis and Caplan, 2012). These postmigratory NCSCs generate all craniofacial bones, the majority of the peripheral nervous system cells and tissues, as well as several non-neural cell types, such as smooth muscle cells of the cardiovascular system, pigment cells in the skin, cartilage, connective tissue, corneal epithelium and dental pulp among them. Although postmigratory, postnatal NCSCs are of restricted developmental potential they maintain functional characteristics resembling their embryonic counterparts and an ability to differentiate into a broad spectrum of cell types (Le Douarin et al., 2004, 2007,

**Figure 1.** Early development of NCSCs. According to current knowledge, migrating neural crest cells are stem cells that

© 2013 Lizier et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Lizier et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

**Dental Pulp Somatic Cells**

Nelson F. Lizier, Irina Kerkis and

Additional information is available at the end of the chapter

2008; Dupin et al., 2007; Le Douarin & Dupin, 2003, 2012).

display almost the same potential as ES cells.

Cristiane V. Wenceslau

http://dx.doi.org/10.5772/55856

**1. Introduction**


## **Generation of Induced Pluripotent Stem Cells from Dental Pulp Somatic Cells**

Nelson F. Lizier, Irina Kerkis and Cristiane V. Wenceslau

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55856

### **1. Introduction**

[81] Keefer, C. L, Pant, D, Blomberg, L, & Talbot, N. C. Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates. Anim Reprod

[82] Brevini, T. A, Antonini, S, Cillo, F, Crestan, M, & Gandolfi, F. Porcine embryonic stem cells: Facts, challenges and hopes. Theriogenology (2007). Suppl 1:S, 206-13.

[83] Talbot, N. C. Blomberg Le A. The pursuit of ES cell lines of domesticated ungulates.

[84] Hall, V. Porcine embryonic stem cells: a possible source for cell replacement therapy.

[85] Prather, R. S, Hawley, R. J, Carter, D. B, Lai, L, & Greenstein, J. L. Transgenic swine

[86] Brandl, U, Michel, S, Erhardt, M, Brenner, P, Burdorf, L, Jockle, H, et al. Transgenic animals in experimental xenotransplantation models: orthotopic heart transplanta‐

[87] Piedrahita, J. A, & Mir, B. Cloning and transgenesis in mammals: implications for

[88] Ezashi, T, Telugu, B. P, Alexenko, A. P, Sachdev, S, Sinha, S, & Roberts, R. M. Deriva‐ tion of induced pluripotent stem cells from pig somatic cells. Proc Natl Acad Sci U S

for biomedicine and agriculture. Theriogenology (2003). , 59, 115-23.

tion in the pig-to-baboon model. Transplant Proc (2007). , 39, 577-8.

xenotransplantation. Am J Transplant (2004). Suppl , 6, 43-50.

Sci (2007). , 98, 147-68.

130 Pluripotent Stem Cells

Stem Cell Rev (2008). , 4, 235-54.

Stem Cell Rev (2008). , 4, 275-82.

A (2009). , 106, 10993-8.

During early development, human dental pulp is originated from neural crest, which is a transient embryonic structure (Fig. 1). According to current knowledge, neural crest stem cells (NCSCs) have the capacity to self-renewal and display a developmental potential almost the same as embryonic stem (ES) cells (Kerkis and Caplan, 2012). These postmigratory NCSCs generate all craniofacial bones, the majority of the peripheral nervous system cells and tissues, as well as several non-neural cell types, such as smooth muscle cells of the cardiovascular system, pigment cells in the skin, cartilage, connective tissue, corneal epithelium and dental pulp among them. Although postmigratory, postnatal NCSCs are of restricted developmental potential they maintain functional characteristics resembling their embryonic counterparts and an ability to differentiate into a broad spectrum of cell types (Le Douarin et al., 2004, 2007, 2008; Dupin et al., 2007; Le Douarin & Dupin, 2003, 2012).

© 2013 Lizier et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Lizier et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The marathon of induced pluripotent stem cells (iPSC) started when Yamanaka in 2006, by forcing the expression of certain pluripotent genes in fibroblasts, reversed them into a pluripotent state similar to ES cells (Takahashi &Yamanaka, 2006). The main goal of iPSC generation is to create patient-specific cells, which would be advantageous for cell therapy due to immune compatibility (Ohnuki et al., 2009). Research involving the production of iPSC is being developed around the world. Production of iPSC opens new avenues for understanding human genetic diseases; embryogenesis and will likely have a great impact in drug screening and toxicological tests. However, fibroblasts, which were firstly used for iPSC production, present low efficiency and slow process of reprogramming. Moreover, these cells throughout all life are exposed to environmental factors, which can compromise their use as genetic models (Liu, 2008; McDevitt & Palecek, 2008; Nishikawa et al., 2008; Yu & Thomson; 2008; Zhao & Daley, 2008; Maherali & Hochedlinger, 2008; Ooi et al., 2012). Indeed, more immature somatic cells such as, postmigratory NCSCs, and adult stem cells isolated from young organism showed high efficiency of reprogramming (Zouboulis et al., 2008; Muchkaeva et al., 2012). Because of the possibility to isolate NCSCs from easily accessible tissue (e.g. baby teeth is discarded), the dental pulp derived somatic cells have become an ideal model system to study stem cell biology in diseases during different stages of the development (childhood, youth, middle-aged and old) with a special focus on non-invasive source of the cells for investigation of pediatric diseases (Kerkis & Caplan, 2012; Lizier et al., 2012).

deciduous teeth (Miura et al., 2003; Kerkis et al., 2006). DPCs (dental pulp cells) and MStCs (mesenchymal/stromal cells) were isolated from human third molars by two independent groups (Takeda et al., 2008; Ikeda et al., 2008). All these cell types present fibroblast-like morphology and however differ in methods of isolation, show significant difference in expression pattern of stem cell markers and in purity of isolated population (Yan et al., 2010;

Generation of Induced Pluripotent Stem Cells from Dental Pulp Somatic Cells

http://dx.doi.org/10.5772/55856

133

**Figure 2.** iPSC technology. The iPSC production can be induced by forcing the expression of certain pluripotent genes.

For reprogramming of SHED/SCAP/DPSCs, heterogeneous primary human dental stem/ progenitor cell population at passages 2 and 3 were used (Yan et al., 2010). These populations were tested for their cell surface marker expression by flow cytometry and they were positive for STRO-1, CD146, CD73, CD90, CD105 and negative for CD14, CD34, and CD45, showing typical immunophenotype of mesenchymal stem cells (MSC) (Friedenstein et al., 1976; Caplan, 1991). The first study used four factor genes for reprogramming, such as c-Myc [Myc protooncogene protein], Klf4 [Krüppel-like factor], Oct4 [octamer-binding transcription factor 4], and Sox2 [(sex determining region Y)-box 2] into pLenti6.2/C-Lumio/V5-DEST vector system. Although the cells started to present morphological changes (fibroblastic to epithelial cell-like transition), the reprogramming process failed. Further, lentiviral vectors pSin-EF2-gene-Pur carrying 1 of the 4 factors Lin28 [Lin-28 homolog A], Nanog (Nanog homeobox), Oct4, and Sox2 were used and first ES-like colonies were obtained. To improve reprogramming efficien‐ cy, human genes c-Myc, Klf4, Oct4, and Sox2 were subcloned into the vector pMXs and

Beltrão-Braga et al., 2011; Tamaoki et al., 2010; Oda et al., 2010).

**2.1. Transduction and reprogramming**

Our group isolated and fully characterized human immature dental pulp stem cells (hIDPSC), which is a very attractive cell type, from deciduous teeth (baby teeth) (Kerkis et al., 2006; Lizier et al., 2012). The hIDPSC present fibroblast-like morphology, retain characteristics of adult multipotent stem cells and express at least one of three transcription factors: Oct4, Nanog and Sox2 (Kerkis et al., 2006; Lizier et al., 2012). We also used these cells as an alternative source for iPSC derivation (Beltrão-Braga et al., 2011) (Fig. 2).Different research groups derived iPSC from dental pulp fibroblasts and stem cells from young, middle aged and old patients. The difference was observed between the protocols and efficiency of iPSC generation in all these studies (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011). The present chapter is focused on comparative investigation of the methods and efficiency of iPSC generation from dental pulp stem cells and fibroblasts (control). Differentiation potential, assuredness and the future perspectives of the use of these iPSC derived from dental pulp stem cells in basic research and in biotechnology will also be broadly discussed.

### **2. Dental pulp somatic cells used for iPSC generation**

Human dental tissues are rich in stem cells (Giordano et al., 2011; Kerkis & Caplan, 2012). Different research groups isolated and characterized several types of stem cells used for iPSC generation: (i) from apical papilla (Yan et al., 2010), (ii) from dental pulp of primary exfoliated deciduous teeth (Yan et al., 2010; Beltrão-Braga et al., 2011) and (iii) from wisdom teeth (Tamaoki et al., 2010; Oda et al., 2010). SCAP (stem cells from apical papilla) were obtained from tissue at the apex of a tooth root (Yan et al., 2010). SHED (stem cells from human exfoliated deciduous) and IDPSC (immature dental pulp stem cells) were derived from exfoliated deciduous teeth (Miura et al., 2003; Kerkis et al., 2006). DPCs (dental pulp cells) and MStCs (mesenchymal/stromal cells) were isolated from human third molars by two independent groups (Takeda et al., 2008; Ikeda et al., 2008). All these cell types present fibroblast-like morphology and however differ in methods of isolation, show significant difference in expression pattern of stem cell markers and in purity of isolated population (Yan et al., 2010; Beltrão-Braga et al., 2011; Tamaoki et al., 2010; Oda et al., 2010).

**Figure 2.** iPSC technology. The iPSC production can be induced by forcing the expression of certain pluripotent genes.

#### **2.1. Transduction and reprogramming**

The marathon of induced pluripotent stem cells (iPSC) started when Yamanaka in 2006, by forcing the expression of certain pluripotent genes in fibroblasts, reversed them into a pluripotent state similar to ES cells (Takahashi &Yamanaka, 2006). The main goal of iPSC generation is to create patient-specific cells, which would be advantageous for cell therapy due to immune compatibility (Ohnuki et al., 2009). Research involving the production of iPSC is being developed around the world. Production of iPSC opens new avenues for understanding human genetic diseases; embryogenesis and will likely have a great impact in drug screening and toxicological tests. However, fibroblasts, which were firstly used for iPSC production, present low efficiency and slow process of reprogramming. Moreover, these cells throughout all life are exposed to environmental factors, which can compromise their use as genetic models (Liu, 2008; McDevitt & Palecek, 2008; Nishikawa et al., 2008; Yu & Thomson; 2008; Zhao & Daley, 2008; Maherali & Hochedlinger, 2008; Ooi et al., 2012). Indeed, more immature somatic cells such as, postmigratory NCSCs, and adult stem cells isolated from young organism showed high efficiency of reprogramming (Zouboulis et al., 2008; Muchkaeva et al., 2012). Because of the possibility to isolate NCSCs from easily accessible tissue (e.g. baby teeth is discarded), the dental pulp derived somatic cells have become an ideal model system to study stem cell biology in diseases during different stages of the development (childhood, youth, middle-aged and old) with a special focus on non-invasive source of the cells for investigation

Our group isolated and fully characterized human immature dental pulp stem cells (hIDPSC), which is a very attractive cell type, from deciduous teeth (baby teeth) (Kerkis et al., 2006; Lizier et al., 2012). The hIDPSC present fibroblast-like morphology, retain characteristics of adult multipotent stem cells and express at least one of three transcription factors: Oct4, Nanog and Sox2 (Kerkis et al., 2006; Lizier et al., 2012). We also used these cells as an alternative source for iPSC derivation (Beltrão-Braga et al., 2011) (Fig. 2).Different research groups derived iPSC from dental pulp fibroblasts and stem cells from young, middle aged and old patients. The difference was observed between the protocols and efficiency of iPSC generation in all these studies (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011). The present chapter is focused on comparative investigation of the methods and efficiency of iPSC generation from dental pulp stem cells and fibroblasts (control). Differentiation potential, assuredness and the future perspectives of the use of these iPSC derived from dental pulp stem

Human dental tissues are rich in stem cells (Giordano et al., 2011; Kerkis & Caplan, 2012). Different research groups isolated and characterized several types of stem cells used for iPSC generation: (i) from apical papilla (Yan et al., 2010), (ii) from dental pulp of primary exfoliated deciduous teeth (Yan et al., 2010; Beltrão-Braga et al., 2011) and (iii) from wisdom teeth (Tamaoki et al., 2010; Oda et al., 2010). SCAP (stem cells from apical papilla) were obtained from tissue at the apex of a tooth root (Yan et al., 2010). SHED (stem cells from human exfoliated deciduous) and IDPSC (immature dental pulp stem cells) were derived from exfoliated

of pediatric diseases (Kerkis & Caplan, 2012; Lizier et al., 2012).

132 Pluripotent Stem Cells

cells in basic research and in biotechnology will also be broadly discussed.

**2. Dental pulp somatic cells used for iPSC generation**

For reprogramming of SHED/SCAP/DPSCs, heterogeneous primary human dental stem/ progenitor cell population at passages 2 and 3 were used (Yan et al., 2010). These populations were tested for their cell surface marker expression by flow cytometry and they were positive for STRO-1, CD146, CD73, CD90, CD105 and negative for CD14, CD34, and CD45, showing typical immunophenotype of mesenchymal stem cells (MSC) (Friedenstein et al., 1976; Caplan, 1991). The first study used four factor genes for reprogramming, such as c-Myc [Myc protooncogene protein], Klf4 [Krüppel-like factor], Oct4 [octamer-binding transcription factor 4], and Sox2 [(sex determining region Y)-box 2] into pLenti6.2/C-Lumio/V5-DEST vector system. Although the cells started to present morphological changes (fibroblastic to epithelial cell-like transition), the reprogramming process failed. Further, lentiviral vectors pSin-EF2-gene-Pur carrying 1 of the 4 factors Lin28 [Lin-28 homolog A], Nanog (Nanog homeobox), Oct4, and Sox2 were used and first ES-like colonies were obtained. To improve reprogramming efficien‐ cy, human genes c-Myc, Klf4, Oct4, and Sox2 were subcloned into the vector pMXs and produced retrovirus was used for second round of transduction. Human fibroblasts, used as a control in this study, were not able to undergo reprogramming under proposed conditions.

efficiency in clonally expanded MStCs was higher and it correlates with cell proliferative ability. The clones, which showed higher proliferative ability, demonstrated a rate of reprog‐ ramming ~ 30–100-fold higher than HDFs and ~ 7-fold higher than clones with lower prolif‐ erative ability. The IDPSC also present high reprogramming efficiency and no difference was observed between the cells from both donors. Therefore, difficulties in reprogramming SHED/ SCAP/DPSCs can be related with cell heterogeneity of original populations (Yan et al., 2010).

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**Figure 3.** hIDPSC-derived iPSC. (A) Representative figure of morphological characteristics of hIDPSC *in vitro* culturing (light microscopy). (B-D) iPSC derivations were shown to be obtained under both conditions: feeder-free, on matrigel-

Furthermore, the aging process influences all organs, tissues and cells of organism. The studies showed that this factor is also important for cells reprogramming (Zouboulis et al., 2008; Banito et al., 2009). SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC were isolated from young donors of variable ages 7, 10, 12, 13, 14 16, 19, 20 and 24 years old. The difference in efficiency of iPSC generation was observed between MStCs isolated from third molars of 10-, 13-, and 16-yearold donors. More efficient reprogramming was observed when MStCs from the 10-year-old donor were used. Similar observation was made by Tamaoki and co-wokers (2010). In our study we used IDPSC from 7 years old donors and we observed rapid and efficient reprog‐

coated dishes (B and C) and on MEF (D).

ramming in both cell populations.

Retroviruses expressing four Oct3/4, Sox2, Klf4, and c-Myc or three (without c-Myc) factors were used for reprogramming DPCs from wisdom teeth and from human dermal fibroblasts (HDFs), which was performed according to the methods previously described (Takahashi et al., 2007). Another group, which used MStCs from wisdom teeth, also demonstrated successful reprogramming of these cells with pMXs retrovirus vectors containing three human Oct3/4, Sox2, and Klf4 factor genes (Oda et al., 2010).

IDPSC is a homogeneous population in respect of the expression of MSCs (Friedenstein et al., 1976; Caplan, 1991) markers, such as CD73, CD105, nestin and vimentin. Within IDPSC population, several cells also express Oct3/4 and Nanog (Kerkis et al., 2006; Lizier et al., 2012). To reprogram IDPSC, our group used four Yamanaka's factors (Klf4, Oct4, c-Myc and Sox2) and previously established protocol (Takahashi et al., 2007; Beltrão-Braga et al., 2011).

#### **2.2. Formation of ES cell-like colonies and expansion**

There are several important points that should be considered when iPSC are isolated and expanded: (i) the use of mouse embryonic fibroblasts (MEF) as a feeder layer, (ii) the efficiency of reprogramming and (iii) the efficiency of expansion (Takahashi & Yamanaka, 2006; Lewitzky & Yamanaka, 2007; Bilic & Belmonte, 2012). Isolation of iPSC on MEF limits the manipulation and further clinical application of these cells. Thus, isolation and expansion of iPSC without MEF is an important step, which avoids contamination of human cells with animal products. Efficiency of reprogramming depends on different factor, such as gene expression profile of cells, which were used in experiments. It has been shown that more immature cells undergo this process more efficiently, then committed or terminally differen‐ tiated cells (Zouboulis et al., 2008; Muchkaeva et al., 2012). And finally, during reprogramming, the cells receive different number of reprogramming factors and/or they did not respond equally to this process, therefore multiple ES-like did not complete reprogramming or non-ES cell-like colonies raised (Aasen et al., 2008; Marchetto et al., 2009). SHED/SCAP/DPSCs/DPCs/ MStCs-derived iPSC were obtained using MEF as a feeder layer (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010). IDPSC-derived iPSC were shown to be obtained under both conditions: feeder-free on matrigel-coated dishes and on MEF (Fig. 3) (Beltrão-Braga et al., 2011). It seems that time-course of reprogramming of different cell types varied in accordance with age of cell donor, cell type and number of factors used. Thus, SHED/SCAP/DPSCsderived iPSC, showed the formation of the first colonies ~2-3 weeks after gene transduction. DPCs-derived iPSC were reprogrammed in ~14 days, when 4 factors were used and in ~ 20-25 days, when reprogramming was performed with only 3 factors. MStCs-derived iPSC were reprogrammed in ~25 days, while IDPSC-derived iPSC demonstrated the formation of first colonies at ~ day 5-11. All studies demonstrated that efficiency of iPSC derivation from dental pulp tissues is higher than that from human dermal fibroblast and primary gingival fibroblasts (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011). Because the MStCs were a heterogeneous cell population, Oda and co-authors (2010) additionally used clonally expanded MStCs in reprogramming experiments. They observed that reprogramming efficiency in clonally expanded MStCs was higher and it correlates with cell proliferative ability. The clones, which showed higher proliferative ability, demonstrated a rate of reprog‐ ramming ~ 30–100-fold higher than HDFs and ~ 7-fold higher than clones with lower prolif‐ erative ability. The IDPSC also present high reprogramming efficiency and no difference was observed between the cells from both donors. Therefore, difficulties in reprogramming SHED/ SCAP/DPSCs can be related with cell heterogeneity of original populations (Yan et al., 2010).

produced retrovirus was used for second round of transduction. Human fibroblasts, used as a control in this study, were not able to undergo reprogramming under proposed conditions.

Retroviruses expressing four Oct3/4, Sox2, Klf4, and c-Myc or three (without c-Myc) factors were used for reprogramming DPCs from wisdom teeth and from human dermal fibroblasts (HDFs), which was performed according to the methods previously described (Takahashi et al., 2007). Another group, which used MStCs from wisdom teeth, also demonstrated successful reprogramming of these cells with pMXs retrovirus vectors containing three human Oct3/4,

IDPSC is a homogeneous population in respect of the expression of MSCs (Friedenstein et al., 1976; Caplan, 1991) markers, such as CD73, CD105, nestin and vimentin. Within IDPSC population, several cells also express Oct3/4 and Nanog (Kerkis et al., 2006; Lizier et al., 2012). To reprogram IDPSC, our group used four Yamanaka's factors (Klf4, Oct4, c-Myc and Sox2)

There are several important points that should be considered when iPSC are isolated and expanded: (i) the use of mouse embryonic fibroblasts (MEF) as a feeder layer, (ii) the efficiency of reprogramming and (iii) the efficiency of expansion (Takahashi & Yamanaka, 2006; Lewitzky & Yamanaka, 2007; Bilic & Belmonte, 2012). Isolation of iPSC on MEF limits the manipulation and further clinical application of these cells. Thus, isolation and expansion of iPSC without MEF is an important step, which avoids contamination of human cells with animal products. Efficiency of reprogramming depends on different factor, such as gene expression profile of cells, which were used in experiments. It has been shown that more immature cells undergo this process more efficiently, then committed or terminally differen‐ tiated cells (Zouboulis et al., 2008; Muchkaeva et al., 2012). And finally, during reprogramming, the cells receive different number of reprogramming factors and/or they did not respond equally to this process, therefore multiple ES-like did not complete reprogramming or non-ES cell-like colonies raised (Aasen et al., 2008; Marchetto et al., 2009). SHED/SCAP/DPSCs/DPCs/ MStCs-derived iPSC were obtained using MEF as a feeder layer (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010). IDPSC-derived iPSC were shown to be obtained under both conditions: feeder-free on matrigel-coated dishes and on MEF (Fig. 3) (Beltrão-Braga et al., 2011). It seems that time-course of reprogramming of different cell types varied in accordance with age of cell donor, cell type and number of factors used. Thus, SHED/SCAP/DPSCsderived iPSC, showed the formation of the first colonies ~2-3 weeks after gene transduction. DPCs-derived iPSC were reprogrammed in ~14 days, when 4 factors were used and in ~ 20-25 days, when reprogramming was performed with only 3 factors. MStCs-derived iPSC were reprogrammed in ~25 days, while IDPSC-derived iPSC demonstrated the formation of first colonies at ~ day 5-11. All studies demonstrated that efficiency of iPSC derivation from dental pulp tissues is higher than that from human dermal fibroblast and primary gingival fibroblasts (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011). Because the MStCs were a heterogeneous cell population, Oda and co-authors (2010) additionally used clonally expanded MStCs in reprogramming experiments. They observed that reprogramming

and previously established protocol (Takahashi et al., 2007; Beltrão-Braga et al., 2011).

Sox2, and Klf4 factor genes (Oda et al., 2010).

134 Pluripotent Stem Cells

**2.2. Formation of ES cell-like colonies and expansion**

**Figure 3.** hIDPSC-derived iPSC. (A) Representative figure of morphological characteristics of hIDPSC *in vitro* culturing (light microscopy). (B-D) iPSC derivations were shown to be obtained under both conditions: feeder-free, on matrigelcoated dishes (B and C) and on MEF (D).

Furthermore, the aging process influences all organs, tissues and cells of organism. The studies showed that this factor is also important for cells reprogramming (Zouboulis et al., 2008; Banito et al., 2009). SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC were isolated from young donors of variable ages 7, 10, 12, 13, 14 16, 19, 20 and 24 years old. The difference in efficiency of iPSC generation was observed between MStCs isolated from third molars of 10-, 13-, and 16-yearold donors. More efficient reprogramming was observed when MStCs from the 10-year-old donor were used. Similar observation was made by Tamaoki and co-wokers (2010). In our study we used IDPSC from 7 years old donors and we observed rapid and efficient reprog‐ ramming in both cell populations.

#### **2.3. Provirus integration**

Viral vectors are commonly used to deliver genetic material into cells, which can be performed *in vivo* (living organism) or *in vitro* (cell culture). Delivery of genes by a virus is efficient, however with respect to safety, it is ideal not to use lentivirus and retrovirus vectors, since they can integrate into the host DNA. As opposed to lentiviruses and retroviruses, adenoviral DNA does not integrate into the genome and are considered to be safer (Tamaoki et al., 2010). All studies performed with stem cells of dental tissue origin used lentivirus and/or retrovirus vectors for reprogramming. Yan and co-workers (2010) examined the presence of transgenes in the genome of iPSC clones isolating genomic DNA and generating primers specific for each transgene. They showed that the 4 factors were all integrated into the genome of the transduced SHED/SCAP/DPSC-iPSC. Other study, which used MStCs from wisdom teeth for reprogram‐ ming, did not provide any records about viral vectors integration (Tamaoki et al., 2010), while another group demonstrated retroviral silencing (Oda et al., 2010). Beltrão-Braga and coauthors (2011), which used retroviral vector for reprogramming, showed the lack of transgene expression by RT-PCR analyses in iPSC-derived from IDPSCs.

higher in iPSC derived from clonally isolated MStCs, when compared with parental cell lines,

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137

**Figure 4.** Expression of Oct3/4, Nanog, Sox2 and TRA-1-81 proteins in two lineages of hIDPS-IPSC five days after trans‐ duction with four factors. In A-C3,G) hIDPS-IPSC1 and D-F3,H) hIDPS-IPSC2 are presented, both showing multiple small colonies, which already express hallmarks of pluripotent cells, such as, A-A3) and D-D3) Oct3/4; B-B3) and E-E3) Nanog, C-C3) and F-F3) Sox2; G) and H) TRA-1-81, respectively. Nucleus stained with DAPI (blue). Note, that Oct3/4, Nanog, Sox2 present nuclear, while TRA-1-81 presents cytoplasm localization. Several cells, which did not present ex‐ pression of these proteins and served as a control, are indicated by white arrows. Confocal Microscopy: A-F) Differen‐ tial interference contrast (DIC); A1-F1 and A2-F2) Fluorescent microscopy (Fm); A3-F3, G, H) DIC+Fm. Scale Bars: A-D3,

Yan et al., (2010) quantified by real-time PCR the expression levels of endogenous Klf4 and c-Myc. Klf4 showed relative higher expression in DPC lines than in HDFs, however lower than in ES cells. Endogenous c-Myc expression in most DPC lines was also slightly higher than that in HDFs and in a few iPSC clones were close to ES cells. In contrast, Oda et al., (2010) observed low expression of Klf4 in high reprogramming cells, which was unexpected, once Klf4 is a reprogramming factor. Yan et al., (2010) showed that endogenous Klf4 expression level determined by real-time PCR did not completely correlate with the reprogramming efficiency of each DPCs (wisdom teeth) line. It is noteworthy that highly expression of KLF4 was

F-F3, H =50μm; E-E3,G=100μm.

iPSC-derived from these lines and HDFs, used as a control.

#### **2.4. Characterization of SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC-derived iPSC**

#### *2.4.1. Expression of pluripotent stem cell markers*

As expected in all studies, the iPSC obtained from tissues of dental origin, which showed ESlike cells morphology, express key markers of pluripotent stem cells in an appropriate manner. Immunofluorescence study demonstrates uniform expression of these antigens in iPSC colonies derived from different types of dental stem cells. Transcription factor proteins as Oct3/4, Nanog, Sox2 demonstrate nuclear localization, while cell surface markers, such as stage specific embryonic antigen (SSEA) 3 and SSEA4, as well as cell surface antigens of human embryonic carcinoma cells (TRA-1-60 and TRA-1-81) show cell surface localization. Appro‐ priate expression of transcription factors Klf4, c-Myc, Lin28, that were part of the transgene used for reprogramming, also was observed (Yan et al. 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011) (Fig. 4).

#### *2.4.2. Expression of molecular markers of pluripotent stem cells*

Only one study performed quantitative PCR analysis before and after reprogramming for endogenous expression of Oct4, Nanog and Sox2 genes and compared the expression level of all these genes with those in pluripotent human ES cells. Albeit we revealed a tendency for increasing of expression of pluripotent factors Oct4, Nanog and Sox2, when compared to nonreprogrammed cells (18%, 1% and 2%, respectively), it was significantly lower 20% (Oct4), 10% (Nanog) and 40% (Sox2) in comparison with human ES cells (100% - Oct4, Nanog, Sox2) (Beltrão-Braga et al., 2011). Other studies did not provide any data about expression of these key markers in SHED/SCAP/DPSCs/DPCs/MStCs – derived iPSC in comparison with ES cells (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010). However, Oda and colleagues (2010) demonstrated that expression levels of Oct4, Nanog, Sox2, Klf4, c-Myc, Lin28 and P53 was higher in iPSC derived from clonally isolated MStCs, when compared with parental cell lines, iPSC-derived from these lines and HDFs, used as a control.

**2.3. Provirus integration**

136 Pluripotent Stem Cells

Viral vectors are commonly used to deliver genetic material into cells, which can be performed *in vivo* (living organism) or *in vitro* (cell culture). Delivery of genes by a virus is efficient, however with respect to safety, it is ideal not to use lentivirus and retrovirus vectors, since they can integrate into the host DNA. As opposed to lentiviruses and retroviruses, adenoviral DNA does not integrate into the genome and are considered to be safer (Tamaoki et al., 2010). All studies performed with stem cells of dental tissue origin used lentivirus and/or retrovirus vectors for reprogramming. Yan and co-workers (2010) examined the presence of transgenes in the genome of iPSC clones isolating genomic DNA and generating primers specific for each transgene. They showed that the 4 factors were all integrated into the genome of the transduced SHED/SCAP/DPSC-iPSC. Other study, which used MStCs from wisdom teeth for reprogram‐ ming, did not provide any records about viral vectors integration (Tamaoki et al., 2010), while another group demonstrated retroviral silencing (Oda et al., 2010). Beltrão-Braga and coauthors (2011), which used retroviral vector for reprogramming, showed the lack of transgene

expression by RT-PCR analyses in iPSC-derived from IDPSCs.

*2.4.1. Expression of pluripotent stem cell markers*

2010; Beltrão-Braga et al., 2011) (Fig. 4).

*2.4.2. Expression of molecular markers of pluripotent stem cells*

**2.4. Characterization of SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC-derived iPSC**

As expected in all studies, the iPSC obtained from tissues of dental origin, which showed ESlike cells morphology, express key markers of pluripotent stem cells in an appropriate manner. Immunofluorescence study demonstrates uniform expression of these antigens in iPSC colonies derived from different types of dental stem cells. Transcription factor proteins as Oct3/4, Nanog, Sox2 demonstrate nuclear localization, while cell surface markers, such as stage specific embryonic antigen (SSEA) 3 and SSEA4, as well as cell surface antigens of human embryonic carcinoma cells (TRA-1-60 and TRA-1-81) show cell surface localization. Appro‐ priate expression of transcription factors Klf4, c-Myc, Lin28, that were part of the transgene used for reprogramming, also was observed (Yan et al. 2010; Tamaoki et al., 2010; Oda et al.,

Only one study performed quantitative PCR analysis before and after reprogramming for endogenous expression of Oct4, Nanog and Sox2 genes and compared the expression level of all these genes with those in pluripotent human ES cells. Albeit we revealed a tendency for increasing of expression of pluripotent factors Oct4, Nanog and Sox2, when compared to nonreprogrammed cells (18%, 1% and 2%, respectively), it was significantly lower 20% (Oct4), 10% (Nanog) and 40% (Sox2) in comparison with human ES cells (100% - Oct4, Nanog, Sox2) (Beltrão-Braga et al., 2011). Other studies did not provide any data about expression of these key markers in SHED/SCAP/DPSCs/DPCs/MStCs – derived iPSC in comparison with ES cells (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010). However, Oda and colleagues (2010) demonstrated that expression levels of Oct4, Nanog, Sox2, Klf4, c-Myc, Lin28 and P53 was

**Figure 4.** Expression of Oct3/4, Nanog, Sox2 and TRA-1-81 proteins in two lineages of hIDPS-IPSC five days after trans‐ duction with four factors. In A-C3,G) hIDPS-IPSC1 and D-F3,H) hIDPS-IPSC2 are presented, both showing multiple small colonies, which already express hallmarks of pluripotent cells, such as, A-A3) and D-D3) Oct3/4; B-B3) and E-E3) Nanog, C-C3) and F-F3) Sox2; G) and H) TRA-1-81, respectively. Nucleus stained with DAPI (blue). Note, that Oct3/4, Nanog, Sox2 present nuclear, while TRA-1-81 presents cytoplasm localization. Several cells, which did not present ex‐ pression of these proteins and served as a control, are indicated by white arrows. Confocal Microscopy: A-F) Differen‐ tial interference contrast (DIC); A1-F1 and A2-F2) Fluorescent microscopy (Fm); A3-F3, G, H) DIC+Fm. Scale Bars: A-D3, F-F3, H =50μm; E-E3,G=100μm.

Yan et al., (2010) quantified by real-time PCR the expression levels of endogenous Klf4 and c-Myc. Klf4 showed relative higher expression in DPC lines than in HDFs, however lower than in ES cells. Endogenous c-Myc expression in most DPC lines was also slightly higher than that in HDFs and in a few iPSC clones were close to ES cells. In contrast, Oda et al., (2010) observed low expression of Klf4 in high reprogramming cells, which was unexpected, once Klf4 is a reprogramming factor. Yan et al., (2010) showed that endogenous Klf4 expression level determined by real-time PCR did not completely correlate with the reprogramming efficiency of each DPCs (wisdom teeth) line. It is noteworthy that highly expression of KLF4 was previously reported in senescent cells and terminally differentiated cells (Shields et al., 1996; Conkright et al., 1999). Taken together, these data suggest that endogenous Klf4 expression may not be the single factor in charge for the reprogramming efficiency to MSCs derived from wisdom teeth.

*2.4.6. Karyotype analysis of dental stem cell-derived iPSC*

Magnification 63X. Differential interference contrast (DIC)

glial fibrillary acidic protein (GFAP) (Fig. 6).

*2.5.1. Embryoid body formation and in vitro differentiation*

Karyotype study has been performed by all authors and demonstrated that karyotype of reprogrammed cells remained unchanged (Fig. 5). Overall, during reprogramming of stem cell from dental pulp, numerical and gross structural chromosomal abnormalities were not detected (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011).

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**Figure 5.** Representative figures of karyotype analysis of both hIDPSC and hIDPS-iPSC: Routine Giemsa staining did not reveal any numerical changes in chromosome number (A and B) and any chromosomal structural changes (B).

Similar to human ES cells, iPSC require the formation of embryoid bodies (EB) in order to undergo *in vitro* differentiation into various cell types. All studies confirm successful differentiation of iPSC-derived from different types of dental stem cells into all three germ layers (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011). As expected, the majority of iPSC formed EBs with cystic cavities. The histological analysis demonstrates that EBs differentiated into different cell types of ectodermal, mesodermal, or endodermal origin, which was demonstrated using antibodies against specific pro‐ teins, which is expressed in each of three germ layers. It has been shown that after reprogramming, cells tend to maintain their original commitment. Dental pulp stem cells are multipotent stem cells derived from neural crest and they showed strong commit‐ ment into neural lineages. Therefore, neural differentiation is widely presented in all iPSCderived from different types of dental stem cells. Under appropriate neurogenic culture medium, the EBs developed into the cells with neural-like morphology (Fig. 6), which express such markers as nestin, β-tubulin III (TUJ1), neuron-specific Enolase (NSE) and

**2.5. Differentiation of SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC-iPSC**

#### *2.4.3. Searching for new factors of reprogramming*

Oda et al., (2010) tried to find the additional unknown factor(s) that could help in the cell reprogramming. They focused their study on practically two genes: PAXIP1 (or PTIP) and PARP. PAXIP1 acts as component of a histone H3 lysine four (H3K4) methyltransferase complex (Cho et al., 2007; Patel et al., 2007) and has a role in DNA double-strand break repair (van Attikum and Gasser, 2009). It was demonstrated that efficient reprogramming of pluripotent gene (Oct3/4, Sox2) expression is associated with H3K4 methylation in mouse somatic cell nuclei transplantation into amphibian oocytes (Murata et al., 2010). The expression of this gene was about 30% more in the high reprogramming cells than in low reprogramming as well as 3–4 times more in iPSC when compared with each parental cell line. PARP-1 belongs to PARP family being the most abundant member and is responsible for > 85% of nuclear PARP activity modifying histone structure through DNA-dependent "PARylation". Higher expres‐ sion of PARP-1 was also seen after induction of reprogramming in cells derived from wisdom teeth. The authors supposed that due to possible conformational change of chromatin by direct/indirect actions of chromatin modification proteins such as PAXIP1 and possibly PARP-1, high iPSC generation clones may be accessible for reprogramming factors. However, further investigation is needed to illuminate the iPSC reprogramming mechanisms using these genes.

#### *2.4.4. Methylation status of cytosine guanine dinucleotides (CpG)*

The methylation status of CpG in the promoter regions of Nanog and Oct4 was examined using bisulfite DNA sequencing method in two studies (Yan et al., 2010; Oda et al., 2010) and of Nanog in one study (Tamaoki et al., 2010). They showed that parental MStCs from wisdom teeth were highly (Oct3/4) or partially (Nanog) methylated and the iPSC-derived from these cells were highly unmethylated, suggesting that these promoters were active after cells reprogramming. In contrast, the analysis of iPSC clones derived from DPSCs (wisdom teeth) and SHED (deciduous teeth) showed that Nanog promoter had similar or slightly higher number of methylated sites, than their non-transduced counterparts. The SHED-/DPSC-iPSC had less methylated sites of Oct3/4 promoter than the non-transduced cells (Yan et al., 2010).

#### *2.4.5. Telomerase activity*

Telomerase activity is known to be highly activated in ES cells in order to maintain the integrity of chromosome structure. After reprogramming, SHED- (deciduous teeth) SCAP-, and DPSC (wisdom teeth) - iPSC showed telomerase activity very close to ES cells and a lot more in comparison to their non-transduced counterparts (Yan et al., 2010). Parental DPSCs (wisdom teeth) showed low telomerase activity whereas in each iPSC telomerase activity was high (Oda et al., 2010).

#### *2.4.6. Karyotype analysis of dental stem cell-derived iPSC*

previously reported in senescent cells and terminally differentiated cells (Shields et al., 1996; Conkright et al., 1999). Taken together, these data suggest that endogenous Klf4 expression may not be the single factor in charge for the reprogramming efficiency to MSCs derived from

Oda et al., (2010) tried to find the additional unknown factor(s) that could help in the cell reprogramming. They focused their study on practically two genes: PAXIP1 (or PTIP) and PARP. PAXIP1 acts as component of a histone H3 lysine four (H3K4) methyltransferase complex (Cho et al., 2007; Patel et al., 2007) and has a role in DNA double-strand break repair (van Attikum and Gasser, 2009). It was demonstrated that efficient reprogramming of pluripotent gene (Oct3/4, Sox2) expression is associated with H3K4 methylation in mouse somatic cell nuclei transplantation into amphibian oocytes (Murata et al., 2010). The expression of this gene was about 30% more in the high reprogramming cells than in low reprogramming as well as 3–4 times more in iPSC when compared with each parental cell line. PARP-1 belongs to PARP family being the most abundant member and is responsible for > 85% of nuclear PARP activity modifying histone structure through DNA-dependent "PARylation". Higher expres‐ sion of PARP-1 was also seen after induction of reprogramming in cells derived from wisdom teeth. The authors supposed that due to possible conformational change of chromatin by direct/indirect actions of chromatin modification proteins such as PAXIP1 and possibly PARP-1, high iPSC generation clones may be accessible for reprogramming factors. However, further investigation is needed to illuminate the iPSC reprogramming mechanisms using these

The methylation status of CpG in the promoter regions of Nanog and Oct4 was examined using bisulfite DNA sequencing method in two studies (Yan et al., 2010; Oda et al., 2010) and of Nanog in one study (Tamaoki et al., 2010). They showed that parental MStCs from wisdom teeth were highly (Oct3/4) or partially (Nanog) methylated and the iPSC-derived from these cells were highly unmethylated, suggesting that these promoters were active after cells reprogramming. In contrast, the analysis of iPSC clones derived from DPSCs (wisdom teeth) and SHED (deciduous teeth) showed that Nanog promoter had similar or slightly higher number of methylated sites, than their non-transduced counterparts. The SHED-/DPSC-iPSC had less methylated sites of Oct3/4 promoter than the non-transduced cells (Yan et al., 2010).

Telomerase activity is known to be highly activated in ES cells in order to maintain the integrity of chromosome structure. After reprogramming, SHED- (deciduous teeth) SCAP-, and DPSC (wisdom teeth) - iPSC showed telomerase activity very close to ES cells and a lot more in comparison to their non-transduced counterparts (Yan et al., 2010). Parental DPSCs (wisdom teeth) showed low telomerase activity whereas in each iPSC telomerase

wisdom teeth.

138 Pluripotent Stem Cells

genes.

*2.4.5. Telomerase activity*

activity was high (Oda et al., 2010).

*2.4.3. Searching for new factors of reprogramming*

*2.4.4. Methylation status of cytosine guanine dinucleotides (CpG)*

Karyotype study has been performed by all authors and demonstrated that karyotype of reprogrammed cells remained unchanged (Fig. 5). Overall, during reprogramming of stem cell from dental pulp, numerical and gross structural chromosomal abnormalities were not detected (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011).

**Figure 5.** Representative figures of karyotype analysis of both hIDPSC and hIDPS-iPSC: Routine Giemsa staining did not reveal any numerical changes in chromosome number (A and B) and any chromosomal structural changes (B). Magnification 63X. Differential interference contrast (DIC)

#### **2.5. Differentiation of SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC-iPSC**

#### *2.5.1. Embryoid body formation and in vitro differentiation*

Similar to human ES cells, iPSC require the formation of embryoid bodies (EB) in order to undergo *in vitro* differentiation into various cell types. All studies confirm successful differentiation of iPSC-derived from different types of dental stem cells into all three germ layers (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010; Beltrão-Braga et al., 2011). As expected, the majority of iPSC formed EBs with cystic cavities. The histological analysis demonstrates that EBs differentiated into different cell types of ectodermal, mesodermal, or endodermal origin, which was demonstrated using antibodies against specific pro‐ teins, which is expressed in each of three germ layers. It has been shown that after reprogramming, cells tend to maintain their original commitment. Dental pulp stem cells are multipotent stem cells derived from neural crest and they showed strong commit‐ ment into neural lineages. Therefore, neural differentiation is widely presented in all iPSCderived from different types of dental stem cells. Under appropriate neurogenic culture medium, the EBs developed into the cells with neural-like morphology (Fig. 6), which express such markers as nestin, β-tubulin III (TUJ1), neuron-specific Enolase (NSE) and glial fibrillary acidic protein (GFAP) (Fig. 6).

neurons and peripheral neuroendocrine tissue, and synaptophysin (a synaptic vesicle glycoprotein), which is found in neuroendocrine cells as well as virtually in all neurons that participate in synaptic transmission in the brain and spinal cord are expressed in

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**Figure 7.** HE stained differentiated tissues from hIDPS-iPSC teratoma seven weeks after transplantation into nude mice right limb. A) Morphogenesis observed during differentiation of hIDPS-iPSC: glomerulus-like structure formation, with Bowman's capsule and convoluted tubule–like structures. B) Cartilage and in (C) Condrocytes-like cells (higher magnification). D) Respiratory-like epithelium. E) Gastrointestinal-like epithelium. F) Neural tubes-like structures. G)

Tamaoki and co-wokers (2010) determined the human leukocyte antigens (HLA) types of 107 dental pulp cells lines in the Japanese population and identified 2 cell lines with homozygous HLA types at all 3 loci (A, B, and DR) examined. They showed that in the Japanese population the frequencies of haplotypes of these 2 homozygous cell lines were estimated to be 8.7% and 1.5%, data provided by the Japanese Red Cross Society (http://www.bmdc.jrc.or.jp/stat.html). Using these frequencies, the coverage rate for a perfect match of iPSC lines, which were established from these 2 lines was calculated. The authors showed that iPSC lines established from these 2 homozygous cell lines would cover 16.6% and 3.0% of the Japanese population,

respectively, which corresponds to approximately 20% of the Japanese population.

One of the major challenge of pluripotent stem cells use in cell therapies is an immunemediated rejection after transplantation. Today, this problem can be overcome by direct reprogramming of patients somatic cells and by creating an iPSC bank consisting of various HLA types thus providing therapeutic tool for the patients, which need cell transplantation free from immune-mediated rejection. Two works reported that the establishment of 50 unique stem cells lines, having homozygous alleles of the 3 HLA loci (A, B, and DR), would cover ~

**2.7. SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC-iPSC bank and therapeutic use**

Blood vessel. H) Adipose-like tissue. Magnifications: A, C) 100x, B, D-G) 20x, Scale Bar (H) = 200μm.

**2.6. HLA typing**

neuronal cells produced by IDPSC-iPSC in teratomas (Beltrão-Braga et al., 2011).

**Figure 6.** Representative figure of *in vitro* differentiation of hIDPS-iPSC. A) EBs adherent on Petri dish showing differ‐ entiation into neural-like cells after culturing in neurobasal+B27 medium. B) Same as in (A) showing neural-like cells in high magnification. C) Neural-like cells present positive immunostaining with anti-nestin antibody. D) positive immu‐ nostaining with anti-TUJ1 antibody. E) Neuron-specific enolase positive immunostaining (green) in hIDPS-iPSC derived neuronal cells. F) Morphological presentation of glial-like cells derived from hIDPS-iPSC. Positive immunostaining for (F) anti-GFAP antibody in glial-like cells. Nucleus stained with DAPI (blue). A, B= Differential interference contrast (DIC). C-E=Epi. F= DIC+Epi. Scale Bars: A-F=20μm.

#### *2.5.2. Teratoma formation*

To test the pluripotency, iPSC were injected into the testis or intramuscularly into the right and/or left hind leg of severe combined immunodeficient (SCID) mouse. Teratomas formation by SHED/SCAP/DPSCs/DPCs/MStCs–iPSC occurs of nine to eleven weeks after injection and histological examination of the tumor shows representative tissues of three embryonic germ layers, such as gut-like epithelium (endoderm), cartilage (mesoderm), and neuroepithelial rosettes (ectoderm) (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010). Teratomas obtained from IDPSC-iPSC were formed between 5 and 7 weeks after reprog‐ rammed cells injection. The mice injected with parental IDPSC, as expected, did not form teratomas. We observed that teratomas were composed by tissues originated from three primary germ layers. Histological characterization of tumor masses showed that these teratomas includes ectodermal: primitive neural tissues, including neural tube and neural rosettes and retinal epithelium; mesodermal: muscle-like cells and gromerulus-like structures and endodermal tissues: respiratory or gastro-intestinal-like epithelium and glandular-like tissue formation (Fig. 7). Similar to *in vitro* differentiation, teratomas derived from IDPSC-iPS cells, display strong neuronal commitment forming rosette-, neuro‐ sphere- and neural tube-like structures. Neuron-specific enolase, which is a marker of neurons and peripheral neuroendocrine tissue, and synaptophysin (a synaptic vesicle glycoprotein), which is found in neuroendocrine cells as well as virtually in all neurons that participate in synaptic transmission in the brain and spinal cord are expressed in neuronal cells produced by IDPSC-iPSC in teratomas (Beltrão-Braga et al., 2011).

**Figure 7.** HE stained differentiated tissues from hIDPS-iPSC teratoma seven weeks after transplantation into nude mice right limb. A) Morphogenesis observed during differentiation of hIDPS-iPSC: glomerulus-like structure formation, with Bowman's capsule and convoluted tubule–like structures. B) Cartilage and in (C) Condrocytes-like cells (higher magnification). D) Respiratory-like epithelium. E) Gastrointestinal-like epithelium. F) Neural tubes-like structures. G) Blood vessel. H) Adipose-like tissue. Magnifications: A, C) 100x, B, D-G) 20x, Scale Bar (H) = 200μm.

#### **2.6. HLA typing**

**Figure 6.** Representative figure of *in vitro* differentiation of hIDPS-iPSC. A) EBs adherent on Petri dish showing differ‐ entiation into neural-like cells after culturing in neurobasal+B27 medium. B) Same as in (A) showing neural-like cells in high magnification. C) Neural-like cells present positive immunostaining with anti-nestin antibody. D) positive immu‐ nostaining with anti-TUJ1 antibody. E) Neuron-specific enolase positive immunostaining (green) in hIDPS-iPSC derived neuronal cells. F) Morphological presentation of glial-like cells derived from hIDPS-iPSC. Positive immunostaining for (F) anti-GFAP antibody in glial-like cells. Nucleus stained with DAPI (blue). A, B= Differential interference contrast (DIC).

To test the pluripotency, iPSC were injected into the testis or intramuscularly into the right and/or left hind leg of severe combined immunodeficient (SCID) mouse. Teratomas formation by SHED/SCAP/DPSCs/DPCs/MStCs–iPSC occurs of nine to eleven weeks after injection and histological examination of the tumor shows representative tissues of three embryonic germ layers, such as gut-like epithelium (endoderm), cartilage (mesoderm), and neuroepithelial rosettes (ectoderm) (Yan et al., 2010; Tamaoki et al., 2010; Oda et al., 2010). Teratomas obtained from IDPSC-iPSC were formed between 5 and 7 weeks after reprog‐ rammed cells injection. The mice injected with parental IDPSC, as expected, did not form teratomas. We observed that teratomas were composed by tissues originated from three primary germ layers. Histological characterization of tumor masses showed that these teratomas includes ectodermal: primitive neural tissues, including neural tube and neural rosettes and retinal epithelium; mesodermal: muscle-like cells and gromerulus-like structures and endodermal tissues: respiratory or gastro-intestinal-like epithelium and glandular-like tissue formation (Fig. 7). Similar to *in vitro* differentiation, teratomas derived from IDPSC-iPS cells, display strong neuronal commitment forming rosette-, neuro‐ sphere- and neural tube-like structures. Neuron-specific enolase, which is a marker of

C-E=Epi. F= DIC+Epi. Scale Bars: A-F=20μm.

*2.5.2. Teratoma formation*

140 Pluripotent Stem Cells

Tamaoki and co-wokers (2010) determined the human leukocyte antigens (HLA) types of 107 dental pulp cells lines in the Japanese population and identified 2 cell lines with homozygous HLA types at all 3 loci (A, B, and DR) examined. They showed that in the Japanese population the frequencies of haplotypes of these 2 homozygous cell lines were estimated to be 8.7% and 1.5%, data provided by the Japanese Red Cross Society (http://www.bmdc.jrc.or.jp/stat.html). Using these frequencies, the coverage rate for a perfect match of iPSC lines, which were established from these 2 lines was calculated. The authors showed that iPSC lines established from these 2 homozygous cell lines would cover 16.6% and 3.0% of the Japanese population, respectively, which corresponds to approximately 20% of the Japanese population.

#### **2.7. SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC-iPSC bank and therapeutic use**

One of the major challenge of pluripotent stem cells use in cell therapies is an immunemediated rejection after transplantation. Today, this problem can be overcome by direct reprogramming of patients somatic cells and by creating an iPSC bank consisting of various HLA types thus providing therapeutic tool for the patients, which need cell transplantation free from immune-mediated rejection. Two works reported that the establishment of 50 unique stem cells lines, having homozygous alleles of the 3 HLA loci (A, B, and DR), would cover ~ 90% of the Japanese population with a faultless match of these loci (Nakajima et al., 2007; Nakatsuji et al., 2008). Considering that iPSC derivation is a time consuming process and of elevated cost, it should be necessary for cell therapies and regenerative medicine to establish iPSC banks with a sufficient collection of HLA types, thus avoiding additional costs which are required for iPSC production for each individual patient.

*2.7.2. Perspectives of iPSC therapeutic use*

**3. Final considerations**

Currently, iPSC are used to understand human diseases, including Alzheimer's disease, Parkinson's disease, cardiovascular disease, diabetes, and amyotrophic lateral sclerosis (ALS), to develop and screen bioactive molecules - candidate to therapeutic drugs and to identify molecules or genes implicated in tissue regeneration. These *in vitro* studies enable researchers to understand fundamental principles of iPSC function and differentiation, which further will provide knowledge, necessary for therapeutic use of iPSC. Based on this knowledge multiple pre-clinical and clinical protocols will be produced optimizing iPSC transplantation in diverse animal and human diseases thus becoming a tool in cellreplacement therapy. Therefore, iPSC in the future may have tremendous clinical poten‐ tial when highly efficient and safe protocols of generation of reprogrammed stem cells will be developed. Increasing our understanding of the molecular mechanisms that underlie reprogramming, we will be able to identify the cell types and methods of reprogram‐ ming, which will minimize DNA alterations, and conditions of iPSC cultivation that will allow widespread use of these cells in clinic. The scientists, however, should answer the question if iPSC are truly equivalent to human ES cells. Although iPSC potential for regenerative medicine is great, our current knowledge about iPSC variability, and utility must also increase greatly before iPSC became a standard tool for regenerative medicine.

Generation of Induced Pluripotent Stem Cells from Dental Pulp Somatic Cells

http://dx.doi.org/10.5772/55856

143

Different dental tissues, which include apical papilla, primary exfoliated deciduous and permanent teeth, as well as wisdom teeth were used to derivate iPSC. The data obtained by different authors indicate that these tissues can be easily isolated and MSCs cells in sufficient quantities can be obtained. MSCs *in vivo* are reversibly arrested cells, which are localized in their niches maintaining their temporarily quiescent state. They differ from terminally differentiated cells by developmental path that involves a set of increasingly committed stages of specialization. The fact that these cells are undifferentiated cells suggests that their reprog‐ ramming will occur more easily than that of terminally differentiated cells. Indeed, all studies demonstrated that in SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC the reprogramming process occurs more easily than in human fibroblasts used as a control, under the similar protocols and vectors used for transduction, which showed to be efficient. It was also possible to reprogram MSCs from wisdom teeth using only three factors (without c-Myc) avoiding future implications with potential risks of oncogene use (Yu et al., 2007). For the clinical applications of stem cells xenogeneic reagents pose the risk of a severe immune response, and the trans‐ mission of viral or bacterial infections, prions, and unidentified zoonosis. We demonstrated that isolation of IDPSC-iPSC can occur under feeder-free conditions on matrigel-coated dishes. Clinical stem cell therapy trials are ongoing, which request a strong focus on the safety and quality of *in vitro* expanded stem cell transplants. By replacing xenogeneic products with a defined xeno-free medium, the safety and quality of the cells with therapeutic potential may be enhanced significantly. Similar to human ES cells, dental MSCs derived iPSC form compact colony and retain immortal growth characteristics in culture. They express markers charac‐

#### *2.7.1. Requirements of iPSC generation*

In spite of optimistic prognosis in respect of how many iPSC should be produced in order to satisfy their immunological matching within definite human population, several requirements must be challenged before establishing iPSC bank. The principal require‐ ment is a method of reprogramming, which should be safe. Therefore, three major concerns exist in the current reprogramming strategies for clinical applications: (i) the low reprog‐ ramming efficiency of human somatic cells makes it difficult to generate patient-specific iPSC, when a small amount of the cells of the patient is used; (ii) carcinogenesis may be caused by genomic integration of retro- or lentiviral fragments into host DNA; and (iii) Myc is an oncogene, which after reactivation might cause malignant tumor formation. Whereas iPSC can be generated by three transcription factors (Oct3/4, Sox2, and Klf4) without Myc, reprogramming efficiency are significantly reduced. Although, several methods of iPSC generation without viral integration have been reported; their efficien‐ cies are extremely low in comparison with viral vectors used for induction of reprogram‐ ming (Okita et al., 2008; Stadtfeld et al., 2008; Fusaki et al., 2009; Kaji et al., 2009; Kim et al., 2009; Soldner et al., 2009; Woltjen et al., 2009; Yu et al., 2009; Yusa et al., 2009; Zhou et al., 2009). Next important issue is availability of donor cells, which can provide high efficiency in the generation of non-integrated human iPSC. Therefore, source of the cells also makes its own demand, such as, it should be easily accessible with minimum discomfort for the patient, the procedure of stem cell isolation should be non-invasive, the tissue should be easily processed, the cells should be rapidly proliferating and produced in sufficient quantities, these cells should be young and collected from healthy volun‐ teers. Furthermore, the possibility of genetic abnormalities in donor cells due to ultravio‐ let (UV) irradiation should be minimized and finally, these cells would be able to be stored in liquid nitrogen for a long time without the loss of their prime characteristics.

Dental pulp stem cells from deciduous and wisdom teeth are an ideal source that meets the majority of aforementioned requirements. The loss of baby (deciduous) teeth occurs naturally and they can be removed with minimal discomfort to the patient during a routine visit to the dentist, in many clinics, as well as wisdom teeth. We also showed that not only cells, but also dental pulp can be cryopreserved and new cells can be obtained later, after thawing (Lizier et al., 2012). Therefore, frozen dental pulp does not require *in vitro* cultivation in order to produce the cells of donor until he needs these cells for iPSC production and/or clinical treatment. Additionally, several dental pulps from the same individual can be cryopreserved. This elevates a probability of successful MSCs isolation in high quantities. These cells are safer, once they can be used by first and second degree relatives and within all family (Kerkis and Caplan, 2012; Lizier et al., 2012).

#### *2.7.2. Perspectives of iPSC therapeutic use*

90% of the Japanese population with a faultless match of these loci (Nakajima et al., 2007; Nakatsuji et al., 2008). Considering that iPSC derivation is a time consuming process and of elevated cost, it should be necessary for cell therapies and regenerative medicine to establish iPSC banks with a sufficient collection of HLA types, thus avoiding additional costs which are

In spite of optimistic prognosis in respect of how many iPSC should be produced in order to satisfy their immunological matching within definite human population, several requirements must be challenged before establishing iPSC bank. The principal require‐ ment is a method of reprogramming, which should be safe. Therefore, three major concerns exist in the current reprogramming strategies for clinical applications: (i) the low reprog‐ ramming efficiency of human somatic cells makes it difficult to generate patient-specific iPSC, when a small amount of the cells of the patient is used; (ii) carcinogenesis may be caused by genomic integration of retro- or lentiviral fragments into host DNA; and (iii) Myc is an oncogene, which after reactivation might cause malignant tumor formation. Whereas iPSC can be generated by three transcription factors (Oct3/4, Sox2, and Klf4) without Myc, reprogramming efficiency are significantly reduced. Although, several methods of iPSC generation without viral integration have been reported; their efficien‐ cies are extremely low in comparison with viral vectors used for induction of reprogram‐ ming (Okita et al., 2008; Stadtfeld et al., 2008; Fusaki et al., 2009; Kaji et al., 2009; Kim et al., 2009; Soldner et al., 2009; Woltjen et al., 2009; Yu et al., 2009; Yusa et al., 2009; Zhou et al., 2009). Next important issue is availability of donor cells, which can provide high efficiency in the generation of non-integrated human iPSC. Therefore, source of the cells also makes its own demand, such as, it should be easily accessible with minimum discomfort for the patient, the procedure of stem cell isolation should be non-invasive, the tissue should be easily processed, the cells should be rapidly proliferating and produced in sufficient quantities, these cells should be young and collected from healthy volun‐ teers. Furthermore, the possibility of genetic abnormalities in donor cells due to ultravio‐ let (UV) irradiation should be minimized and finally, these cells would be able to be stored

in liquid nitrogen for a long time without the loss of their prime characteristics.

relatives and within all family (Kerkis and Caplan, 2012; Lizier et al., 2012).

Dental pulp stem cells from deciduous and wisdom teeth are an ideal source that meets the majority of aforementioned requirements. The loss of baby (deciduous) teeth occurs naturally and they can be removed with minimal discomfort to the patient during a routine visit to the dentist, in many clinics, as well as wisdom teeth. We also showed that not only cells, but also dental pulp can be cryopreserved and new cells can be obtained later, after thawing (Lizier et al., 2012). Therefore, frozen dental pulp does not require *in vitro* cultivation in order to produce the cells of donor until he needs these cells for iPSC production and/or clinical treatment. Additionally, several dental pulps from the same individual can be cryopreserved. This elevates a probability of successful MSCs isolation in high quantities. These cells are safer, once they can be used by first and second degree

required for iPSC production for each individual patient.

*2.7.1. Requirements of iPSC generation*

142 Pluripotent Stem Cells

Currently, iPSC are used to understand human diseases, including Alzheimer's disease, Parkinson's disease, cardiovascular disease, diabetes, and amyotrophic lateral sclerosis (ALS), to develop and screen bioactive molecules - candidate to therapeutic drugs and to identify molecules or genes implicated in tissue regeneration. These *in vitro* studies enable researchers to understand fundamental principles of iPSC function and differentiation, which further will provide knowledge, necessary for therapeutic use of iPSC. Based on this knowledge multiple pre-clinical and clinical protocols will be produced optimizing iPSC transplantation in diverse animal and human diseases thus becoming a tool in cellreplacement therapy. Therefore, iPSC in the future may have tremendous clinical poten‐ tial when highly efficient and safe protocols of generation of reprogrammed stem cells will be developed. Increasing our understanding of the molecular mechanisms that underlie reprogramming, we will be able to identify the cell types and methods of reprogram‐ ming, which will minimize DNA alterations, and conditions of iPSC cultivation that will allow widespread use of these cells in clinic. The scientists, however, should answer the question if iPSC are truly equivalent to human ES cells. Although iPSC potential for regenerative medicine is great, our current knowledge about iPSC variability, and utility must also increase greatly before iPSC became a standard tool for regenerative medicine.

#### **3. Final considerations**

Different dental tissues, which include apical papilla, primary exfoliated deciduous and permanent teeth, as well as wisdom teeth were used to derivate iPSC. The data obtained by different authors indicate that these tissues can be easily isolated and MSCs cells in sufficient quantities can be obtained. MSCs *in vivo* are reversibly arrested cells, which are localized in their niches maintaining their temporarily quiescent state. They differ from terminally differentiated cells by developmental path that involves a set of increasingly committed stages of specialization. The fact that these cells are undifferentiated cells suggests that their reprog‐ ramming will occur more easily than that of terminally differentiated cells. Indeed, all studies demonstrated that in SHED/SCAP/DPSCs/DPCs/MStCs/IDPSC the reprogramming process occurs more easily than in human fibroblasts used as a control, under the similar protocols and vectors used for transduction, which showed to be efficient. It was also possible to reprogram MSCs from wisdom teeth using only three factors (without c-Myc) avoiding future implications with potential risks of oncogene use (Yu et al., 2007). For the clinical applications of stem cells xenogeneic reagents pose the risk of a severe immune response, and the trans‐ mission of viral or bacterial infections, prions, and unidentified zoonosis. We demonstrated that isolation of IDPSC-iPSC can occur under feeder-free conditions on matrigel-coated dishes. Clinical stem cell therapy trials are ongoing, which request a strong focus on the safety and quality of *in vitro* expanded stem cell transplants. By replacing xenogeneic products with a defined xeno-free medium, the safety and quality of the cells with therapeutic potential may be enhanced significantly. Similar to human ES cells, dental MSCs derived iPSC form compact colony and retain immortal growth characteristics in culture. They express markers charac‐ teristic of pluripotency including Nanog, Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, exhibit high telomerase activity and have stable karyotype. Controversies were observed in methyl‐ ation status of CpG in the promoter regions of Nanog and Oct4 in dental tissue derived iPSC. Thus iPSC-derived from MStCs from wisdom teeth have their promoters of Oct4 and Nanog highly unmethylated after reprogramming (Yan et al., 2010; Oda et al., 2010). The SHED-/ DPSC-iPSC had less methylated sites of Oct4 promoter than the non-transduced cells (Yan et al., 2010). What ensues at the molecular level during the reprogramming process, however, is not fully understood and is the current focus in iPSC research (Amabile and Meissner, 2009). Furthermore, dental tissue derived iPSC exhibits differentiation potential like human ES cells and can differentiate *in vitro* and *in vivo* into cells of all three primary germ layers (Takahashi et al., 2007; Yu et al., 2007).

**Acknowledgements**

our research.

**Author details**

**References**

21.

33-41.

20395-406. Epub 2007 May 11.

Nelson F. Lizier, Irina Kerkis and Cristiane V. Wenceslau

Biotechnol. Nov; 26(11):1276-84. Epub 2008 Oct 17.

Federal University of Sao Paulo, Brazil

The authors thank Dr. Alysson Muotri from University of California San Diego, Dr. Patricia Beltrao-Braga and Dr. Paulo Maiorka from University of Sao Paulo for their contribution in

Generation of Induced Pluripotent Stem Cells from Dental Pulp Somatic Cells

http://dx.doi.org/10.5772/55856

145

Laboratory of Genetics, Butantan Institute and Department of Morphology and Genetics,

[1] Aasen T, Raya A, Barrero MJ, Garreta E, Consiglio A, Gonzalez F, Vassena R, Bilić J, Pekarik V, Tiscornia G, Edel M, Boué S, Izpisúa Belmonte JC. (2008) Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat

[2] Amabile G, Meissner A. (2009) Induced pluripotent stem cells: current progress and potential for regenerative medicine. Trends Mol Med. Feb; 15(2):59-68. Epub 2009 Jan

[3] Banito A, Rashid ST, Acosta JC, Li S, Pereira CF, Geti I, Pinho S, Silva JC, Azuara V, Walsh M, Vallier L, Gil J. (2009) Senescence impairs successful reprogramming to

[4] Beltrão-Braga PC, Pignatari GC, Maiorka PC, Oliveira NA, Lizier NF, Wenceslau CV, Miglino MA, Muotri AR, Kerkis I. (2011) Feeder-free derivation of induced pluripo‐

[5] Bilic J, Izpisua Belmonte JC. (2012) Concise review: Induced pluripotent stem cells versus embryonic stem cells: close enough or yet too far apart? Stem Cells. Jan; 30(1):

[7] Cho YW, Hong T, Hong S, Guo H, Yu H, Kim D, Guszczynski T, Dressler GR, Cope‐ land TD, Kalkum M, Ge K. (2007) PTIP associates with MLL3- and MLL4-containing histone H3 lysine 4 methyltransferase complex. J Biol Chem. Jul 13; 282(28):

tent stem cells from human immature dental pulp stem cells. Cell Transplant.

[6] Caplan AI. (1991) Mesenchymal stem cells. J Orthop Res. Sep; 9(5):641-50.

pluripotent stem cells. Genes Dev. Sep 15; 23(18):2134-9. Epub 2009 Aug 20.

In cell types with endogenously expression of one or more of the factors that induce pluripo‐ tency, such as neural cells that strongly express Sox2, pluripotency may be induced more easily or even with only a subset of factors (de Souza, 2010). In accordance, we observed that in hIDPSC, which express these factors, but at low level, the reprogramming was speedier, when compared with other dental tissue derived stem cells. During reprogramming, the integration the 4 factors into the genome of the transduced SHED/SCAP/DPSC-iPSC occurred (Yan et al., 2010). Currently, the nonintegrating reprogramming approaches, which include adenoviruses, plasmid- and episomal vector-based methods, and delivery of reprogramming factors directly as proteins have been developed. Additionally, other factors have been identified that can substitute the four Yamanaka's traditional transcription factors. Thus, Klf227 and Klf5 can replace Klf4, Sox1 and Sox3 can replace Sox2, and n-Myc and I-Myc can replace c-Myc (Nakagawa et al., 2008). Nr5a2 (Nuclear receptor subfamily 5, group A, member 2) can be used to substitute Oct-4 in the reprogramming of murine somatic cells (Heng et al., 2010). Some small molecules as the histone deacetylase inhibitor valproic acid can replace Klf4 and c-Myc for reprogramming human fibroblasts (Huangfu et al., 2008; Lin et al., 2009).

The creation of patient-specific stem cell lines is relevant for the study of basic biology, molecular mechanisms of various diseases, for drug discovery and for treating a number of human degenerative diseases without evoking immune rejection. HLA typing of DPC lines (Tamaoki et al., 2010) is of extreme importance because allows to limit the number of human iPSC, which should be obtained for each definite human population, thus avoiding unnecessary elevated costs of iPSC for cell therapies and regenerative medicine. So far, human iPSC have been used for the study of the reprogramming process itself and establishment of disease-specific cell lines and the differentiation of these cell lines into the different cell types affected by the disease, such as, spinal motor neurons, dopaminergic neurons and cardiomyocytes derived from patients suffering from amyotrophic lateral sclerosis (Dimos et al., 2008), spinal muscular atrophy (Ebert et al., 2009), sporadic Parkinson's disease (Soldner et al., 2009). Exploration of iPSC is still in its infancy, and understanding the true potential of these cells requires continued research, comprehen‐ sion and profound comparisons with human ES cells.

## **Acknowledgements**

teristic of pluripotency including Nanog, Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, exhibit high telomerase activity and have stable karyotype. Controversies were observed in methyl‐ ation status of CpG in the promoter regions of Nanog and Oct4 in dental tissue derived iPSC. Thus iPSC-derived from MStCs from wisdom teeth have their promoters of Oct4 and Nanog highly unmethylated after reprogramming (Yan et al., 2010; Oda et al., 2010). The SHED-/ DPSC-iPSC had less methylated sites of Oct4 promoter than the non-transduced cells (Yan et al., 2010). What ensues at the molecular level during the reprogramming process, however, is not fully understood and is the current focus in iPSC research (Amabile and Meissner, 2009). Furthermore, dental tissue derived iPSC exhibits differentiation potential like human ES cells and can differentiate *in vitro* and *in vivo* into cells of all three primary germ layers (Takahashi

In cell types with endogenously expression of one or more of the factors that induce pluripo‐ tency, such as neural cells that strongly express Sox2, pluripotency may be induced more easily or even with only a subset of factors (de Souza, 2010). In accordance, we observed that in hIDPSC, which express these factors, but at low level, the reprogramming was speedier, when compared with other dental tissue derived stem cells. During reprogramming, the integration the 4 factors into the genome of the transduced SHED/SCAP/DPSC-iPSC occurred (Yan et al., 2010). Currently, the nonintegrating reprogramming approaches, which include adenoviruses, plasmid- and episomal vector-based methods, and delivery of reprogramming factors directly as proteins have been developed. Additionally, other factors have been identified that can substitute the four Yamanaka's traditional transcription factors. Thus, Klf227 and Klf5 can replace Klf4, Sox1 and Sox3 can replace Sox2, and n-Myc and I-Myc can replace c-Myc (Nakagawa et al., 2008). Nr5a2 (Nuclear receptor subfamily 5, group A, member 2) can be used to substitute Oct-4 in the reprogramming of murine somatic cells (Heng et al., 2010). Some small molecules as the histone deacetylase inhibitor valproic acid can replace Klf4 and c-Myc

for reprogramming human fibroblasts (Huangfu et al., 2008; Lin et al., 2009).

sion and profound comparisons with human ES cells.

The creation of patient-specific stem cell lines is relevant for the study of basic biology, molecular mechanisms of various diseases, for drug discovery and for treating a number of human degenerative diseases without evoking immune rejection. HLA typing of DPC lines (Tamaoki et al., 2010) is of extreme importance because allows to limit the number of human iPSC, which should be obtained for each definite human population, thus avoiding unnecessary elevated costs of iPSC for cell therapies and regenerative medicine. So far, human iPSC have been used for the study of the reprogramming process itself and establishment of disease-specific cell lines and the differentiation of these cell lines into the different cell types affected by the disease, such as, spinal motor neurons, dopaminergic neurons and cardiomyocytes derived from patients suffering from amyotrophic lateral sclerosis (Dimos et al., 2008), spinal muscular atrophy (Ebert et al., 2009), sporadic Parkinson's disease (Soldner et al., 2009). Exploration of iPSC is still in its infancy, and understanding the true potential of these cells requires continued research, comprehen‐

et al., 2007; Yu et al., 2007).

144 Pluripotent Stem Cells

The authors thank Dr. Alysson Muotri from University of California San Diego, Dr. Patricia Beltrao-Braga and Dr. Paulo Maiorka from University of Sao Paulo for their contribution in our research.

## **Author details**

Nelson F. Lizier, Irina Kerkis and Cristiane V. Wenceslau

Laboratory of Genetics, Butantan Institute and Department of Morphology and Genetics, Federal University of Sao Paulo, Brazil

## **References**


[8] Conkright, M. D., Wani, M. A., Anderson, K. P., and Lingrel, J. B. (1999) A gene en‐ coding an intestinal-enriched member of the Krüppel-like factor family expressed in intestinal epithelial cells. Nucleic Acids Res. 27, 1263–1270

mature dental pulp stem cells expressing OCT-4 and other embryonic stem cell

Generation of Induced Pluripotent Stem Cells from Dental Pulp Somatic Cells

http://dx.doi.org/10.5772/55856

147

[22] Kim D, Kim CH, Moon JI, Chung YG, Chang MY, Han BS, Ko S, Yang E, Cha KY, Lanza R, et al. (2009) Generation of human induced pluripotent stem cells by direct

[23] Le Douarin NM, Brito JM, Creuzet S (2007) Role of the neural crest in face and brain

[24] Le Douarin NM, Calloni GW, Dupin E (2008) The stem cells of the neural crest. Cell

[25] Le Douarin NM, Creuzet S, Dupin E (2004) Neural crest cell plasticity and its limits.

[26] Le Douarin NM, Dupin E (2003) Multipotentiality of the neural crest. Curr Opin Gen‐

[27] Le Douarin NM, Dupin E. (2012)The neural crest in vertebrate evolution. Curr Opin

[28] Lewitzky M, Yamanaka S. (2007) Reprogramming somatic cells towards pluripoten‐

[29] Lin T, Ambasudhan R, Yuan X, Li W, Hilcove S, Abujarour R, Lin X, Hahm HS, Hao E, Hayek A, et al. (2009) A chemical platform for improved induction of human

[31] Lizier NF, Kerkis A, Gomes CM, Hebling J, Oliveira CF, Caplan AI, Kerkis I (2012) Scaling-up of dental pulp stem cells isolated from multiple niches. PLoS One;

[32] Maherali N, Hochedlinger K (2008) Induced pluripotency of mouse and human so‐

[33] Marchetto MC, Yeo GW, Kainohana O, Marsala M, Gage FH, Muotri AR. (2009) Transcriptional signature and memory retention of human-induced pluripotent stem

[34] McDevitt TC, Palecek SP (2008) Innovation in the culture and derivation of pluripo‐

[35] Miura, M., Gronthos, S., Zhao, M., Lu, B., Fisher, L.W., et al. (2003) SHED: Stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A 100, 5807.

[36] Muchkaeva IA, Dashinimaev EB, Terskikh VV, Sukhanov YV, Vasiliev AV (2012) Molecular mechanisms of induced pluripotency. Acta Naturae. Jan; 4(1):12-22.

[30] Liu SV (2008) iPS cells: a more critical review. Stem Cells Dev. Jun;17(3):391-7.

cy by defined factors. Curr Opin Biotechnol. Oct; 18(5):467-73.

matic cells. Cold Spring Harb Symp Quant Biol. 8; 73:157-62.

tent human stem cells. Curr Opin Biotechnol. Oct; 19(5):527-33

delivery of reprogramming proteins. Cell Stem Cell 4: 472–476.

markers. Cells Tissues Organs; 184(3-4):105-16.

development. Brain Res Rev. Oct; 55(2):237-47.

Cycle 7: 1013–9.

et Dev 13: 529–36.

Development 131: 4637-50

Genet Dev. Jul 6. [Epub ahead of print]

iPSCs. Nat Methods 6: 805–808.

7(6):e39885. Epub 2012 Jun 29.

cells. PLoS One. Sep 18; 4(9):e7076.


mature dental pulp stem cells expressing OCT-4 and other embryonic stem cell markers. Cells Tissues Organs; 184(3-4):105-16.

[22] Kim D, Kim CH, Moon JI, Chung YG, Chang MY, Han BS, Ko S, Yang E, Cha KY, Lanza R, et al. (2009) Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4: 472–476.

[8] Conkright, M. D., Wani, M. A., Anderson, K. P., and Lingrel, J. B. (1999) A gene en‐ coding an intestinal-enriched member of the Krüppel-like factor family expressed in

[10] Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF, Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K. (2008) In‐ duced pluripotent stem cells generated from patients with ALS can be differentiated

[11] Dupin E, Calloni G, Real C, Gonçalves-Trentin A, Le Douarin NM. (2007) Neural

[12] Ebert AD, Yu J, Rose FF Jr, Mattis VB, Lorson CL, Thomson JA, Svendsen CN. (2009) Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature. Jan

[13] Friedenstein AJ, Gorskaja JF, Kulagina NN. (1976) Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol. Sep; 4(5):267-74.

[14] Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M. (2009) Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad 85: 348–362.

[15] Giordano G, La Monaca G, Annibali S, Cicconetti A, Ottolenghi L. (2011) Stem cells from oral niches: a review. Ann Stomatol (Roma). Jan; 2(1-2):3-8. Epub 2011 Jul 18. [16] Heng JC, Feng B, Han J, Jiang J, Kraus P, Ng JH, Orlov YL, Huss M, Yang L, Lufkin T, Lim B, Ng HH. (2010) The nuclear receptor Nr5a2 can replace Oct4 in the reprog‐ ramming of murine somatic cells to pluripotent cells. Cell Stem Cell. Feb 5; 6(2):

[17] Huangfu D, Osafune K, Maehr R, Guo W, Eijkelenboom A, Chen S, Muhlestein W, Melton DA. (2008) Induction of pluripotent stem cells from primary human fibro‐

[18] Ikeda E, Yagi K, Kojima M, Yagyuu T, Ohshima A, Sobajima S, Tadokoro M, Katsube Y, Isoda K, Kondoh M, Kawase M, Go MJ, Adachi H, Yokota Y, Kirita T, Ohgushi H. (2008) Multipotent cells from the human third molar: feasibility of cell-based therapy

[19] Kaji K, Norrby K, Paca A, Mileikovsky M, Mohseni P, Woltjen K. (2009) Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature

[20] Kerkis I, Caplan AI. (2012) Stem cells in dental pulp of deciduous teeth. Tissue Eng

[21] Kerkis I, Kerkis A, Dozortsev D, Stukart-Parsons GC, Gomes Massironi SM, Pereira LV, Caplan AI, Cerruti HF (2006)Isolation and characterization of a population of im‐

blasts with only Oct4 and Sox2. Nat Biotechnol. Nov; 26(11):1269-75.

for liver disease. Differentiation. May; 76(5):495-505.

[9] de Souza N. (2010) Primer: induced pluripotency. Nat Methods. Jan; 7(1):20-1.

intestinal epithelial cells. Nucleic Acids Res. 27, 1263–1270

into motor neurons. Science. Aug 29; 321(5893):1218-21.

15; 457(7227):277-80. Epub 2008 Dec 21.

146 Pluripotent Stem Cells

167-74. Epub 2010 Jan 21.

458: 771–775.

Part B Rev. Apr;18(2):129-38

crest progenitors and stem cells. C R Biol. Jun-Jul; 330(6-7):521-9.


[37] Murata K, Kouzarides T, Bannister AJ, Gurdon JB (2010) Histone H3 lysine 4 methyl‐ ation is associated with the transcriptional reprogramming efficiency of somatic nu‐ clei by oocytes. Epigenetics Chromatin. Feb 4; 3(1):4.

[49] Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K (2008) Induced pluripotent

Generation of Induced Pluripotent Stem Cells from Dental Pulp Somatic Cells

http://dx.doi.org/10.5772/55856

149

[50] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined

[51] Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse em‐ bryonic and adult fibroblast cultures by defined factors. Cell. 126(4): 663-76.

[52] Takeda T, Tezuka Y, Horiuchi M, Hosono K, Iida K, Hatakeyama D, Miyaki S, Kuni‐ sada T, Shibata T, Tezuka K. (2008) Characterization of dental pulp stem cells of hu‐

[53] Tamaoki N, Takahashi K, Tanaka T, Ichisaka T, Aoki H, Takeda-Kawaguchi T, Iida K, Kunisada T, Shibata T, Yamanaka S, Tezuka K (2010) Dental pulp cells for induced

[54] van Attikum H, Gasser SM (2009) Crosstalk between histone modifications during

[55] Woltjen K, Michael IP, Mohseni P, Desai R, Mileikovsky M, Hamalainen R, Cowling R, Wang W, Liu P, Gertsenstein M, Stadtfeld and Hochedlinger et al. (2009) piggyBac trans‐ position reprograms fibroblasts to induced pluripotent stem cells. Nature 458: 766–770

[56] Yan, X., Qin, H., Qu, C., Tuan, R.S., Shi, S., and Huang, G.T (2010) iPS cell reprogram‐ med from human mesenchymal-like stem/progenitor cells of dental tissue origin.

[57] Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson JA (2009) Human in‐ duced pluripotent stem cells free of vector and transgene sequences. Science 324: 797–

[59] Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, et al. (2007) Induced pluripotent stem cell lines

[60] Yusa K, Rad R, Takeda J, Bradley A (2009) Generation of transgene-free induced plu‐ ripotent mouse stem cells by the piggyBac transposon. Nat Methods 6: 363–369.

[61] Zhao R, Daley GQ (2008) From fibroblasts to iPS cells: induced pluripotency by de‐

[62] Zhou W, Freed CR (2009) Adenoviral gene delivery can reprogram human fibro‐

[63] Zouboulis CC, Adjaye J, Akamatsu H, Moe-Behrens G, Niemann C (2008) Human

skin stem cells and the ageing process. Exp Gerontol. Nov; 43(11):986-97.

blasts to induced pluripotent stem cells. Stem Cells 27: 2667–2674.

[58] Yu J, Thomson JA. (2008) Pluripotent stem cell lines. Genes Dev.; 22(15):1987-97.

derived from human somatic cells. Science 318: 1917–1920.

fined factors. J Cell Biochem.; 105(4): 949-55

the DNA damage response. Trends Cell Biol. May; 19(5):207-17. Review.

stem cells generated without viral integration. Science 322: 945–949.

factors. Cell 131: 861–872.

Stem Cells Dev 19, 469.

801.

man tooth germs. J Dent Res. Jul; 87(7):676-81.

pluripotent stem cell banking. J Dent Res. Aug; 89(8):773-8.


[49] Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K (2008) Induced pluripotent stem cells generated without viral integration. Science 322: 945–949.

[37] Murata K, Kouzarides T, Bannister AJ, Gurdon JB (2010) Histone H3 lysine 4 methyl‐ ation is associated with the transcriptional reprogramming efficiency of somatic nu‐

[38] Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mo‐ chiduki Y, Takizawa N, Yamanaka S (2008) Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol. Jan; 26(1):

[39] Nakajima F, Tokunaga K, Nakatsuji N (2007) Human leukocyte antigen matching es‐ timations in a hypothetical bank of human embryonic stem cell lines in the Japanese population for use in cell transplantation therapy. Stem Cells. Apr; 25(4):983-5. Epub

[40] Nakatsuji N, Nakajima F, Tokunaga K (2008) HLA-haplotype banking and iPS cells.

[41] Nishikawa S, Goldstein RA, Nierras CR (2008) The promise of human induced pluri‐ potent stem cells for research and therapy. Nat Rev Mol Cell Biol. Sep; 9(9):725-9.

[42] Oda Y, Yoshimura Y, Ohnishi H, Tadokoro M, Katsube Y, Sasao M, Kubo Y, Hattori K, Saito S, Horimoto K, Yuba S, Ohgushi H (2010) Induction of pluripotent stem cells from human third molar mesenchymal stromal cells. J Biol Chem. Sep 17; 285(38):

[43] Ohnuki M, Takahashi K, Yamanaka S (2009) Generation and characterization of hu‐ man induced pluripotent stem cells. Curr Protoc Stem Cell Biol. Jun; Chapter 4: Unit

[44] Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S (2008) Generation of mouse induced pluripotent stem cells without viral vectors. Science. Nov 7;

[45] Ooi L, Sidhu K, Poljak A, Sutherland G, O'Connor MD, Sachdev P, Münch G (2012) Induced pluripotent stem cells as tools for disease modelling and drug discovery in

[46] Patel SR, Kim D, Levitan I, Dressler GR (2007) The BRCT-domain containing protein PTIP links PAX2 to a histone H3, lysine 4 methyltransferase complex. Dev Cell. Oct;

[47] Shields, J. M., Christy, R. J., and Yang, V. W. (1996) Identification and characteriza‐ tion of a gene encoding a gut-enriched Krüppel-like factor expressed during growth

[48] Soldner F, Hockemeyer D, Beard C, Gao Q, Bell GW, Cook EG, Hargus G, Blak A, Cooper O, Mitalipova M, Isacson O, Jaenisch R (2009) Parkinson's disease patient-de‐ rived induced pluripotent stem cells free of viral reprogramming factors. Cell. Mar 6;

clei by oocytes. Epigenetics Chromatin. Feb 4; 3(1):4.

101-6. Epub 2007 Nov 30.

Nat Biotechnol. Jul; 26(7):739-40.

322(5903):949-53. Epub 2008 Oct 9.

Alzheimer's disease. J Neural Transm. Jun 13.

arrest. J. Biol. Chem. 271, 20009–20017.

2006 Dec 21.

148 Pluripotent Stem Cells

29270-8.

4A.2.

13(4):580-92.

136(5):964-77.


**Section 2**

**Mechanistic Underpinning**

## **Mechanistic Underpinning**

**Chapter 8**

**Molecular Mechanisms Underlying Pluripotency**

Pluripotency and self renewal are the two primary characteristics of pluripotent stem cells (PSCs) [1]-[4]. **Pluripotency** refers to the capacity of a single cell to give rise to any cell type of an embryo or an adult animal [5],[6]. A mammalian organism is developed from a single fertilized egg, the zygote, in an extremely ordered and error-proof fashion [7]. The zygote and the subsequent 2 to 4-cell stage blastomere are considered to be **totipotent** since they can give rise to the entire fetus, including the embryo and the extra embryonic tissue such as the placenta and the umbilical cord (Fig.1) [7]. As embryo development proceeds to 8-cell stage and beyond depending on the species, the cells in the blastomere gradually lose their totipotency. At about embryonic day 3.5 (E3.5) in mouse (about E5 in human) the blastomere compacts into a blastocyst in which two distinct cell populations reside. Cells in the outer layer of the blastocyst form the trophectoderm (TE) which eventually give rise to the extra embryonic tissue, trophoblast of the placenta, whereas cells in the inside of the blastocyst form the inner cell mass (ICM). The ICM then gives rise to additional two lineages of cells, the primitive endoderm (PrEn or hypoblast) and the primitive ectoderm (PrEc or epiblast) (Fig.1). The PrEn produces the secondary extra embryonic tissues, such as yolk sac, allantois and amnion, while the PrEc gives rise to all three germ layers of the embryo, namely the ectoderm, the mesoderm and the endoderm (Fig.1). Although the extra embryonic tissues are indispensible for mammalian embryonic development, it is the ICM derived PrEc (or epiblast) cells that form all the cells of

an embryo and adult animal, thus these cells are defined as **pluripotent** [5],[8].

As the embryo implants into the uterus and development further commences to E5-E6.5 days in mouse, some of the post-implantation epiblast cells are found to maintain the capability of producing all derivatives of the three embryonic germ layers [1],[5]. The difference between cells derived from ICM of the pre-implantation blastocyst and those from post-plantation epiblast is that the ICM derived cells express stage-specific embryonic antigen 1 (SSEA1), give rise to all three embryonic germ layers, and most importantly, contribute to chimeric mouse

> © 2013 Bieberich and Wang; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Bieberich and Wang; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Erhard Bieberich and Guanghu Wang

http://dx.doi.org/10.5772/55596

**1. Introduction**

Additional information is available at the end of the chapter

## **Molecular Mechanisms Underlying Pluripotency**

Erhard Bieberich and Guanghu Wang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55596

## **1. Introduction**

Pluripotency and self renewal are the two primary characteristics of pluripotent stem cells (PSCs) [1]-[4]. **Pluripotency** refers to the capacity of a single cell to give rise to any cell type of an embryo or an adult animal [5],[6]. A mammalian organism is developed from a single fertilized egg, the zygote, in an extremely ordered and error-proof fashion [7]. The zygote and the subsequent 2 to 4-cell stage blastomere are considered to be **totipotent** since they can give rise to the entire fetus, including the embryo and the extra embryonic tissue such as the placenta and the umbilical cord (Fig.1) [7]. As embryo development proceeds to 8-cell stage and beyond depending on the species, the cells in the blastomere gradually lose their totipotency. At about embryonic day 3.5 (E3.5) in mouse (about E5 in human) the blastomere compacts into a blastocyst in which two distinct cell populations reside. Cells in the outer layer of the blastocyst form the trophectoderm (TE) which eventually give rise to the extra embryonic tissue, trophoblast of the placenta, whereas cells in the inside of the blastocyst form the inner cell mass (ICM). The ICM then gives rise to additional two lineages of cells, the primitive endoderm (PrEn or hypoblast) and the primitive ectoderm (PrEc or epiblast) (Fig.1). The PrEn produces the secondary extra embryonic tissues, such as yolk sac, allantois and amnion, while the PrEc gives rise to all three germ layers of the embryo, namely the ectoderm, the mesoderm and the endoderm (Fig.1). Although the extra embryonic tissues are indispensible for mammalian embryonic development, it is the ICM derived PrEc (or epiblast) cells that form all the cells of an embryo and adult animal, thus these cells are defined as **pluripotent** [5],[8].

As the embryo implants into the uterus and development further commences to E5-E6.5 days in mouse, some of the post-implantation epiblast cells are found to maintain the capability of producing all derivatives of the three embryonic germ layers [1],[5]. The difference between cells derived from ICM of the pre-implantation blastocyst and those from post-plantation epiblast is that the ICM derived cells express stage-specific embryonic antigen 1 (SSEA1), give rise to all three embryonic germ layers, and most importantly, contribute to chimeric mouse

and germ line transmission, while those from post-implantation epiblast do not express SSEA1 and do not contribute to chimeric mouse and germ line [1],[5],[9]-[11]. Thus the ICM derived cells are defined to be in a "naive (or ground, primordial)" state of pluripotency, and those from post-implantation epiblast are defined to be in a "primed (or refined)" state of pluripo‐ tency [1],[5],[12],[13]. Other *in vivo* sources of pluripotent cells include the germline cells extracted either from embryonic or adult male reproduction organs [5],[8] (Fig. 1).

are. However, hESCs resemble more closely mouse epiblast stem cells (mEpiSCs), which were derived from post-implantation epiblast [16],[22], indicating that the hESCs are probably in a

Decoding the molecular basis of pluripotency and self renewal is fundamental to the under‐ standing of stem cell biology, embryonic development, and clinical application of regenerative medicine. The *in vitro* culture of these PSCs, especially those from induced pluripotent stem cells (iPSCs), has provided an unprecedented tool to investigate deeper into the molecular mechanism governing pluripotency. Gradually we have uncovered that pluripotency is regulated by a complex network of factors, including transcription factors and epigenetic regulators, which trigger multiple signaling transduction pathways, such as the TGF-β pathway and Wnt pathway. Since the concise molecular mechanism controlling pluripotency varies among the different kinds of PSCs, we will first give a brief introduction of their

**mECCs mESCs miPSCs mEpiSCs hESCs hiPSCs**

Yes Yes Yes Yes Yes Yes

XaXa XaXa XaXa XaXi XaXi XaXi

Differentiation Self renewal and pluripotency

Naive state Primed state Not determined, possibly primed

Yes Yes Yes No Not determined

Domed shape

conditions LIF, FBS LIF, BMP4 LIF Fgf2, Activin Fgf2, Activin,

factors Oct4, Nanog, Sox2, Stat3, Klf2, Klf4, Oct4, Nanog, Sox2

2i Self renewal and pluripotency Differentiation and cell death References 7, 17-19 10-14, 22-28 32-35 3,9,16 4,15 51, 53, 54

LIF Self renewal and pluripotency None

Somatic cells Late epiblast ICM of Blastocyst Somatic cells

MEF CM

Molecular Mechanisms Underlying Pluripotency

http://dx.doi.org/10.5772/55596

155

Flat Flat Flat

state

Not determined

Fgf2, Activin, MEF CM

primed state of pluripotency (Table 1).

properties.

Teratoma formation

Chimera and germ line contribution

Culture

Morphology

X chromosome

Pluripotency status

Pluripotency

Response to

Response to Fgf2

Response to

**Table 1.** Properties of some PSCs

Origin Teratoma

Domed

shape Domed shape

ICM of Blastocyst

**Figure 1.** Early embryonic development and sources of PSCs. ICM, inner cell mass; TE, trophectoderm or Trophoblast; EpiSCs, epiblast stem cells; EGC, embryonic germ cells; iPSCs, induced pluripotent stem cells; MGSCs, male germ stem cells; PrEn, primitive endoderm; PrEc, primitive ectoderm; ESCs, embryonic stem cells.

Both the innate totipotency and pluripotency are transient developmental stages in the beginning of embryogenesis [5],[7]. Because of their finite number and transient nature, these cells are very challenging to study, although scientists have showed immense interest to understand them since they hold key answers to many aspects of biology and life.

Intriguingly, pluripotency can be captured or induced in cell cultures with defined growing conditions [14]-[16]. Mouse ESCs (mESCs) are one of the first and best-established ICMderived cells (Fig.1 and Table 1). Well defined culture conditions allow mESCs to self renew infinitely while maintaining a pluripotent state *in vitro*, providing an invaluable source of cells for molecular studies and differentiation into a variety of desired cell types (Table 1) [17]-[21].

Despite the genetic similarities between mouse and human, it was until two decades later that the first human ESCs (hESCs) were established in cell culture [9]. These cells give rise to all lineages of the primary germ layers and form teratomas (Table 1). Although they were also derived from ICM of pre-implantation embryos, the hESCs demonstrate many striking differences from mESCs (discussed in detail below). Since germ line transmission studies cannot be performed with these cells, it is not known at what exact pluripotency state the hESCs are. However, hESCs resemble more closely mouse epiblast stem cells (mEpiSCs), which were derived from post-implantation epiblast [16],[22], indicating that the hESCs are probably in a primed state of pluripotency (Table 1).

Decoding the molecular basis of pluripotency and self renewal is fundamental to the under‐ standing of stem cell biology, embryonic development, and clinical application of regenerative medicine. The *in vitro* culture of these PSCs, especially those from induced pluripotent stem cells (iPSCs), has provided an unprecedented tool to investigate deeper into the molecular mechanism governing pluripotency. Gradually we have uncovered that pluripotency is regulated by a complex network of factors, including transcription factors and epigenetic regulators, which trigger multiple signaling transduction pathways, such as the TGF-β pathway and Wnt pathway. Since the concise molecular mechanism controlling pluripotency varies among the different kinds of PSCs, we will first give a brief introduction of their properties.


**Table 1.** Properties of some PSCs

and germ line transmission, while those from post-implantation epiblast do not express SSEA1 and do not contribute to chimeric mouse and germ line [1],[5],[9]-[11]. Thus the ICM derived cells are defined to be in a "naive (or ground, primordial)" state of pluripotency, and those from post-implantation epiblast are defined to be in a "primed (or refined)" state of pluripo‐ tency [1],[5],[12],[13]. Other *in vivo* sources of pluripotent cells include the germline cells

**Figure 1.** Early embryonic development and sources of PSCs. ICM, inner cell mass; TE, trophectoderm or Trophoblast; EpiSCs, epiblast stem cells; EGC, embryonic germ cells; iPSCs, induced pluripotent stem cells; MGSCs, male germ stem

Both the innate totipotency and pluripotency are transient developmental stages in the beginning of embryogenesis [5],[7]. Because of their finite number and transient nature, these cells are very challenging to study, although scientists have showed immense interest to

Intriguingly, pluripotency can be captured or induced in cell cultures with defined growing conditions [14]-[16]. Mouse ESCs (mESCs) are one of the first and best-established ICMderived cells (Fig.1 and Table 1). Well defined culture conditions allow mESCs to self renew infinitely while maintaining a pluripotent state *in vitro*, providing an invaluable source of cells for molecular studies and differentiation into a variety of desired cell types (Table 1) [17]-[21].

Despite the genetic similarities between mouse and human, it was until two decades later that the first human ESCs (hESCs) were established in cell culture [9]. These cells give rise to all lineages of the primary germ layers and form teratomas (Table 1). Although they were also derived from ICM of pre-implantation embryos, the hESCs demonstrate many striking differences from mESCs (discussed in detail below). Since germ line transmission studies cannot be performed with these cells, it is not known at what exact pluripotency state the hESCs

understand them since they hold key answers to many aspects of biology and life.

cells; PrEn, primitive endoderm; PrEc, primitive ectoderm; ESCs, embryonic stem cells.

extracted either from embryonic or adult male reproduction organs [5],[8] (Fig. 1).

154 Pluripotent Stem Cells

## **2. Properties of PSCs**

**Mouse ECCs:** Mouse embryonic carcinoma cells (mECCs), the first PSCs established in cell culture, were derived in 1964 from teratomas from an inbred mouse line, which produces spontaneous testicular teratomas (Table 1) [14],[23]. These cells show many aspects of naive PSCs, such as the expression of antigen SSEA1, differentiation into all three germ layers when stimulated, and generation of chimeric mice when injected into blastocysts [14],[23]-[25]. However, since the ECCs carry many mutations, the chimeric mice derived from these cells develop spontaneous tumors [26].

of other pluripotency factors that have been shown to be essential for mESCs, such as Klf4 and Stella [5],[13],[16]. This suggests fundamental differences in the mechanisms that maintain

Molecular Mechanisms Underlying Pluripotency

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157

Epigenetically, mEpiSCs display X chromosome inactivation (XCI) as well as stability of the genetic imprint [3],[5],[8]. This epigenetic status is shared with the late epiblast of the postimplantation embryo, which reinforces the similarity between cultured mEpiSCs and their *in vivo* counterpart. This similarity has been confirmed by gene expression profile experiments, which show that EpiSCs are closely related to the pluripotent cells located in the epiblast of a

Mouse EpiSCs also differ with mESCs regarding growing conditions, phenotypes and function. To keep them in a self renewing state, activin, fibroblast growth factor 2 (Fgf2), and transforming growth factor β (TGF-β) are needed, whereas LIF is dispensable and BMP4 leads to differentiation (Table 1) [16],[22]. Instead of growing in a dome-shaped morphology as mESCs, mEpiSCs exhibit a flattened shape and do not propagate well as single cells. Whereas cell cycle regulation in mEpiSCs remains to be investigated in detail, the doubling time of mEpiSCs is 18 hours, compared with only 10–14 hours doubling time of mESCs, suggesting

**Human ESCs:** Like mESCs, human ESCs (hESCs) were isolated from the ICM of the preimplantation blastocyst almost two decades after the isolation of mESCs [9]. hESCs possess the potential to differentiae into all three primary germ layers and to produce teratomas when injected into blastocysts [1],[9]. They express high levels of pluripotency factors Oct4, Nanog, and Sox2, and are positive for SSEA3/4 and AP. However, hESCs share multiple defining features with mouse EpiSCs rather than mESCs. These characteristics include flat morphology, dependence on FGF2/Activin signaling to self renew, inclination for XCI, and reduced tolerance to single-cell dissociation by trypsinization (Table 1). These molecular and biological similarities with mEpiSCs suggest that hESCs correspond, at least partially, to the primed

**iPSCs:** In 2006, Shinya Yamanaka's research group at Kyoto University made a milestone achievement by converting adult mouse cells back to a ground pluripotent stem cell-like state through exogenous expression of only four transcription factors, Oct4, Sox2, Klf4, and c-Myc [39]. These miPSCs exhibit all characteristics of mESCs, including expression of pluripotency marker protein, activation of both X chromosomes, and most importantly, the ability to generate chimeric animals and contributing to germ line transmission (Table 1). Later on, similar cells were also induced from human somatic cells. hiPSCs resemble more hESCs and mEpiSCs than mESCs (Table 1)[40]-[42]. As their production efficiency rapidly improved, iPSCs have soon been able to compete with traditional embryonic and adult stem cells [39]- [50]. The primary advantages of iPSCs compared to other stem cells are: a) iPSCs can be created from the tissue of the same patient that will receive the transplantation, thus avoiding immune rejection, and b) the lack of ethical implications because cells are harvested from a consent individual. These patient-specific cells can be used to study diseases *in vitro*, to test drugs on a human model without ethical concerns, and to hopefully be used as a source of tissue

pluripotency in mESCs and mEpiSCs [8],[16].

that a normal G1–S transition occurs in mEpiSCs [16],[22].

pluripotency state rather than to the naive state.

replacement for diseased and damaged cells.

post-implantation embryo [16],[22]

**Mouse ESCs:** Based on the findings made from mECCs, derivation of mESCs directly from ICM of normal developing embryos became possible and faster (Fig.1). Two groups, Kauf‐ man's and Martin's, isolated such cells in culture from the ICM of pre-implantation blastocysts using different protocols in 1981 [15],[27]. And it was Martin who coined the term ESCs [27].

mESCs satisfy all the characteristics of naive pluripotency with a normal karyotype, resem‐ bling their *in vivo* counterparts in terms of expressing the pluripotency factors Oct4, Sox2, and Nanog, and SSEA1 and alkaline phosphatase (AP) (Table 1). They can be differentiated into all derivatives of the three germ layers; grow in a dome-shaped morphology; display a high nuclei/cytoplasm ratio; and most importantly, form teratomas and give rise to germ line transmission when injected into blastocysts (Table 1) [17]-[21].

The cell cycle control in mESCs also seems to be unique. They have an unusually short G1 phase and no regulation at the G1–S transition, the presence of hyperphosphorylated retino‐ blastoma (RB) protein, and unresponsiveness to activity of cyclin-dependent kinase 4 (CDK4) [11],[28]. Epigenetically, mESCs possess a hypermethylated genome and both X-chromosomes are activated if isolated from female embryos [29]-[31].

To grow mESCs, a feeder cell layer of mouse embryonic fibroblasts is typically used, with medium containing ES qualified fetal bovine serum or knockout serum replacement, leukemia inhibitory factor (LIF), non-essential amino acid, and β-mercaptoethanol [20],[21],[32],[33]. LIF serves as the extrinsic factor for pluripotency and self renewal by activating the signal transducer and activator of transcription 3 (Stat3) pathway [21],[34],[35]. Later it was discov‐ ered that LIF and Bone morphogenic protein 4 (BMP4) can support mESCs pluripotency without serum (Table 1) [36],[37]. More recently, it has been demonstrated that mESCs can be derived and maintained using medium containing small molecule inhibitors of glycogen synthase kinase (GSK3) (which functions to activate the Wnt signaling pathway) and the mitogen-activated protein kinase (MAPK) signaling pathways, completely devoid of serum and extrinsic growth factors [5],[38]. This culture media with defined components is termed the 2i (2 inhibitors) system [5],[38].

**Mouse EpiSCs:** Mouse Epiblast Stem cells (mEpiSCs) have been derived from post-implan‐ tation blastocysts (E5–E6.5) (Fig.1) [16],[22]. These cells demonstrate the properties of selfrenewal and pluripotency, but they cannot colonize the ICM of a blastocyst and produce germ line transmission [13],[16],[22]. Thus mEpiSCs are in the "primed" state of pluripotency. Furthermore, mEpiSCs express high levels of Oct4, Sox2, and Nanog but relatively low levels of other pluripotency factors that have been shown to be essential for mESCs, such as Klf4 and Stella [5],[13],[16]. This suggests fundamental differences in the mechanisms that maintain pluripotency in mESCs and mEpiSCs [8],[16].

**2. Properties of PSCs**

156 Pluripotent Stem Cells

develop spontaneous tumors [26].

transmission when injected into blastocysts (Table 1) [17]-[21].

are activated if isolated from female embryos [29]-[31].

the 2i (2 inhibitors) system [5],[38].

**Mouse ECCs:** Mouse embryonic carcinoma cells (mECCs), the first PSCs established in cell culture, were derived in 1964 from teratomas from an inbred mouse line, which produces spontaneous testicular teratomas (Table 1) [14],[23]. These cells show many aspects of naive PSCs, such as the expression of antigen SSEA1, differentiation into all three germ layers when stimulated, and generation of chimeric mice when injected into blastocysts [14],[23]-[25]. However, since the ECCs carry many mutations, the chimeric mice derived from these cells

**Mouse ESCs:** Based on the findings made from mECCs, derivation of mESCs directly from ICM of normal developing embryos became possible and faster (Fig.1). Two groups, Kauf‐ man's and Martin's, isolated such cells in culture from the ICM of pre-implantation blastocysts using different protocols in 1981 [15],[27]. And it was Martin who coined the term ESCs [27]. mESCs satisfy all the characteristics of naive pluripotency with a normal karyotype, resem‐ bling their *in vivo* counterparts in terms of expressing the pluripotency factors Oct4, Sox2, and Nanog, and SSEA1 and alkaline phosphatase (AP) (Table 1). They can be differentiated into all derivatives of the three germ layers; grow in a dome-shaped morphology; display a high nuclei/cytoplasm ratio; and most importantly, form teratomas and give rise to germ line

The cell cycle control in mESCs also seems to be unique. They have an unusually short G1 phase and no regulation at the G1–S transition, the presence of hyperphosphorylated retino‐ blastoma (RB) protein, and unresponsiveness to activity of cyclin-dependent kinase 4 (CDK4) [11],[28]. Epigenetically, mESCs possess a hypermethylated genome and both X-chromosomes

To grow mESCs, a feeder cell layer of mouse embryonic fibroblasts is typically used, with medium containing ES qualified fetal bovine serum or knockout serum replacement, leukemia inhibitory factor (LIF), non-essential amino acid, and β-mercaptoethanol [20],[21],[32],[33]. LIF serves as the extrinsic factor for pluripotency and self renewal by activating the signal transducer and activator of transcription 3 (Stat3) pathway [21],[34],[35]. Later it was discov‐ ered that LIF and Bone morphogenic protein 4 (BMP4) can support mESCs pluripotency without serum (Table 1) [36],[37]. More recently, it has been demonstrated that mESCs can be derived and maintained using medium containing small molecule inhibitors of glycogen synthase kinase (GSK3) (which functions to activate the Wnt signaling pathway) and the mitogen-activated protein kinase (MAPK) signaling pathways, completely devoid of serum and extrinsic growth factors [5],[38]. This culture media with defined components is termed

**Mouse EpiSCs:** Mouse Epiblast Stem cells (mEpiSCs) have been derived from post-implan‐ tation blastocysts (E5–E6.5) (Fig.1) [16],[22]. These cells demonstrate the properties of selfrenewal and pluripotency, but they cannot colonize the ICM of a blastocyst and produce germ line transmission [13],[16],[22]. Thus mEpiSCs are in the "primed" state of pluripotency. Furthermore, mEpiSCs express high levels of Oct4, Sox2, and Nanog but relatively low levels

Epigenetically, mEpiSCs display X chromosome inactivation (XCI) as well as stability of the genetic imprint [3],[5],[8]. This epigenetic status is shared with the late epiblast of the postimplantation embryo, which reinforces the similarity between cultured mEpiSCs and their *in vivo* counterpart. This similarity has been confirmed by gene expression profile experiments, which show that EpiSCs are closely related to the pluripotent cells located in the epiblast of a post-implantation embryo [16],[22]

Mouse EpiSCs also differ with mESCs regarding growing conditions, phenotypes and function. To keep them in a self renewing state, activin, fibroblast growth factor 2 (Fgf2), and transforming growth factor β (TGF-β) are needed, whereas LIF is dispensable and BMP4 leads to differentiation (Table 1) [16],[22]. Instead of growing in a dome-shaped morphology as mESCs, mEpiSCs exhibit a flattened shape and do not propagate well as single cells. Whereas cell cycle regulation in mEpiSCs remains to be investigated in detail, the doubling time of mEpiSCs is 18 hours, compared with only 10–14 hours doubling time of mESCs, suggesting that a normal G1–S transition occurs in mEpiSCs [16],[22].

**Human ESCs:** Like mESCs, human ESCs (hESCs) were isolated from the ICM of the preimplantation blastocyst almost two decades after the isolation of mESCs [9]. hESCs possess the potential to differentiae into all three primary germ layers and to produce teratomas when injected into blastocysts [1],[9]. They express high levels of pluripotency factors Oct4, Nanog, and Sox2, and are positive for SSEA3/4 and AP. However, hESCs share multiple defining features with mouse EpiSCs rather than mESCs. These characteristics include flat morphology, dependence on FGF2/Activin signaling to self renew, inclination for XCI, and reduced tolerance to single-cell dissociation by trypsinization (Table 1). These molecular and biological similarities with mEpiSCs suggest that hESCs correspond, at least partially, to the primed pluripotency state rather than to the naive state.

**iPSCs:** In 2006, Shinya Yamanaka's research group at Kyoto University made a milestone achievement by converting adult mouse cells back to a ground pluripotent stem cell-like state through exogenous expression of only four transcription factors, Oct4, Sox2, Klf4, and c-Myc [39]. These miPSCs exhibit all characteristics of mESCs, including expression of pluripotency marker protein, activation of both X chromosomes, and most importantly, the ability to generate chimeric animals and contributing to germ line transmission (Table 1). Later on, similar cells were also induced from human somatic cells. hiPSCs resemble more hESCs and mEpiSCs than mESCs (Table 1)[40]-[42]. As their production efficiency rapidly improved, iPSCs have soon been able to compete with traditional embryonic and adult stem cells [39]- [50]. The primary advantages of iPSCs compared to other stem cells are: a) iPSCs can be created from the tissue of the same patient that will receive the transplantation, thus avoiding immune rejection, and b) the lack of ethical implications because cells are harvested from a consent individual. These patient-specific cells can be used to study diseases *in vitro*, to test drugs on a human model without ethical concerns, and to hopefully be used as a source of tissue replacement for diseased and damaged cells.

## **3. Embryonic Germ stem Cells (mEGC) and Male Germ Stem Cell (MGSCs)**

PSCs have also been derived from reproductive system cells. When cultivated in adequate growth conditions, reproductive system cells generate ES-like stem cells (it is termed embry‐ onic germ cells (EGCs) if isolated from mouse embryonic day 8.5 embryos [51], or male germ stem cells (MGSCs) if derived from postnatal male gonads [52]) (Fig.1). The EGCs and MGSCs are both naive stem cells, capable of generating all three embryonic germ layer cells, teratomas and chimeras. EGCs and MGSCs have also been derived from human sources, but their characteristics are not as well defined [53]-[55].

Next we will focus on the mESCs, which are in the naive state, and hESCs, which are probably in the primed state, to discuss the molecular mechanism of pluripotency maintenance.

## **4. Transcription factors regulatating pluripotency**

An interplay of transcription factors and epigenetic factors participates in the maintenance of pluripotency of stem cells [34],[35],[56]-[62]. Among them Oct4 (or POU5F1), Nanog, and Sox2 are generally accepted as the core pluripotency factors, since they are vital to maintian the pluripotency of both the hESCs and mESCs, which are in a different pluripotency state [1],[8], [63]. These three factors also collectively bind to an array of genes that are essential for pluripotency and differentiation [1],[8],[63].

**Figure 2.** Schematic representation of the transcriptional regulation of core pluripotency factors and the extended

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These three core factors do not function by themselves. Instead they are involved in a multiplegene complex to regulate stem cell pluripotency (Fig.2). Their interacting partners have been extensively studied by coimmunoprecipitation (Co-IP) or chromatin immunoprecipitation (ChIP) assays in both mESCs and hESCs. Oct4 has been found to associate with Sox2, Nanog, Smad1, Stat3, TCF3, Rest, Hsp90, etc. [4],[21],[71]-[73]. Nanog seems to interact with Oct4, Smad1, Nac1, Zfp281, and Hsp90 [21],[72]-[74]. And Sox2 associates with Oct4, Nanog, Klf4,

One unique aspect of the regulation of these core factors is that they act together to regulate their own promoters, forming an interconnected auto-regulatory feedback loop (Fig.2)[5], [51], [71]. Another unique aspect is that they co-occupy and active/enhance expression of other genes necessary to maintain ESC status, while contributing to repression of genes encoding differentiation signals (Fig.2) [5],[51],[71],[77]-[80]. For example, binding of Oct4 to a promoter region of a gene increases the likelihood of Nanog, Sox2, and other regulatory factors to bind

Apart from transcriptional control of these core factors, post-translational modifications also play an essential role. Oct4 has been reported to be ubiquitinated in differentiating mouse embryonic carcinoma cells but not in mESCs [81],[82]. Phosphorylation of Nanog promotes its interaction with the prolyl isomerase Pin1, leading to increased Nanog stability by suppressing its ubiquitination[83]. In addition, a recent report demonstrates that Hsp90, a molecular chaperone, associates with Oct4 and Nanog and maintains their cellular level, possibly through protecting them against degradation by the ubiquitin protesome pathway [21]. These studies demonstrate that post-translational modifications and protein stability of the pluripotency

factors

Rpa1, Sall4, and Npm1 [75],[76].

to the same promoter [5],[51],[71],[77]-[80].

factors is also vital for stem cell pluripotency maintenance.

#### **4.1. Core pluripotency factors and their transcription cotrol**

The POU transcription factor Oct4 is a central player for stem cell pluripotency (Fig.2). Its expression is strictly confined to the totipotent, pluripotent, and germ cells during early development. *In vitro*, the cellular level of Oct4 must be tightly controlled to maintain the pluripotency status, up- or down-regulation by 50% leads to ESC differentiation [64],[65]. *In vivo*, Oct4 deletion in mice leads to ICM failure [57].

The homeoprotein Nanog is another central factor for pluripotency (Fig.2) [66]. The ICM in Nanog-deficient mice fails to generate epiblast and only produces endoderm-like cells [66]. Furthermore, ESCs derived from Nanog-deficient mice cannot maintain pluripotency and instead differentiate into extraembryonic endoderm lineages [66]. Mechanistically, Nanog functions by inhibiting NFκB and cooperating with Stat3 to inhibit cell differentiation in mESCs [66]-[68].

The third central factor is Sox2 (Fig.2) [3],[5],[63],[69]. Sox2 exhibits an expression pattern similar to that of Oct4 during development [70]. Genetic ablation studies indicate that silencing of *Sox2* affects a somewhat later stage of embryogenesis, possibly because of a stronger maternal contribution of Sox2 protein. Key feature of acute *Sox2* loss appears to be an inability to sustain appropriate Oct4 levels [70].

**3. Embryonic Germ stem Cells (mEGC) and Male Germ Stem Cell (MGSCs)**

PSCs have also been derived from reproductive system cells. When cultivated in adequate growth conditions, reproductive system cells generate ES-like stem cells (it is termed embry‐ onic germ cells (EGCs) if isolated from mouse embryonic day 8.5 embryos [51], or male germ stem cells (MGSCs) if derived from postnatal male gonads [52]) (Fig.1). The EGCs and MGSCs are both naive stem cells, capable of generating all three embryonic germ layer cells, teratomas and chimeras. EGCs and MGSCs have also been derived from human sources, but their

Next we will focus on the mESCs, which are in the naive state, and hESCs, which are probably in the primed state, to discuss the molecular mechanism of pluripotency maintenance.

An interplay of transcription factors and epigenetic factors participates in the maintenance of pluripotency of stem cells [34],[35],[56]-[62]. Among them Oct4 (or POU5F1), Nanog, and Sox2 are generally accepted as the core pluripotency factors, since they are vital to maintian the pluripotency of both the hESCs and mESCs, which are in a different pluripotency state [1],[8], [63]. These three factors also collectively bind to an array of genes that are essential for

The POU transcription factor Oct4 is a central player for stem cell pluripotency (Fig.2). Its expression is strictly confined to the totipotent, pluripotent, and germ cells during early development. *In vitro*, the cellular level of Oct4 must be tightly controlled to maintain the pluripotency status, up- or down-regulation by 50% leads to ESC differentiation [64],[65]. *In*

The homeoprotein Nanog is another central factor for pluripotency (Fig.2) [66]. The ICM in Nanog-deficient mice fails to generate epiblast and only produces endoderm-like cells [66]. Furthermore, ESCs derived from Nanog-deficient mice cannot maintain pluripotency and instead differentiate into extraembryonic endoderm lineages [66]. Mechanistically, Nanog functions by inhibiting NFκB and cooperating with Stat3 to inhibit cell differentiation in

The third central factor is Sox2 (Fig.2) [3],[5],[63],[69]. Sox2 exhibits an expression pattern similar to that of Oct4 during development [70]. Genetic ablation studies indicate that silencing of *Sox2* affects a somewhat later stage of embryogenesis, possibly because of a stronger maternal contribution of Sox2 protein. Key feature of acute *Sox2* loss appears to be an inability

characteristics are not as well defined [53]-[55].

158 Pluripotent Stem Cells

pluripotency and differentiation [1],[8],[63].

**4. Transcription factors regulatating pluripotency**

**4.1. Core pluripotency factors and their transcription cotrol**

*vivo*, Oct4 deletion in mice leads to ICM failure [57].

to sustain appropriate Oct4 levels [70].

mESCs [66]-[68].

**Figure 2.** Schematic representation of the transcriptional regulation of core pluripotency factors and the extended factors

These three core factors do not function by themselves. Instead they are involved in a multiplegene complex to regulate stem cell pluripotency (Fig.2). Their interacting partners have been extensively studied by coimmunoprecipitation (Co-IP) or chromatin immunoprecipitation (ChIP) assays in both mESCs and hESCs. Oct4 has been found to associate with Sox2, Nanog, Smad1, Stat3, TCF3, Rest, Hsp90, etc. [4],[21],[71]-[73]. Nanog seems to interact with Oct4, Smad1, Nac1, Zfp281, and Hsp90 [21],[72]-[74]. And Sox2 associates with Oct4, Nanog, Klf4, Rpa1, Sall4, and Npm1 [75],[76].

One unique aspect of the regulation of these core factors is that they act together to regulate their own promoters, forming an interconnected auto-regulatory feedback loop (Fig.2)[5], [51], [71]. Another unique aspect is that they co-occupy and active/enhance expression of other genes necessary to maintain ESC status, while contributing to repression of genes encoding differentiation signals (Fig.2) [5],[51],[71],[77]-[80]. For example, binding of Oct4 to a promoter region of a gene increases the likelihood of Nanog, Sox2, and other regulatory factors to bind to the same promoter [5],[51],[71],[77]-[80].

Apart from transcriptional control of these core factors, post-translational modifications also play an essential role. Oct4 has been reported to be ubiquitinated in differentiating mouse embryonic carcinoma cells but not in mESCs [81],[82]. Phosphorylation of Nanog promotes its interaction with the prolyl isomerase Pin1, leading to increased Nanog stability by suppressing its ubiquitination[83]. In addition, a recent report demonstrates that Hsp90, a molecular chaperone, associates with Oct4 and Nanog and maintains their cellular level, possibly through protecting them against degradation by the ubiquitin protesome pathway [21]. These studies demonstrate that post-translational modifications and protein stability of the pluripotency factors is also vital for stem cell pluripotency maintenance.

#### **4.2. Extended network of regulatory factors**

Besides the core pluripotency factors, many other transcription factors participate in the regulation of stem cell pluripotency, including cMyc, Klf2, Klf4, Stat3, Rex1, Sall4, Zfp281, and the proteins associated with the three core factors [1],[3],[5]. These transcription factors participate in the pluripotency regulation in a state or species-specific fashion. For example, Stat3 plays an important role in mESC pluripotency since target deletion of Stat3 resulted in early embryonic lethality, and ectopic expression of a dominant-negative Stat3 in ESCs leads to loss of pluripotency [34],[35]. However, Stat3 is not suficient to maintain the pluripotency of hESCs and mEpiSCs [21],[84].

sustained (Figs. 3 and 4); and 3), ectopic expressing of an constitutively active form of β-catenin maintains the expression levels of Oct4 and Nanog and thus self renewal and pluripotency in

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Moreover, Wnt signaling inhibits the differentiation of ESCs, especially to neural differentia‐ tion [90],[97],[98]. Mutation of Apc, an important mediator in the Wnt pathway, leads to impaired differentiation both *in vitro* and in teratomas [99]. Furthermore, ESCs with highly

**Figure 3.** Signaling pathways regulating pluripotency of the primed stem cells, such as mEpiSCs and possibly hESCs

The role of the Wnt signaling in stem cell pluripotency is further confirmed by its down-stream effectors, the TCF/LEF family of transcription factors [72],[88],[92],[100],[101]. In ESCs, TCF3 is the most abundantly expressed member of this transcription factor family. TCF3-null ESCs have an increased resistance to differentiation and up-regulation of various Oct4 and Nanogregulated genes [74],[102],[103]. Activation of Wnt converts TCF3 into an activator, elevating the expression of these same targets and suppressing differentiation [103]. TCF3 may also suppress the expression of Oct4 and Nanog, although its ability to activate these targets is

However, Wnt signaling alone is not sufficient to support the ground state pluripotency [38]. It has been shown that inhibition of GSK3 in mESCs enhances growth capacity and suppresses neural differentiation, but it also promotes non-neural differentiation [37], [38]. To block differentiation of mESCs, the combination of a GSK3 inhibitor and an FGF-Erk inhibitor (the

elevated β-catenin levels also have a compromised ability to differentiate [10].

ESCs [96].

unclear [104],[105].

2i system) [5],[38] is necessary.

## **5. Signal transduction pathways in pluripotency maintenance**

Innate signal transduction pathways are crucially important for understanding the regulation of the stem cell pluripotency. Extensive efforts, including high throughput genetic and chemical screening, have been invested into identifying genes and pathways that affect the core pluripotency factors Oct4, Nanog, and Sox2, or their associated genes. We have now gradually obtained a glimpse of the intrinsic signalling pathways that are involved in the regulation of stem cell pluripotency and differentiation. These signallng pathways include the Wnt pathway, TGF-β pathway, LIF/Stat3 pathway, Fgf pathway, insulin geowth factor (IGF) pathway, Notch pathway, Hedgehog pathway, etc [3]-[5],[71],[74],[85],[86] (Fig.3). Among them, the Wnt and TGF-β pathways are most heavily studied and best understood in terms of their roles in stem cell pluripotency maintenance. Here, we will focus on discussing the Wnt pathway, TGF-β signaling, LIF/Stat3 pathway, and the Fgf pathway (Figs. 3 and 4).

#### **5.1. WNT signaling pathway**

The Wnt pathway plays an important role in tissue development by regulating a wide range of cellular processes such as proliferation, adhesion, morphology, and migration [87]-[91]. It consists of over 30 extracellular ligands that bind to Frizzled (FZD) and low-density lipoprotein receptor related protein (LRP) receptors at the cell surface (Fig.3) [91]. The Wnt ligands are able to activate both the canonical pathway and the non-canonical pathway [88],[92]. The activation of Wnt pathway in the canonical pathway results in the preservation of β-catenin and its subsequent nuclear translocation, which enables downstream gene activation by the TCF/LEF family transcription factors [87]-[95]. The non-canonical pathway is independent of β-catenin and involves the activation of several other signaling pathways, such as the JNK pathway [3]-[5],[88],[91],[92].

The Wnt signaling pathway is directly linked to the core transcriptional network of pluripo‐ tency and is demonstrated to be essential for self renewal and pluripotency of both naïve and primed PSCs, when LIF is absent. Evidences for this notion include: 1), Wnt signaling is activated in both mESCs and hESCs, and is down-regulated during differentiation [90]; 2), activation of the canonical Wnt pathway is required to maintain the expression level of core pluripotency factors Oct4 and Nanog, through which the self-renewal and pluripotency are sustained (Figs. 3 and 4); and 3), ectopic expressing of an constitutively active form of β-catenin maintains the expression levels of Oct4 and Nanog and thus self renewal and pluripotency in ESCs [96].

**4.2. Extended network of regulatory factors**

of hESCs and mEpiSCs [21],[84].

160 Pluripotent Stem Cells

**5.1. WNT signaling pathway**

pathway [3]-[5],[88],[91],[92].

Besides the core pluripotency factors, many other transcription factors participate in the regulation of stem cell pluripotency, including cMyc, Klf2, Klf4, Stat3, Rex1, Sall4, Zfp281, and the proteins associated with the three core factors [1],[3],[5]. These transcription factors participate in the pluripotency regulation in a state or species-specific fashion. For example, Stat3 plays an important role in mESC pluripotency since target deletion of Stat3 resulted in early embryonic lethality, and ectopic expression of a dominant-negative Stat3 in ESCs leads to loss of pluripotency [34],[35]. However, Stat3 is not suficient to maintain the pluripotency

Innate signal transduction pathways are crucially important for understanding the regulation of the stem cell pluripotency. Extensive efforts, including high throughput genetic and chemical screening, have been invested into identifying genes and pathways that affect the core pluripotency factors Oct4, Nanog, and Sox2, or their associated genes. We have now gradually obtained a glimpse of the intrinsic signalling pathways that are involved in the regulation of stem cell pluripotency and differentiation. These signallng pathways include the Wnt pathway, TGF-β pathway, LIF/Stat3 pathway, Fgf pathway, insulin geowth factor (IGF) pathway, Notch pathway, Hedgehog pathway, etc [3]-[5],[71],[74],[85],[86] (Fig.3). Among them, the Wnt and TGF-β pathways are most heavily studied and best understood in terms of their roles in stem cell pluripotency maintenance. Here, we will focus on discussing the Wnt

**5. Signal transduction pathways in pluripotency maintenance**

pathway, TGF-β signaling, LIF/Stat3 pathway, and the Fgf pathway (Figs. 3 and 4).

The Wnt pathway plays an important role in tissue development by regulating a wide range of cellular processes such as proliferation, adhesion, morphology, and migration [87]-[91]. It consists of over 30 extracellular ligands that bind to Frizzled (FZD) and low-density lipoprotein receptor related protein (LRP) receptors at the cell surface (Fig.3) [91]. The Wnt ligands are able to activate both the canonical pathway and the non-canonical pathway [88],[92]. The activation of Wnt pathway in the canonical pathway results in the preservation of β-catenin and its subsequent nuclear translocation, which enables downstream gene activation by the TCF/LEF family transcription factors [87]-[95]. The non-canonical pathway is independent of β-catenin and involves the activation of several other signaling pathways, such as the JNK

The Wnt signaling pathway is directly linked to the core transcriptional network of pluripo‐ tency and is demonstrated to be essential for self renewal and pluripotency of both naïve and primed PSCs, when LIF is absent. Evidences for this notion include: 1), Wnt signaling is activated in both mESCs and hESCs, and is down-regulated during differentiation [90]; 2), activation of the canonical Wnt pathway is required to maintain the expression level of core pluripotency factors Oct4 and Nanog, through which the self-renewal and pluripotency are

Moreover, Wnt signaling inhibits the differentiation of ESCs, especially to neural differentia‐ tion [90],[97],[98]. Mutation of Apc, an important mediator in the Wnt pathway, leads to impaired differentiation both *in vitro* and in teratomas [99]. Furthermore, ESCs with highly elevated β-catenin levels also have a compromised ability to differentiate [10].

**Figure 3.** Signaling pathways regulating pluripotency of the primed stem cells, such as mEpiSCs and possibly hESCs

The role of the Wnt signaling in stem cell pluripotency is further confirmed by its down-stream effectors, the TCF/LEF family of transcription factors [72],[88],[92],[100],[101]. In ESCs, TCF3 is the most abundantly expressed member of this transcription factor family. TCF3-null ESCs have an increased resistance to differentiation and up-regulation of various Oct4 and Nanogregulated genes [74],[102],[103]. Activation of Wnt converts TCF3 into an activator, elevating the expression of these same targets and suppressing differentiation [103]. TCF3 may also suppress the expression of Oct4 and Nanog, although its ability to activate these targets is unclear [104],[105].

However, Wnt signaling alone is not sufficient to support the ground state pluripotency [38]. It has been shown that inhibition of GSK3 in mESCs enhances growth capacity and suppresses neural differentiation, but it also promotes non-neural differentiation [37], [38]. To block differentiation of mESCs, the combination of a GSK3 inhibitor and an FGF-Erk inhibitor (the 2i system) [5],[38] is necessary.

### **5.2. TGF-β signaling pathway**

The TGF-β signaling pathway plays a vital role in both the developmental and adult life of a mammalian organism by regulating many processes including apoptosis, proliferation, senescence, inflammation, cell fate, and tissue repair [94],[106],[107]. The TGF-β super family contains more than 30 growth factors including TGF-βs, BMPs, growth and differentiation factors (GDFs), Activin, and Nodal [2],[106],[108]. The canonical signaling cascade of TGF-β pathway involves the ligands of the TGF-β super family binding to cell surface receptors that activate the Smad proteins in the cytoplasm, which leads to their nuclear translocation and transcriptional activation of target genes [108]. The noncanonical TGF-β signaling includes intracellular signaling pathways activated by TGF-β family members that do not activate Smad proteins [108]. The TGF-β pathway can also be regulated by other key signaling pathways such as Wnt signaling pathways.

early differentiation (Fig.3) [111]. Activin A is demonstrated to be able to support long-term feeder-free culture and maintenance of pluripotency in hESCs by inducing the expression of Oct4 and Nanog, and suppressing BMP (Fig.3) [87]. Nodal expression also plays a role in the maintenance of human ES cell pluripotency through the inhibition of neuroectodermal differentiation, a default differentiation pathway of ESCs (Fig.3) [112]. Furthermore, inhibition of the TGF-β/Activin/Nodal pathways initiated differentiation and resulted in the decreased

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In addition, Activin and Nodal signaling has been shown to promote mESC self-renewal in serum-free conditions [114]. It is therefore clear that TGF-β signaling plays an important role in the maintenance of self-renewal and pluripotency, although the exact mechanism of action for this family of growth factors appears to differ between family members, pluripotency state,

BMPs are also potent inhibitors of differentiation in mouse embryos. Knockdown of their down-stream mediator, Smad1 and Smad4, in mESCs leads to a change of the expression pattern of germ layer markers during differentiation [37],[115],[116]. TGF-β signaling also participates in the cell fate decision making of mESCs. Multiple cell lineages, including neural, hematopoietic, cardiomyogenic, and hepatic, have been found to be affected by the TGF-β family [115],[117]. For example, BMP4 regulates mesodermal cell commitment to the hemato‐ poietic lineage and specifies blood lineages at the later stages of differentiation [118]-[120]. Another study found that BMP4 and Activin induce mesoderm differentiation into cardiac lineage [121]. In addition, BMP2-induced mesodermal and cardiac specification results in full cardiogenic differentiation, leading to an enrichment of cardiomyocytes within embryoid bodies [122]. This ability of the TGF-β family members to commit mESCs toward a mesodermal fate is thought to be due to Smad-mediated regulation of the Oct4 promoter, further implicat‐ ing a role for Smad signaling in the regulation of the core self-renewal network in ESCs [123], [124]. Consistent with this notion, it is found that several Smad target genes overlap with genes bound by the key pluripotency factors, for example, Smad4-regulated genes have a substantial overlap with those of Sox2, NR0B1/Dax1, and Klf4 [116]. In addition, another study demon‐ strated that several Smad targets were mapped to Nanog, Oct4, and TCF3-bound genes [116].

**Jak/Stat3 pathway** The self renewal and pluripotency of mESCs are initially maintained by an extrinsic factor, leukemia inhibitory factor (LIF) [7]. The key downstream mediators of LIF are the Jak/Stat pathway [7]. Stat3 has multiple roles in the regulation of mESC pluripotency including gene activation, cell cycle regulation, and inhibition of differentiation pathways [35], [125]. The activation of the Stat3 pathway by LIF induces transcription of self-renewal and pluripotency genes such as Nanog [35],[125]. Furthermore, constitutively active Stat3 pro‐ motes mESC self-renewal in the absence of LIF [35],[125]. Stat3 has also been reported to function through the regulation of c-Myc and Klf family proteins [126],[127], although these target genes have not been shown to be completely sufficient to replace the effect of LIF. This ability of LIF-mediated activation of Stat3 to support the long-term self-renewal of mESCs *in vitro* has been supported *in vivo* by the requirement of this pathway in gp130-deficient

expression of stem cell marker proteins [111],[113].

blastocysts, an embryonic diapause case [128].

and species (Figs. 3 and 4).

**Figure 4.** Signaling pathways regulating pluripotency of the naive stem cells, such as mESCs and miPSCs

All members of this family are important for stem cell pluripotency and self-renewal of both mESCs and hESCs, although the role of these signaling molecules appears to differ between the two types of cells [109]. In mESCs, BMP4 maintains self-renewal through inhibition of the MAPK/ERK pathway and the expression of Id protein [36],[37], and promotes mESC prolif‐ eration via an increase in Wnt expression (Fig.4) [86]. In contrast, BMP4 promotes hESC differentiation through down-regulation of Nanog and Oct4 [110]. Long-term maintenance of hESC pluripotency therefore requires down-regulation of BMP activity by Noggin and Fgf2 [85]. In hESCs, on the other hand, it is other members of the TGF-β super family that maintain their pluripotency. Phosphorylation and nuclear localization of Smad2 induced by TGF-β, Activin, or Nodal signaling was observed in undifferentiated hESCs and is decreased upon early differentiation (Fig.3) [111]. Activin A is demonstrated to be able to support long-term feeder-free culture and maintenance of pluripotency in hESCs by inducing the expression of Oct4 and Nanog, and suppressing BMP (Fig.3) [87]. Nodal expression also plays a role in the maintenance of human ES cell pluripotency through the inhibition of neuroectodermal differentiation, a default differentiation pathway of ESCs (Fig.3) [112]. Furthermore, inhibition of the TGF-β/Activin/Nodal pathways initiated differentiation and resulted in the decreased expression of stem cell marker proteins [111],[113].

**5.2. TGF-β signaling pathway**

162 Pluripotent Stem Cells

as Wnt signaling pathways.

The TGF-β signaling pathway plays a vital role in both the developmental and adult life of a mammalian organism by regulating many processes including apoptosis, proliferation, senescence, inflammation, cell fate, and tissue repair [94],[106],[107]. The TGF-β super family contains more than 30 growth factors including TGF-βs, BMPs, growth and differentiation factors (GDFs), Activin, and Nodal [2],[106],[108]. The canonical signaling cascade of TGF-β pathway involves the ligands of the TGF-β super family binding to cell surface receptors that activate the Smad proteins in the cytoplasm, which leads to their nuclear translocation and transcriptional activation of target genes [108]. The noncanonical TGF-β signaling includes intracellular signaling pathways activated by TGF-β family members that do not activate Smad proteins [108]. The TGF-β pathway can also be regulated by other key signaling pathways such

**Figure 4.** Signaling pathways regulating pluripotency of the naive stem cells, such as mESCs and miPSCs

All members of this family are important for stem cell pluripotency and self-renewal of both mESCs and hESCs, although the role of these signaling molecules appears to differ between the two types of cells [109]. In mESCs, BMP4 maintains self-renewal through inhibition of the MAPK/ERK pathway and the expression of Id protein [36],[37], and promotes mESC prolif‐ eration via an increase in Wnt expression (Fig.4) [86]. In contrast, BMP4 promotes hESC differentiation through down-regulation of Nanog and Oct4 [110]. Long-term maintenance of hESC pluripotency therefore requires down-regulation of BMP activity by Noggin and Fgf2 [85]. In hESCs, on the other hand, it is other members of the TGF-β super family that maintain their pluripotency. Phosphorylation and nuclear localization of Smad2 induced by TGF-β, Activin, or Nodal signaling was observed in undifferentiated hESCs and is decreased upon

In addition, Activin and Nodal signaling has been shown to promote mESC self-renewal in serum-free conditions [114]. It is therefore clear that TGF-β signaling plays an important role in the maintenance of self-renewal and pluripotency, although the exact mechanism of action for this family of growth factors appears to differ between family members, pluripotency state, and species (Figs. 3 and 4).

BMPs are also potent inhibitors of differentiation in mouse embryos. Knockdown of their down-stream mediator, Smad1 and Smad4, in mESCs leads to a change of the expression pattern of germ layer markers during differentiation [37],[115],[116]. TGF-β signaling also participates in the cell fate decision making of mESCs. Multiple cell lineages, including neural, hematopoietic, cardiomyogenic, and hepatic, have been found to be affected by the TGF-β family [115],[117]. For example, BMP4 regulates mesodermal cell commitment to the hemato‐ poietic lineage and specifies blood lineages at the later stages of differentiation [118]-[120]. Another study found that BMP4 and Activin induce mesoderm differentiation into cardiac lineage [121]. In addition, BMP2-induced mesodermal and cardiac specification results in full cardiogenic differentiation, leading to an enrichment of cardiomyocytes within embryoid bodies [122]. This ability of the TGF-β family members to commit mESCs toward a mesodermal fate is thought to be due to Smad-mediated regulation of the Oct4 promoter, further implicat‐ ing a role for Smad signaling in the regulation of the core self-renewal network in ESCs [123], [124]. Consistent with this notion, it is found that several Smad target genes overlap with genes bound by the key pluripotency factors, for example, Smad4-regulated genes have a substantial overlap with those of Sox2, NR0B1/Dax1, and Klf4 [116]. In addition, another study demon‐ strated that several Smad targets were mapped to Nanog, Oct4, and TCF3-bound genes [116].

**Jak/Stat3 pathway** The self renewal and pluripotency of mESCs are initially maintained by an extrinsic factor, leukemia inhibitory factor (LIF) [7]. The key downstream mediators of LIF are the Jak/Stat pathway [7]. Stat3 has multiple roles in the regulation of mESC pluripotency including gene activation, cell cycle regulation, and inhibition of differentiation pathways [35], [125]. The activation of the Stat3 pathway by LIF induces transcription of self-renewal and pluripotency genes such as Nanog [35],[125]. Furthermore, constitutively active Stat3 pro‐ motes mESC self-renewal in the absence of LIF [35],[125]. Stat3 has also been reported to function through the regulation of c-Myc and Klf family proteins [126],[127], although these target genes have not been shown to be completely sufficient to replace the effect of LIF. This ability of LIF-mediated activation of Stat3 to support the long-term self-renewal of mESCs *in vitro* has been supported *in vivo* by the requirement of this pathway in gp130-deficient blastocysts, an embryonic diapause case [128].

In addition to Stat3 homodimers, Stat1 is able to heterodimerize with Stat3 in mESCs. However, Stat1 is unlikely to be required for self-renewal and pluripotency as LIF still maintains undifferentiated growth of Stat1-deficient cells [129].

suppresses its expression, forming a negative feedback loop involving mir-145, Oct4, Nanog,

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165

Another very important member of the miRNA family is let7 which has differentiation promoting activities itself, and also targets some of the pluripotency-associated genes [143], [144]. Let7 expression is negatively regulated by the RNA binding protein Lin28 [145]. Upon differentiation of pluripotent cells, Lin28 is down-regulated, resulting in stabilization and increase in the level of let7 [146]. This in turn provides the basis for establishment of negative feedback loops in which let7 expression is negatively regulated by the RNA binding protein

Recently, Oct4 has been shown to control and activate the expression of another type of noncoding RNA, the large intergenic noncoding RNAs [147]. Interestingly, knockdown of the expression of such RNAs caused growth defects and apoptosis, implying that these noncoding

Chromatin remodeling factors are recruited to the DNA to modify the density of the nucleo‐ somes, thereby affecting gene expression [149],[150]. Some of these factors are essential for PSC viability, stability, and differentiation [31],[151],[152]. The ones known to have the most profound impact on ESC pluripotency are histone-modifying enzymes, such as Polycomb group (PcG) protein complexes, SetDB1, and Tip60-p400 [153],[154]. These enzymes repress genes that encode lineage-specific differentiation regulators by catalyzing methylation or

It is demonstrated that sumoylated SetDB1 binds to Oct4 and represses its expression [153], [156]. Loss of the Tip60-p400 complex affects ESC morphology and state [154]. The Tip60-p400 complex is shown to associate with active promoters in ESCs and appears to be recruited directly by the H3K4me3 mark and indirectly by Nanog [154]. Interestingly, the complex is also associated with nucleosomes with H3K4me3 at PcG-occupied genes encoding lineage specific regulators, where it apparently facilitates repression of these poised genes [154].

Our understanding of the nature of pluripotency has been formulated extensively by the recent development of different lines of PSCs, especially the iPSCs. Although differences exist between them, the naïve and primed PSCs share certain similarities. For example, they both express the core pluripotency factors, Oct4, Nanog, and Sox2. The core transcrip‐ tion factors frequently share enhancers and autoregulate themselves. They also collective‐ ly bind to the promoters of an expanded network of proteins, including pluripotencyassociated factors and lineage-specific factors, to enhance or repress their gene expression, through which the fate of the cells is determined. The epigenetic studies have added another layer of complexity of the regulation of these core pluripotency factors and hence

RNAs are involved in self-renewal and reprogramming of stem cells [147],[148].

ubiquitination of the histones in their promoters [153]-[155].

and Klf4 [142].

Lin28 [145].

**7. Conclusion**

**6.2. Chromatin remodeling factors**

Although it is sufficient to maintain the pluripotency of mESCs, the Jak/Stat3 pathway does not appear to maintain pluripotency of hESCs, which are possibly in the primed state of pluripotency [90], indicating that the signaling pathways responsible for maintaining pluri‐ potency is species (or pluripotency state)-specific.

#### **5.3. Fgf and the MAPK pathway**

Fgf2 (or basic Fgf) is the first growth factor identified as being crucial for hESC pluripotency maintenance and self renewal. It is widely accepted that a serum-free culture of hESCs on mouse feeder cells requires soluble Fgf2 [88],[107],[130],[131]. In hESCs, exogenous Fgf2 activates the ERK/MAPK pathway, which is thought to be necessary for the maintenance of pluripotency, although the mechanism of action is still unclear [130],[132],[133]. In contrast to hESCs, mESCs and miPSCs do not require the Fgf2 or the ERK/MAPK pathway for pluripo‐ tency and self-renewal (Table 1) [5],[11]. Actually, ERK signaling triggers mESCs to differen‐ tiate towards the primitive endoderm lineage (Table 1) [1],[134]. Interestingly, inhibition of ERK activity has been shown to enhance the efficiency of mESC derivation from mouse embryos [135]. The mechanism underlying this seems to be that ERK1/2 activation triggers mESCs to exit the self-renewal program and enter lineage differentiation [136]. As mentioned earlier, the direct consequence of this is that blocking the ERK/MAPK-mediated differentiation pathway can help the derivation and maintenance of naive state PSCs, such as mESCs.

## **6. Epigenetic factors regulating stem cell pluripotency**

Apart from the aforementioned transcription factors, epigenetics factors have also been found to play a vital role in stem cell pluripotency. These mechanisms include covalent modification of histone, DNA methylation and acetylation, and non-coding RNAs [31],[62],[137],[138]. Here we will discuss the functions of noncoding RNAs and chromatin remodeling factors in stem cell pluripotency.

#### **6.1. Noncoding RNAs**

The best understood class of noncoding RNA is the family of microRNAs (miRNAs), short RNAs capable of destabilizing and repressing specific target RNAs. These miRNAs are generally generated by the enzymes Dicer and Dcgr8 [139]-[141]. As for their function in stem cell pluripotency, it has been shown that genetic ablation of these enzymes affects the cell cycle and differentiation of ESCs [139]-[141]. Furthermore, some specific miRNAs are involved in pluripotency regulation. For example, mir-302 and mir-290–295 bind directly to and modulate the core pluripotency factors Oct4, Sox2, and Nanog [6]. miR-145 represses the 3' untranslated regions of Oct4, Sox2, and Klf4, thus increasing the amount of mir-145 leads to loss of pluri‐ potency [142]. It is further demonstrated that Oct4 also binds to the promoter of mir-145 and suppresses its expression, forming a negative feedback loop involving mir-145, Oct4, Nanog, and Klf4 [142].

Another very important member of the miRNA family is let7 which has differentiation promoting activities itself, and also targets some of the pluripotency-associated genes [143], [144]. Let7 expression is negatively regulated by the RNA binding protein Lin28 [145]. Upon differentiation of pluripotent cells, Lin28 is down-regulated, resulting in stabilization and increase in the level of let7 [146]. This in turn provides the basis for establishment of negative feedback loops in which let7 expression is negatively regulated by the RNA binding protein Lin28 [145].

Recently, Oct4 has been shown to control and activate the expression of another type of noncoding RNA, the large intergenic noncoding RNAs [147]. Interestingly, knockdown of the expression of such RNAs caused growth defects and apoptosis, implying that these noncoding RNAs are involved in self-renewal and reprogramming of stem cells [147],[148].

#### **6.2. Chromatin remodeling factors**

In addition to Stat3 homodimers, Stat1 is able to heterodimerize with Stat3 in mESCs. However, Stat1 is unlikely to be required for self-renewal and pluripotency as LIF still maintains

Although it is sufficient to maintain the pluripotency of mESCs, the Jak/Stat3 pathway does not appear to maintain pluripotency of hESCs, which are possibly in the primed state of pluripotency [90], indicating that the signaling pathways responsible for maintaining pluri‐

Fgf2 (or basic Fgf) is the first growth factor identified as being crucial for hESC pluripotency maintenance and self renewal. It is widely accepted that a serum-free culture of hESCs on mouse feeder cells requires soluble Fgf2 [88],[107],[130],[131]. In hESCs, exogenous Fgf2 activates the ERK/MAPK pathway, which is thought to be necessary for the maintenance of pluripotency, although the mechanism of action is still unclear [130],[132],[133]. In contrast to hESCs, mESCs and miPSCs do not require the Fgf2 or the ERK/MAPK pathway for pluripo‐ tency and self-renewal (Table 1) [5],[11]. Actually, ERK signaling triggers mESCs to differen‐ tiate towards the primitive endoderm lineage (Table 1) [1],[134]. Interestingly, inhibition of ERK activity has been shown to enhance the efficiency of mESC derivation from mouse embryos [135]. The mechanism underlying this seems to be that ERK1/2 activation triggers mESCs to exit the self-renewal program and enter lineage differentiation [136]. As mentioned earlier, the direct consequence of this is that blocking the ERK/MAPK-mediated differentiation pathway can help the derivation and maintenance of naive state PSCs, such as mESCs.

Apart from the aforementioned transcription factors, epigenetics factors have also been found to play a vital role in stem cell pluripotency. These mechanisms include covalent modification of histone, DNA methylation and acetylation, and non-coding RNAs [31],[62],[137],[138]. Here we will discuss the functions of noncoding RNAs and chromatin remodeling factors in stem

The best understood class of noncoding RNA is the family of microRNAs (miRNAs), short RNAs capable of destabilizing and repressing specific target RNAs. These miRNAs are generally generated by the enzymes Dicer and Dcgr8 [139]-[141]. As for their function in stem cell pluripotency, it has been shown that genetic ablation of these enzymes affects the cell cycle and differentiation of ESCs [139]-[141]. Furthermore, some specific miRNAs are involved in pluripotency regulation. For example, mir-302 and mir-290–295 bind directly to and modulate the core pluripotency factors Oct4, Sox2, and Nanog [6]. miR-145 represses the 3' untranslated regions of Oct4, Sox2, and Klf4, thus increasing the amount of mir-145 leads to loss of pluri‐ potency [142]. It is further demonstrated that Oct4 also binds to the promoter of mir-145 and

undifferentiated growth of Stat1-deficient cells [129].

potency is species (or pluripotency state)-specific.

**6. Epigenetic factors regulating stem cell pluripotency**

**5.3. Fgf and the MAPK pathway**

164 Pluripotent Stem Cells

cell pluripotency.

**6.1. Noncoding RNAs**

Chromatin remodeling factors are recruited to the DNA to modify the density of the nucleo‐ somes, thereby affecting gene expression [149],[150]. Some of these factors are essential for PSC viability, stability, and differentiation [31],[151],[152]. The ones known to have the most profound impact on ESC pluripotency are histone-modifying enzymes, such as Polycomb group (PcG) protein complexes, SetDB1, and Tip60-p400 [153],[154]. These enzymes repress genes that encode lineage-specific differentiation regulators by catalyzing methylation or ubiquitination of the histones in their promoters [153]-[155].

It is demonstrated that sumoylated SetDB1 binds to Oct4 and represses its expression [153], [156]. Loss of the Tip60-p400 complex affects ESC morphology and state [154]. The Tip60-p400 complex is shown to associate with active promoters in ESCs and appears to be recruited directly by the H3K4me3 mark and indirectly by Nanog [154]. Interestingly, the complex is also associated with nucleosomes with H3K4me3 at PcG-occupied genes encoding lineage specific regulators, where it apparently facilitates repression of these poised genes [154].

### **7. Conclusion**

Our understanding of the nature of pluripotency has been formulated extensively by the recent development of different lines of PSCs, especially the iPSCs. Although differences exist between them, the naïve and primed PSCs share certain similarities. For example, they both express the core pluripotency factors, Oct4, Nanog, and Sox2. The core transcrip‐ tion factors frequently share enhancers and autoregulate themselves. They also collective‐ ly bind to the promoters of an expanded network of proteins, including pluripotencyassociated factors and lineage-specific factors, to enhance or repress their gene expression, through which the fate of the cells is determined. The epigenetic studies have added another layer of complexity of the regulation of these core pluripotency factors and hence

pluripotency. In addition, a recent study of our/my laboratory shows that Hsp90 main‐ tains stem cell pluripotency by associating with and sustaining the cellular levels of Oct4 and Nanog, implying that the maturation or stability of these core pluripotency factors are crucially important for stem cell pluripotency [21].

[3] Xue, K., Ng, J. H. & Ng, H. H. Mapping the networks for pluripotency. *Philos Trans R Soc Lond B Biol Sci* 366, 2238-2246, doi:366/1575/2238 [pii]10.1098/rstb.2011.0005

Molecular Mechanisms Underlying Pluripotency

http://dx.doi.org/10.5772/55596

167

[4] Greenow, K. & Clarke, A. R. Controlling the stem cell compartment and regeneration in vivo: the role of pluripotency pathways. *Physiol Rev* 92, 75-99, doi:92/1/75

[5] Dejosez, M. & Zwaka, T. P. Pluripotency and nuclear reprogramming. *Annu Rev Bio‐*

[6] Marson, A. *et al.* Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. *Cell* 134, 521-533, doi:S0092-8674(08)00938-0

[7] Mitalipov, S. & Wolf, D. Totipotency, pluripotency and nuclear reprogramming. *Adv*

[8] Pauklin, S., Pedersen, R. A. & Vallier, L. Mouse pluripotent stem cells at a glance. *J*

[9] Thomson, J. A. *et al.* Embryonic stem cell lines derived from human blastocysts. *Sci‐*

[10] Vescovi, A. L., Reynolds, B. A., Rietze, R. L. & Bjornson, C. Ground-breaking stem-

[11] Burdon, T., Smith, A. & Savatier, P. Signalling, cell cycle and pluripotency in embry‐ onic stem cells. *Trends Cell Biol* 12, 432-438, doi:S0962892402023528 [pii] (2002).

[12] Krishnamurthy, K., Wang, G., Silva, J., Condie, B. G. & Bieberich, E. Ceramide regu‐ lates atypical PKCzeta/lambda-mediated cell polarity in primitive ectoderm cells. A novel function of sphingolipids in morphogenesis. *J Biol Chem* 282, 3379-3390,

[13] Nichols, J. & Smith, A. Naive and primed pluripotent states. *Cell Stem Cell* 4, 487-492,

[14] Evans, M. Discovering pluripotency: 30 years of mouse embryonic stem cells. *Nat Rev*

[15] Evans, M. J. & Kaufman, M. H. Establishment in culture of pluripotential cells from

[16] Tesar, P. J. *et al.* New cell lines from mouse epiblast share defining features with hu‐ man embryonic stem cells. *Nature* 448, 196-199, doi:nature05972 [pii]10.1038/

*chem* 81, 737-765, doi:10.1146/annurev-biochem-052709-104948 (2012).

*Biochem Eng Biotechnol* 114, 185-199, doi:10.1007/10\_2008\_45 (2009).

*Cell Sci* 124, 3727-3732, doi:124/22/3727 [pii]10.1242/jcs.074120 (2011).

cell work has been reproduced. *Nature* 447, 259 (2007).

doi:M607779200 [pii]10.1074/jbc.M607779200 (2007).

mouse embryos. *Nature* 292, 154-156 (1981).

nature05972 (2007).

doi:S1934-5909(09)00224-0 [pii]10.1016/j.stem.2009.05.015 (2009).

*Mol Cell Biol* 12, 680-686, doi:nrm3190 [pii]10.1038/nrm3190 (2011).

(2011).

[pii]10.1152/physrev.00040.2010 (2012).

[pii]10.1016/j.cell.2008.07.020 (2008).

*ence* 282, 1145-1147 (1998).

Many of the methodologies to induce or convert somatic cells into PSCs involve using chemical inhibitors targeting specific pathways. This highlights the importance of understanding the roles of signaling pathways in stem cell pluripotency and self-renewal.

Furthermore, an in-depth understanding of pluripotency is highly applicable to regenerative medicine. Knowledge of their culture condition, state of pluripotency, and signal transduction pathways could greatly facilitate *in vitro* culture, manipulation, and differentiation, either from autologous or allogeneic sources. This knowledge will also guide a more effective generation of iPSCs, which will ultimately lead to individualized regenerative medicine.

## **Acknowledgements**

This work is supported by a grant from American Heart Association and an intramural grant from Georgia Health Sciences University to GW, and in part by NIH (R01AG034389) and NSF (1121579) to EB. We are also thankful for the support by the Institute of Molecular Medicine and Genetics (under the directorship of Dr. Lin Mei) of Genetics of Georgia Health Sciences University (soon to be Georgia Regents University Augusta).

## **Author details**

Erhard Bieberich and Guanghu Wang\*

\*Address all correspondence to: gwang@gru.edu

Institute of Molecular Medicine and Genetics, Medical College of Georgia, Georgia Regents University, Augusta, GA, USA

## **References**


[3] Xue, K., Ng, J. H. & Ng, H. H. Mapping the networks for pluripotency. *Philos Trans R Soc Lond B Biol Sci* 366, 2238-2246, doi:366/1575/2238 [pii]10.1098/rstb.2011.0005 (2011).

pluripotency. In addition, a recent study of our/my laboratory shows that Hsp90 main‐ tains stem cell pluripotency by associating with and sustaining the cellular levels of Oct4 and Nanog, implying that the maturation or stability of these core pluripotency factors are

Many of the methodologies to induce or convert somatic cells into PSCs involve using chemical inhibitors targeting specific pathways. This highlights the importance of understanding the

Furthermore, an in-depth understanding of pluripotency is highly applicable to regenerative medicine. Knowledge of their culture condition, state of pluripotency, and signal transduction pathways could greatly facilitate *in vitro* culture, manipulation, and differentiation, either from autologous or allogeneic sources. This knowledge will also guide a more effective generation

This work is supported by a grant from American Heart Association and an intramural grant from Georgia Health Sciences University to GW, and in part by NIH (R01AG034389) and NSF (1121579) to EB. We are also thankful for the support by the Institute of Molecular Medicine and Genetics (under the directorship of Dr. Lin Mei) of Genetics of Georgia Health Sciences

Institute of Molecular Medicine and Genetics, Medical College of Georgia, Georgia Regents

[1] Young, R. A. Control of the embryonic stem cell state. *Cell* 144, 940-954,

[2] Tiedemann, H., Asashima, M., Grunz, H. & Knochel, W. Pluripotent cells (stem cells) and their determination and differentiation in early vertebrate embryogenesis. *Dev*

doi:S0092-8674(11)00071-7 [pii]10.1016/j.cell.2011.01.032 (2011).

*Growth Differ* 43, 469-502, doi:dgd599 [pii] (2001).

crucially important for stem cell pluripotency [21].

**Acknowledgements**

166 Pluripotent Stem Cells

**Author details**

**References**

Erhard Bieberich and Guanghu Wang\*

University, Augusta, GA, USA

\*Address all correspondence to: gwang@gru.edu

roles of signaling pathways in stem cell pluripotency and self-renewal.

of iPSCs, which will ultimately lead to individualized regenerative medicine.

University (soon to be Georgia Regents University Augusta).


[17] Kuijk, E. W., Chuva de Sousa Lopes, S. M., Geijsen, N., Macklon, N. & Roelen, B. A. The different shades of mammalian pluripotent stem cells. *Hum Reprod Update* 17, 254-271, doi:dmq035 [pii]10.1093/humupd/dmq035 (2011).

[31] Meissner, A. Epigenetic modifications in pluripotent and differentiated cells. *Nat Bio‐*

Molecular Mechanisms Underlying Pluripotency

http://dx.doi.org/10.5772/55596

169

[32] Wang, G. *et al.* Direct binding to ceramide activates protein kinase Czeta before the formation of a pro-apoptotic complex with PAR-4 in differentiating stem cells. *J Biol*

[33] Bieberich, E., MacKinnon, S., Silva, J., Noggle, S. & Condie, B. G. Regulation of cell death in mitotic neural progenitor cells by asymmetric distribution of prostate apop‐ tosis response 4 (PAR-4) and simultaneous elevation of endogenous ceramide. *J Cell*

[34] Raz, R., Lee, C. K., Cannizzaro, L. A., d'Eustachio, P. & Levy, D. E. Essential role of STAT3 for embryonic stem cell pluripotency. *Proc Natl Acad Sci U S A* 96, 2846-2851

[35] Niwa, H., Burdon, T., Chambers, I. & Smith, A. Self-renewal of pluripotent embryon‐ ic stem cells is mediated via activation of STAT3. *Genes Dev* 12, 2048-2060 (1998).

[36] Qi, X. *et al.* BMP4 supports self-renewal of embryonic stem cells by inhibiting mito‐ gen-activated protein kinase pathways. *Proc Natl Acad Sci U S A* 101, 6027-6032, doi:

[37] Ying, Q. L., Nichols, J., Chambers, I. & Smith, A. BMP induction of Id proteins sup‐ presses differentiation and sustains embryonic stem cell self-renewal in collaboration

[38] Ying, Q. L. *et al.* The ground state of embryonic stem cell self-renewal. *Nature* 453,

[39] Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse em‐ bryonic and adult fibroblast cultures by defined factors. *Cell* 126, 663-676,

[40] Takahashi, K. *et al.* Induction of pluripotent stem cells from adult human fibroblasts by defined factors. *Cell* 131, 861-872, doi:S0092-8674(07)01471-7 [pii]10.1016/j.cell.

[41] Takahashi, K., Okita, K., Nakagawa, M. & Yamanaka, S. Induction of pluripotent stem cells from fibroblast cultures. *Nat Protoc* 2, 3081-3089, doi:nprot.2007.418

[42] Yamanaka, S. Induction of pluripotent stem cells from mouse fibroblasts by four transcription factors. *Cell Prolif* 41 Suppl 1, 51-56, doi:CPR493 [pii]10.1111/j.

[43] Cheng, D., Lei, L., Lu, Z., Li, Z. & Wang, H. [Induction and characterization of in‐ duced pluripotent stem (iPS) cells: a review]. *Sheng Wu Gong Cheng Xue Bao* 26,

with STAT3. *Cell* 115, 281-292, doi:S009286740300847X [pii] (2003).

519-523, doi:nature06968 [pii]10.1038/nature06968 (2008).

doi:S0092-8674(06)00976-7 [pii]10.1016/j.cell.2006.07.024 (2006).

10.1073/pnas.04013671010401367101 [pii] (2004).

*technol* 28, 1079-1088, doi:nbt.1684 [pii]10.1038/nbt.1684 (2010).

*Chem* 280, 26415-26424 (2005).

*Biol* 162, 469-479 (2003).

2007.11.019 (2007).

421-430 (2010).

[pii]10.1038/nprot.2007.418 (2007).

1365-2184.2008.00493.x (2008).

(1999).


[31] Meissner, A. Epigenetic modifications in pluripotent and differentiated cells. *Nat Bio‐ technol* 28, 1079-1088, doi:nbt.1684 [pii]10.1038/nbt.1684 (2010).

[17] Kuijk, E. W., Chuva de Sousa Lopes, S. M., Geijsen, N., Macklon, N. & Roelen, B. A. The different shades of mammalian pluripotent stem cells. *Hum Reprod Update* 17,

[18] Ginis, I. *et al.* Differences between human and mouse embryonic stem cells. *Dev Biol* 269, 360-380, doi:10.1016/j.ydbio.2003.12.034S0012160604000193 [pii] (2004).

[19] Singh, S. K., Kagalwala, M. N., Parker-Thornburg, J., Adams, H. & Majumder, S. REST maintains self-renewal and pluripotency of embryonic stem cells. *Nature* 453,

[20] Bieberich, E., Silva, J., Wang, G., Krishnamurthy, K. & Condie, B. G. Selective apopto‐ sis of pluripotent mouse and human stem cells by novel ceramide analogues pre‐ vents teratoma formation and enriches for neural precursors in ES cell-derived

[21] Bradley, E., Bieberich, E., Mivechi, N. F., Tangpisuthipongsa, D. & Wang, G. Regula‐ tion of embryonic stem cell pluripotency by heat shock protein 90. *Stem Cells* 30,

[22] Brons, I. G. *et al.* Derivation of pluripotent epiblast stem cells from mammalian em‐ bryos. *Nature* 448, 191-195, doi:nature05950 [pii]10.1038/nature05950 (2007).

[23] Kleinsmith, L. J. & Pierce, G. B., Jr. Multipotentiality of Single Embryonal Carcinoma

[24] Gooi, H. C. *et al.* Stage-specific embryonic antigen involves alpha 1 goes to 3 fucosy‐

[25] Solter, D. & Knowles, B. B. Monoclonal antibody defining a stage-specific mouse em‐

[26] Papaioannou, V. E., McBurney, M. W., Gardner, R. L. & Evans, M. J. Fate of teratocar‐

[27] Martin, G. R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. *Proc Natl Acad Sci U S A* 78,

[28] Savatier, P., Lapillonne, H., van Grunsven, L. A., Rudkin, B. B. & Samarut, J. With‐ drawal of differentiation inhibitory activity/leukemia inhibitory factor up-regulates D-type cyclins and cyclin-dependent kinase inhibitors in mouse embryonic stem

[29] Minkovsky, A., Patel, S. & Plath, K. Concise review: Pluripotency and the transcrip‐ tional inactivation of the female Mammalian X chromosome. *Stem Cells* 30, 48-54, doi:

[30] Bernstein, B. E., Meissner, A. & Lander, E. S. The mammalian epigenome. *Cell* 128,

669-681, doi:S0092-8674(07)00128-6 [pii]10.1016/j.cell.2007.01.033 (2007).

bryonic antigen (SSEA-1). *Proc Natl Acad Sci U S A* 75, 5565-5569 (1978).

cinoma cells injected into early mouse embryos. *Nature* 258, 70-73 (1975).

254-271, doi:dmq035 [pii]10.1093/humupd/dmq035 (2011).

223-227, doi:nature06863 [pii]10.1038/nature06863 (2008).

lated type 2 blood group chains. *Nature* 292, 156-158 (1981).

neural transplants. *J Cell Biol* 167, 723-734 (2004).

1624-1633, doi:10.1002/stem.1143 (2012).

Cells. *Cancer Res* 24, 1544-1551 (1964).

7634-7638 (1981).

168 Pluripotent Stem Cells

cells. *Oncogene* 12, 309-322 (1996).

10.1002/stem.755 (2012).


[44] Huangfu, D. *et al.* Induction of pluripotent stem cells by defined factors is greatly im‐ proved by small-molecule compounds. *Nat Biotechnol* 26, 795-797, doi:nbt1418 [pii]10.1038/nbt1418 (2008).

[57] Nichols, J. *et al.* Formation of pluripotent stem cells in the mammalian embryo de‐ pends on the POU transcription factor Oct4. *Cell* 95, 379-391,

Molecular Mechanisms Underlying Pluripotency

http://dx.doi.org/10.5772/55596

171

[58] Kim, J. B. *et al.* Oct4-induced pluripotency in adult neural stem cells. *Cell* 136,

[59] Torres, J. & Watt, F. M. Nanog maintains pluripotency of mouse embryonic stem cells by inhibiting NFkappaB and cooperating with Stat3. *Nat Cell Biol* 10, 194-201,

[60] Ebert, A. D. *et al.* Induced pluripotent stem cells from a spinal muscular atrophy pa‐ tient. *Nature* 457, 277-280, doi:nature07677 [pii]10.1038/nature07677 (2009).

[61] Yu, J. *et al.* Human induced pluripotent stem cells free of vector and transgene se‐ quences. *Science* 324, 797-801, doi:1172482 [pii]10.1126/science.1172482 (2009).

[62] Gan, Q., Yoshida, T., McDonald, O. G. & Owens, G. K. Concise review: epigenetic mechanisms contribute to pluripotency and cell lineage determination of embryonic stem cells. *Stem Cells* 25, 2-9, doi:2006-0383 [pii]10.1634/stemcells.2006-0383 (2007).

[63] Hanna, J. H., Saha, K. & Jaenisch, R. Pluripotency and cellular reprogramming: facts, hypotheses, unresolved issues. *Cell* 143, 508-525, doi:S0092-8674(10)01144-X

[64] Niwa, H., Miyazaki, J. & Smith, A. G. Quantitative expression of Oct-3/4 defines dif‐ ferentiation, dedifferentiation or self-renewal of ES cells. *Nat Genet* 24, 372-376, doi:

[65] Shimozaki, K., Nakashima, K., Niwa, H. & Taga, T. Involvement of Oct3/4 in the en‐ hancement of neuronal differentiation of ES cells in neurogenesis-inducing cultures.

[66] Mitsui, K. *et al.* The homeoprotein Nanog is required for maintenance of pluripoten‐ cy in mouse epiblast and ES cells. *Cell* 113, 631-642, doi:S0092867403003933 [pii]

[67] Chambers, I. *et al.* Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. *Cell* 113, 643-655, doi:S0092867403003921 [pii] (2003).

[68] Suzuki, A. *et al.* Nanog binds to Smad1 and blocks bone morphogenetic protein-in‐ duced differentiation of embryonic stem cells. *Proc Natl Acad Sci U S A* 103,

[69] Avilion, A. A. *et al.* Multipotent cell lineages in early mouse development depend on

[70] Masui, S. *et al.* Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. *Nat Cell Biol* 9, 625-635, doi:ncb1589 [pii]10.1038/

10294-10299, doi:0506945103 [pii]10.1073/pnas.0506945103 (2006).

SOX2 function. *Genes Dev* 17, 126-140, doi:10.1101/gad.224503 (2003).

411-419, doi:S0092-8674(09)00071-3 [pii]10.1016/j.cell.2009.01.023 (2009).

doi:S0092-8674(00)81769-9 [pii] (1998).

doi:ncb1680 [pii]10.1038/ncb1680 (2008).

[pii]10.1016/j.cell.2010.10.008 (2010).

*Development* 130, 2505-2512 (2003).

10.1038/74199 (2000).

(2003).

ncb1589 (2007).


[57] Nichols, J. *et al.* Formation of pluripotent stem cells in the mammalian embryo de‐ pends on the POU transcription factor Oct4. *Cell* 95, 379-391, doi:S0092-8674(00)81769-9 [pii] (1998).

[44] Huangfu, D. *et al.* Induction of pluripotent stem cells by defined factors is greatly im‐ proved by small-molecule compounds. *Nat Biotechnol* 26, 795-797, doi:nbt1418

[45] Cho, H. J. *et al.* Induction of pluripotent stem cells from adult somatic cells by pro‐ tein-based reprogramming without genetic manipulation. *Blood* 116, 386-395,

[46] Hjelm, B. E. *et al.* Induction of pluripotent stem cells from autopsy donor-derived so‐ matic cells. *Neurosci Lett* 502, 219-224, doi:S0304-3940(11)01125-6 [pii]10.1016/j.neulet.

[47] Okahara-Narita, J. *et al.* Induction of pluripotent stem cells from fetal and adult cyno‐ molgus monkey fibroblasts using four human transcription factors. *Primates*, doi:

[48] Oda, Y. *et al.* Induction of pluripotent stem cells from human third molar mesenchy‐ mal stromal cells. *J Biol Chem* 285, 29270-29278, doi:M109.055889 [pii]10.1074/

[49] Shi, Y. *et al.* Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. *Cell Stem Cell* 3, 568-574,

[50] Huangfu, D. *et al.* Induction of pluripotent stem cells from primary human fibro‐ blasts with only Oct4 and Sox2. *Nat Biotechnol* 26, 1269-1275, doi:nbt.1502

[51] Surani, M. A. Reprogramming a somatic nucleus by trans-modification activity in germ cells. *Semin Cell Dev Biol* 10, 273-277, doi:10.1006/scdb.

[52] Kanatsu-Shinohara, M. *et al.* Generation of pluripotent stem cells from neonatal mouse testis. *Cell* 119, 1001-1012, doi:S0092867404010578 [pii]10.1016/j.cell.

[53] Shamblott, M. J. *et al.* Derivation of pluripotent stem cells from cultured human pri‐

[54] Conrad, S. *et al.* Generation of pluripotent stem cells from adult human testis. *Nature*

[55] Ko, K. *et al.* Human adult germline stem cells in question. *Nature* 465, E1; discussion

[56] Mountford, P., Nichols, J., Zevnik, B., O'Brien, C. & Smith, A. Maintenance of pluri‐ potential embryonic stem cells by stem cell selection. *Reprod Fertil Dev* 10, 527-533

mordial germ cells. *Proc Natl Acad Sci U S A* 95, 13726-13731 (1998).

456, 344-349, doi:nature07404 [pii]10.1038/nature07404 (2008).

E3, doi:nature09089 [pii]10.1038/nature09089 (2010).

doi:S1934-5909(08)00527-4 [pii] 10.1016/j.stem.2008.10.004 (2008).

doi:blood-2010-02-269589 [pii]10.1182/blood-2010-02-269589 (2010).

[pii]10.1038/nbt1418 (2008).

170 Pluripotent Stem Cells

2011.07.048 (2011).

jbc.M109.055889 (2010).

[pii]10.1038/nbt.1502 (2008).

2004.11.011 (2004).

(1998).

1998.0285S1084-9521(98)90285-3 [pii] (1999).

10.1007/s10329-011-0283-1 (2011).


[71] Chen, X. *et al.* Integration of external signaling pathways with the core transcription‐ al network in embryonic stem cells. *Cell* 133, 1106-1117, doi:S0092-8674(08)00617-X [pii]10.1016/j.cell.2008.04.043 (2008).

[83] Moretto-Zita, M. *et al.* Phosphorylation stabilizes Nanog by promoting its interaction with Pin1. *Proc Natl Acad Sci U S A* 107, 13312-13317, doi:1005847107 [pii]10.1073/

Molecular Mechanisms Underlying Pluripotency

http://dx.doi.org/10.5772/55596

173

[84] Daheron, L. *et al.* LIF/STAT3 signaling fails to maintain self-renewal of human em‐ bryonic stem cells. *Stem Cells* 22, 770-778, doi:10.1634/stemcells.22-5-770 22/5/770 [pii]

[85] Xu, R. H. *et al.* Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. *Nat Methods* 2, 185-190, doi:nmeth744 [pii] 10.1038/

[86] Lee, M. Y., Lim, H. W., Lee, S. H. & Han, H. J. Smad, PI3K/Akt, and Wnt-dependent signaling pathways are involved in BMP-4-induced ESC self-renewal. *Stem Cells* 27,

[87] Xiao, L., Yuan, X. & Sharkis, S. J. Activin A maintains self-renewal and regulates fi‐ broblast growth factor, Wnt, and bone morphogenic protein pathways in human em‐ bryonic stem cells. *Stem Cells* 24, 1476-1486, doi:2005-0299 [pii] 10.1634/stemcells.

[88] Ding, V. M. *et al.* FGF-2 modulates Wnt signaling in undifferentiated hESC and iPS cells through activated PI3-K/GSK3beta signaling. *J Cell Physiol* 225, 417-428, doi:

[89] Kim, L. & Kimmel, A. R. GSK3 at the edge: regulation of developmental specification

[90] Sato, N., Meijer, L., Skaltsounis, L., Greengard, P. & Brivanlou, A. H. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. *Nat Med* 10, 55-63, doi:

[91] Bejsovec, A. Wnt pathway activation: new relations and locations. *Cell* 120, 11-14,

[92] Widelitz, R. Wnt signaling through canonical and non-canonical pathways: recent progress. *Growth Factors* 23, 111-116, doi:T416183G77101V73 [pii]

[93] Woodgett, J. R. Judging a protein by more than its name: GSK-3. *Sci STKE* 2001, RE12

[94] Huang, J. *et al.* More synergetic cooperation of Yamanaka factors in induced pluripo‐ tent stem cells than in embryonic stem cells. *Cell Res* 19, 1127-1138, doi:cr2009106 [pii]

[95] Polakis, P. The oncogenic activation of beta-catenin. *Curr Opin Genet Dev* 9, 15-21

and cell polarization. *Curr Drug Targets* 7, 1411-1419 (2006).

doi:S0092867404012462 [pii] 10.1016/j.cell.2004.12.021 (2005).

pnas.1005847107 (2010).

(2004).

nmeth744 (2005).

2005-0299 (2006).

10.1002/jcp.22214 (2010).

10.1038/nm979 nm979 [pii] (2004).

10.1080/08977190500125746 (2005).

10.1038/cr.2009.106 (2009).

(2001).

(1999).

1858-1868, doi:10.1002/stem.124 (2009).


[83] Moretto-Zita, M. *et al.* Phosphorylation stabilizes Nanog by promoting its interaction with Pin1. *Proc Natl Acad Sci U S A* 107, 13312-13317, doi:1005847107 [pii]10.1073/ pnas.1005847107 (2010).

[71] Chen, X. *et al.* Integration of external signaling pathways with the core transcription‐ al network in embryonic stem cells. *Cell* 133, 1106-1117, doi:S0092-8674(08)00617-X

[72] van den Berg, D. L. *et al.* An Oct4-centered protein interaction network in embryonic stem cells. *Cell Stem Cell* 6, 369-381, doi:S1934-5909(10)00091-3 [pii]10.1016/j.stem.

[73] Liang, J. *et al.* Nanog and Oct4 associate with unique transcriptional repression com‐ plexes in embryonic stem cells. *Nat Cell Biol* 10, 731-739, doi:ncb1736 [pii]10.1038/

[74] Boyer, L. A. *et al.* Core transcriptional regulatory circuitry in human embryonic stem cells. *Cell* 122, 947-956, doi:S0092-8674(05)00825-1 [pii]10.1016/j.cell.2005.08.020

[75] Johansson, H. & Simonsson, S. Core transcription factors, Oct4, Sox2 and Nanog, in‐ dividually form complexes with nucleophosmin (Npm1) to control embryonic stem (ES) cell fate determination. *Aging (Albany NY)* 2, 815-822, doi:100222 [pii] (2010).

[76] Gao, Z. *et al.* Determination of protein interactome of transcription factor sox2 in em‐ bryonic stem cells engineered for inducible expression of four reprogramming fac‐ tors. *J Biol Chem* 287, 11384-11397, doi:M111.320143 [pii]10.1074/jbc.M111.320143

[77] Ng, H. H. & Surani, M. A. The transcriptional and signalling networks of pluripoten‐ cy. *Nat Cell Biol* 13, 490-496, doi:ncb0511-490 [pii]10.1038/ncb0511-490 (2011).

[78] Loh, Y. H. *et al.* The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. *Nat Genet* 38, 431-440, doi:ng1760 [pii]10.1038/ng1760

[79] Boyer, L. A. *et al.* Polycomb complexes repress developmental regulators in murine embryonic stem cells. *Nature* 441, 349-353, doi:nature04733 [pii]10.1038/nature04733

[80] Lee, T. I. *et al.* Control of developmental regulators by Polycomb in human embryon‐ ic stem cells. *Cell* 125, 301-313, doi:S0092-8674(06)00384-9 [pii]10.1016/j.cell.

[81] Liao, B. & Jin, Y. Wwp2 mediates Oct4 ubiquitination and its own auto-ubiquitina‐ tion in a dosage-dependent manner. *Cell Res* 20, 332-344, doi:cr2009136

[82] Li, H. *et al.* Wwp2-mediated ubiquitination of the RNA polymerase II large subunit in mouse embryonic pluripotent stem cells. *Mol Cell Biol* 27, 5296-5305, doi:MCB.

[pii]10.1016/j.cell.2008.04.043 (2008).

2010.02.014 (2010).

ncb1736 (2008).

(2005).

172 Pluripotent Stem Cells

(2012).

(2006).

(2006).

2006.02.043 (2006).

[pii]10.1038/cr.2009.136 (2010).

01667-06 [pii]10.1128/MCB.01667-06 (2007).


[96] Takao, Y., Yokota, T. & Koide, H. Beta-catenin up-regulates Nanog expression through interaction with Oct-3/4 in embryonic stem cells. *Biochem Biophys Res Com‐ mun* 353, 699-705, doi:S0006-291X(06)02748-3 [pii] 10.1016/j.bbrc.2006.12.072 (2007).

[108] Wu, M. Y. & Hill, C. S. Tgf-beta superfamily signaling in embryonic development and homeostasis. *Dev Cell* 16, 329-343, doi:S1534-5807(09)00086-0 [pii] 10.1016/

Molecular Mechanisms Underlying Pluripotency

http://dx.doi.org/10.5772/55596

175

[109] Watabe, T. & Miyazono, K. Roles of TGF-beta family signaling in stem cell renewal and differentiation. *Cell Res* 19, 103-115, doi:cr2008323 [pii] 10.1038/cr.2008.323 (2009).

[110] Xu, R. H. *et al.* BMP4 initiates human embryonic stem cell differentiation to tropho‐ blast. *Nat Biotechnol* 20, 1261-1264, doi:10.1038/nbt761 nbt761 [pii] (2002).

[111] James, D., Levine, A. J., Besser, D. & Hemmati-Brivanlou, A. TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem

cells. *Development* 132, 1273-1282, doi:dev.01706 [pii] 10.1242/dev.01706 (2005).

[112] Vallier, L., Reynolds, D. & Pedersen, R. A. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. *Dev Biol* 275,

[113] Vallier, L., Alexander, M. & Pedersen, R. A. Activin/Nodal and FGF pathways coop‐ erate to maintain pluripotency of human embryonic stem cells. *J Cell Sci* 118,

[114] Ogawa, K. *et al.* Activin-Nodal signaling is involved in propagation of mouse embry‐ onic stem cells. *J Cell Sci* 120, 55-65, doi:120/1/55 [pii] 10.1242/jcs.03296 (2007).

[115] Wilson, P. A. & Hemmati-Brivanlou, A. Induction of epidermis and inhibition of

[116] Fei, T. *et al.* Genome-wide mapping of SMAD target genes reveals the role of BMP signaling in embryonic stem cell fate determination. *Genome Res* 20, 36-44, doi:gr.

[117] Yuasa, S. *et al.* Transient inhibition of BMP signaling by Noggin induces cardiomyo‐ cyte differentiation of mouse embryonic stem cells. *Nat Biotechnol* 23, 607-611,

[118] Lengerke, C. *et al.* BMP and Wnt specify hematopoietic fate by activation of the Cdx-Hox pathway. *Cell Stem Cell* 2, 72-82, doi:S1934-5909(07)00239-1 [pii] 10.1016/j.stem.

[119] Park, C. *et al.* A hierarchical order of factors in the generation of FLK1- and SCL-ex‐ pressing hematopoietic and endothelial progenitors from embryonic stem cells. *De‐*

[120] Sadlon, T. J., Lewis, I. D. & D'Andrea, R. J. BMP4: its role in development of the hem‐ atopoietic system and potential as a hematopoietic growth factor. *Stem Cells* 22,

*velopment* 131, 2749-2762, doi:10.1242/dev.01130 131/11/2749 [pii] (2004).

457-474, doi:22/4/457 [pii] 10.1634/stemcells.22-4-457 (2004).

neural fate by Bmp-4. *Nature* 376, 331-333, doi:10.1038/376331a0 (1995).

403-421, doi:S0012-1606(04)00587-1 [pii] 10.1016/j.ydbio.2004.08.031 (2004).

4495-4509, doi:118/19/4495 [pii] 10.1242/jcs.02553 (2005).

092114.109 [pii] 10.1101/gr.092114.109 (2010).

doi:nbt1093 [pii] 10.1038/nbt1093 (2005).

2007.10.022 (2008).

j.devcel.2009.02.012 (2009).


[108] Wu, M. Y. & Hill, C. S. Tgf-beta superfamily signaling in embryonic development and homeostasis. *Dev Cell* 16, 329-343, doi:S1534-5807(09)00086-0 [pii] 10.1016/ j.devcel.2009.02.012 (2009).

[96] Takao, Y., Yokota, T. & Koide, H. Beta-catenin up-regulates Nanog expression through interaction with Oct-3/4 in embryonic stem cells. *Biochem Biophys Res Com‐ mun* 353, 699-705, doi:S0006-291X(06)02748-3 [pii] 10.1016/j.bbrc.2006.12.072 (2007).

[97] Haegele, L. *et al.* Wnt signalling inhibits neural differentiation of embryonic stem cells by controlling bone morphogenetic protein expression. *Mol Cell Neurosci* 24,

[98] Aubert, J., Dunstan, H., Chambers, I. & Smith, A. Functional gene screening in em‐ bryonic stem cells implicates Wnt antagonism in neural differentiation. *Nat Biotechnol*

[99] Kielman, M. F. *et al.* Apc modulates embryonic stem-cell differentiation by control‐ ling the dosage of beta-catenin signaling. *Nat Genet* 32, 594-605, doi:10.1038/ng1045

[100] Doble, B. W., Patel, S., Wood, G. A., Kockeritz, L. K. & Woodgett, J. R. Functional re‐ dundancy of GSK-3alpha and GSK-3beta in Wnt/beta-catenin signaling shown by us‐ ing an allelic series of embryonic stem cell lines. *Dev Cell* 12, 957-971,

[101] Liu, Z., Scannell, D. R., Eisen, M. B. & Tjian, R. Control of embryonic stem cell lineage commitment by core promoter factor, TAF3. *Cell* 146, 720-731,

[102] Sun, Y., Li, H., Yang, H., Rao, M. S. & Zhan, M. Mechanisms controlling embryonic stem cell self-renewal and differentiation. *Crit Rev Eukaryot Gene Expr* 16, 211-231,

[103] Cole, M. F., Johnstone, S. E., Newman, J. J., Kagey, M. H. & Young, R. A. Tcf3 is an integral component of the core regulatory circuitry of embryonic stem cells. *Genes*

[104] Tam, W. L. *et al.* T-cell factor 3 regulates embryonic stem cell pluripotency and selfrenewal by the transcriptional control of multiple lineage pathways. *Stem Cells* 26,

[105] Pereira, L., Yi, F. & Merrill, B. J. Repression of Nanog gene transcription by Tcf3 lim‐ its embryonic stem cell self-renewal. *Mol Cell Biol* 26, 7479-7491, doi:MCB.00368-06

[106] Santibanez, J. F., Quintanilla, M. & Bernabeu, C. TGF-beta/TGF-beta receptor system and its role in physiological and pathological conditions. *Clin Sci (Lond)* 121, 233-251,

[107] Xu, R. H. *et al.* NANOG is a direct target of TGFbeta/activin-mediated SMAD signal‐ ing in human ESCs. *Cell Stem Cell* 3, 196-206, doi:S1934-5909(08)00335-4 [pii] 10.1016/

doi:S1534-5807(07)00148-7 [pii] 10.1016/j.devcel.2007.04.001 (2007).

doi:S0092-8674(11)00881-6 [pii] 10.1016/j.cell.2011.08.005 (2011).

*Dev* 22, 746-755, doi:22/6/746 [pii] 10.1101/gad.1642408 (2008).

2019-2031, doi:2007-1115 [pii] 10.1634/stemcells.2007-1115 (2008).

doi:5671614a1bbedf32,278d0bf81ca44a82 [pii] (2006).

[pii] 10.1128/MCB.00368-06 (2006).

j.stem.2008.07.001 (2008).

doi:CS20110086 [pii] 10.1042/CS20110086 (2011).

696-708, doi:S104474310300232X [pii] (2003).

ng1045 [pii] (2002).

174 Pluripotent Stem Cells

20, 1240-1245, doi:10.1038/nbt763 nbt763 [pii] (2002).


[121] Laflamme, M. A. *et al.* Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. *Nat Biotechnol* 25, 1015-1024, doi:nbt1327 [pii] 10.1038/nbt1327 (2007).

[134] Chazaud, C., Yamanaka, Y., Pawson, T. & Rossant, J. Early lineage segregation be‐ tween epiblast and primitive endoderm in mouse blastocysts through the Grb2- MAPK pathway. *Dev Cell* 10, 615-624, doi:S1534-5807(06)00125-0 [pii] 10.1016/

Molecular Mechanisms Underlying Pluripotency

http://dx.doi.org/10.5772/55596

177

[135] Buehr, M. & Smith, A. Genesis of embryonic stem cells. *Philos Trans R Soc Lond B Biol*

[136] Kunath, T. *et al.* FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. *Devel‐*

[137] Atkinson, S. & Armstrong, L. Epigenetics in embryonic stem cells: regulation of plu‐ ripotency and differentiation. *Cell Tissue Res* 331, 23-29, doi:10.1007/s00441-007-0536-x

[138] Marks, H. *et al.* The transcriptional and epigenomic foundations of ground state plu‐ ripotency. *Cell* 149, 590-604, doi:S0092-8674(12)00409-6 [pii] 10.1016/j.cell.2012.03.026

[139] Murchison, E. P., Partridge, J. F., Tam, O. H., Cheloufi, S. & Hannon, G. J. Characteri‐ zation of Dicer-deficient murine embryonic stem cells. *Proc Natl Acad Sci U S A* 102,

[140] Wang, Y., Medvid, R., Melton, C., Jaenisch, R. & Blelloch, R. DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal. *Nat Genet*

[141] Melton, C., Judson, R. L. & Blelloch, R. Opposing microRNA families regulate selfrenewal in mouse embryonic stem cells. *Nature* 463, 621-626, doi:nature08725 [pii]

[142] Xu, N., Papagiannakopoulos, T., Pan, G., Thomson, J. A. & Kosik, K. S. Micro‐ RNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. *Cell* 137, 647-658, doi:S0092-8674(09)00252-9 [pii] 10.1016/j.cell.

[143] Helland, A. *et al.* Deregulation of MYCN, LIN28B and LET7 in a molecular subtype of aggressive high-grade serous ovarian cancers. *PLoS One* 6, e18064, doi:10.1371/

[144] Judson, R. L., Babiarz, J. E., Venere, M. & Blelloch, R. Embryonic stem cell-specific microRNAs promote induced pluripotency. *Nat Biotechnol* 27, 459-461, doi:nbt.1535

[145] Thornton, J. E. & Gregory, R. I. How does Lin28 let-7 control development and dis‐ ease? *Trends Cell Biol*, doi:S0962-8924(12)00104-3 [pii] 10.1016/j.tcb.2012.06.001 (2012).

12135-12140, doi:0505479102 [pii] 10.1073/pnas.0505479102 (2005).

39, 380-385, doi:ng1969 [pii] 10.1038/ng1969 (2007).

10.1038/nature08725 (2010).

journal.pone.0018064 (2011).

[pii] 10.1038/nbt.1535 (2009).

2009.02.038 (2009).

*Sci* 358, 1397-1402; discussion 1402, doi:10.1098/rstb.2003.1327 (2003).

*opment* 134, 2895-2902, doi:134/16/2895 [pii] 10.1242/dev.02880 (2007).

j.devcel.2006.02.020 (2006).

(2008).

(2012).


[134] Chazaud, C., Yamanaka, Y., Pawson, T. & Rossant, J. Early lineage segregation be‐ tween epiblast and primitive endoderm in mouse blastocysts through the Grb2- MAPK pathway. *Dev Cell* 10, 615-624, doi:S1534-5807(06)00125-0 [pii] 10.1016/ j.devcel.2006.02.020 (2006).

[121] Laflamme, M. A. *et al.* Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. *Nat Biotechnol* 25,

[122] Behfar, A. *et al.* Stem cell differentiation requires a paracrine pathway in the heart.

[123] Zeineddine, D. *et al.* Oct-3/4 dose dependently regulates specification of embryonic stem cells toward a cardiac lineage and early heart development. *Dev Cell* 11,

[124] Narazaki, G. *et al.* Directed and systematic differentiation of cardiovascular cells from mouse induced pluripotent stem cells. *Circulation* 118, 498-506, doi:CIRCULA‐

[125] Niwa, H., Ogawa, K., Shimosato, D. & Adachi, K. A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells. *Nature* 460, 118-122, doi:na‐

[126] Li, Y. *et al.* Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4. *Blood* 105, 635-637, doi:10.1182/

[127] Cartwright, P. *et al.* LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. *Development* 132, 885-896, doi:dev.01670 [pii] 10.1242/

[128] Nichols, J., Chambers, I., Taga, T. & Smith, A. Physiological rationale for responsive‐ ness of mouse embryonic stem cells to gp130 cytokines. *Development* 128, 2333-2339

[129] Durbin, J. E., Hackenmiller, R., Simon, M. C. & Levy, D. E. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. *Cell* 84,

[130] Amit, M. *et al.* Clonally derived human embryonic stem cell lines maintain pluripo‐ tency and proliferative potential for prolonged periods of culture. *Dev Biol* 227,

[131] Xu, C. *et al.* Feeder-free growth of undifferentiated human embryonic stem cells. *Nat*

[132] Dvorak, P. *et al.* Expression and potential role of fibroblast growth factor 2 and its re‐ ceptors in human embryonic stem cells. *Stem Cells* 23, 1200-1211, doi:2004-0303 [pii]

[133] Li, J. *et al.* MEK/ERK signaling contributes to the maintenance of human embryonic stem cell self-renewal. *Differentiation* 75, 299-307, doi:S0301-4681(09)60124-9 [pii]

271-278, doi:10.1006/dbio.2000.9912 S0012-1606(00)99912-3 [pii] (2000).

*Biotechnol* 19, 971-974, doi:10.1038/nbt1001-971 nbt1001-971 [pii] (2001).

TIONAHA.108.769562 [pii] 10.1161/CIRCULATIONAHA.108.769562 (2008).

535-546, doi:S1534-5807(06)00314-5 [pii] 10.1016/j.devcel.2006.07.013 (2006).

*FASEB J* 16, 1558-1566, doi:10.1096/fj.02-0072com 16/12/1558 [pii] (2002).

1015-1024, doi:nbt1327 [pii] 10.1038/nbt1327 (2007).

ture08113 [pii] 10.1038/nature08113 (2009).

blood-2004-07-2681 2004-07-2681 [pii] (2005).

443-450, doi:S0092-8674(00)81289-1 [pii] (1996).

10.1634/stemcells.2004-0303 (2005).

10.1111/j.1432-0436.2006.00143.x (2007).

dev.01670 (2005).

(2001).

176 Pluripotent Stem Cells


[146] Subramanyam, D. *et al.* Multiple targets of miR-302 and miR-372 promote reprog‐ ramming of human fibroblasts to induced pluripotent stem cells. *Nat Biotechnol* 29, 443-448, doi:nbt.1862 [pii] 10.1038/nbt.1862 (2011).

**Chapter 9**

**Stem Cells and Epigenetic Reprogramming**

In general stem cells have to fulfill two characteristics: self-renewal and the ability to differ‐ entiate into different cell/tissue types. Depending on their limitations in differentiation (pluripotent vs. multipotent) stem cells can be divided in embryonic or adult stem cells, depending on their limitations in differentiation [1]. This chapter will focus only on embryonic stem cells (ESCs) and their cognate artificial derivatives known as induced pluripotent stem cells (iPSCs). Embryonic stem cells, have been the center of much attention because of their pluripotency or ability to differentiate into any cell type in the body [2,3]. Induced pluripotent stem cells (iPSCs) are reprogrammed into the pluripotent state by the introduction of exoge‐ nous factors. These factors change the potency state of terminally differentiated somatic cells to by interacting with cellular chromatin and protein/RNA networks with the somatic cell. Following reprogramming, the newly formed stem cell resembles the ESC [4]. The recent development of these artificial or "man-made" cells has delivered two key potential upsides: (a) the ability to avoid the ethical issues associated with embryo-derived cells, and (b) the ability to generate autologous (i.e. patient derived) cells for regenerative medicine, tissue engineering, and disease modeling purposes [4]. Compared to ESCs, which are derived from the limited resource of assisted fertility by-products, iPSCs can potentially provide an

One of the applications of iPSCs is the ability to model diseases for drug screening, toxicology testing, and cell therapy among others [4]. For basic biomedical research, cell culture has been a key element for every approach. However one drawback when studying human cells is that they have limited life span in culture. Many cell lines have not been faithfully adapted for growth *in vitro*. Hence the lack of accessible models of normal and pathologic tissue has left many important questions in human pathogenesis inaccessible [5]. In contrast due to their selfrenewal and pluripotency patient derived iPSCs can be extremely useful for patient research and diagnostic purposes. Every iPSC that is compromised in disease can be restructured into

> © 2013 Cota et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Cota et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

Perla Cota, Mehdi Shafa and Derrick E. Rancourt

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55983

unlimited source of pluripotent cells.

**1. Introduction**


## **Stem Cells and Epigenetic Reprogramming**

Perla Cota, Mehdi Shafa and Derrick E. Rancourt

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55983

## **1. Introduction**

[146] Subramanyam, D. *et al.* Multiple targets of miR-302 and miR-372 promote reprog‐ ramming of human fibroblasts to induced pluripotent stem cells. *Nat Biotechnol* 29,

[147] Loewer, S. *et al.* Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells. *Nat Genet* 42, 1113-1117, doi:ng.710 [pii]

[148] Sheik Mohamed, J., Gaughwin, P. M., Lim, B., Robson, P. & Lipovich, L. Conserved long noncoding RNAs transcriptionally regulated by Oct4 and Nanog modulate plu‐ ripotency in mouse embryonic stem cells. *RNA* 16, 324-337, doi:rna.1441510 [pii]

[149] Knezetic, J. A. & Luse, D. S. The presence of nucleosomes on a DNA template pre‐ vents initiation by RNA polymerase II in vitro. *Cell* 45, 95-104, doi:

[150] Li, B., Carey, M. & Workman, J. L. The role of chromatin during transcription. *Cell* 128, 707-719, doi:S0092-8674(07)00109-2 [pii] 10.1016/j.cell.2007.01.015 (2007).

[151] Kagey, M. H. *et al.* Mediator and cohesin connect gene expression and chromatin ar‐ chitecture. *Nature* 467, 430-435, doi:nature09380 [pii] 10.1038/nature09380 (2010).

[152] Leeb, M. *et al.* Polycomb complexes act redundantly to repress genomic repeats and genes. *Genes Dev* 24, 265-276, doi:24/3/265 [pii] 10.1101/gad.544410 (2010).

[153] Bilodeau, S., Kagey, M. H., Frampton, G. M., Rahl, P. B. & Young, R. A. SetDB1 con‐ tributes to repression of genes encoding developmental regulators and maintenance of ES cell state. *Genes Dev* 23, 2484-2489, doi:23/21/2484 [pii] 10.1101/gad.1837309

[154] Fazzio, T. G., Huff, J. T. & Panning, B. An RNAi screen of chromatin proteins identi‐ fies Tip60-p400 as a regulator of embryonic stem cell identity. *Cell* 134, 162-174,

[155] Stielow, B. *et al.* Identification of SUMO-dependent chromatin-associated transcrip‐ tional repression components by a genome-wide RNAi screen. *Mol Cell* 29, 742-754,

[156] Yuan, P. *et al.* Eset partners with Oct4 to restrict extraembryonic trophoblast lineage potential in embryonic stem cells. *Genes Dev* 23, 2507-2520, doi:23/21/2507 [pii]

doi:S0092-8674(08)00692-2 [pii] 10.1016/j.cell.2008.05.031 (2008).

doi:S1097-2765(08)00164-0 [pii] 10.1016/j.molcel.2007.12.032 (2008).

443-448, doi:nbt.1862 [pii] 10.1038/nbt.1862 (2011).

10.1038/ng.710 (2010).

178 Pluripotent Stem Cells

10.1261/rna.1441510 (2010).

10.1101/gad.1831909 (2009).

(2009).

0092-8674(86)90541-6 [pii] (1986).

In general stem cells have to fulfill two characteristics: self-renewal and the ability to differ‐ entiate into different cell/tissue types. Depending on their limitations in differentiation (pluripotent vs. multipotent) stem cells can be divided in embryonic or adult stem cells, depending on their limitations in differentiation [1]. This chapter will focus only on embryonic stem cells (ESCs) and their cognate artificial derivatives known as induced pluripotent stem cells (iPSCs). Embryonic stem cells, have been the center of much attention because of their pluripotency or ability to differentiate into any cell type in the body [2,3]. Induced pluripotent stem cells (iPSCs) are reprogrammed into the pluripotent state by the introduction of exoge‐ nous factors. These factors change the potency state of terminally differentiated somatic cells to by interacting with cellular chromatin and protein/RNA networks with the somatic cell. Following reprogramming, the newly formed stem cell resembles the ESC [4]. The recent development of these artificial or "man-made" cells has delivered two key potential upsides: (a) the ability to avoid the ethical issues associated with embryo-derived cells, and (b) the ability to generate autologous (i.e. patient derived) cells for regenerative medicine, tissue engineering, and disease modeling purposes [4]. Compared to ESCs, which are derived from the limited resource of assisted fertility by-products, iPSCs can potentially provide an unlimited source of pluripotent cells.

One of the applications of iPSCs is the ability to model diseases for drug screening, toxicology testing, and cell therapy among others [4]. For basic biomedical research, cell culture has been a key element for every approach. However one drawback when studying human cells is that they have limited life span in culture. Many cell lines have not been faithfully adapted for growth *in vitro*. Hence the lack of accessible models of normal and pathologic tissue has left many important questions in human pathogenesis inaccessible [5]. In contrast due to their selfrenewal and pluripotency patient derived iPSCs can be extremely useful for patient research and diagnostic purposes. Every iPSC that is compromised in disease can be restructured into

© 2013 Cota et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Cota et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

tissue in culture giving researchers an unlimited source of cells/tissue for the study of the disease [6, 4].

When applied to cell therapy, autologous iPSCs are differentiated into a chosen cell type and then transplanted to the damaged tissue with the advantage that immune rejection can be avoided. Furthermore iPSCs be used as a conduit for somatic gene therapy. For example a disease-causing mutation can be repaired in patient iPSCs by homologous DNA recombination prior to transplantation [4]. A study on engineered mice that suffer from human sickle cell anemia showed that when applying hematopoietic progenitor cells produced from autologous iPSCs, animals were rescued from systemic hematological symptoms. In this case, for the production of the hematopoietic progenitor cells, a biopsy of adult fibroblast was taken from the afflicted mouse and reprogrammed into iPSCs. Derived iPSCs were repaired by homolo‐ gous recombination. These cells were then differentiated to hematopoietic progenitor cells *in vitro* and transplanted back into the affected mouse [7].

Despite the success in animals, there are still some drawbacks with using iPSCs for human benefit. Since the derivation of the iPSCs commonly involves integrating viral vectors for introducing reprogramming factor, this represents a risk to the human health. Moreover certain epigenetic abnormalities in the iPSCs including the epigenetic memory of their donor cells could lead to mutation in prolonged culture [4]. Such epigenetic differences are one reason why research has begun to focus on the epigenetics of cellular reprograming. Although iPSCs are the functional equivalent of ESCs, epigenetic differences have been noted, including differences in gene expression, DNA methylation, histone marks and telomere/telomerase status [8, 9]. Moreover, researchers have also recognized a role for chromatin remodeling during reprogramming and have recently applied small molecules to circumvent epigenetic blocks and enhance reprogramming efficiency [10,11].

**2. Induced pluripotent stem cells**

upon blastocyst implantation and pass through germline [13].

**Figure 1.** Two state process for reprogramming somatic cells (Adapted from 6)

It was recently discovered that a terminally differentiated cell could be reprogrammed into an ESC-like cell using four transcription factors. Having pluripotent characteristics, these iPSCs are capable of becoming one of more than 200 cell types [12]. In order to be consider ESCs, they must fulfill certain criteria: (1) to express pluripotency factors such as Oct4, Sox2, Nanog, and SSEA1, however this criteria only apply for mouse ESCs, since in human ESCs SSEA3 and SSEA4 are expressed in stead however this criteria only apply for mouse ESCs, since in human ESCs SSEA3 and SSEA4 are expressed in stead (2) in female cells there must be the reactivation of the inactive X chromosome, (3) they should be able to differentiate into the three germ layers (ectoderm, endoderm and mesoderm) and in the case of mouse, be able to generate chimeras

Stem Cells and Epigenetic Reprogramming http://dx.doi.org/10.5772/55983 181

Takahashi and Yamanaka (2006) were the first that found a way to circumvent two of the most important drawbacks when using ESCs related to immune rejection and ethical. In their study they first hypothesized that the factors that play a role in maintaining ESC pluripotency could potentially turn somatic cells back into a pluripotent state. Starting with 24 candidate genes known to be involved in pluripotency and a herculean combinatorial effort they reduced this original 24 down to four factors: Oct3/4, Klf4, Sox2, and c-Myc able to reprogram mouse fibroblasts into an ESC-like state. Yamanaka and colleagues also reported the same results one

After the four factor derivation of the iPSCs, much interest was focused on the process of somatic cell reprogramming. Although still not well understood, Scheper and Copray (2009) proposed one approach that divided reprogramming in two broad stages. First Oct4 and Sox2 repressed genes associated with the host cell lineage and reset the epigenome of the cell towards a permissive chromatin mode putting the cell in an embryonic-like state. The second stage allowed the reprogramming factors to reactivate the endogenous autoregulatory loop

year later for the human (2007) when using this same combination of factors [14].

that triggers the pluripotency transcriptional network [6] (Figure 1).

Given that there is a huge interest in using iPSCs, mainly in regenerative medicine; researchers want to understand the exact mechanism of reprogramming, as any error in this process could cause tumor formation once applied to patients. Understanding the fundamentals of this reprogramming process by comparing it to the pluripotent state of ESCs will give us many tools to be able to manipulate the reprogramming process within a controlled environment.

Since iPSC are being compared at all times to ESCs, a basic concept that must be kept in mind is that ESCs rely on a complex network of interacting pluripotency transcription factors, and different "epigenetic landscapes" in order to maintain their "open" chromatin to regulate either self-renewal or differentiation [1]. Moreover, when a somatic cell is subjected to reprogramming, it suffers large-scale epigenetic alterations, carried on as if they were different multiple layers of epigenetic events that control the expression and accommodation of important pluripotency transcription factors [1].

In this chapter, a deeper explanation about iPSCs together with the basic concepts of epige‐ netics and the different levels of regulation will be provided. Insight into some of the recently discovered epigenetic events of cellular reprogramming will be discussed.

**Figure 1.** Two state process for reprogramming somatic cells (Adapted from 6)

## **2. Induced pluripotent stem cells**

tissue in culture giving researchers an unlimited source of cells/tissue for the study of the

When applied to cell therapy, autologous iPSCs are differentiated into a chosen cell type and then transplanted to the damaged tissue with the advantage that immune rejection can be avoided. Furthermore iPSCs be used as a conduit for somatic gene therapy. For example a disease-causing mutation can be repaired in patient iPSCs by homologous DNA recombination prior to transplantation [4]. A study on engineered mice that suffer from human sickle cell anemia showed that when applying hematopoietic progenitor cells produced from autologous iPSCs, animals were rescued from systemic hematological symptoms. In this case, for the production of the hematopoietic progenitor cells, a biopsy of adult fibroblast was taken from the afflicted mouse and reprogrammed into iPSCs. Derived iPSCs were repaired by homolo‐ gous recombination. These cells were then differentiated to hematopoietic progenitor cells *in*

Despite the success in animals, there are still some drawbacks with using iPSCs for human benefit. Since the derivation of the iPSCs commonly involves integrating viral vectors for introducing reprogramming factor, this represents a risk to the human health. Moreover certain epigenetic abnormalities in the iPSCs including the epigenetic memory of their donor cells could lead to mutation in prolonged culture [4]. Such epigenetic differences are one reason why research has begun to focus on the epigenetics of cellular reprograming. Although iPSCs are the functional equivalent of ESCs, epigenetic differences have been noted, including differences in gene expression, DNA methylation, histone marks and telomere/telomerase status [8, 9]. Moreover, researchers have also recognized a role for chromatin remodeling during reprogramming and have recently applied small molecules to circumvent epigenetic

Given that there is a huge interest in using iPSCs, mainly in regenerative medicine; researchers want to understand the exact mechanism of reprogramming, as any error in this process could cause tumor formation once applied to patients. Understanding the fundamentals of this reprogramming process by comparing it to the pluripotent state of ESCs will give us many tools to be able to manipulate the reprogramming process within a controlled environment.

Since iPSC are being compared at all times to ESCs, a basic concept that must be kept in mind is that ESCs rely on a complex network of interacting pluripotency transcription factors, and different "epigenetic landscapes" in order to maintain their "open" chromatin to regulate either self-renewal or differentiation [1]. Moreover, when a somatic cell is subjected to reprogramming, it suffers large-scale epigenetic alterations, carried on as if they were different multiple layers of epigenetic events that control the expression and accommodation of

In this chapter, a deeper explanation about iPSCs together with the basic concepts of epige‐ netics and the different levels of regulation will be provided. Insight into some of the recently

discovered epigenetic events of cellular reprogramming will be discussed.

*vitro* and transplanted back into the affected mouse [7].

blocks and enhance reprogramming efficiency [10,11].

important pluripotency transcription factors [1].

disease [6, 4].

180 Pluripotent Stem Cells

It was recently discovered that a terminally differentiated cell could be reprogrammed into an ESC-like cell using four transcription factors. Having pluripotent characteristics, these iPSCs are capable of becoming one of more than 200 cell types [12]. In order to be consider ESCs, they must fulfill certain criteria: (1) to express pluripotency factors such as Oct4, Sox2, Nanog, and SSEA1, however this criteria only apply for mouse ESCs, since in human ESCs SSEA3 and SSEA4 are expressed in stead however this criteria only apply for mouse ESCs, since in human ESCs SSEA3 and SSEA4 are expressed in stead (2) in female cells there must be the reactivation of the inactive X chromosome, (3) they should be able to differentiate into the three germ layers (ectoderm, endoderm and mesoderm) and in the case of mouse, be able to generate chimeras upon blastocyst implantation and pass through germline [13].

Takahashi and Yamanaka (2006) were the first that found a way to circumvent two of the most important drawbacks when using ESCs related to immune rejection and ethical. In their study they first hypothesized that the factors that play a role in maintaining ESC pluripotency could potentially turn somatic cells back into a pluripotent state. Starting with 24 candidate genes known to be involved in pluripotency and a herculean combinatorial effort they reduced this original 24 down to four factors: Oct3/4, Klf4, Sox2, and c-Myc able to reprogram mouse fibroblasts into an ESC-like state. Yamanaka and colleagues also reported the same results one year later for the human (2007) when using this same combination of factors [14].

After the four factor derivation of the iPSCs, much interest was focused on the process of somatic cell reprogramming. Although still not well understood, Scheper and Copray (2009) proposed one approach that divided reprogramming in two broad stages. First Oct4 and Sox2 repressed genes associated with the host cell lineage and reset the epigenome of the cell towards a permissive chromatin mode putting the cell in an embryonic-like state. The second stage allowed the reprogramming factors to reactivate the endogenous autoregulatory loop that triggers the pluripotency transcriptional network [6] (Figure 1).

After the first proposed cocktail to reprogram differentiated cells into iPSCs, many research‐ ers started to ask the question of how these factors were interacting in orderto modify existing epigenetic marks and return to a pluripotent state. To date it has been reported that differentiated cells have been successfully reprogrammed by substituting some of the factors such as Klf4 or c-Myc with other transcription factors such as Nanog or Lin28 or molecules (valproic acid or Wnt ligand). It tells us that there are different pathways involved in this process and that epigenetic enzymes are being activated in every case [9] all to one end‐ point of pluripotency.

Waddington referred to epigenetics for the first time as genetic interactions that can affect the phenotype. Later, he proposed a model based on how cells followed a developmental differentiation path much like traveling down a series of canals that start from a fertilized totipotent embryo and ending up as a specific lineage committed cell [15]. In this model, cells committed to a specific lineage cannot be recommitted to another lineage or canal. Howev‐ er, with the recent milestone of iPSC generation, Yamanaka suggested that cells could be pushed back up the canal towards the pluripotent state. During reprogramming, cells can experience other events. They can be stopped by some epigenetic bump and remain incompletely reprogrammed. In this situation cells return to their specific lineage or transition to another lineage. Finally, instead of moving they can undergo apoptosis or cellular senescence. This model proposed by Yamanaka (2009) is known as the stochastic model of iPSC generation [16] (Figure 2).

Since the development of iPSCs, many researchers have focused their attention on the epigenetics changes that iPSCs acquire, together with the chromatin dynamics that occurs during cellular reprogramming. It has been already proposed that one way to ease cell destiny is by having less lineage epigenetic patterns [17]. The most used protocol for the production of iPSCs is the one that involves the application of the four transcription factors previously described by Yamanaka (2006) [15]. Thus initial studies have focused on how these four factors worked together to initiate the reprogramming cascade. In this regard it has been proposed that Oct4 and Sox2 are totally indispensable for reprogramming while Klf4 and c-Myc enhance the efficiency and alter the structure of the chromatin to enable Oct4 and Sox2 to target more genes that are important for pluripotency [15].

**3. Epigenetics**

complete reprogramming. (Adapted from 16)

inactive regions such as H3K9me and H3k27me [23].

Epigenetics is defined in general as heritable changes in gene expression that do not affect DNA-sequence [19, 20]. In the nucleus DNA is wrapped into a protein complex known as chromatin. This protein complex, known as the nucleosome, is formed by proteins called histones (H2A, H2B, H3, H4) (Figure 4) [21] into a structure resembling beads on a string. Histone H1 in turn play a role in assembling higher order chromatin structure by interacting with the "inter-bead" regions of chromation. Via changes in histone post-translational modifications (acetylation, methylation, ubiquitination, and phosphorylation), chromatin becomes very dynamic, controlling the expression or repression of specific genes in specific cells, as well as during the cell cycle or in response to environmental cues. These changes in histone via reversible post-translational marks (as well as reversible marks to primary DNA sequence) are considered to be epigenetic modifications. Additionally, changes in the nucle‐ osome moving through DNA can be facilitated by chromatin remodeling enzymes [22, 21]. Histone modifications associated with active transcription, such as acetylation of histones 3 and 4 or di-/trimethylation of H3K4, are usually referred as euchromatin modifications. On the other hand there are the heterochromatin modifications which are characteristic to be on

**Figure 2.** Stochastic model for iPSC generation. All of the cells initiate reprogramming, but only a few can achieve

Stem Cells and Epigenetic Reprogramming http://dx.doi.org/10.5772/55983 183

Finally, it is crucial to find the best method of reprogramming in order to approximate ESCs as much as possible. For this purpose, there are several variables that have to be taken into consideration in order to have reproducible and efficient reprogramming. First is the selection of reprogramming factors where the combination (and efficiency) can vary depending on the cell type. Second is the type of method used for factor delivery, be it viral vectors, RNA, protein, or small molecules, among others. Third is the selection of cell type, since the efficiency and kinetics reprogramming changes between cell types. Understanding how reprogramming factors coordinate the cascade to orchestrate reprogramming means it is important to know the right timing and stoichiometry for optimal reprogramming. Culture environment likewise is very important. Finally the selection of a method to identify and characterize iPSCs is very critical (Figure 3) [18].

**Figure 2.** Stochastic model for iPSC generation. All of the cells initiate reprogramming, but only a few can achieve complete reprogramming. (Adapted from 16)

## **3. Epigenetics**

After the first proposed cocktail to reprogram differentiated cells into iPSCs, many research‐ ers started to ask the question of how these factors were interacting in orderto modify existing epigenetic marks and return to a pluripotent state. To date it has been reported that differentiated cells have been successfully reprogrammed by substituting some of the factors such as Klf4 or c-Myc with other transcription factors such as Nanog or Lin28 or molecules (valproic acid or Wnt ligand). It tells us that there are different pathways involved in this process and that epigenetic enzymes are being activated in every case [9] all to one end‐

Waddington referred to epigenetics for the first time as genetic interactions that can affect the phenotype. Later, he proposed a model based on how cells followed a developmental differentiation path much like traveling down a series of canals that start from a fertilized totipotent embryo and ending up as a specific lineage committed cell [15]. In this model, cells committed to a specific lineage cannot be recommitted to another lineage or canal. Howev‐ er, with the recent milestone of iPSC generation, Yamanaka suggested that cells could be pushed back up the canal towards the pluripotent state. During reprogramming, cells can experience other events. They can be stopped by some epigenetic bump and remain incompletely reprogrammed. In this situation cells return to their specific lineage or transition to another lineage. Finally, instead of moving they can undergo apoptosis or cellular senescence. This model proposed by Yamanaka (2009) is known as the stochastic model of

Since the development of iPSCs, many researchers have focused their attention on the epigenetics changes that iPSCs acquire, together with the chromatin dynamics that occurs during cellular reprogramming. It has been already proposed that one way to ease cell destiny is by having less lineage epigenetic patterns [17]. The most used protocol for the production of iPSCs is the one that involves the application of the four transcription factors previously described by Yamanaka (2006) [15]. Thus initial studies have focused on how these four factors worked together to initiate the reprogramming cascade. In this regard it has been proposed that Oct4 and Sox2 are totally indispensable for reprogramming while Klf4 and c-Myc enhance the efficiency and alter the structure of the chromatin to enable Oct4 and Sox2 to target more

Finally, it is crucial to find the best method of reprogramming in order to approximate ESCs as much as possible. For this purpose, there are several variables that have to be taken into consideration in order to have reproducible and efficient reprogramming. First is the selection of reprogramming factors where the combination (and efficiency) can vary depending on the cell type. Second is the type of method used for factor delivery, be it viral vectors, RNA, protein, or small molecules, among others. Third is the selection of cell type, since the efficiency and kinetics reprogramming changes between cell types. Understanding how reprogramming factors coordinate the cascade to orchestrate reprogramming means it is important to know the right timing and stoichiometry for optimal reprogramming. Culture environment likewise is very important. Finally the selection of a method to identify and characterize iPSCs is very

point of pluripotency.

182 Pluripotent Stem Cells

iPSC generation [16] (Figure 2).

critical (Figure 3) [18].

genes that are important for pluripotency [15].

Epigenetics is defined in general as heritable changes in gene expression that do not affect DNA-sequence [19, 20]. In the nucleus DNA is wrapped into a protein complex known as chromatin. This protein complex, known as the nucleosome, is formed by proteins called histones (H2A, H2B, H3, H4) (Figure 4) [21] into a structure resembling beads on a string. Histone H1 in turn play a role in assembling higher order chromatin structure by interacting with the "inter-bead" regions of chromation. Via changes in histone post-translational modifications (acetylation, methylation, ubiquitination, and phosphorylation), chromatin becomes very dynamic, controlling the expression or repression of specific genes in specific cells, as well as during the cell cycle or in response to environmental cues. These changes in histone via reversible post-translational marks (as well as reversible marks to primary DNA sequence) are considered to be epigenetic modifications. Additionally, changes in the nucle‐ osome moving through DNA can be facilitated by chromatin remodeling enzymes [22, 21]. Histone modifications associated with active transcription, such as acetylation of histones 3 and 4 or di-/trimethylation of H3K4, are usually referred as euchromatin modifications. On the other hand there are the heterochromatin modifications which are characteristic to be on inactive regions such as H3K9me and H3k27me [23].

of mSWI/SNF in mouse ESCs and observed that the majority of the binding does not occur in transcriptional start sites, but at distal enhancers and silencer sites. In another study of Ho and collaborators, they showed that mSWI/SNF complex binds to promoters/enhancers of pluri‐ potency transcription factors such as Oct4 and Sox2, in accordance with studies that have shown enhanced reprogramming when they add mSWI/SNF together with reprogramming

Stem Cells and Epigenetic Reprogramming http://dx.doi.org/10.5772/55983 185

**Figure 4.** Nucleosome with histone posttranslational modifications (Adapted from 1)

that activate Oct4, Sox2 and Nanog [31].

**•** Histone Modification

The chromodomain helicase DNA binding protein 1 (CHD1) is well known for its remodeling activity in the maintenance of stemness. It also has main function in recognizing a substrate of transcription regulatory histone acetylation complex SAGA. CHD1 has been suggested to act as a molecular adaptor, which bring several epigenetic complexes together [29]. In ESCs, this adaptor has been suggested to be indispensable for the maintenance of pluripotent chromatin state where it is highly expressed when compared to differentiated cells. After knockdown of the CHD1 with RNAi, the pattern of diffuse ESCs heterochromatin disappears showing a higher amount of heterochromatin. In turn, CHD1 knockdown fibroblasts reprogrammed less efficiently [30]. The nature of CHD1 in pluripotent cells specifies that it can prevent the formation of heterochromatin foci [30]. CHD1 has also been reported to be one of the genes

Histones are highly positively charged proteins that bind to DNA and have a major role in DNA packaging and gene expression. As mentioned earlier, they are subjected to a variety of post-translational modifications that alter the interaction of the histone protein with the bound DNA. These modifications include acetylation and methylation of the N-terminus tails as well as phosphorylation, poly ADP-ribosylation, ubiquitination and sumoylation. Differential modification of the core histones yields different chromatin structure. These patterns of modification form a kind of "histone code" that will ultimately govern gene expression [1].

factors [28].

**Figure 3.** Overview of the iPSC Derivation Process (Adapted from 18)

## **4. Chromatin phases (epigenetic regulation)**

**•** Chromatin Remodelling

Chromatin remodeling refers to the architectural change of chromatin by the movement of the nucleosomes along the DNA, giving rise to change the condensation of the chromatin. Protein complexes use ATP-hydrolysis to alter the histone DNA interaction, suggesting that there is a transient separation of the DNA from histone complexes, moving nucleosomes to a different position in the DNA or forming a DNA loop. These movements adjust the accessibility of DNA to transcription factors [23]. The many chromatin remodeling com‐ plexes are divided into families depending on their composition and biochemical activity. In this chapter, two of the most well studied ATP chromatin remodeling enzymes are dis‐ cussed: SWI/SNF and CHD1 [24].

The basic assembling of the SWI/SNF chromatin remodeling enzyme in mammals (also known as BAF) is with the genes that code for the 9-12 subunits of the mammal SWI/SNF (mSWI/SNF) in combination with one catalytic ATPase subunit called brahma homolog (BRM or SMAR‐ CA2, BRM/SWI2-related gene 1[25]. mSWI/SNF uses the energy from ATPase hydrolysis to move along in the DNA. One way this works to move along is to bind the DNA into an internal site of the nucleosome, then pull it in order to weaken the nucleosome (Figure 5) [26].

One of the characteristics of mSWI/SNF chromatin remodelers is the subunit change during the transition from a pluripotent to a multipotent state and then from a multipotent state to differentiation. Ho and colleagues (2009) [27] did a whole genome study to observe the binding of mSWI/SNF in mouse ESCs and observed that the majority of the binding does not occur in transcriptional start sites, but at distal enhancers and silencer sites. In another study of Ho and collaborators, they showed that mSWI/SNF complex binds to promoters/enhancers of pluri‐ potency transcription factors such as Oct4 and Sox2, in accordance with studies that have shown enhanced reprogramming when they add mSWI/SNF together with reprogramming factors [28].

**Figure 4.** Nucleosome with histone posttranslational modifications (Adapted from 1)

The chromodomain helicase DNA binding protein 1 (CHD1) is well known for its remodeling activity in the maintenance of stemness. It also has main function in recognizing a substrate of transcription regulatory histone acetylation complex SAGA. CHD1 has been suggested to act as a molecular adaptor, which bring several epigenetic complexes together [29]. In ESCs, this adaptor has been suggested to be indispensable for the maintenance of pluripotent chromatin state where it is highly expressed when compared to differentiated cells. After knockdown of the CHD1 with RNAi, the pattern of diffuse ESCs heterochromatin disappears showing a higher amount of heterochromatin. In turn, CHD1 knockdown fibroblasts reprogrammed less efficiently [30]. The nature of CHD1 in pluripotent cells specifies that it can prevent the formation of heterochromatin foci [30]. CHD1 has also been reported to be one of the genes that activate Oct4, Sox2 and Nanog [31].

**•** Histone Modification

**Figure 3.** Overview of the iPSC Derivation Process (Adapted from 18)

**4. Chromatin phases (epigenetic regulation)**

Chromatin remodeling refers to the architectural change of chromatin by the movement of the nucleosomes along the DNA, giving rise to change the condensation of the chromatin. Protein complexes use ATP-hydrolysis to alter the histone DNA interaction, suggesting that there is a transient separation of the DNA from histone complexes, moving nucleosomes to a different position in the DNA or forming a DNA loop. These movements adjust the accessibility of DNA to transcription factors [23]. The many chromatin remodeling com‐ plexes are divided into families depending on their composition and biochemical activity. In this chapter, two of the most well studied ATP chromatin remodeling enzymes are dis‐

The basic assembling of the SWI/SNF chromatin remodeling enzyme in mammals (also known as BAF) is with the genes that code for the 9-12 subunits of the mammal SWI/SNF (mSWI/SNF) in combination with one catalytic ATPase subunit called brahma homolog (BRM or SMAR‐ CA2, BRM/SWI2-related gene 1[25]. mSWI/SNF uses the energy from ATPase hydrolysis to move along in the DNA. One way this works to move along is to bind the DNA into an internal

One of the characteristics of mSWI/SNF chromatin remodelers is the subunit change during the transition from a pluripotent to a multipotent state and then from a multipotent state to differentiation. Ho and colleagues (2009) [27] did a whole genome study to observe the binding

site of the nucleosome, then pull it in order to weaken the nucleosome (Figure 5) [26].

**•** Chromatin Remodelling

184 Pluripotent Stem Cells

cussed: SWI/SNF and CHD1 [24].

Histones are highly positively charged proteins that bind to DNA and have a major role in DNA packaging and gene expression. As mentioned earlier, they are subjected to a variety of post-translational modifications that alter the interaction of the histone protein with the bound DNA. These modifications include acetylation and methylation of the N-terminus tails as well as phosphorylation, poly ADP-ribosylation, ubiquitination and sumoylation. Differential modification of the core histones yields different chromatin structure. These patterns of modification form a kind of "histone code" that will ultimately govern gene expression [1].

**Figure 5.** Chromatin Remodeling Enzyme SWI/SNFN (Adapted from 1)

Histone acetylation is the addition of acetyl moieties onto each of the histones of the nucleo‐ some and is regulated by the activity of histone acetyltransferases (HAT) and histone deace‐ tylases (HDAC). HATs and HDACs operate as coactivators and corepressors and together they dynamically change the activation and repression of genes in both a site specific as well as global manner. There are four families of HATs; Gcn5-related N-acetyltransferase (GNAT), MYST and p300/CBP. These HATs share highly similar motifs including an acetyl-CoA binding domain with the Arg/Gln-X-XGly-X-Gly/Ala sequence [32]. HAT activity and specificity are highly dependent on the complexes they form with other HATs and transcriptional coactivators. Lysine, found at the amino terminus of the histone, is the primary targeted site of acetylation. At physiological pH, lysine is positively charged and contributes to the overall positive charge of the histone. However, the amount of lysine acetylation is directly correlated with the accessibility of the amino terminus or "histone tail" [32]. More accessibility means greater degree of acetylation. Upon acetylation, the residue is neutralized, reducing the positive charge of the histone, decreasing the interaction with the negatively charged DNA and directly influencing chromatin structure. HDACs are broken down into two families; classical HDACs and NAD+ HDACs. Like their HAT counterparts, HDACs share a conserved active site [33] and also require the need to complex with co-repressors in order to function properly. Once bound, active HDACs serve to remove the acetyl moiety from the histone tail through a charge-relay system of residues found within the active site [33, 34]. Once removed, the histones bind tighter to the DNA as well as enabling tighter packing of adjacent histones leading to more transcriptional repression (Figure 6) [35].

compared to arginine HMT. Arginine N-methyltransferases (PRMT) are less defined in terms of the targeted sites of methylation, with multiple target residues in histone H3 and H4 [37].

Stem Cells and Epigenetic Reprogramming http://dx.doi.org/10.5772/55983 187

The process of methylation does not alter the charge properties of the targeted residue, unlike those of acetylation. Instead, the addition of methyl groups serves as a recognition site for regulatory proteins to bind and elicit additional modification. This allows for a great deal of complexity depending on the target region of the MMT and the resultant recognition effector protein. In essence, it allows additional information to be encoded in the histones beyond just stearic and charge hindrance of acetylation and phosphorylation [38]. Thus, this places a great deal of importance on the proteins that interact with the methylated residue. There are a variety of motifs that are able to recognize both single and double methylated lysine residues and even one methylated lysine and methylated arginine. The basic conformation that recognizes single methylated lysine residues is a cage with polar and non-polar regions that envelops the methylated lysine residues [39]. A few of the common motifs include ankyrin repeats, chromodomain, MBT repeats, PHD finger, and double tudor [40]. An example of a motif that is able to recognize methylated lysine and arginine residues is RAG2-PHD. These varieties of recognition motifs underline the great deal of complexity behind methylated residues and even hints at potential cross talk between methylated lysine and arginine residues. There have been studies showing that the methylation of one residue, H3R2, precluded the recognition of

Methylation of lysine and arginine residues has recently been discovered to also undergo demethylation via histone demehtylases (HDM). These enzymes are divided into two classes: amine oxidases, which are able to demethylate the first and second methyl lysine groups, and JmjC domain-containing proteins, which are able to demthylate all three methyl lysine groups. As well, it was also found recently that a JmjC domain-containing protein, JMJD6, was able to reverse arginine methylation [41]. HDM became a very key regulator of pluripotency after it was found that KDM3A and KMD4C are direct transcriptional targets of the pluripotency

a neighbouring methylated residue, H3K4me3 (Figure 7) [37].

**Figure 6.** Histone acetylation and deacetylation mechanism (Adapted from 36)

Histone methylation is the addition of methyl groups onto lysine and arginine residues of histones in both transcriptionally active as well as silenced regions of the chromatin. The patterns of modification of lysine residues within histones are more defined as compared to arginine. Methylation is catalyzed by Histone methyltransferases (HMTs) and demethylation by histone demethylases (HDMs). Lysine residues can be methylated up to three times whereas arginine can only be methylated twice. Moreover, the symmetry of the methyl groups on each of the residues also plays an important role in the function of chromatin. HMT all share a common SET domain within their catalytic core [37]. Lysine HMTs are very well defined as

**Figure 6.** Histone acetylation and deacetylation mechanism (Adapted from 36)

Histone acetylation is the addition of acetyl moieties onto each of the histones of the nucleo‐ some and is regulated by the activity of histone acetyltransferases (HAT) and histone deace‐ tylases (HDAC). HATs and HDACs operate as coactivators and corepressors and together they dynamically change the activation and repression of genes in both a site specific as well as global manner. There are four families of HATs; Gcn5-related N-acetyltransferase (GNAT), MYST and p300/CBP. These HATs share highly similar motifs including an acetyl-CoA binding domain with the Arg/Gln-X-XGly-X-Gly/Ala sequence [32]. HAT activity and specificity are highly dependent on the complexes they form with other HATs and transcriptional coactivators. Lysine, found at the amino terminus of the histone, is the primary targeted site of acetylation. At physiological pH, lysine is positively charged and contributes to the overall positive charge of the histone. However, the amount of lysine acetylation is directly correlated with the accessibility of the amino terminus or "histone tail" [32]. More accessibility means greater degree of acetylation. Upon acetylation, the residue is neutralized, reducing the positive charge of the histone, decreasing the interaction with the negatively charged DNA and directly influencing chromatin structure. HDACs are broken down into two families; classical HDACs and NAD+ HDACs. Like their HAT counterparts, HDACs share a conserved active site [33] and also require the need to complex with co-repressors in order to function properly. Once bound, active HDACs serve to remove the acetyl moiety from the histone tail through a charge-relay system of residues found within the active site [33, 34]. Once removed, the histones bind tighter to the DNA as well as enabling tighter packing of adjacent histones

Histone methylation is the addition of methyl groups onto lysine and arginine residues of histones in both transcriptionally active as well as silenced regions of the chromatin. The patterns of modification of lysine residues within histones are more defined as compared to arginine. Methylation is catalyzed by Histone methyltransferases (HMTs) and demethylation by histone demethylases (HDMs). Lysine residues can be methylated up to three times whereas arginine can only be methylated twice. Moreover, the symmetry of the methyl groups on each of the residues also plays an important role in the function of chromatin. HMT all share a common SET domain within their catalytic core [37]. Lysine HMTs are very well defined as

leading to more transcriptional repression (Figure 6) [35].

**Figure 5.** Chromatin Remodeling Enzyme SWI/SNFN (Adapted from 1)

186 Pluripotent Stem Cells

compared to arginine HMT. Arginine N-methyltransferases (PRMT) are less defined in terms of the targeted sites of methylation, with multiple target residues in histone H3 and H4 [37].

The process of methylation does not alter the charge properties of the targeted residue, unlike those of acetylation. Instead, the addition of methyl groups serves as a recognition site for regulatory proteins to bind and elicit additional modification. This allows for a great deal of complexity depending on the target region of the MMT and the resultant recognition effector protein. In essence, it allows additional information to be encoded in the histones beyond just stearic and charge hindrance of acetylation and phosphorylation [38]. Thus, this places a great deal of importance on the proteins that interact with the methylated residue. There are a variety of motifs that are able to recognize both single and double methylated lysine residues and even one methylated lysine and methylated arginine. The basic conformation that recognizes single methylated lysine residues is a cage with polar and non-polar regions that envelops the methylated lysine residues [39]. A few of the common motifs include ankyrin repeats, chromodomain, MBT repeats, PHD finger, and double tudor [40]. An example of a motif that is able to recognize methylated lysine and arginine residues is RAG2-PHD. These varieties of recognition motifs underline the great deal of complexity behind methylated residues and even hints at potential cross talk between methylated lysine and arginine residues. There have been studies showing that the methylation of one residue, H3R2, precluded the recognition of a neighbouring methylated residue, H3K4me3 (Figure 7) [37].

Methylation of lysine and arginine residues has recently been discovered to also undergo demethylation via histone demehtylases (HDM). These enzymes are divided into two classes: amine oxidases, which are able to demethylate the first and second methyl lysine groups, and JmjC domain-containing proteins, which are able to demthylate all three methyl lysine groups. As well, it was also found recently that a JmjC domain-containing protein, JMJD6, was able to reverse arginine methylation [41]. HDM became a very key regulator of pluripotency after it was found that KDM3A and KMD4C are direct transcriptional targets of the pluripotency

**Figure 7.** Methyl-lysine binding effector proteins (Adapted from 37)

promoting transcription factor Oct4 [42]. When these two enzymes were knocked down, the resulting cells lost the ability to self-renew as well as showed an altered morphology. More‐ over, another high-ranking HDM JARID2 is highly expressed in ESCs but becomes rapidly downregulated upon differentiation [43]. The level of regulatory complexity of the genes, not only in ESCs or iPSCs, but also somatic cells, Hence, further examination is needed to elucidate how these enzymes contribute to the epigenetic regulation of genes.

**•** DNA methylation

DNA Methylation is the classical example of epigenetic regulation of gene expression. This process, catalyzed by DNA methyl transferase (DNMT) enzymes, involves the addition of a methyl group onto the carbon 5 position of cytosine residues within DNA, forming 5-meth‐ ylcytosine. There are three main members of the DNMT family: Dmnt1, Dmnt3a and Dmnt3b. Dmnt1 is the best studied of the three and its primary role is to copy DNA methylation patterns during DNA synthesis as well as repair of DNA methylation patterns [44]. Dmnt3a and Dmnt3b are similar enzymes both in structure as well as function. These two DMNTs are capable of methylating native DNA, regardless of whether the DNA is in a replicative state or not [45]. Since they are able to write DNA methylation patterns onto "naked" DNA, they are termed *de novo* DMNTs.

Patterns of DNA methylation can be "read" through the recruitment of three different protein families: MBD, zinc-finger, and UHRF proteins. The most well-known are the MBD proteins, which interact with the DNA via a methyl-CpG-binding domain. Once the MBD proteins bind to the 5'site of the methylated cytosine, they repress transcription. Zinc finger proteins, like MBD proteins, also recognize and bind to methylcytosine, however they have a preference for consecutively methylated cytosine residues as well as non-methylated residues. Interestingly they are still able to repress transcription of DNA in a similar manner. Ubiquitin like containing PHD and RING finger Domain (UHRF) proteins use their intrinsic RING and SET DNA binding domains to interact with the methylated cytosine. However, the purpose of UHRF is not to repress transcription, but actually to aid DMNT, especially during DNA replication, in order to conserve and maintain the DNA methylation [46].

DNA methylation can be achieved via two mechanisms, either actively or passively (Figure 8). Passive demethylation involves the inhibition of the DMNT protein during DNA replication and allows for newly synthesized cytosine to escape methyl imprinting from its parent DNA strand. This process usually occurs during cellular replication. Active demethylation can occur in both dividing and non-dividing cells [47]. Currently, there is no known enzyme that is able to remove the strong covalent bond of the methyl group from the cytosine residue. Instead, the methylated cytosine is thought to undergo a series of further modifications (AID/APOBEC) that ultimately change the 5mC into a thymine [48]. This elicits a base mismatch and activates the base excision repair pathway to replace the residue with a naked cytosine. Another

Stem Cells and Epigenetic Reprogramming http://dx.doi.org/10.5772/55983 189

**Figure 8.** Active and passive DNA methylation mechanism (Adapted from 1)

**Figure 8.** Active and passive DNA methylation mechanism (Adapted from 1)

promoting transcription factor Oct4 [42]. When these two enzymes were knocked down, the resulting cells lost the ability to self-renew as well as showed an altered morphology. More‐ over, another high-ranking HDM JARID2 is highly expressed in ESCs but becomes rapidly downregulated upon differentiation [43]. The level of regulatory complexity of the genes, not only in ESCs or iPSCs, but also somatic cells, Hence, further examination is needed to elucidate

DNA Methylation is the classical example of epigenetic regulation of gene expression. This process, catalyzed by DNA methyl transferase (DNMT) enzymes, involves the addition of a methyl group onto the carbon 5 position of cytosine residues within DNA, forming 5-meth‐ ylcytosine. There are three main members of the DNMT family: Dmnt1, Dmnt3a and Dmnt3b. Dmnt1 is the best studied of the three and its primary role is to copy DNA methylation patterns during DNA synthesis as well as repair of DNA methylation patterns [44]. Dmnt3a and Dmnt3b are similar enzymes both in structure as well as function. These two DMNTs are capable of methylating native DNA, regardless of whether the DNA is in a replicative state or not [45]. Since they are able to write DNA methylation patterns onto "naked" DNA, they are

Patterns of DNA methylation can be "read" through the recruitment of three different protein families: MBD, zinc-finger, and UHRF proteins. The most well-known are the MBD proteins, which interact with the DNA via a methyl-CpG-binding domain. Once the MBD proteins bind to the 5'site of the methylated cytosine, they repress transcription. Zinc finger proteins, like MBD proteins, also recognize and bind to methylcytosine, however they have a preference for consecutively methylated cytosine residues as well as non-methylated residues. Interestingly they are still able to repress transcription of DNA in a similar manner. Ubiquitin like containing PHD and RING finger Domain (UHRF) proteins use their intrinsic RING and SET DNA binding domains to interact with the methylated cytosine. However, the purpose of UHRF is not to repress transcription, but actually to aid DMNT, especially during DNA replication, in

how these enzymes contribute to the epigenetic regulation of genes.

**Figure 7.** Methyl-lysine binding effector proteins (Adapted from 37)

order to conserve and maintain the DNA methylation [46].

**•** DNA methylation

188 Pluripotent Stem Cells

termed *de novo* DMNTs.

DNA methylation can be achieved via two mechanisms, either actively or passively (Figure 8). Passive demethylation involves the inhibition of the DMNT protein during DNA replication and allows for newly synthesized cytosine to escape methyl imprinting from its parent DNA strand. This process usually occurs during cellular replication. Active demethylation can occur in both dividing and non-dividing cells [47]. Currently, there is no known enzyme that is able to remove the strong covalent bond of the methyl group from the cytosine residue. Instead, the methylated cytosine is thought to undergo a series of further modifications (AID/APOBEC) that ultimately change the 5mC into a thymine [48]. This elicits a base mismatch and activates the base excision repair pathway to replace the residue with a naked cytosine. Another proposed demethylation pathway involves the use of the ten-eleven translocation (Tet) enzymes. This family of proteins are able to add a hydroxyl group onto the methyl moiety of 5mC to form 5hmC. Once in this state, 5hmC can return to an unmodified cytosine residue through either further oxidation by Tet enzymes or deamination by AID/APOBEC. Unlike the deacetylation and demethylation of histones, DNA demethylation is much more complex and involves a number of enzymatic processes which has contributed to the great deal of debate about which pathway is more dominant [50].

is determined by the locus, the type of transcription factor and on the context [14]. Hence the

Stem Cells and Epigenetic Reprogramming http://dx.doi.org/10.5772/55983 191

It has been reported that Oct4 is indispensable in the reprogramming process. In some cases, such as what has been observed in neural stem cell reprogramming, the presence of Oct4 is sufficient for reprogramming in [51]. Moreover, as Oct4 can work alone, it also has a great effect in reprogramming when combined with Sox2. Oct4 and Sox2 form a heterodimer that interact with some promoters. In addition this heterodimer has been shown to interact with Nanog. Nanog is another transcription factor that participates in the ESCs regulatory circuitry together with Oct4 and Sox2 to maintain pluripotency [52]. In this context they activate transcription in a chromatin independent manner by interacting with transcriptional coactivators [53, 54]. Moreover, it has been shown in mouse ESCs that Oct4 and Nanog can repress gene expression through interaction with histone deacetylase such as Mta1 [55]. It has been elucidated that in the first stage of reprogramming, a cascade of differentiation genes are turned off, while pluripotency genes progressively become upregulated in order to push the

c-Myc is an important participant in recruiting multiple chromatin modifications, such as histone acetyltransferases (GCN5, p300) and histones deacetylases (HDACs). In this regard, c-Myc increases the methylation site H3K4me3 and the global acetylation [56]. In the reprog‐ ramming process c-Myc activates its target before other core pluripotency transcription factors are activated, facilitating the opening of the chromatin for other factors [57, 58]. An example of c-Myc's potential in opening chromatin is its association with Tip60-p400 complex, which acetylate and remodel nucleosomes respectively. p400 is a member of the Swi2/Snt2 family which is well known among the ATPase chromatin remodelling enzymes, exchanging histones H2AZ-H2B within nucleosomes [57]. It also functions to release paused RNA polymerase from about one-third of the genes that are being actively transcribed. This activity could enhance cellular reprogramming [59]. At the same time, the transcription factor Klf4, activates the

Based on the Yamanaka's stochastic model (Figure 2), cells need to overcome the epigenetic barrier in order to become pluripotent [16, 17]. Nowadays, one of the major focuses in the iPSC field is to understand the epigenetic molecules that orchestrate chromatin remodelling in order to organize it into a pluripotent state similar to ESCs. While some somatic cell reprogramming

Once the differentiated cells have been reprogrammed, the epigenetic marks in iPSCs resemble ESCs [61]. For a great amount of eukaryotic DNA methylation is a mark that serves to define different cellular functions such as X chromosome inactivation, aging, imprinting, genome stability, tissue specific gene regulation, and so on [63, 64]. DNA methylation is one of the epigenetic marks that is modified during reprogramming (Figure 9). In this case, the process of demethylation is most common taking the methyl group from the promoters of

transcription of Sox2 which participates in the pluripotency cascade [60].

mechanisms are being unveiled, many are still yet unknown [9].

function of the four Yamanaka factors, Oct4, Sox2, Klf4 and c-Myc, [13] is crucial.

differentiated cell toward an ESC-like state.

**5.2. Chromatin remodelling**

**•** DNA methylation

## **5. Epigenetic reprogramming**

In order to understand the interactions and mechanisms involved in reprogramming a differentiated cell into an iPSC, a great effort has been made to study the ESC pluripotent state, in particular the means by which pluripotency transcription factors interact with each other or with other proteins such as chromatin remodeling enzymes and histone modifying enzymes. Moreover researchers have focused on finding the networks in iPSCs once the endogenous pluripotency factors have been activated by the exogenous Yamanaka factors [13]. Understanding the interactions between the core pluripotency transcription factors and the previously mentioned epigenetic enzymes will provide some advantages to the iPSC field. One such advantage is the possible discovery of new cocktails that enhance reprogramming. In addition, it could explain the chronology of the epigenetic events for reprogramming on a molecular level. This section will cover some of the known molecular interactions among the pluripotent transcription factors and some of the epigenetic enzymes.

#### **5.1. Pluripotency gene networks**

The first event toward transition from a differentiated to an iPSC state is the establishment of a proper chromatin state. Once the cells have found the correct chromatin state, the second event is to maintain and inherit it as they divide and proliferate [14]. The natural state of an ESC chromatin is known as "open", where the heterochromatin is disperse and dynamic, which at the same time reflects a hyperactive transcriptional status [49]. The molecular structure for ESC to maintain pluripotency requires an interconnection of transcription factors with epigenetic proteins that are also interacting with the DNA. Due to fact that iPSCs are like ESC, they have to sustain the same molecular structure. In addition, they have to overcome an epigenetic barrier during the reprogramming process. The reprogramming process involves a chain reaction involving transcription factors, chromatin modifying enzymes and other histone related enzymes.

An approach of how reprogramming occurs, suggests that the first step maybe interaction of transcription factors with the naked DNA, via histone modifiers or together with chromatin remodeling factors [14]. There are not time points or an order to follow for each specific transcription factor. Certain transcription factors are able to interact with DNA or with a chromatin remodeling enzyme depending what gene is activating. This molecular mechanism is determined by the locus, the type of transcription factor and on the context [14]. Hence the function of the four Yamanaka factors, Oct4, Sox2, Klf4 and c-Myc, [13] is crucial.

It has been reported that Oct4 is indispensable in the reprogramming process. In some cases, such as what has been observed in neural stem cell reprogramming, the presence of Oct4 is sufficient for reprogramming in [51]. Moreover, as Oct4 can work alone, it also has a great effect in reprogramming when combined with Sox2. Oct4 and Sox2 form a heterodimer that interact with some promoters. In addition this heterodimer has been shown to interact with Nanog. Nanog is another transcription factor that participates in the ESCs regulatory circuitry together with Oct4 and Sox2 to maintain pluripotency [52]. In this context they activate transcription in a chromatin independent manner by interacting with transcriptional coactivators [53, 54]. Moreover, it has been shown in mouse ESCs that Oct4 and Nanog can repress gene expression through interaction with histone deacetylase such as Mta1 [55]. It has been elucidated that in the first stage of reprogramming, a cascade of differentiation genes are turned off, while pluripotency genes progressively become upregulated in order to push the differentiated cell toward an ESC-like state.

c-Myc is an important participant in recruiting multiple chromatin modifications, such as histone acetyltransferases (GCN5, p300) and histones deacetylases (HDACs). In this regard, c-Myc increases the methylation site H3K4me3 and the global acetylation [56]. In the reprog‐ ramming process c-Myc activates its target before other core pluripotency transcription factors are activated, facilitating the opening of the chromatin for other factors [57, 58]. An example of c-Myc's potential in opening chromatin is its association with Tip60-p400 complex, which acetylate and remodel nucleosomes respectively. p400 is a member of the Swi2/Snt2 family which is well known among the ATPase chromatin remodelling enzymes, exchanging histones H2AZ-H2B within nucleosomes [57]. It also functions to release paused RNA polymerase from about one-third of the genes that are being actively transcribed. This activity could enhance cellular reprogramming [59]. At the same time, the transcription factor Klf4, activates the transcription of Sox2 which participates in the pluripotency cascade [60].

#### **5.2. Chromatin remodelling**

proposed demethylation pathway involves the use of the ten-eleven translocation (Tet) enzymes. This family of proteins are able to add a hydroxyl group onto the methyl moiety of 5mC to form 5hmC. Once in this state, 5hmC can return to an unmodified cytosine residue through either further oxidation by Tet enzymes or deamination by AID/APOBEC. Unlike the deacetylation and demethylation of histones, DNA demethylation is much more complex and involves a number of enzymatic processes which has contributed to the great deal of debate

In order to understand the interactions and mechanisms involved in reprogramming a differentiated cell into an iPSC, a great effort has been made to study the ESC pluripotent state, in particular the means by which pluripotency transcription factors interact with each other or with other proteins such as chromatin remodeling enzymes and histone modifying enzymes. Moreover researchers have focused on finding the networks in iPSCs once the endogenous pluripotency factors have been activated by the exogenous Yamanaka factors [13]. Understanding the interactions between the core pluripotency transcription factors and the previously mentioned epigenetic enzymes will provide some advantages to the iPSC field. One such advantage is the possible discovery of new cocktails that enhance reprogramming. In addition, it could explain the chronology of the epigenetic events for reprogramming on a molecular level. This section will cover some of the known molecular interactions among the

The first event toward transition from a differentiated to an iPSC state is the establishment of a proper chromatin state. Once the cells have found the correct chromatin state, the second event is to maintain and inherit it as they divide and proliferate [14]. The natural state of an ESC chromatin is known as "open", where the heterochromatin is disperse and dynamic, which at the same time reflects a hyperactive transcriptional status [49]. The molecular structure for ESC to maintain pluripotency requires an interconnection of transcription factors with epigenetic proteins that are also interacting with the DNA. Due to fact that iPSCs are like ESC, they have to sustain the same molecular structure. In addition, they have to overcome an epigenetic barrier during the reprogramming process. The reprogramming process involves a chain reaction involving transcription factors, chromatin modifying enzymes and other

An approach of how reprogramming occurs, suggests that the first step maybe interaction of transcription factors with the naked DNA, via histone modifiers or together with chromatin remodeling factors [14]. There are not time points or an order to follow for each specific transcription factor. Certain transcription factors are able to interact with DNA or with a chromatin remodeling enzyme depending what gene is activating. This molecular mechanism

pluripotent transcription factors and some of the epigenetic enzymes.

about which pathway is more dominant [50].

**5. Epigenetic reprogramming**

190 Pluripotent Stem Cells

**5.1. Pluripotency gene networks**

histone related enzymes.

Based on the Yamanaka's stochastic model (Figure 2), cells need to overcome the epigenetic barrier in order to become pluripotent [16, 17]. Nowadays, one of the major focuses in the iPSC field is to understand the epigenetic molecules that orchestrate chromatin remodelling in order to organize it into a pluripotent state similar to ESCs. While some somatic cell reprogramming mechanisms are being unveiled, many are still yet unknown [9].

**•** DNA methylation

Once the differentiated cells have been reprogrammed, the epigenetic marks in iPSCs resemble ESCs [61]. For a great amount of eukaryotic DNA methylation is a mark that serves to define different cellular functions such as X chromosome inactivation, aging, imprinting, genome stability, tissue specific gene regulation, and so on [63, 64]. DNA methylation is one of the epigenetic marks that is modified during reprogramming (Figure 9). In this case, the process of demethylation is most common taking the methyl group from the promoters of some genes that are responsible for pluripotency which in turn allows them to return to a pluripotent state.

27). These marks in the histones occur by S-adenosylmethionine (SAM-dependent) protein methylation. Due to this bivalent mark in the histones, these cells have the capacity to activate

Stem Cells and Epigenetic Reprogramming http://dx.doi.org/10.5772/55983 193

Since the histone-lysine N-methyltransferase (MLL) catalytic subunits are well known to introduce the H3k4me3 mark and activate transcription, this makes them potential regulators in reprogramming. An example of their activity is shown with Wdr5, a subunit in common with H3k4 methyltransferases. Wdr5 has been proposed to play an important role in main‐ taining pluripotency and has been proposed as one possible mechanism occurring during reprograming. Wdr5 is activated by exogenous Oct4 when mouse embryonic fibroblast are transfected with the four Yamanaka factors. Wdr5 directly binds to loci where self-renewal genes are encoded such as Oct4 and Nanog, in order to re-establish an H3k4 mark. Wdr5 is thus defined as an indispensable subunit which proportions H3k4 methylation [68]. Similarly, MLL interacts with some other chromatin remodeling enzymes, such as CHD1 and NURF, in

It has been already mentioned that ESCs and iPSCs are known to have bivalent chromatin bearing both the active mark H3k4me3, and the repressive mark H3k27me3. One of the mechanism that controls the bivalency is through the activity of the polycomb proteins found in two major complexes PRC1 and PRC2 [70]. It is thought that PRC1 and PRC2 act as antagonists and are intrinsically involved in establishing the fate of ESC development. PRC2 is in charge of the H3k27me3 mark [71] and known to silence the HOX genes used and other regulators during ESC differentiation [72]. One of the basic mechanisms in ESC/iPSC differ‐

Utx demethylase has been reported to be a significant regulator of cellular reprogramming [73]. Utx is encoded by an X-chromosome gene and belongs to the small family of Jmjc proteins, mediating the demethylation of H3k27 tri- and di-methyl repressive chromatin marks. In this study, it was found that Utx was dispensable for the maintenance of pluripotency, since pluripotency marker expression was maintained in knockout ESC lines. However fibroblasts derived from Utx knockout mice failed to be reprogrammed. This result indicated that the absence of Utx prevented the demethylation of H3k27me3 marks needed to re-establish

H3K9me3 and H3k79me2 have also been reported to be important for the maintenance of pluripotency in ESCs. These marks are usually left by repressive methyltransferases via the identification of specific motifs in heterochromatin. Reprogramming has been facilitated by

Histone acetylation is another important histone mark that has usually been correlated with gene activation (Figure 10). This mark has been reported to transform chromatin during reprogramming. Little is known about histone acetyltransferases for pluripotency mainte‐ nance or reprogramming, however Tip60/p400 has been reported as a histone acetyltransferase important for maintaining the ESC state. Here Tip60/p400 also works as a chromatin remod‐

the inhibiting two methyltransferases (GLP and G9a) that methylate H3k9 [74].

or repress genes them in order to change their fate [67].

order to achieve H3k4 methylation [29, 69].

pluripotency *in vitro* [73].

entiation is the demethylation of this H3k27me3 mark.

eling enzyme, since it has a SWI2/SNF2 subunit [75].

Currently there is not too much evidence about the process of demethylation and the enzymes that catalyze this event. However, DNA demethylation events have been classified as passive or active. Passive DNA demethylation occurs during the process of DNA replication when maintenance methytransferases are inactive, and thus they are not able to methylate newly released strands [62]. On the other hand for active DNA demethylation, the main protagonists are enzymes that work regardless of DNA replication [62].

**Figure 9.** DNA demethylation as a reprogramming process when going to iPSC (Adapted from 13)

An example of an active DNA demethylation event is shown by Pereira and colleagues (2008), where they have studied the efficiency of reprogramming of human lymphocytes by fusing them with mouse ESCs. They found that one of the first events occurring was the demethyla‐ tion of the Oct4 gene. They suggest that this event is a result of an active chromatin remodeling locus before its actual expression [65].

Two mechanisms have been proposed as candidates for the active DNA demethylation mechanism. The first one involves the deamination of 5-methylcytosine in DNA by an enzyme called activation-induce deaminase (AID) [62]. The other mechanism is based on the oxidiza‐ tion of the 5-methyl group (-CH3) followed by conversion into 5-carboxylcytosine (-COOH). This conversion is catalyzed by the enzyme TET1 in a Fe(II) and α-ketoglutarate dependent reaction [66]. To date neither of these mechanisms has been proven *in vitro.* Hence there are still a number of unknown molecular mechanisms that govern the reprogramming process. Is there an active DNA demethylation? How are the DNS demethylating enzymes activated? Are they recruited by other processes of chromatin remodeling or do transcription factors initialize the process?

**•** Histone modifications

The most common marks in ESC and iPSCs are the active mark of H3k4me3 (histone 3, trimethylated at lysine 4) and the repressive mark H3k27me3 (histone 3, trimethylated at lysine 27). These marks in the histones occur by S-adenosylmethionine (SAM-dependent) protein methylation. Due to this bivalent mark in the histones, these cells have the capacity to activate or repress genes them in order to change their fate [67].

some genes that are responsible for pluripotency which in turn allows them to return to a

Currently there is not too much evidence about the process of demethylation and the enzymes that catalyze this event. However, DNA demethylation events have been classified as passive or active. Passive DNA demethylation occurs during the process of DNA replication when maintenance methytransferases are inactive, and thus they are not able to methylate newly released strands [62]. On the other hand for active DNA demethylation, the main protagonists

are enzymes that work regardless of DNA replication [62].

**Figure 9.** DNA demethylation as a reprogramming process when going to iPSC (Adapted from 13)

locus before its actual expression [65].

the process?

**•** Histone modifications

An example of an active DNA demethylation event is shown by Pereira and colleagues (2008), where they have studied the efficiency of reprogramming of human lymphocytes by fusing them with mouse ESCs. They found that one of the first events occurring was the demethyla‐ tion of the Oct4 gene. They suggest that this event is a result of an active chromatin remodeling

Two mechanisms have been proposed as candidates for the active DNA demethylation mechanism. The first one involves the deamination of 5-methylcytosine in DNA by an enzyme called activation-induce deaminase (AID) [62]. The other mechanism is based on the oxidiza‐ tion of the 5-methyl group (-CH3) followed by conversion into 5-carboxylcytosine (-COOH). This conversion is catalyzed by the enzyme TET1 in a Fe(II) and α-ketoglutarate dependent reaction [66]. To date neither of these mechanisms has been proven *in vitro.* Hence there are still a number of unknown molecular mechanisms that govern the reprogramming process. Is there an active DNA demethylation? How are the DNS demethylating enzymes activated? Are they recruited by other processes of chromatin remodeling or do transcription factors initialize

The most common marks in ESC and iPSCs are the active mark of H3k4me3 (histone 3, trimethylated at lysine 4) and the repressive mark H3k27me3 (histone 3, trimethylated at lysine

pluripotent state.

192 Pluripotent Stem Cells

Since the histone-lysine N-methyltransferase (MLL) catalytic subunits are well known to introduce the H3k4me3 mark and activate transcription, this makes them potential regulators in reprogramming. An example of their activity is shown with Wdr5, a subunit in common with H3k4 methyltransferases. Wdr5 has been proposed to play an important role in main‐ taining pluripotency and has been proposed as one possible mechanism occurring during reprograming. Wdr5 is activated by exogenous Oct4 when mouse embryonic fibroblast are transfected with the four Yamanaka factors. Wdr5 directly binds to loci where self-renewal genes are encoded such as Oct4 and Nanog, in order to re-establish an H3k4 mark. Wdr5 is thus defined as an indispensable subunit which proportions H3k4 methylation [68]. Similarly, MLL interacts with some other chromatin remodeling enzymes, such as CHD1 and NURF, in order to achieve H3k4 methylation [29, 69].

It has been already mentioned that ESCs and iPSCs are known to have bivalent chromatin bearing both the active mark H3k4me3, and the repressive mark H3k27me3. One of the mechanism that controls the bivalency is through the activity of the polycomb proteins found in two major complexes PRC1 and PRC2 [70]. It is thought that PRC1 and PRC2 act as antagonists and are intrinsically involved in establishing the fate of ESC development. PRC2 is in charge of the H3k27me3 mark [71] and known to silence the HOX genes used and other regulators during ESC differentiation [72]. One of the basic mechanisms in ESC/iPSC differ‐ entiation is the demethylation of this H3k27me3 mark.

Utx demethylase has been reported to be a significant regulator of cellular reprogramming [73]. Utx is encoded by an X-chromosome gene and belongs to the small family of Jmjc proteins, mediating the demethylation of H3k27 tri- and di-methyl repressive chromatin marks. In this study, it was found that Utx was dispensable for the maintenance of pluripotency, since pluripotency marker expression was maintained in knockout ESC lines. However fibroblasts derived from Utx knockout mice failed to be reprogrammed. This result indicated that the absence of Utx prevented the demethylation of H3k27me3 marks needed to re-establish pluripotency *in vitro* [73].

H3K9me3 and H3k79me2 have also been reported to be important for the maintenance of pluripotency in ESCs. These marks are usually left by repressive methyltransferases via the identification of specific motifs in heterochromatin. Reprogramming has been facilitated by the inhibiting two methyltransferases (GLP and G9a) that methylate H3k9 [74].

Histone acetylation is another important histone mark that has usually been correlated with gene activation (Figure 10). This mark has been reported to transform chromatin during reprogramming. Little is known about histone acetyltransferases for pluripotency mainte‐ nance or reprogramming, however Tip60/p400 has been reported as a histone acetyltransferase important for maintaining the ESC state. Here Tip60/p400 also works as a chromatin remod‐ eling enzyme, since it has a SWI2/SNF2 subunit [75].

spreading at an early stage of reprogramming as a form of physical rearrangement prior to the

Stem Cells and Epigenetic Reprogramming http://dx.doi.org/10.5772/55983 195

As previously mentioned, chromatin remodeling is caused by catalytic modification where ATPases use the energy from the ATP to move along in DNA. Thus they regulate gene expression by spacing nucleosome arrays, exchanging histone variants, disassembling or sliding the nucleosome [14]. One example of the importance of the chromatin remodeling enzymes is observed in a study of Brg, part of a family of DNA ATPases homologous to the catalytic subunit of yeast SWI2/SNT2 ATPase [78]. Brg is assembled to 11 other Brg/Brahma associated factors (BAFs). In ESCs, BAF complexes have an exclusive subunit which is called esBAF. The authors observed that esBAF facilitates STAT3 to access binding sites that will respond to LIF, which will further activate the pluripotency transcription factor Klf4 [78]. Therefore, it is thought that the LIF signaling pathway is dependent on prior chromatin remodeling [78]. A previous study showed that overexpressing of esBAF in addition to the four Yamanaka was able to acquire a euchromatic chromatin by increasing the kinetics of Oct4, Nanog and Rex1 promoter demethylation. This facilitated the accessibility of the reprogram‐

Moreover, Onder and colleagues (2012) [79] focused on the study of chromatin-modifying enzymes during the reprogramming process in iPSCs. They used a loss of function approach with shRNA where they selected 22 genes involved in DNA and histone methylation path‐ ways. From their results, they found that inhibition of the histone methyltransferase DOT1L, reprogramming was enhanced resulting in more iPSCs colonies. DOT1L inhibition does not enhance the upregulation of the pluripotency gene network but can be used to substitute for

euchromatin epigenetic alterations which occur after day 7 (Figure 11) [77].

**Figure 11.** Global epigenetic changes in iPSCs (Adapted from 80).

ming factors and hence the process was enhanced [28].

**•** Chromatin remodeling

**Figure 10.** Histone acetylation as an activating mark for reprogramming. (Adapted from 13)

On the other hand the histone deacetylases (HDACs) are known to repress the expression of genes, therefore there is an increased interest on their inhibition. An example of their impor‐ tance in reprogramming of somatic cells was shown by Hadas Hezroni and collaborators (2011) [76]. In this study they used hybrid cell lines by fusing mouse embryonic fibroblast with ESCs and found that low H3k9 acetylation correlated with low reprogramming capacity. When they tried to overcome this effect using histone deacetylase inhibitors, they found an increase in the reprogramming efficiency. They reported that genes involved in extracellular matrix (ECM) activity were enriched during reprograming and concluded that H3K9ac is a mark intrinsically related to pluripotency and that promoting its increase using HDACs inhibitors promote ECM activity, which co-relates positively affect pluripotency and self-renewal [76].

Most epigenetic reprogramming studies have focused on isolated chromatin marks, revealing the down regulation of somatic genes. However there are more than some marks that lead to an "open" dynamic chromatin. Anna Mattout and colleagues [77] presented a study where for the first time they showed chromatin dimensions as global changes occurring during reprog‐ ramming. They analyzed a battery of histone modifications (H3ac, H4ac, H4k5ac, H3k27ac, H3k4me3, H3k36me2, H3k9me3, and H3k27me3 also γH2AX, HP1α and lamin A, by immu‐ nofluorescence and biochemical fractionations comparing mouse ESCs to fully- and partiallyreprogrammed mouse iPSCs. They first identified that H3k36me2, H4k5ac and H3k4me3 have the highest correlation with pluripotency. Later, they showed that most of the euchromatin/ active marks (H3ac, H3k9ac, H3k27 ac, H4ac, H4k5ac, H3k4me3 and H3k36me2) are higher in the ESCs and fully reprogrammed iPSCs, whereas in partially reprogrammed cells these marks more closely resembled that of mouse embryonic fibroblasts. On the other hand they observed that the marks in heterochromatin, such as HP1α and H3k9me3 rearrange during reprogram‐ ming towards a more diffused pattern. This was seen in all of the cells lines including partially reprogrammed iPSCs. With these two phenomena they presented a time line suggesting that marks in heterochromatin start changing at a very early stage (by day 6 during reprogram‐ ming) compared to the histone changes occurring in the active euchromatin. They concluded that during reprogramming global histone heterochromatin defining marks start changing and spreading at an early stage of reprogramming as a form of physical rearrangement prior to the euchromatin epigenetic alterations which occur after day 7 (Figure 11) [77].

**Figure 11.** Global epigenetic changes in iPSCs (Adapted from 80).

**•** Chromatin remodeling

**Figure 10.** Histone acetylation as an activating mark for reprogramming. (Adapted from 13)

194 Pluripotent Stem Cells

activity, which co-relates positively affect pluripotency and self-renewal [76].

On the other hand the histone deacetylases (HDACs) are known to repress the expression of genes, therefore there is an increased interest on their inhibition. An example of their impor‐ tance in reprogramming of somatic cells was shown by Hadas Hezroni and collaborators (2011) [76]. In this study they used hybrid cell lines by fusing mouse embryonic fibroblast with ESCs and found that low H3k9 acetylation correlated with low reprogramming capacity. When they tried to overcome this effect using histone deacetylase inhibitors, they found an increase in the reprogramming efficiency. They reported that genes involved in extracellular matrix (ECM) activity were enriched during reprograming and concluded that H3K9ac is a mark intrinsically related to pluripotency and that promoting its increase using HDACs inhibitors promote ECM

Most epigenetic reprogramming studies have focused on isolated chromatin marks, revealing the down regulation of somatic genes. However there are more than some marks that lead to an "open" dynamic chromatin. Anna Mattout and colleagues [77] presented a study where for the first time they showed chromatin dimensions as global changes occurring during reprog‐ ramming. They analyzed a battery of histone modifications (H3ac, H4ac, H4k5ac, H3k27ac, H3k4me3, H3k36me2, H3k9me3, and H3k27me3 also γH2AX, HP1α and lamin A, by immu‐ nofluorescence and biochemical fractionations comparing mouse ESCs to fully- and partiallyreprogrammed mouse iPSCs. They first identified that H3k36me2, H4k5ac and H3k4me3 have the highest correlation with pluripotency. Later, they showed that most of the euchromatin/ active marks (H3ac, H3k9ac, H3k27 ac, H4ac, H4k5ac, H3k4me3 and H3k36me2) are higher in the ESCs and fully reprogrammed iPSCs, whereas in partially reprogrammed cells these marks more closely resembled that of mouse embryonic fibroblasts. On the other hand they observed that the marks in heterochromatin, such as HP1α and H3k9me3 rearrange during reprogram‐ ming towards a more diffused pattern. This was seen in all of the cells lines including partially reprogrammed iPSCs. With these two phenomena they presented a time line suggesting that marks in heterochromatin start changing at a very early stage (by day 6 during reprogram‐ ming) compared to the histone changes occurring in the active euchromatin. They concluded that during reprogramming global histone heterochromatin defining marks start changing and

As previously mentioned, chromatin remodeling is caused by catalytic modification where ATPases use the energy from the ATP to move along in DNA. Thus they regulate gene expression by spacing nucleosome arrays, exchanging histone variants, disassembling or sliding the nucleosome [14]. One example of the importance of the chromatin remodeling enzymes is observed in a study of Brg, part of a family of DNA ATPases homologous to the catalytic subunit of yeast SWI2/SNT2 ATPase [78]. Brg is assembled to 11 other Brg/Brahma associated factors (BAFs). In ESCs, BAF complexes have an exclusive subunit which is called esBAF. The authors observed that esBAF facilitates STAT3 to access binding sites that will respond to LIF, which will further activate the pluripotency transcription factor Klf4 [78]. Therefore, it is thought that the LIF signaling pathway is dependent on prior chromatin remodeling [78]. A previous study showed that overexpressing of esBAF in addition to the four Yamanaka was able to acquire a euchromatic chromatin by increasing the kinetics of Oct4, Nanog and Rex1 promoter demethylation. This facilitated the accessibility of the reprogram‐ ming factors and hence the process was enhanced [28].

Moreover, Onder and colleagues (2012) [79] focused on the study of chromatin-modifying enzymes during the reprogramming process in iPSCs. They used a loss of function approach with shRNA where they selected 22 genes involved in DNA and histone methylation path‐ ways. From their results, they found that inhibition of the histone methyltransferase DOT1L, reprogramming was enhanced resulting in more iPSCs colonies. DOT1L inhibition does not enhance the upregulation of the pluripotency gene network but can be used to substitute for Klf4 and c-Myc during reprogramming. Inhibition of this molecule is associated with an increase of Nanog and Lin28, factors, which are necessary for reprogramming. Finally using ChIP-seq they found that the H3K79me2 mark was lost in genes that participate in epithelial mesenchymal transition. Among some of the mesenchymal regulators were SNAI1, SNAI2, ZEB1, ZEB2 and TGFB2, that at the same time where strongly repressed during reprogram‐ ming. Together with this, they also reported that epithelial genes such as CDH1 (E-cadherin) and OCLN were upregulated. The above was a clear example of how chromatin modifying enzymes are critical in the molecular process of reprogramming enhancing the cascade that begins with the four Yamanaka factors [79].

to interconnect transcription factors, chromatin, and histone modifier enzymes. What is the kinetics of this process? This is one of the first questions that puzzle most researchers. While there is firm understanding that the exogenous transcription factors are first to prompt these changes, reprogramming is not an efficient process. From the extensive interest in making reprogramming an efficient process, there have been a lot of remarkable results using different type of molecules that target chromatin enzymes. Thus understanding of the reprogramming process, including the timing of chromatin remodeling, interactions with transcription factors, increase or decrease of histone acetylation and most important, the precise interconnection of factors that break the epigenetic barrier, will give us a base line to design a better protocol for

Stem Cells and Epigenetic Reprogramming http://dx.doi.org/10.5772/55983 197

There will come a point where researchers will manipulate chromatin kinetics in order to promote the reprogramming of somatic cells into iPSCs. This achievement will bring a cell that reprograms efficiently; in a short period of time, which will have an epigenetic signature identical to ESCs. In the future, with a better understanding of chromatin not only in reprog‐ ramming but also in differentiation, the iPSC field could become an area of synthetic biology. In any case, the iPSC field still has a long way to go before it is fruitful. A clearer understanding of the epigenetics of the reprogramming must come forward before iPSCs can be fully accepted

Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB,

[1] Tollervey, J R, & Lunyak, V V. Epigenetics Judge, jury and executioner of stem cell fate. Landes Bioscience (2012). http://www-ncbi-nlm-nih-gov.ezproxy.lib.ucalga‐

[2] Evans, M J, & Kaufman, M H. Establishment in culture of pluripotential cells from mouse embryos. Nature (1981). http://www-ncbi-nlm-nih-gov.ezproxy.lib.ucalga‐

[3] Thomson, J. A, & Odorico, J. S. Human embryonic stem cell and embryonic germ cell lines. Trends Biotechnol (2000). http://www.ncbi.nlm.nih.gov/pubmed/10652509

the develop of iPSCs.

for regenerative medicine.

Perla Cota, Mehdi Shafa and Derrick E. Rancourt\*

ry.ca/pubmed?term=22805743

ry.ca/pubmed/7242681, 5819, 154-6.

\*Address all correspondence to: rancourt@ucalgary.ca

**Author details**

Canada

**References**

The above are just some of the studies of many that have been reported. They lead us to question whether remodeling enzymes are in charge of the major chromatin opening that occurs during reprogramming, or if histone marks lead the process.

#### **5.3. MicroRNAs in reprogramming**

MicroRNAs (miRs) are small RNAs involved in the inhibition of the gene expression by destabilizing target RNAs. They are usually formed by the proteins Dicer and Drosha with its cofactor Dgcr8. The importance of miRs arises from the observation that some miRs induce reprogramming of somatic cells into iPSCs [80]. Among some of the miRs found to positively regulate ESC pluripotency are: ESC cell-cycle regulating miR291a-3p, miR291b-3p, miR294, miR295 and miR302. Interestingly, miR302 has been reported regulate some of the epigenetic modifications that occur during reprogramming. miR302 is a family of four highly homologous microRNAs that are transcribed together and form a noncoding RNA cluster [81]. They are highly expressed in human ESCs and absent in differentiated cells. Lin and collaborators (2011) have focus in how the miR302 controls several enzymes that are involved in active demethy‐ lation [81]. MiR302 targets and represses AOF2/1 histone demethylases and MECP1/2 (methyl CpG binding proteins). At the same time it blocks cytosine methyltransferase 1 (DNMT1). During reprograming miR302 coordinates DNA demethylation, together with a histone methylation on the active mark H3k4 that will alter the chromatin structure and the gene activity. The example above highlights the critical role of miRs in founding and sustaining pluripotency in cells [82].

### **6. Conclusion**

The biology of pluripotent stem cells is still in a very early stage; even understanding what is the best way to obtain a true embryonic stem cell remains unclear. During reprogramming a number of changes occur in the cell. These changes start usually by the stimulation of exoge‐ nous transcription factors that consequentially trigger a large number of other reactions: signalling, gene transcription, and most importantly epigenetic changes, including chromatin remodeling, histone modification, and DNA methylation [1, 12, 13].

During reprogramming, chromatin changes to an "open" dynamic configuration resembling the epigenetic landscape as in ESCs. In order to reach this configuration, the somatic cell has to interconnect transcription factors, chromatin, and histone modifier enzymes. What is the kinetics of this process? This is one of the first questions that puzzle most researchers. While there is firm understanding that the exogenous transcription factors are first to prompt these changes, reprogramming is not an efficient process. From the extensive interest in making reprogramming an efficient process, there have been a lot of remarkable results using different type of molecules that target chromatin enzymes. Thus understanding of the reprogramming process, including the timing of chromatin remodeling, interactions with transcription factors, increase or decrease of histone acetylation and most important, the precise interconnection of factors that break the epigenetic barrier, will give us a base line to design a better protocol for the develop of iPSCs.

There will come a point where researchers will manipulate chromatin kinetics in order to promote the reprogramming of somatic cells into iPSCs. This achievement will bring a cell that reprograms efficiently; in a short period of time, which will have an epigenetic signature identical to ESCs. In the future, with a better understanding of chromatin not only in reprog‐ ramming but also in differentiation, the iPSC field could become an area of synthetic biology. In any case, the iPSC field still has a long way to go before it is fruitful. A clearer understanding of the epigenetics of the reprogramming must come forward before iPSCs can be fully accepted for regenerative medicine.

## **Author details**

Klf4 and c-Myc during reprogramming. Inhibition of this molecule is associated with an increase of Nanog and Lin28, factors, which are necessary for reprogramming. Finally using ChIP-seq they found that the H3K79me2 mark was lost in genes that participate in epithelial mesenchymal transition. Among some of the mesenchymal regulators were SNAI1, SNAI2, ZEB1, ZEB2 and TGFB2, that at the same time where strongly repressed during reprogram‐ ming. Together with this, they also reported that epithelial genes such as CDH1 (E-cadherin) and OCLN were upregulated. The above was a clear example of how chromatin modifying enzymes are critical in the molecular process of reprogramming enhancing the cascade that

The above are just some of the studies of many that have been reported. They lead us to question whether remodeling enzymes are in charge of the major chromatin opening that

MicroRNAs (miRs) are small RNAs involved in the inhibition of the gene expression by destabilizing target RNAs. They are usually formed by the proteins Dicer and Drosha with its cofactor Dgcr8. The importance of miRs arises from the observation that some miRs induce reprogramming of somatic cells into iPSCs [80]. Among some of the miRs found to positively regulate ESC pluripotency are: ESC cell-cycle regulating miR291a-3p, miR291b-3p, miR294, miR295 and miR302. Interestingly, miR302 has been reported regulate some of the epigenetic modifications that occur during reprogramming. miR302 is a family of four highly homologous microRNAs that are transcribed together and form a noncoding RNA cluster [81]. They are highly expressed in human ESCs and absent in differentiated cells. Lin and collaborators (2011) have focus in how the miR302 controls several enzymes that are involved in active demethy‐ lation [81]. MiR302 targets and represses AOF2/1 histone demethylases and MECP1/2 (methyl CpG binding proteins). At the same time it blocks cytosine methyltransferase 1 (DNMT1). During reprograming miR302 coordinates DNA demethylation, together with a histone methylation on the active mark H3k4 that will alter the chromatin structure and the gene activity. The example above highlights the critical role of miRs in founding and sustaining

The biology of pluripotent stem cells is still in a very early stage; even understanding what is the best way to obtain a true embryonic stem cell remains unclear. During reprogramming a number of changes occur in the cell. These changes start usually by the stimulation of exoge‐ nous transcription factors that consequentially trigger a large number of other reactions: signalling, gene transcription, and most importantly epigenetic changes, including chromatin

During reprogramming, chromatin changes to an "open" dynamic configuration resembling the epigenetic landscape as in ESCs. In order to reach this configuration, the somatic cell has

remodeling, histone modification, and DNA methylation [1, 12, 13].

occurs during reprogramming, or if histone marks lead the process.

begins with the four Yamanaka factors [79].

**5.3. MicroRNAs in reprogramming**

196 Pluripotent Stem Cells

pluripotency in cells [82].

**6. Conclusion**

Perla Cota, Mehdi Shafa and Derrick E. Rancourt\*

\*Address all correspondence to: rancourt@ucalgary.ca

Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada

## **References**


[4] Verma, A. Verma N: Induced pluripotent stem cells and promises of neuroregenera‐ tive medicine. Neurol India (2011). http://www.ncbi.nlm.nih.gov/pubmed/21891933

[16] Yamanaka, S. Elite and stochastic models for induced pluripotent stem cells genera‐ tion. Nature (2009). http://www.ncbi.nlm.nih.gov/pubmed/19571877, 460, 49-52.

Stem Cells and Epigenetic Reprogramming http://dx.doi.org/10.5772/55983 199

[17] Okita, K, Ichisaka, T, & Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature (2007). http://www.nature.com/nature/journal/

[18] Maherali, N, & Hochedlinger, K. Guidelines and Techniques for the Generation of In‐ duced Pluripotent Stem Cells. Cell Stem Cell (2008). http://www.ncbi.nlm.nih.gov/

[19] Wolffe, A P, & Matzke, M A. Epigenetics: regulation through repression. Science

[20] Strahl, B. D, & Allis, C. D. The language of covalent histone modifications. Nature

[21] Orkin, S H, & Hochedlinger, K. Chromatin connections to pluripotency and cellular reprogramming. Cell (2011). http://www.ncbi.nlm.nih.gov/pubmed/21663790

[22] Han, J W, & Yoon, Y-s. u. p. Epigenetic Landscape of pluripotent stem cells. Antioxi‐ dants & Redox Signaling (2012). http://www.ncbi.nlm.nih.gov/pubmed/22044221, 17,

[23] Li, B, Carey, M, & Workman, J L. The Role of Chromatin during Transcription. Cell

[24] Hargreaves, D C, & Crabtree, G R. ATP-dependent chromatin remodeling genetics, genomics and mechanisms. Cell Res (2011). http://www.ncbi.nlm.nih.gov/pubmed/

[25] Phelan, M L, Sif, S, Narlikar, G J, & Kingston, R E. Reconstitution of a core chromatin remodeling complex from SWI/SNF subunits. Mol Cell (1999). http://

[26] Saha, A, Wittmeyer, J, & Cairns, B R. Chromatin remodeling through directional DNA translocation from an internal nucleosomal site. Nat Struct Mol Biol (2005).

[27] Ho, L, Ronan, J L, Wu, J, Staahl, B T, Chen, L, Kuo, a, Lessard, J, Nesvizhskii, A. I, Ranish, J, & Crabtree, G R. An embryonic stem cell chromatin remodeling complex, esBAF, is essential for embryonic stem cell self-renewal and pluripotency. Proc Natl Acad Sci USA (2009). http://www.pnas.org/content/early/2009/03/10/0812889106, 106,

[28] Singhal, N, Graumann, J, Wu, G, Araúzo-bravo, M J, Han, D W, Greber, B, Gentile, L, Mann, M, & Schöler, H R. Chromatin-remodeling components of the BAF complex facilitates reprogramming. Cell (2010). http://www.cell.com/abstract/

(2007). http://www.ncbi.nlm.nih.gov/pubmed/17320508, 128, 707-719.

www.ncbi.nlm.nih.gov/pubmed/10078207, 3, 247-253.

http://www.ncbi.nlm.nih.gov/pubmed/16086025, 12, 747-755.

(1999). http://www.ncbi.nlm.nih.gov/pubmed/10521337, 288, 481-486.

(2000). http://www.ncbi.nlm.nih.gov/pubmed/10638745, 403, 41-45.

n7151/abs/nature05934.html

pubmed/19041776, 3, 595-605.

205-223.

5181-5186.

S0092-8674(10)00491-5, 141, 943-955.

21358755, 21, 396-420.


[16] Yamanaka, S. Elite and stochastic models for induced pluripotent stem cells genera‐ tion. Nature (2009). http://www.ncbi.nlm.nih.gov/pubmed/19571877, 460, 49-52.

[4] Verma, A. Verma N: Induced pluripotent stem cells and promises of neuroregenera‐ tive medicine. Neurol India (2011). http://www.ncbi.nlm.nih.gov/pubmed/21891933

[5] Park In-HArora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch M W, Cow‐ an C, Hochedlinger K and Daley G Q. Disease-Specific Induced Pluripotent Stem

[6] Scheper, W, & Copray, S. The Molecular Mechanism of Induced Pluripotency: A two-Stage Switch Stem Cell. Rev and Rep (2009). http://www.ncbi.nlm.nih.gov/pubmed/

[7] Hanna, J, Wernig, M, Markoulaki, S, Sun, C-W, Meissner, A, Cassady, J P, Beard, C, Brambrink, T, Wu, L-C, Townes, T M, & Jaenisch, R. Treatment of Sickle Cell Anemia Mouse Model with iPS Cells Generated from Autologous Skin. Science (2007). http://

[8] Maherali, N, Sridharan, R, Xie, W, Utikal, J, Eminli, S, Arnold, K, Stadfeld, M, Ya‐ chechko, R, Tchieu, J, Jaenisch, R, Plath, K, & Hochedlinger, K. Directly Reprogram‐ med Fibroblasts Show Global Epigenetic Remodeling and Widespread Tissue Contribution. Cell Stem Cell (2007). http://www.ncbi.nlm.nih.gov/pubmed/18371336,

[9] Hochedlinger, K, & Plath, K. Epigenetic reprogramming and induced pluripotency. Development (2009). http://www.ncbi.nlm.nih.gov/pubmed/19168672, 136(4), 509-23.

[10] Shafa, M, & Krawetz, R. and Rancourt D E: Returning to the stem state. Epigenetics of recapitulating pre-differentiation chromatin structure. BioEssays (2010). http://

[11] Shi, Y, Desponts, C, Do, J T, Hahm, H S, Schöler, H. R, & Ding, S. Induction of pluri‐ potent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with smallmolecule compounds. Cell Stem Cell (2008). http://www.ncbi.nlm.nih.gov/pubmed/

[12] Takahashi, K. Yamanaka S: Induction of Pluripotent Stem Cells from Mouse Embry‐ onic and Adult Fibroblast Cultures by Defined Factors. Cell (2006). http://

[13] Ehrensberger, A H, & Svejstrup, J Q. Reprogramming chromatin. Critical Reviews in Biochemistry and Molecular Biology (2012). Early Online http://

[14] Takahashi, K, Tanabe, K, Ohnuki, M, Narita, M, Ichisaka, T, Tomoda, K, & Yamana‐ ka, S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell (2007). http://www.ncbi.nlm.nih.gov/pubmed/18035408, 31(5), 861-72.

[15] Waddington, C. The strategy of the genes; a discussion of some aspects of theoretical

Cells. Cell (2008). http://www.ncbi.nlm.nih.gov/pubmed/18691744, 134, 1-10.

19551525, 5, 204-223.

198 Pluripotent Stem Cells

1, 55-70.

18983970

www.ncbi.nlm.nih.gov/pubmed/18063756

www.ncbi.nlm.nih.gov/pubmed/20652894, 32, 791-799.

www.ncbi.nlm.nih.gov/pubmed/16904174, 126, 663-676.

www.ncbi.nlm.nih.gov/pubmed/22757592, 1-19.

biology. London, Allen and Unwin (1957).


[29] Sims, R J. rd, Chen C F, Santos-Rosa H, Kouzarides T, Patel S S, Reinberg D. Human but not yeast CHD1 binds directly and selectively to histone H3 methylated at lysine 4 via its tandem chromodomains. J Biol Chem (2005). http://www.ncbi.nlm.nih.gov/ pubmed/16263726, 280, 41789-41792.

[39] Lee, J, Thompson, J R, Botuyan, M V, & Mer, G. Distinct binding modes specifies the recognition of methylated histones H3K4 and H4K20 by JMJD2A-tudor. Nat. Struct.

Stem Cells and Epigenetic Reprogramming http://dx.doi.org/10.5772/55983 201

Mol. Biol. (2008). http://www.ncbi.nlm.nih.gov/pubmed/18084306, 15, 109-111.

[40] Collins, R E, Northrop, J P, Horton, J R, Lee, D Y, Zhang, X, Stallcup, M R, & Cheng, X. The ankyrin repeats of G9a and GLP histone methyltransferases are mono- and di‐ methyllysine binding modules. Nat. Struct. Mol. Biol. (2008). http://

[41] Chang, B, Chen, Y, & Zhao, . . JMJD6 is a histone arginine demethylase. Science 2007;

[42] Loh, Y H, Zhang, W, Chen, X, George, J, Ng, H H, & Jmjd, a. and Jmjd2c histone H3 Lys 9 demethylases regulate self-renewal in embryonic stem cells. Genes Dev. (2007).

[43] Tsukada, Y, Fang, J, Erdjument-bromage, H, Warren, M E, Borchers, C H, Tempst, P, & Zhang, Y. Histone demethylation by a family of JmjC domain-containing proteins. Nature (2006). http://www.nature.com/nature/journal/n7078/abs/nature04433.html

[44] Leonhardt, H, Page, A W, Weier, H U, & Bestor, T H. A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell (1992).

[45] Okano, M, Bell, D W, Haber, D A, & Li, E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell

[46] Achour, M, Jacq, X, Rondé, P, Alhosin, M, Charlot, C, Chataigneau, T, Jeanblanc, M, Macaluso, M, Giordano, A, Hughes, A. D, Schini-kerth, V. B, & Bronner, C. The inter‐ action of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression. Oncogene (2008). http://

[47] Zhang, F, Pomerantz, J H, Sen, G, Palermo, A T, & Blau, H M. Active tissue-specific DNA demethylation conferred by somatic cell nuclei in stable heterokaryons. Proc Natl Acad Sci USA (2007). http://www.pnas.org/content/104/11/4395.short, 104,

[48] Rai, K, Huggins, I J, James, S R, Karpf, A R, Jones, D A, & Cairns, B R. DNA deme‐ thylation in zebrafish involves the coupling of a deaminase, a glycosylase, and gadd45. Cell (2008). http://www.ncbi.nlm.nih.gov/pubmed/19109892, 135, 1201-1212.

[49] Efroni, S, Duttagupta, R, Cheng, J, Dehghani, H, Hoeppner, D J, Dash, C, & Bazetjones, D P. Le Grice S, McKay R D, Buetow K, Gingeras T R, Misteli T, Mehorer E. Global transcription in pluripotent embryonic stem cells. Cell Stem Cell (2008). http://

318:444- 447. http://www.sciencemag.org/content/318/5849/444.short

http://www.ncbi.nlm.nih.gov/pubmed/17938240, 21, 2545-2557.

http://www.ncbi.nlm.nih.gov/pubmed/1423634, 71, 865-873.

www.ncbi.nlm.nih.gov/pubmed/17934516, 27, 2187-2197.

www.ncbi.nlm.nih.gov/pubmed/18462694, 2, 437-447.

4395-4400.

(1999). http://www.ncbi.nlm.nih.gov/pubmed/10555141, 99, 247-257.

www.ncbi.nlm.nih.gov/pubmed/18264113, 15, 245-250.


[39] Lee, J, Thompson, J R, Botuyan, M V, & Mer, G. Distinct binding modes specifies the recognition of methylated histones H3K4 and H4K20 by JMJD2A-tudor. Nat. Struct. Mol. Biol. (2008). http://www.ncbi.nlm.nih.gov/pubmed/18084306, 15, 109-111.

[29] Sims, R J. rd, Chen C F, Santos-Rosa H, Kouzarides T, Patel S S, Reinberg D. Human but not yeast CHD1 binds directly and selectively to histone H3 methylated at lysine 4 via its tandem chromodomains. J Biol Chem (2005). http://www.ncbi.nlm.nih.gov/

[30] Gaspar-maia, A. Alajem, Polesso F, Sridharan R, Mason M J, Heidersbach A, Ramal‐ ho-Santos J, McManus M T, Plath K, Meshorer E, Ramalho-Santos M. Chd1 regulates open chromatin and pluripotency of embryonic stem cells. Nature (2009). http://

[31] Chen, X, Xu, H, Yuan, P, Fang, F, Huss, M, Vega, V B, Wong, E, Orlove, Y L, Zhang, W, Jiang, J, Loh, Y H, Yeo, H C, Yeo, Z X, Narang, V, Govindarajan, K R, Leong, B, Shahab, A, Ruan, Y, Bourque, G, Sung, W K, Clarke, N D, Wei, C L, & Ng, H H. Inte‐ gration of external signalling pathways with the core transcriptional network in em‐ bryonic stem cells. Cell (2008). http://www.ncbi.nlm.nih.gov/pubmed/18555785, 133,

[32] Roth, S Y, Denu, J M, & Allis, C D. Histone Acetyltransferases. Annu. Rev. Biochem.

[33] Finnin, M S, Donigian, J R, Cohen, A, Richon, V M, Rifkind, R A, Marks, P A, Bre‐ slow, R, & Pavletich, N P. Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors. Nature (1999). http://www.ncbi.nlm.nih.gov/pubmed/

[34] Buggy, J J, Sideris, M L, Mak, P, Lorimer, D D, Mcintosh, B, & Clark, J M. Cloning and characterization of a novel human histone deacetylase, HDAC8.Biochem J

[35] Wade, P A. Transcriptional control at regulatory checkpoints by histone deacetylases: molecular connections between cancer and chromatin. Hum. Mol.Genet (2001).

[36] De Ruijter, A, Van Gennip, M, Caron, A H, Kemp, H N, & Van Kuilenburg, S. A B P. Histone deacetylases (HDACs) : Characterization of the classical HDAC family. Bio‐ chem J (2003). Pt 3): 737-749. http://www.ncbi.nlm.nih.gov/pmc/articles/

[37] Ng, S S, Yue, W W, Oppermann, U, & Klose, R J. Dynamic protein methylation in chromatin biology. Cell. Mol. Life Sci. (2009). http://www.ncbi.nlm.nih.gov/pmc/arti‐

[38] Lan, F, Collins, R E, De Cegli, R, Alpatov, R, Horton, J R, Shi, X, Gozani, O, Cheng, X, & Shi, Y. Recognition of unmethylated histone H3 lysine 4 links BHC80 to LSD1 mediated gene repression. Nature (2007). http://www.ncbi.nlm.nih.gov/pubmed/

(2001). http://www.ncbi.nlm.nih.gov/pubmed/11395403, 70, 81-120.

(2008). http://www.ncbi.nlm.nih.gov/pubmed/10926844, 350, 199-205.

http://hmg.oxfordjournals.org/content/10/7/693.short, 10, 693-698.

pubmed/16263726, 280, 41789-41792.

1106-1117.

200 Pluripotent Stem Cells

10490031, 401, 188-193.

PMC1223209/

cles/PMC2794343/, 66, 407-422.

17687328, 448, 718-722.

www.ncbi.nlm.nih.gov/pubmed/19587682, 460, 863-868.


[50] Moore, L D, Le, T, & Fan, G. DNA Methylation and Its Basic Function. Neuropsycho‐ pharmacology Reviews (2012). http://www.ncbi.nlm.nih.gov/pubmed/22781841, 1-16.

Development (2011). http://genesdev.cshlp.org/content/25/10/1035.short, 25,

Stem Cells and Epigenetic Reprogramming http://dx.doi.org/10.5772/55983 203

[62] Zhu, J-K. Active DNA Demethylation Mediated by DNA Glycosylases. Annu Rev Genet (2009). http://www.ncbi.nlm.nih.gov/pubmed/19659441, 43, 143-166.

[63] Bender, J. DNA methylation and epigenetics. Annu Rev Plant Biol (2004). http://

[64] Bird, A. DNA methylation patterns and epigenetic memory. Genes Dev (2002). http://

[65] Pereira, C F, Terranova, R, Ryan, N. K, Santos, J, Morris, K J, Cui, W, Merkenschlag‐ er, M, & Fisher, A G. Heterokaryo-based reprogramming of human B lymphocytes for pluripotency requires Oct4 but not Sox2. PLoS Genet (2008). e1000170. http://

[66] Ito, S, Shen, L, Dai, Q, Wu, S C, Collins, L B, Swenberg, J A, He, C, & Zhang, Y. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science (2011). http://www.sciencemag.org/content/333/6047/1300.short, 333,

[67] Bernstein, B E, Mikkelsen, T S, Xie, X, Kamal, M, Huebert, D J, Cuff, J, Fry, B, Meiss‐ ner, A, Wernig, M, Plath, K, Jaenish, R, Wagschal, A, Feil, R, Schreiber, S L, & Lander, E S. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell (2006). http://www.ncbi.nlm.nih.gov/pubmed/16630819, 125, 315-326.

[68] Ang, Y S, Tsai, S Y, Lee, D F, Monk, J, Su, J, Ratnakumar, K, Ding, J, Ge, Y, Darr, H, Chang, B, Wang, J, Rendl, M, Berstein, E, Schaniel, C, & Lemischka, I R. Wdr5 medi‐ ates self-renewal and reprogramming via the embryonic stem cell core transcription‐ al network. Cell (2011). http://www.ncbi.nlm.nih.gov/pubmed/21477851, 145,

[69] Wysocka, J, Swigut, T, Xiao, H, Milne, T A, Kwon, S Y, Landry, J, Kauer, M, Tackett, A J, Chair, B T, Badenhorst, P, Wu, C, & Allis, C D. A PHD finger of NURF couples histone H3 lysine 4 Trymethylation with chromatin remodeling. Nature (2006).

[70] Schuettengruber, B, Chourrout, D, Vervoort, M, Leblanc, B, & Cavalli, G. Genome regulation by polycomband trithorax proteins. Cell (2007). http://www.cell.com/

[71] Shen, X, Liu, Y, Hsu, Y J, Fujiwara, Y, Kim, J, Mao, X, Yuan, G. C, & Orkin, S H. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol Cell (2008). http://

[72] Boyer, L A, Plath, K, Seitlinger, J, Brambrink, T, Medeiros, L A, Lee, T I, Levine, S S, Wernig, M, Tajonar, A, Ray, M K, Bell, G W, Otte, A P, Vidal, M, Gifford, D K,

http://www.ncbi.nlm.nih.gov/pubmed/16728976, 442, 86-90.

www.ncbi.nlm.nih.gov/pubmed/19026780, 32, 491-502.

abstract/S0092-8674(07)00190-0, 128, 735-745.

www.ncbi.nlm.nih.gov/pubmed/15725056, 55, 41-68.

www.ncbi.nlm.nih.gov/pubmed/11782440, 16, 6-21.

www.ncbi.nlm.nih.gov/pubmed/18773085

1035-1040.

1300-1303.

183-197.


Development (2011). http://genesdev.cshlp.org/content/25/10/1035.short, 25, 1035-1040.

[62] Zhu, J-K. Active DNA Demethylation Mediated by DNA Glycosylases. Annu Rev Genet (2009). http://www.ncbi.nlm.nih.gov/pubmed/19659441, 43, 143-166.

[50] Moore, L D, Le, T, & Fan, G. DNA Methylation and Its Basic Function. Neuropsycho‐ pharmacology Reviews (2012). http://www.ncbi.nlm.nih.gov/pubmed/22781841, 1-16.

[51] Kim, J B, Greber, B, Araúzo-bravo, M J, Meyer, J, Park, K I, Zaehres, H, & Schöler, H R. Direct reprogramming of human neural stem cells by Oct4. Nature (2009). http://

[52] Boyer, L A, Lee, T I, Cole, M F, Johnstone, S E, Levine, S S, Zucker, J P, Guenther, M G, Kumar, R M, Murray, H L, Jenner, R G, Gifford, D K, Melton, D A, Jaenish, R, & Young, R A. Core Transcriptional regulatory circuitry in human embryonic stem cells. Cell (2005). http://www.ncbi.nlm.nih.gov/pubmed/16153702, 122, 947-956.

[53] Fong, Y W, Inouye, C, Yamaguchi, T, Cattoglio, C, Grubisic, I, & Tjian, R. A DNA re‐ pair complex functions as an Oct4/Sox2 coactivator in embryonic stem cells. Cell

[54] Levasseur, D N, Wang, J, Dorschner, M O, Stamatoyannopoulos, J A, & Orkin, S H. Oct4 dependence of chromatin structure within the extended Nanog locus in ES cells. Gene Dev (2008). http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2259027/, 22, 575-580.

[55] Liang, J, Wan, M, Zhang, Y, Gu, P, Xin, H, Jung, S Y, Qin, J, Wong, J, Cooney, A J, Liu, D, & Songyang, Z. Nanog and Oct4 associate with unique transcriptional repres‐ sion complexes in embryonic stem cells. Nat Cell Biol (2008). http://

[56] Lin, C H, Lin, C, Tanaka, H, Fero, M L, & Eisenman, R N. Gene regulation and epige‐ netic remodeling in murine embryonic stem cells by c-myc. PLoS ONE (2009). e7839. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0007839

[57] Kim, J, Woo, A J, Chu, J, Snow, J W, Fujiwara, Y, Kim, C G, Cantor, A B, & Orkin, S H. A Myc network accounts for similarities between embryonic stem and cancer cell transcription programs. Cell (2010). http://www.cell.com/retrieve/pii/

[58] Stadtfeld, M, & Hochedlinger, K. Induced pluripotency: history, mechanisms, and applications. Genes Dev. (2010). http://www.ncbi.nlm.nih.gov/pubmed/20952534,

[59] Rahl, P B, Lin, C Y, Seila, A C, Flynn, R A, Mccuine, S, Burge, C. B, & Sharp, P A. Young R A. c-Myc regulates transcriptional pause release. Cell (2010). http://

[60] Niwa, H, Ogawa, K, Shimosato, D, & Adachi, K. A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells. Nature (2009). http://

[61] Pawlak, M, & Jaenisch, R. De novo DNA methylation by Dnmt3a and Dnmt3b is dis‐ pensable for nuclear reprogramming of somatic cells to a pluripotent state. Genes &

www.ncbi.nlm.nih.gov/pmc/articles/PMC2864022/, 141, 432-445.

www.ncbi.nlm.nih.gov/pubmed/19571885, 460, 118-122.

(2011). http://www.ncbi.nlm.nih.gov/pubmed/21962512, 147, 120-131.

www.ncbi.nlm.nih.gov/pubmed/18454139, 10, 731-739.

S0092867410010585, 143, 313-324.

24(20), 2239-2263.

202 Pluripotent Stem Cells

www.nature.com/nature/journal/n7264/abs/nature08436.html


Young, R A, & Jaenish, R. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature (2006). http://www.ncbi.nlm.nih.gov/pubmed/ 16625203, 441, 349-353.

**Chapter 10**

**Epigenetic Reprogramming in Stem Cells**

This chapter will be focused on epigenetic mechanisms known to affect self-renewal and developmental potency of embryonic-like stem cells, and germ cells which mimic similar epigenetic signatures as pluripotent stem cells. Examples of epigenetic regulation have proven crucial for defining the stem cell state. In particular, a wealth of knowledge regarding stem cell-specific epigenetic modifications has occurred over the past decade with discoveries that include describing unique stem cell-specific chromosome structure, DNA and histone modifications and noncoding RNAs. The impact of these findings and the better understand‐ ing of epigenetic regulation in pluripotent stem cells provides a foundation for discovering mechanisms which regulate human development and differentiation in addition to those that

In eukaryotes, chromosomes consist of repeating chromatin units called nucleosomes, which encompass segments of DNA (~147 bp) wound around a central core of eight histone (**H**) proteins (two each of core histone proteins H2A, H2B, H3, and H4). These units are separated by a linker DNA associated with histone protein, H1. Post-translational modifications of these histone residues regulate gene expression [1, 2]. The types of modification of specific amino acids within these proteins include acetylation (*Ac*), mono- di- or tri-methylation (*Me1, 2, 3*), and ubiquitylation (**Ub**). Although these mechanism in general appear to work independently, recent evidence has demonstrated that crosstalk does exist between some of these modifica‐ tions [1, 3]. Chromatin can be distinguished based on its anatomical structure as either heterochromatin or euchromatin. Heterochromatin is characterized by tightly packed nucle‐ osomes that occur at centromeres, telomeres, and areas of repetitive DNA and is associated with low gene transcription while euchromatin constitute less compacted areas of chromatin and associated with active gene expression. In general, these areas of chromatin are nonrandomly distributed within the nucleus and cell-type and cell-cycle dependent [4, 5].

> © 2013 Eckert et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Eckert et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

Richard L. Eckert, Yasin Kizilyer and Candace L. Kerr

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56327

can facilitate cellular reprogramming.

**1. Introduction**


## **Epigenetic Reprogramming in Stem Cells**

Richard L. Eckert, Yasin Kizilyer and Candace L. Kerr

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56327

## **1. Introduction**

Young, R A, & Jaenish, R. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature (2006). http://www.ncbi.nlm.nih.gov/pubmed/

[73] Mansour, A A, Gafni, O, Weinberger, L, Zviran, A, Ayyash, M, Rais, Y, Krupalnik, V, Zerbib, M, Amann-zalcenstein, D, Maza, I, Geula, S, Viukov, S, Holtzman, L, Priblu‐ da, A, Canaani, E, Horn-saban, S, Amit, I, Novershtern, N, Hanna, J H, & The, H. K27 demethylase Utx regulates somatic and germ cell epigenetic reprogramming. Nature (2012). http://www.nature.com/nature/journal/vaop/ncurrent/full/nature11272.html

[74] Shi, Y, Do, J T, Desponts, C, Hahm, H S, Schöler, H R, & Ding, S. A combined chemi‐ cal and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell, (2008b). http://www.ncbi.nlm.nih.gov/pubmed/18522845, 2, 525-528.

[75] Doyon, Y, & Coté, J. The highly conserved and multifunctional NuA4 HAT complex. Curr Opin Genet Dev (2004). http://www.ncbi.nlm.nih.gov/pubmed/15196461, 14,

[76] Hezroni, H, Tzchori, I, Davidi, A, Mattout, A, Biran, A, Nissim-rafinia, M, Westphal, H, & Meshorer, E. H. K9 histone acetylation predicts pluripotency and reprogram‐ ming capacity of ES cells. Nucelus (2011). http://www.ncbi.nlm.nih.gov/pubmed/

[77] Mattout, A, Biran, A, & Meshorer, E. Global epigenetic changes during somatic cell reprogramming to iPS cells. J Mol Cell Biol (2011). http://www.ncbi.nlm.nih.gov/

[78] Ho, L, Miller, E L, Ronan, J L, Ho, W Q, Jothi, R, & Crabtree, G R. esBAF facilitates pluripotency by conditioning the genome for LIF/STAT3 signalling and by regulat‐ ing polycomb function. Nat Cell Biol (2011). http://www.ncbi.nlm.nih.gov/pubmed/

[79] Onder, T. T, Kara, N, Cherry, A, Sinha, A U, Zhu, N, Bernt, K M, Cahan, P, Marcarci, B O, Unternaehrer, J, Gupta, P B, Lander, E S, Armstrong, S A, & Daley, G Q. Chro‐ matin-modifying enzymes as modulators of reprogramming. Nature (2012). http://

[80] Judson, R L, Babiarz, J E, Venere, M, & Blelloch, R. Embryonic stem cell-specific mi‐ croRNAs promote induced pluripotency. Nat. Biotechnol (2009). http://

[81] Lin, S-L, Chang, D C, Lin, C H, Ying, S Y, Leu, D, & Wu, D T. S. Regulation of somat‐ ic cell reprogramming through inducible mir-302 expression. Nucleic Acids Research

[82] Suh, M R, Lee, Y, Kim, J Y, Kim, S K, Moon, S H, Lee, J Y, Cha, K Y, Chung, H M, Yoon, H S, & Moon, S Y. Human embryonic stem cells express a unique set of microRNAs. Dev.

(2011). http://nar.oxfordjournals.org/content/39/3/1054.short, 39, 1054-1065.

Biol. (2004). http://www.ncbi.nlm.nih.gov/pubmed/15183728, 270, 488-498.

www.ncbi.nlm.nih.gov/pubmed/22388813, 483, 598-602.

www.ncbi.nlm.nih.gov/pubmed/19363475, 27, 459-461.

16625203, 441, 349-353.

204 Pluripotent Stem Cells

147-154.

21941115

pubmed/22044880, 3, 341-350.

21785422, 13, 903-913.

This chapter will be focused on epigenetic mechanisms known to affect self-renewal and developmental potency of embryonic-like stem cells, and germ cells which mimic similar epigenetic signatures as pluripotent stem cells. Examples of epigenetic regulation have proven crucial for defining the stem cell state. In particular, a wealth of knowledge regarding stem cell-specific epigenetic modifications has occurred over the past decade with discoveries that include describing unique stem cell-specific chromosome structure, DNA and histone modifications and noncoding RNAs. The impact of these findings and the better understand‐ ing of epigenetic regulation in pluripotent stem cells provides a foundation for discovering mechanisms which regulate human development and differentiation in addition to those that can facilitate cellular reprogramming.

In eukaryotes, chromosomes consist of repeating chromatin units called nucleosomes, which encompass segments of DNA (~147 bp) wound around a central core of eight histone (**H**) proteins (two each of core histone proteins H2A, H2B, H3, and H4). These units are separated by a linker DNA associated with histone protein, H1. Post-translational modifications of these histone residues regulate gene expression [1, 2]. The types of modification of specific amino acids within these proteins include acetylation (*Ac*), mono- di- or tri-methylation (*Me1, 2, 3*), and ubiquitylation (**Ub**). Although these mechanism in general appear to work independently, recent evidence has demonstrated that crosstalk does exist between some of these modifica‐ tions [1, 3]. Chromatin can be distinguished based on its anatomical structure as either heterochromatin or euchromatin. Heterochromatin is characterized by tightly packed nucle‐ osomes that occur at centromeres, telomeres, and areas of repetitive DNA and is associated with low gene transcription while euchromatin constitute less compacted areas of chromatin and associated with active gene expression. In general, these areas of chromatin are nonrandomly distributed within the nucleus and cell-type and cell-cycle dependent [4, 5].

© 2013 Eckert et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Eckert et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In mammals, heterochromatin is associated with high levels of some methylation marks, including lysine (**K**)-9, K-27 and K-20 on histone (**H**) -3 (H3K9me3, H3K27me3, H3K20me3), low levels of acetylation, and its associated proteins including heterochromatin protein 1 (**HP1**) [6]. In contrast, euchromatin is associated with high levels of acetylation and methylation marks, such as K-4, K-36, and K79 on Histone 3 (H3K4, H3K36 and H3K7)(reviewed by [1]). These modifications occur through the activity of the following enzymes, histone acetyl transferases (**HATs**), histone deacetylases (**HDACs**), histone methyltransferases (**HMT**) and histone demethylases (**HDMT**). Further chromatin regulation is also possible through modifications of the histone tails which are suspected to produce a 'histone code' that initiate higher order chromatin folding [7].

H3**K**9me2 marks, gene-poor and increased in size and abundance from ESCs to differentiated cells (from 4% genome coverage in mouse ESCs to at most 46%, in liver cells) [14]. Wen, Wu et al. described these LOCKs as large regions with K9 modifications up to 4.9 Mb that are conserved between human and mouse. Like LADS, LOCKS also show tissue-specific distri‐ butions and inversely correlate with gene expression. Specific knockouts of the H3K9 meth‐ yltransferase, G9a, abolished LOCK formation in mouse ESCs and caused gene derepression without any peripheral localization alteration [14, 15]. Although LADS and LOCKS are similar in their effects on gene expression and localization, the relationship between them and their

Epigenetic Reprogramming in Stem Cells http://dx.doi.org/10.5772/56327 207

Since the discovery of generating induced pluripotent stem (**iPS**) cells, the ability to reprogram a differentiated cell back towards a more embryonic-like state, it has been shown by a variety of laboratories that there is an extensive amount of epigenetic variability between different iPS lines and even among different clones. As a result, much research has been involved in understanding the global demethylation and methylation patterns in ESC to identify particular epigenetic marks and their effected genes to study their role in pluripotency and reprogram‐ ming. Methylation of DNA cytosine residues (**mCG**), particularly in CpG islands of promoters, is a well-established mechanism that represses gene transcription in adult cells [16]. Several studies have specifically shown in ESCs, that loss of DNA methyltransferases also compro‐ mises their ability to differentiate into mature cells without affecting their self-renewal [17].

In embryonic stem cells, lineage-specific gene expression program regulators are repressed, but poised for a rapid response to differentiate [18]. These areas of chromatin, have so called bivalent domains, consist of opposing chromatin marks; i.e. H3K4me for activation and H3K27me for silencing. In ESCs, these domains are believed to be responsible for preventing the transcription responsible for their early differentiation to a specific lineage while priming the area for activation when the appropriate cues are expressed. Consistent with this belief is the findings that the bulk of the protein-encoding genes of human ESCs, including transcrip‐ tionally inactive genes have H3K4me, H3K9ac and H3K14ac rich promoter regions in areas of the nucleosome adjacent and downstream of transcription start sites [19, 20]. Moreover, in ESCs, genes with bivalent gene promoters tend to have unmethylated CpG islands [21]. The initial step of active DNA demethylation in mammals occurs by the conversion of 5-methyl‐ cytosine of DNA (5mC) to 5-hydroxymethylcytosine (5hmC). A prime example of this in pluripotent stem cells has been shown in regulating the expression of the stem cell transcription factor, Nanog. Here, the demethylated state is critical for the upregulation of Nanog which is an essential regulator of ESC pluripotency and self-renewal, while its downregulation attributed to methylation of its promoter is required for ICM specification [22]. Recent studies demonstrate that demethylation of Nanog is in part contributed to the expression of the Tet methylcytosine dioxygenase 1 (TET1) enzyme which is a TET family member of enzymes that catalyze the conversion of 5mC to 5hmC. This enzyme has been found to demethylate Nanog promoter sites in mouse ES cells [23, 24]. Both TET1 and TET2 expression have also been shown to be regulated by Oct4 expression in mouse ES cells, downregulated following differentiation

function at the nuclear periphery is presently unclear.

**2.2. Bivalency**

## **2. Epigenetic control in pluripotent stem cells**

#### **2.1. Changes in chromatin ultrastructure in pluripotent stem cells**

With the discovery of culturing embryonic stem cells (ESCs), several groups have been able to show the progression of global changes in the chromatin architecture of these cells. Through these studies, it has been shown that undifferentiated pluripotent stem cells contain less heterochromatic regions and express less chromatin structural proteins. Moreover, binding of these proteins (i.e. HP1α, lamin B) to heterochromatic regions is weaker compared to lineagecommitted cells [8]. Additionally, pluripotent transcription factors and chromatin remodeling proteins are overexpressed in ESCs compared to more differentiated progenitor cells [9]. One study further showed that chromatin remodeler Chd1 knockdown results with heterochro‐ matin accumulation and skewed differentiation in mouse ESCs, which suggests functional relevancy to the 'open' chromatin structure [10]. Together, these studies show that pluripotent ESCs has an open and hyperdynamic chromatin structure which transforms into a more compact, repressive-like, chromatin state during differentiation.

As ESC chromatin is more transcriptionally permissive, it is also more sensitive to nuclease activity. This may also be in part due to differences that are seen in the chromatin localization in the nucleus. For instance, one study using the DamID (DNA adenine methyltransferase identification) technique showed that pluripotency genes, including Oct4 and Nanog, move to the nuclear lamina and are silenced while lineage-specific genes disassociate from the lamina and are expressed. This was specifically shown during the differentiation of mouse ESCs into terminal astrocytes which demonstrated cell type-specific relocations of these areas during differentiation [11]. These areas near the nuclear periphery were called Lamina Associated Domains (**LADS**). Further study showed that these areas were enriched with repressive histone modifications, H3K9me2 and H3K27me3 which had tissue-specific distributions. Additionally, they consisted of few, minimally expressed genes, which were also marked by these repressive histone modifications [11-13].

Genome-wide ChIP analyses have also described other lamina associated domains with significant overlap with the LADS domains. These regions referred as **L**arge **O**rganized **C**hromatin domains of H3**K**9me2 or **LOCKS,** which are also hallmarked with increased H3**K**9me2 marks, gene-poor and increased in size and abundance from ESCs to differentiated cells (from 4% genome coverage in mouse ESCs to at most 46%, in liver cells) [14]. Wen, Wu et al. described these LOCKs as large regions with K9 modifications up to 4.9 Mb that are conserved between human and mouse. Like LADS, LOCKS also show tissue-specific distri‐ butions and inversely correlate with gene expression. Specific knockouts of the H3K9 meth‐ yltransferase, G9a, abolished LOCK formation in mouse ESCs and caused gene derepression without any peripheral localization alteration [14, 15]. Although LADS and LOCKS are similar in their effects on gene expression and localization, the relationship between them and their function at the nuclear periphery is presently unclear.

#### **2.2. Bivalency**

In mammals, heterochromatin is associated with high levels of some methylation marks, including lysine (**K**)-9, K-27 and K-20 on histone (**H**) -3 (H3K9me3, H3K27me3, H3K20me3), low levels of acetylation, and its associated proteins including heterochromatin protein 1 (**HP1**) [6]. In contrast, euchromatin is associated with high levels of acetylation and methylation marks, such as K-4, K-36, and K79 on Histone 3 (H3K4, H3K36 and H3K7)(reviewed by [1]). These modifications occur through the activity of the following enzymes, histone acetyl transferases (**HATs**), histone deacetylases (**HDACs**), histone methyltransferases (**HMT**) and histone demethylases (**HDMT**). Further chromatin regulation is also possible through modifications of the histone tails which are suspected to produce a 'histone code' that initiate

With the discovery of culturing embryonic stem cells (ESCs), several groups have been able to show the progression of global changes in the chromatin architecture of these cells. Through these studies, it has been shown that undifferentiated pluripotent stem cells contain less heterochromatic regions and express less chromatin structural proteins. Moreover, binding of these proteins (i.e. HP1α, lamin B) to heterochromatic regions is weaker compared to lineagecommitted cells [8]. Additionally, pluripotent transcription factors and chromatin remodeling proteins are overexpressed in ESCs compared to more differentiated progenitor cells [9]. One study further showed that chromatin remodeler Chd1 knockdown results with heterochro‐ matin accumulation and skewed differentiation in mouse ESCs, which suggests functional relevancy to the 'open' chromatin structure [10]. Together, these studies show that pluripotent ESCs has an open and hyperdynamic chromatin structure which transforms into a more

As ESC chromatin is more transcriptionally permissive, it is also more sensitive to nuclease activity. This may also be in part due to differences that are seen in the chromatin localization in the nucleus. For instance, one study using the DamID (DNA adenine methyltransferase identification) technique showed that pluripotency genes, including Oct4 and Nanog, move to the nuclear lamina and are silenced while lineage-specific genes disassociate from the lamina and are expressed. This was specifically shown during the differentiation of mouse ESCs into terminal astrocytes which demonstrated cell type-specific relocations of these areas during differentiation [11]. These areas near the nuclear periphery were called Lamina Associated Domains (**LADS**). Further study showed that these areas were enriched with repressive histone modifications, H3K9me2 and H3K27me3 which had tissue-specific distributions. Additionally, they consisted of few, minimally expressed genes, which were also marked by

Genome-wide ChIP analyses have also described other lamina associated domains with significant overlap with the LADS domains. These regions referred as **L**arge **O**rganized **C**hromatin domains of H3**K**9me2 or **LOCKS,** which are also hallmarked with increased

higher order chromatin folding [7].

206 Pluripotent Stem Cells

**2. Epigenetic control in pluripotent stem cells**

**2.1. Changes in chromatin ultrastructure in pluripotent stem cells**

compact, repressive-like, chromatin state during differentiation.

these repressive histone modifications [11-13].

Since the discovery of generating induced pluripotent stem (**iPS**) cells, the ability to reprogram a differentiated cell back towards a more embryonic-like state, it has been shown by a variety of laboratories that there is an extensive amount of epigenetic variability between different iPS lines and even among different clones. As a result, much research has been involved in understanding the global demethylation and methylation patterns in ESC to identify particular epigenetic marks and their effected genes to study their role in pluripotency and reprogram‐ ming. Methylation of DNA cytosine residues (**mCG**), particularly in CpG islands of promoters, is a well-established mechanism that represses gene transcription in adult cells [16]. Several studies have specifically shown in ESCs, that loss of DNA methyltransferases also compro‐ mises their ability to differentiate into mature cells without affecting their self-renewal [17].

In embryonic stem cells, lineage-specific gene expression program regulators are repressed, but poised for a rapid response to differentiate [18]. These areas of chromatin, have so called bivalent domains, consist of opposing chromatin marks; i.e. H3K4me for activation and H3K27me for silencing. In ESCs, these domains are believed to be responsible for preventing the transcription responsible for their early differentiation to a specific lineage while priming the area for activation when the appropriate cues are expressed. Consistent with this belief is the findings that the bulk of the protein-encoding genes of human ESCs, including transcrip‐ tionally inactive genes have H3K4me, H3K9ac and H3K14ac rich promoter regions in areas of the nucleosome adjacent and downstream of transcription start sites [19, 20]. Moreover, in ESCs, genes with bivalent gene promoters tend to have unmethylated CpG islands [21]. The initial step of active DNA demethylation in mammals occurs by the conversion of 5-methyl‐ cytosine of DNA (5mC) to 5-hydroxymethylcytosine (5hmC). A prime example of this in pluripotent stem cells has been shown in regulating the expression of the stem cell transcription factor, Nanog. Here, the demethylated state is critical for the upregulation of Nanog which is an essential regulator of ESC pluripotency and self-renewal, while its downregulation attributed to methylation of its promoter is required for ICM specification [22]. Recent studies demonstrate that demethylation of Nanog is in part contributed to the expression of the Tet methylcytosine dioxygenase 1 (TET1) enzyme which is a TET family member of enzymes that catalyze the conversion of 5mC to 5hmC. This enzyme has been found to demethylate Nanog promoter sites in mouse ES cells [23, 24]. Both TET1 and TET2 expression have also been shown to be regulated by Oct4 expression in mouse ES cells, downregulated following differentiation alongside other stem cell markers, and is induced concomitantly with 5-hmC during fibroblast reprogramming into iPS cells [25].

PHF1, MTF2, Eed, YY1, RBBP4, RBBP7, PCL1, PCL2, PCL3, JARID2, AEBP2, and PHF19 and Suz12 [34, 35]. Initial steps in stem cell reprogramming involves the recruitment of histone deacetylase by activity of the PRC2 complex, which causes local deacetylation of chromatin and subsequent methylation of K27 of histone H3. This H3K27 methylation then recruits the Bmi-1 complex to the site, which leads to the monoubiquitination of Lys119 histone H2A, and in turn suppresses gene expression [36]. The coordinative action of these two complexes plays an important role in the regulation and maintenance of gene expression during development

Epigenetic Reprogramming in Stem Cells http://dx.doi.org/10.5772/56327 209

BAF complex > Nature Neuroscience 13, 1330–1337 (2010) doi:10.1038/nn.2671 Published online 26 October 2010

**Figure 1.** Chromatin remodeling factors of the TrxG, PRC1/2 and BAF complexes work together to regulate stem cell status. In pluripotent stem cells, genes necessary for lineage-specific regulation consist of 'bivalent' chromatin do‐ mains that contain repressed H3K27me3 marks, as well as active H3K4me3 marks. These genes are then 'primed' for rapid induction of expression upon receiving differentiation cues. Proteins of the TrxG family tri-methylate H3K4 lead‐ ing to active chromatin marks. PRC2 activity leads to repressive tri-methylation of H3K27 and subsequent recruitment of PRC1 to the nucleosome region. Upon recruitment, PRC1 transfers a mono-ubiquitin residue to histone 2A (H2AK119). Together, the binding of PRC1 and the ubiquitylation of H2AK119ub silences gene expression. BAF com‐ plexes directly unwind nucleosomal DNA by using ATP and helicase-like subunits. Together, these complexes coexist

Polycomb repressive complexes have been shown associated with many developmental regulator regions in ESCs, and many of the PcG repressed targets of ESCs are also 'bivalent' [30, 39]. For instance, PRC2 target genes have been shown to be preferentially turned on during ESC differentiation and that the pluripotent stem cell regulating genes Oct4, Sox2, and Nanog co-occupy a significant subset of these genes. Therefore, it has been suggested that the PRC2

TrxG & PRCs : *Cell Stem Cell.* 2012 Jul 6;11(1):16-21. doi: 10.1016/j.stem.2012.06.005.

and/or work hierarchically to regulate pluripotency and bivalency in stem cells.

and contributes to the epigenetic memory of stem cells [37, 38].

In addition to promoter regulation, methylC-Seq genome-wide analysis has also discovered novel types of DNA methylation regulation at non-CG sites (CHG and CHH sites where H = A, C, or T residues). These analyses showed that non-CG methylation accounted for 25% of the total ESC methylome and that these sites were more commonly found within gene bodies than within promoter sites [26]. Furthermore, the methylation of these sites was lost when differentiation was induced in ESCs, and restored during the generation of in induced pluripotent stem cells. This included many differentially methylated regions associated with genes involved in pluripotency and differentiation.

#### **2.3. Polycomb and trithorax group proteins in pluripotent stem cells**

Recent studies have established that developmental gene priming and bivalency are crucial for pluripotency whereby the chromatin of pluripotent stem cells are transcriptionally permissive, with normally silent DNA repeat regions, transcriptionally related histone modifications such as H3K9ac, H3K4me3, H3K36me3 and low stochastic transcription of lineage-restricted genes [8, 9]. The poised state is believed to inhibit the activity of RNA Polymerase II (**RNAP II**) and thereby deregulate elongation. In the poised state, RNAP II demonstrates high Ser5 phosphorylation and low levels of Ser2 phosphorylation which is in part controlled by a number of complexes involved in this process. For this, several groups or families of complexes involved in epigenetic regulation have been studied in pluripotent stem cells. These proteins include those which regulate histone modifications, DNA methylation and ATP-dependent chromatin remodelling and include the polycomb group (**PcG**) proteins, trithorax group (**TrxG**) proteins, and ATP-dependent enzymes of the BAF complex. In general, PcG proteins are usually associated with epigenetic gene silencing, while their antagonists TrxG and BAF complexes proteins are involved in epigenetic maintenance or activation of differentiation. The importance of these proteins in developmental regulation of gene expres‐ sion is well-established [27], while, their functions in adult and pluripotent stem cells is only recently been understood [28]. This includes studies which show that PcG, TrxG, and other chromatin remodeling factors including ATP-dependent enzymes are interconnected in their roles to regulate pluripotency (see Figure 1) [29-33].

PcG, TrxG, and BAF complex associated genes are conserved from fly to man and are important in the regulation of organogenesis and development. PcG proteins were initially discovered as repressors of the Hox or homoeotic genes in *Drosophila,* while TrxG and BAP (BAF *Droso‐ philia* homolog) proteins maintained Hox gene expression in the appropriate spatial domains. Hox genes encode a family of evolutionarily conserved regulators, which are involved in establishing body segmentation patterns during the development of the fly. In mammalians, these proteins also regulate genes involved in development and differentiation.

PcG proteins produce two distinct protein complexes that act sequentially to regulate gene expression – the "Bmi-1 complex" also known as Polycomb Repressive Complexes (**PRC1**) and the "Eed complex" also known as **PRC2**. The PRC1 or Bmi-1 complex includes Bmi-1, Ring1A/ B, Mph1/Rae28, Mel-18, M33, and Scmh1. The PRC2 or Eed complex includes Ezh1, Ezh2, PHF1, MTF2, Eed, YY1, RBBP4, RBBP7, PCL1, PCL2, PCL3, JARID2, AEBP2, and PHF19 and Suz12 [34, 35]. Initial steps in stem cell reprogramming involves the recruitment of histone deacetylase by activity of the PRC2 complex, which causes local deacetylation of chromatin and subsequent methylation of K27 of histone H3. This H3K27 methylation then recruits the Bmi-1 complex to the site, which leads to the monoubiquitination of Lys119 histone H2A, and in turn suppresses gene expression [36]. The coordinative action of these two complexes plays an important role in the regulation and maintenance of gene expression during development and contributes to the epigenetic memory of stem cells [37, 38].

alongside other stem cell markers, and is induced concomitantly with 5-hmC during fibroblast

In addition to promoter regulation, methylC-Seq genome-wide analysis has also discovered novel types of DNA methylation regulation at non-CG sites (CHG and CHH sites where H = A, C, or T residues). These analyses showed that non-CG methylation accounted for 25% of the total ESC methylome and that these sites were more commonly found within gene bodies than within promoter sites [26]. Furthermore, the methylation of these sites was lost when differentiation was induced in ESCs, and restored during the generation of in induced pluripotent stem cells. This included many differentially methylated regions associated with

Recent studies have established that developmental gene priming and bivalency are crucial for pluripotency whereby the chromatin of pluripotent stem cells are transcriptionally permissive, with normally silent DNA repeat regions, transcriptionally related histone modifications such as H3K9ac, H3K4me3, H3K36me3 and low stochastic transcription of lineage-restricted genes [8, 9]. The poised state is believed to inhibit the activity of RNA Polymerase II (**RNAP II**) and thereby deregulate elongation. In the poised state, RNAP II demonstrates high Ser5 phosphorylation and low levels of Ser2 phosphorylation which is in part controlled by a number of complexes involved in this process. For this, several groups or families of complexes involved in epigenetic regulation have been studied in pluripotent stem cells. These proteins include those which regulate histone modifications, DNA methylation and ATP-dependent chromatin remodelling and include the polycomb group (**PcG**) proteins, trithorax group (**TrxG**) proteins, and ATP-dependent enzymes of the BAF complex. In general, PcG proteins are usually associated with epigenetic gene silencing, while their antagonists TrxG and BAF complexes proteins are involved in epigenetic maintenance or activation of differentiation. The importance of these proteins in developmental regulation of gene expres‐ sion is well-established [27], while, their functions in adult and pluripotent stem cells is only recently been understood [28]. This includes studies which show that PcG, TrxG, and other chromatin remodeling factors including ATP-dependent enzymes are interconnected in their

PcG, TrxG, and BAF complex associated genes are conserved from fly to man and are important in the regulation of organogenesis and development. PcG proteins were initially discovered as repressors of the Hox or homoeotic genes in *Drosophila,* while TrxG and BAP (BAF *Droso‐ philia* homolog) proteins maintained Hox gene expression in the appropriate spatial domains. Hox genes encode a family of evolutionarily conserved regulators, which are involved in establishing body segmentation patterns during the development of the fly. In mammalians,

PcG proteins produce two distinct protein complexes that act sequentially to regulate gene expression – the "Bmi-1 complex" also known as Polycomb Repressive Complexes (**PRC1**) and the "Eed complex" also known as **PRC2**. The PRC1 or Bmi-1 complex includes Bmi-1, Ring1A/ B, Mph1/Rae28, Mel-18, M33, and Scmh1. The PRC2 or Eed complex includes Ezh1, Ezh2,

these proteins also regulate genes involved in development and differentiation.

reprogramming into iPS cells [25].

208 Pluripotent Stem Cells

genes involved in pluripotency and differentiation.

roles to regulate pluripotency (see Figure 1) [29-33].

**2.3. Polycomb and trithorax group proteins in pluripotent stem cells**

BAF complex > Nature Neuroscience 13, 1330–1337 (2010) doi:10.1038/nn.2671 Published online 26 October 2010 TrxG & PRCs : *Cell Stem Cell.* 2012 Jul 6;11(1):16-21. doi: 10.1016/j.stem.2012.06.005.

**Figure 1.** Chromatin remodeling factors of the TrxG, PRC1/2 and BAF complexes work together to regulate stem cell status. In pluripotent stem cells, genes necessary for lineage-specific regulation consist of 'bivalent' chromatin do‐ mains that contain repressed H3K27me3 marks, as well as active H3K4me3 marks. These genes are then 'primed' for rapid induction of expression upon receiving differentiation cues. Proteins of the TrxG family tri-methylate H3K4 lead‐ ing to active chromatin marks. PRC2 activity leads to repressive tri-methylation of H3K27 and subsequent recruitment of PRC1 to the nucleosome region. Upon recruitment, PRC1 transfers a mono-ubiquitin residue to histone 2A (H2AK119). Together, the binding of PRC1 and the ubiquitylation of H2AK119ub silences gene expression. BAF com‐ plexes directly unwind nucleosomal DNA by using ATP and helicase-like subunits. Together, these complexes coexist and/or work hierarchically to regulate pluripotency and bivalency in stem cells.

Polycomb repressive complexes have been shown associated with many developmental regulator regions in ESCs, and many of the PcG repressed targets of ESCs are also 'bivalent' [30, 39]. For instance, PRC2 target genes have been shown to be preferentially turned on during ESC differentiation and that the pluripotent stem cell regulating genes Oct4, Sox2, and Nanog co-occupy a significant subset of these genes. Therefore, it has been suggested that the PRC2 complex represses a distinct group of developmental genes that have to be repressed to maintain pluripotency. This would promote a poised or primed state which could be readily activated during early differentiation [40]. For example, the histone methyltransferase Ezh2 is known to catalyze H3K27me3. In fact, bivalency domains at PRC2 regulated promoters are roughly five times more likely to become DNA methylated during differentiation than those with non-PRC2 regulated promoters [21] suggesting that the PRC2 complex plays a pivotal role in the switch for early lineage commitment [41]. Jarid2, a member of the Jumonji family of histone demethylases, has also been shown to play an important role in properly recruiting PRC1 and PRC2 and initiating the RNA Polymerase II activating form (Ser5P-RNAPII) [42] to bivalent loci to promote differentiation [43-45]. While Jarid2 is enzymatically inactive in ESCs, recent evidence has shown that Jarid2 is regulated by pluripotency factors in ESCs [43]. In null ESCs lacking Jarid2 expression were able to self-renew but unable to differentiate despite expressing appropriate PRC2 target genes demonstrating that transcriptional priming of bivalent genes in ESCs was dependent on Jarid2 expression.

**3. Epigenetic control in primordial germ cells and pluripotent embryonic**

Epigenetic Reprogramming in Stem Cells http://dx.doi.org/10.5772/56327 211

Primordial germ cells (**PGCs**) are the progenitors of sperm and egg. In humans, these cells first appear around the fifth week after conception and in mouse, during embryonic day (**E**) 5. At this time, PGCs emerge from differentiated epiblast cells which have already begun to undergo major epigenetic changes including DNA methylation and X inactivation [57]. Thus, epigenetic regulation or reprogramming must occur in PGCs in order to achieve an undifferentiated totipotent-like state [57, 58]. X activation and demethylation in PGCs is similar to that seen in the process of generating ESCs and reprogramming somatic cells into pluripotent iPS cells [59]. Together, these epigenetic changes in germ cell development is to prevent the transmission of aberrant epigenetic modifications to the next generation and to promote epigenetic equiva‐ lency in the germ line of male and female embryos, which is necessary for proper imprinting. This is the only time in which homologous chromosomes are epigenetically indistinguishable

Key initiators of PGC induction in the epiblast include the Blimp1 (**B-lymphocyte-induced maturation protein 1**), also known as PR domain zinc finger protein 1 (**Prdm1**), Prdm14 and protein arginine methyltransferase 5 (**Prmt5**). These factors have been shown to initiate epigenetic reprogramming and induce repression of the somatic program during germ cell specification [62-64], and in a similar fashion to facilitate somatic cell reprogramming in iPS cells and in epiblast stem cell generation [65, 66]. Their effects occur through both direct and indirect targets. For instance, it is known that BLIMP1 associates with the arginine methyl‐ transferase PRMT5 to reduce expression of Hox-family genes and other somatic genes in PGCs

In addition to pluripotent associated genes, early germ-cell development and imprinted genes also undergo demethylation during this time. These include well-established postmigratory germ cell genes Mvh (also known as Ddx4), Sycp3 (synaptonemal complex protein 3) and Dazl (deleted in azoospermia-like). These genes are demethylated in germ cells and repressed in somatic cells. This demethylation occurs during the migration of PGCs into the gonad at CpG islands of their promoters as well as at differentially methylated regions (**DMRs**) of imprinted genes [68, 69]. Whether DNA methylation in PGCs is erased by an active or a passive mecha‐ nism is currently unclear. However, two DNA deaminases, activation-induced cytidine deaminase (**AID**) and apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (**APOBEC1**) may demonstrate a key role in this process. While both enzymes have been shown *in vitro* to deaminate 5-methylcytosine in DNA in mouse PGCs, deficiencies in AID expression has also shown that it is essential for erasure of DNA methylation [70]. Both, Aid and Apobec1 are located in a cluster of genes which comprise Stella, Growth differentiation factor 3 (**GDF3**) and Nanog. Stella, Gdf3 and Nanog are all expressed in pluripotent tissues as well as in germ cells. [71]. While Stella is a known constituent of germ cell development, Gdf3 and Nanog have important roles in conferring stem-cell identity on ES cells. It has also been suggested that *in vivo* targeting in the zygote of AID to the methylated DMR of the imprinted gene H19 results in efficient and substantial demethylation of this region [72]. Together these findings suggest

and in PGCs occurs primarily in the developing embryonic gonad [60, 61].

**germ cells**

via H2A/H4 R3 methylation [67].

In addition to the bivalent marks associated with PRC2 associated H3 modifications in pluripotent stem cells, bivalent genes in stem cells also exhibit repressive marks of H2AK119Ub1 at their promoter and throughout the coding region. For this reason, members of the PRC1 complex Ring1A and Ring1B proteins which catalyze H2AK119ub1 have also reveal a role in regulating pluripotency [30, 46]. For instance, in ESCs, double mutants of Ring1A/B demonstrate reduced levels of H2AK119Ub1, repression of known stemness genes (including Oct4 targets), increased expression of developmental regulator targets, and spontaneous differentiation. Furthermore, upon differentiation, Ring1A/B lost binding to their target loci suggesting that a Ring 1/B mediated complex functions downstream of the stem cell core transcriptional machinery to maintain the ESC state [47].

In addition to PcG complexes, core members of the TrxG and BAF chromatin remodeling complexes have also been shown to contribute to the bivalent mark in stem cells by acting in concert to establish and preserve H3K4me3 [48, 49]. Another core member, WD repeat domain 5 (**Wdr5**) of the TrxG complex, has also been associated with the undifferentiated state and shown to regulate self-renewal in mouse ESCs [50]. This study went further to show that Wdr5 expression can promote efficient derivation of pluripotent iPS cells. Along with PcG and TrxG complexes, other chromatin remodeling complexes have been shown to have crucial roles in maintaining pluripotency. For instance, it has been shown that ESCs contain a unique BAF complex, which distinguishes them from differentiated cell types. This ESC complex consists of BAF60a, BAF155 and BRG subunits in the absence of BAF60c, BAF170 or BRM [48, 51]. Components of this ESC BAF complex also appear in RNAi screens for pluripotent genes [52, 53]. In mouse ESCs, it was also shown that BRG removal causes decreased self-renewal and aberrant differentiation, whereas BRG withdrawal from differentiated cells had very little effect [51]. Additionally, the pluripotency regulator genes Oct4, Sox2 and Nanog, have been shown to be targeted by components of the ESC-specific BAF complex [48, 51, 54-56] as well as facilitate IPS cell reprogramming [33].

## **3. Epigenetic control in primordial germ cells and pluripotent embryonic germ cells**

complex represses a distinct group of developmental genes that have to be repressed to maintain pluripotency. This would promote a poised or primed state which could be readily activated during early differentiation [40]. For example, the histone methyltransferase Ezh2 is known to catalyze H3K27me3. In fact, bivalency domains at PRC2 regulated promoters are roughly five times more likely to become DNA methylated during differentiation than those with non-PRC2 regulated promoters [21] suggesting that the PRC2 complex plays a pivotal role in the switch for early lineage commitment [41]. Jarid2, a member of the Jumonji family of histone demethylases, has also been shown to play an important role in properly recruiting PRC1 and PRC2 and initiating the RNA Polymerase II activating form (Ser5P-RNAPII) [42] to bivalent loci to promote differentiation [43-45]. While Jarid2 is enzymatically inactive in ESCs, recent evidence has shown that Jarid2 is regulated by pluripotency factors in ESCs [43]. In null ESCs lacking Jarid2 expression were able to self-renew but unable to differentiate despite expressing appropriate PRC2 target genes demonstrating that transcriptional priming of

In addition to the bivalent marks associated with PRC2 associated H3 modifications in pluripotent stem cells, bivalent genes in stem cells also exhibit repressive marks of H2AK119Ub1 at their promoter and throughout the coding region. For this reason, members of the PRC1 complex Ring1A and Ring1B proteins which catalyze H2AK119ub1 have also reveal a role in regulating pluripotency [30, 46]. For instance, in ESCs, double mutants of Ring1A/B demonstrate reduced levels of H2AK119Ub1, repression of known stemness genes (including Oct4 targets), increased expression of developmental regulator targets, and spontaneous differentiation. Furthermore, upon differentiation, Ring1A/B lost binding to their target loci suggesting that a Ring 1/B mediated complex functions downstream of the stem cell

In addition to PcG complexes, core members of the TrxG and BAF chromatin remodeling complexes have also been shown to contribute to the bivalent mark in stem cells by acting in concert to establish and preserve H3K4me3 [48, 49]. Another core member, WD repeat domain 5 (**Wdr5**) of the TrxG complex, has also been associated with the undifferentiated state and shown to regulate self-renewal in mouse ESCs [50]. This study went further to show that Wdr5 expression can promote efficient derivation of pluripotent iPS cells. Along with PcG and TrxG complexes, other chromatin remodeling complexes have been shown to have crucial roles in maintaining pluripotency. For instance, it has been shown that ESCs contain a unique BAF complex, which distinguishes them from differentiated cell types. This ESC complex consists of BAF60a, BAF155 and BRG subunits in the absence of BAF60c, BAF170 or BRM [48, 51]. Components of this ESC BAF complex also appear in RNAi screens for pluripotent genes [52, 53]. In mouse ESCs, it was also shown that BRG removal causes decreased self-renewal and aberrant differentiation, whereas BRG withdrawal from differentiated cells had very little effect [51]. Additionally, the pluripotency regulator genes Oct4, Sox2 and Nanog, have been shown to be targeted by components of the ESC-specific BAF complex [48, 51, 54-56] as well

bivalent genes in ESCs was dependent on Jarid2 expression.

210 Pluripotent Stem Cells

core transcriptional machinery to maintain the ESC state [47].

as facilitate IPS cell reprogramming [33].

Primordial germ cells (**PGCs**) are the progenitors of sperm and egg. In humans, these cells first appear around the fifth week after conception and in mouse, during embryonic day (**E**) 5. At this time, PGCs emerge from differentiated epiblast cells which have already begun to undergo major epigenetic changes including DNA methylation and X inactivation [57]. Thus, epigenetic regulation or reprogramming must occur in PGCs in order to achieve an undifferentiated totipotent-like state [57, 58]. X activation and demethylation in PGCs is similar to that seen in the process of generating ESCs and reprogramming somatic cells into pluripotent iPS cells [59]. Together, these epigenetic changes in germ cell development is to prevent the transmission of aberrant epigenetic modifications to the next generation and to promote epigenetic equiva‐ lency in the germ line of male and female embryos, which is necessary for proper imprinting. This is the only time in which homologous chromosomes are epigenetically indistinguishable and in PGCs occurs primarily in the developing embryonic gonad [60, 61].

Key initiators of PGC induction in the epiblast include the Blimp1 (**B-lymphocyte-induced maturation protein 1**), also known as PR domain zinc finger protein 1 (**Prdm1**), Prdm14 and protein arginine methyltransferase 5 (**Prmt5**). These factors have been shown to initiate epigenetic reprogramming and induce repression of the somatic program during germ cell specification [62-64], and in a similar fashion to facilitate somatic cell reprogramming in iPS cells and in epiblast stem cell generation [65, 66]. Their effects occur through both direct and indirect targets. For instance, it is known that BLIMP1 associates with the arginine methyl‐ transferase PRMT5 to reduce expression of Hox-family genes and other somatic genes in PGCs via H2A/H4 R3 methylation [67].

In addition to pluripotent associated genes, early germ-cell development and imprinted genes also undergo demethylation during this time. These include well-established postmigratory germ cell genes Mvh (also known as Ddx4), Sycp3 (synaptonemal complex protein 3) and Dazl (deleted in azoospermia-like). These genes are demethylated in germ cells and repressed in somatic cells. This demethylation occurs during the migration of PGCs into the gonad at CpG islands of their promoters as well as at differentially methylated regions (**DMRs**) of imprinted genes [68, 69]. Whether DNA methylation in PGCs is erased by an active or a passive mecha‐ nism is currently unclear. However, two DNA deaminases, activation-induced cytidine deaminase (**AID**) and apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (**APOBEC1**) may demonstrate a key role in this process. While both enzymes have been shown *in vitro* to deaminate 5-methylcytosine in DNA in mouse PGCs, deficiencies in AID expression has also shown that it is essential for erasure of DNA methylation [70]. Both, Aid and Apobec1 are located in a cluster of genes which comprise Stella, Growth differentiation factor 3 (**GDF3**) and Nanog. Stella, Gdf3 and Nanog are all expressed in pluripotent tissues as well as in germ cells. [71]. While Stella is a known constituent of germ cell development, Gdf3 and Nanog have important roles in conferring stem-cell identity on ES cells. It has also been suggested that *in vivo* targeting in the zygote of AID to the methylated DMR of the imprinted gene H19 results in efficient and substantial demethylation of this region [72]. Together these findings suggest an important role of AID in facilitating demethylation and reprogramming the pluripotent state. Similar to AID, TET1 and TET2 may also play a facilitative role in PGC demethylation as both TET enzymes are expressed in mouse PGCs during imprint erasure, concurrent with 5hmC enrichment [73].

associated with repressing differentiation in ESCs. In a similar fashion, another PRC2 associ‐ ated factor, the transcription factor YY1 has also been shown to directly regulate miR-29 transcription through the recruitment of HDAC1 and EZH2 to the regulatory regions of the

Epigenetic Reprogramming in Stem Cells http://dx.doi.org/10.5772/56327 213

Studies in mice have shown that induction of neural differentiation in mouse ESCs with retinoic acid results in increased miR-134, miR-296, and miR-470 which in turn interact with the coding sequences of the pluripotency transcription factors Oct4, Sox2, and Nanog. These results suggest that through interaction of the miRNAs these pluripotent stem cells genes are downregulated thereby permitting differentiation to proceed [91]. Additionally, the ESCspecific miR-290 cluster has also been shown to regulate Oct4 methylation in differentiating ESCs [94]. Other studies have shown that mouse ESCs deficient in proteins of the miRNA processing apparatus such as Dicer, Drosha, DGCR8, and Ddx5 exhibit differentiation and

Interestingly, PcG proteins have been shown to be both regulators of miRNA expression as well as their targets. For instance, miRNA-101 has been shown to directly regulate the

Progenitor cells and adult stem cells are thought to be predecessors of pluripotent or multi‐ potent stem cells that are generated during early differentiation. During their transition in development, bivalently marked stem cell genes can become either active, or inactive, or remain bivalent, dependent in part, on the activity of key enzymes which drive these chromatin modifications such as lysine demethylases (**KDMs**), histone deubiquitylases (**DUBs**), and DNA methyltransferases (**DNMTs**). Bivalent chromatin, although present in progenitor cells, is less frequent than in pluripotent stem cells. This has been shown in mesenchymal stem cells, hematopoietic stem cells and neural progenitors, in which cases the bivalency continues to resolve upon further differentiation [100]. During this process in pluripotent stem cells, active genes exhibit diminishing suppressive chromatin marks, an increase in H3K4me3, gain of H3K36me3 within coding sequences of DNA, and contain RNAP II that carries high Ser5 phosphorylation and low levels of Ser2 phosphorylation near promoter and within coding regions. Moreover, inactive genes show loss of active chromatin marks while retaining

Specific differences occur in the chromatin states between pluripotent stem cells, progenitor cells and more differentiated cell types which include active, repressed and poised states of chromatin. Several lines of evidence suggest that priming in the poised state enables genes to respond rapidly when differentiation cues are presented [30]. For example, during neural induction, several hundred genes including those required to maintain stem cell-ness become de novo mCG and therefore transcriptionally silenced. Furthermore, the observation was made that neural precursors that are derived from ESCs acquired more mCG than terminal

expression of the PRC protein EZH2 in highly aggressive cancers [98, 99].

**5. Epigenetic regulation in progenitor and adult stem cells**

repressive ones, and in some cases gaining CpG methylation (mCpG).

miR-29 promoter [93].

developmental defects [95-97].

Another important epigenetic process required for germ cell development and cellular reprogramming to the pluripotent state involves the X chromosome. In female adult cells, one of the two X chromosomes is inactivated to compensate for the differences in gene expression between sexes. For this purpose, X chromosome inactivation is initiated in early embryos by noncoding X-inactive specific transcript (Xist) RNA followed by chromatin modifications on the inactive X chromosome which leads to stable gene repression in somatic cells. Likewise, reactivation of the X chromosome is required for the totipotency of the female blastocyst and germ cell development. Reactivation of the X chromosome also occurs to establish pluripo‐ tency in iPS cells. During development, epigenetic reprogramming or re-activation of the inactive X-chromosome commences in PGCs during their migration through the hindgut along their route to the developing gonads where imprint erasure is completed [74]. In mouse PGCs, decreased Xist expression, and the displacement of PcG repressor proteins EED and SUZ12, results in the loss of the inactive X associated histone modification, H3K27me3 [74]. In humans, PcG proteins YY1, EZH2, and EED have also been found in the ovarian follicles, oocytes and preimplantation embryos. YY1 and EZH2 transcripts were additionally detected in human metaphase II oocytes suggesting they may be play a similar role in human germ cell reprog‐ ramming [75].

*In vitro*, PGCs cultured under specific conditions can also demonstrate epigenetic reprogram‐ ming with pluripotent cell-like characteristics. In these cases, PGCs form pluripotent stem cell colonies called embryonic germ cells (EGCs) which have notable similarities in their epige‐ nomes [58, 76-82], and like ESCs, EGCs have been shown to induce epigenetic reprogramming of somatic nucleus in hybrid cells [60].

## **4. MicroRNAs and stem cells**

MicroRNAs (**miRNAs**) are a family of non-protein coding RNAs with transcripts of ~20–25 nucleotides that play essential roles in regulating gene expression (see [83-85]. A subset of miRNAs have been shown to be preferentially expressed in undifferentiated stem cells and for some, have been shown to play essential roles in pluripotency, proliferation, and modula‐ tion of expression patterns that are related to differentiation [86-89]. The promoter regions of these miRNAs are often occupied by the pluripotency transcription factors, including Oct4, Sox2, and Nanog [90]. In addition, during ESC differentiation, proteins modulated by miRNAs [91] have also been shown to be modulated by PcG proteins [92]. For instance, Marson *et al.* showed that approximately one quarter of the Oct4/Sox2/Nanog/Tcf3-occupied miRNAs belonged to a set of repressed miRNA genes bound by Suz12 in murine ES cells [90]. Here, the PRC2 complex protein SUZ12 was bound to a subset of inactive miRNAs controlling differ‐ entiation in mouse ESCs [90]. In this study, SUZ12 bound to the promoters of several miRNAs associated with repressing differentiation in ESCs. In a similar fashion, another PRC2 associ‐ ated factor, the transcription factor YY1 has also been shown to directly regulate miR-29 transcription through the recruitment of HDAC1 and EZH2 to the regulatory regions of the miR-29 promoter [93].

an important role of AID in facilitating demethylation and reprogramming the pluripotent state. Similar to AID, TET1 and TET2 may also play a facilitative role in PGC demethylation as both TET enzymes are expressed in mouse PGCs during imprint erasure, concurrent with

Another important epigenetic process required for germ cell development and cellular reprogramming to the pluripotent state involves the X chromosome. In female adult cells, one of the two X chromosomes is inactivated to compensate for the differences in gene expression between sexes. For this purpose, X chromosome inactivation is initiated in early embryos by noncoding X-inactive specific transcript (Xist) RNA followed by chromatin modifications on the inactive X chromosome which leads to stable gene repression in somatic cells. Likewise, reactivation of the X chromosome is required for the totipotency of the female blastocyst and germ cell development. Reactivation of the X chromosome also occurs to establish pluripo‐ tency in iPS cells. During development, epigenetic reprogramming or re-activation of the inactive X-chromosome commences in PGCs during their migration through the hindgut along their route to the developing gonads where imprint erasure is completed [74]. In mouse PGCs, decreased Xist expression, and the displacement of PcG repressor proteins EED and SUZ12, results in the loss of the inactive X associated histone modification, H3K27me3 [74]. In humans, PcG proteins YY1, EZH2, and EED have also been found in the ovarian follicles, oocytes and preimplantation embryos. YY1 and EZH2 transcripts were additionally detected in human metaphase II oocytes suggesting they may be play a similar role in human germ cell reprog‐

*In vitro*, PGCs cultured under specific conditions can also demonstrate epigenetic reprogram‐ ming with pluripotent cell-like characteristics. In these cases, PGCs form pluripotent stem cell colonies called embryonic germ cells (EGCs) which have notable similarities in their epige‐ nomes [58, 76-82], and like ESCs, EGCs have been shown to induce epigenetic reprogramming

MicroRNAs (**miRNAs**) are a family of non-protein coding RNAs with transcripts of ~20–25 nucleotides that play essential roles in regulating gene expression (see [83-85]. A subset of miRNAs have been shown to be preferentially expressed in undifferentiated stem cells and for some, have been shown to play essential roles in pluripotency, proliferation, and modula‐ tion of expression patterns that are related to differentiation [86-89]. The promoter regions of these miRNAs are often occupied by the pluripotency transcription factors, including Oct4, Sox2, and Nanog [90]. In addition, during ESC differentiation, proteins modulated by miRNAs [91] have also been shown to be modulated by PcG proteins [92]. For instance, Marson *et al.* showed that approximately one quarter of the Oct4/Sox2/Nanog/Tcf3-occupied miRNAs belonged to a set of repressed miRNA genes bound by Suz12 in murine ES cells [90]. Here, the PRC2 complex protein SUZ12 was bound to a subset of inactive miRNAs controlling differ‐ entiation in mouse ESCs [90]. In this study, SUZ12 bound to the promoters of several miRNAs

5hmC enrichment [73].

212 Pluripotent Stem Cells

ramming [75].

of somatic nucleus in hybrid cells [60].

**4. MicroRNAs and stem cells**

Studies in mice have shown that induction of neural differentiation in mouse ESCs with retinoic acid results in increased miR-134, miR-296, and miR-470 which in turn interact with the coding sequences of the pluripotency transcription factors Oct4, Sox2, and Nanog. These results suggest that through interaction of the miRNAs these pluripotent stem cells genes are downregulated thereby permitting differentiation to proceed [91]. Additionally, the ESCspecific miR-290 cluster has also been shown to regulate Oct4 methylation in differentiating ESCs [94]. Other studies have shown that mouse ESCs deficient in proteins of the miRNA processing apparatus such as Dicer, Drosha, DGCR8, and Ddx5 exhibit differentiation and developmental defects [95-97].

Interestingly, PcG proteins have been shown to be both regulators of miRNA expression as well as their targets. For instance, miRNA-101 has been shown to directly regulate the expression of the PRC protein EZH2 in highly aggressive cancers [98, 99].

## **5. Epigenetic regulation in progenitor and adult stem cells**

Progenitor cells and adult stem cells are thought to be predecessors of pluripotent or multi‐ potent stem cells that are generated during early differentiation. During their transition in development, bivalently marked stem cell genes can become either active, or inactive, or remain bivalent, dependent in part, on the activity of key enzymes which drive these chromatin modifications such as lysine demethylases (**KDMs**), histone deubiquitylases (**DUBs**), and DNA methyltransferases (**DNMTs**). Bivalent chromatin, although present in progenitor cells, is less frequent than in pluripotent stem cells. This has been shown in mesenchymal stem cells, hematopoietic stem cells and neural progenitors, in which cases the bivalency continues to resolve upon further differentiation [100]. During this process in pluripotent stem cells, active genes exhibit diminishing suppressive chromatin marks, an increase in H3K4me3, gain of H3K36me3 within coding sequences of DNA, and contain RNAP II that carries high Ser5 phosphorylation and low levels of Ser2 phosphorylation near promoter and within coding regions. Moreover, inactive genes show loss of active chromatin marks while retaining repressive ones, and in some cases gaining CpG methylation (mCpG).

Specific differences occur in the chromatin states between pluripotent stem cells, progenitor cells and more differentiated cell types which include active, repressed and poised states of chromatin. Several lines of evidence suggest that priming in the poised state enables genes to respond rapidly when differentiation cues are presented [30]. For example, during neural induction, several hundred genes including those required to maintain stem cell-ness become de novo mCG and therefore transcriptionally silenced. Furthermore, the observation was made that neural precursors that are derived from ESCs acquired more mCG than terminal neurons, suggesting that the transition from pluripotent to lineage-committed cells is associ‐ ated with these changes [17, 21, 101, 102].

tumor suppressor genes [121]. In this case, EZH2 was shown to target a pro-differentiation tumor suppressor gene, retinoic acid receptor β2 (**RARβ2**) [120], which is reduced or lost in

Epigenetic Reprogramming in Stem Cells http://dx.doi.org/10.5772/56327 215

The pluripotent stem cells have a chromatin that is hyperdynamic, with a preponderance of modified histones and chromatin remodelers that ensures low-level transcription and tight regulation. Losing pluripotency is accompanied with a more compact, repressive, chromatin structure, which leads to cellular differentiation. Chromatin architecture is regulated at multiple levels in conjunction with known pluripotent genes to constitute an interwoven pluripotency network. Although there are many gaps in our knowledge of how epigenetic modifications regulate the pluripotent state, it is known that PcG repressor proteins prevent the precocious expression of lineage-restricted gene expression in pluripotent stem cells and germ cells by contributing to a unique 'primed' bivalent state of the chromatin. Future studies will provide mechanistic insights into the signaling cues required to maintain this state and inhibit differentiation while iPS cells and adult stem cells provide a renewed opportunity to study the role of chromatin architecture for controlling the pluripotent state. This will include understanding the mechanisms that interplay between pluripotent transcription factors, epigenetic regulators, and miRNAs to balance self-renewal and differentiation, properties

cdk, cyclin-dependent kinase; H2A-K119-Ub, ubiquitinylated histone H2A lysine 119; H3K27me3, tri-methylated histone H3 lysine K27; PcG, Polycomb group genes; ESC, embry‐ onic stem cells; EGC, embryonic germ cells; PGC, primordial germ cells; iPS, induced pluri‐ potent stem cells; CSC, cancer stem cells; RARβ2, retinoic acid receptor β2; Hh, Hedghog; KDMs, lysine demethylases; DUBs, histone deubiquitylases; DNMT, DNA methyltransferas‐ es; YY1, Ying Yang 1; EZH2 Enhancer of Zeste-2; EED, embryonic ectoderm development; GDF3, growth differentiation factor 3; DMR, differentially methylated regions; DAZL. deleted in azoospermia-like; Mvh, deadhead box 4; Sycp3, synaptonemal complex protein 3; E, embryonic day; RNAP II, RNA Polymerase II; trxG, trithorax group proteins; AID, activationinduced cytidine deaminase; **APOBEC1,** apolipoprotein B mRNA editing enzyme, catalytic

This work was supported by NIH grants R01 AR053851 and R01 CA131074 to R Eckert and R21HD057487 awarded to C Kerr as well as by the State of Maryland Stem Cell Research Fund

2010-MSCRFI-0110-00 to R Eckert and 2007-MSCRFII-0159-00 awarded to C Kerr.

many human malignancies.

which regulate reprogramming and carcinogenesis.

**7. Conclusion**

**Nomenclature**

polypeptide 1

**Acknowledgements**

Polycomb group proteins also appear to play a unique role in defining the progenitor or adult stem cell state. It has been shown that the PRC1 complex protein Bmi-1 activates multiple pathways that are important for regulating the stem cell-like state. For example, it has been shown that Bmi-1 is potentially upregulated via the pluripotent stem cell marker SALL4 signaling and has been shown to regulate stem cell self-renewal by repressing Hox genes, as well as INK4a locus genes, p16INK4a and p19ARF. BMI1 has also been shown to facilitate stem cell-like features in adult stem cells such as increased telomerase activity, transcriptional factor GATA3, and NF-kB pathways. These pathways are associated with the prevention of senes‐ cence, differentiation and apoptosis, while promoting immortalization and proliferation (for review see [103]).

## **6. Epigenetic dysregulation in cancer stem cells**

Cancer stem/initiating cells (CSC) have been defined as a subset of cancer cells that have clonal ability or self-renewal and are resilient against cancer therapies [104, 105]. As such CSCs are implicated in cancer initiation, metastasis, and recurrence of some cancers [106]. Although the most well established pluripotent stem cell genes OCT4, NANOG, cMYC and SOX2 are implicated in many poorly differentiated or metastatic cancers [107-109], they are not ex‐ pressed in all and they are not all elevated concordantly. In addition, targets of NANOG, OCT4, SOX2, and c-MYC are often overexpressed in tumors that are poorly differentiated, more so than in those that are well differentiated [110]. These genes also play a significant role in the induction of pluripotency into iPS cells from differentiated cell types and are thus involved in regulating epigenetic reprogramming [111-113]. More specifically, it is found that c-MYC, which is also an oncogene is sufficient for the reactivation of ESC-like transcriptional program in both, normal and cancer cells [114]. Additionally, studies have shown that one of the inherent issues with generating iPS cells is their propensity to become cancer stem cell-like [115, 116]. Taken together, these results indicate that aberrant activation of an ESC or iPS-like transcriptional program might cause induction of pathological self-renewal in adult differen‐ tiated cells, characteristic of cancer stem cells.

Aberrant function of PcG proteins has also been established in the malignancy of various cancers [117]. This is not surprising as it is well known that polycomb complexes contribute to the epigenetic regulation of key networks associated with self-renewal [118], differentiation, and proliferation [92, 119-123]. These roles for polycombs have been demonstrated in cancer cells and normal stem cells [124] and more recently studied for their targeted function in CSCs [125]. For instance, there is much evidence that overexpression of the EZH2 polycomb gene occurs in multiple human malignancies (see [117, 126]). One study showed that this may in part be atributed to a genomic loss of miR-101 which has been shown to lead to increased EZH2 levels [99, 127]. Although how EZH2 contributes to carcinogenesis remains poorly defined, recent evidence suggests that overexpression of EZH2 can contribute to improper silencing of tumor suppressor genes [121]. In this case, EZH2 was shown to target a pro-differentiation tumor suppressor gene, retinoic acid receptor β2 (**RARβ2**) [120], which is reduced or lost in many human malignancies.

## **7. Conclusion**

neurons, suggesting that the transition from pluripotent to lineage-committed cells is associ‐

Polycomb group proteins also appear to play a unique role in defining the progenitor or adult stem cell state. It has been shown that the PRC1 complex protein Bmi-1 activates multiple pathways that are important for regulating the stem cell-like state. For example, it has been shown that Bmi-1 is potentially upregulated via the pluripotent stem cell marker SALL4 signaling and has been shown to regulate stem cell self-renewal by repressing Hox genes, as well as INK4a locus genes, p16INK4a and p19ARF. BMI1 has also been shown to facilitate stem cell-like features in adult stem cells such as increased telomerase activity, transcriptional factor GATA3, and NF-kB pathways. These pathways are associated with the prevention of senes‐ cence, differentiation and apoptosis, while promoting immortalization and proliferation (for

Cancer stem/initiating cells (CSC) have been defined as a subset of cancer cells that have clonal ability or self-renewal and are resilient against cancer therapies [104, 105]. As such CSCs are implicated in cancer initiation, metastasis, and recurrence of some cancers [106]. Although the most well established pluripotent stem cell genes OCT4, NANOG, cMYC and SOX2 are implicated in many poorly differentiated or metastatic cancers [107-109], they are not ex‐ pressed in all and they are not all elevated concordantly. In addition, targets of NANOG, OCT4, SOX2, and c-MYC are often overexpressed in tumors that are poorly differentiated, more so than in those that are well differentiated [110]. These genes also play a significant role in the induction of pluripotency into iPS cells from differentiated cell types and are thus involved in regulating epigenetic reprogramming [111-113]. More specifically, it is found that c-MYC, which is also an oncogene is sufficient for the reactivation of ESC-like transcriptional program in both, normal and cancer cells [114]. Additionally, studies have shown that one of the inherent issues with generating iPS cells is their propensity to become cancer stem cell-like [115, 116]. Taken together, these results indicate that aberrant activation of an ESC or iPS-like transcriptional program might cause induction of pathological self-renewal in adult differen‐

Aberrant function of PcG proteins has also been established in the malignancy of various cancers [117]. This is not surprising as it is well known that polycomb complexes contribute to the epigenetic regulation of key networks associated with self-renewal [118], differentiation, and proliferation [92, 119-123]. These roles for polycombs have been demonstrated in cancer cells and normal stem cells [124] and more recently studied for their targeted function in CSCs [125]. For instance, there is much evidence that overexpression of the EZH2 polycomb gene occurs in multiple human malignancies (see [117, 126]). One study showed that this may in part be atributed to a genomic loss of miR-101 which has been shown to lead to increased EZH2 levels [99, 127]. Although how EZH2 contributes to carcinogenesis remains poorly defined, recent evidence suggests that overexpression of EZH2 can contribute to improper silencing of

ated with these changes [17, 21, 101, 102].

**6. Epigenetic dysregulation in cancer stem cells**

tiated cells, characteristic of cancer stem cells.

review see [103]).

214 Pluripotent Stem Cells

The pluripotent stem cells have a chromatin that is hyperdynamic, with a preponderance of modified histones and chromatin remodelers that ensures low-level transcription and tight regulation. Losing pluripotency is accompanied with a more compact, repressive, chromatin structure, which leads to cellular differentiation. Chromatin architecture is regulated at multiple levels in conjunction with known pluripotent genes to constitute an interwoven pluripotency network. Although there are many gaps in our knowledge of how epigenetic modifications regulate the pluripotent state, it is known that PcG repressor proteins prevent the precocious expression of lineage-restricted gene expression in pluripotent stem cells and germ cells by contributing to a unique 'primed' bivalent state of the chromatin. Future studies will provide mechanistic insights into the signaling cues required to maintain this state and inhibit differentiation while iPS cells and adult stem cells provide a renewed opportunity to study the role of chromatin architecture for controlling the pluripotent state. This will include understanding the mechanisms that interplay between pluripotent transcription factors, epigenetic regulators, and miRNAs to balance self-renewal and differentiation, properties which regulate reprogramming and carcinogenesis.

## **Nomenclature**

cdk, cyclin-dependent kinase; H2A-K119-Ub, ubiquitinylated histone H2A lysine 119; H3K27me3, tri-methylated histone H3 lysine K27; PcG, Polycomb group genes; ESC, embry‐ onic stem cells; EGC, embryonic germ cells; PGC, primordial germ cells; iPS, induced pluri‐ potent stem cells; CSC, cancer stem cells; RARβ2, retinoic acid receptor β2; Hh, Hedghog; KDMs, lysine demethylases; DUBs, histone deubiquitylases; DNMT, DNA methyltransferas‐ es; YY1, Ying Yang 1; EZH2 Enhancer of Zeste-2; EED, embryonic ectoderm development; GDF3, growth differentiation factor 3; DMR, differentially methylated regions; DAZL. deleted in azoospermia-like; Mvh, deadhead box 4; Sycp3, synaptonemal complex protein 3; E, embryonic day; RNAP II, RNA Polymerase II; trxG, trithorax group proteins; AID, activationinduced cytidine deaminase; **APOBEC1,** apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1

### **Acknowledgements**

This work was supported by NIH grants R01 AR053851 and R01 CA131074 to R Eckert and R21HD057487 awarded to C Kerr as well as by the State of Maryland Stem Cell Research Fund 2010-MSCRFI-0110-00 to R Eckert and 2007-MSCRFII-0159-00 awarded to C Kerr.

## **Author details**

Richard L. Eckert1,2,3,4, Yasin Kizilyer1 and Candace L. Kerr1\*

\*Address all correspondence to: ckerr@som.umaryland.edu

1 Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA

[9] Efroni S, Duttagupta R, Cheng J, Dehghani H, Hoeppner DJ, Dash C, et al. Global transcription in pluripotent embryonic stem cells. Cell Stem Cell. 2008 May 8;2(5):

Epigenetic Reprogramming in Stem Cells http://dx.doi.org/10.5772/56327 217

[10] Gaspar-Maia A, Alajem A, Polesso F, Sridharan R, Mason MJ, Heidersbach A, et al. Chd1 regulates open chromatin and pluripotency of embryonic stem cells. Nature.

[11] Peric-Hupkes D, Meuleman W, Pagie L, Bruggeman SW, Solovei I, Brugman W, et al. Molecular maps of the reorganization of genome-nuclear lamina interactions during

[12] Guelen L, Pagie L, Brasset E, Meuleman W, Faza MB, Talhout W, et al. Domain or‐ ganization of human chromosomes revealed by mapping of nuclear lamina interac‐

[13] Pickersgill H, Kalverda B, de Wit E, Talhout W, Fornerod M, van Steensel B. Charac‐ terization of the Drosophila melanogaster genome at the nuclear lamina. Nat Genet.

[14] Wen B, Wu H, Shinkai Y, Irizarry RA, Feinberg AP. Large histone H3 lysine 9 dime‐ thylated chromatin blocks distinguish differentiated from embryonic stem cells. Nat

[15] Yokochi T, Poduch K, Ryba T, Lu J, Hiratani I, Tachibana M, et al. G9a selectively re‐ presses a class of late-replicating genes at the nuclear periphery. Proc Natl Acad Sci

[16] Cedar H, Bergman Y. Linking DNA methylation and histone modification: patterns

[17] Trowbridge JJ, Orkin SH. DNA methylation in adult stem cells: New insights into

[18] Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell.

[19] Guenther MG, Levine SS, Boyer LA, Jaenisch R, Young RA. A chromatin landmark and transcription initiation at most promoters in human cells. Cell. 2007 Jul 13;130(1):

[20] Roelen BA, Lopes SM. Of stem cells and gametes: similarities and differences. Curr

[21] Mohn F, Weber M, Rebhan M, Roloff TC, Richter J, Stadler MB, et al. Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of

437-47.

2009 Aug 13;460(7257):863-8.

2006 Sep;38(9):1005-14.

Genet. 2009 Feb;41(2):246-50.

2006 Apr 21;125(2):315-26.

Med Chem. 2008;15(13):1249-56.

77-88.

U S A. 2009 Nov 17;106(46):19363-8.

self-renewal. Epigenetics. 2010 Apr 31;5(3).

and paradigms. Nat Rev Genet. 2009 May;10(5):295-304.

neuronal progenitors. Mol Cell. 2008 Jun 20;30(6):755-66.

differentiation. Mol Cell. 2010 May 28;38(4):603-13.

tions. Nature. 2008 Jun 12;453(7197):948-51.

2 Department of Reproductive Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA

3 Department of Dermatology, University of Maryland School of Medicine, Baltimore, Maryland, USA

4 Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA

#### **References**


[9] Efroni S, Duttagupta R, Cheng J, Dehghani H, Hoeppner DJ, Dash C, et al. Global transcription in pluripotent embryonic stem cells. Cell Stem Cell. 2008 May 8;2(5): 437-47.

**Author details**

216 Pluripotent Stem Cells

Richard L. Eckert1,2,3,4, Yasin Kizilyer1

Medicine, Baltimore, Maryland, USA

Medicine, Baltimore, Maryland, USA

Baltimore, Maryland, USA

Maryland, USA

**References**

693-705.

3;142(5):682-5.

Oct;24(10):511-7.

Jan 6;403(6765):41-5.

\*Address all correspondence to: ckerr@som.umaryland.edu

and their readers. Cell. 2010 Sep 17;142(6):967-80.

the genome. Curr Opin Genet Dev. 2010 Oct;20(5):562-9.

rammed cells. Curr Opin Genet Dev. 2011 Apr;21(2):140-6.

and Candace L. Kerr1\*

1 Department of Biochemistry and Molecular Biology, University of Maryland School of

2 Department of Reproductive Sciences, University of Maryland School of Medicine,

3 Department of Dermatology, University of Maryland School of Medicine, Baltimore,

4 Department of Microbiology and Immunology, University of Maryland School of

[1] Kouzarides T. Chromatin modifications and their function. Cell. 2007 Feb 23;128(4):

[2] Vermeulen M, Eberl HC, Matarese F, Marks H, Denissov S, Butter F, et al. Quantita‐ tive interaction proteomics and genome-wide profiling of epigenetic histone marks

[3] Lee JS, Smith E, Shilatifard A. The language of histone crosstalk. Cell. 2010 Sep

[4] Joffe B, Leonhardt H, Solovei I. Differentiation and large scale spatial organization of

[5] Fisher CL, Fisher AG. Chromatin states in pluripotent, differentiated, and reprog‐

[6] Kloc A, Martienssen R. RNAi, heterochromatin and the cell cycle. Trends Genet. 2008

[7] Strahl BD, Allis CD. The language of covalent histone modifications. Nature. 2000

[8] Mattout A, Meshorer E. Chromatin plasticity and genome organization in pluripo‐

tent embryonic stem cells. Curr Opin Cell Biol. 2010 Jun;22(3):334-41.


[22] Ito S, D'Alessio AC, Taranova OV, Hong K, Sowers LC, Zhang Y. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature. 2010 Aug 26;466(7310):1129-33.

[36] Fischle W, Wang Y, Jacobs SA, Kim Y, Allis CD, Khorasanizadeh S. Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb

Epigenetic Reprogramming in Stem Cells http://dx.doi.org/10.5772/56327 219

[37] Jacobs SA, Khorasanizadeh S. Structure of HP1 chromodomain bound to a lysine 9-

[38] Orlando V. Polycomb, epigenomes, and control of cell identity. Cell. 2003 Mar

[39] Spivakov M, Fisher AG. Epigenetic signatures of stem-cell identity. Nat Rev Genet.

[40] Lee TI, Jenner RG, Boyer LA, Guenther MG, Levine SS, Kumar RM, et al. Control of developmental regulators by Polycomb in human embryonic stem cells. Cell. 2006

[41] Landeira D, Fisher AG. Inactive yet indispensable: the tale of Jarid2. Trends Cell Biol.

[42] Brookes E, Pombo A. Modifications of RNA polymerase II are pivotal in regulating

[43] Landeira D, Sauer S, Poot R, Dvorkina M, Mazzarella L, Jorgensen HF, et al. Jarid2 is a PRC2 component in embryonic stem cells required for multi-lineage differentiation and recruitment of PRC1 and RNA Polymerase II to developmental regulators. Nat

[44] Pasini D, Cloos PA, Walfridsson J, Olsson L, Bukowski JP, Johansen JV, et al. JARID2 regulates binding of the Polycomb repressive complex 2 to target genes in ES cells.

[45] Shen X, Kim W, Fujiwara Y, Simon MD, Liu Y, Mysliwiec MR, et al. Jumonji modu‐ lates polycomb activity and self-renewal versus differentiation of stem cells. Cell.

[46] Eskeland R, Leeb M, Grimes GR, Kress C, Boyle S, Sproul D, et al. Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquiti‐

[47] Endoh M, Endo TA, Endoh T, Fujimura Y, Ohara O, Toyoda T, et al. Polycomb group proteins Ring1A/B are functionally linked to the core transcriptional regulatory cir‐

[48] Ho L, Jothi R, Ronan JL, Cui K, Zhao K, Crabtree GR. An embryonic stem cell chro‐ matin remodeling complex, esBAF, is an essential component of the core pluripoten‐ cy transcriptional network. Proc Natl Acad Sci U S A. 2009 Mar 31;106(13):5187-91.

[49] Lessard JA, Crabtree GR. Chromatin regulatory mechanisms in pluripotency. Annu

cuitry to maintain ES cell identity. Development. 2008 Apr;135(8):1513-24.

gene expression states. EMBO Rep. 2009 Nov;10(11):1213-9.

and HP1 chromodomains. Genes Dev. 2003 Aug 1;17(15):1870-81.

methylated histone H3 tail. Science. 2002 Mar 15;295(5562):2080-3.

7;112(5):599-606.

2007 Apr;8(4):263-71.

Apr 21;125(2):301-13.

2011 Feb;21(2):74-80.

Cell Biol. 2010 Jun;12(6):618-24.

2009 Dec 24;139(7):1303-14.

Nature. 2010 Mar 11;464(7286):306-10.

nation. Mol Cell. 2010 May 14;38(3):452-64.

Rev Cell Dev Biol. 2010 Nov 10;26:503-32.


[36] Fischle W, Wang Y, Jacobs SA, Kim Y, Allis CD, Khorasanizadeh S. Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains. Genes Dev. 2003 Aug 1;17(15):1870-81.

[22] Ito S, D'Alessio AC, Taranova OV, Hong K, Sowers LC, Zhang Y. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification.

[23] Ito S, Shen L, Dai Q, Wu SC, Collins LB, Swenberg JA, et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science. 2010 Sep

[24] Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, et al. Conver‐ sion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL

[25] Koh KP, Yabuuchi A, Rao S, Huang Y, Cunniff K, Nardone J, et al. Tet1 and Tet2 reg‐ ulate 5-hydroxymethylcytosine production and cell lineage specification in mouse

[26] Lister R, Pelizzola M, Dowen RH, Hawkins RD, Hon G, Tonti-Filippini J, et al. Hu‐ man DNA methylomes at base resolution show widespread epigenomic differences.

[27] Simon JA, Kingston RE. Mechanisms of polycomb gene silencing: knowns and un‐

[28] Sauvageau M, Sauvageau G. Polycomb group genes: keeping stem cell activity in

[29] Cole MF, Young RA. Mapping key features of transcriptional regulatory circuitry in embryonic stem cells. Cold Spring Harb Symp Quant Biol. 2008;73:183-93.

[30] Surface LE, Thornton SR, Boyer LA. Polycomb group proteins set the stage for early

[31] Ho L, Crabtree GR. Chromatin remodelling during development. Nature. 2010 Jan

[32] Pietersen AM, van Lohuizen M. Stem cell regulation by polycomb repressors: post‐

[33] Singhal N, Graumann J, Wu G, Arauzo-Bravo MJ, Han DW, Greber B, et al. Chroma‐ tin-Remodeling Components of the BAF Complex Facilitate Reprogramming. Cell.

[34] Sauvageau M, Sauvageau G. Polycomb group proteins: multi-faceted regulators of

[35] Wu H, D'Alessio AC, Ito S, Wang Z, Cui K, Zhao K, et al. Genome-wide analysis of 5 hydroxymethylcytosine distribution reveals its dual function in transcriptional regu‐

lation in mouse embryonic stem cells. Genes Dev. 2011 Apr 1;25(7):679-84.

somatic stem cells and cancer. Cell Stem Cell. 2010 Sep 3;7(3):299-313.

Nature. 2010 Aug 26;466(7310):1129-33.

Nature. 2009 Nov 19;462(7271):315-22.

balance. PLoS Biol. 2008 Apr 29;6(4):e113.

partner TET1. Science. 2009 May 15;324(5929):930-5.

embryonic stem cells. Cell Stem Cell. 2011 Feb 4;8(2):200-13.

knowns. Nat Rev Mol Cell Biol. 2009 Oct;10(10):697-708.

lineage commitment. Cell Stem Cell. 2010 Sep 3;7(3):288-98.

poning commitment. Curr Opin Cell Biol. 2008 Apr;20(2):201-7.

2;333(6047):1300-3.

218 Pluripotent Stem Cells

28;463(7280):474-84.

2010 Jun 11;141(6):943-55.


[50] Ang YS, Tsai SY, Lee DF, Monk J, Su J, Ratnakumar K, et al. Wdr5 mediates self-re‐ newal and reprogramming via the embryonic stem cell core transcriptional network. Cell. 2010 Apr 15;145(2):183-97.

formation but is required for specification of primordial germ cells in the mouse. De‐

Epigenetic Reprogramming in Stem Cells http://dx.doi.org/10.5772/56327 221

[64] Yamaji M, Seki Y, Kurimoto K, Yabuta Y, Yuasa M, Shigeta M, et al. Critical function of Prdm14 for the establishment of the germ cell lineage in mice. Nat Genet. 2008

[65] Gillich A, Bao S, Grabole N, Hayashi K, Trotter MW, Pasque V, et al. Epiblast stem cell-based system reveals reprogramming synergy of germline factors. Cell Stem

[66] Nagamatsu G, Kosaka T, Kawasumi M, Kinoshita T, Takubo K, Akiyama H, et al. A germ cell-specific gene, Prmt5, works in somatic cell reprogramming. J Biol Chem.

[67] Ancelin K, Lange UC, Hajkova P, Schneider R, Bannister AJ, Kouzarides T, et al. Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse

[68] Maatouk DM, Kellam LD, Mann MR, Lei H, Li E, Bartolomei MS, et al. DNA methyl‐ ation is a primary mechanism for silencing postmigratory primordial germ cell genes in both germ cell and somatic cell lineages. Development. 2006 Sep;133(17):3411-8.

[70] Popp C, Dean W, Feng S, Cokus SJ, Andrews S, Pellegrini M, et al. Genome-wide era‐ sure of DNA methylation in mouse primordial germ cells is affected by AID deficien‐

[71] Morgan HD, Dean W, Coker HA, Reik W, Petersen-Mahrt SK. Activation-induced cytidine deaminase deaminates 5-methylcytosine in DNA and is expressed in pluri‐ potent tissues: implications for epigenetic reprogramming. J Biol Chem. 2004 Dec

[72] Reik W. Stability and flexibility of epigenetic gene regulation in mammalian develop‐

[73] Hackett JA, Sengupta R, Zylicz JJ, Murakami K, Lee C, Down TA, et al. Germline DNA Demethylation Dynamics and Imprint Erasure Through 5-Hydroxymethylcy‐

[74] de Napoles M, Nesterova T, Brockdorff N. Early loss of Xist RNA expression and in‐ active X chromosome associated chromatin modification in developing primordial

[75] Hinkins M, Huntriss J, Miller D, Picton HM. Expression of Polycomb-group genes in human ovarian follicles, oocytes and preimplantation embryos. Reproduction. 2005

[76] Labosky PA, Barlow DP, Hogan BL. Mouse embryonic germ (EG) cell lines: transmis‐ sion through the germline and differences in the methylation imprint of insulin-like

[69] Reik W, Dean W. Back to the beginning. Nature. 2002 Nov 14;420(6912):127.

velopment. 2005 Mar;132(6):1315-25.

Aug;40(8):1016-22.

Cell. 2012 Apr 6;10(4):425-39.

2011 Mar 25;286(12):10641-8.

germ cells. Nat Cell Biol. 2006;8(6):623-30.

cy. Nature. 2010 Feb 25;463(7284):1101-5.

ment. Nature. 2007 May 24;447(7143):425-32.

10;279(50):52353-60.

Dec;130(6):883-8.

tosine. Science. 2012 Dec 13.

germ cells. PLoS One. 2007;2(9):e860.


formation but is required for specification of primordial germ cells in the mouse. De‐ velopment. 2005 Mar;132(6):1315-25.

[64] Yamaji M, Seki Y, Kurimoto K, Yabuta Y, Yuasa M, Shigeta M, et al. Critical function of Prdm14 for the establishment of the germ cell lineage in mice. Nat Genet. 2008 Aug;40(8):1016-22.

[50] Ang YS, Tsai SY, Lee DF, Monk J, Su J, Ratnakumar K, et al. Wdr5 mediates self-re‐ newal and reprogramming via the embryonic stem cell core transcriptional network.

[51] Ho L, Ronan JL, Wu J, Staahl BT, Chen L, Kuo A, et al. An embryonic stem cell chro‐ matin remodeling complex, esBAF, is essential for embryonic stem cell self-renewal

[52] Fazzio TG, Huff JT, Panning B. An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity. Cell. 2008 Jul 11;134(1):

[53] Schaniel C, Ang YS, Ratnakumar K, Cormier C, James T, Bernstein E, et al. Smarcc1/ Baf155 couples self-renewal gene repression with changes in chromatin structure in

[54] Liang J, Wan M, Zhang Y, Gu P, Xin H, Jung SY, et al. Nanog and Oct4 associate with unique transcriptional repression complexes in embryonic stem cells. Nat Cell Biol.

[55] Pardo M, Lang B, Yu L, Prosser H, Bradley A, Babu MM, et al. An expanded Oct4 interaction network: implications for stem cell biology, development, and disease.

[56] van den Berg DL, Snoek T, Mullin NP, Yates A, Bezstarosti K, Demmers J, et al. An Oct4-centered protein interaction network in embryonic stem cells. Cell Stem Cell.

[57] Hayashi K, Surani MA. Resetting the epigenome beyond pluripotency in the germ‐

[58] Surani MA, Hayashi K, Hajkova P. Genetic and epigenetic regulators of pluripoten‐

[59] Monk M, Boubelik M, Lehnert S. Temporal and regional changes in DNA methyla‐ tion in the embryonic, extraembryonic and germ cell lineages during mouse embryo

[60] Tada M, Tada T, Lefebvre L, Barton SC, Surani MA. Embryonic germ cells induce ep‐ igenetic reprogramming of somatic nucleus in hybrid cells. Embo J. 1997 Nov

[61] Hajkova P, Ancelin K, Waldmann T, Lacoste N, Lange UC, Cesari F, et al. Chromatin dynamics during epigenetic reprogramming in the mouse germ line. Nature. 2008

[62] Ohinata Y, Payer B, O'Carroll D, Ancelin K, Ono Y, Sano M, et al. Blimp1 is a critical determinant of the germ cell lineage in mice. Nature. 2005 Jul 14;436(7048):207-13. [63] Vincent SD, Dunn NR, Sciammas R, Shapiro-Shalef M, Davis MM, Calame K, et al. The zinc finger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis

and pluripotency. Proc Natl Acad Sci U S A. 2009 Mar 31;106(13):5181-6.

mouse embryonic stem cells. Stem Cells. 2009 Dec;27(12):2979-91.

Cell. 2010 Apr 15;145(2):183-97.

162-74.

220 Pluripotent Stem Cells

2008 Jun;10(6):731-9.

2010 Apr 2;6(4):369-81.

3;16(21):6510-20.

Apr 17;452(7189):877-81.

Cell Stem Cell. 2010 Apr 2;6(4):382-95.

line. Cell Stem Cell. 2009 Jun 5;4(6):493-8.

development. Development. 1987 Mar;99(3):371-82.

cy. Cell. 2007 Feb 23;128(4):747-62.


growth factor 2 receptor (Igf2r) gene compared with embryonic stem (ES) cell lines. Development. 1994 Nov;120(11):3197-204.

[89] Lakshmipathy U, Love B, Goff LA, Jornsten R, Graichen R, Hart RP, et al. MicroRNA Expression Pattern of Undifferentiated and Differentiated Human Embryonic Stem

Epigenetic Reprogramming in Stem Cells http://dx.doi.org/10.5772/56327 223

[90] Marson A, Levine SS, Cole MF, Frampton GM, Brambrink T, Johnstone S, et al. Con‐ necting microRNA genes to the core transcriptional regulatory circuitry of embryonic

[91] Tay Y, Zhang J, Thomson AM, Lim B, Rigoutsos I. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature. 2008 Oct

[92] Pasini D, Bracken AP, Helin K. Polycomb group proteins in cell cycle progression

[93] Wang H, Garzon R, Sun H, Ladner KJ, Singh R, Dahlman J, et al. NF-kappaB-YY1 miR-29 regulatory circuitry in skeletal myogenesis and rhabdomyosarcoma. Cancer

[94] Sinkkonen L, Hugenschmidt T, Berninger P, Gaidatzis D, Mohn F, Artus-Revel CG, et al. MicroRNAs control de novo DNA methylation through regulation of transcrip‐ tional repressors in mouse embryonic stem cells. Nat Struct Mol Biol. 2008 Mar;15(3):

[95] Kanellopoulou C, Muljo SA, Kung AL, Ganesan S, Drapkin R, Jenuwein T, et al. Dic‐ er-deficient mouse embryonic stem cells are defective in differentiation and centro‐

[96] Wang Y, Medvid R, Melton C, Jaenisch R, Blelloch R. DGCR8 is essential for micro‐ RNA biogenesis and silencing of embryonic stem cell self-renewal. Nat Genet. 2007

[97] Fukuda T, Yamagata K, Fujiyama S, Matsumoto T, Koshida I, Yoshimura K, et al. DEAD-box RNA helicase subunits of the Drosha complex are required for processing

[98] Banerjee R, Mani RS, Russo N, Scanlon CS, Tsodikov A, Jing X, et al. The tumor sup‐ pressor gene rap1GAP is silenced by miR-101-mediated EZH2 overexpression in in‐

[99] Varambally S, Cao Q, Mani RS, Shankar S, Wang X, Ateeq B, et al. Genomic loss of microRNA-101 leads to overexpression of histone methyltransferase EZH2 in cancer.

[100] Cui K, Zang C, Roh TY, Schones DE, Childs RW, Peng W, et al. Chromatin signatures in multipotent human hematopoietic stem cells indicate the fate of bivalent genes

of rRNA and a subset of microRNAs. Nat Cell Biol. 2007 May;9(5):604-11.

vasive squamous cell carcinoma. Oncogene. 2011 Oct 20;30(42):4339-49.

during differentiation. Cell Stem Cell. 2009 Jan 9;4(1):80-93.

Cells. Stem Cells Dev. 2007 Dec;16(6):1003-16.

stem cells. Cell. 2008 Aug 8;134(3):521-33.

and cancer. Cell Cycle. 2004 Apr;3(4):396-400.

meric silencing. Genes Dev. 2005 Feb 15;19(4):489-501.

Science. 2008 Dec 12;322(5908):1695-9.

23;455(7216):1124-8.

259-67.

Mar;39(3):380-5.

Cell. 2008 Nov 4;14(5):369-81.


[89] Lakshmipathy U, Love B, Goff LA, Jornsten R, Graichen R, Hart RP, et al. MicroRNA Expression Pattern of Undifferentiated and Differentiated Human Embryonic Stem Cells. Stem Cells Dev. 2007 Dec;16(6):1003-16.

growth factor 2 receptor (Igf2r) gene compared with embryonic stem (ES) cell lines.

[77] Tada T, Tada M, Hilton K, Barton SC, Sado T, Takagi N, et al. Epigenotype switching of imprintable loci in embryonic germ cells. Dev Genes Evol. 1998 Feb;207(8):551-61.

[78] Onyango P, Jiang S, Uejima H, Shamblott MJ, Gearhart JD, Cui H, et al. Monoallelic expression and methylation of imprinted genes in human and mouse embryonic

[79] Matsui Y, Zsebo K, Hogan BL. Derivation of pluripotential embryonic stem cells

[80] Stewart CL, Gadi I, Bhatt H. Stem cells from primordial germ cells can reenter the

[81] Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW, Donovan PJ, et al. Deri‐ vation of pluripotent stem cells from cultured human primordial germ cells. Pro‐ ceedings of the National Academy of Sciences of the United States of America.

[82] Hiller M, Liu C, Blumenthal PD, Gearhart JD, Kerr CL. Bone morphogenetic protein 4 mediates human embryonic germ cell derivation. Stem Cells Dev. 2011 Feb;20(2):

[83] Kashyap V, Rezende NC, Scotland KB, Shaffer SM, Persson JL, Gudas LJ, et al. Regu‐ lation of stem cell pluripotency and differentiation involves a mutual regulatory cir‐ cuit of the NANOG, OCT4, and SOX2 pluripotency transcription factors with polycomb repressive complexes and stem cell microRNAs. Stem Cells Dev. 2009 Sep;

[84] Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009 Jan

[85] Gangaraju VK, Lin H. MicroRNAs: key regulators of stem cells. Nat Rev Mol Cell Bi‐

[86] Houbaviy HB, Murray MF, Sharp PA. Embryonic stem cell-specific MicroRNAs. Dev

[87] Barroso-delJesus A, Romero-Lopez C, Lucena-Aguilar G, Melen GJ, Sanchez L, Li‐ gero G, et al. Embryonic stem cell-specific miR302-367 cluster: human gene structure and functional characterization of its core promoter. Mol Cell Biol. 2008 Nov;28(21):

[88] Letzen B, Liu C, Thakor N, Gearhart J, All A, Kerr C. MicroRNA Expression Profiling of Oligodendrocyte Differentiation from Human Embryonic Stem Cells. PLoS one.

2010;5(5):e10480 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2864763/.

germ cell lineages. Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10599-604.

from murine primordial germ cells in culture. Cell. 1992;70(5):841-7.

Development. 1994 Nov;120(11):3197-204.

germ line. Dev Biol. 1994 Feb;161(2):626-8.

1998;95(23):13726-31.

351-61.

222 Pluripotent Stem Cells

18(7):1093-108.

23;136(2):215-33.

6609-19.

ol. 2009 Feb;10(2):116-25.

Cell. 2003 Aug;5(2):351-8.


[101] Collas P. Programming differentiation potential in mesenchymal stem cells. Epige‐ netics. 2010 Aug 16;5(6):476-82.

[115] Ohm J, Mali P, Van Neste L, Berman D, Liang L, Pandiyan K, et al. Cancer-Related Epigenome Changes Associated with Reprogramming to Induced Pluripotent Stem

Epigenetic Reprogramming in Stem Cells http://dx.doi.org/10.5772/56327 225

[116] Mali P, Chou BK, Yen J, Ye Z, Zou J, Dowey S, et al. Butyrate greatly enhances deri‐ vation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes. Stem Cells. 2010 Apr;28(4):

[117] Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-Sinha C, Sanda MG, et al. The polycomb group protein EZH2 is involved in progression of prostate can‐

[118] Kamminga LM, Bystrykh LV, de Boer A, Houwer S, Douma J, Weersing E, et al. The Polycomb group gene Ezh2 prevents hematopoietic stem cell exhaustion. Blood. 2006

[119] Pasini D, Bracken AP, Jensen MR, Lazzerini Denchi E, Helin K. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity. Embo J. 2004

[120] Bracken AP, Dietrich N, Pasini D, Hansen KH, Helin K. Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes Dev. 2006

[121] Bracken AP, Kleine-Kohlbrecher D, Dietrich N, Pasini D, Gargiulo G, Beekman C, et al. The Polycomb group proteins bind throughout the INK4A-ARF locus and are dis‐

[122] Dietrich N, Bracken AP, Trinh E, Schjerling CK, Koseki H, Rappsilber J, et al. Bypass of senescence by the polycomb group protein CBX8 through direct binding to the

[123] Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K. EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. Embo J.

[124] Widschwendter M, Fiegl H, Egle D, Mueller-Holzner E, Spizzo G, Marth C, et al. Epi‐

[125] Liu S, Dontu G, Mantle ID, Patel S, Ahn NS, Jackson KW, et al. Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem

[126] Raman JD, Mongan NP, Tickoo SK, Boorjian SA, Scherr DS, Gudas LJ. Increased ex‐ pression of the polycomb group gene, EZH2, in transitional cell carcinoma of the

genetic stem cell signature in cancer. Nat Genet. 2007 Feb;39(2):157-8.

associated in senescent cells. Genes Dev. 2007 Mar 1;21(5):525-30.

INK4A-ARF locus. Embo J. 2007 Mar 21;26(6):1637-48.

cells. Cancer Res. 2006 Jun 15;66(12):6063-71.

bladder. Clin Cancer Res. 2005 Dec 15;11(24 Pt 1):8570-6.

Cells. Cancer Research. 2010;70(19):7662.

cer. Nature. 2002 Oct 10;419(6907):624-9.

713-20.

Mar 1;107(5):2170-9.

Oct 13;23(20):4061-71.

May 1;20(9):1123-36.

2003 Oct 15;22(20):5323-35.


[115] Ohm J, Mali P, Van Neste L, Berman D, Liang L, Pandiyan K, et al. Cancer-Related Epigenome Changes Associated with Reprogramming to Induced Pluripotent Stem Cells. Cancer Research. 2010;70(19):7662.

[101] Collas P. Programming differentiation potential in mesenchymal stem cells. Epige‐

[102] Meissner A, Mikkelsen TS, Gu H, Wernig M, Hanna J, Sivachenko A, et al. Genomescale DNA methylation maps of pluripotent and differentiated cells. Nature. 2008

[103] Jiang L, Li J, Song L. Bmi-1, stem cells and cancer. Acta Biochim Biophys Sin (Shang‐

[104] Clarke MF, Fuller M. Stem cells and cancer: two faces of eve. Cell. 2006 Mar 24;124(6):

[105] Hill RP, Perris R. "Destemming" cancer stem cells. J Natl Cancer Inst. 2007 Oct

[106] Dalerba P, Cho RW, Clarke MF. Cancer stem cells: models and concepts. Annu Rev

[107] Mongan NP, Martin KM, Gudas LJ. The putative human stem cell marker, Rex-1 (Zfp42): structural classification and expression in normal human epithelial and car‐

[108] Chiou SH, Yu CC, Huang CY, Lin SC, Liu CJ, Tsai TH, et al. Positive correlations of Oct-4 and Nanog in oral cancer stem-like cells and high-grade oral squamous cell

[109] Raman JD, Mongan NP, Liu L, Tickoo SK, Nanus DM, Scherr DS, et al. Decreased ex‐ pression of the human stem cell marker, Rex-1 (zfp-42), in renal cell carcinoma. Car‐

[110] Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, Regev A, et al. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human

[111] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007

[112] Yu J, Vodyanik M, Smuga-Otto K, Antosiewicz-Bourget J, Frane J, Tian S, et al. In‐ duced pluripotent stem cell lines derived from human somatic cells. Science.

[113] Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, et al. In vi‐ tro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature. 2007 Jul

[114] Wong DJ, Liu H, Ridky TW, Cassarino D, Segal E, Chang HY. Module map of stem cell genes guides creation of epithelial cancer stem cells. Cell Stem Cell. 2008 Apr

cinoma cell cultures. Mol Carcinog. 2006 Dec;45(12):887-900.

carcinoma. Clin Cancer Res. 2008 Jul 1;14(13):4085-95.

cinogenesis. 2006 Mar;27(3):499-507.

tumors. Nat Genet. 2008 May;40(5):499-507.

netics. 2010 Aug 16;5(6):476-82.

Aug 7;454(7205):766-70.

hai). 2009 Jul;41(7):527-34.

1111-5.

224 Pluripotent Stem Cells

3;99(19):1435-40.

Med. 2007;58:267-84.

Nov 30;131(5):861-72.

2007;318(5858):1917.

19;448(7151):318-24.

10;2(4):333-44.


[127] Friedman JM, Liang G, Liu CC, Wolff EM, Tsai YC, Ye W, et al. The putative tumor suppressor microRNA-101 modulates the cancer epigenome by repressing the poly‐ comb group protein EZH2. Cancer Res. 2009 Mar 15;69(6):2623-9.

**Chapter 11**

**Multiple Paths to Reprogramming**

Antonio Lo Nigro and Catherine M. Verfaillie

Additional information is available at the end of the chapter

through a process, known as "reprogramming".

not work with mammalian cells [3].

For a long time, differentiation was considered a "one way process"; Conrad Waddington, in the 1950s, described cellular differentiation and development as a ball rolling towards different one-way ramified valleys, giving rise to specific cell fates, irreversibly [1]. However, in the last decades, a series of studies have shown that somatic cells and stem cells are more plastic than previously believed. Using different technical approaches, the epigenetic barriers imposed during development in differentiated cells can be erased, and cells can re-acquire pluripotency

The first evidence came at the end of the 1950's from the pivotal experiments performed by J.B. Gordon in the zoology department at Oxford University [2]. At that time, embryologists, not aware of epigenetic regulation, i.e. the role of chromatin and its crucial modifications in cell fate determination, wondered whether development and cellular differentiation arise upon specific restriction of the genetic information contained in their nuclei. To answer this basic but intriguing question, Gordon used a technique, now known as somatic cellular nuclear transfer (SCNT) in *Xenopus laevis laevis*. For these experiments, nuclei from intestinal epithelial cells of albino tadpoles were transferred into unfertilized and enucleated wild-type frog oocytes. This resulted in the development of normal albino frogs, which in some cases were also fertile. They concluded from these studies that adult nuclei contain the genetic information necessary for the development of a frog. Moreover, cellular differentiation, during develop‐ ment, does not occur through loss of genetic information. These findings were exciting for the scientific community but at the same time controversial for two reasons: I) the efficiency with which a 'cloned' frog reached the adult stage was around 1% and II) the same technique did

For forty years, the scientific community was not able to use SCNT in other species. Finally, in 1997, by using the same technique, Ian Wilmut and colleagues, at the Roslin Institute in

> © 2013 Lo Nigro and Verfaillie; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Lo Nigro and Verfaillie; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

http://dx.doi.org/10.5772/55104

**1. Introduction**

## **Chapter 11**

## **Multiple Paths to Reprogramming**

Antonio Lo Nigro and Catherine M. Verfaillie

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55104

## **1. Introduction**

[127] Friedman JM, Liang G, Liu CC, Wolff EM, Tsai YC, Ye W, et al. The putative tumor suppressor microRNA-101 modulates the cancer epigenome by repressing the poly‐

comb group protein EZH2. Cancer Res. 2009 Mar 15;69(6):2623-9.

226 Pluripotent Stem Cells

For a long time, differentiation was considered a "one way process"; Conrad Waddington, in the 1950s, described cellular differentiation and development as a ball rolling towards different one-way ramified valleys, giving rise to specific cell fates, irreversibly [1]. However, in the last decades, a series of studies have shown that somatic cells and stem cells are more plastic than previously believed. Using different technical approaches, the epigenetic barriers imposed during development in differentiated cells can be erased, and cells can re-acquire pluripotency through a process, known as "reprogramming".

The first evidence came at the end of the 1950's from the pivotal experiments performed by J.B. Gordon in the zoology department at Oxford University [2]. At that time, embryologists, not aware of epigenetic regulation, i.e. the role of chromatin and its crucial modifications in cell fate determination, wondered whether development and cellular differentiation arise upon specific restriction of the genetic information contained in their nuclei. To answer this basic but intriguing question, Gordon used a technique, now known as somatic cellular nuclear transfer (SCNT) in *Xenopus laevis laevis*. For these experiments, nuclei from intestinal epithelial cells of albino tadpoles were transferred into unfertilized and enucleated wild-type frog oocytes. This resulted in the development of normal albino frogs, which in some cases were also fertile. They concluded from these studies that adult nuclei contain the genetic information necessary for the development of a frog. Moreover, cellular differentiation, during develop‐ ment, does not occur through loss of genetic information. These findings were exciting for the scientific community but at the same time controversial for two reasons: I) the efficiency with which a 'cloned' frog reached the adult stage was around 1% and II) the same technique did not work with mammalian cells [3].

For forty years, the scientific community was not able to use SCNT in other species. Finally, in 1997, by using the same technique, Ian Wilmut and colleagues, at the Roslin Institute in

© 2013 Lo Nigro and Verfaillie; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Lo Nigro and Verfaillie; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Edinburgh, Scotland, succeeded in generating the sheep named "Dolly" by SCNT, further confirming that genetic modifications, leading to cellular differentiation, are not irreversible. Two key improvements in his technical strategy led to the first cloning of a mammal: unfer‐ tilized oocytes were used as recipients and donor cells were induced to exit from the normal cellular cycle, by serum withdrawal [4, 5].

develop till blastocyst stage with a good efficiency (20-50% depending on the species), but most of them die in the post-implantation stage, without reaching birth (1-5% of survival rate

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 229

Analysis of the cloned animals also showed several abnormalities: increased telomere shortening (which may have caused the premature death of Dolly), altered gene expression during development, prolonged gestation, fetal or placental edema, increased risk of obesity and cancer. The reasons for these pathologies remain not fully understood. The defects may be due to infidelity of the reprogramming: residual epigenetic memory of the donor cell may be present an/or imprinting of important developmental genes may be altered. Nevertheless, reproductive cloning remains attractive and may have potential implications in agriculture and industrial biotechnology. However, as it relates to humans, cloning (also therapeutic)

Evidence that differentiation is reversible also comes from another technique, known as cell fusion [10]. In cell fusion experiments, two or more cells can be fused together (by using polyethylene glycol (PEG) or electrofusion) to generate a single cell, called heterokaryon or hybrid. The larger or more dividing cell type is the "dominant" one, and the "recessive" cell will convert its gene expression profile to the one imposed by the dominant cell type. Obvi‐ ously, alteration in the ratio of the two cell types during fusion will affect the final fate of the

A heterokaryon, produced by inhibiting cell division, is a fused cell that becomes multinucleat‐ ed and survives only short-term. If the cell cycle is not blocked, the fused cells will form a hybrid, because upon the first division the two different nuclei will become a single nucleus, having 4n chromosomes (see Figure 2). Its karyotype can be: 1) euploid, when fused cells are from same species (the two cell types have the same number of chromosomes, thus, their segregation will be balanced); 2) aneuploid, when cells fused are from different species (the two cell types have a

different number of chromosomes, thus, they will be lost and/or rearranged).

remains controversial as theoretically, it may allow the cloning of a human being.

to birth, depending on the species).

fused cells [11].

**Figure 2.** Cell fusion

One year later, Wakayama and colleagues [6] reported that SCNT also allowed the cloning of the most used animal model, the mouse. Again, another technical advance led to this progress: the use of an enucleation pipette, which allowed for the removal of the nucleus from the oocytes. This advance also allowed the conclusion that reprogramming factors are not oocytespecific, meaning that SCNT can be done also using zygotes and fertilized eggs [7], and that the molecules responsible for reprogramming were present in the cell cytoplasm. In general, nuclear transfer (see Figure 1) involves two steps: a) de-differentiation of a somatic donor cell to an embryonic state and the *in vitro* maturation till the blastocyst stage (also known as therapeutic cloning); b) the further development of the cloned blastocyst, after the implantation in the maternal uterus (reproductive cloning).

**Figure 1.** SCNT, Therapeutic and Reproductive cloning

Therapeutic cloning permits the derivation of nuclear transfer derived embryonic stem cells (ntESCs). Recently, the efficiency of isolation of ntESCs drastically increased, at least in mice, from 1% to 20% [8]. It has also been possible to derive similar cells in cats, dogs, wolves, goats and monkeys. Although the isolation of human ntESCs has been reported, this paper has been retracted later on [9]. Thus, the possibility of therapeutic cloning with human cells needs to still be demonstrated. However, the therapeutic cloning remains a promising technology for regenerative medicine, considering that ntESCs, from other species, were able to differentiate into all the cell types of an adult body.

Reproductive cloning is technically more difficult than therapeutic cloning, as it involves the further development *in vivo* in a pseudopregnant female. Embryos, derived after SCNT, develop till blastocyst stage with a good efficiency (20-50% depending on the species), but most of them die in the post-implantation stage, without reaching birth (1-5% of survival rate to birth, depending on the species).

Analysis of the cloned animals also showed several abnormalities: increased telomere shortening (which may have caused the premature death of Dolly), altered gene expression during development, prolonged gestation, fetal or placental edema, increased risk of obesity and cancer. The reasons for these pathologies remain not fully understood. The defects may be due to infidelity of the reprogramming: residual epigenetic memory of the donor cell may be present an/or imprinting of important developmental genes may be altered. Nevertheless, reproductive cloning remains attractive and may have potential implications in agriculture and industrial biotechnology. However, as it relates to humans, cloning (also therapeutic) remains controversial as theoretically, it may allow the cloning of a human being.

Evidence that differentiation is reversible also comes from another technique, known as cell fusion [10]. In cell fusion experiments, two or more cells can be fused together (by using polyethylene glycol (PEG) or electrofusion) to generate a single cell, called heterokaryon or hybrid. The larger or more dividing cell type is the "dominant" one, and the "recessive" cell will convert its gene expression profile to the one imposed by the dominant cell type. Obvi‐ ously, alteration in the ratio of the two cell types during fusion will affect the final fate of the fused cells [11].

A heterokaryon, produced by inhibiting cell division, is a fused cell that becomes multinucleat‐ ed and survives only short-term. If the cell cycle is not blocked, the fused cells will form a hybrid, because upon the first division the two different nuclei will become a single nucleus, having 4n chromosomes (see Figure 2). Its karyotype can be: 1) euploid, when fused cells are from same species (the two cell types have the same number of chromosomes, thus, their segregation will be balanced); 2) aneuploid, when cells fused are from different species (the two cell types have a different number of chromosomes, thus, they will be lost and/or rearranged).

Edinburgh, Scotland, succeeded in generating the sheep named "Dolly" by SCNT, further confirming that genetic modifications, leading to cellular differentiation, are not irreversible. Two key improvements in his technical strategy led to the first cloning of a mammal: unfer‐ tilized oocytes were used as recipients and donor cells were induced to exit from the normal

One year later, Wakayama and colleagues [6] reported that SCNT also allowed the cloning of the most used animal model, the mouse. Again, another technical advance led to this progress: the use of an enucleation pipette, which allowed for the removal of the nucleus from the oocytes. This advance also allowed the conclusion that reprogramming factors are not oocytespecific, meaning that SCNT can be done also using zygotes and fertilized eggs [7], and that the molecules responsible for reprogramming were present in the cell cytoplasm. In general, nuclear transfer (see Figure 1) involves two steps: a) de-differentiation of a somatic donor cell to an embryonic state and the *in vitro* maturation till the blastocyst stage (also known as therapeutic cloning); b) the further development of the cloned blastocyst, after the implantation

Therapeutic cloning permits the derivation of nuclear transfer derived embryonic stem cells (ntESCs). Recently, the efficiency of isolation of ntESCs drastically increased, at least in mice, from 1% to 20% [8]. It has also been possible to derive similar cells in cats, dogs, wolves, goats and monkeys. Although the isolation of human ntESCs has been reported, this paper has been retracted later on [9]. Thus, the possibility of therapeutic cloning with human cells needs to still be demonstrated. However, the therapeutic cloning remains a promising technology for regenerative medicine, considering that ntESCs, from other species, were able to differentiate

Reproductive cloning is technically more difficult than therapeutic cloning, as it involves the further development *in vivo* in a pseudopregnant female. Embryos, derived after SCNT,

cellular cycle, by serum withdrawal [4, 5].

228 Pluripotent Stem Cells

in the maternal uterus (reproductive cloning).

**Figure 1.** SCNT, Therapeutic and Reproductive cloning

into all the cell types of an adult body.

Cell fusion experiments advanced medical knowledge on cell plasticity. In 1969, Harris et al. fused tumor cells with normal cells and demonstrated that there are trans-acting oncosup‐ pressor genes. Upon fusion, malignancy was suppressed and this was not due to the loss of an oncogene, as after prolonged *in vitro* culture malignancy re-emerged [12]. Fibroblasts can be induced to produce albumin or melanin if fused with hepatocytes [13, 14] or melanocytes [15, 16], respectively.

In 1983, Blau et al. [17] produced for the first time heterokaryons from diploid human amniocytes fused with differentiated mouse muscle cells. She demonstrated that the hetero‐ karyons express many human muscle-related genes and that this activation was mediated by factors present in the cytoplasm (as non-dividing heterokaryons have distinct nuclei). Similar heterokaryons with muscle cells can be produced not only by fusing them with amniocytes but also with cells of the three embryonic lineages (mesoderm, ectoderm, endoderm) [18].

In 1997, Surani, Tada and colleagues demonstrated, by producing proliferative hybrids that cell fusion not only "switches" the fate of different cell types but also "reprograms" them to a pluripotent state. Thymocytes from adult mice were fused to embryonic germ cells, pluripotent stem cells derived from primordial germ cells (PGCs). By using DNA sensitive restriction enzymes, they demonstrated that the genome of the somatic cell underwent a general deme‐ thylation, with reactivation of imprinted and non-imprinted genes, resembling the reprog‐ ramming events occurring in germ cell development [19].

**Figure 3.** iPSC technology and applications

Finally, It has also become clear that cell plasticity and reprogramming may be partially achieved or enhanced by the culture microenvironment. An increasing number of studies is showing how small molecules, including epigenetic modifiers and signaling pathway modulators, play a crucial role in cell-fate determination [24]. All together, these studies highlight that culture media influences the epigenetic-state of the cells in which they are

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 231

**1.** *Reprogramming to the pluripotency-state*, describing transcription factors used in mouse and

**2.** *Lineage conversion*, illustrating the differentiated cell types/precursors obtained and the

**3.** *Culture mediated reprogramming*, providing the published data which highlight the influence of culture media and small molecules on stem cells fate and features

In 1987, two key discoveries highlighted how crucial the role is of some "master" TFs in cell fate determination. During *Drosophila Melanogaster* development, the gene *Antennopedia (Antp)* specifies the formation of the thoracic segment, which will then form the legs of the adult fly. Heat-induced overexpression of *Atnp,* at specific larval stage, led to the formation of additional legs instead of antennae [26]. Similarly, ectopic expression of *eyeless* (the homolo‐ gous of *Pax6* in mice) caused the development of functional eyes on the wings, antennae and

Twenty years after these pivotal experiments, Shinya Yamanaka's group used the same TFbased technology to reprogram adult fibroblasts to pluripotent state [20]. Mouse embryonic

cultured and thus their features [25]. In this chapter, we will discuss:

differences of this approach with the iPSC technology

**2. Reprogramming to the pluripotency-state**

legs of drosophila [27].

human, different methodologies and potentiality of iPSC technolology

They also fused female thymocytes, derived from Oct4-GFP mice, with mouse ESCs [17]. Two days after fusion, expression of GFP, from the thymocyte, was detected. The X chromosome, normally silenced in adult female cells, was reactivated. Moreover, hybrids had developmental potential, like ESCs, as they contributed to the three germ layers of chimeric animals, upon blastocyst aggregation [18]. Using the same approach, in 2005, Cowan succeeded in creating hybrids between human somatic cells and human ESCs [19].

This further elucidated that the differentiation state of cells is plastic and reversible; both SCNT and cell fusion experiments clearly demonstrated that it is possible to reset the epigenetic landscape of somatic cells. Despite all these studies were already present in the literature, the field of reprogramming only became jumpstarted in 2006 when Takahashi and Yamanaka [20] demonstrated that the overexpression of pluripotency-related transcrip‐ tion factors (TFs) can dedifferentiate adult fibroblasts to induced pluripotent stem cells (iPSCs), iPSCs strongly resemble ESCs. iPSC technology is an inefficient process, but differently from SCNT or cell fusion, may have in the near future therapeutic applica‐ tions, including human disease modeling, drug screening and patient-specific cell thera‐ py (see Figure 3).

After this publication [20], several studies demonstrated the potential of epigenetic reprog‐ ramming. Indeed, there is now evidence that use of different "cocktails" of TFs allows not only to redirect fibroblasts to an ESC-fate but also to a lineage-specific cell types/precursors, like cardiomyocytes, neuronal precursors, hepatocytes and blood cells, from a tissue different than the tissue from which the somatic cell was isolated [21-23].

**Figure 3.** iPSC technology and applications

Cell fusion experiments advanced medical knowledge on cell plasticity. In 1969, Harris et al. fused tumor cells with normal cells and demonstrated that there are trans-acting oncosup‐ pressor genes. Upon fusion, malignancy was suppressed and this was not due to the loss of an oncogene, as after prolonged *in vitro* culture malignancy re-emerged [12]. Fibroblasts can be induced to produce albumin or melanin if fused with hepatocytes [13, 14] or melanocytes

In 1983, Blau et al. [17] produced for the first time heterokaryons from diploid human amniocytes fused with differentiated mouse muscle cells. She demonstrated that the hetero‐ karyons express many human muscle-related genes and that this activation was mediated by factors present in the cytoplasm (as non-dividing heterokaryons have distinct nuclei). Similar heterokaryons with muscle cells can be produced not only by fusing them with amniocytes but also with cells of the three embryonic lineages (mesoderm, ectoderm, endoderm) [18].

In 1997, Surani, Tada and colleagues demonstrated, by producing proliferative hybrids that cell fusion not only "switches" the fate of different cell types but also "reprograms" them to a pluripotent state. Thymocytes from adult mice were fused to embryonic germ cells, pluripotent stem cells derived from primordial germ cells (PGCs). By using DNA sensitive restriction enzymes, they demonstrated that the genome of the somatic cell underwent a general deme‐ thylation, with reactivation of imprinted and non-imprinted genes, resembling the reprog‐

They also fused female thymocytes, derived from Oct4-GFP mice, with mouse ESCs [17]. Two days after fusion, expression of GFP, from the thymocyte, was detected. The X chromosome, normally silenced in adult female cells, was reactivated. Moreover, hybrids had developmental potential, like ESCs, as they contributed to the three germ layers of chimeric animals, upon blastocyst aggregation [18]. Using the same approach, in 2005, Cowan succeeded in creating

This further elucidated that the differentiation state of cells is plastic and reversible; both SCNT and cell fusion experiments clearly demonstrated that it is possible to reset the epigenetic landscape of somatic cells. Despite all these studies were already present in the literature, the field of reprogramming only became jumpstarted in 2006 when Takahashi and Yamanaka [20] demonstrated that the overexpression of pluripotency-related transcrip‐ tion factors (TFs) can dedifferentiate adult fibroblasts to induced pluripotent stem cells (iPSCs), iPSCs strongly resemble ESCs. iPSC technology is an inefficient process, but differently from SCNT or cell fusion, may have in the near future therapeutic applica‐ tions, including human disease modeling, drug screening and patient-specific cell thera‐

After this publication [20], several studies demonstrated the potential of epigenetic reprog‐ ramming. Indeed, there is now evidence that use of different "cocktails" of TFs allows not only to redirect fibroblasts to an ESC-fate but also to a lineage-specific cell types/precursors, like cardiomyocytes, neuronal precursors, hepatocytes and blood cells, from a tissue different than

ramming events occurring in germ cell development [19].

hybrids between human somatic cells and human ESCs [19].

the tissue from which the somatic cell was isolated [21-23].

[15, 16], respectively.

230 Pluripotent Stem Cells

py (see Figure 3).

Finally, It has also become clear that cell plasticity and reprogramming may be partially achieved or enhanced by the culture microenvironment. An increasing number of studies is showing how small molecules, including epigenetic modifiers and signaling pathway modulators, play a crucial role in cell-fate determination [24]. All together, these studies highlight that culture media influences the epigenetic-state of the cells in which they are cultured and thus their features [25]. In this chapter, we will discuss:


## **2. Reprogramming to the pluripotency-state**

In 1987, two key discoveries highlighted how crucial the role is of some "master" TFs in cell fate determination. During *Drosophila Melanogaster* development, the gene *Antennopedia (Antp)* specifies the formation of the thoracic segment, which will then form the legs of the adult fly. Heat-induced overexpression of *Atnp,* at specific larval stage, led to the formation of additional legs instead of antennae [26]. Similarly, ectopic expression of *eyeless* (the homolo‐ gous of *Pax6* in mice) caused the development of functional eyes on the wings, antennae and legs of drosophila [27].

Twenty years after these pivotal experiments, Shinya Yamanaka's group used the same TFbased technology to reprogram adult fibroblasts to pluripotent state [20]. Mouse embryonic and adult fibroblasts, transduced with retroviral vectors encoding for Oct4, Sox2, Klf4 and c-Myc and cultured in ESC-medium, erased their differentiated epigenetic state and reestab‐ lished the pluripotent state; these cells were named induced pluripotent stem cells (iPSCs). Murine iPSCs exhibited morphological and growth properties of ESCs, and expressed alkaline phosphatase and SSEA1. No differences, if compared with ESCs, could be detected in their methylation status, X activation status, embryoid body (EB) formation, *in vitro* differentiation capacity (ectodermal, mesodermal and endodermal), teratoma formation and *in vivo* devel‐ opmental potential (contribution to the three germ layers of chimeric animals).

implantation [37]. Moreover, it is well known that Oct4 and Sox2 form a complex together, which regulates synergistically the trascription of among others, Fbx15, Fgf4 and Utf1 [38-41]. Nanog, (from Tír na nÓg, the Land of Ever-Young in the old Irish mythology) is another homeobox TF essential for pluripotency. Nanog expression can be detected in the late morula, in the ICM of the blastocyst and in the epiblast. Nanog knockout mice are lethal and the ICM fails to further progress to the epiblast-stage [42-44]. In contrast to Oct4, Nanog overexpression maintains ESC selferewal and pluripotency in a feeder-free and LIF-independent way.

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 233

Lin28 is a negative regulator of micro (mi)RNA processing. It blocks the posttranscriptional processing of several primary miRNA transcripts (pri-miRNAs). It is responsible for miRNA biogenesis in both cancer cells and ESCs; so it plays a key role in tumorigenesis and develop‐ ment. Lin28 KO mice have decreased weight at birth and increased postnatal lethality [45]. Different from the above genes, Klf4 and c-Myc are not ESC-specific but are required for their direct or indirect effect on cell proliferation. Of note, iPSCs can also be produced without c-

Although the combination of Oct4, Sox2, Klf4 and c-Myc (OSKM) consistently allows the reprogramming, this it is not an efficient process (0.01-0.1%). For this reason, subsequent studies were focused on improving iPSC efficiency and since the first iPSC publication, many papers have reported several other genes which enhance the efficiency of iPSC generation. Inclusion of Utf1, another TF involved in ESC pluripotency, together with the inhibition of p53, increases iPSC generation by 200-fold [47]. Similarly, other factors (like the SV40 large T antigen, SV40LT; the telomerase reverse transcriptase, TERT) and microRNAs (miRNAs) controlling cell proliferation, senescence and apoptosis also affects the efficiency and the speed of reprogramming [48-52]. Other studies have reported important roles for Sall4 [53], Esrrb (which can replace Klf4] [54] and Tbx3 (which improves the germline contribution) [55].

The starting cell, used for reprogramming, is a key parameter that influences the kinetics, the efficiency and the quality of the iPSCs. Fibroblasts are the most commonly used somatic cells because they can be easily isolated. In mouse studies, embryonic fibroblasts (MEFs) are commonly used as iPSCs can be generated in 10-12 days; MEF-derived iPSC generation is therefore recommended for studies aimed at understanding the mechanisms underlying iPSC

To generate iPSCs from human foreskin fibroblasts (HFFs), three weeks are required and the efficiency is 100 fold less compared with human primary keratinocytes, in which reprogram‐ ming also occurs faster [56]. When using CD133<sup>+</sup> cord blood cells, iPSCs can be produced by overexpression of only Oct4 and Sox2. As cord blood banks exist, it is believed that this cell source may be useful to make an iPSC bank representing a wide panel of haplotypes for

Another crucial parameter is the differentiation status; Hematopoietic stem cells and progen‐ itors have a higher efficiency of reprogramming than terminally differentiated B- and T-

generation, as well as the TFs and the chemicals that may enhance this process.

Myc and this is clinically relevant, considering the oncogenic features of c-Myc [46].

**2.2. The donor cell type and epigenetic memory**

regenerative medicine [57].

Noteworthy, pluripotent stem cells (PSCs) possess mechanisms that lead to the silencing of the integrated transgenes. Therefore, the expression of the four TFs is necessary for generating iPSCs but dispensable for maintenance of the iPSC fate; hence, pluripotency and selfrenewal capacity rely on the trans-activation of the endogenous genes, suggesting a true and complete reprogramming. Mouse iPSCs, like ESCs, were germline competent [28] and supported the development of a mice in tetraploid complementation assay [29]. In this assay, embryonic cells at the two cell-stage are fused together. This results in a tetraploid blastocyst in which just the extraembryonic tissues will further develop; by complementing the tetraploid embryo with normal diploid PSCs, it is possible to generate an individual, completely derived from the diploid PSCs. Interestingly, the same combination of TFs [30] or a somewhat different one (Oct4, Sox2, Nanog and Lin28) can be used for the reprogramming of human cells [31].

This discovery is groundbreaking because with iPSC technology, PSCs can now be induced/ derived for autologous cell transplantation, avoiding immunological problems and ethical issues related to the use of human ESCs. In addition, iPSCs from patients carrying a disease can be derived and used to better understand the biological problem leading to the disease as well as for drug-screening.

#### **2.1. Rationale behind iPSCs**

The rationale for the selection of the genes for this "reprogramming" cocktail was obviously based on the studies, done in the preceding decade, aimed at understanding the network of TFs responsible for ESC pluripotency and selfrenewal.

The Oct4 (also known as Pou5f1) gene encodes for a TF that belongs to the POU homeodo‐ main DNA binding domain family [32]. It plays a key role in the development and in maintenance of both ESCs self-renewal and pluripotency. Misregulation of its levels triggers loss of the ESC fate; a 50% loss of expression drives ESCs to trophectoderm while a 50% greater expression induces primitive endoderm or mesoderm [33]. Knockout (KO) experiments in mouse demonstrated lethality at the preimplantation stage *in vivo* and failure of ESC derivation *in vitro* [34].

Sox2 belongs to a family of TFs, having the high mobility group (HMG) DNA-binding domain, identified for the first time in the SRY (sex determining Y region) protein, which is the testis determining factor. In general, the genes from the Sox family are involved in different and crucial steps of mammalian development [35, 36]. Sox2 KO embryos die immediately after implantation [37]. Moreover, it is well known that Oct4 and Sox2 form a complex together, which regulates synergistically the trascription of among others, Fbx15, Fgf4 and Utf1 [38-41].

Nanog, (from Tír na nÓg, the Land of Ever-Young in the old Irish mythology) is another homeobox TF essential for pluripotency. Nanog expression can be detected in the late morula, in the ICM of the blastocyst and in the epiblast. Nanog knockout mice are lethal and the ICM fails to further progress to the epiblast-stage [42-44]. In contrast to Oct4, Nanog overexpression maintains ESC selferewal and pluripotency in a feeder-free and LIF-independent way.

Lin28 is a negative regulator of micro (mi)RNA processing. It blocks the posttranscriptional processing of several primary miRNA transcripts (pri-miRNAs). It is responsible for miRNA biogenesis in both cancer cells and ESCs; so it plays a key role in tumorigenesis and develop‐ ment. Lin28 KO mice have decreased weight at birth and increased postnatal lethality [45].

Different from the above genes, Klf4 and c-Myc are not ESC-specific but are required for their direct or indirect effect on cell proliferation. Of note, iPSCs can also be produced without c-Myc and this is clinically relevant, considering the oncogenic features of c-Myc [46].

Although the combination of Oct4, Sox2, Klf4 and c-Myc (OSKM) consistently allows the reprogramming, this it is not an efficient process (0.01-0.1%). For this reason, subsequent studies were focused on improving iPSC efficiency and since the first iPSC publication, many papers have reported several other genes which enhance the efficiency of iPSC generation.

Inclusion of Utf1, another TF involved in ESC pluripotency, together with the inhibition of p53, increases iPSC generation by 200-fold [47]. Similarly, other factors (like the SV40 large T antigen, SV40LT; the telomerase reverse transcriptase, TERT) and microRNAs (miRNAs) controlling cell proliferation, senescence and apoptosis also affects the efficiency and the speed of reprogramming [48-52]. Other studies have reported important roles for Sall4 [53], Esrrb (which can replace Klf4] [54] and Tbx3 (which improves the germline contribution) [55].

#### **2.2. The donor cell type and epigenetic memory**

and adult fibroblasts, transduced with retroviral vectors encoding for Oct4, Sox2, Klf4 and c-Myc and cultured in ESC-medium, erased their differentiated epigenetic state and reestab‐ lished the pluripotent state; these cells were named induced pluripotent stem cells (iPSCs). Murine iPSCs exhibited morphological and growth properties of ESCs, and expressed alkaline phosphatase and SSEA1. No differences, if compared with ESCs, could be detected in their methylation status, X activation status, embryoid body (EB) formation, *in vitro* differentiation capacity (ectodermal, mesodermal and endodermal), teratoma formation and *in vivo* devel‐

Noteworthy, pluripotent stem cells (PSCs) possess mechanisms that lead to the silencing of the integrated transgenes. Therefore, the expression of the four TFs is necessary for generating iPSCs but dispensable for maintenance of the iPSC fate; hence, pluripotency and selfrenewal capacity rely on the trans-activation of the endogenous genes, suggesting a true and complete reprogramming. Mouse iPSCs, like ESCs, were germline competent [28] and supported the development of a mice in tetraploid complementation assay [29]. In this assay, embryonic cells at the two cell-stage are fused together. This results in a tetraploid blastocyst in which just the extraembryonic tissues will further develop; by complementing the tetraploid embryo with normal diploid PSCs, it is possible to generate an individual, completely derived from the diploid PSCs. Interestingly, the same combination of TFs [30] or a somewhat different one (Oct4, Sox2, Nanog and Lin28) can be used for the reprogramming of human cells [31].

This discovery is groundbreaking because with iPSC technology, PSCs can now be induced/ derived for autologous cell transplantation, avoiding immunological problems and ethical issues related to the use of human ESCs. In addition, iPSCs from patients carrying a disease can be derived and used to better understand the biological problem leading to the disease as

The rationale for the selection of the genes for this "reprogramming" cocktail was obviously based on the studies, done in the preceding decade, aimed at understanding the network of

The Oct4 (also known as Pou5f1) gene encodes for a TF that belongs to the POU homeodo‐ main DNA binding domain family [32]. It plays a key role in the development and in maintenance of both ESCs self-renewal and pluripotency. Misregulation of its levels triggers loss of the ESC fate; a 50% loss of expression drives ESCs to trophectoderm while a 50% greater expression induces primitive endoderm or mesoderm [33]. Knockout (KO) experiments in mouse demonstrated lethality at the preimplantation stage *in vivo* and

Sox2 belongs to a family of TFs, having the high mobility group (HMG) DNA-binding domain, identified for the first time in the SRY (sex determining Y region) protein, which is the testis determining factor. In general, the genes from the Sox family are involved in different and crucial steps of mammalian development [35, 36]. Sox2 KO embryos die immediately after

well as for drug-screening.

232 Pluripotent Stem Cells

**2.1. Rationale behind iPSCs**

failure of ESC derivation *in vitro* [34].

TFs responsible for ESC pluripotency and selfrenewal.

opmental potential (contribution to the three germ layers of chimeric animals).

The starting cell, used for reprogramming, is a key parameter that influences the kinetics, the efficiency and the quality of the iPSCs. Fibroblasts are the most commonly used somatic cells because they can be easily isolated. In mouse studies, embryonic fibroblasts (MEFs) are commonly used as iPSCs can be generated in 10-12 days; MEF-derived iPSC generation is therefore recommended for studies aimed at understanding the mechanisms underlying iPSC generation, as well as the TFs and the chemicals that may enhance this process.

To generate iPSCs from human foreskin fibroblasts (HFFs), three weeks are required and the efficiency is 100 fold less compared with human primary keratinocytes, in which reprogram‐ ming also occurs faster [56]. When using CD133<sup>+</sup> cord blood cells, iPSCs can be produced by overexpression of only Oct4 and Sox2. As cord blood banks exist, it is believed that this cell source may be useful to make an iPSC bank representing a wide panel of haplotypes for regenerative medicine [57].

Another crucial parameter is the differentiation status; Hematopoietic stem cells and progen‐ itors have a higher efficiency of reprogramming than terminally differentiated B- and T- lymphocytes [58]. Similarly, Sox2+ neural progenitor cells form iPSCs just by forced overexpression of Oct4 [59]. Many other cell types, such as adipose stem cells [60], dental pulp cells [61], oral mucosa cells [62] and peripheral blood cells [63] can also be used to generate iPSCs.

[71]. This was possible by including in between the different cDNAs, the 2A self-deleting peptide. This permits the continuous translation of downstream cDNA after the release of the previous protein [72]. In general, viral vector-based methods are quite efficient and reprodu‐ cible (>0.1% in mouse cells, <0.01 in human cells). However, clones generated by viral delivery are not clinically safe. The transgenes may be reactivated during iPSC differentiation; more‐ over long terminal repeats (LTR) may activate proto-oncogenes increasing their tumorigenicity

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 235

Another possible strategy for iPSC generation is the transient delivery of OSKM by Piggyback (PB) transposon [74]. This system consists of a donor vector, containing the cassette (OSKM), flanked by a 5' and a 3' inverted repeat, and a helper plasmid, expressing the PB transposase. When cotransfected, the cassette of the donor plasmid is pasted into the TTAA sequences present in the genome, but can be remobilized after the reprogramming [75, 76]. The PB transposon-mediated generation of iPSCs occurs with high efficiency and, among the integra‐ tive methods, this is the only one that allows a precise deletion of the cassette. However, alterations at the integration sites were found; therefore, sequences at the integrations sites

Adenoviral vectors do not integrate into the host genome and can, thus, be used for making iPSCs [77, 78]. Adenoviral vectors can carry up to 36kB and can infect both dividing and nondividing cells. However, the efficiency of iPSC generation is extremely low (0.002 to 0.0001%), probably because the premature dilution of adenoviral vectors during cell replication.

A more efficient alternative has been reported by Fusaki and colleagues [79], using F-deficient Sendai viral vectors. Sendai viruses are minus strand RNA virus, which replicate their genome in the host cytoplasm. Because these viral vectors replicate ubiquitously, their efficiency of reprogramming is similar to retroviral vectors. However, to obtain viral vector-free iPSCs, elimination of the vector using temperature sensitive mutant or antiviral compounds is

OriP/Epstain-Barr nuclear antigen-1-based (OriP/EBNA1) vectors can be transfected into host cells and maintained stably episomically (because they replicate during cell divisions through their oriP element) using a drug in the culture medium [80]. Yu and colleagues [81] used the combination of three OriP/EBNA1 vectors (having a combination of 10 reprogramming factors) to generate iPSCs from HFF. By removing the drug selection, episomal vectors are eliminated

[28, 73].

*2.3.1.2. Transposon delivery*

must be verified.

required.

*2.3.2. Non-integrative method*

*2.3.2.1. Viral vector-based delivery*

*2.3.2.2. Episomal and minicircle vectors*

from proliferating iPSCs.

iPSCs from different origins have a similar, if not identical, gene expression profile in their pluripotent state. However, it has become clear that some genomic regions are differentially methylated [64]; they retain an epigenetic memory of the cell of origin and this is reflected in their differentiation capacity. For example, iPSCs generated from blood poorly differentiated into neuronal cells but had a higher capacity to differentiate into hematopoietic cells [65].

The cell of origin to be used for iPSC generation, also has impact on safety issues; iPSC lines, generated from tail tip fibroblasts, have shown a higher propensity to form teratomas than lines obtained from stomach, hepatocyte or MEF, due to the persistence of undifferentiated cells even after iPSC differentiation [66].

#### **2.3. Methods for iPSC generation**

The method of transgene delivery is a crucial factor in determining the efficiency but also the clinical relevance of iPSCs. Although initial reports were based on retroviral vectors, later publications described several other methods, which allow the generation of iPSCs. They can be divided into two main groups: integrative and non-integrative methods [67, 68]. Integrative methods are in general more efficient but they are less safe than the non-integrative methods, which are, however, inefficient.

When choosing the strategy of reprogramming, it is important to consider the aim of the study; integrative methods, the most efficient ones, should be used for elucidating mechanisms underlying iPSC generation, and TFs and chemicals that may enhance this process, while, nonintegrative methods will be required to generate clinical-grade iPSCs.

#### *2.3.1. Integrative method*

#### *2.3.1.1. Viral vector-based delivery*

Mouse and human iPSCs were initially produced by transduction of Moloney murine leukemia virus (MMLV)-derived retroviral vectors. Vectors, based on this system, allow cargoes of up to 8Kb fragments, can efficiently infect (although only dividing cells) and are generally silenced in pluripotent cells [69].

Lentiviral vectors, derived from HIV, have also been used. Differently from the retroviral vectors, the latter have a higher cloning capacity (up to 10Kb of DNA) and can infect both dividing and non-dividing cells. However, transgenes introduced using lentiviral vectors are less-silenced and this can result in a more laborious identification of bona fide iPSC clones (having the transgenes silenced). The lentiviral vector system allowed the Tet-inducible expression of the transgenes in a tightly controlled way [70]. Subsequently, polycistronic lentiviral vectors, having the OSKM cDNA under the control of a unique promoter were used [71]. This was possible by including in between the different cDNAs, the 2A self-deleting peptide. This permits the continuous translation of downstream cDNA after the release of the previous protein [72]. In general, viral vector-based methods are quite efficient and reprodu‐ cible (>0.1% in mouse cells, <0.01 in human cells). However, clones generated by viral delivery are not clinically safe. The transgenes may be reactivated during iPSC differentiation; more‐ over long terminal repeats (LTR) may activate proto-oncogenes increasing their tumorigenicity [28, 73].

#### *2.3.1.2. Transposon delivery*

lymphocytes [58]. Similarly, Sox2+

cells even after iPSC differentiation [66].

**2.3. Methods for iPSC generation**

which are, however, inefficient.

*2.3.1.1. Viral vector-based delivery*

generally silenced in pluripotent cells [69].

*2.3.1. Integrative method*

iPSCs.

234 Pluripotent Stem Cells

neural progenitor cells form iPSCs just by forced

overexpression of Oct4 [59]. Many other cell types, such as adipose stem cells [60], dental pulp cells [61], oral mucosa cells [62] and peripheral blood cells [63] can also be used to generate

iPSCs from different origins have a similar, if not identical, gene expression profile in their pluripotent state. However, it has become clear that some genomic regions are differentially methylated [64]; they retain an epigenetic memory of the cell of origin and this is reflected in their differentiation capacity. For example, iPSCs generated from blood poorly differentiated into neuronal cells but had a higher capacity to differentiate into hematopoietic cells [65].

The cell of origin to be used for iPSC generation, also has impact on safety issues; iPSC lines, generated from tail tip fibroblasts, have shown a higher propensity to form teratomas than lines obtained from stomach, hepatocyte or MEF, due to the persistence of undifferentiated

The method of transgene delivery is a crucial factor in determining the efficiency but also the clinical relevance of iPSCs. Although initial reports were based on retroviral vectors, later publications described several other methods, which allow the generation of iPSCs. They can be divided into two main groups: integrative and non-integrative methods [67, 68]. Integrative methods are in general more efficient but they are less safe than the non-integrative methods,

When choosing the strategy of reprogramming, it is important to consider the aim of the study; integrative methods, the most efficient ones, should be used for elucidating mechanisms underlying iPSC generation, and TFs and chemicals that may enhance this process, while, non-

Mouse and human iPSCs were initially produced by transduction of Moloney murine leukemia virus (MMLV)-derived retroviral vectors. Vectors, based on this system, allow cargoes of up to 8Kb fragments, can efficiently infect (although only dividing cells) and are

Lentiviral vectors, derived from HIV, have also been used. Differently from the retroviral vectors, the latter have a higher cloning capacity (up to 10Kb of DNA) and can infect both dividing and non-dividing cells. However, transgenes introduced using lentiviral vectors are less-silenced and this can result in a more laborious identification of bona fide iPSC clones (having the transgenes silenced). The lentiviral vector system allowed the Tet-inducible expression of the transgenes in a tightly controlled way [70]. Subsequently, polycistronic lentiviral vectors, having the OSKM cDNA under the control of a unique promoter were used

integrative methods will be required to generate clinical-grade iPSCs.

Another possible strategy for iPSC generation is the transient delivery of OSKM by Piggyback (PB) transposon [74]. This system consists of a donor vector, containing the cassette (OSKM), flanked by a 5' and a 3' inverted repeat, and a helper plasmid, expressing the PB transposase. When cotransfected, the cassette of the donor plasmid is pasted into the TTAA sequences present in the genome, but can be remobilized after the reprogramming [75, 76]. The PB transposon-mediated generation of iPSCs occurs with high efficiency and, among the integra‐ tive methods, this is the only one that allows a precise deletion of the cassette. However, alterations at the integration sites were found; therefore, sequences at the integrations sites must be verified.

#### *2.3.2. Non-integrative method*

#### *2.3.2.1. Viral vector-based delivery*

Adenoviral vectors do not integrate into the host genome and can, thus, be used for making iPSCs [77, 78]. Adenoviral vectors can carry up to 36kB and can infect both dividing and nondividing cells. However, the efficiency of iPSC generation is extremely low (0.002 to 0.0001%), probably because the premature dilution of adenoviral vectors during cell replication.

A more efficient alternative has been reported by Fusaki and colleagues [79], using F-deficient Sendai viral vectors. Sendai viruses are minus strand RNA virus, which replicate their genome in the host cytoplasm. Because these viral vectors replicate ubiquitously, their efficiency of reprogramming is similar to retroviral vectors. However, to obtain viral vector-free iPSCs, elimination of the vector using temperature sensitive mutant or antiviral compounds is required.

#### *2.3.2.2. Episomal and minicircle vectors*

OriP/Epstain-Barr nuclear antigen-1-based (OriP/EBNA1) vectors can be transfected into host cells and maintained stably episomically (because they replicate during cell divisions through their oriP element) using a drug in the culture medium [80]. Yu and colleagues [81] used the combination of three OriP/EBNA1 vectors (having a combination of 10 reprogramming factors) to generate iPSCs from HFF. By removing the drug selection, episomal vectors are eliminated from proliferating iPSCs.

Another alternative are the minicircle vectors, that differently from the above vectors, are nonreplicative [82]. These vectors have a better transfection efficiency than OriP/EBNA1, due to their reduced length (they lack the bacterial origin of replication and the antibiotic resistance gene). However, both strategies have a three-fold lower efficiency (<0.001%) than retroviral vector-based reprogramming.

**2.4. iPSC technology, unsolved questions and emerging technologies**

the efficiency of iPSC technology [91].

least some iPSC clones are indistinguishable from ESCs.

and is thus not caused by the reprogramming process.

burgeoning field of research (see next section).

iPSCs were reproducibly derived from most, if not all, somatic tissues; however the efficiency reported is always less than 1%. It is the consensus of scientific community, that many more than 1% of the transfected/transduced cells start the reprogramming process. Using a live cell imaging approach, it was demonstrated [87] that almost all the transduced cells undergo symmetric divisions within 48 hours, retaining a fibroblast-morphology. At later stages, reprogramming cells undergo asymmetric divisions: one descendant becomes an iPSC while the other one undegoes cell death. Still unknown, stochastic and clonal events appear to control this process at later stages; in fact most of the cells do not complete the initiated process. Several studies have demonstrated key roles for demethylation [88], telomerase length [89] and mesenchymal to epithelial transition [90], during the reprogramming. A better understanding, especially of the later stochastic mechanisms, is still needed to fully understand and improve

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 237

Another important question has been whether or not ESCs and iPSCs are similar and if not, whether differences are functionally important for their application. Conclusions from different studies are conflicting. Several papers have reported that there are remarkable differences in gene expression and DNA methylation [92, 93], while other studies, which included a large number of ESC and iPSC lines, concluded that it is quite difficult to distinguish between ESCs and iPSCs [94-96]. Considering that there are differences among different ESC lines [97], it is now believed that iPSCs clones have a higher variability than ESCs but that at

Furthermore, the recent work of Young and colleagues [98] demonstrated that most of the genetic variability in between different iPSC clones is already present in the starting cell line

Interestingly, in the last five years, several studies have clearly demonstrated the potential of iPSC technology in regenerative medicine. Hanna et al [99] generated iPSCs from a humanized model of sickle cell anemia. After correcting the hemoglobin gene, by gene targeting, iPSCs were able to generate hematopoietic stem cells and to rescue the disease. Similarly, the potential of iPSCs for cell therapy was demonstrated for macular degeneration [100], Parkin‐

iPSCs derived from patients with specific diseases have been used for studying the mecha‐ nisms involved in these diseases and for drug screening [105, 106]. *In vitro* disease modeling is not only possible for monogenic disease but also for more complex polygenic diseases,

As a result of the success with reprogramming of somatic cells to a pluripotency-state, lineage reprogramming (trans-differentiation) between different somatic cell types has also become a

In conclusion, iPSC technology will, in the near future, have a drastic impact on science, regenerative medicine and business. Precise selection of "clean" clones, through the evaluation of their genomic and epigenetic integrity, as well as their gene expression profile, will be crucial

son's disease [101], platelet deficiencies [102] and spinal cord injury [103, 104].

having a late onset, like schizophrenia [107] and Alzheimer [108, 109].

#### *2.3.2.3. Protein/RNA based delivery*

Previous studies have shown that proteins can be directly delivered into cells by fusing them with peptides [83], which mediates their transduction, such as poly-arginine or the HIV transactivator of transcription (TAT). Zhou et al. [84] produced recombinant OSKM proteins fused with poly-arginine in *Escherichia coli* and generated iPSCs from Oct4-GFP MEF, including valproic acid (a histone deacetylase inhibitor) in the medium. Kim et al. [85], succeeded in reprogramming human neonatal fibroblasts by producing OSKM, fused to a Myc tag and nine arginines.

Similarly, *in vitro* transcribed ssRNA, modified by phosphatase treatment and by substituting cytidine and uridine for 5-methylcytidine and pseudouridine, can be delivered into different human cells [86]. This method also requires a recombinant B18R protein, which improves cell viability and protein stability. Differently from protein delivery, the latter strategy has a fast kinetics and a higher efficiency [0.01-0.1%, depending on the cell type). However, also in this case, a careful screening for mutations in different iPSC clones will be needed before an eventual clinical application.


**Table 1.** Comparison of different strategies for iPSC generation

#### **2.4. iPSC technology, unsolved questions and emerging technologies**

Another alternative are the minicircle vectors, that differently from the above vectors, are nonreplicative [82]. These vectors have a better transfection efficiency than OriP/EBNA1, due to their reduced length (they lack the bacterial origin of replication and the antibiotic resistance gene). However, both strategies have a three-fold lower efficiency (<0.001%) than retroviral

Previous studies have shown that proteins can be directly delivered into cells by fusing them with peptides [83], which mediates their transduction, such as poly-arginine or the HIV transactivator of transcription (TAT). Zhou et al. [84] produced recombinant OSKM proteins fused with poly-arginine in *Escherichia coli* and generated iPSCs from Oct4-GFP MEF, including valproic acid (a histone deacetylase inhibitor) in the medium. Kim et al. [85], succeeded in reprogramming human neonatal fibroblasts by producing OSKM, fused to a Myc tag and nine

Similarly, *in vitro* transcribed ssRNA, modified by phosphatase treatment and by substituting cytidine and uridine for 5-methylcytidine and pseudouridine, can be delivered into different human cells [86]. This method also requires a recombinant B18R protein, which improves cell viability and protein stability. Differently from protein delivery, the latter strategy has a fast kinetics and a higher efficiency [0.01-0.1%, depending on the cell type). However, also in this case, a careful screening for mutations in different iPSC clones will be needed before an

Viral Retrovirus + 4X

Viral Lentivirus + 3X

Viral Adenovirus - 1X

Viral Sendai Virus - 4X

DNA Transposon - 2X

DNA Minicircle - X

DNA Episomal plasmid - X

RNA Recombinant RNA - 3X

PROTEIN Recombinant protein - X

**Genomic Integration** **Efficiency reported**

vector-based reprogramming.

eventual clinical application.

**Type of vector Method**

4x= >0.1%; 3X= <0.1%; 2X= <0.01%;1X= <0.001%

**Table 1.** Comparison of different strategies for iPSC generation

arginines.

236 Pluripotent Stem Cells

*2.3.2.3. Protein/RNA based delivery*

iPSCs were reproducibly derived from most, if not all, somatic tissues; however the efficiency reported is always less than 1%. It is the consensus of scientific community, that many more than 1% of the transfected/transduced cells start the reprogramming process. Using a live cell imaging approach, it was demonstrated [87] that almost all the transduced cells undergo symmetric divisions within 48 hours, retaining a fibroblast-morphology. At later stages, reprogramming cells undergo asymmetric divisions: one descendant becomes an iPSC while the other one undegoes cell death. Still unknown, stochastic and clonal events appear to control this process at later stages; in fact most of the cells do not complete the initiated process. Several studies have demonstrated key roles for demethylation [88], telomerase length [89] and mesenchymal to epithelial transition [90], during the reprogramming. A better understanding, especially of the later stochastic mechanisms, is still needed to fully understand and improve the efficiency of iPSC technology [91].

Another important question has been whether or not ESCs and iPSCs are similar and if not, whether differences are functionally important for their application. Conclusions from different studies are conflicting. Several papers have reported that there are remarkable differences in gene expression and DNA methylation [92, 93], while other studies, which included a large number of ESC and iPSC lines, concluded that it is quite difficult to distinguish between ESCs and iPSCs [94-96]. Considering that there are differences among different ESC lines [97], it is now believed that iPSCs clones have a higher variability than ESCs but that at least some iPSC clones are indistinguishable from ESCs.

Furthermore, the recent work of Young and colleagues [98] demonstrated that most of the genetic variability in between different iPSC clones is already present in the starting cell line and is thus not caused by the reprogramming process.

Interestingly, in the last five years, several studies have clearly demonstrated the potential of iPSC technology in regenerative medicine. Hanna et al [99] generated iPSCs from a humanized model of sickle cell anemia. After correcting the hemoglobin gene, by gene targeting, iPSCs were able to generate hematopoietic stem cells and to rescue the disease. Similarly, the potential of iPSCs for cell therapy was demonstrated for macular degeneration [100], Parkin‐ son's disease [101], platelet deficiencies [102] and spinal cord injury [103, 104].

iPSCs derived from patients with specific diseases have been used for studying the mecha‐ nisms involved in these diseases and for drug screening [105, 106]. *In vitro* disease modeling is not only possible for monogenic disease but also for more complex polygenic diseases, having a late onset, like schizophrenia [107] and Alzheimer [108, 109].

As a result of the success with reprogramming of somatic cells to a pluripotency-state, lineage reprogramming (trans-differentiation) between different somatic cell types has also become a burgeoning field of research (see next section).

In conclusion, iPSC technology will, in the near future, have a drastic impact on science, regenerative medicine and business. Precise selection of "clean" clones, through the evaluation of their genomic and epigenetic integrity, as well as their gene expression profile, will be crucial for downstream applications. Despite these remaining hurdles, it is believed that clinical applications for iPSCs are not far off.

**3.1. Conversion into mesodermal types**

uptake after transplantation into mice).

potential, as shown by transplantation experiments.

Hand2 is added to the above cocktail of genes [131].

**3.2. Conversion into endodermal types**

Seale and colleagues have recently found that Myf5+

fat cells *in vivo* and *in vitro* [124], while studying the role of PRDM16 during development. Overexpression of PRDM16 differentiated primary mouse myoblasts with nearly 100% efficiency to brown fat. *Vice versa,* downregulation of PRDM16 in primary brown fat cells resulted in the expression of MyoD and Myogenin and in a myotube-like morphology. Interestingly, forced expression PRDM16 was not able to induce the same conversion in nonmyogenic cell lines, like fibroblasts. Performing proteomic studies, they identified C/EBP*α* as a partner of PRDM16 in brown fat. Subsequently, they used combined overexpression of C/ EBP*α* and PRDM16 [125] and demonstrated that mouse and human dermal fibroblasts could differentiate into brown-fat cells, which functional features (fat pad formation and glucose

Human dermal fibroblasts were converted into multipotent blood progenitors by just Oct4 overexpression [126] in combination with treatment with a hematopoietic permissive medium, containing growth factors and cytokines. Oct4 is a key TF for pluripotency but it is not expressed in the hematopoieic system [127]; probably, Oct4, in this case of lineage reprogram‐ ming, is mimicking the effect of Oct1 and Oct2, two other members of Pou family of TFs expressed in lymphoid development [128]. The induced progenitors express CD45 and express adult globin protein (unlike hematopoietic cells derived from human ESCs and iPSCs). Multipotent blood progenitors have myeloid, erythroid and megakaryocytic but not lymphoid

The forced overexpression of TFs involved in cardiac development (Tbx5, Mef2c and Gata4) converts mouse cardiac and dermal fibroblasts into cardiomyocyte-like cells, termed induced cardiomyocyte (iCMs) [129]. Around 20% of the cells appear to be 'converted' in three days as measured by the expression of alpha-myosin heavy chain (αMHC), although one month is required for their complete maturation, which resulted in spontaneous beating capacity. Transplantation of iCMs, the day after the viral transduction, in injured hearts results in their engraftment and differentiation *in vivo*. Interestingly, the same strategy is able to convert cardiac fibroblasts *in vivo* [130]. When retroviral vectors, carrying the above factors, are injected after myocardial infarction, this results in the efficient conversion (>50%) of cardiac fibroblasts into functionally beating cardiomyocytes. Efficiency of cardiac-conversion is increased when

The lineage reprogramming into β-cells is of particular interest, considering the potential for the treatment of diabetes. Zhou et al., [132] were able to *in vivo* convert exocrine acinar cells into functional β-like cells, combining three genes essential for pancreatic development (Ngn3, Pdx1 and MafA). Adenoviral vectors, carrying the pancreatic cocktail, were injected; again, conversion occurred in three day and efficiency of conversion was relatively high (20%). Analysis, one month later, showed that induced β-like cells produced insulin and rescued the hyperglycemic level after streptozotocin-treatment. However, the same combination of factors

muscle precursors can convert into brown

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 239

## **3. Lineage conversion**

The discovery of iPSCs, together with previous experiments involving SCNT and cell fusion, clearly showed that differentiation is a reversible process and that cells are more 'plastic' than previously believed. Therefore, a new field, called lineage reprogramming, emerged rapidly in the last five years. Recent attempts have demonstrated that, the forced overexpression of TFs can also convert one cell type to another of the same or of other somatic germ layers. Lineage reprogramming depends on the capacity of certain TFs to overcome the existing epigenetic barriers and to rapidly initiate the new identity-specific gene network [110-113].

Examples of direct lineage conversion were described already in 1986; Davis, Lassar et al. [114, 115] converted different fibroblast lines into myogenic cells by overexpression of MyoD, a basic helix-loop-helix transcription factor, in combination with the demethylat‐ ing agent, 5-azacytidine. Subsequent studies confirmed that myogenic conversion, as shown by presence of desmin and myosin heavy chain, could be achieved *in vitro* starting from a variety of cell types (adipose, melanoma, neuroblastoma and liver cell lines). However, the complete downregulation of the 'original' tissue-specific genes was only seen when starting with mesodermal cells and not with endodermal or ectodermal cell lines [116, 117].

A similar transdifferantiation was also seen in the blood system. The deletion of Pax5 in pro-B cells resulted in their switch into the T-cell lineage [118, 119]. Later on, the same group investigated this transdifferentiation more extensively; mature B cells were isolated from Pax5 knockout mice and transplanted back into a lymphocyte deficient recipient. Surprisingly, they could detect in the reconstituted mice donor pro B cells, which then gradually converted into T cells [120]. This demonstrated that lost of Pax5 led to a T cell phenotype through dedifferentiation rather than direct transdifferentiation. Another example of direct conversion came from the work done by Graf and colleagues [121]; overexpression of C/EBP*α or* C/ EBP*β,* a basic leucine zipper TF binding CCAAT enhancers, induced a macrophage phenotype (as shown by Mac1 expression) in bone marrow or spleen-derived B cells. In the induced macrophages almost all the B cell genes analyzed were downregulated and cells acquired phagocytic function *in vitro*.

The above examples describe experimental conversions but there were also cases in which this conversion occurs naturally. Jarriault et al. [122], demonstrated that the epithelial rectal cell 'Y', migrates anterodorsally from the rectum to become a 'PDA' motor neuron. This conversion from Y to PDA is not direct but occurs through a de-differentiation state, in which the initial (Y-cell) and the final (PDA-cell) identity are not present [123]. In this section, we will describe the relevant cell types, recently, obtained by lineage reprogramming.

#### **3.1. Conversion into mesodermal types**

for downstream applications. Despite these remaining hurdles, it is believed that clinical

The discovery of iPSCs, together with previous experiments involving SCNT and cell fusion, clearly showed that differentiation is a reversible process and that cells are more 'plastic' than previously believed. Therefore, a new field, called lineage reprogramming, emerged rapidly in the last five years. Recent attempts have demonstrated that, the forced overexpression of TFs can also convert one cell type to another of the same or of other somatic germ layers. Lineage reprogramming depends on the capacity of certain TFs to overcome the existing epigenetic barriers and to rapidly initiate the new identity-specific

Examples of direct lineage conversion were described already in 1986; Davis, Lassar et al. [114, 115] converted different fibroblast lines into myogenic cells by overexpression of MyoD, a basic helix-loop-helix transcription factor, in combination with the demethylat‐ ing agent, 5-azacytidine. Subsequent studies confirmed that myogenic conversion, as shown by presence of desmin and myosin heavy chain, could be achieved *in vitro* starting from a variety of cell types (adipose, melanoma, neuroblastoma and liver cell lines). However, the complete downregulation of the 'original' tissue-specific genes was only seen when starting with mesodermal cells and not with endodermal or ectodermal cell

A similar transdifferantiation was also seen in the blood system. The deletion of Pax5 in pro-B cells resulted in their switch into the T-cell lineage [118, 119]. Later on, the same group investigated this transdifferentiation more extensively; mature B cells were isolated from Pax5 knockout mice and transplanted back into a lymphocyte deficient recipient. Surprisingly, they could detect in the reconstituted mice donor pro B cells, which then gradually converted into T cells [120]. This demonstrated that lost of Pax5 led to a T cell phenotype through dedifferentiation rather than direct transdifferentiation. Another example of direct conversion came from the work done by Graf and colleagues [121]; overexpression of C/EBP*α or* C/ EBP*β,* a basic leucine zipper TF binding CCAAT enhancers, induced a macrophage phenotype (as shown by Mac1 expression) in bone marrow or spleen-derived B cells. In the induced macrophages almost all the B cell genes analyzed were downregulated and cells acquired

The above examples describe experimental conversions but there were also cases in which this conversion occurs naturally. Jarriault et al. [122], demonstrated that the epithelial rectal cell 'Y', migrates anterodorsally from the rectum to become a 'PDA' motor neuron. This conversion from Y to PDA is not direct but occurs through a de-differentiation state, in which the initial (Y-cell) and the final (PDA-cell) identity are not present [123]. In this section, we will describe

the relevant cell types, recently, obtained by lineage reprogramming.

applications for iPSCs are not far off.

**3. Lineage conversion**

238 Pluripotent Stem Cells

gene network [110-113].

lines [116, 117].

phagocytic function *in vitro*.

Seale and colleagues have recently found that Myf5+ muscle precursors can convert into brown fat cells *in vivo* and *in vitro* [124], while studying the role of PRDM16 during development. Overexpression of PRDM16 differentiated primary mouse myoblasts with nearly 100% efficiency to brown fat. *Vice versa,* downregulation of PRDM16 in primary brown fat cells resulted in the expression of MyoD and Myogenin and in a myotube-like morphology. Interestingly, forced expression PRDM16 was not able to induce the same conversion in nonmyogenic cell lines, like fibroblasts. Performing proteomic studies, they identified C/EBP*α* as a partner of PRDM16 in brown fat. Subsequently, they used combined overexpression of C/ EBP*α* and PRDM16 [125] and demonstrated that mouse and human dermal fibroblasts could differentiate into brown-fat cells, which functional features (fat pad formation and glucose uptake after transplantation into mice).

Human dermal fibroblasts were converted into multipotent blood progenitors by just Oct4 overexpression [126] in combination with treatment with a hematopoietic permissive medium, containing growth factors and cytokines. Oct4 is a key TF for pluripotency but it is not expressed in the hematopoieic system [127]; probably, Oct4, in this case of lineage reprogram‐ ming, is mimicking the effect of Oct1 and Oct2, two other members of Pou family of TFs expressed in lymphoid development [128]. The induced progenitors express CD45 and express adult globin protein (unlike hematopoietic cells derived from human ESCs and iPSCs). Multipotent blood progenitors have myeloid, erythroid and megakaryocytic but not lymphoid potential, as shown by transplantation experiments.

The forced overexpression of TFs involved in cardiac development (Tbx5, Mef2c and Gata4) converts mouse cardiac and dermal fibroblasts into cardiomyocyte-like cells, termed induced cardiomyocyte (iCMs) [129]. Around 20% of the cells appear to be 'converted' in three days as measured by the expression of alpha-myosin heavy chain (αMHC), although one month is required for their complete maturation, which resulted in spontaneous beating capacity. Transplantation of iCMs, the day after the viral transduction, in injured hearts results in their engraftment and differentiation *in vivo*. Interestingly, the same strategy is able to convert cardiac fibroblasts *in vivo* [130]. When retroviral vectors, carrying the above factors, are injected after myocardial infarction, this results in the efficient conversion (>50%) of cardiac fibroblasts into functionally beating cardiomyocytes. Efficiency of cardiac-conversion is increased when Hand2 is added to the above cocktail of genes [131].

#### **3.2. Conversion into endodermal types**

The lineage reprogramming into β-cells is of particular interest, considering the potential for the treatment of diabetes. Zhou et al., [132] were able to *in vivo* convert exocrine acinar cells into functional β-like cells, combining three genes essential for pancreatic development (Ngn3, Pdx1 and MafA). Adenoviral vectors, carrying the pancreatic cocktail, were injected; again, conversion occurred in three day and efficiency of conversion was relatively high (20%). Analysis, one month later, showed that induced β-like cells produced insulin and rescued the hyperglycemic level after streptozotocin-treatment. However, the same combination of factors failed to reprogram myocytes *in vivo* and mouse embryonic fibroblast *in vitro*, indicating that additional factors will be needed to achieve this conversion from unrelated cell types.

fibroblasts were converted with Ascl1, Brn2, Mytl1, Lhx3, Ngn2, Isl1 and Hb9 whereas human cells also required NeuroD1 [143]. iMNs displayed markers, electrophysiological features and gene expression profile, which strongly resemble motor neurons. Moreover, iMNs engrafted into the developing chick spinal cord, forming axonal and dentritic projections toward the

Of note, Qiang et al [144] demonstrated that lineage reprogramming is also useful for drug screening and disease modeling. iNs, again with glutamatergic features, were induced by overexpressing Ascl1, Bm2, Mytl1 together with Zic1 and Olig2. iNs were produced from both healthy donors and Alzheimer's patients. iNs produced from patients displayed the typical accumulation of beta amyloid peptides (Aβ40 and Aβ42). Combining lineage reprogramming with gene-targeting technology, similar cells could also be used for autologous transplantation.

**Starting cell type Conversion into Factors**

(m/h) dermal fibroblasts, myoblasts Brown-fat cells C/EBPα and PRDM16 (125) (m) embryonic fibroblasts Myoblasts MyoD (114, 115)

(m) cardiac and tail tip fibroblasts Cardiomyocytes Tbx5, Mef2c, Gata4 ± Hand2 (129) (131) (m) embryonic fibroblasts Cardiomyocytes Oct4, Sox2, Klf4 and cMyc (145) (m) exocrine cells β-like cells Ngn3, Pdx1 and MafA (132) (m) embryonic and dermal fibroblasts Hepatocyte-like cells Hnf4α, FoxA1, FoxA2 and FoxA3 (133) (m) embryonic and tail tip fibroblasts Hepatocyte-like cells Gata4, Hnf1α, Foxa3 and p19Arf KD (134)

(m/h) embryonic fibroblasts Motor neurons Ascl1, Brn2, Mytl1, Lhx3, Ngn2, Isl1, Hb9 and

cells C/EBPα or β, PU.1 (121)

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 241

cells Oct4, Sox2, Klf4 and cMyc (146)

cells Sox2, Klf4, c-Myc,Tcf3 and Brn4 (138)

Neurons Ascl1, Mytl1, Bm2 and NeuroD1 (135) (136)

neurons Ascl1, Mytl1, Bm2, Lmx1a and FoxA2 (141)

neurons Ascl1, Bm2, Mytl1, Zic1 and Olig2 (144)

NeuroD1 (143)

neurons Ascl1, Lmx1a and Nurr1 (142)

cells Oct4, Sox2, Klf4 and Zic3 (139)

cells Sox2 (140)

progenitors Oct4 (126)

(m) Β-Cells Macrophage-like

(h) dermal fibroblasts Multipotent blood

(m) embryonic fibroblasts Neural progenitor

(m/h) fibroblasts Neural progenitor

(h) fibroblasts Neural progenitor

(m) embryonic and (h) fetal fibroblasts Neural progenitor

(m) tail and (h) embryonic fibroblasts Dopaminergic

(h) embryonic and fetal lung fibroblasts Dopaminergic

**Table 2.** Examples of lineage conversion. (m)= mouse (h) = human

(h) skin fibroblasts Glutamatergic

(m) embryonic and (h) fetal, postnatal and dermal fibroblasts

adjacent musculature.

More recently, mouse fibroblasts were converted into hepatocyte-like cells by overexpressing Hnf4α, FoxA1, FoxA2 and FoxA3 [133] or by Gata4, Hnf1α, Foxa3 together with p19Arf inactivation [134]. The reprogrammed cells were termed induced hepatocytes (iHeps) and had a gene expression profile and features (albumin production and cytochrome P450 activity), which closely resemble mature hepatocyte. iHeps *in vivo* were able to reconstitute hepatic tissues and to support hepatic function in the fumoaryl-acetate hydrolase (FAH-/-) deficient mice.

#### **3.3. Conversion into neuronal types**

The conversion into neuronal types is, probably, the one that received more attention in the field of lineage reprogramming. The increasing attention is due to their possible application for the treatment of diseases involving the nervous system.

In 2010, Vierbuchen et al. [135] were the first to describe how overexpression of Ascl1, Mytl1 and Bm2 (also known as Pou3f2, again a member of Pou family) can convert embryonic and tail-tip fibroblasts into a mixed populations of induced neurons (iNs). iNs generate functional synapses with mouse cortical neurons and have action potentials; the detailed electrophysio‐ logical analysis showed that iNs contains mainly cells with features of glutamatergic neurons (with just a small percentage of GABAergic neurons). Remarkably, the addition of NeuroD1 to the above set of genes was necessary to achieve the same conversion in human cells [136]. The enriched cocktail of factors was able not only convert fibroblasts but also mouse hepatocyte into iNs [137].

Several groups, differently, converted fibroblasts into induced neural stem cells (iNSCs), that differently from the previous examples, can still self-renew and differentiate into different neuronal subtypes (multipotent). Different cocktail of factors and inductive media have been used to obtain multipotent neuronal stem cells from human and mouse fibroblasts: the group of Scholer [138] used Sox2, Klf4, c-Myc, together with Tcf3 and Brn4 (also known as Pou3f4); our group [139] by adding Zic3 to Oct4, Sox2 and Klf4; Ring et al. [140], by just overexpressing Sox2.

Different laboratories focused on a more direct conversion into specific neuronal subtypes, with a particular interest on neuronal cell types affected in neurodegenerative diseases. Two groups have been able to convert mouse and human fibroblasts into induced Dopaminergic Neurons (iDAs), the subtype affected in Parkinson's disease. The first laboratory [141] achieved this by adding FoxA2 and Lmx1a to Ascl1, Mytl1 and Bm2; the second [142] by overexpressing Ascl1, Lmx1a and Nurr1 (also known as Nr4a2). iDA cells, upon transplanta‐ tion in mice, were capable to integrate into the host neuronal circuitry and express markers typical for mature dopaminergic neurons.

Lineage conversion was also achieved into spinal motor neurons, the subtypes involved in amyotrophic lateral sclerosis and spinal muscular atrophy. Conversion into induced Motor Neurons (iMNs) was achieved for both mouse and human fibroblasts; mouse embryonic fibroblasts were converted with Ascl1, Brn2, Mytl1, Lhx3, Ngn2, Isl1 and Hb9 whereas human cells also required NeuroD1 [143]. iMNs displayed markers, electrophysiological features and gene expression profile, which strongly resemble motor neurons. Moreover, iMNs engrafted into the developing chick spinal cord, forming axonal and dentritic projections toward the adjacent musculature.

failed to reprogram myocytes *in vivo* and mouse embryonic fibroblast *in vitro*, indicating that

More recently, mouse fibroblasts were converted into hepatocyte-like cells by overexpressing Hnf4α, FoxA1, FoxA2 and FoxA3 [133] or by Gata4, Hnf1α, Foxa3 together with p19Arf inactivation [134]. The reprogrammed cells were termed induced hepatocytes (iHeps) and had a gene expression profile and features (albumin production and cytochrome P450 activity), which closely resemble mature hepatocyte. iHeps *in vivo* were able to reconstitute hepatic tissues and to support hepatic function in the fumoaryl-acetate hydrolase (FAH-/-) deficient

The conversion into neuronal types is, probably, the one that received more attention in the field of lineage reprogramming. The increasing attention is due to their possible application

In 2010, Vierbuchen et al. [135] were the first to describe how overexpression of Ascl1, Mytl1 and Bm2 (also known as Pou3f2, again a member of Pou family) can convert embryonic and tail-tip fibroblasts into a mixed populations of induced neurons (iNs). iNs generate functional synapses with mouse cortical neurons and have action potentials; the detailed electrophysio‐ logical analysis showed that iNs contains mainly cells with features of glutamatergic neurons (with just a small percentage of GABAergic neurons). Remarkably, the addition of NeuroD1 to the above set of genes was necessary to achieve the same conversion in human cells [136]. The enriched cocktail of factors was able not only convert fibroblasts but also mouse hepatocyte

Several groups, differently, converted fibroblasts into induced neural stem cells (iNSCs), that differently from the previous examples, can still self-renew and differentiate into different neuronal subtypes (multipotent). Different cocktail of factors and inductive media have been used to obtain multipotent neuronal stem cells from human and mouse fibroblasts: the group of Scholer [138] used Sox2, Klf4, c-Myc, together with Tcf3 and Brn4 (also known as Pou3f4); our group [139] by adding Zic3 to Oct4, Sox2 and Klf4; Ring et al. [140], by just overexpressing

Different laboratories focused on a more direct conversion into specific neuronal subtypes, with a particular interest on neuronal cell types affected in neurodegenerative diseases. Two groups have been able to convert mouse and human fibroblasts into induced Dopaminergic Neurons (iDAs), the subtype affected in Parkinson's disease. The first laboratory [141] achieved this by adding FoxA2 and Lmx1a to Ascl1, Mytl1 and Bm2; the second [142] by overexpressing Ascl1, Lmx1a and Nurr1 (also known as Nr4a2). iDA cells, upon transplanta‐ tion in mice, were capable to integrate into the host neuronal circuitry and express markers

Lineage conversion was also achieved into spinal motor neurons, the subtypes involved in amyotrophic lateral sclerosis and spinal muscular atrophy. Conversion into induced Motor Neurons (iMNs) was achieved for both mouse and human fibroblasts; mouse embryonic

additional factors will be needed to achieve this conversion from unrelated cell types.

mice.

240 Pluripotent Stem Cells

into iNs [137].

Sox2.

**3.3. Conversion into neuronal types**

typical for mature dopaminergic neurons.

for the treatment of diseases involving the nervous system.

Of note, Qiang et al [144] demonstrated that lineage reprogramming is also useful for drug screening and disease modeling. iNs, again with glutamatergic features, were induced by overexpressing Ascl1, Bm2, Mytl1 together with Zic1 and Olig2. iNs were produced from both healthy donors and Alzheimer's patients. iNs produced from patients displayed the typical accumulation of beta amyloid peptides (Aβ40 and Aβ42). Combining lineage reprogramming with gene-targeting technology, similar cells could also be used for autologous transplantation.


#### **3.4. Direct versus indirect strategy**

Most of the examples, given in the previous section, describe the direct conversion from one cell type to another, in which the reprogramming is achieved without any intermediate state. However, other reports clearly demonstrated the possibility to achieve similar results, by using an alternative strategy, in which lineage conversion is indirect. Indirect conversion is achieved passing through a limited de-differentiation state by overexpressing Yamanaka factors for a shorter time. Like for the direct conversion, the indirect conversion is strongly dependent on the specific culture medium (growth factors and cytokines) given during the reprogramming phase.

or maintenance during embryonic development, overcome the pre-existing epigenetic marks and generate a new state. In the indirect conversion, the TFs, which allow the reprogramming to the pluripotency-state, are temporally overerexpressed together with fate-specific signals to convert original cell type into a new state. Differently than iPSC technology, the efficiencies are much higher (even 20 % in some cases) and the kinetics of conversion are rapid (a few day

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 243

**Strategy Efficiency Kinetic Potentiality Expandibility Tumorigenicity Cell Proliferation** iPSC Low Weeks-Months Pluripotent Yes High Required Direct conversion High Hours-Days Unipotent No Low Not required Indirect conversion High Days-Weeks Multi/Unipotent Yes Moderate Required

As for iPSCs, many questions still remain unsolved in lineage conversion. It is not clear whether the new cell type, generated upon conversion, is a hybrid between the original and the new cell. It is intriguing that, in direct conversion, TFs erase partially or completely the previous epigenetic marks, without cell divisions (in which chromatin marks are lost) but it is totally unknown how this is possible. Remarkably, in both iPSCs and lineage conversion, efficiencies are lower with human cells, if compared with mouse. It is unknown whether this is due to the

Reprogramming to the pluripotency-state and lineage conversion are achieved through the forced expression of TFs. However, in the last decade, several reports have highlighted how culture medium per se can be responsible for (partial) reprogramming. Moreover, there is an increasing amout of evidences showing that small molecules, including epigenetic modifiers and signaling pathway inhibitors, enhance the efficiency and kinetics of reprogramming.

Epiblast stem cells (EpiSCs) are isolated from post-implantation embryos between E5.5-E7.5. EpiSCs are the post-implantation equivalent of ESCs; they still express Oct4, Nanog and Sox2 but express lower levels of Stella and Rex1 [147]. ESCs and EpiSCs have also different culture requirements and features. While ESC selfrenewal is LIF dependent, EpiSC proliferation requires bFGF and Activin signaling. EpiSC female lines, but not ESC lines, have one of the X chromosome inactive. Importantly, EpiSCs, differently from ESCs, do not have the ability to

In 2009, Bao et al. [148] demonstrated that established EpiSC lines could de-differentiate/ revert into an ESC-like state by culturing EpiSCs in ESC medium (cointaining LIF) for 2-5 weeks. Once 'reverted' cells lost all the features of the original EpiSCs and acquired all ESCcharacteristics ( X was reactivated, growth was LIF-dependent and cells were capable to

contribute to chimeras *in vivo*, when aggregated into recipient morula/blastocysts.

intrinsic karyotypic instability of mouse cells in culture or to molecular mechanisms.

to a week maximum, and not two weeks to a month, like for iPSC, see Table 3).

**Table 3.** Comparison of different strategy of TF-based Reprogramming

**4. Culture mediated reprogramming**

The laboratory of Sheng Ding, at the Gladstone Institute of San Francisco, was the first to describe the possibility of lineage reprogramming through an indirect strategy. Short temporal overexpression of the Yamanaka factors induced a partial dedifferentiatied state, that allowed the subsequent conversion into cardiomyocytes-like cells by applying extracellular factors [145]. OSKM factors were overexpressed for six days in a medium free of signals necessary for pluripotency (i.e. leukemia inhibitory factor). After this short priming phase, cells were then cultured in media promoting cardiogenesis, i.e. cointaining BMP4. Three day after the cardiac induction, the expression of Nkx2.5, Gata4 and Flk1 (mid-stage markers of cardiac develop‐ ments) could be detected. The further development into more mature cardiomyocytes, showing sarcomeric structures and cardiac features (expression of cardiac markers and cellcell interaction) required at least two more days.

Interestingly, the authors also demonstrated that this indirect lineage conversion does not pass through a pluripotency-state, i.e. ESC/iPSC culture media in the induction phase drastically decrease the efficiency of conversion; *vice versa*, the addiction of a Jak inhibitor (which blocks the most important pluripotency-pathway) increased the efficiency of the process. The same group also demonstrated that a similar strategy induced expandable Neural Progenitor Cells (NPCs), having multipotent potential [146].

Both direct and indirect lineage conversions have pros and cons. The direct conversion, as in case of SCNT or cell fusions, occurs in hours-days. Induced cells are unipotent, are produced with a high efficiency, without the requirement of cell proliferation and with a lower risk for teratoma. The indirect strategy requires days-weeks and produces cells, which can be unipo‐ tent or multipotent. Cells induced by this strategy can be expanded but have a moderate risk for teratoma.

#### **3.5. Mechanisms, differences with iPSC technology and unsolved questions**

Reprogramming to the pluripotency-state occurs via a gradual and genome-wide de-differ‐ entiation, involving a first phase where epigenetic marks of differentiation are erased and a second phase in which the epigenetic marks of pluripotency are established to initiate the endogenous pluripotency-network. In lineage conversion, specific TFs are able to modulate cell fate in two different ways (direct or indirect), which does not involve a pluripotent-state and is associated with a lower tumor risk, still a major obstacle to achieve clinical applications with ESCs and iPSCs. In the direct conversion, ectopic TFs, involved in cell fate determination or maintenance during embryonic development, overcome the pre-existing epigenetic marks and generate a new state. In the indirect conversion, the TFs, which allow the reprogramming to the pluripotency-state, are temporally overerexpressed together with fate-specific signals to convert original cell type into a new state. Differently than iPSC technology, the efficiencies are much higher (even 20 % in some cases) and the kinetics of conversion are rapid (a few day to a week maximum, and not two weeks to a month, like for iPSC, see Table 3).


**Table 3.** Comparison of different strategy of TF-based Reprogramming

**3.4. Direct versus indirect strategy**

cell interaction) required at least two more days.

(NPCs), having multipotent potential [146].

phase.

242 Pluripotent Stem Cells

for teratoma.

Most of the examples, given in the previous section, describe the direct conversion from one cell type to another, in which the reprogramming is achieved without any intermediate state. However, other reports clearly demonstrated the possibility to achieve similar results, by using an alternative strategy, in which lineage conversion is indirect. Indirect conversion is achieved passing through a limited de-differentiation state by overexpressing Yamanaka factors for a shorter time. Like for the direct conversion, the indirect conversion is strongly dependent on the specific culture medium (growth factors and cytokines) given during the reprogramming

The laboratory of Sheng Ding, at the Gladstone Institute of San Francisco, was the first to describe the possibility of lineage reprogramming through an indirect strategy. Short temporal overexpression of the Yamanaka factors induced a partial dedifferentiatied state, that allowed the subsequent conversion into cardiomyocytes-like cells by applying extracellular factors [145]. OSKM factors were overexpressed for six days in a medium free of signals necessary for pluripotency (i.e. leukemia inhibitory factor). After this short priming phase, cells were then cultured in media promoting cardiogenesis, i.e. cointaining BMP4. Three day after the cardiac induction, the expression of Nkx2.5, Gata4 and Flk1 (mid-stage markers of cardiac develop‐ ments) could be detected. The further development into more mature cardiomyocytes, showing sarcomeric structures and cardiac features (expression of cardiac markers and cell-

Interestingly, the authors also demonstrated that this indirect lineage conversion does not pass through a pluripotency-state, i.e. ESC/iPSC culture media in the induction phase drastically decrease the efficiency of conversion; *vice versa*, the addiction of a Jak inhibitor (which blocks the most important pluripotency-pathway) increased the efficiency of the process. The same group also demonstrated that a similar strategy induced expandable Neural Progenitor Cells

Both direct and indirect lineage conversions have pros and cons. The direct conversion, as in case of SCNT or cell fusions, occurs in hours-days. Induced cells are unipotent, are produced with a high efficiency, without the requirement of cell proliferation and with a lower risk for teratoma. The indirect strategy requires days-weeks and produces cells, which can be unipo‐ tent or multipotent. Cells induced by this strategy can be expanded but have a moderate risk

Reprogramming to the pluripotency-state occurs via a gradual and genome-wide de-differ‐ entiation, involving a first phase where epigenetic marks of differentiation are erased and a second phase in which the epigenetic marks of pluripotency are established to initiate the endogenous pluripotency-network. In lineage conversion, specific TFs are able to modulate cell fate in two different ways (direct or indirect), which does not involve a pluripotent-state and is associated with a lower tumor risk, still a major obstacle to achieve clinical applications with ESCs and iPSCs. In the direct conversion, ectopic TFs, involved in cell fate determination

**3.5. Mechanisms, differences with iPSC technology and unsolved questions**

As for iPSCs, many questions still remain unsolved in lineage conversion. It is not clear whether the new cell type, generated upon conversion, is a hybrid between the original and the new cell. It is intriguing that, in direct conversion, TFs erase partially or completely the previous epigenetic marks, without cell divisions (in which chromatin marks are lost) but it is totally unknown how this is possible. Remarkably, in both iPSCs and lineage conversion, efficiencies are lower with human cells, if compared with mouse. It is unknown whether this is due to the intrinsic karyotypic instability of mouse cells in culture or to molecular mechanisms.

## **4. Culture mediated reprogramming**

Reprogramming to the pluripotency-state and lineage conversion are achieved through the forced expression of TFs. However, in the last decade, several reports have highlighted how culture medium per se can be responsible for (partial) reprogramming. Moreover, there is an increasing amout of evidences showing that small molecules, including epigenetic modifiers and signaling pathway inhibitors, enhance the efficiency and kinetics of reprogramming.

Epiblast stem cells (EpiSCs) are isolated from post-implantation embryos between E5.5-E7.5. EpiSCs are the post-implantation equivalent of ESCs; they still express Oct4, Nanog and Sox2 but express lower levels of Stella and Rex1 [147]. ESCs and EpiSCs have also different culture requirements and features. While ESC selfrenewal is LIF dependent, EpiSC proliferation requires bFGF and Activin signaling. EpiSC female lines, but not ESC lines, have one of the X chromosome inactive. Importantly, EpiSCs, differently from ESCs, do not have the ability to contribute to chimeras *in vivo*, when aggregated into recipient morula/blastocysts.

In 2009, Bao et al. [148] demonstrated that established EpiSC lines could de-differentiate/ revert into an ESC-like state by culturing EpiSCs in ESC medium (cointaining LIF) for 2-5 weeks. Once 'reverted' cells lost all the features of the original EpiSCs and acquired all ESCcharacteristics ( X was reactivated, growth was LIF-dependent and cells were capable to

contribute to chimeras). This report showed that the simple manipulation of culture medium can dedifferentiate EpiSCs to a more primitive ESC-state but this is not the only case reported in literature.

germline trasmission. Subsequently analysis on rodent MAPC lines [171, 172] showed a lineage marker profile (Oct4, Gata4, Gata6, Sox7 and SSEA1) found also in the nascent hypoblast of the blastocyst and in rat blastocyst-derived Extraembryonic Endoderm Precursor cells (XEN-Ps) [173]. Recently, we demonstrated that similar cells are not present in fresh BM but appear after prolonged *in vitro* culture. To ascertain whether the MAPC culture system reprograms BM cells to the equivalent of XEN-P, we, first, showed that rMAPC and XEN-P cells exhibit similar features under reciprocal culture conditions. Second, we reported, using the same MAPC medium, the quick and efficient isolation of new cell lines directly from blastocyst, which we termed Hypoblast Stem Cells (HypoSCs) and which strongly resemble XEN-P in

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 245

Moreover, specific culture media may also be responsible for the broader differentiation potential described for some adult stem cell types [175] and this should be more considered

Small molecules are acquiring, on a daily basis, more relevance in the stem cell field because they can control protein functions selectively, reversibly and in a tunable way. Strikingly many reports have also shown how pathway inhibitors and epigenetic modifiers play a crucial role in the reprogramming process [177]. In 2010, the group of Ding reported that human primary somatic cells can be reprogrammed into human iPSCs with only Oct4 and a cocktail of small

Mouse (m)ESCs were first isolated more then three decades ago [179, 180]. mESCs have been derived and cultured in LIF and bone morphogenetic protein (BMP, contained in the serum) to inhibit their differentiation [181]. However, the efficiency of mESCs derivation was low in general and almost not possible from some mouse strains (like C57BL/6). More recently, several reports have now demonstrated that mESC culture in MEF or feeder-free are heterogeneous and fluctuates between a pre-implantation ESC and a post-implantation EpiSC-state [182, 183].

Ying and colleagues [184] demonstrated that mESCs can be maintained in an homogenous ground-state without the requirements of external stimuli, provided by growth factors and/or feeders. This achievement was possible by using two signaling modulators that regulate pathways involved in mESC differentiation: *PD0325901*, which blocks the differentiationinducing signalling from mitogen-activated protein kinase (MEK), inhibiting the phosphory‐ lation of ERK1/2; and *CHIR99021*, which inhibits the glycogen synthase kinase 3 (GSK3) and decreases the phosphorylation of β-Catenin, supporting their growth and further suppressing residual differentiation. The isolation of mESCs, with the two inhibitors (2i), together with LIF, allows now the efficient derivation of ESCs regardless of the mouse strain as well as from rat for the first time [185]. ESC lines cultured in 2i and LIF can be clonally propagated without feeders and support superior chimerism and germline transmission. The two inhibitors have

also been used to increase the efficiency of iPSCs generation [178].

in stem cell research, especially when reaching clinical trials phases [176].

features and developmental potential [174].

molecules [178].

*4.1.1. Signaling modulators*

**4.1. Small molecules in stem cells and reprogramming**

In 2004, Kanatsu-Shinohara et al. [149] descibed that mouse germline stem cells (GSCs) isolated from neonatal testis reverted occasionally into cells with ESC-like colonies morphol‐ ogy within 4-7 weeks if cultured in LIF, epidermal growth factor (EGF), glial cell linederived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2). The reverted cells were named multipotent germ stem cells (mGSs); they expressed not only Oct4 (already present in GSCs) but also Nanog and Sox2 at ESC-level. Analysis on mGSs showed the loss of spermatogonial properties (although the erasure of the androgenic imprinting was not complete) and the gain of ESC features (teratoma formation and contribution to chimeras with germline transmission). However, despite their siimilarity to ESCs, mGSs were not able to form offspring, after tetraploid complementation. Unipotent germline stem cells, but this time from adult testis, were converted into germline-derived pluripotent stem cells (gPSs) by Ko and colleagues [150]. Reprogrammed cells, like in the above case, were higly similar to ESCs but again, they could not form live animals in tetraploid complementation assay. The reason for this is most likely the residual persistence of androgenetic imprinting. The possibility to reprogram a germline stem cell into a cell with pluripotent features, even without the capacity of forming chimeric animal, is interesting because it might allow autologous cell therapy without embryo-manipulation. Similar conversions with mouse cells were also described by other laboratories [151, 152].

In 2008, Conrad and colleagues [153], showed that cells derived from human testis can be converted into cells with human ESC-like features. Cells isolated from human testis were cultured in GDNF-containing medium for 4 days and then selected based on the expression of CD49f and further selection on laminin matrix in medium cointaining LIF. 3-4 weeks later colonies with ESC-morphology appeared; human adult GSCs (haGSCs), like human ESCs, expressed SSEA4, TRA 1-60, TRA 1-81 and generated EBs and teratomas. However, a later report [154] questioned the previous finding of Conrad, arguing whether haGSCs really expressed Oct4, Nanog and Sox2; moreover, microarray data comparison further showed that haGSCs are similar to fibroblasts-derived from human testic biopsies but not to hESCs.

These studies strongly suggest that stem/progenitor cells derived from testis can to some extent be converted, by long-term culture, to cells with ESC-like properties, without any reprogramming factors; however, converted cells differ significantly from ESCs. The propensity of GSCs to be converted to ESC-like cells may depend on their Oct4 expres‐ sion. Although gonads are the only place where Oct4 is functionally expressed in adult healthy-rodents [127], many reports described the isolation of Oct4+ cells from rodents [155-169]. It remains to be determined whether culture mediated reprogramming is responsible for the Oct4 re-activation in such cell lines.

In 2002, our group [170] isolated multipotent adult progenitor cells (MAPCs) from rodent bone marrow (BM), upon prolonged culture at low density in a medium cointaining LIF, PDGF and EGF. Murine MAPCs differentiated *in vitro* into cells of the three germ layers and one murine line was also able to contribute to chimeric mice, although at low efficiency and without germline trasmission. Subsequently analysis on rodent MAPC lines [171, 172] showed a lineage marker profile (Oct4, Gata4, Gata6, Sox7 and SSEA1) found also in the nascent hypoblast of the blastocyst and in rat blastocyst-derived Extraembryonic Endoderm Precursor cells (XEN-Ps) [173]. Recently, we demonstrated that similar cells are not present in fresh BM but appear after prolonged *in vitro* culture. To ascertain whether the MAPC culture system reprograms BM cells to the equivalent of XEN-P, we, first, showed that rMAPC and XEN-P cells exhibit similar features under reciprocal culture conditions. Second, we reported, using the same MAPC medium, the quick and efficient isolation of new cell lines directly from blastocyst, which we termed Hypoblast Stem Cells (HypoSCs) and which strongly resemble XEN-P in features and developmental potential [174].

Moreover, specific culture media may also be responsible for the broader differentiation potential described for some adult stem cell types [175] and this should be more considered in stem cell research, especially when reaching clinical trials phases [176].

#### **4.1. Small molecules in stem cells and reprogramming**

Small molecules are acquiring, on a daily basis, more relevance in the stem cell field because they can control protein functions selectively, reversibly and in a tunable way. Strikingly many reports have also shown how pathway inhibitors and epigenetic modifiers play a crucial role in the reprogramming process [177]. In 2010, the group of Ding reported that human primary somatic cells can be reprogrammed into human iPSCs with only Oct4 and a cocktail of small molecules [178].

#### *4.1.1. Signaling modulators*

contribute to chimeras). This report showed that the simple manipulation of culture medium can dedifferentiate EpiSCs to a more primitive ESC-state but this is not the only case

In 2004, Kanatsu-Shinohara et al. [149] descibed that mouse germline stem cells (GSCs) isolated from neonatal testis reverted occasionally into cells with ESC-like colonies morphol‐ ogy within 4-7 weeks if cultured in LIF, epidermal growth factor (EGF), glial cell linederived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2). The reverted cells were named multipotent germ stem cells (mGSs); they expressed not only Oct4 (already present in GSCs) but also Nanog and Sox2 at ESC-level. Analysis on mGSs showed the loss of spermatogonial properties (although the erasure of the androgenic imprinting was not complete) and the gain of ESC features (teratoma formation and contribution to chimeras with germline transmission). However, despite their siimilarity to ESCs, mGSs were not able to form offspring, after tetraploid complementation. Unipotent germline stem cells, but this time from adult testis, were converted into germline-derived pluripotent stem cells (gPSs) by Ko and colleagues [150]. Reprogrammed cells, like in the above case, were higly similar to ESCs but again, they could not form live animals in tetraploid complementation assay. The reason for this is most likely the residual persistence of androgenetic imprinting. The possibility to reprogram a germline stem cell into a cell with pluripotent features, even without the capacity of forming chimeric animal, is interesting because it might allow autologous cell therapy without embryo-manipulation. Similar conversions with mouse cells

In 2008, Conrad and colleagues [153], showed that cells derived from human testis can be converted into cells with human ESC-like features. Cells isolated from human testis were cultured in GDNF-containing medium for 4 days and then selected based on the expression of CD49f and further selection on laminin matrix in medium cointaining LIF. 3-4 weeks later colonies with ESC-morphology appeared; human adult GSCs (haGSCs), like human ESCs, expressed SSEA4, TRA 1-60, TRA 1-81 and generated EBs and teratomas. However, a later report [154] questioned the previous finding of Conrad, arguing whether haGSCs really expressed Oct4, Nanog and Sox2; moreover, microarray data comparison further showed that haGSCs are similar to fibroblasts-derived from human testic biopsies but not to hESCs.

These studies strongly suggest that stem/progenitor cells derived from testis can to some extent be converted, by long-term culture, to cells with ESC-like properties, without any reprogramming factors; however, converted cells differ significantly from ESCs. The propensity of GSCs to be converted to ESC-like cells may depend on their Oct4 expres‐ sion. Although gonads are the only place where Oct4 is functionally expressed in adult healthy-rodents [127], many reports described the isolation of Oct4+ cells from rodents [155-169]. It remains to be determined whether culture mediated reprogramming is

In 2002, our group [170] isolated multipotent adult progenitor cells (MAPCs) from rodent bone marrow (BM), upon prolonged culture at low density in a medium cointaining LIF, PDGF and EGF. Murine MAPCs differentiated *in vitro* into cells of the three germ layers and one murine line was also able to contribute to chimeric mice, although at low efficiency and without

reported in literature.

244 Pluripotent Stem Cells

were also described by other laboratories [151, 152].

responsible for the Oct4 re-activation in such cell lines.

Mouse (m)ESCs were first isolated more then three decades ago [179, 180]. mESCs have been derived and cultured in LIF and bone morphogenetic protein (BMP, contained in the serum) to inhibit their differentiation [181]. However, the efficiency of mESCs derivation was low in general and almost not possible from some mouse strains (like C57BL/6). More recently, several reports have now demonstrated that mESC culture in MEF or feeder-free are heterogeneous and fluctuates between a pre-implantation ESC and a post-implantation EpiSC-state [182, 183].

Ying and colleagues [184] demonstrated that mESCs can be maintained in an homogenous ground-state without the requirements of external stimuli, provided by growth factors and/or feeders. This achievement was possible by using two signaling modulators that regulate pathways involved in mESC differentiation: *PD0325901*, which blocks the differentiationinducing signalling from mitogen-activated protein kinase (MEK), inhibiting the phosphory‐ lation of ERK1/2; and *CHIR99021*, which inhibits the glycogen synthase kinase 3 (GSK3) and decreases the phosphorylation of β-Catenin, supporting their growth and further suppressing residual differentiation. The isolation of mESCs, with the two inhibitors (2i), together with LIF, allows now the efficient derivation of ESCs regardless of the mouse strain as well as from rat for the first time [185]. ESC lines cultured in 2i and LIF can be clonally propagated without feeders and support superior chimerism and germline transmission. The two inhibitors have also been used to increase the efficiency of iPSCs generation [178].

Mesenchymal-to-epithelial transition (MET) is a reversible process which drives cells from a multipolar, spindle and motile mesenchymal shape to a planar and polarized epithelial shape. MET is an important process during embryo development but also in reprogramming; i.e. fibroblasts change shape towards an epithelial morphology at the early stage of iPSC genera‐ tion. TGFβ pathway negatively regulates an epithelial phenotype. The block of TGFβ1-2-3 receptors, using *SB431542*, in combination with PD0325901, enhances both the kinetics and the efficiency of reprogramming, during iPSC generation [186].

Similarly, *Trichostatin A* [137], suberoylanilide hydroxamic acid [71] and valproic acid (VPA), inhibitors of histone deacetylases (HDACs) also increases efficiencies of reprogramming, even

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 247

**Small molecules Category Effect on** PD0325901 Signaling modulators MEK inhibitor CHIR99021 Signaling modulators WNT/β-Catenin SB431542 Signaling modulators MET

Vitamin C Signaling modulators Cellular Senescence PS48 Signaling modulators Glycolysis 5-aza, RG108 Epigenetic modifiers DNMT inhibitor BIX-01294 Epigenetic modifiers HMTase inhibitor Parnate Epigenetic modifiers LSD1 inhibitor TSA, SAHA, VPA Epigenetic modifiers HDAC inhibitor

The importance and the impact on society of reprogramming has been recently recognised by the Nobel Assembly at the Karolinka Institute of Stockholm, which co-awarded John Gurdon and Shiniya Yamanaka with the Nobel Price in Medicine 2012. Their outstanding reports demonstrated that cellular fate is plastic and that differentiation is a reversible process. Epigenetic markers imposed by development can be erased through the multiple pathways to reprogramming. This means the epigenetic landscape as described by Waddington should be revised, as balls are capable of rolling back up and over the hill. The SCNT and the forced expression of TFs show that somatic cells can re-acquire all the features, lost upon their differentiation. Adult somatic cells can be redirected to the pluripotency-state or can be

Although the precise mechanism via which the phenotype of all these cells can be changed remains to be fully elucidated, the iPSC technology is drastically changing and boosting the stem cell field; it allows one to obtain pluripotent stem cells for autologous therapy, avoiding the problems of immune rejection as well as the ethical issues related to the use of human embryo for scientific purposes. The possibility to also obtain precursors, with restricted differentiation potential, may be another alternative to reach the bedside, as it is likely associated with lower tumorigenicity. It is also clear that culture conditions have such a significant effect on cell fate, not only during reprogramming but also in establishing the potential of different adult stem cells, that this should be kept in mind when comparing studies across laboratories, and definitely when contemplating clinical trials with cultured stem cells.

with only Oct4 and Sox2 overexpression. [190, 194].

**Table 4.** Small molecules in reprogramming

converted into cells of another lineage.

**5. Conclusions**

Cellular senescence is a pathway that negatively interferes with reprogramming. Expres‐ sion of OSKM increases oxidative stress and DNA damage, inducing senescence. *Vitamin C* (or ascorbic acid), is an important cofactor for metabolic processes but also has a strong antioxidant effect; i.e. Vitamin C reduces reactive oxidandant species (ROS). In iPSC reprogramming, Vitamin C enhances the conversion from a partial reprogrammed to a fully reprogrammed-state [187], capable of forming completely iPSC-derived mice in tetraploid complementation assay [188].

Stem cells have a different metabolism if compared to differentiated cells [189]. Stem cells have a strong energetic and metabolic demand to meet their self-renewal and to do this, they mainly rely on glycolysis followed by fermentation of lactic acid in the cytosol. Differently, differentiated cells mainly rely on a low rate of glycolysis followed by oxida‐ tion of pyruvate in the mitochondria, which results in the production of ROS. Consistent with this, *PS48*, an activator of 3-phosphoinositide dependent protein kinase-1 (PDK1) that activates the PI3/Akt pathway, results in the upregulation of glycolytic genes and strongly facilitates iPSC reprogramming [178].

#### *4.1.2. Epigenetic modifiers*

The structure of eukaryotic genome is higly organized; genomic DNA is wrapped around structural proteines, called histones. DNA and histones, together, form the chromatin. Protein complexes are responsible for chromatin modifications. Histones then determine the tran‐ scriptional status; i.e. in an open and closed form. In somatic cells, chromatin is mainly in a closed conformation, while in pluripotent cells, chromatin is in an open conformation and it is dynamically associated with chromatin proteins. Obviously, during iPSC generation, the chromatin must change from a somatic to a pluripotent state. Therefore, many small molecules, which modulates chromatin have been described to enhance the efficiency of reprogramming and even to substitute for some of the reprogramming factors.

Pluripotent stem cells have, in general, a more demethylated DNA, in comparison with somatic cells; in fact, *5-azacytidine* [5-aza) and *RG108*, two inhibitors of DNA methyltransferases (DNMTs), are responsible of DNA methylation and methylation maintanance, increases efficiency of reprogramming [190, 191].

G9a is an histone methyltransferase (HMTase), which induce silencing of Oct4, through methylation of H3K9. *BIX-01294*, an inhibitor of G9a, enhances reprogramming [192]. Recently, *parnate*, an inhibitor of LSD1, a H3K4 demethylase, was used to reprogram human somatic cells with only Oct4 and Klf4 [193].

Similarly, *Trichostatin A* [137], suberoylanilide hydroxamic acid [71] and valproic acid (VPA), inhibitors of histone deacetylases (HDACs) also increases efficiencies of reprogramming, even with only Oct4 and Sox2 overexpression. [190, 194].


**Table 4.** Small molecules in reprogramming

## **5. Conclusions**

Mesenchymal-to-epithelial transition (MET) is a reversible process which drives cells from a multipolar, spindle and motile mesenchymal shape to a planar and polarized epithelial shape. MET is an important process during embryo development but also in reprogramming; i.e. fibroblasts change shape towards an epithelial morphology at the early stage of iPSC genera‐ tion. TGFβ pathway negatively regulates an epithelial phenotype. The block of TGFβ1-2-3 receptors, using *SB431542*, in combination with PD0325901, enhances both the kinetics and the

Cellular senescence is a pathway that negatively interferes with reprogramming. Expres‐ sion of OSKM increases oxidative stress and DNA damage, inducing senescence. *Vitamin C* (or ascorbic acid), is an important cofactor for metabolic processes but also has a strong antioxidant effect; i.e. Vitamin C reduces reactive oxidandant species (ROS). In iPSC reprogramming, Vitamin C enhances the conversion from a partial reprogrammed to a fully reprogrammed-state [187], capable of forming completely iPSC-derived mice in tetraploid

Stem cells have a different metabolism if compared to differentiated cells [189]. Stem cells have a strong energetic and metabolic demand to meet their self-renewal and to do this, they mainly rely on glycolysis followed by fermentation of lactic acid in the cytosol. Differently, differentiated cells mainly rely on a low rate of glycolysis followed by oxida‐ tion of pyruvate in the mitochondria, which results in the production of ROS. Consistent with this, *PS48*, an activator of 3-phosphoinositide dependent protein kinase-1 (PDK1) that activates the PI3/Akt pathway, results in the upregulation of glycolytic genes and strongly

The structure of eukaryotic genome is higly organized; genomic DNA is wrapped around structural proteines, called histones. DNA and histones, together, form the chromatin. Protein complexes are responsible for chromatin modifications. Histones then determine the tran‐ scriptional status; i.e. in an open and closed form. In somatic cells, chromatin is mainly in a closed conformation, while in pluripotent cells, chromatin is in an open conformation and it is dynamically associated with chromatin proteins. Obviously, during iPSC generation, the chromatin must change from a somatic to a pluripotent state. Therefore, many small molecules, which modulates chromatin have been described to enhance the efficiency of reprogramming

Pluripotent stem cells have, in general, a more demethylated DNA, in comparison with somatic cells; in fact, *5-azacytidine* [5-aza) and *RG108*, two inhibitors of DNA methyltransferases (DNMTs), are responsible of DNA methylation and methylation maintanance, increases

G9a is an histone methyltransferase (HMTase), which induce silencing of Oct4, through methylation of H3K9. *BIX-01294*, an inhibitor of G9a, enhances reprogramming [192]. Recently, *parnate*, an inhibitor of LSD1, a H3K4 demethylase, was used to reprogram human somatic

efficiency of reprogramming, during iPSC generation [186].

and even to substitute for some of the reprogramming factors.

complementation assay [188].

246 Pluripotent Stem Cells

facilitates iPSC reprogramming [178].

efficiency of reprogramming [190, 191].

cells with only Oct4 and Klf4 [193].

*4.1.2. Epigenetic modifiers*

The importance and the impact on society of reprogramming has been recently recognised by the Nobel Assembly at the Karolinka Institute of Stockholm, which co-awarded John Gurdon and Shiniya Yamanaka with the Nobel Price in Medicine 2012. Their outstanding reports demonstrated that cellular fate is plastic and that differentiation is a reversible process. Epigenetic markers imposed by development can be erased through the multiple pathways to reprogramming. This means the epigenetic landscape as described by Waddington should be revised, as balls are capable of rolling back up and over the hill. The SCNT and the forced expression of TFs show that somatic cells can re-acquire all the features, lost upon their differentiation. Adult somatic cells can be redirected to the pluripotency-state or can be converted into cells of another lineage.

Although the precise mechanism via which the phenotype of all these cells can be changed remains to be fully elucidated, the iPSC technology is drastically changing and boosting the stem cell field; it allows one to obtain pluripotent stem cells for autologous therapy, avoiding the problems of immune rejection as well as the ethical issues related to the use of human embryo for scientific purposes. The possibility to also obtain precursors, with restricted differentiation potential, may be another alternative to reach the bedside, as it is likely associated with lower tumorigenicity. It is also clear that culture conditions have such a significant effect on cell fate, not only during reprogramming but also in establishing the potential of different adult stem cells, that this should be kept in mind when comparing studies across laboratories, and definitely when contemplating clinical trials with cultured stem cells.

## **Acknowledgements**

C.M.V. was supported by an Odysseus grant from FWO, Flanders, by a CoE and GOA KU Leuven award; A.L.N. was supported by an IWT grant, Flanders.

[10] Yamanaka, S, et al. Nuclear reprogramming to a pluripotent state by three ap‐

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 249

[11] Pavlath, G. K, et al. Expression of muscle genes in heterokaryons depends on gene

[12] Harris, H, et al. Suppression of malignancy by cell fusion. Nature. (1969). Jul 26;,

[13] Peterson, J. A, et al. Expression of differentiated functions in hepatoma cell hybrids: induction of mouse albumin production in rat hepatoma-mouse fibroblast hybrids. Proceedings of the National Academy of Sciences of the United States of America.

[14] Weiss, M. C, et al. Expression of differentiated functions in hepatoma cell hybrids: re‐ appearance of tyrosine aminotransferase inducibility after the loss of chromosomes. Proceedings of the National Academy of Sciences of the United States of America.

[15] Davidson, R. L. Regulation of malanin synthesis in mammalian cells: effect of gene dosage on the expression of differentiation. Proceedings of the National Academy of

[16] Davidson, R. L, et al. Regulation of pigment synthesis in mammalian cells, as studied by somatic hybridization. Proceedings of the National Academy of Sciences of the

[17] Tada, M, et al. Nuclear reprogramming of somatic cells by in vitro hybridization

[18] Ying, Q. L, et al. Changing potency by spontaneous fusion. Nature. (2002). Apr 4;,

[19] Cowan, C. A, et al. Nuclear reprogramming of somatic cells after fusion with human

[20] Takahashi, K, et al. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. (2006). Aug 25;, 126(4), 663-76.

[21] Vierbuchen, T, et al. Direct lineage conversions: unnatural but useful? Nature bio‐

[22] Sancho-martinez, I, et al. Lineage conversion methodologies meet the reprogram‐

[23] Asuelime, G. E, et al. A case of cellular alchemy: lineage reprogramming and its po‐ tential in regenerative medicine. Journal of molecular cell biology. (2012). Aug;, 4(4),

Sciences of the United States of America. (1972). Apr;, 69(4), 951-5.

with ES cells. Current biology : CB. (2001). Oct 2;, 11(19), 1553-8.

embryonic stem cells. Science. (2005). Aug 26;, 309(5739), 1369-73.

ming toolbox. Nature cell biology. (2012). Sep 3;, 14(9), 892-9.

United States of America. (1966). Nov;, 56(5), 1437-40.

technology. (2011). Oct;, 29(10), 892-907.

proaches. Nature. (2010). Jun 10;, 465(7299), 704-12.

223(5204), 363-8.

416(6880), 545-8.

190-6.

(1972). Mar;, 69(3), 571-5.

(1971). Dec;, 68(12), 3026-30.

dosage. The Journal of cell biology. (1986). Jan;, 102(1), 124-30.

## **Author details**

Antonio Lo Nigro\* and Catherine M. Verfaillie

\*Address all correspondence to: antonio.lonigro@med.kuleuven.be

\*Address all correspondence to: catherine.verfaillie@med.kuleuven.be

Interdepartmental Stem Cell Institute Leuven (SCIL) & Department of Development and Regeneration, KU Leuven, Leuven, Belgium

### **References**


[10] Yamanaka, S, et al. Nuclear reprogramming to a pluripotent state by three ap‐ proaches. Nature. (2010). Jun 10;, 465(7299), 704-12.

**Acknowledgements**

248 Pluripotent Stem Cells

**Author details**

Antonio Lo Nigro\*

**References**

C.M.V. was supported by an Odysseus grant from FWO, Flanders, by a CoE and GOA KU

Interdepartmental Stem Cell Institute Leuven (SCIL) & Department of Development and

[1] Waddington, C. H. The strategy of the genes; a discussion of some aspects of theoret‐

[2] Gurdon, J. B. The developmental capacity of nuclei taken from differentiating endo‐ derm cells of Xenopus laevis. Journal of embryology and experimental morphology.

[3] Gurdon, J. B. Adult frogs derived from the nuclei of single somatic cells. Develop‐

[4] Campbell, K. H, et al. Sheep cloned by nuclear transfer from a cultured cell line. Na‐

[5] Wilmut, I, et al. Viable offspring derived from fetal and adult mammalian cells. Na‐

[6] Wakayama, T, et al. Full-term development of mice from enucleated oocytes injected

[7] Egli, D, et al. Developmental reprogramming after chromosome transfer into mitotic

[8] Yang, X, et al. Nuclear reprogramming of cloned embryos and its implications for

with cumulus cell nuclei. Nature. (1998). Jul 23;, 394(6691), 369-74.

therapeutic cloning. Nature genetics. (2007). Mar;, 39(3), 295-302.

[9] Kennedy, D. Editorial retraction. Science. (2006). Jan 20;311(5759):335.

mouse zygotes. Nature. (2007). Jun 7;, 447(7145), 679-85.

Leuven award; A.L.N. was supported by an IWT grant, Flanders.

and Catherine M. Verfaillie

\*Address all correspondence to: antonio.lonigro@med.kuleuven.be

ical biology. London,: Allen & Unwin; (1957).

mental biology. (1962). Apr;, 4, 256-73.

ture. (1996). Mar 7;, 380(6569), 64-6.

ture. (1997). Feb 27;, 385(6619), 810-3.

Regeneration, KU Leuven, Leuven, Belgium

(1960). Dec;, 8, 505-26.

\*Address all correspondence to: catherine.verfaillie@med.kuleuven.be


[24] Yuan, X, et al. Small molecules in cellular reprogramming and differentiation. Prog‐ ress in drug research Fortschritte der Arzneimittelforschung Progres des recherches pharmaceutiques. (2011). , 67, 253-66.

[39] Nishimoto, M, et al. The gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 251

[40] Ambrosetti, D. C, et al. Modulation of the activity of multiple transcriptional activa‐ tion domains by the DNA binding domains mediates the synergistic action of Sox2 and Oct-3 on the fibroblast growth factor-4 enhancer. The Journal of biological chem‐

[41] Ambrosetti, D. C, et al. Synergistic activation of the fibroblast growth factor 4 en‐ hancer by Sox2 and Oct-3 depends on protein-protein interactions facilitated by a specific spatial arrangement of factor binding sites. Molecular and cellular biology.

[42] Mitsui, K, et al. The homeoprotein Nanog is required for maintenance of pluripoten‐

[43] Chambers, I, et al. Functional expression cloning of Nanog, a pluripotency sustaining

[44] Silva, J, et al. Nanog is the gateway to the pluripotent ground state. Cell. (2009). Aug

[45] Viswanathan, S. R, et al. Selective blockade of microRNA processing by Lin28. Sci‐

[46] Nakagawa, M, et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nature biotechnology. (2008). Jan;, 26(1), 101-6.

[47] Zhao, Y, et al. Two supporting factors greatly improve the efficiency of human iPSC

[48] Li, H, et al. The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature. (2009).

[49] Kawamura, T, et al. Linking the tumour suppressor pathway to somatic cell reprog‐

[50] Judson, R. L, et al. Embryonic stem cell-specific microRNAs promote induced pluri‐

[51] Mallanna, S. K, et al. Emerging roles of microRNAs in the control of embryonic stem cells and the generation of induced pluripotent stem cells. Developmental biology.

[52] Park, I. H, et al. Reprogramming of human somatic cells to pluripotency with de‐

cy in mouse epiblast and ES cells. Cell. (2003). May 30;, 113(5), 631-42.

factor in embryonic stem cells. Cell. (2003). May 30;, 113(5), 643-55.

and Sox-2. Molecular and cellular biology. (1999). Aug;, 19(8), 5453-65.

istry. (2000). Jul 28;, 275(30), 23387-97.

ence. (2008). Apr 4;, 320(5872), 97-100.

Aug 27;, 460(7259), 1136-9.

(2010). Aug 1;, 344(1), 16-25.

generation. Cell stem cell. (2008). Nov 6;, 3(5), 475-9.

ramming. Nature. (2009). Aug 27;460(7259):1140-4., 53.

fined factors. Nature. (2008). Jan 10;, 451(7175), 141-6.

potency. Nature biotechnology. (2009). May;, 27(5), 459-61.

(1997). Nov;, 17(11), 6321-9.

21;, 138(4), 722-37.


[39] Nishimoto, M, et al. The gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4 and Sox-2. Molecular and cellular biology. (1999). Aug;, 19(8), 5453-65.

[24] Yuan, X, et al. Small molecules in cellular reprogramming and differentiation. Prog‐ ress in drug research Fortschritte der Arzneimittelforschung Progres des recherches

[25] Roobrouck, V. D, et al. Concise review: culture mediated changes in fate and/or po‐

[26] Schneuwly, S, et al. Redesigning the body plan of Drosophila by ectopic expression of the homoeotic gene Antennapedia. Nature. (1987). Feb 26-Mar 4;, 325(6107), 816-8.

[27] Gehring, W. J. The master control gene for morphogenesis and evolution of the eye. Genes to cells : devoted to molecular & cellular mechanisms. (1996). Jan;, 1(1), 11-5.

[28] Okita, K, et al. Generation of germline-competent induced pluripotent stem cells. Na‐

[29] Kang, L, et al. iPS cells can support full-term development of tetraploid blastocyst-

[30] Takahashi, K, et al. Induction of pluripotent stem cells from adult human fibroblasts

[31] Yu, J, et al. Induced pluripotent stem cell lines derived from human somatic cells.

[32] Scholer, H. R, et al. Octamer binding proteins confer transcriptional activity in early

[33] Niwa, H, et al. Quantitative expression of Oct-3/4 defines differentiation, dedifferen‐ tiation or self-renewal of ES cells. Nature genetics. (2000). Apr;, 24(4), 372-6.

[34] Nichols, J, et al. Formation of pluripotent stem cells in the mammalian embryo de‐ pends on the POU transcription factor Oct4. Cell. (1998). Oct 30;, 95(3), 379-91.

[35] Gubbay, J, et al. A gene mapping to the sex-determining region of the mouse Y chro‐ mosome is a member of a novel family of embryonically expressed genes. Nature.

[36] Bowles, J, et al. Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators. Developmental biology. (2000). Nov 15;,

[37] Avilion, A. A, et al. Multipotent cell lineages in early mouse development depend on

[38] Tokuzawa, Y, et al. Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development. Molecular and cellular biology.

SOX2 function. Genes & development. (2003). Jan 1;, 17(1), 126-40.

complemented embryos. Cell stem cell. (2009). Aug 7;, 5(2), 135-8.

mouse embryogenesis. The EMBO journal. (1989). Sep;, 8(9), 2551-7.

by defined factors. Cell. (2007). Nov 30;, 131(5), 861-72.

Science. (2007). Dec 21;, 318(5858), 1917-20.

(1990). Jul 19;, 346(6281), 245-50.

(2003). Apr;, 23(8), 2699-708.

227(2), 239-55.

pharmaceutiques. (2011). , 67, 253-66.

250 Pluripotent Stem Cells

ture. (2007). Jul 19;, 448(7151), 313-7.

tency of stem cells. Stem cells. (2011). Apr;, 29(4), 583-9.


[53] Tsubooka, N, et al. Roles of Sall4 in the generation of pluripotent stem cells from blastocysts and fibroblasts. Genes to cells : devoted to molecular & cellular mecha‐ nisms. (2009). Jun;, 14(6), 683-94.

[68] Okita, K, et al. Induced pluripotent stem cells: opportunities and challenges. Philo‐ sophical transactions of the Royal Society of London Series B, Biological sciences.

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 253

[69] Hotta, A, et al. Retroviral vector silencing during iPS cell induction: an epigenetic beacon that signals distinct pluripotent states. Journal of cellular biochemistry.

[70] Wu, Z, et al. Generation of pig induced pluripotent stem cells with a drug-inducible

[71] Carey, B. W, et al. Reprogramming of murine and human somatic cells using a single polycistronic vector. Proceedings of the National Academy of Sciences of the United

[72] Ryan, M. D, et al. Virus-encoded proteinases of the picornavirus super-group. The

[73] Hacein-bey-abina, S, et al. LMO2-associated clonal T cell proliferation in two patients

[74] Lacoste, A, et al. An efficient and reversible transposable system for gene delivery and lineage-specific differentiation in human embryonic stem cells. Cell stem cell.

[75] Woltjen, K, et al. piggyBac transposition reprograms fibroblasts to induced pluripo‐

[76] Yusa, K, et al. Generation of transgene-free induced pluripotent mouse stem cells by

[77] Stadtfeld, M, et al. Induced pluripotent stem cells generated without viral integra‐

[78] Zhou, W, et al. Adenoviral gene delivery can reprogram human fibroblasts to in‐

[79] Fusaki, N, et al. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proceedings of the Japan Academy Series B, Physical and biological sci‐

[80] Yates, J, et al. A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proceedings of the National Academy of Sciences of the United States of America. (1984). Jun;,

[81] Yu, J, et al. Human induced pluripotent stem cells free of vector and transgene se‐

the piggyBac transposon. Nature methods. (2009). May;, 6(5), 363-9.

duced pluripotent stem cells. Stem cells. (2009). Nov;, 27(11), 2667-74.

after gene therapy for SCID-X1. Science. (2003). Oct 17;, 302(5644), 415-9.

system. Journal of molecular cell biology. (2009). Oct;, 1(1), 46-54.

States of America. (2009). Jan 6;, 106(1), 157-62.

Journal of general virology. (1997). Apr;78 ( Pt 4):699-723.

tent stem cells. Nature. (2009). Apr 9;, 458(7239), 766-70.

tion. Science. (2008). Nov 7;, 322(5903), 945-9.

quences. Science. (2009). May 8;, 324(5928), 797-801.

(2011). Aug 12;, 366(1575), 2198-207.

(2008). Nov 1;, 105(4), 940-8.

(2009). Sep 4;, 5(3), 332-42.

ences. (2009). , 85(8), 348-62.

81(12), 3806-10.


[68] Okita, K, et al. Induced pluripotent stem cells: opportunities and challenges. Philo‐ sophical transactions of the Royal Society of London Series B, Biological sciences. (2011). Aug 12;, 366(1575), 2198-207.

[53] Tsubooka, N, et al. Roles of Sall4 in the generation of pluripotent stem cells from blastocysts and fibroblasts. Genes to cells : devoted to molecular & cellular mecha‐

[54] Feng, B, et al. Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb. Nature cell biology. (2009). Feb;, 11(2), 197-203.

[55] Han, J, et al. Tbx3 improves the germ-line competency of induced pluripotent stem

[56] Aasen, T, et al. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nature biotechnology. (2008). Nov;, 26(11), 1276-84.

[57] Giorgetti, A, et al. Generation of induced pluripotent stem cells from human cord

[58] Eminli, S, et al. Differentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells. Nature genetics. (2009). Sep;,

[59] Kim, J. B, et al. Pluripotent stem cells induced from adult neural stem cells by reprog‐

[60] Sun, N, et al. Feeder-free derivation of induced pluripotent stem cells from adult hu‐ man adipose stem cells. Proceedings of the National Academy of Sciences of the

[61] Tamaoki, N, et al. Dental pulp cells for induced pluripotent stem cell banking. Jour‐

[62] Miyoshi, K, et al. Generation of human induced pluripotent stem cells from oral mu‐ cosa. Journal of bioscience and bioengineering. (2010). Sep;, 110(3), 345-50.

[63] Loh, Y. H, et al. Generation of induced pluripotent stem cells from human blood.

[64] Doi, A, et al. Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibro‐

[65] Kim, K, et al. Donor cell type can influence the epigenome and differentiation poten‐ tial of human induced pluripotent stem cells. Nature biotechnology. (2011). Dec;,

[66] Miura, K, et al. Variation in the safety of induced pluripotent stem cell lines. Nature

[67] Gonzalez, F, et al. Methods for making induced pluripotent stem cells: reprogram‐

ming a la carte. Nature reviews Genetics. (2011). Apr;, 12(4), 231-42.

blood using OCT4 and SOX2. Cell stem cell. (2009). Oct 2;, 5(4), 353-7.

ramming with two factors. Nature. (2008). Jul 31;, 454(7204), 646-50.

United States of America. (2009). Sep 15;, 106(37), 15720-5.

nal of dental research. (2010). Aug;, 89(8), 773-8.

blasts. Nature genetics. (2009). Dec;, 41(12), 1350-3.

Blood. (2009). May 28;, 113(22), 5476-9.

biotechnology. (2009). Aug;, 27(8), 743-5.

nisms. (2009). Jun;, 14(6), 683-94.

41(9), 968-76.

252 Pluripotent Stem Cells

29(12), 1117-9.

cells. Nature. (2010). Feb 25;, 463(7284), 1096-100.


[82] Jia, F, et al. A nonviral minicircle vector for deriving human iPS cells. Nature meth‐ ods. (2010). Mar;, 7(3), 197-9.

[98] Young, M. A, et al. Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells. Cell stem cell. (2012). May 4;,

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 255

[99] Hanna, J, et al. Treatment of sickle cell anemia mouse model with iPS cells generated

[100] Okamoto, S, et al. Induction of retinal pigment epithelial cells from monkey iPS cells.

[101] Kriks, S, et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature. (2011). Dec 22;, 480(7378), 547-51.

[102] Takayama, N, et al. Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells. The Journal of exper‐

[103] Nori, S, et al. Grafted human-induced pluripotent stem-cell-derived neurospheres promote motor functional recovery after spinal cord injury in mice. Proceedings of the National Academy of Sciences of the United States of America. (2011). Oct 4;,

[104] Tsuji, O, et al. Therapeutic potential of appropriately evaluated safe-induced pluripo‐ tent stem cells for spinal cord injury. Proceedings of the National Academy of Scien‐

[105] Dimos, J. T, et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science. (2008). Aug 29;, 321(5893), 1218-21.

[106] Park, I. H, et al. Disease-specific induced pluripotent stem cells. Cell. (2008). Sep 5;,

[107] Brennand, K. J, et al. Modelling schizophrenia using human induced pluripotent

[108] Israel, M. A, et al. Probing sporadic and familial Alzheimer's disease using induced

[109] Yahata, N, et al. Anti-Abeta drug screening platform using human iPS cell-derived neurons for the treatment of Alzheimer's disease. PloS one. (2011). e25788.

[110] Xu, J, et al. Pioneer factor interactions and unmethylated CpG dinucleotides mark si‐ lent tissue-specific enhancers in embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America. (2007). Jul 24;, 104(30),

[111] Xu, J, et al. Transcriptional competence and the active marking of tissue-specific en‐ hancers by defined transcription factors in embryonic and induced pluripotent stem

ces of the United States of America. (2010). Jul 13;, 107(28), 12704-9.

from autologous skin. Science. (2007). Dec 21;, 318(5858), 1920-3.

imental medicine. (2010). Dec 20;, 207(13), 2817-30.

stem cells. Nature. (2011). May 12;, 473(7346), 221-5.

pluripotent stem cells. Nature. (2012). Feb 9;, 482(7384), 216-20.

cells. Genes & development. (2009). Dec 15;, 23(24), 2824-38.

Investigative ophthalmology & visual science. (2011). , 52(12), 8785-90.

10(5), 570-82.

108(40), 16825-30.

134(5), 877-86.

12377-82.


[98] Young, M. A, et al. Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells. Cell stem cell. (2012). May 4;, 10(5), 570-82.

[82] Jia, F, et al. A nonviral minicircle vector for deriving human iPS cells. Nature meth‐

[83] Wadia, J. S, et al. Protein transduction technology. Current opinion in biotechnology.

[84] Zhou, H, et al. Generation of induced pluripotent stem cells using recombinant pro‐

[85] Kim, D, et al. Generation of human induced pluripotent stem cells by direct delivery

[86] Warren, L, et al. Highly efficient reprogramming to pluripotency and directed differ‐ entiation of human cells with synthetic modified mRNA. Cell stem cell. (2010). Nov

[87] Araki, R, et al. Conversion of ancestral fibroblasts to induced pluripotent stem cells.

[88] Rideout, W. M, et al. Nuclear cloning and epigenetic reprogramming of the genome.

[89] Marion, R. M, et al. Telomeres acquire embryonic stem cell characteristics in induced

[90] Chen, J, et al. EMT and MET as paradigms for cell fate switching. Journal of molecu‐

[91] Yamanaka, S. Induced pluripotent stem cells: past, present, and future. Cell stem cell.

[92] Chin, M. H, et al. Induced pluripotent stem cells and embryonic stem cells are distin‐ guished by gene expression signatures. Cell stem cell. (2009). Jul 2;, 5(1), 111-23.

[93] Marchetto, M. C, et al. Transcriptional signature and memory retention of human-in‐

[94] Bock, C, et al. Reference Maps of human ES and iPS cell variation enable highthroughput characterization of pluripotent cell lines. Cell. (2011). Feb 4;, 144(3),

[95] Guenther, M. G, et al. Chromatin structure and gene expression programs of human embryonic and induced pluripotent stem cells. Cell stem cell. (2010). Aug 6;, 7(2),

[96] Newman, A. M, et al. Lab-specific gene expression signatures in pluripotent stem

[97] Osafune, K, et al. Marked differences in differentiation propensity among human embryonic stem cell lines. Nature biotechnology. (2008). Mar;, 26(3), 313-5.

pluripotent stem cells. Cell stem cell. (2009). Feb 6;, 4(2), 141-54.

duced pluripotent stem cells. PloS one. (2009). e7076.

cells. Cell stem cell. (2010). Aug 6;, 7(2), 258-62.

of reprogramming proteins. Cell stem cell. (2009). Jun 5;, 4(6), 472-6.

ods. (2010). Mar;, 7(3), 197-9.

teins. Cell stem cell. (2009). May 8;, 4(5), 381-4.

Stem cells. (2010). Feb;, 28(2), 213-20.

Science. (2001). Aug 10;, 293(5532), 1093-8.

lar cell biology. (2012). Apr;, 4(2), 66-9.

(2012). Jun 14;, 10(6), 678-84.

439-52.

249-57.

(2002). Feb;, 13(1), 52-6.

254 Pluripotent Stem Cells

5;, 7(5), 618-30.


[112] Zaret, K. S, et al. Pioneer transcription factors: establishing competence for gene ex‐ pression. Genes & development. (2011). Nov 1;, 25(21), 2227-41.

[128] Emslie, D, et al. Oct2 enhances antibody-secreting cell differentiation through regula‐ tion of IL-5 receptor alpha chain expression on activated B cells. The Journal of exper‐

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 257

[129] Ieda, M, et al. Direct reprogramming of fibroblasts into functional cardiomyocytes by

[130] Qian, L, et al. In vivo reprogramming of murine cardiac fibroblasts into induced car‐

[131] Song, K, et al. Heart repair by reprogramming non-myocytes with cardiac transcrip‐

[132] Zhou, Q, et al. In vivo reprogramming of adult pancreatic exocrine cells to beta-cells.

[133] Sekiya, S, et al. Direct conversion of mouse fibroblasts to hepatocyte-like cells by de‐

[134] Huang, P, et al. Induction of functional hepatocyte-like cells from mouse fibroblasts

[135] Vierbuchen, T, et al. Direct conversion of fibroblasts to functional neurons by defined

[136] Pang, Z. P, et al. Induction of human neuronal cells by defined transcription factors.

[137] Marro, S, et al. Direct lineage conversion of terminally differentiated hepatocytes to

[138] Han, D. W, et al. Direct reprogramming of fibroblasts into neural stem cells by de‐

[139] Kumar, A, et al. Zic3 induces conversion of human fibroblasts to stable neural pro‐ genitor-like cells. Journal of molecular cell biology. (2012). Aug;, 4(4), 252-5.

[140] Ring, K. L, et al. Direct reprogramming of mouse and human fibroblasts into multi‐ potent neural stem cells with a single factor. Cell stem cell. (2012). Jul 6;, 11(1), 100-9.

[141] Pfisterer, U, et al. Direct conversion of human fibroblasts to dopaminergic neurons. Proceedings of the National Academy of Sciences of the United States of America.

[142] Caiazzo, M, et al. Direct generation of functional dopaminergic neurons from mouse

[143] Son, E. Y, et al. Conversion of mouse and human fibroblasts into functional spinal

and human fibroblasts. Nature. (2011). Aug 11;, 476(7359), 224-7.

motor neurons. Cell stem cell. (2011). Sep 2;, 9(3), 205-18.

imental medicine. (2008). Feb 18;, 205(2), 409-21.

defined factors. Cell. (2010). Aug 6;, 142(3), 375-86.

diomyocytes. Nature. (2012). May 31;, 485(7400), 593-8.

tion factors. Nature. (2012). May 31;, 485(7400), 599-604.

fined factors. Nature. (2011). Jul 21;, 475(7356), 390-3.

factors. Nature. (2010). Feb 25;, 463(7284), 1035-41.

Nature. (2011). Aug 11;, 476(7359), 220-3.

(2011). Jun 21;, 108(25), 10343-8.

by defined factors. Nature. (2011). Jul 21;, 475(7356), 386-9.

functional neurons. Cell stem cell. (2011). Oct 4;, 9(4), 374-82.

fined factors. Cell stem cell. (2012). Apr 6;, 10(4), 465-72.

Nature. (2008). Oct 2;, 455(7213), 627-32.


[128] Emslie, D, et al. Oct2 enhances antibody-secreting cell differentiation through regula‐ tion of IL-5 receptor alpha chain expression on activated B cells. The Journal of exper‐ imental medicine. (2008). Feb 18;, 205(2), 409-21.

[112] Zaret, K. S, et al. Pioneer transcription factors: establishing competence for gene ex‐

[113] Schafer, B. W, et al. Effect of cell history on response to helix-loop-helix family of

[114] Davis, R. L, et al. Expression of a single transfected cDNA converts fibroblasts to my‐

[115] Lassar, A. B, et al. Transfection of a DNA locus that mediates the conversion of

[116] Choi, J, et al. MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes. Proceedings of the National Academy of Sciences of

[117] Weintraub, H, et al. Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proceedings of the National Academy of Sciences of the United States of America. (1989). Jul;, 86(14), 5434-8.

[118] Rolink, A. G, et al. Long-term in vivo reconstitution of T-cell development by Pax5-

[119] Mikkola, I, et al. Reversion of B cell commitment upon loss of Pax5 expression. Sci‐

[120] Cobaleda, C, et al. Conversion of mature B cells into T cells by dedifferentiation to

[121] Xie, H, et al. Stepwise reprogramming of B cells into macrophages. Cell. (2004). May

[122] Jarriault, S, et al. A Caenorhabditis elegans model for epithelial-neuronal transdiffer‐ entiation. Proceedings of the National Academy of Sciences of the United States of

[123] Richard, J. P, et al. Direct in vivo cellular reprogramming involves transition through discrete, non-pluripotent steps. Development. (2011). Apr;, 138(8), 1483-92.

[124] Seale, P, et al. PRDM16 controls a brown fat/skeletal muscle switch. Nature. (2008).

[125] Kajimura, S, et al. Initiation of myoblast to brown fat switch by a PRDM16-C/EBP-

[126] Szabo, E, et al. Direct conversion of human fibroblasts to multilineage blood progeni‐

[127] Lengner, C. J, et al. Oct4 expression is not required for mouse somatic stem cell self-

beta transcriptional complex. Nature. (2009). Aug 27;, 460(7259), 1154-8.

deficient B-cell progenitors. Nature. (1999). Oct 7;, 401(6753), 603-6.

uncommitted progenitors. Nature. (2007). Sep 27;, 449(7161), 473-7.

pression. Genes & development. (2011). Nov 1;, 25(21), 2227-41.

myogenic regulators. Nature. (1990). Mar 29;, 344(6265), 454-8.

10T1/2 fibroblasts to myoblasts. Cell. (1986). Dec 5;, 47(5), 649-56.

the United States of America. (1990). Oct;, 87(20), 7988-92.

oblasts. Cell. (1987). Dec 24;, 51(6), 987-1000.

256 Pluripotent Stem Cells

ence. (2002). Jul 5;, 297(5578), 110-3.

America. (2008). Mar 11;, 105(10), 3790-5.

tors. Nature. (2010). Nov 25;, 468(7323), 521-6.

renewal. Cell stem cell. (2007). Oct 11;, 1(4), 403-15.

28;, 117(5), 663-76.

Aug 21;, 454(7207), 961-7.


[144] Qiang, L, et al. Directed conversion of Alzheimer's disease patient skin fibroblasts in‐ to functional neurons. Cell. (2011). Aug 5;, 146(3), 359-71.

[160] Kucia, M, et al. A population of very small embryonic-like (VSEL) CXCR4(+)SSEA-1(+)Oct-4+ stem cells identified in adult bone marrow. Leukemia : of‐ ficial journal of the Leukemia Society of America, Leukemia Research Fund, UK.

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 259

[161] Lamoury, F. M, et al. Undifferentiated mouse mesenchymal stem cells spontaneously express neural and stem cell markers Oct-4 and Rex-1. Cytotherapy. (2006). , 8(3),

[162] Ling, T. Y, et al. Identification of pulmonary Oct-4+ stem/progenitor cells and dem‐ onstration of their susceptibility to SARS coronavirus (SARS-CoV) infection in vitro. Proceedings of the National Academy of Sciences of the United States of America.

[163] Nayernia, K, et al. Derivation of male germ cells from bone marrow stem cells. Labo‐ ratory investigation; a journal of technical methods and pathology. (2006). Jul;, 86(7),

[164] Pallante, B. A, et al. Bone marrow Oct3/4+ cells differentiate into cardiac myocytes via age-dependent paracrine mechanisms. Circulation research. (2007). Jan 5;100(1):e,

[165] Redvers, R. P, et al. Side population in adult murine epidermis exhibits phenotypic and functional characteristics of keratinocyte stem cells. Proceedings of the National Academy of Sciences of the United States of America. (2006). Aug 29;, 103(35),

[166] Ren, H, et al. Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions. Biochemical and biophysical research communications. (2006).

[167] Romero-ramos, M, et al. Neuronal differentiation of stem cells isolated from adult

[168] Sagrinati, C, et al. Isolation and characterization of multipotent progenitor cells from the Bowman's capsule of adult human kidneys. Journal of the American Society of

[169] Wang, R, et al. Phenotypic analysis of c-Kit expression in epithelial monolayers de‐ rived from postnatal rat pancreatic islets. The Journal of endocrinology. (2004). Jul;,

[170] Jiang, Y, et al. Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Experimental hematology. (2002). Aug;, 30(8),

[171] Subramanian, K, et al. Isolation procedure and characterization of multipotent adult progenitor cells from rat bone marrow. Methods in molecular biology. (2010). , 636,

muscle. Journal of neuroscience research. (2002). Sep 15;, 69(6), 894-907.

Nephrology : JASN. (2006). Sep;, 17(9), 2443-56.

(2006). May;, 20(5), 857-69.

(2006). Jun 20;, 103(25), 9530-5.

228-42.

654-63.

1-11.

13168-73.

Aug 18;, 347(1), 12-21.

182(1), 113-22.

896-904.

55-78.


[160] Kucia, M, et al. A population of very small embryonic-like (VSEL) CXCR4(+)SSEA-1(+)Oct-4+ stem cells identified in adult bone marrow. Leukemia : of‐ ficial journal of the Leukemia Society of America, Leukemia Research Fund, UK. (2006). May;, 20(5), 857-69.

[144] Qiang, L, et al. Directed conversion of Alzheimer's disease patient skin fibroblasts in‐

[145] Efe, J. A, et al. Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nature cell biology. (2011). Mar;, 13(3), 215-22.

[146] Kim, J, et al. Direct reprogramming of mouse fibroblasts to neural progenitors. Pro‐ ceedings of the National Academy of Sciences of the United States of America.

[147] Brons, I. G, et al. Derivation of pluripotent epiblast stem cells from mammalian em‐

[148] Bao, S, et al. Epigenetic reversion of post-implantation epiblast to pluripotent embry‐

[149] Kanatsu-shinohara, M, et al. Generation of pluripotent stem cells from neonatal

[150] Ko, K, et al. Induction of pluripotency in adult unipotent germline stem cells. Cell

[151] Guan, K, et al. Pluripotency of spermatogonial stem cells from adult mouse testis.

[152] Seandel, M, et al. Generation of functional multipotent adult stem cells from GPR125+ germline progenitors. Nature. (2007). Sep 20;, 449(7160), 346-50.

[153] Conrad, S, et al. Generation of pluripotent stem cells from adult human testis. Na‐

[154] Ko, K, et al. Human adult germline stem cells in question. Nature. (2010). Jun

[155] Anjos-afonso, F, et al. Nonhematopoietic/endothelial SSEA-1+ cells define the most primitive progenitors in the adult murine bone marrow mesenchymal compartment.

[156] Danner, S, et al. Derivation of oocyte-like cells from a clonal pancreatic stem cell line.

[157] Goolsby, J, et al. Hematopoietic progenitors express neural genes. Proceedings of the National Academy of Sciences of the United States of America. (2003). Dec 9;,

[158] Gupta, S, et al. Isolation and characterization of kidney-derived stem cells. Journal of the American Society of Nephrology : JASN. (2006). Nov;, 17(11), 3028-40.

[159] Johnson, J, et al. Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood. Cell. (2005). Jul 29;, 122(2), 303-15.

to functional neurons. Cell. (2011). Aug 5;, 146(3), 359-71.

(2011). May 10;, 108(19), 7838-43.

258 Pluripotent Stem Cells

stem cell. (2009). Jul 2;, 5(1), 87-96.

bryos. Nature. (2007). Jul 12;, 448(7150), 191-5.

mouse testis. Cell. (2004). Dec 29;, 119(7), 1001-12.

Nature. (2006). Apr 27;, 440(7088), 1199-203.

ture. (2008). Nov 20;, 456(7220), 344-9.

Blood. (2007). Feb 1;, 109(3), 1298-306.

Molecular human reproduction. (2007). Jan;, 13(1), 11-20.

24;465(7301):E1; discussion E3.

100(25), 14926-31.

onic stem cells. Nature. (2009). Oct 29;, 461(7268), 1292-5.


[172] Ulloa-montoya, F, et al. Comparative transcriptome analysis of embryonic and adult stem cells with extended and limited differentiation capacity. Genome biology. (2007). R163.

[186] Lin, T, et al. A chemical platform for improved induction of human iPSCs. Nature

Multiple Paths to Reprogramming http://dx.doi.org/10.5772/55104 261

[187] Esteban, M. A, et al. Vitamin C enhances the generation of mouse and human in‐

[188] Stadtfeld, M, et al. Ascorbic acid prevents loss of Dlk1-Dio3 imprinting and facilitates generation of all-iPS cell mice from terminally differentiated B cells. Nature genetics.

[189] Zhang, J, et al. UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells. The EMBO journal. (2011). Dec 14;, 30(24), 4860-73.

[190] Huangfu, D, et al. Induction of pluripotent stem cells by defined factors is greatly im‐ proved by small-molecule compounds. Nature biotechnology. (2008). Jul;, 26(7),

[191] Shi, Y, et al. Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell stem cell. (2008). Nov 6;, 3(5),

[192] Shi, Y, et al. A combined chemical and genetic approach for the generation of in‐

[193] Li, W, et al. Generation of human-induced pluripotent stem cells in the absence of

[194] Huangfu, D, et al. Induction of pluripotent stem cells from primary human fibro‐ blasts with only Oct4 and Sox2. Nature biotechnology. (2008). Nov;, 26(11), 1269-75.

duced pluripotent stem cells. Cell stem cell. (2008). Jun 5;, 2(6), 525-8.

exogenous Sox2. Stem cells. (2009). Dec;, 27(12), 2992-3000.

duced pluripotent stem cells. Cell stem cell. (2010). Jan 8;, 6(1), 71-9.

methods. (2009). Nov;, 6(11), 805-8.

(2012). Apr;S1-2., 44(4), 398-405.

795-7.

568-74.


[186] Lin, T, et al. A chemical platform for improved induction of human iPSCs. Nature methods. (2009). Nov;, 6(11), 805-8.

[172] Ulloa-montoya, F, et al. Comparative transcriptome analysis of embryonic and adult stem cells with extended and limited differentiation capacity. Genome biology.

[173] Debeb, B. G, et al. Isolation of Octexpressing extraembryonic endoderm precursor

[174] Lo Nigro Aet al. MAPC culture conditions support the derivation of cells with nas‐ cent hypoblast features from bone marrow and blastocysts. Journal of molecular cell

[175] Roobrouck, V. D, et al. Differentiation potential of human postnatal mesenchymal stem cells, mesoangioblasts, and multipotent adult progenitor cells reflected in their transcriptome and partially influenced by the culture conditions. Stem cells. (2011).

[176] Fuchs, E. The impact of cell culture on stem cell research. Cell stem cell. (2012). Jun

[177] Nie, B, et al. Cellular reprogramming: a small molecule perspective. Current opinion

[178] Zhu, S, et al. Reprogramming of human primary somatic cells by OCT4 and chemical

[179] Martin, G. R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proceedings of the National Academy of Sciences of the United States of America. (1981). Dec;, 78(12), 7634-8.

[180] Evans, M. J, et al. Establishment in culture of pluripotential cells from mouse em‐

[181] Ying, Q. L, et al. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell. (2003). Oct 31;,

[182] Hayashi, K, et al. Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states. Cell stem cell. (2008). Oct 9;, 3(4),

[183] Toyooka, Y, et al. Identification and characterization of subpopulations in undiffer‐

[184] Ying, Q. L, et al. The ground state of embryonic stem cell self-renewal. Nature.

[185] Buehr, M, et al. Capture of authentic embryonic stem cells from rat blastocysts. Cell.

entiated ES cell culture. Development. (2008). Mar;, 135(5), 909-18.

(2007). R163.

260 Pluripotent Stem Cells

biology. (2012). Aug 9.

May;, 29(5), 871-82.

in cell biology. (2012). Sep 6.

compounds. Cell stem cell. (2010). Dec 3;, 7(6), 651-5.

bryos. Nature. (1981). Jul 9;, 292(5819), 154-6.

(2008). May 22;, 453(7194), 519-23.

(2008). Dec 26;, 135(7), 1287-98.

14;, 10(6), 640-1.

115(3), 281-92.

391-401.

cell lines. PloS one. (2009). e7216., 4.


**Chapter 12**

**Induced Pluripotent Stem Cells: Current and Emerging**

Induced pluripotent stem (iPS) cells are somatic cells which have been imbued with pluripo‐ tent differentiation potential through some form of artificial treatment. On a general level, these treatments involve modifications in the expression of keystone genes associated with pluripotency in embryonic stem cells (ESCs) or their downstream expression products. Despite the conceptual simplicity of iPS cell technology, the 2006 development of the first iPS cell line by Yamanaka and Takahashi [1] has led to an exponential increase in the volume of pluripo‐

tency research and a new perspective from which to approach regenerative medicine.

ESCs in some therapeutic contexts due to reduced risk factors for the patient.

the host to transcribe and generate the products independently.

iPS cells are a potential alternative to ESCs in therapeutic contexts, retaining the regenerative potential of ESCs inherent in pluripotent phenotypes, while bypassing some of the risks associated with ESC transplants. A number of studies have demonstrated that iPS cells and ESCs have effectively indistinguishable pluripotent capability, implying that iPS cells maintain the same therapeutic potential long associated with natural ESCs. However, unlike ESCs, iPS cells do not carry a risk of immunorejection due to their patient specific nature, and are not affected by the same ethical concerns as ESCs. As such, iPS cells may actually be preferable to

Since Yamanaka's hallmark 2006 paper and methodology, numerous iPS cell generation technologies have been developed. Most methods rely upon epigenetic expression of genes determined to be pluripotency regulators. Expression is most commonly induced through viral integration into the host genome, though other episomal methods do exist. Non-genetic induced pluripotency methods generally utilize the downstream expression products of the same keystone genes to generate the same effect as epigenetic expression, without requiring

> © 2013 Kimmel and Sugaya; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Kimmel and Sugaya; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Technologies**

http://dx.doi.org/10.5772/55101

**1. Introduction**

Jacob Kimmel and Kiminobu Sugaya

Additional information is available at the end of the chapter

## **Induced Pluripotent Stem Cells: Current and Emerging Technologies**

Jacob Kimmel and Kiminobu Sugaya

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55101

## **1. Introduction**

Induced pluripotent stem (iPS) cells are somatic cells which have been imbued with pluripo‐ tent differentiation potential through some form of artificial treatment. On a general level, these treatments involve modifications in the expression of keystone genes associated with pluripotency in embryonic stem cells (ESCs) or their downstream expression products. Despite the conceptual simplicity of iPS cell technology, the 2006 development of the first iPS cell line by Yamanaka and Takahashi [1] has led to an exponential increase in the volume of pluripo‐ tency research and a new perspective from which to approach regenerative medicine.

iPS cells are a potential alternative to ESCs in therapeutic contexts, retaining the regenerative potential of ESCs inherent in pluripotent phenotypes, while bypassing some of the risks associated with ESC transplants. A number of studies have demonstrated that iPS cells and ESCs have effectively indistinguishable pluripotent capability, implying that iPS cells maintain the same therapeutic potential long associated with natural ESCs. However, unlike ESCs, iPS cells do not carry a risk of immunorejection due to their patient specific nature, and are not affected by the same ethical concerns as ESCs. As such, iPS cells may actually be preferable to ESCs in some therapeutic contexts due to reduced risk factors for the patient.

Since Yamanaka's hallmark 2006 paper and methodology, numerous iPS cell generation technologies have been developed. Most methods rely upon epigenetic expression of genes determined to be pluripotency regulators. Expression is most commonly induced through viral integration into the host genome, though other episomal methods do exist. Non-genetic induced pluripotency methods generally utilize the downstream expression products of the same keystone genes to generate the same effect as epigenetic expression, without requiring the host to transcribe and generate the products independently.

© 2013 Kimmel and Sugaya; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Kimmel and Sugaya; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

iPS cells promise a new paradigm in regenerative medicine. Developing iPS technologies have the potential to generate patient specific stem cells, for use in generating any target phenotype within the human body for transplant. In the research context as well, iPS cells have the potential to greatly advance existing disease models. Patient specific iPS cells could be used to create individualized disease models, potentially allowing for more specialized treatment of patients. Here, we discuss a number of the technologies in development seeking to fulfill these promises, as well as their potential applications in both therapeutic and research settings.

**3.1. Sox2, Oct4, Lin28, Nanog**

for reprogramming [7].

**3.2. iPS-S: Sox2, Oct4, Lin28, Nanog, Klf4, Myc-c**

Yu et. al. demonstrated in 2007 that iPS cells could be generated from pre-natal and post-natal fibroblasts without transduction of the protooncogene Myc-c, using a combination of Sox2, Oct4, Lin28, and Nanog (SOLN). Factors were selected based on their high expression in ESCs, in comparison to myeloid progenitors. Removal of Myc-c from the gene cocktail eliminated the risk of transcription factor induced tumorogenesis, overcoming one of the fundamental issues with Yamanaka and Takahashi's initial methodology. Additionally, Yu et. al. recognized the potential usefulness of Nanog in iPS technologies, noting that it could lead to an increased recovery rate for iPS cell clones generated using the SOLN method. This is potentially due to Nanog's action upstream of Oct4 and Sox2. Lin28 did not integrate in one iPS clone from each of the two cell lines tested, suggesting that while Lin28 may improve efficacy, it is not necessary

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Combining the SOLN and SOKM transcription factor cocktails, Liao et al demonstrated in 2008 that the efficacy of transfection could be improved by using all 6 previously demonstrated transcription factors in a single transduction and deemed their method iPS-S. Combination of the 6 factors was attempted based on empirical speculation, and proved successful. Trans‐ duced colonies also developed more rapidly, within 17 days post-transduction, as opposed to 26 days using the standard SOLN factors. The iPS-S method also increased efficacy roughly 10 fold, which combined with the more rapid development of iPS colonies, partially addressed the inefficiency issues with the canonical iPS technology [8]. As with other transcription factor combinations utilizing Myc-c, the iPS-S method carries with it a risk of tumorogenesis due to

random transgene reactivation, inhibiting the use of iPS-S in some applications.

cells (NSCs) due to their endogenous expression of Sox2, Myc-c, and Klf4 [9].

**3.3. Combined epigentic, small-compound, and endogenous expression approaches**

One of the first approaches to reduce the number of factors required, it was demonstrated that treatment with the epigenetic small compound BIX-01294 could substitute transduction of Sox2 or Oct4, using the traditional SOKM combination, in neural progenitor cells (NPCs) [5]. The study was notable for multiple reasons, both the use of chemical conditions to remove transcription factors, and the reliance upon endogenous gene expression in the target somatic cell line. While reliance upon Sox2 expression in NPCs ultimately limits the applicability of the BIX-OKM combination, it set a precedent for use of endogenous gene expressions to reduce the number of necessary transcription factors in certain cell lines, potentially allowing for safer, more efficient iPS generation in specific contexts. This concept was further explored by Kim et. al., who demonstrated that Oct4 alone was capable of inducing pluripotency in neural stem

Shi et al. improved upon their original small compound approach, eliminating the need for Myc-c transfection and endogenous Sox2 expression. BIX-01294 and non-genetic calcium channel agonist BayK8644 were identified via a phenotypic compound screen of known drugs, and combined with the transduction of Oct4 and Klf4 (OK), were able to induce pluripotency

## **2. Canonical methodology**

The seminal event in the development of iPS technology, Yamanaka and Takahashi's 2006 publication demonstrated for the first time that the pluripotent phenotype could be induced in somatic cells and was not exclusive to ESCs. In their initial approach, Yamanaka et al. screened 24 genes as potential candidates to induce pluripotency in mouse embryonic fibroblasts (MEFs). The candidate genes were chosen for their perceived roles in regulating pluripotencty in ESC cultures. From the initial candidates, four genes were eventually identified to be necessary for induction of pluripotency, each shown to play a role in ESC pluripotency regulation: Sox2 [2], Oct4 [3], Klf4, and Myc-c [4], often abbreviated as SOKM. MEF cultures were transduced using four pMXs-based retroviral vectors, each containing one of the target transcription factors. MEFs transduced with these factors formed colonies exhibiting ESC morphology and the pluripotent phenotype, as demonstrated by their differ‐ entiation ability and teratoma formation *in vivo.*

Groundbreaking as it was, this initial iPS technology had multiple issues preventing imme‐ diate use in downstream applications. Despite its effectiveness, the early SOKM method had a decidedly low efficacy [1, 5], inhibiting generation of large scale iPS cultures for use in potential clinical applications or in the laboratory. The viral integration method also presented challenges, as genome integration could lead to random gene reactivation within the iPS culture, potentially causing deleterious effects. Myc-c itself acts as a protooncogene, which led to tumorogenesis in 50% of mice chimeric mice derived using the SOKM method [6]. Com‐ pounded with the risk for random gene reactivation, the use of Myc-c could lead to tumoro‐ genesis in potential patients.

## **3. Improved epigenetic methods**

In the interim since the development of the initial induction methodology, many improve‐ ments and variations on the technology have been made. Most of these improved methods utilize a similar epigenetic pathway to that of the original study, relying upon the host culture to express downstream products which induce the pluripotent phenotype. We discuss in brief some of these improved epigenetic methods, their potential niche applications, and their delivery vectors.

#### **3.1. Sox2, Oct4, Lin28, Nanog**

iPS cells promise a new paradigm in regenerative medicine. Developing iPS technologies have the potential to generate patient specific stem cells, for use in generating any target phenotype within the human body for transplant. In the research context as well, iPS cells have the potential to greatly advance existing disease models. Patient specific iPS cells could be used to create individualized disease models, potentially allowing for more specialized treatment of patients. Here, we discuss a number of the technologies in development seeking to fulfill these promises, as well as their potential applications in both therapeutic and research settings.

The seminal event in the development of iPS technology, Yamanaka and Takahashi's 2006 publication demonstrated for the first time that the pluripotent phenotype could be induced in somatic cells and was not exclusive to ESCs. In their initial approach, Yamanaka et al. screened 24 genes as potential candidates to induce pluripotency in mouse embryonic fibroblasts (MEFs). The candidate genes were chosen for their perceived roles in regulating pluripotencty in ESC cultures. From the initial candidates, four genes were eventually identified to be necessary for induction of pluripotency, each shown to play a role in ESC pluripotency regulation: Sox2 [2], Oct4 [3], Klf4, and Myc-c [4], often abbreviated as SOKM. MEF cultures were transduced using four pMXs-based retroviral vectors, each containing one of the target transcription factors. MEFs transduced with these factors formed colonies exhibiting ESC morphology and the pluripotent phenotype, as demonstrated by their differ‐

Groundbreaking as it was, this initial iPS technology had multiple issues preventing imme‐ diate use in downstream applications. Despite its effectiveness, the early SOKM method had a decidedly low efficacy [1, 5], inhibiting generation of large scale iPS cultures for use in potential clinical applications or in the laboratory. The viral integration method also presented challenges, as genome integration could lead to random gene reactivation within the iPS culture, potentially causing deleterious effects. Myc-c itself acts as a protooncogene, which led to tumorogenesis in 50% of mice chimeric mice derived using the SOKM method [6]. Com‐ pounded with the risk for random gene reactivation, the use of Myc-c could lead to tumoro‐

In the interim since the development of the initial induction methodology, many improve‐ ments and variations on the technology have been made. Most of these improved methods utilize a similar epigenetic pathway to that of the original study, relying upon the host culture to express downstream products which induce the pluripotent phenotype. We discuss in brief some of these improved epigenetic methods, their potential niche applications, and their

**2. Canonical methodology**

264 Pluripotent Stem Cells

entiation ability and teratoma formation *in vivo.*

genesis in potential patients.

delivery vectors.

**3. Improved epigenetic methods**

Yu et. al. demonstrated in 2007 that iPS cells could be generated from pre-natal and post-natal fibroblasts without transduction of the protooncogene Myc-c, using a combination of Sox2, Oct4, Lin28, and Nanog (SOLN). Factors were selected based on their high expression in ESCs, in comparison to myeloid progenitors. Removal of Myc-c from the gene cocktail eliminated the risk of transcription factor induced tumorogenesis, overcoming one of the fundamental issues with Yamanaka and Takahashi's initial methodology. Additionally, Yu et. al. recognized the potential usefulness of Nanog in iPS technologies, noting that it could lead to an increased recovery rate for iPS cell clones generated using the SOLN method. This is potentially due to Nanog's action upstream of Oct4 and Sox2. Lin28 did not integrate in one iPS clone from each of the two cell lines tested, suggesting that while Lin28 may improve efficacy, it is not necessary for reprogramming [7].

#### **3.2. iPS-S: Sox2, Oct4, Lin28, Nanog, Klf4, Myc-c**

Combining the SOLN and SOKM transcription factor cocktails, Liao et al demonstrated in 2008 that the efficacy of transfection could be improved by using all 6 previously demonstrated transcription factors in a single transduction and deemed their method iPS-S. Combination of the 6 factors was attempted based on empirical speculation, and proved successful. Trans‐ duced colonies also developed more rapidly, within 17 days post-transduction, as opposed to 26 days using the standard SOLN factors. The iPS-S method also increased efficacy roughly 10 fold, which combined with the more rapid development of iPS colonies, partially addressed the inefficiency issues with the canonical iPS technology [8]. As with other transcription factor combinations utilizing Myc-c, the iPS-S method carries with it a risk of tumorogenesis due to random transgene reactivation, inhibiting the use of iPS-S in some applications.

#### **3.3. Combined epigentic, small-compound, and endogenous expression approaches**

One of the first approaches to reduce the number of factors required, it was demonstrated that treatment with the epigenetic small compound BIX-01294 could substitute transduction of Sox2 or Oct4, using the traditional SOKM combination, in neural progenitor cells (NPCs) [5]. The study was notable for multiple reasons, both the use of chemical conditions to remove transcription factors, and the reliance upon endogenous gene expression in the target somatic cell line. While reliance upon Sox2 expression in NPCs ultimately limits the applicability of the BIX-OKM combination, it set a precedent for use of endogenous gene expressions to reduce the number of necessary transcription factors in certain cell lines, potentially allowing for safer, more efficient iPS generation in specific contexts. This concept was further explored by Kim et. al., who demonstrated that Oct4 alone was capable of inducing pluripotency in neural stem cells (NSCs) due to their endogenous expression of Sox2, Myc-c, and Klf4 [9].

Shi et al. improved upon their original small compound approach, eliminating the need for Myc-c transfection and endogenous Sox2 expression. BIX-01294 and non-genetic calcium channel agonist BayK8644 were identified via a phenotypic compound screen of known drugs, and combined with the transduction of Oct4 and Klf4 (OK), were able to induce pluripotency in MEFs. The elimination of multiple transcription factors suggests that it may be possible to further replicate the effects of epigenetic transduction using chemical conditions, reducing the risk for random gene reactivation and potentially allowing for more controlled iPS generation temporality [10].

over time, unlike some episomal vectors. Retroviral technologies are very mature, allowing for rapid development of vectors and efficient production of vectors in the laboratory. While standard retroviral vectors are only capable of infecting dividing cells, the lentiviral subclass of retroviruses are indeed capable of infecting non-dividing cells, an important consideration when infecting cell types that divide rarely, such as neurons. A combination of these attributes

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However, retroviral vectors and the lentiviral sub-class also have certain inherent risk factors. Most prominently, viral integration into the host genome can cause random gene reactivation, as discussed in section 2. Using the original SOKM transcription factors, this risk is exaggerated due to the protooncogenetic nature of Myc-c. Although various epigenetic methods have eliminated the necessity of Myc-c, random gene reactivation may still lead to tumorogenesis and deleterious effects in potential transplant patients. As a case study, an FDA clinical trial involving the retroviral transduction of non-protooncogenes led to the development of lymphoma in two patients [13]. Residual expression of transgenes may also lead to phenotypic expression differences between iPS cells and ESCs, leading to a less accurate model of human ESCs for research use or some clinical applications [7, 14]. To reduce the risk of random transgene reactivation and minimize remnant transgene expression, transgenes can be excised

Episomal factors, by definition, allow for the introduction of genetic factors without integration into the host genome. A lack of host genome integration inherently removes the risk of random transgene reactivation associated with viral vectors, but presents functional challenges in some contexts. The most common type of episomal vector in the context of iPS technology is the plasmid, a DNA library separate from the host's nucleic genome, first confirmed as a viable reprogramming vector using the original SOKM factors [15]. The plasmid method has several advantages, both in the laboratory and in downstream applications. Plasmids are a welldeveloped technology, are very easy to generate in great quantity in the laboratory, and have a relatively low cost-of-use compared to comparable viral vectors. For these reasons, plasmids

The most prominent advantage of plasmids is the lack of integration inherent in episomal vectors. Although there is a potential for spontaneous integration of transgenes during the reprogramming process, iPS clones generated from plasmid vectors can be screened to select only integration-free clones [17]. As such, plasmid vectors are unaffected by issues related to transgene integration, such as residual transgene expression and random transgene reactiva‐ tion. Although these advantages make plasmids a desirable vector for reprogramming, their efficacy remains well below that of viral integration, limiting the potential for large scale iPS cell generation using plasmids [18]. This reduced efficacy could potentially be due to the temporary nature of plasmids, and the speculated ongoing nature of the reprogramming process [18]; transcription factor expression may be reduced before the iPS reprogramming process is complete, altering the stoichiometric balance of factors and ending reprogramming

makes retroviral vectors a highly functional candidate for iPS cell induction.

using a Cre/Lox system, as demonstrated by multiple groups [12, 14].

are the favored vector in Yamanaka's laboratory [16].

**3.6. Plasmid vectors**


**Table 1.** Epigenetic methods covered in this section: year of publication, vectors used, and required transcription factors. \* Reliant upon endogenous expression of certain somatic cell phenotypes.

#### **3.4. Nanog**

While the majority of epigenetic approaches rely upon multiple transcription factors, chemical conditions, or endogenous expression, we patented technology capable of generating iPS cells through transfection of Nanog alone in 2006 [11]. Nanog is capable of inducing pluripotency without the aid of other factors due to its role upstream of Oct4 and Sox2. We demonstrated this interaction in bone marrow derived mesenchymal stem cells (MSCs), in which Nanog transfection successfully increased Sox2 and Oct4 levels [12]. Nanog has been demonstrated to induce pluripotency when delivered through lentiviral or plasmid vectors, providing both integrated and episomal gene expressions pathways.

Eliminating the need for multiple transcription factors has various benefits. As with other modified gene cocktails, the elimination of Myc-c greatly reduces the risk of tumorogenesis. Transfection of a single genetic factor may have higher efficacy than that of multiple factors and could lead to a lower overall cost per iPS cell generated. The improved efficiency and reduced cost of this method could allow for more rapid production of iPS cells for use in therapeutic treatments at a lower eventual cost to the patient.

#### **3.5. Retroviral vectors**

Beginning with the seminal paper by Yamanaka and Takahashi, the majority of improved epigenetic methods have utilized retroviral vectors to deliver their target transcription factors. In the context of induced pluripotency, retroviral vectors provide a number of distinct advantages, leading to their widespread use. Due to integration with the host genome, retroviral vectors are capable of generating stable iPS clones that maintain their phenotype over time, unlike some episomal vectors. Retroviral technologies are very mature, allowing for rapid development of vectors and efficient production of vectors in the laboratory. While standard retroviral vectors are only capable of infecting dividing cells, the lentiviral subclass of retroviruses are indeed capable of infecting non-dividing cells, an important consideration when infecting cell types that divide rarely, such as neurons. A combination of these attributes makes retroviral vectors a highly functional candidate for iPS cell induction.

However, retroviral vectors and the lentiviral sub-class also have certain inherent risk factors. Most prominently, viral integration into the host genome can cause random gene reactivation, as discussed in section 2. Using the original SOKM transcription factors, this risk is exaggerated due to the protooncogenetic nature of Myc-c. Although various epigenetic methods have eliminated the necessity of Myc-c, random gene reactivation may still lead to tumorogenesis and deleterious effects in potential transplant patients. As a case study, an FDA clinical trial involving the retroviral transduction of non-protooncogenes led to the development of lymphoma in two patients [13]. Residual expression of transgenes may also lead to phenotypic expression differences between iPS cells and ESCs, leading to a less accurate model of human ESCs for research use or some clinical applications [7, 14]. To reduce the risk of random transgene reactivation and minimize remnant transgene expression, transgenes can be excised using a Cre/Lox system, as demonstrated by multiple groups [12, 14].

#### **3.6. Plasmid vectors**

in MEFs. The elimination of multiple transcription factors suggests that it may be possible to further replicate the effects of epigenetic transduction using chemical conditions, reducing the risk for random gene reactivation and potentially allowing for more controlled iPS generation

 Shi et. al. Retroviral, small-compound \* Oct4, Klf4, Myc-c, small-compound BIX-01294 Shi et. al. Retroviral, small-compound Oct4, Klf4, small-compounds BIX-01294, BayK8644 Liao et. al. Lentiviral Sox2, Oct4, Klf4, Myc-c, Lin28, Nanog Okita et. al. Plasmid Sox2, Oct4, Klf4, Myc-c

**Year Group Vector Transcription Factors** 2006 Yamanaka et. al. Retroviral Sox2, Oct4, Klf4, Myc-c 2007 Yu et. al. Lentiviral Sox2, Oct4, LIN28, Nanog

2009 Kim et. al. Retroviral \* Oct4

factors. \* Reliant upon endogenous expression of certain somatic cell phenotypes.

integrated and episomal gene expressions pathways.

therapeutic treatments at a lower eventual cost to the patient.

2009 Fusaki et. al. Sendai virus Sox2, Oct4, Klf4, Myc-c 2010 Sugaya et. al. Retroviral, plasmid Nanog

**Table 1.** Epigenetic methods covered in this section: year of publication, vectors used, and required transcription

While the majority of epigenetic approaches rely upon multiple transcription factors, chemical conditions, or endogenous expression, we patented technology capable of generating iPS cells through transfection of Nanog alone in 2006 [11]. Nanog is capable of inducing pluripotency without the aid of other factors due to its role upstream of Oct4 and Sox2. We demonstrated this interaction in bone marrow derived mesenchymal stem cells (MSCs), in which Nanog transfection successfully increased Sox2 and Oct4 levels [12]. Nanog has been demonstrated to induce pluripotency when delivered through lentiviral or plasmid vectors, providing both

Eliminating the need for multiple transcription factors has various benefits. As with other modified gene cocktails, the elimination of Myc-c greatly reduces the risk of tumorogenesis. Transfection of a single genetic factor may have higher efficacy than that of multiple factors and could lead to a lower overall cost per iPS cell generated. The improved efficiency and reduced cost of this method could allow for more rapid production of iPS cells for use in

Beginning with the seminal paper by Yamanaka and Takahashi, the majority of improved epigenetic methods have utilized retroviral vectors to deliver their target transcription factors. In the context of induced pluripotency, retroviral vectors provide a number of distinct advantages, leading to their widespread use. Due to integration with the host genome, retroviral vectors are capable of generating stable iPS clones that maintain their phenotype

temporality [10].

266 Pluripotent Stem Cells

**3.4. Nanog**

**3.5. Retroviral vectors**

Episomal factors, by definition, allow for the introduction of genetic factors without integration into the host genome. A lack of host genome integration inherently removes the risk of random transgene reactivation associated with viral vectors, but presents functional challenges in some contexts. The most common type of episomal vector in the context of iPS technology is the plasmid, a DNA library separate from the host's nucleic genome, first confirmed as a viable reprogramming vector using the original SOKM factors [15]. The plasmid method has several advantages, both in the laboratory and in downstream applications. Plasmids are a welldeveloped technology, are very easy to generate in great quantity in the laboratory, and have a relatively low cost-of-use compared to comparable viral vectors. For these reasons, plasmids are the favored vector in Yamanaka's laboratory [16].

The most prominent advantage of plasmids is the lack of integration inherent in episomal vectors. Although there is a potential for spontaneous integration of transgenes during the reprogramming process, iPS clones generated from plasmid vectors can be screened to select only integration-free clones [17]. As such, plasmid vectors are unaffected by issues related to transgene integration, such as residual transgene expression and random transgene reactiva‐ tion. Although these advantages make plasmids a desirable vector for reprogramming, their efficacy remains well below that of viral integration, limiting the potential for large scale iPS cell generation using plasmids [18]. This reduced efficacy could potentially be due to the temporary nature of plasmids, and the speculated ongoing nature of the reprogramming process [18]; transcription factor expression may be reduced before the iPS reprogramming process is complete, altering the stoichiometric balance of factors and ending reprogramming


genome alteration. Non-genetic induction removes the risk of genetic factor reactivation and consequent genetic modification inherent with these epigenetic methods. To circumvent genetic transfection, technologies have been developed which utilize downstream RNA and

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Warren et. al. have demonstrated that modified mRNAs transcribing for the four SOKM factor proteins are capable of reprogramming when passed into the cytosol of various human cell types with a catatonic delivery vehicle [23]. Initially, cytotoxicity of transfected mRNAs inhibited effective reprogramming, requiring modifications to the mRNA. In a novel approach, Warren et. al. modified the ribonucleotide bases of vector mRNAs by substituting 5-methyl‐ cytidine for cytidine and pseudouridine for uridine, reducing the immunogenicity of the mRNAs [24]. Combined with interferon inhibitor media supplements, the modifications

mRNA induced pluripotent stem (RiPS) cell generation is highly efficient relative to other technologies, with an efficacy of 1.34% in Warren's initial study. However, the modified mRNAs are difficult to generate in the laboratory and the techniques are labor-intensive. Repeated mRNA administrations are also required, increasing the labor-intensive nature of

Multiple groups have also demonstrated reprogramming utilizing the protein products of the SOKM factors [25, 26]. In order for the target proteins to pass through a lipid bilayer, both groups attached each target protein to a cell penetrating peptide (CPP). At this stage, cells are treated with CPP-conjugated proteins multiple times to ensure a continuous supply of reprogramming factors. The protein induced pluripotent stem (piPS) cell induction method is significantly less efficient than epigenetic methods, with an efficacy of ~0.001%. In addition to the transduction inefficiencies, the temporality of the process is relatively slow and the multiple treatment protocols are very labor intensive, making volume production of piPS clones difficult. Although inefficient, the piPS method does eliminate the risk of transgene

While each induction method has an inherent relative efficacy, it must be noted that a number of external factors affect reprogramming efficacy as well. Multiple groups have reported that O2 concentrations play a role in reprogramming efficacy [27, 28], with hypoxia noted to increase efficiency. The presence of methylation inhibitors, such as 5'-azacytidine, in culture medium have also been noted increase efficiency [29, 30]. Hanna et. al. have also demonstrated that cell

protein phases of the desired genetic factors to induce expression.

the technique and complicating volume production of RiPS clones.

reactivation and genome integration, just as the RiPS method.

**5. Optimization of induction methods**

**5.1. Factors affecting efficacy**

**4.1. mRNA transduction**

**4.2. Protein transduction**

allowed for generation of viable iPS clones.

**Table 2.** Comparison of different epigenetic vector technologies

in cells that may have otherwise formed colonies. Depending upon the chosen transcription factor combination and somatic cell phenotype, plasmid transduction may also require multiple transfections to effectively reprogram cells, increasing the difficulty and laborintensiveness of the technique.

#### **3.7. Sendai virus**

Sendai virus is widely known to replicate in the cytoplasm of host cells without integrating into the host genome. As such, it has been widely studied as an efficient expression vector and is known to effectively express transgene without integration [19-21]. Fusaki et. al. have demonstrated that a sendai virus vector carrying the four SOKM factors is sufficient to successfully generate iPS colonies [22]. Sendai virus reprogramming was shown to be as or more effective than traditional retroviral reprogramming, with a ~1% efficacy. Even though sendai viruses do not integrate into the genome, the persistence of a viral genome within iPS clones remains a concern for downstream applications. However, Fusaki et. al. were able to isolate clones that had no remnant presence of viral genomes. As such, the sendai viral vector is very attractive for use in downstream clinical applications. In the laboratory, sendai viral vectors leave something to be desired. Pluripotent gene expression of sendai induced iPS cells has been shown to degrade over the course of 18-20 passages, making long term iPS clone maintenance difficult.

### **4. Non-genetic reprograming methods**

Complimenting research into genetic induction of pluripotency, a number of avenues into nongenetic iPS generation have been studied. Although non-integration epigenetic methods have been developed, many are inefficient, and cannot completely eliminate the possibility of genome alteration. Non-genetic induction removes the risk of genetic factor reactivation and consequent genetic modification inherent with these epigenetic methods. To circumvent genetic transfection, technologies have been developed which utilize downstream RNA and protein phases of the desired genetic factors to induce expression.

#### **4.1. mRNA transduction**

Warren et. al. have demonstrated that modified mRNAs transcribing for the four SOKM factor proteins are capable of reprogramming when passed into the cytosol of various human cell types with a catatonic delivery vehicle [23]. Initially, cytotoxicity of transfected mRNAs inhibited effective reprogramming, requiring modifications to the mRNA. In a novel approach, Warren et. al. modified the ribonucleotide bases of vector mRNAs by substituting 5-methyl‐ cytidine for cytidine and pseudouridine for uridine, reducing the immunogenicity of the mRNAs [24]. Combined with interferon inhibitor media supplements, the modifications allowed for generation of viable iPS clones.

mRNA induced pluripotent stem (RiPS) cell generation is highly efficient relative to other technologies, with an efficacy of 1.34% in Warren's initial study. However, the modified mRNAs are difficult to generate in the laboratory and the techniques are labor-intensive. Repeated mRNA administrations are also required, increasing the labor-intensive nature of the technique and complicating volume production of RiPS clones.

#### **4.2. Protein transduction**

in cells that may have otherwise formed colonies. Depending upon the chosen transcription factor combination and somatic cell phenotype, plasmid transduction may also require multiple transfections to effectively reprogram cells, increasing the difficulty and labor-

**Vector Advantages Disadvantages**

Genome integration may lead to random gene reactivation, cannot infect non-dividing cells, residual transgene expression concerns, can potentially induce immunogenicity

Genome integration may lead to random gene reactivation, residual transgene expression concerns

Lower efficacy than viral integration methods, clones must be screened to check for integration

Pluripotent gene expression degrades over the course of 18-20 passages, clones must be screened for viral genome remnants

Retroviral Genome integration allows single transduction iPS

Lentiviral Genome integration allows single transduction iPS

Plasmid Produces integration-free iPS clones, relatively low

Sendai Virus Produces integration-free iPS clones, relatively

**Table 2.** Comparison of different epigenetic vector technologies

clone generation, well-developed technology, relatively efficient transduction rates

clone generation, well-developed technology, relatively efficient transduction rates

cost, volume production is easily scalable

high efficacy

Sendai virus is widely known to replicate in the cytoplasm of host cells without integrating into the host genome. As such, it has been widely studied as an efficient expression vector and is known to effectively express transgene without integration [19-21]. Fusaki et. al. have demonstrated that a sendai virus vector carrying the four SOKM factors is sufficient to successfully generate iPS colonies [22]. Sendai virus reprogramming was shown to be as or more effective than traditional retroviral reprogramming, with a ~1% efficacy. Even though sendai viruses do not integrate into the genome, the persistence of a viral genome within iPS clones remains a concern for downstream applications. However, Fusaki et. al. were able to isolate clones that had no remnant presence of viral genomes. As such, the sendai viral vector is very attractive for use in downstream clinical applications. In the laboratory, sendai viral vectors leave something to be desired. Pluripotent gene expression of sendai induced iPS cells has been shown to degrade over the course of 18-20 passages, making long term iPS clone

Complimenting research into genetic induction of pluripotency, a number of avenues into nongenetic iPS generation have been studied. Although non-integration epigenetic methods have been developed, many are inefficient, and cannot completely eliminate the possibility of

intensiveness of the technique.

**3.7. Sendai virus**

268 Pluripotent Stem Cells

maintenance difficult.

**4. Non-genetic reprograming methods**

Multiple groups have also demonstrated reprogramming utilizing the protein products of the SOKM factors [25, 26]. In order for the target proteins to pass through a lipid bilayer, both groups attached each target protein to a cell penetrating peptide (CPP). At this stage, cells are treated with CPP-conjugated proteins multiple times to ensure a continuous supply of reprogramming factors. The protein induced pluripotent stem (piPS) cell induction method is significantly less efficient than epigenetic methods, with an efficacy of ~0.001%. In addition to the transduction inefficiencies, the temporality of the process is relatively slow and the multiple treatment protocols are very labor intensive, making volume production of piPS clones difficult. Although inefficient, the piPS method does eliminate the risk of transgene reactivation and genome integration, just as the RiPS method.

## **5. Optimization of induction methods**

#### **5.1. Factors affecting efficacy**

While each induction method has an inherent relative efficacy, it must be noted that a number of external factors affect reprogramming efficacy as well. Multiple groups have reported that O2 concentrations play a role in reprogramming efficacy [27, 28], with hypoxia noted to increase efficiency. The presence of methylation inhibitors, such as 5'-azacytidine, in culture medium have also been noted increase efficiency [29, 30]. Hanna et. al. have also demonstrated that cell division rate plays a role in the kinetics of iPS induction [31]. Findings such as these suggest that the specific culture environment play a major role in pluripotency induction and may effect downstream development of iPS clones.

**6. Clinical applications**

applications (Figure 1).

**6.2. Parkinson's disease**

induce functional recovery [43].

**6.1. Acute neurological damage**

iPS cells can theoretically become any tissue in the body, which opens a number of possibilities for the use of iPS derived cells in graft and transplant based treatments. A key advantage of iPS cells is patient-specificity. iPS cells could be generated from a patient's own somatic cells and differentiated into the desired phenotype, allowing for an effectively autologous trans‐ plant which attenuates the risk of immunorejection. In the manner, iPS technology can be used as a pathway of sorts to generate desired tissues for transplant and tissue engineering

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Induced pluripotent stem cell technologies have provided an exciting avenue for potential treatment of many neurological diseases, many of which have few treatment options at present. Among these disorders, acute neurological damage has an exceedingly direct treatment model through the iPS pathway. In many cases, such as stroke or spinal cord injury, direct trans‐ plantation of neuronal cells derived from patient-specific iPS cells to the damaged region could potentially aid in convalescence. Studies have already demonstrated functional recovery in spinal cord injury models of mice treated with iPS derived neuronal cells [37]. Groups have also confirmed functional recovery in peripheral nerve regions [38] and murine ischemia models [39, 40], with promising graft cell growth rates in ischemia models, and notable integration with existing neural networks. These findings show promise for the potential of iPS cell therapies in acute neurological damage conditions; however, further research is needed

Some higher cognitive disorders could also be addressed using similar direct transplantation therapies. Parkinson's disease is perhaps the most direct of the higher cognitive disorders to address in this manner, as the primary cause of functional degradation can be traced to a single cell phenotype. The loss of dopamine secreting neurons in the *substantia nigra* region of the brain has been established as the leading cause of many Parkinson's symptoms, suggesting that direct replacement of lost dopamine secreting neurons through iPS cell derived neurons could aid in recovery. Through the use of various methods, multiple groups have efficiently differentiated iPS cells into dopaminergic neurons [41, 42], overcoming the first obstacle in the implementation of a transplantation therapy. In a rodent model, transplantation of dopama‐ nergic neurons and other neuronal phenotypes into Parkinson's disease model were able to

These results demonstrate the potential of iPS cells to provide functional recovery in Parkin‐ son's disease patients. However, further research is needed to establish the degree of recovery post-transplant, to improve the efficacy of transplantation, and to assess the long-term benefit of transplantation. It has been suggested that transplanted neuronal populations derived from iPS cells of hereditary Parkinson's patients may be inclined to exhibit similar degenerative

phenotypes after implantation and this potential must be explored.

to ascertain the efficacy, safety, and long term effects of such transplantations.

Of prominent concern, it has been reasoned that the stoichiometric abundances of reprogram‐ ming factors in relation to one another plays a role in reprogramming efficacy [18]. This rationale is based upon the differential effects of some pluripotency factors when expressed in different levels; for instance, expression of Oct4 and Sox2 at median levels can maintain pluripotency of ESCs, but overexpression of Oct4 can induce differentiation [32]. A similar action has been demonstrated in the context of iPS induction, in which a threefold increase of Oct4 increased efficacy, but further increases reduced the efficiency of reprogramming [33]. As such, the ability to monitor and manipulate the stoichiometric expression levels of tran‐ scription factors may play a role in selection of vectors and induction technologies in the future.

iPS cells have recently been shown to possess preferential differentiation based on their somatic cell origin, referred to as epigenetic memory [34-36]. It is believed that variations in DNA methylation status allow differentiation preferences to persist beyond the boundaries of reprogramming. It may be possible to exploit this epigenetic memory to increase the terminal differentiation efficiency of iPS cells based on the desired differentiated phenotype. By selecting cells of origin in the same lineage, or tissues known to have limited transdifferentia‐ tion ability into the target cell type, it may be possible to augment the efficacy of current induction protocols.

#### **5.2. Application specific induced pluripotent stem cells**

Until now, the majority of iPS cell research has sought to increase the efficacy at which stable pluripotent iPS clones could be developed. However, for the optimal production of a desired differentiated phenotype, solely optimizing the efficacy at which iPS clones can be developed may not be the best strategy. First touched on by Yamanaka in 2009 as the concept of "func‐ tional pluripotency" [6], it may be more effective to optimize for the generation of a target differentiated phenotype in the context of downstream applications. As such, a number of the efficacy factors mentioned above could be considered and optimized for each target phenotype and each downstream application.

Until such a time as reprogramming efficacy improves dramatically, the optimization of reprogramming in the context of specific downstream applications may be a way to increase efficiency. For each application, specific factors regulate the optimal induction method and environment, such as the acceptability of genome integration, the temporality in which desired phenotypes are needed, and the volume in which the target phenotype is required. Based on application specific factors such as these, it may be optimal to utilize various induction methods combined with an optimized set of efficacy conditions described above to generate iPS cell products on an application by application basis, rather than focusing solely on improving the generation of iPS clones.

## **6. Clinical applications**

division rate plays a role in the kinetics of iPS induction [31]. Findings such as these suggest that the specific culture environment play a major role in pluripotency induction and may

Of prominent concern, it has been reasoned that the stoichiometric abundances of reprogram‐ ming factors in relation to one another plays a role in reprogramming efficacy [18]. This rationale is based upon the differential effects of some pluripotency factors when expressed in different levels; for instance, expression of Oct4 and Sox2 at median levels can maintain pluripotency of ESCs, but overexpression of Oct4 can induce differentiation [32]. A similar action has been demonstrated in the context of iPS induction, in which a threefold increase of Oct4 increased efficacy, but further increases reduced the efficiency of reprogramming [33]. As such, the ability to monitor and manipulate the stoichiometric expression levels of tran‐ scription factors may play a role in selection of vectors and induction technologies in the future.

iPS cells have recently been shown to possess preferential differentiation based on their somatic cell origin, referred to as epigenetic memory [34-36]. It is believed that variations in DNA methylation status allow differentiation preferences to persist beyond the boundaries of reprogramming. It may be possible to exploit this epigenetic memory to increase the terminal differentiation efficiency of iPS cells based on the desired differentiated phenotype. By selecting cells of origin in the same lineage, or tissues known to have limited transdifferentia‐ tion ability into the target cell type, it may be possible to augment the efficacy of current

Until now, the majority of iPS cell research has sought to increase the efficacy at which stable pluripotent iPS clones could be developed. However, for the optimal production of a desired differentiated phenotype, solely optimizing the efficacy at which iPS clones can be developed may not be the best strategy. First touched on by Yamanaka in 2009 as the concept of "func‐ tional pluripotency" [6], it may be more effective to optimize for the generation of a target differentiated phenotype in the context of downstream applications. As such, a number of the efficacy factors mentioned above could be considered and optimized for each target phenotype

Until such a time as reprogramming efficacy improves dramatically, the optimization of reprogramming in the context of specific downstream applications may be a way to increase efficiency. For each application, specific factors regulate the optimal induction method and environment, such as the acceptability of genome integration, the temporality in which desired phenotypes are needed, and the volume in which the target phenotype is required. Based on application specific factors such as these, it may be optimal to utilize various induction methods combined with an optimized set of efficacy conditions described above to generate iPS cell products on an application by application basis, rather than focusing solely on

effect downstream development of iPS clones.

**5.2. Application specific induced pluripotent stem cells**

induction protocols.

270 Pluripotent Stem Cells

and each downstream application.

improving the generation of iPS clones.

iPS cells can theoretically become any tissue in the body, which opens a number of possibilities for the use of iPS derived cells in graft and transplant based treatments. A key advantage of iPS cells is patient-specificity. iPS cells could be generated from a patient's own somatic cells and differentiated into the desired phenotype, allowing for an effectively autologous trans‐ plant which attenuates the risk of immunorejection. In the manner, iPS technology can be used as a pathway of sorts to generate desired tissues for transplant and tissue engineering applications (Figure 1).

#### **6.1. Acute neurological damage**

Induced pluripotent stem cell technologies have provided an exciting avenue for potential treatment of many neurological diseases, many of which have few treatment options at present. Among these disorders, acute neurological damage has an exceedingly direct treatment model through the iPS pathway. In many cases, such as stroke or spinal cord injury, direct trans‐ plantation of neuronal cells derived from patient-specific iPS cells to the damaged region could potentially aid in convalescence. Studies have already demonstrated functional recovery in spinal cord injury models of mice treated with iPS derived neuronal cells [37]. Groups have also confirmed functional recovery in peripheral nerve regions [38] and murine ischemia models [39, 40], with promising graft cell growth rates in ischemia models, and notable integration with existing neural networks. These findings show promise for the potential of iPS cell therapies in acute neurological damage conditions; however, further research is needed to ascertain the efficacy, safety, and long term effects of such transplantations.

#### **6.2. Parkinson's disease**

Some higher cognitive disorders could also be addressed using similar direct transplantation therapies. Parkinson's disease is perhaps the most direct of the higher cognitive disorders to address in this manner, as the primary cause of functional degradation can be traced to a single cell phenotype. The loss of dopamine secreting neurons in the *substantia nigra* region of the brain has been established as the leading cause of many Parkinson's symptoms, suggesting that direct replacement of lost dopamine secreting neurons through iPS cell derived neurons could aid in recovery. Through the use of various methods, multiple groups have efficiently differentiated iPS cells into dopaminergic neurons [41, 42], overcoming the first obstacle in the implementation of a transplantation therapy. In a rodent model, transplantation of dopama‐ nergic neurons and other neuronal phenotypes into Parkinson's disease model were able to induce functional recovery [43].

These results demonstrate the potential of iPS cells to provide functional recovery in Parkin‐ son's disease patients. However, further research is needed to establish the degree of recovery post-transplant, to improve the efficacy of transplantation, and to assess the long-term benefit of transplantation. It has been suggested that transplanted neuronal populations derived from iPS cells of hereditary Parkinson's patients may be inclined to exhibit similar degenerative phenotypes after implantation and this potential must be explored.

**6.3. Alzheimer's disease**

have positive effects on patient phenotype.

**6.4. Cardiovascular treatments**

the source of cardiomyocytes.

therapies to treat hemophilia.

**6.5. Hemophilia**

Unlike Parkinson's disease, Alzheimer's disease cannot be traced to the loss of single cell phenotype in a distinct region. In Alzheimer's, damage is diffuse throughout the brain, forming neurofibrillary tangles characterized by high levels of amyloid precursor protein (APP) expression. It has been demonstrated that high levels of APP expression influence differen‐ tiation toward the glial phenotype [44], inhibiting direct replacement of neurons through nonterminally differentiated stem cells. As such, Alzheimer's does not lend itself to transplantation therapy as readily as acute neurological injury or Parkinson's. However, there is a potential that transplantation of terminally differentiated neuronal populations derived from iPS cells could have beneficial effects. Transplanted cells may not necessarily replace damaged neurons, but increased neurotrophic factor production from transplanted neuronal populations may

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It has been demonstrated that pluripotent stem cells have the potential to differentiate into cardiomyocyte [45, 46]. Utilizing a number of various culture conditions, including co-culture with stromal cells and cytokine supplementation, differentiation into cardiomyocytes can be made relatively efficient [47]. There is a potential that transplantation of iPS derived cario‐ myocytes may be able to assist patients who have suffered a myocardial infraction, as has been demonstrated when transplanting other related phenotypes [48]. Studies in a murine model have shown that transplantation of ESC derived cariomyocytes mitigated the functional damage of myocardial infraction [49]. Due to the similarity of iPS cell and ESC phenotypes, there is a potential that similar results would be possible utilizing patient specific iPS cells as

Hemophilia is caused by a genetic mutation that reduces the production of coagulant factor VIII or XI depending on the type. Therefore, it's possible that transplantation of iPS derived endothelial cells which express coagulant factors could correct the hemophilia phenotype in patients [50]. In a murine model, transplantation of iPS derived endothelial cells positive for factor VIII expression was able to mitigate the hemophilia A phenotype to a large degree. Endothelial cells were transplanted by injection directly into the liver of hemophiliac mice and functionality was assessed by a tail cutting assay. After treatment, mice with transplant‐ ed endothelial cells survived for 3+ months after tail cutting, while control mice died within hours. Factor VIII expression was increased to 8%-12% of normal, indicating that full restoration of factor VIII expression may not be necessary to effectively mitigate the hemophilia phenotype [51]. These findings show promise for the development of cell based

**Figure 1.** Induced pluripotent treatment pathway: Somatic cells are isolated from the patient, reprogrammed into iPS cells, and then differentiated into the target phenotype for treatment

#### **6.3. Alzheimer's disease**

Unlike Parkinson's disease, Alzheimer's disease cannot be traced to the loss of single cell phenotype in a distinct region. In Alzheimer's, damage is diffuse throughout the brain, forming neurofibrillary tangles characterized by high levels of amyloid precursor protein (APP) expression. It has been demonstrated that high levels of APP expression influence differen‐ tiation toward the glial phenotype [44], inhibiting direct replacement of neurons through nonterminally differentiated stem cells. As such, Alzheimer's does not lend itself to transplantation therapy as readily as acute neurological injury or Parkinson's. However, there is a potential that transplantation of terminally differentiated neuronal populations derived from iPS cells could have beneficial effects. Transplanted cells may not necessarily replace damaged neurons, but increased neurotrophic factor production from transplanted neuronal populations may have positive effects on patient phenotype.

#### **6.4. Cardiovascular treatments**

It has been demonstrated that pluripotent stem cells have the potential to differentiate into cardiomyocyte [45, 46]. Utilizing a number of various culture conditions, including co-culture with stromal cells and cytokine supplementation, differentiation into cardiomyocytes can be made relatively efficient [47]. There is a potential that transplantation of iPS derived cario‐ myocytes may be able to assist patients who have suffered a myocardial infraction, as has been demonstrated when transplanting other related phenotypes [48]. Studies in a murine model have shown that transplantation of ESC derived cariomyocytes mitigated the functional damage of myocardial infraction [49]. Due to the similarity of iPS cell and ESC phenotypes, there is a potential that similar results would be possible utilizing patient specific iPS cells as the source of cardiomyocytes.

#### **6.5. Hemophilia**

**Figure 1.** Induced pluripotent treatment pathway: Somatic cells are isolated from the patient, reprogrammed into iPS

cells, and then differentiated into the target phenotype for treatment

272 Pluripotent Stem Cells

Hemophilia is caused by a genetic mutation that reduces the production of coagulant factor VIII or XI depending on the type. Therefore, it's possible that transplantation of iPS derived endothelial cells which express coagulant factors could correct the hemophilia phenotype in patients [50]. In a murine model, transplantation of iPS derived endothelial cells positive for factor VIII expression was able to mitigate the hemophilia A phenotype to a large degree. Endothelial cells were transplanted by injection directly into the liver of hemophiliac mice and functionality was assessed by a tail cutting assay. After treatment, mice with transplant‐ ed endothelial cells survived for 3+ months after tail cutting, while control mice died within hours. Factor VIII expression was increased to 8%-12% of normal, indicating that full restoration of factor VIII expression may not be necessary to effectively mitigate the hemophilia phenotype [51]. These findings show promise for the development of cell based therapies to treat hemophilia.

#### **6.6. Blood supply**

Blood supply shortages are an ever-present concern in many regions, leading to demand for additional sources of red blood cells (RBCs). iPS cells could theoretically be used to generate RBCs as a supplemental source and it has been demonstrated that iPS cells are capable of direct erythrocytic differentiation [52]. Although the technology exists, the use of iPS cells to generate RBCs may not always been practical due to the cost of iPS generation, culture, and subsequent differentiation. As such, until technologies are developed which allow for industrial scale iPS cell culture and differentiation, the use of iPS cells to augment the blood supply will be fairly limited. In certain circumstances, such as a patient in need of a rare blood type in advance of surgery, generation of iPS derived RBCs may be a viable option for treatment.

Alzheimer's disease phenotype, after months in culture [56]. Utilizing a similar approach, it may be possible to emulate other age related disease phenotypes through variable gene expression, providing a second avenue from which to approach the issue. iPS line have also been derived from Huntington's patients, in which differentiated neurons main‐ tained some portions of the Huntington's phenotype [57, 58]. CDKL5 mutant iPS lines have also been generated from Rett syndrome patients, and may allow for investigation of CDKL5's underlying mechanism within patient cells [59]. Amyotrophic lateral sclerosis (ALS) has also been effectively modeled using an iPS line derived from familial ALS

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Once generated, these disease models can provide insight into the underlying mechanisms of the disease. *In vitro* research of molecular level cellular mechanisms is much cheaper and more efficient than similar research in mammal models, potentially allowing for increased research throughput. Established *in vitro* models also remove confounding factors related to animal models, potentially making direct identification of mechanisms easier. In the context of phenotypic identification and the discovery of underlying mechanisms, it is important to consider the controls necessary for using these iPS derived disease models. Due to potential phenotypic differences in iPS clones, even from the same isolation, it would be necessary to generate models using multiple iPS lines from each patient in a diverse group. This spread would allow for adequate confirmation that the identified phenotype or mechanism is indeed consistent for all patients with the disease, rather than an artifact of reprogramming or a trait

While these results in summary are very promising, substantial challenges remain before iPS cell cultures can be used as disease models in every instance. Although diseases with limited temporal dependency, such as spinal muscular atrophy, and clear monogenic origin, such as Huntington's, are replicated relatively easily *in vitro*, there remain unsolved problems in replicating diseases influenced by multiple factors. As demonstrated in attempts to replicate the Parkinson's phenotype *in vitro*, time related factors can also play a large role in disease phenotype, complicating modeling. Other diseases, such as Alzheimer's, may be dependent upon cellular interactions between multiple cell phenotypes in addition to age related factors. The homogenous nature of iPS derived cell cultures complicates accurate replication of these interactions *in vitro*. In some cases, it may be possible to model some of these cell to cell interactions using coculture, as demonstrated in ALS models that incorporate both astrocytes and neurons [61]. Further research is needed to overcome these barriers before iPS cell based

As a corollary to disease modeling, drug discovery is a promising research application for iPS cells. Developing new drugs is exceedingly expensive and many drug candidates are rejected in the final human trial stage due to toxicology concerns [46]. At present, 90% of all drugs candidates that enter clinical trials fail to be approved, leading to a low drug candidate to successful drug ratio [62]. If drugs could be screened for human toxicology earlier in the development cycle, a number of these candidates could be eliminated earlier, allowing for

patients [60].

specific to a single individual [53].

**7.2. Drug discovery**

disease modeling can be exploited to its full potential.

## **7. Research applications**

#### **7.1. Disease modeling**

Accurate disease modeling is a biotechnological problem of fundamental importance. Most current disease models rely upon murine model organisms, which are capable of providing insight, but are less than ideal due to interspecies differences [53]. iPS technologies could allow for *in vitro* disease modeling, using cultures isolated from those suffering with a given condition. If widely applied, patient specific iPS cultures could potentially be created to analyze the nuances of a disease in a particular patient, determining which course of treatment would be best. Using skin fibroblasts isolated from a patient with spinal muscular atrophy, Ebert et. al. demonstrated that iPS derived motor neurons could be effectively grown in culture and maintained the disease phenotype of the patient [54]. These findings indicate that iPS cells derived from patients with genetic disorders may exhibit the disease phenotype, allowing for their use as a disease model.

Similar isolations have also occurred with Parkinson's patients, in which iPS clones were generated from patients and subsequently differentiated into dopamanergic neurons. However, in the context of Parkinson's disease, the disease phenotype was not as readily presented *in vitro* due to the relative age of the neurons. While cultured neurons have a lifespan in weeks, Parkinson's develops over a period of years due in conjunction with age related factors, possibly requiring a form of artificial stress treatment to accurately reproduce the phenotype *in vitro* [14]. However, early stage metabolic dysfunction has already been identified and corrected *in vitro* using neurons generated from familial Parkinson's patient derived iPS cells, indicating that some early stage phenotypes may be identifiable without full phenotypic replication [55]. Alzheimer's disease, like Parkinson's, is strongly influenced by a number of age related factor which complicate the creation of an accurate model. Recently, Shi et. al. demonstrated one potential approach to this problem by using iPS cells derived from Down syndrome patients. Down syndrome patients overexpress a gene known to encode for amyloid precursor protein (APP), a major component of the Alzheimer's phenotype. Cortical neurons generated from these iPS lines expressed amyloid aggregates and hyperphosphorylated tau protein, both hallmarks of the

Alzheimer's disease phenotype, after months in culture [56]. Utilizing a similar approach, it may be possible to emulate other age related disease phenotypes through variable gene expression, providing a second avenue from which to approach the issue. iPS line have also been derived from Huntington's patients, in which differentiated neurons main‐ tained some portions of the Huntington's phenotype [57, 58]. CDKL5 mutant iPS lines have also been generated from Rett syndrome patients, and may allow for investigation of CDKL5's underlying mechanism within patient cells [59]. Amyotrophic lateral sclerosis (ALS) has also been effectively modeled using an iPS line derived from familial ALS patients [60].

Once generated, these disease models can provide insight into the underlying mechanisms of the disease. *In vitro* research of molecular level cellular mechanisms is much cheaper and more efficient than similar research in mammal models, potentially allowing for increased research throughput. Established *in vitro* models also remove confounding factors related to animal models, potentially making direct identification of mechanisms easier. In the context of phenotypic identification and the discovery of underlying mechanisms, it is important to consider the controls necessary for using these iPS derived disease models. Due to potential phenotypic differences in iPS clones, even from the same isolation, it would be necessary to generate models using multiple iPS lines from each patient in a diverse group. This spread would allow for adequate confirmation that the identified phenotype or mechanism is indeed consistent for all patients with the disease, rather than an artifact of reprogramming or a trait specific to a single individual [53].

While these results in summary are very promising, substantial challenges remain before iPS cell cultures can be used as disease models in every instance. Although diseases with limited temporal dependency, such as spinal muscular atrophy, and clear monogenic origin, such as Huntington's, are replicated relatively easily *in vitro*, there remain unsolved problems in replicating diseases influenced by multiple factors. As demonstrated in attempts to replicate the Parkinson's phenotype *in vitro*, time related factors can also play a large role in disease phenotype, complicating modeling. Other diseases, such as Alzheimer's, may be dependent upon cellular interactions between multiple cell phenotypes in addition to age related factors. The homogenous nature of iPS derived cell cultures complicates accurate replication of these interactions *in vitro*. In some cases, it may be possible to model some of these cell to cell interactions using coculture, as demonstrated in ALS models that incorporate both astrocytes and neurons [61]. Further research is needed to overcome these barriers before iPS cell based disease modeling can be exploited to its full potential.

#### **7.2. Drug discovery**

**6.6. Blood supply**

274 Pluripotent Stem Cells

**7. Research applications**

their use as a disease model.

**7.1. Disease modeling**

Blood supply shortages are an ever-present concern in many regions, leading to demand for additional sources of red blood cells (RBCs). iPS cells could theoretically be used to generate RBCs as a supplemental source and it has been demonstrated that iPS cells are capable of direct erythrocytic differentiation [52]. Although the technology exists, the use of iPS cells to generate RBCs may not always been practical due to the cost of iPS generation, culture, and subsequent differentiation. As such, until technologies are developed which allow for industrial scale iPS cell culture and differentiation, the use of iPS cells to augment the blood supply will be fairly limited. In certain circumstances, such as a patient in need of a rare blood type in advance of

Accurate disease modeling is a biotechnological problem of fundamental importance. Most current disease models rely upon murine model organisms, which are capable of providing insight, but are less than ideal due to interspecies differences [53]. iPS technologies could allow for *in vitro* disease modeling, using cultures isolated from those suffering with a given condition. If widely applied, patient specific iPS cultures could potentially be created to analyze the nuances of a disease in a particular patient, determining which course of treatment would be best. Using skin fibroblasts isolated from a patient with spinal muscular atrophy, Ebert et. al. demonstrated that iPS derived motor neurons could be effectively grown in culture and maintained the disease phenotype of the patient [54]. These findings indicate that iPS cells derived from patients with genetic disorders may exhibit the disease phenotype, allowing for

Similar isolations have also occurred with Parkinson's patients, in which iPS clones were generated from patients and subsequently differentiated into dopamanergic neurons. However, in the context of Parkinson's disease, the disease phenotype was not as readily presented *in vitro* due to the relative age of the neurons. While cultured neurons have a lifespan in weeks, Parkinson's develops over a period of years due in conjunction with age related factors, possibly requiring a form of artificial stress treatment to accurately reproduce the phenotype *in vitro* [14]. However, early stage metabolic dysfunction has already been identified and corrected *in vitro* using neurons generated from familial Parkinson's patient derived iPS cells, indicating that some early stage phenotypes may be identifiable without full phenotypic replication [55]. Alzheimer's disease, like Parkinson's, is strongly influenced by a number of age related factor which complicate the creation of an accurate model. Recently, Shi et. al. demonstrated one potential approach to this problem by using iPS cells derived from Down syndrome patients. Down syndrome patients overexpress a gene known to encode for amyloid precursor protein (APP), a major component of the Alzheimer's phenotype. Cortical neurons generated from these iPS lines expressed amyloid aggregates and hyperphosphorylated tau protein, both hallmarks of the

surgery, generation of iPS derived RBCs may be a viable option for treatment.

As a corollary to disease modeling, drug discovery is a promising research application for iPS cells. Developing new drugs is exceedingly expensive and many drug candidates are rejected in the final human trial stage due to toxicology concerns [46]. At present, 90% of all drugs candidates that enter clinical trials fail to be approved, leading to a low drug candidate to successful drug ratio [62]. If drugs could be screened for human toxicology earlier in the development cycle, a number of these candidates could be eliminated earlier, allowing for increased funding to more promising drugs. This redistribution of funding could eventually lead to more drug candidates developed in a more rapid fashion.

To assess for toxicology, iPS clones could be generated from a broad cross-section of potential patients, representing various patient backgrounds. Due to the immortalized nature of iPS cultures, these cells could be expanded and maintained indefinitely at relatively low expense to drug developers. As a consequence of effective cell storage technologies, an iPS clone bank would only expand overtime, allowing for the aggregation of clones generated during multiple studies. From these clones, tissues could be generated for toxicology testing early in the development cycle, potentially identifying toxic drug candidates before further testing takes place (Figure 2). In this manner, a diverse donor population could effectively provide each type of human tissue with a relatively small amount of tissue collection.

Using iPS disease models as described above, the effectiveness of new drug therapies could also be tested *in vitro.* The overall cost of testing using these *in vitro* models is less than that of animal modeling, and could allow large scale screening of potential drug candidates early in the development cycle. Due to the elimination of certain confounding factors present in animal models, drug testing in iPS derived disease models may also yield unique insights not demonstrated using traditional models. The iPS clone bank described above could be expand‐ ed to include similarly diverse clone populations from patients with a specific disease. Similar to its benefits in toxicology testing, an iPS clone bank could allow for testing on a broad crosssection of disease patients at a relatively low cost. Recently, studies have utilized iPS disease models to assess the efficacy of Alzheimer's disease drug candidates *in vitro* [56] and to successfully screen for new drugs to potentially treat ALS [60]. These studies demonstrate the potential for the use of iPS cells in the context of drug development, both to improve the efficiency of existing drug development pipelines and to screen for entirely new compounds in a relatively low cost model.

However, drug discovery and toxicology screening using iPS cells is limited by their ability to accurately replicate *in vivo* conditions. As discussed above, the homogenous nature of iPS cultures neglects many influential factors related to cellular interaction, and the temporally naive nature of iPS cultures neglects many age related factors. As such, further research is necessary before iPS derived tissues are suitable for use in toxicology testing. For drug therapy screening, the current state of disease models as discussed above is a limiting factor. Although not all diseases can be effectively modeled for screening today, some disorders that have well characterized iPS models may benefit from broad drug screening in the near future.

Efficiency remains an issue, especially with regards to technologies that do not integrate transgenes into the host genome. To address efficiency concerns, it is possible that application specific optimization of induction technologies could improve the efficacy of current induction

**Figure 2.** Drug discovery and toxicology workflow. Somatic cells are isolated from a broad cross section of donors,

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reprogrammed, and differentiated into relevant tissues for toxicology screening and drug testing

As disease models, iPS cells are limited by the neglect of several influential factors. Most prominently, the homogenous populations derived from iPS cells inherently neglect interac‐ tions between multiple cell phenotypes, and these interactions may be critical to understanding disease mechanisms [6]. iPS cells could potentially be differentiated into various cell types and cocultured to replicate interactions between cell types, but it may be difficult to generate an

technologies.

### **8. Conclusion: Challenges to the road ahead**

A number of roadblocks remain before iPS cells are ready for the clinic. At present, there still remains a risk of teratoma formation in the event that a subpopulation of iPS cells is not terminally differentiated prior to transplantation. In the context of a patient-specific autolo‐ gous treatment using iPS cells, methods must be developed by which iPS cells can be generated in sufficient quantity, reliably, and in a time frame appropriate for the targeted disease.

increased funding to more promising drugs. This redistribution of funding could eventually

To assess for toxicology, iPS clones could be generated from a broad cross-section of potential patients, representing various patient backgrounds. Due to the immortalized nature of iPS cultures, these cells could be expanded and maintained indefinitely at relatively low expense to drug developers. As a consequence of effective cell storage technologies, an iPS clone bank would only expand overtime, allowing for the aggregation of clones generated during multiple studies. From these clones, tissues could be generated for toxicology testing early in the development cycle, potentially identifying toxic drug candidates before further testing takes place (Figure 2). In this manner, a diverse donor population could effectively provide each

Using iPS disease models as described above, the effectiveness of new drug therapies could also be tested *in vitro.* The overall cost of testing using these *in vitro* models is less than that of animal modeling, and could allow large scale screening of potential drug candidates early in the development cycle. Due to the elimination of certain confounding factors present in animal models, drug testing in iPS derived disease models may also yield unique insights not demonstrated using traditional models. The iPS clone bank described above could be expand‐ ed to include similarly diverse clone populations from patients with a specific disease. Similar to its benefits in toxicology testing, an iPS clone bank could allow for testing on a broad crosssection of disease patients at a relatively low cost. Recently, studies have utilized iPS disease models to assess the efficacy of Alzheimer's disease drug candidates *in vitro* [56] and to successfully screen for new drugs to potentially treat ALS [60]. These studies demonstrate the potential for the use of iPS cells in the context of drug development, both to improve the efficiency of existing drug development pipelines and to screen for entirely new compounds

However, drug discovery and toxicology screening using iPS cells is limited by their ability to accurately replicate *in vivo* conditions. As discussed above, the homogenous nature of iPS cultures neglects many influential factors related to cellular interaction, and the temporally naive nature of iPS cultures neglects many age related factors. As such, further research is necessary before iPS derived tissues are suitable for use in toxicology testing. For drug therapy screening, the current state of disease models as discussed above is a limiting factor. Although not all diseases can be effectively modeled for screening today, some disorders that have well

A number of roadblocks remain before iPS cells are ready for the clinic. At present, there still remains a risk of teratoma formation in the event that a subpopulation of iPS cells is not terminally differentiated prior to transplantation. In the context of a patient-specific autolo‐ gous treatment using iPS cells, methods must be developed by which iPS cells can be generated in sufficient quantity, reliably, and in a time frame appropriate for the targeted disease.

characterized iPS models may benefit from broad drug screening in the near future.

**8. Conclusion: Challenges to the road ahead**

lead to more drug candidates developed in a more rapid fashion.

type of human tissue with a relatively small amount of tissue collection.

in a relatively low cost model.

276 Pluripotent Stem Cells

**Figure 2.** Drug discovery and toxicology workflow. Somatic cells are isolated from a broad cross section of donors, reprogrammed, and differentiated into relevant tissues for toxicology screening and drug testing

Efficiency remains an issue, especially with regards to technologies that do not integrate transgenes into the host genome. To address efficiency concerns, it is possible that application specific optimization of induction technologies could improve the efficacy of current induction technologies.

As disease models, iPS cells are limited by the neglect of several influential factors. Most prominently, the homogenous populations derived from iPS cells inherently neglect interac‐ tions between multiple cell phenotypes, and these interactions may be critical to understanding disease mechanisms [6]. iPS cells could potentially be differentiated into various cell types and cocultured to replicate interactions between cell types, but it may be difficult to generate an accurate interaction model, even with multiple cell types. iPS cell cultures also neglect various age related factors, which may be particularly problematic in modeling certain diseases. These same issues act as barriers to the use of iPS cells for drug discovery and toxicology screening, as both applications rely upon accurate iPS models of *in vivo* cellular activity.

[8] Liao, J., et al., *Enhanced efficiency of generating induced pluripotent stem (iPS) cells from human somatic cells by a combination of six transcription factors.* Cell Research, 2008. 18:

Induced Pluripotent Stem Cells: Current and Emerging Technologies

http://dx.doi.org/10.5772/55101

279

[9] Kim, J.B., et al., *Oct4-Induced Pluripotency in Adult Neural Stem Cells.* Cell, 2009. 136(3):

[10] Shi, Y., et al., *Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds.* Cell Stem Cell, 2008. 3(5): p. 568-74.

[11] Sugaya, K. and A. Alvarez, *Methods and materials for increasing potency of cells* U.P.a.T.

[12] Alvarez, A., et al., *Nanog overexpression allows human mesenchymal stem cells to differen‐ tiate into neural cells——Nanog transdifferentiates mesenchymal stem cells.* Neuroscience

[13] Hacein-Bey-Abina, S., et al., *LMO2-associated clonal T cell proliferation in two patients af‐*

[14] Soldner, F., et al., *Parkinson's Disease Patient-Derived Induced Pluripotent Stem Cells Free*

[15] Okita, K., et al., *Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vec‐*

[16] Yamanaka, S., *Induced Pluripotent Stem Cells: Past, Present, and Future.* Cell Stem Cell,

[17] Yu, J., et al., *Human induced pluripotent stem cells free of vector and transgene sequences.*

[18] Okita, K. and S. Yamanaka, *Induced pluripotent stem cells: opportunities and challenges.*

[19] Armeanu, S., et al., *Cell cycle independent infection and gene transfer by recombinant Sen‐*

[20] Bernloehr, C., et al., *Efficient propagation of single gene deleted recombinant Sendai virus*

[21] Tokusumi, T., et al., *Recombinant Sendai viruses expressing different levels of a foreign re‐*

[22] Fusaki, N., et al., *Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome.* Proceedings of the Japan Academy, Series B. Physical and Biological Sciences, 2009.

*ter gene therapy for SCID-X1.* Science, 2003. 302(5644): p. 415-9.

*of Viral Reprogramming Factors.* Cell, 2009. 136(5): p. 964-977.

Philos Trans R Soc Lond B Biol Sci, 2011. 366(1575): p. 2198-207.

*dai viruses.* Journal of Virological Methods, 2003. 108(2): p. 229-233.

*tors.* Science (Washington), 2008. 322(5903): p. 949-953.

p. 600-603.

p. 411-419.

Office, Editor 2006: USA.

and Medicine, 2010: p. 1-13.

2012. 10(6): p. 678-684.

85B(8): p. 348-362.

Science, 2009. 324(5928): p. 797-801.

*vectors.* Virus Research, 2004. 99(2): p. 193-197.

*porter gene.* Virus Research, 2002. 86(1–2): p. 33-38.

Induced pluripotent stem cell technologies have progressed rapidly in recent years. Various induction methods have eliminated or reduced many of the fundamental issues with iPS cells, opening the door to a variety of possible applications. Though there remain a number of challenges facing the development of iPS cells in the clinic and the laboratory, the potential benefits to regenerative medicine are profound.

### **Author details**

Jacob Kimmel and Kiminobu Sugaya

\*Address all correspondence to: kiminobu.sugaya@ucf.edu

University of Central Florida, Orlando, FL, USA

#### **References**


[8] Liao, J., et al., *Enhanced efficiency of generating induced pluripotent stem (iPS) cells from human somatic cells by a combination of six transcription factors.* Cell Research, 2008. 18: p. 600-603.

accurate interaction model, even with multiple cell types. iPS cell cultures also neglect various age related factors, which may be particularly problematic in modeling certain diseases. These same issues act as barriers to the use of iPS cells for drug discovery and toxicology screening,

Induced pluripotent stem cell technologies have progressed rapidly in recent years. Various induction methods have eliminated or reduced many of the fundamental issues with iPS cells, opening the door to a variety of possible applications. Though there remain a number of challenges facing the development of iPS cells in the clinic and the laboratory, the potential

[1] Yamanaka, S. and K. Takahashi, *Induction of Pluripotent Stem Cells from Mouse Embry‐ onic and Adult Fibroblast Cultures by Defined Factors.* Cell, 2006. 126(4): p. 663-676.

[2] Avilion, A.A., et al., *Multipotent cell lineages in early mouse development depend on SOX2*

[3] Hong, Y., et al., *Activation of the mouse Oct4 promoter in medaka embryonic stem cells and its use for ablation of spontaneous differentiation.* Mechanisms of Development, 2004.

[4] Cartwright, P.P., et al., *LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism.* Development (Cambridge, England), 2005. 132(5): p.

[5] Shi, Y., et al., *A Combined Chemical and Genetic Approach for the Generation of Induced*

[7] Junying Yu, et al., *Induced Pluripotent Stem Cell Lines Derived from Human Somatic*

as both applications rely upon accurate iPS models of *in vivo* cellular activity.

benefits to regenerative medicine are profound.

University of Central Florida, Orlando, FL, USA

\*Address all correspondence to: kiminobu.sugaya@ucf.edu

*function.* Genes & Development, 2003. 17(1): p. 126-140.

*Pluripotent Stem Cells.* Cell Stem Cell, 2008. 2(6): p. 525-528.

[6] Yamanaka, S., *A fresh look at iPS cells.* Cell, 2009. 137(1): p. 13-7.

*Cells.* Science Express, 2007. 10: p. 1-4.

Jacob Kimmel and Kiminobu Sugaya

121(7–8): p. 933-943.

885-896.

**Author details**

278 Pluripotent Stem Cells

**References**


[23] Warren, L., et al., *Highly Efficient Reprogramming to Pluripotency and Directed Differen‐ tiation of Human Cells with Synthetic Modified mRNA.* Cell Stem Cell, 2010. 7(5): p. 618-630.

[37] Nori, S., et al., *Transplantation of human iPS cell-derived neurospheres for the treatment of spinal cord injury in NOD-scid mice.* Neuroscience Research, 2010. 68, Supplement 1(0):

Induced Pluripotent Stem Cells: Current and Emerging Technologies

http://dx.doi.org/10.5772/55101

281

[38] Uemura, T., et al., *Transplantation of induced pluripotent stem cell-derived neurospheres for peripheral nerve repair.* Biochemical and Biophysical Research Communications,

[39] Gomi, M., et al., *Functional recovery of the murine brain ischemia model using human in‐ duced pluripotent stem cell-derived telencephalic progenitors.* Brain Research, 2012.

[40] Jensen, M.B., et al., *Survival and Differentiation of Transplanted Neural Stem Cells De‐ rived from Human Induced Pluripotent Stem Cells in A Rat Stroke Model.* Journal of

[41] Cooper, O., et al., *Differentiation of human ES and Parkinson's disease iPS cells into ven‐ tral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specif‐ ic regionalization by retinoic acid.* Molecular and Cellular Neuroscience, 2010. 45(3): p.

[42] Swistowski, A., et al., *Efficient generation of functional dopaminergic neurons from human induced pluripotent stem cells under defined conditions.* Stem Cells, 2010. 28(10): p.

[43] Wernig, M., et al., *Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease.* Proceedings of

[44] Kiminobu Sugaya, S.M., *How to approach alzheimer's disease with stem cell technologies.*

[45] Taha, M.F. and V. Hedayati, *Isolation, identification and multipotential differentiation of*

[46] Nsair, A. and W.R. MacLellan, *Induced pluripotent stem cells for regenerative cardiovas‐ cular therapies and biomedical discovery.* Advanced Drug Delivery Reviews, 2011. 63(4–

[47] Yoshida, Y. and S. Yamanaka, *iPS cells: A source of cardiac regeneration.* Journal of Mo‐

[48] Galli, D., et al., *Mesoangioblasts, vessel-associated multipotent stem cells, repair the infarct‐ ed heart by multiple cellular mechanisms: a comparison with bone marrow progenitors, fibro‐*

[49] Christoforou, N.N., et al., *Implantation of mouse embryonic stem cell-derived cardiac pro‐ genitor cells preserves function of infarcted murine hearts.* PLoS ONE, 2010. 5(7): p.

*blasts, and endothelial cells.* Arterioscler Thromb Vasc Biol, 2005. 25(4): p. 692-7.

the National Academy of Sciences, USA, 2008. 105(15): p. 5856-5861.

*mouse adipose tissue-derived stem cells.* Tissue Cell, 2010. 42(4): p. 211-6.

Journal of Alzheimer's Disease, 2008. 15: p. 241–254.

lecular and Cellular Cardiology, 2011. 50(2): p. 327-332.

p. e427.

2012. 419(1): p. 130-135.

Stroke and Cerebrovascular Diseases, (0).

1459(0): p. 52-60.

258-266.

1893-904.

5): p. 324-330.

e11536-e11536.


[37] Nori, S., et al., *Transplantation of human iPS cell-derived neurospheres for the treatment of spinal cord injury in NOD-scid mice.* Neuroscience Research, 2010. 68, Supplement 1(0): p. e427.

[23] Warren, L., et al., *Highly Efficient Reprogramming to Pluripotency and Directed Differen‐ tiation of Human Cells with Synthetic Modified mRNA.* Cell Stem Cell, 2010. 7(5): p.

[24] Rosa, A. and A.H. Brivanlou, *Synthetic mRNAs: Powerful Tools for Reprogramming and*

[25] Zhao, T.T., et al., *Immunogenicity of induced pluripotent stem cells.* Nature, 2011.

[26] Kim, D., et al., *Generation of Human Induced Pluripotent Stem Cells by Direct Delivery of*

[27] Shimada, H., et al., *Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia.* Biochemical

[28] Yoshida, Y., et al., *Hypoxia enhances the generation of induced pluripotent stem cells.* Cell

[29] Lyssiotis, C.A., et al., *Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4.* Proc Natl Acad Sci U S A, 2009. 106(22): p.

[30] Huangfu, D., et al., *Induction of pluripotent stem cells by defined factors is greatly im‐ proved by small-molecule compounds.* Nat Biotechnol, 2008. 26(7): p. 795-797.

[31] Hanna, J., et al., *Direct cell reprogramming is a stochastic process amenable to acceleration.*

[32] Loh, Y.H., et al., *The Oct4 and Nanog transcription network regulates pluripotency in*

[33] Papapetrou, E.P., et al., *Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation.* Proceedings of

[34] Bar-Nur, O., et al., *Epigenetic Memory and Preferential Lineage-Specific Differentiation in Induced Pluripotent Stem Cells Derived from Human Pancreatic Islet Beta Cells.* Cell Stem

[35] Hu, Q.Q., et al., *Memory in induced pluripotent stem cells: reprogrammed human retinalpigmented epithelial cells show tendency for spontaneous redifferentiation.* Stem cells (Day‐

[36] Kim, K.K., et al., *Epigenetic memory in induced pluripotent stem cells.* Nature, 2010.

the National Academy of Sciences, USA, 2009. 106(31): p. 12759-12764.

*mouse embryonic stem cells.* Nat Genet, 2006. 38(4): p. 431-40.

*Differentiation of Human Cells.* Cell Stem Cell, 2010. 7(5): p. 549-550.

*Reprogramming Proteins.* Cell Stem Cell, 2009. 4(6): p. 472-476.

and Biophysical Research Communications, 2012. 417(2): p. 659-664.

618-630.

280 Pluripotent Stem Cells

8912-7.

474(7350): p. 212-215.

Stem Cell, 2009. 5(3): p. 237-41.

Nature, 2009. 462(7273): p. 595-601.

Cell, 2011. 9(1): p. 17-23.

467(7313): p. 285-290.

ton, Ohio), 2010. 28(11): p. 1981-1991.


[50] Liras, A., *Induced human pluripotent stem cells and advanced therapies: Future perspectives for the treatment of haemophilia?* Thrombosis Research, 2011. 128(1): p. 8-13.

**Chapter 13**

**Molecular Mechanisms of Embryonic Stem Cell**

Embryonic stem (ES) cells isolated from the inner cell mass (ICM) of blastocysts possess the defining pluroptency: unlimited self-renewal and giving rise to all cells of the organ‐ ism[1, 2]. Thus, ES cells hold great promise for regenerative medicine to treat many dis‐ eases including heart failure, diabetes, Alzheimer's and Parkinson's disease by replacing the damaged cells with ES cell-derived healthy ones. The recent advent of induced pluri‐ potent stem (iPS) cells reprogrammed from somatic cells has the potential to revolution‐ ize the field of regenerative medicine since patient-derived iPS cells, in principle, circumvent the ethical problems and immune rejection associated with human ES cells[3]. Nevertheless, the future clinical translation of ES cells and iPS cells is facing nu‐ merous hurdles. Understanding the molecular mechanisms that impart ES cells with plu‐ ripotency may help address some of these challenges. The past few years have seen tremendous progress in understanding of mechanisms which govern ES cell pluripoten‐ cy. In this chapter, we will review critical signaling and transcription factor networks

ES cells require extrinsic growth factors to maintain their pluripotency in culture. These extrinsic growth factors act on different signaling pathways to regulate intrinsic tran‐ scription factor networks to sustain ES cells in the undifferentiated state. The signaling pathways required to support pluripotency in mouse ES cell are distinct from those in

> © 2013 Han et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Han et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

Guofeng Han, Hongtao Wang and Jijun Hao

Additional information is available at the end of the chapter

that have been identified to maintain ES cell pluripotency.

**2. Signaling pathways of ES cells**

human ES cells (Figure 1).

**Pluripotency**

http://dx.doi.org/10.5772/54365

**1. Introduction**


## **Molecular Mechanisms of Embryonic Stem Cell Pluripotency**

Guofeng Han, Hongtao Wang and Jijun Hao

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54365

## **1. Introduction**

[50] Liras, A., *Induced human pluripotent stem cells and advanced therapies: Future perspectives for the treatment of haemophilia?* Thrombosis Research, 2011. 128(1): p. 8-13.

[51] Xu, D.D., et al., *Phenotypic correction of murine hemophilia A using an iPS cell-based thera‐ py.* Proceedings of the National Academy of Sciences of the United States of America,

[52] Peyrard, T., et al., *Banking of Pluripotent Adult Stem Cells as an Unlimited Source for Red Blood Cell Production: Potential Applications for Alloimmunized Patients and Rare Blood*

[53] Saha, K. and R. Jaenisch, *Technical Challenges in Using Human Induced Pluripotent Stem*

[54] Ebert, A.D., et al., *Induced pluripotent stem cells from a spinal muscular atrophy patient.*

[55] Cooper, O., et al., *Pharmacological Rescue of Mitochondrial Deficits in iPSC-Derived Neu‐ ral Cells from Patients with Familial Parkinson's Disease.* Science Translational Medicine,

[56] Shi, Y., et al., *A Human Stem Cell Model of Early Alzheimer's Disease Pathology in Down*

[57] Castiglioni, V., et al., *Induced pluripotent stem cell lines from Huntington's disease mice undergo neuronal differentiation while showing alterations in the lysosomal pathway.* Neu‐

[58] Camnasio, S., et al., *The first reported generation of several induced pluripotent stem cell lines from homozygous and heterozygous Huntington's disease patients demonstrates muta‐ tion related enhanced lysosomal activity.* Neurobiology of Disease, 2012. 46(1): p. 41-51.

[59] Amenduni, M., et al., *iPS cells to model CDKL5-related disorders.* Eur J Hum Genet,

[60] Egawa, N., et al., *Drug Screening for ALS Using Patient-Specific Induced Pluripotent Stem*

[61] Di Giorgio, F.P., et al., *Non-cell autonomous effect of glia on motor neurons in an embryon‐*

[62] Rubin, L.L., *Stem Cells and Drug Discovery: The Beginning of a New Era?* Cell, 2008.

*Cells.* Science Translational Medicine, 2012. 4(145): p. 145ra104.

*ic stem cell-based ALS model.* Nat Neurosci, 2007. 10(5): p. 608-614.

*Challenges.* Transfusion Medicine Reviews, 2011. 25(3): p. 206-216.

*Syndrome.* Science Translational Medicine, 2012. 4(124): p. 124ra29.

*Cells to Model Disease.* Cell Stem Cell, 2009. 5(6): p. 584-595.

2009. 106(3): p. 808-813.

282 Pluripotent Stem Cells

2012. 4(141): p. Article.

2011. 19(12): p. 1246-55.

132(4): p. 549-552.

Nature, 2009. 457(7227): p. 277-280.

robiology of Disease, 2012. 46(1): p. 30-40.

Embryonic stem (ES) cells isolated from the inner cell mass (ICM) of blastocysts possess the defining pluroptency: unlimited self-renewal and giving rise to all cells of the organ‐ ism[1, 2]. Thus, ES cells hold great promise for regenerative medicine to treat many dis‐ eases including heart failure, diabetes, Alzheimer's and Parkinson's disease by replacing the damaged cells with ES cell-derived healthy ones. The recent advent of induced pluri‐ potent stem (iPS) cells reprogrammed from somatic cells has the potential to revolution‐ ize the field of regenerative medicine since patient-derived iPS cells, in principle, circumvent the ethical problems and immune rejection associated with human ES cells[3]. Nevertheless, the future clinical translation of ES cells and iPS cells is facing nu‐ merous hurdles. Understanding the molecular mechanisms that impart ES cells with plu‐ ripotency may help address some of these challenges. The past few years have seen tremendous progress in understanding of mechanisms which govern ES cell pluripoten‐ cy. In this chapter, we will review critical signaling and transcription factor networks that have been identified to maintain ES cell pluripotency.

## **2. Signaling pathways of ES cells**

ES cells require extrinsic growth factors to maintain their pluripotency in culture. These extrinsic growth factors act on different signaling pathways to regulate intrinsic tran‐ scription factor networks to sustain ES cells in the undifferentiated state. The signaling pathways required to support pluripotency in mouse ES cell are distinct from those in human ES cells (Figure 1).

enriched in ES cells were downregulated in differentiated cells by mapping STAT3 binding targets in mouse ES cells and differentiated embryoid bodies (EBs)[13]. Along with these re‐ sults, it has been demonstrated that knocking down STAT3-target genes induces activation of endodermal and mesodermal genes, supporting the conclusion that STAT3 prevents

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http://dx.doi.org/10.5772/54365

285

Interestingly, the LIF receptor and gp130 are also expressed in human ES cells and hu‐ man LIF can induce STAT3 phosphorylation and nuclear translocation in human ES cells. However, human LIF is unable to maintain the pluripotent state of human ESs, suggest‐ ing that mouse and human ES cells require distinct signaling mechanisms to govern

TGF-β superfamily consists of more than 40 members, including TGF-β, Activin, Nodal, and bone morphogenetic proteins (BMPs). The TGF-β members transduce signals by binding to heteromeric complexes of serine/threonine kinase receptors, type I and type II receptors, which subsequently activate intracellular Smad proteins. Smads 2 and 3 are specifically acti‐ vated by activin, nodal and TGF-β ligands, whereas Smads 1, 5 and 8 are activated by BMP ligands[16, 17] (Figure 1). The TGF-β-related signaling pathways play complex roles in regu‐

Bone Morphogenetic Protein (BMP) is a subset of the TGF-β superfamily[18]. When BMP li‐ gands bind to type II BMP receptors (BMPRII), BMPRII then recruits and phosphorylates type I BMP receptors (BMPRI). Activated type I receptors subsequently phosphorylate BMPresponsive SMAD1/5/8 which then forms a complex with SMAD4 and translocates into nu‐ cleus to regulate target gene expression (Figure 1). In mouse ES cells, LIF can substitute MEF feeder layers in maintaining pluripotency in the presence of animal serum by activating the transcription factor STAT3. However, in serum-free cultures, LIF is insufficient to block neu‐ ral differentiation and maintain pluripotency. Recently, Ying et al reported that BMP was able to replace serum to maintain pluripotency of mouse ES cells in the presence of LIF. BMP has been shown to phosphorylate SMAD1/5 and activate inhibitors of differentiation (*Id*) genes, which block neural differentiation by antagonizing neurogenic transcription fac‐ tors[19]. In the absence of MEF and serum, exogenous LIF, in combination with BMP4 pro‐ teins, can sufficiently maintain the pluripotency of mouse ES cells derived from

In contrast to a maintenance role in mouse ES cell pluripotency, BMP has been shown to promote human ES cells differentiation to trophoblasts, and inhibiting BMP signaling with the BMP antagonist, Noggin, sustains the undifferentiated state of human ES cells[20, 21]. In consistence, dorsomorphin and DMH1, small molecule BMP inhibitors previously identified in our lab, were shown to promote long-term self-renewal an pluripotency of human ES cells, presumably by inhibiting BMP induced extraembryonic lineage differentiation[22-25].

mESC differentiation by suppressing lineage-specific genes[14].

their pluripotency[15].

**2.2. TGF-β signaling**

*2.2.1. BMP signaling pathway*

"permissive" mouse strains.

lating the pluripotency and cell fate of ES cells.

**Figure 1.** Exogenous growth factors signal through distinct signaling pathways to regulate transcription factors for ES cell pluripotency.

#### **2.1. LIF/JAK/STAT3 pathway**

Mouse ES cells were originally cultured on feeder layers derived from mouse embryonic fi‐ broblasts (MEF). Later it was found that Leukaemia Inhibitory Factor (LIF), a member of the Interleukin-6 cytokines produced by MEFs, was the key factor to maintain pluripotency of mouse ES cells by inhibiting their differentiation[4]. Upon LIF binding, the LIF receptor re‐ cruits gp130 to form a heterodimer which subsequently activates Janus kinase (JAK) through transphosphorylation[5]. Activated JAK then phosphorylate gp130, creating a dock‐ ing site to bind the SH2 domain of Signal Transducers and Activators of Transcription 3 (STAT3)[6-9]. Once STAT3 binds to the gp130 docking site, JAK then phosphorylates the re‐ cruited STAT3. Phosphorylated STAT3 forms a homodimer, which subsequently translocate into the nucleus, where it binds to gene enhancers to regulate target gene expression[10-12].

Although the LIF/JAK/STAT3 pathway has been well documented to maintain pluripotency of mouse ES cells in the presence of serum, the mechanisms by which activated STAT3 func‐ tions in this regard is poorly understood. Recently, studies in identification of STAT3 target genes have improved our understanding of activated STAT3 in maintaining pluripotency. Chen et al identified 718 STAT3-bound genomic sites that were co-occupied by pluripotency transcription markers (Oct4, Sox2 and Nanog) by using chromatin immunoprecipitation se‐ quencing (ChIP-seq)[12]. In addition, Kidder and colleagues found that STAT3 target genes enriched in ES cells were downregulated in differentiated cells by mapping STAT3 binding targets in mouse ES cells and differentiated embryoid bodies (EBs)[13]. Along with these re‐ sults, it has been demonstrated that knocking down STAT3-target genes induces activation of endodermal and mesodermal genes, supporting the conclusion that STAT3 prevents mESC differentiation by suppressing lineage-specific genes[14].

Interestingly, the LIF receptor and gp130 are also expressed in human ES cells and hu‐ man LIF can induce STAT3 phosphorylation and nuclear translocation in human ES cells. However, human LIF is unable to maintain the pluripotent state of human ESs, suggest‐ ing that mouse and human ES cells require distinct signaling mechanisms to govern their pluripotency[15].

#### **2.2. TGF-β signaling**

TGF-β superfamily consists of more than 40 members, including TGF-β, Activin, Nodal, and bone morphogenetic proteins (BMPs). The TGF-β members transduce signals by binding to heteromeric complexes of serine/threonine kinase receptors, type I and type II receptors, which subsequently activate intracellular Smad proteins. Smads 2 and 3 are specifically acti‐ vated by activin, nodal and TGF-β ligands, whereas Smads 1, 5 and 8 are activated by BMP ligands[16, 17] (Figure 1). The TGF-β-related signaling pathways play complex roles in regu‐ lating the pluripotency and cell fate of ES cells.

#### *2.2.1. BMP signaling pathway*

**Figure 1.** Exogenous growth factors signal through distinct signaling pathways to regulate transcription factors for ES

Mouse ES cells were originally cultured on feeder layers derived from mouse embryonic fi‐ broblasts (MEF). Later it was found that Leukaemia Inhibitory Factor (LIF), a member of the Interleukin-6 cytokines produced by MEFs, was the key factor to maintain pluripotency of mouse ES cells by inhibiting their differentiation[4]. Upon LIF binding, the LIF receptor re‐ cruits gp130 to form a heterodimer which subsequently activates Janus kinase (JAK) through transphosphorylation[5]. Activated JAK then phosphorylate gp130, creating a dock‐ ing site to bind the SH2 domain of Signal Transducers and Activators of Transcription 3 (STAT3)[6-9]. Once STAT3 binds to the gp130 docking site, JAK then phosphorylates the re‐ cruited STAT3. Phosphorylated STAT3 forms a homodimer, which subsequently translocate into the nucleus, where it binds to gene enhancers to regulate target gene expression[10-12].

Although the LIF/JAK/STAT3 pathway has been well documented to maintain pluripotency of mouse ES cells in the presence of serum, the mechanisms by which activated STAT3 func‐ tions in this regard is poorly understood. Recently, studies in identification of STAT3 target genes have improved our understanding of activated STAT3 in maintaining pluripotency. Chen et al identified 718 STAT3-bound genomic sites that were co-occupied by pluripotency transcription markers (Oct4, Sox2 and Nanog) by using chromatin immunoprecipitation se‐ quencing (ChIP-seq)[12]. In addition, Kidder and colleagues found that STAT3 target genes

cell pluripotency.

284 Pluripotent Stem Cells

**2.1. LIF/JAK/STAT3 pathway**

Bone Morphogenetic Protein (BMP) is a subset of the TGF-β superfamily[18]. When BMP li‐ gands bind to type II BMP receptors (BMPRII), BMPRII then recruits and phosphorylates type I BMP receptors (BMPRI). Activated type I receptors subsequently phosphorylate BMPresponsive SMAD1/5/8 which then forms a complex with SMAD4 and translocates into nu‐ cleus to regulate target gene expression (Figure 1). In mouse ES cells, LIF can substitute MEF feeder layers in maintaining pluripotency in the presence of animal serum by activating the transcription factor STAT3. However, in serum-free cultures, LIF is insufficient to block neu‐ ral differentiation and maintain pluripotency. Recently, Ying et al reported that BMP was able to replace serum to maintain pluripotency of mouse ES cells in the presence of LIF. BMP has been shown to phosphorylate SMAD1/5 and activate inhibitors of differentiation (*Id*) genes, which block neural differentiation by antagonizing neurogenic transcription fac‐ tors[19]. In the absence of MEF and serum, exogenous LIF, in combination with BMP4 pro‐ teins, can sufficiently maintain the pluripotency of mouse ES cells derived from "permissive" mouse strains.

In contrast to a maintenance role in mouse ES cell pluripotency, BMP has been shown to promote human ES cells differentiation to trophoblasts, and inhibiting BMP signaling with the BMP antagonist, Noggin, sustains the undifferentiated state of human ES cells[20, 21]. In consistence, dorsomorphin and DMH1, small molecule BMP inhibitors previously identified in our lab, were shown to promote long-term self-renewal an pluripotency of human ES cells, presumably by inhibiting BMP induced extraembryonic lineage differentiation[22-25].

#### *2.2.2. TGF-β/activin/nodal signaling pathway*

Although MEFs feeder layers were initially used to co-culture both mouse and human ES cells, signal factors secreted from MEFs to maintain pluripotency of the two types of ES cells are fundamentally different. Sato et al first discoveried that TGF-β and Nodal genes were highly expressed in undifferentiated human ES cells[26]. Beattie et al later reported that Ac‐ tivin A, a member of the TGF-β superfamily, was secreted by MEFs, and medium enriched with activin A can replace MEF feeder-layers or MEF-conditioned media to maintain human ES cells in an undifferentiated state[27]. In consistence, James et al demonstrated that the TGF-β/Activin/Nodal pathway was activated through the transcription factors Smad2/3 in undifferentiated human ES cells[28]. The notion that TGF-β/Activin/Nodal signaling sup‐ ports human ES self-renewal and pluripotency is further supported by the fact that recombi‐ nant Activin or Nodal stimulation induces higher levels of pluripotent protein expression (Oct4 and Nanog), while inhibition of TGF-β/Activin/Nodal signaling with Lefty or Follista‐ tin decreases expression of these pluripotent proteins in human ES cells[29, 30].

**2.3. FGF/MEK signaling**

human ES cell differentiation[40-42].

The importance of Fibroblast growth factor (FGF) signaling for human ES cells pluripo‐ tency is highlighted by the facts that human ES cells are traditionally cultured in the presence of Fibroblast growth factors (FGFs) either on fibroblast feeder layers or in fibro‐ blast-conditioned medium[35, 36]. Studies have demonstrated that all four FGF receptors (FGFR1, FGFR3 and FGFR4) and several components (SOS1, PTPN11 and RAF1) of their downstream activation cascade are significantly upregulated in undifferentiated human ES cells, in comparison to differentiated human ES cells[37-39]. In consistence, withdraw‐ al of FGFs or inhibition of FGF signaling by a FGFR inhibitor, SU5402, rapidly induces

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Although the pluirpotency maintenance role of exogenous FGFs in human ES cell has been known for a long time, the molecular mechanisms by which they function remain unclear. FGFs signal by binding to FGF receptors (FGFRs), and activate multiple signaling cascades, including Mitogen-Activated Protein Kinases (MAPKs), the Janus kinase/signal transducer and activator of transcription (Jak/Stat), phosphatidylinositol 3-kinase (PI3K) and phosphoi‐ nositide phospholipase C (PLCg) pathway[43]. Several studies have highlighted the FGF contribution to the maintenance of human ES cells mainly through the FGF/MEK pathway (Figure 1), [44, 45]. Studies have showed that FGF2 induces feeder layer cells to secret TGFβ1 and insulin-like growth factor 2 (IGF2), which can subsequently promote the undif‐ ferentiated state of human ES cells[46, 47]. Bendall et al further reported that the function of exogenous FGFs in promoting ES self-renewal could be replaced by addition of IGF2 alone, suggesting an indirect role of FGFs for human ES cell growth. However, this model was challenged in subsequent publications from Wang et al who reported that exogenous IGF2 alone was insufficient to maintain undifferentiated growth of human ES cells, and they pro‐ posed that FGFs may play a direct role in blocking caspase-activated apoptosis through anoikis in human ES cells[48]. Recently, Eiselleova and colleagues postulated a new model whereby endogenous FGF-2 signaling maintained the undifferentiated state and survival of human ESCs, while exogenous FGF-2 mainly suppress cell death and apoptosis genes, thus

indirectly contributing to the maintenance of human ES cell pluripotency[49].

FGF signaling in mouse ES cells has also been extensively investigated. Mouse ES cells geneti‐ cally deficient in Fgf4 and extracellular-signal regulated kinase 2 (Erk2) differentiate ineffi‐ ciently. These results can be reproduced using inhibitors of FGF receptor and ERK, suggesting blockage of the FGF/MEK signaling pathway promotes mouse ES cell pluripotency[50-52]. In‐ deed, serum-free mouse ES cell medium supplemented with FGF/MEK inhibitors and LIF per‐ mits the derivation of mouse ES cells in the absence of feeders from strains normally considered non-permissive[53]. In addition, a recently identified compound, Pluripotin/SC1, has been shown to maintain mouse ES pluripotency by inhibiting ERK1 and activating the pho‐ phoinositide-3 kinase (PI3K) pathway through blocking RasGAP[54-56] [57, 58]. Although in‐ hibition of FGF/MEK pathway can attenuate ES cell differentiation, it is insufficient to support mouse ES cell self-renewal. Combination of the MEK inhibitor PD0325901 with the Glycogen synthase kinase-3 (GSK-3) inhibitor CHIR99021 (known as 2i) can efficiently sustain the pluri‐ potency of mouse ES cells in the absence of exogenous cytokines[59, 60]. Several groups dem‐

Recent studies have focused on understanding the molecular mechanisms of TGF-β/Activin/ Nodal signaling in retaining human ES cells pluripotency. Xu and colleagues showed that TGF-β/Activin/Nodal signaling activated Smad2/3 which subsequently binds to the Nanog promoter in undifferentiated human ES cells to induce expression of Nanog, a pluripotent transcription factor[31]. Additionally, mutating the putative Smad-binding sites reduced the response of Nanog to modulation of TGF-β signaling[31]. Nanog was also shown to coordi‐ nate with Smad2 in a negative-feedback loop to inhibit human ES cell differentiation[32]. In contrast to its important role in maintaining human ES cell pluripotency, the TGF-β/Activin/ Nodal signaling is not essential for pluripotency of mouse ES cells. Although this pathway was shown to be active in undifferentiated mouse ES cells as assessed by phosphorylation of smad 2/3, inhibition of smad 2/3 phosphorylation by SB431542 had no effect on the undiffer‐ entiated state of mouse ES cells[28]. However, the TGF-β/Activin/Nodal signaling may play a role in mouse ES proliferation. A recent study showed that Inhibition of TGF-β/Activin/ Nodal signaling by Smad7 or SB-431542 dramatically decreased mouse ES cell proliferation without effect on their pluripotency[33].

#### *2.2.3. Growth and Differentiation factor 3 (GDF-3)*

GDF-3 is another TGF-beta superfamily member that plays opposite roles in mouse and human ES cells. GDF-3, which acts as a BMP antagonist by direct binding to BMP-4, is specifically expressed in the pluripotent state of both mouse and human ES cells[34]. Ec‐ topic expression of GDF-3 leads to the maintenance of pluripotency in human ES cells, whereas a similar effect is observed in mouse ES cells when GDF-3 levels are decreased. In the absence of LIF, GDF-3-deficient mouse ES cells can still sustain pluripotent mark‐ ers[34]. These results are consistent with previously discussed BMP signals which can promote pluripotency of mouse ES cells, but cause differentiation of human ES cells. Thus lower concentrations of BMP antagonists, such as GDF-3, may enhance pluripoten‐ cy in mouse ES cells, whereas higher levels of GDF-3 may favor pluripotency of human ES cells by abrogating BMP signaling.

#### **2.3. FGF/MEK signaling**

*2.2.2. TGF-β/activin/nodal signaling pathway*

286 Pluripotent Stem Cells

without effect on their pluripotency[33].

ES cells by abrogating BMP signaling.

*2.2.3. Growth and Differentiation factor 3 (GDF-3)*

Although MEFs feeder layers were initially used to co-culture both mouse and human ES cells, signal factors secreted from MEFs to maintain pluripotency of the two types of ES cells are fundamentally different. Sato et al first discoveried that TGF-β and Nodal genes were highly expressed in undifferentiated human ES cells[26]. Beattie et al later reported that Ac‐ tivin A, a member of the TGF-β superfamily, was secreted by MEFs, and medium enriched with activin A can replace MEF feeder-layers or MEF-conditioned media to maintain human ES cells in an undifferentiated state[27]. In consistence, James et al demonstrated that the TGF-β/Activin/Nodal pathway was activated through the transcription factors Smad2/3 in undifferentiated human ES cells[28]. The notion that TGF-β/Activin/Nodal signaling sup‐ ports human ES self-renewal and pluripotency is further supported by the fact that recombi‐ nant Activin or Nodal stimulation induces higher levels of pluripotent protein expression (Oct4 and Nanog), while inhibition of TGF-β/Activin/Nodal signaling with Lefty or Follista‐

tin decreases expression of these pluripotent proteins in human ES cells[29, 30].

Recent studies have focused on understanding the molecular mechanisms of TGF-β/Activin/ Nodal signaling in retaining human ES cells pluripotency. Xu and colleagues showed that TGF-β/Activin/Nodal signaling activated Smad2/3 which subsequently binds to the Nanog promoter in undifferentiated human ES cells to induce expression of Nanog, a pluripotent transcription factor[31]. Additionally, mutating the putative Smad-binding sites reduced the response of Nanog to modulation of TGF-β signaling[31]. Nanog was also shown to coordi‐ nate with Smad2 in a negative-feedback loop to inhibit human ES cell differentiation[32]. In contrast to its important role in maintaining human ES cell pluripotency, the TGF-β/Activin/ Nodal signaling is not essential for pluripotency of mouse ES cells. Although this pathway was shown to be active in undifferentiated mouse ES cells as assessed by phosphorylation of smad 2/3, inhibition of smad 2/3 phosphorylation by SB431542 had no effect on the undiffer‐ entiated state of mouse ES cells[28]. However, the TGF-β/Activin/Nodal signaling may play a role in mouse ES proliferation. A recent study showed that Inhibition of TGF-β/Activin/ Nodal signaling by Smad7 or SB-431542 dramatically decreased mouse ES cell proliferation

GDF-3 is another TGF-beta superfamily member that plays opposite roles in mouse and human ES cells. GDF-3, which acts as a BMP antagonist by direct binding to BMP-4, is specifically expressed in the pluripotent state of both mouse and human ES cells[34]. Ec‐ topic expression of GDF-3 leads to the maintenance of pluripotency in human ES cells, whereas a similar effect is observed in mouse ES cells when GDF-3 levels are decreased. In the absence of LIF, GDF-3-deficient mouse ES cells can still sustain pluripotent mark‐ ers[34]. These results are consistent with previously discussed BMP signals which can promote pluripotency of mouse ES cells, but cause differentiation of human ES cells. Thus lower concentrations of BMP antagonists, such as GDF-3, may enhance pluripoten‐ cy in mouse ES cells, whereas higher levels of GDF-3 may favor pluripotency of human

The importance of Fibroblast growth factor (FGF) signaling for human ES cells pluripo‐ tency is highlighted by the facts that human ES cells are traditionally cultured in the presence of Fibroblast growth factors (FGFs) either on fibroblast feeder layers or in fibro‐ blast-conditioned medium[35, 36]. Studies have demonstrated that all four FGF receptors (FGFR1, FGFR3 and FGFR4) and several components (SOS1, PTPN11 and RAF1) of their downstream activation cascade are significantly upregulated in undifferentiated human ES cells, in comparison to differentiated human ES cells[37-39]. In consistence, withdraw‐ al of FGFs or inhibition of FGF signaling by a FGFR inhibitor, SU5402, rapidly induces human ES cell differentiation[40-42].

Although the pluirpotency maintenance role of exogenous FGFs in human ES cell has been known for a long time, the molecular mechanisms by which they function remain unclear. FGFs signal by binding to FGF receptors (FGFRs), and activate multiple signaling cascades, including Mitogen-Activated Protein Kinases (MAPKs), the Janus kinase/signal transducer and activator of transcription (Jak/Stat), phosphatidylinositol 3-kinase (PI3K) and phosphoi‐ nositide phospholipase C (PLCg) pathway[43]. Several studies have highlighted the FGF contribution to the maintenance of human ES cells mainly through the FGF/MEK pathway (Figure 1), [44, 45]. Studies have showed that FGF2 induces feeder layer cells to secret TGFβ1 and insulin-like growth factor 2 (IGF2), which can subsequently promote the undif‐ ferentiated state of human ES cells[46, 47]. Bendall et al further reported that the function of exogenous FGFs in promoting ES self-renewal could be replaced by addition of IGF2 alone, suggesting an indirect role of FGFs for human ES cell growth. However, this model was challenged in subsequent publications from Wang et al who reported that exogenous IGF2 alone was insufficient to maintain undifferentiated growth of human ES cells, and they pro‐ posed that FGFs may play a direct role in blocking caspase-activated apoptosis through anoikis in human ES cells[48]. Recently, Eiselleova and colleagues postulated a new model whereby endogenous FGF-2 signaling maintained the undifferentiated state and survival of human ESCs, while exogenous FGF-2 mainly suppress cell death and apoptosis genes, thus indirectly contributing to the maintenance of human ES cell pluripotency[49].

FGF signaling in mouse ES cells has also been extensively investigated. Mouse ES cells geneti‐ cally deficient in Fgf4 and extracellular-signal regulated kinase 2 (Erk2) differentiate ineffi‐ ciently. These results can be reproduced using inhibitors of FGF receptor and ERK, suggesting blockage of the FGF/MEK signaling pathway promotes mouse ES cell pluripotency[50-52]. In‐ deed, serum-free mouse ES cell medium supplemented with FGF/MEK inhibitors and LIF per‐ mits the derivation of mouse ES cells in the absence of feeders from strains normally considered non-permissive[53]. In addition, a recently identified compound, Pluripotin/SC1, has been shown to maintain mouse ES pluripotency by inhibiting ERK1 and activating the pho‐ phoinositide-3 kinase (PI3K) pathway through blocking RasGAP[54-56] [57, 58]. Although in‐ hibition of FGF/MEK pathway can attenuate ES cell differentiation, it is insufficient to support mouse ES cell self-renewal. Combination of the MEK inhibitor PD0325901 with the Glycogen synthase kinase-3 (GSK-3) inhibitor CHIR99021 (known as 2i) can efficiently sustain the pluri‐ potency of mouse ES cells in the absence of exogenous cytokines[59, 60]. Several groups dem‐ onstrated that improvement of mouse ES cell pluripotency by inhibition of GSK-3 occurred via Wnt/β-catenin signaling, whereas many others argued that GSK3 was likely to exert β-catenin independent effects in ES cells[59, 61-67].

**3. The regulatory network of pluripotency factors**

tors of the core pluripotency transcriptional network.

**3.1. OCT4 and SOX2**

**3.2. Nanog**

ES cell pluripotency is conferred by a unique transcriptional network[79]. Early global tran‐ scriptional profiles and genetic studies have identified several critical transcription factors that are required for the pluripotency of ES cells, such as Oct4, Sox2, Nanog, Foxd3 and Id, etc [80-88]. Here we will mainly focus on Oct4, Sox2 and Nanog, three key transcription fac‐

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289

OCT4 (also known as Oct3), a POU domain-containing transcription factor, was one of the first transcription factors identified as essential for both early embryo development and plu‐ ripotency maintenance in ES cells[84, 89]. The expression of Oct4 is activated at the 8-cell stage and is later restricted to the inner cell mass (ICM) and germ cells in early mouse em‐ bryogenesis *in vivo* [89-92]. Oct4 is highly expressed in both human and mouse ES cells, and its expression diminishes when these cells differentiate and lose pluripotency. Oct4 regu‐ lates a broad range of target genes including Fgf4, Utf1, Opn, Rex1/ Zfp42, Fbx15, Sox2 and Cdx2[93-95]. Repression of Oct4 activity in ES cells upregulates Cdx2 expression, leading to ES cell differentiation into trophectoderm[96]. Oct4 is also known to activate downstream genes by binding to enhancers carrying the octamer–sox motif (Oct–Sox enhancer), for syn‐ ergistic activation with Sox2. In contrast with its target genes, little is known about Oct4 up‐ stream regulators. The Oct4 promoter contains conserved distal and proximal enhancers that can either repress or activate its expression depending on the binding factors occupying these sites[97, 98]. The precise level of Oct4 is important for ES cell fate determination. Loss of Oct4 causes inappropriate differentiation of ES cells into trophectoderm, whereas overex‐ pression of Oct4 results in differentiation into primitive endoderm and mesoderm[99, 100]. Sox2 is an HMG-box transcription factor that is detected in pluripotent cell lineages and the nervous system[101-103]. Inactivate Sox2 *in vivo* results in early embryonic lethality due to the failure of ICM maintenance[102]. Sox2 can form a complex with the Oct4 protein to occu‐ py Oct–Sox enhancers to regulate target gene expression. Oct–Sox enhancers are found in the regulatory region of most of the genes that are specifically expressed in pluripotent stem

cells, such as Oct4, Sox2, Nanog, Utf1, Lefty, Fgf4 and Fbx15[93, 94, 104-108].

Nanog is another homeobox-containing transcription factor that is specifically expressed in pluripotent ES cells. The essential role of Nanog in maintaining the pluripotency of ES cells is highlighted by the facts that Nanog-deficient ES cells are prone to differentiation, whereas forced expression of Nanog partially renders ES cells self-renewal potential in the absence of LIF[85, 86, 109]. How Nanog regulates stem cell pluripotency remains entirely unknown. Studies have indicated that Nanog may maintain ES cell pluripotency by 1) downregulating downstream genes essential for cell differentiation such as Gata4 and Gata6 and 2) activat‐ ing the expression of genes necessary for self-renewal such as Rex1 and Id[19, 85, 86]. Al‐ though it is widely accepted that Nanog, like Oct4 and Sox2, play a central role in

As demonstrated above, human and mouse ES cells are both derived from blastocyst-stage embryos, but they require different biological signals for maintaining pluripotency. In gen‐ eral, mouse ES cells maintain their pluripotency by activating LIF/STAT3 and BMP signal‐ ing, while human ES cells require TGF-β/Nodal and FGF/MEK pathways. Interestingly, several pathways, such as BMP and FGF/MEK, have completely oppositing effects on main‐ taining the pluriotency of mouse and human ES cells. Activation of BMP signaling and in‐ hibition of the FGF/MEK pathway promote mouse ES self-renewal, whereas inhibition of BMP signaling and activation of FGF/MEK pathway sustain human ES cell pluripotency. These distinct signaling effects on pluripotency may reflect intrinsic differences between mouse and human ES cells. Recent studies have demonstrated that conventional human ES cells do not represent the "ground or naïve state" of stemness, but rather a more develop‐ mentally mature "primed state" resembling mouse epiblast stem cells (mEpiSCs) found in the post-implantation, pre-gastrulation stage of embryos [68-74]. Conventional human ES cells exhibit numerous similarities to the mouse EpiSCs over mouse ES cells (Table 1). For instance, conventional human ES cells and mouse EpiSCs display flattened cell colonies and epigenetic X-chromosome inactivation (XiXa), and require Activin and FGF for pluripotency maintanince. In contrast, mouse ES cells exhibit dome-shaped colony morphology and epi‐ genetic activation of both X-chromosome (XaXa), and require LIF/STAT3 signaling to pro‐ mote self-renewal. Subsequent studies have demonstrated that the medium containing "2i" (MEK inhibitor and GSK-3 inhibitor), when supplemented with other factors (such as for‐ skolin), can efficiently convert conventional human ES cells into a ground or "naïve" state with display of hallmark features of mouse ES cells. This medium can also maintain human ES cell pluriptoency at the naïve state [69, 70, 72, 75-78].


**Table 1.** Comparison of the properties of mouse ES cells (mESCs), mouse epiblast stem cells (mEpiSCs), human ES cells (hESCs) and human iPS cells (hiPSCs).

## **3. The regulatory network of pluripotency factors**

ES cell pluripotency is conferred by a unique transcriptional network[79]. Early global tran‐ scriptional profiles and genetic studies have identified several critical transcription factors that are required for the pluripotency of ES cells, such as Oct4, Sox2, Nanog, Foxd3 and Id, etc [80-88]. Here we will mainly focus on Oct4, Sox2 and Nanog, three key transcription fac‐ tors of the core pluripotency transcriptional network.

#### **3.1. OCT4 and SOX2**

onstrated that improvement of mouse ES cell pluripotency by inhibition of GSK-3 occurred via Wnt/β-catenin signaling, whereas many others argued that GSK3 was likely to exert β-catenin

As demonstrated above, human and mouse ES cells are both derived from blastocyst-stage embryos, but they require different biological signals for maintaining pluripotency. In gen‐ eral, mouse ES cells maintain their pluripotency by activating LIF/STAT3 and BMP signal‐ ing, while human ES cells require TGF-β/Nodal and FGF/MEK pathways. Interestingly, several pathways, such as BMP and FGF/MEK, have completely oppositing effects on main‐ taining the pluriotency of mouse and human ES cells. Activation of BMP signaling and in‐ hibition of the FGF/MEK pathway promote mouse ES self-renewal, whereas inhibition of BMP signaling and activation of FGF/MEK pathway sustain human ES cell pluripotency. These distinct signaling effects on pluripotency may reflect intrinsic differences between mouse and human ES cells. Recent studies have demonstrated that conventional human ES cells do not represent the "ground or naïve state" of stemness, but rather a more develop‐ mentally mature "primed state" resembling mouse epiblast stem cells (mEpiSCs) found in the post-implantation, pre-gastrulation stage of embryos [68-74]. Conventional human ES cells exhibit numerous similarities to the mouse EpiSCs over mouse ES cells (Table 1). For instance, conventional human ES cells and mouse EpiSCs display flattened cell colonies and epigenetic X-chromosome inactivation (XiXa), and require Activin and FGF for pluripotency maintanince. In contrast, mouse ES cells exhibit dome-shaped colony morphology and epi‐ genetic activation of both X-chromosome (XaXa), and require LIF/STAT3 signaling to pro‐ mote self-renewal. Subsequent studies have demonstrated that the medium containing "2i" (MEK inhibitor and GSK-3 inhibitor), when supplemented with other factors (such as for‐ skolin), can efficiently convert conventional human ES cells into a ground or "naïve" state with display of hallmark features of mouse ES cells. This medium can also maintain human

**Table 1.** Comparison of the properties of mouse ES cells (mESCs), mouse epiblast stem cells (mEpiSCs), human ES cells

independent effects in ES cells[59, 61-67].

288 Pluripotent Stem Cells

ES cell pluriptoency at the naïve state [69, 70, 72, 75-78].

(hESCs) and human iPS cells (hiPSCs).

OCT4 (also known as Oct3), a POU domain-containing transcription factor, was one of the first transcription factors identified as essential for both early embryo development and plu‐ ripotency maintenance in ES cells[84, 89]. The expression of Oct4 is activated at the 8-cell stage and is later restricted to the inner cell mass (ICM) and germ cells in early mouse em‐ bryogenesis *in vivo* [89-92]. Oct4 is highly expressed in both human and mouse ES cells, and its expression diminishes when these cells differentiate and lose pluripotency. Oct4 regu‐ lates a broad range of target genes including Fgf4, Utf1, Opn, Rex1/ Zfp42, Fbx15, Sox2 and Cdx2[93-95]. Repression of Oct4 activity in ES cells upregulates Cdx2 expression, leading to ES cell differentiation into trophectoderm[96]. Oct4 is also known to activate downstream genes by binding to enhancers carrying the octamer–sox motif (Oct–Sox enhancer), for syn‐ ergistic activation with Sox2. In contrast with its target genes, little is known about Oct4 up‐ stream regulators. The Oct4 promoter contains conserved distal and proximal enhancers that can either repress or activate its expression depending on the binding factors occupying these sites[97, 98]. The precise level of Oct4 is important for ES cell fate determination. Loss of Oct4 causes inappropriate differentiation of ES cells into trophectoderm, whereas overex‐ pression of Oct4 results in differentiation into primitive endoderm and mesoderm[99, 100].

Sox2 is an HMG-box transcription factor that is detected in pluripotent cell lineages and the nervous system[101-103]. Inactivate Sox2 *in vivo* results in early embryonic lethality due to the failure of ICM maintenance[102]. Sox2 can form a complex with the Oct4 protein to occu‐ py Oct–Sox enhancers to regulate target gene expression. Oct–Sox enhancers are found in the regulatory region of most of the genes that are specifically expressed in pluripotent stem cells, such as Oct4, Sox2, Nanog, Utf1, Lefty, Fgf4 and Fbx15[93, 94, 104-108].

#### **3.2. Nanog**

Nanog is another homeobox-containing transcription factor that is specifically expressed in pluripotent ES cells. The essential role of Nanog in maintaining the pluripotency of ES cells is highlighted by the facts that Nanog-deficient ES cells are prone to differentiation, whereas forced expression of Nanog partially renders ES cells self-renewal potential in the absence of LIF[85, 86, 109]. How Nanog regulates stem cell pluripotency remains entirely unknown. Studies have indicated that Nanog may maintain ES cell pluripotency by 1) downregulating downstream genes essential for cell differentiation such as Gata4 and Gata6 and 2) activat‐ ing the expression of genes necessary for self-renewal such as Rex1 and Id[19, 85, 86]. Al‐ though it is widely accepted that Nanog, like Oct4 and Sox2, play a central role in pluripotency maintenance, this dogma has been challenged by a subsequent report that Nanog protein levels are undetectable in a fraction of ES cells that express Oct4, and the pure populations of Nanog−/− ES cells can be propagated without losing expression of other pluripotency markers[110].

**Author details**

University, Xi'an, China

, Hongtao Wang2

ty of Health Sciences, Pomona, CA, USA

1998;282(5391):1145-7.

ture. 1988;336(6200):688-90.

12991-8.

cytokine receptors. Science. 1995;267(5202):1349-53.

\*Address all correspondence to: jhao@westernu.edu

embryos. Nature. 1981;292(5819):154-6.

and Jijun Hao3\*

2 Department of Burn and Cutaneous Surgery, XiJing Hospital, the Fourth Military Medical

Molecular Mechanisms of Embryonic Stem Cell Pluripotency

http://dx.doi.org/10.5772/54365

291

3 College of Veterinary Medicine, Western University of Health Sciences, Western Universi‐

[1] Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse

[2] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science.

[3] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryon‐

[4] Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl M, et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Na‐

[5] Niwa H, Burdon T, Chambers I, Smith A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. 1998;12(13):2048-60. [6] Stahl N, Farruggella TJ, Boulton TG, Zhong Z, Darnell JE, Jr., Yancopoulos GD. Choice of STATs and other substrates specified by modular tyrosine-based motifs in

[7] Hemmann U, Gerhartz C, Heesel B, Sasse J, Kurapkat G, Grotzinger J, et al. Differen‐ tial activation of acute phase response factor/STAT3 and Stat1 via the cytoplasmic domain of the interleukin 6 signal transducer gp130. II. Src homology SH2 domains define the specificity of stat factor activation. J Biol Chem. 1996;271(22):12999-3007. [8] Gerhartz C, Heesel B, Sasse J, Hemmann U, Landgraf C, Schneider-Mergener J, et al. Differential activation of acute phase response factor/STAT3 and STAT1 via the cyto‐ plasmic domain of the interleukin 6 signal transducer gp130. I. Definition of a novel phosphotyrosine motif mediating STAT1 activation. J Biol Chem. 1996;271(22):

ic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663-76.

1 Department of Nephrology, Shanghai Jimin Hospital, Shanghai, China

Guofeng Han1

**References**

Little is known about the mechanism by which Nanog is regulated in ES cells. Recently, Su‐ zuki et al showed that Nanog expression was upregulated by BrachyuryT and STAT3 in mouse ES cells[111]. In human ES cells and in mouse EpiSCs, Vallier et al reported that Acti‐ vin/Nodal signaling stimulated expression of Nanog, which in turn prevents FGF-induced neuroectoderm differentiation [112]. In addition, several studies indicated that the Oct4/ Sox2 complex was directly bound to the Nanog promoter to regulate target gene expression [106, 107, 113]. Genomic studies have revealed that Oct4, Sox2, and Nanog frequently bind the same regulatory regions in undifferentiated mouse and human ESCs, and that these binding sites are often in close proximity to one another[113-116]. These results indicate that Oct4, Sox2, and Nanog may physically interact with each other and coordinately regulate target genes in some cases. Additionally, Goke and colleagues reported that combinatorial binding sites of the Oct4/Sox2/Nanog were more conserved between mouse and human ES cells than individual binding sites were [113, 114, 117-119].

### **4. Summary**

Understanding the molecular mechanism of pluripotency can greatly expand our knowl‐ edge of ES cell biology and facilitate future stem cell clinical applications. In the past few years, we have seen tremendous advances in understanding ES cell pluripotency. Although mouse ES cells and conventional human ES cells require distinct signaling pathways to maintain pluripotency, they display similar gene expression profiles, activities of transcrip‐ tion factors (such as Oct4, Nanog and Sox2) and transcription factor networks. Our under‐ standing of pluripotency has been further expanded by the advent of iPS cells and the very recent discovery that conventional human ES cells are more equivalent to mouse EpiSCs, but rather "naïve state" of mouse ES cells. Nevertheless, our knowledge of the molecular mechanisms of ES cell pluripotency is still very limited. For instance, it remains unknown how growth factors establish and control transcriptional networks to regulate pluripoency and how ES cells respond so precisely to exogenous cues. Given the rapid advance in ES cell biology, we anticipate the molecular mechanisms underlying pluripotency of ES cells will soon be uncovered and pluripotent stem cells, such as ES cells and iPS cells, will be widely used for clinical applications in the near future.

#### **Acknowledgements**

This work is funded by the seed fund of the Western University of Health Sciences.

## **Author details**

pluripotency maintenance, this dogma has been challenged by a subsequent report that Nanog protein levels are undetectable in a fraction of ES cells that express Oct4, and the pure populations of Nanog−/− ES cells can be propagated without losing expression of other

Little is known about the mechanism by which Nanog is regulated in ES cells. Recently, Su‐ zuki et al showed that Nanog expression was upregulated by BrachyuryT and STAT3 in mouse ES cells[111]. In human ES cells and in mouse EpiSCs, Vallier et al reported that Acti‐ vin/Nodal signaling stimulated expression of Nanog, which in turn prevents FGF-induced neuroectoderm differentiation [112]. In addition, several studies indicated that the Oct4/ Sox2 complex was directly bound to the Nanog promoter to regulate target gene expression [106, 107, 113]. Genomic studies have revealed that Oct4, Sox2, and Nanog frequently bind the same regulatory regions in undifferentiated mouse and human ESCs, and that these binding sites are often in close proximity to one another[113-116]. These results indicate that Oct4, Sox2, and Nanog may physically interact with each other and coordinately regulate target genes in some cases. Additionally, Goke and colleagues reported that combinatorial binding sites of the Oct4/Sox2/Nanog were more conserved between mouse and human ES

Understanding the molecular mechanism of pluripotency can greatly expand our knowl‐ edge of ES cell biology and facilitate future stem cell clinical applications. In the past few years, we have seen tremendous advances in understanding ES cell pluripotency. Although mouse ES cells and conventional human ES cells require distinct signaling pathways to maintain pluripotency, they display similar gene expression profiles, activities of transcrip‐ tion factors (such as Oct4, Nanog and Sox2) and transcription factor networks. Our under‐ standing of pluripotency has been further expanded by the advent of iPS cells and the very recent discovery that conventional human ES cells are more equivalent to mouse EpiSCs, but rather "naïve state" of mouse ES cells. Nevertheless, our knowledge of the molecular mechanisms of ES cell pluripotency is still very limited. For instance, it remains unknown how growth factors establish and control transcriptional networks to regulate pluripoency and how ES cells respond so precisely to exogenous cues. Given the rapid advance in ES cell biology, we anticipate the molecular mechanisms underlying pluripotency of ES cells will soon be uncovered and pluripotent stem cells, such as ES cells and iPS cells, will be widely

This work is funded by the seed fund of the Western University of Health Sciences.

pluripotency markers[110].

290 Pluripotent Stem Cells

**4. Summary**

cells than individual binding sites were [113, 114, 117-119].

used for clinical applications in the near future.

**Acknowledgements**

Guofeng Han1 , Hongtao Wang2 and Jijun Hao3\*

\*Address all correspondence to: jhao@westernu.edu

1 Department of Nephrology, Shanghai Jimin Hospital, Shanghai, China

2 Department of Burn and Cutaneous Surgery, XiJing Hospital, the Fourth Military Medical University, Xi'an, China

3 College of Veterinary Medicine, Western University of Health Sciences, Western Universi‐ ty of Health Sciences, Pomona, CA, USA

## **References**


[9] Ihle JN, Kerr IM. Jaks and Stats in signaling by the cytokine receptor superfamily. Trends Genet. 1995;11(2):69-74.

[23] Hao J, Ho JN, Lewis JA, Karim KA, Daniels RN, Gentry PR, et al. In vivo structureactivity relationship study of dorsomorphin analogues identifies selective VEGF and

Molecular Mechanisms of Embryonic Stem Cell Pluripotency

http://dx.doi.org/10.5772/54365

293

[24] Gonzalez R, Lee JW, Snyder EY, Schultz PG. Dorsomorphin promotes human embry‐ onic stem cell self-renewal. Angew Chem Int Ed Engl. 2011;50(15):3439-41.

[25] Hao J, Sawyer DB, Hatzopoulos AK, Hong CC. Recent Progress on Chemical Biology of Pluripotent Stem Cell Self-renewal, Reprogramming and Cardiomyogenesis. Rec

[26] Sato N, Sanjuan IM, Heke M, Uchida M, Naef F, Brivanlou AH. Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev Biol.

[27] Beattie GM, Lopez AD, Bucay N, Hinton A, Firpo MT, King CC, et al. Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder lay‐

[28] James D, Levine AJ, Besser D, Hemmati-Brivanlou A. TGF beta/activin/nodal signal‐ ing is necessary for the maintenance of pluripotency in human embryonic stem. De‐

[29] Xiao L, Yuan X, Sharkis SJ. Activin A maintains self-renewal and regulates fibroblast growth factor, Wnt, and bone morphogenic protein pathways in human embryonic

[30] Vallier L, Alexander M, Pedersen RA. Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J Cell Sci. 2005;118(Pt 19):

[31] Xu RH, Sampsell-Barron TL, Gu F, Root S, Peck RM, Pan GJ, et al. NANOG is a direct target of TGF beta/Activin-mediated SMAD signaling in human ESCs. Cell Stem

[32] Vallier L, Mendjan S, Brown S, Chng Z, Teo A, Smithers LE, et al. Activin/Nodal sig‐ nalling maintains pluripotency by controlling Nanog expression. Development.

[33] Ogawa K, Saito A, Matsui H, Suzuki H, Ohtsuka S, Shimosato D, et al. Activin-Nodal signaling is involved in propagation of mouse embryonic stem cells. J Cell Sci.

[34] Levine AJ, Brivanlou AH. GDF3, a BMP inhibitor, regulates cell fate in stem cells and

[35] Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, et al. Clo‐ nally derived human embryonic stem cell lines maintain pluripotency and prolifera‐

tive potential for prolonged periods of culture. Dev Biol. 2000;227(2):271-8.

BMP inhibitors. ACS Chem Biol. 2010;5(2):245-53.

Pat Regen Med. 2011;1(3):263-74.

ers. Stem Cells. 2005;23(4):489-95.

velopment. 2005;132(6):1273-82.

stem cells. Stem Cells. 2006;24(6):1476-86.

early embryos. Development. 2006;133(2):209-16.

2003;260(2):404-13.

4495-509.

Cell. 2008;3(2):196-206.

2009;136(8):1339-49.

2007;120(Pt 1):55-65.


[23] Hao J, Ho JN, Lewis JA, Karim KA, Daniels RN, Gentry PR, et al. In vivo structureactivity relationship study of dorsomorphin analogues identifies selective VEGF and BMP inhibitors. ACS Chem Biol. 2010;5(2):245-53.

[9] Ihle JN, Kerr IM. Jaks and Stats in signaling by the cytokine receptor superfamily.

[10] Auernhammer CJ, Melmed S. Leukemia-inhibitory factor-neuroimmune modulator

[11] Reich NC, Liu L. Tracking STAT nuclear traffic. Nat Rev Immunol. 2006;6(8):602-12.

[12] Chen X, Xu H, Yuan P, Fang F, Huss M, Vega VB, et al. Integration of external signal‐ ing pathways with the core transcriptional network in embryonic stem cells. Cell.

[13] Kidder BL, Yang J, Palmer S. STAT3 and c-Myc genome-wide promoter occupancy in

[14] Bourillot PY, Aksoy I, Schreiber V, Wianny F, Schulz H, Hummel O, et al. Novel STAT3 target genes exert distinct roles in the inhibition of mesoderm and endoderm

[15] Daheron L, Opitz SL, Zaehres H, Lensch MW, Andrews PW, Itskovitz-Eldor J, et al. LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells.

[16] Wrana JL, Attisano L, Carcamo J, Zentella A, Doody J, Laiho M, et al. Tgf-Beta Sig‐ nals through a Heteromeric Protein-Kinase Receptor Complex. Cell. 1992;71(6):

[17] Schmierer B, Hill CS. TGF beta-SMAD signal transduction: molecular specificity and

[18] Sebald W, Nickel J, Zhang JL, Mueller TD. Molecular recognition in bone morphoge‐

[19] Ying QL, Nichols J, Chambers I, Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with

[20] Xu RH, Chen X, Li DS, Li R, Addicks GC, Glennon C, et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat Biotechnol. 2002;20(12):

[21] Xu RH, Peck RM, Li DS, Feng X, Ludwig T, Thomson JA. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Meth‐

[22] Yu PB, Hong CC, Sachidanandan C, Babitt JL, Deng DY, Hoyng SA, et al. Dorsomor‐ phin inhibits BMP signals required for embryogenesis and iron metabolism. Nat

netic protein (BMP)/receptor interaction. Biol Chem. 2004;385(8):697-710.

functional flexibility. Nat Rev Mol Cell Bio. 2007;8(12):970-82.

differentiation in cooperation with Nanog. Stem Cells. 2009;27(8):1760-71.

Trends Genet. 1995;11(2):69-74.

2008;133(6):1106-17.

292 Pluripotent Stem Cells

Stem Cells. 2004;22(5):770-8.

STAT3. Cell. 2003;115(3):281-92.

1003-14.

1261-4.

ods. 2005;2(3):185-90.

Chem Biol. 2008;4(1):33-41.

of endocrine function. Endocr Rev. 2000;21(3):313-45.

embryonic stem cells. PLoS One. 2008;3(12):e3932.


[36] Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol. 2001;19(10):971-4.

[50] Burdon T, Stracey C, Chambers I, Nichols J, Smith A. Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells. Dev Biol.

Molecular Mechanisms of Embryonic Stem Cell Pluripotency

http://dx.doi.org/10.5772/54365

295

[51] Kunath T, Saba-El-Leil MK, Almousailleakh M, Wray J, Meloche S, Smith A. FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embry‐ onic stem cells from self-renewal to lineage commitment. Development. 2007;134(16):

[52] Stavridis MP, Lunn JS, Collins BJ, Storey KG. A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification. Development.

[53] Batlle-Morera L, Smith A, Nichols J. Parameters Influencing Derivation of Embryonic

[54] Qi X, Li TG, Hao J, Hu J, Wang J, Simmons H, et al. BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways. Proc

[55] Burdon T, Stracey C, Chambers I, Nichols J, Smith A. Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells. Dev Biol.

[56] Paling NR, Wheadon H, Bone HK, Welham MJ. Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling. J Biol Chem.

[57] Chen S, Do JT, Zhang Q, Yao S, Yan F, Peters EC, et al. Self-renewal of embryonic stem cells by a small molecule. Proc Natl Acad Sci U S A. 2006;103(46):17266-71. [58] Chen S, Ding S, Yan F, Schultz P, inventors; Compounds that maintain pluripotency

[59] Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, et al. The ground

[60] Smith A, Ying Q, inventors; Culture medium containing kinase inhibitors, and uses

[61] Aubert J, Dunstan H, Chambers I, Smith A. Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation. Nat Biotechnol.

[62] Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH. Maintenance of pluripo‐ tency in human and mouse embryonic stem cells through activation of Wnt signaling

[63] Ogawa K, Nishinakamura R, Iwamatsu Y, Shimosato D, Niwa H. Synergistic action of Wnt and LIF in maintaining pluripotency of mouse ES cells. Biochem Biophys Res

by a pharmacological GSK-3-specific inhibitor. Nat Med. 2004;10(1):55-63.

state of embryonic stem cell self-renewal. Nature. 2008;453(7194):519-23.

Stem Cells From Murine Embryos. Genesis. 2008;46(12):758-67.

Natl Acad Sci U S A. 2004;101(16):6027-32.

of embryonic stem cells patent US20100234400. 2010.

thereof patent US20080014638. 2008.

1999;210(1):30-43.

2007;134(16):2889-94.

1999;210(1):30-43.

2004;279(46):48063-70.

2002;20(12):1240-5.

Commun. 2006;343(1):159-66.

2895-902.


[50] Burdon T, Stracey C, Chambers I, Nichols J, Smith A. Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells. Dev Biol. 1999;210(1):30-43.

[36] Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol. 2001;19(10):971-4.

[37] Brandenberger R, Wei H, Zhang S, Lei S, Murage J, Fisk GJ, et al. Transcriptome characterization elucidates signaling networks that control human ES cell growth

[38] Dvash T, Mayshar Y, Darr H, McElhaney M, Barker D, Yanuka O, et al. Temporal gene expression during differentiation of human embryonic stem cells and embryoid

[39] Ginis I, Luo YQ, Miura T, Thies S, Brandenberger R, Gerecht-Nir S, et al. Differences between human and mouse embryonic stem cells. Dev Biol. 2004;269(2):360-80. [40] Kim SJ, Cheon SH, Yoo SJ, Kwon J, Park JH, Kim CG, et al. Contribution of the PI3K/Akt/PKB signal pathway to maintenance of self-renewal in human embryonic stem cells (Retracted Article. See vol 580, pg 1529, 2006). Febs Lett. 2005;579(2):534-40.

[41] Dvorak P. Basic fibroblast growth factor and its receptors in human embryonic stem

[42] Dvorak P, Dvorakova D, Koskova S, Vodinska M, Najvirtova M, Krekac D, et al. Ex‐ pression and potential role of fibroblast growth factor 2 and its receptors in human

[43] Dailey L, Ambrosetti D, Mansukhani A, Basilico C. Mechanisms underlying differen‐ tial responses to FGF signaling. Cytokine Growth Factor Rev. 2005;16(2):233-47. [44] Kang HB, Kim JS, Kwon HJ, Nam KH, Youn HS, Sok DE, et al. Basic fibroblast growth factor activates ERK and induces c-fos in human embryonic stem cell line

[45] Li J, Wang GW, Wang CY, Zhao Y, Zhang H, Tan ZJ, et al. MEK/ERK signaling con‐ tributes to the maintenance of human embryonic stem cell self-renewal. Differentia‐

[46] Bendall SC, Stewart MH, Menendez P, George D, Vijayaragavan K, Werbowetski-Ogilvie T, et al. IGF and FGF cooperatively establish the regulatory stem cell niche of

[47] Greber B, Lehrach H, Adjaye J. Fibroblast growth factor 2 modulates transforming growth factor beta signaling in mouse embryonic fibroblasts and human ESCs

[48] Wang XF, Lin G, Martins-Taylor K, Zeng H, Xu RH. Inhibition of Caspase-mediated Anoikis Is Critical for Basic Fibroblast Growth Factor-sustained Culture of Human

Pluripotent Stem Cells. Journal of Biological Chemistry. 2009;284(49):34054-64. [49] Eiselleova L, Matulka K, Kriz V, Kunova M, Schmidtova Z, Neradil J, et al. A Com‐ plex Role for FGF-2 in Self-Renewal, Survival, and Adhesion of Human Embryonic

pluripotent human cells in vitro. Nature. 2007;448(7157):1015-21.

(hESCs) to support hESC self-renewal. Stem Cells. 2007;25(2):455-64.

and differentiation. Nat Biotechnol. 2004;22(6):707-16.

bodies. Hum Reprod. 2004;19(12):2875-83.

294 Pluripotent Stem Cells

cells. Folia Histochem Cyto. 2005;43(4):203-8.

MizhES1. Stem Cells Dev. 2005;14(4):395-401.

Stem Cells. Stem Cells. 2009;27(8):1847-57.

tion. 2007;75(4):299-307.

embryonic stem cells. Stem Cells. 2005;23(8):1200-11.


[64] Kielman MF, Rindapaa M, Gaspar C, van Poppel N, Breukel C, van Leeuwen S, et al. Apc modulates embryonic stem-cell differentiation by controlling the dosage of betacatenin signaling. Nat Genet. 2002;32(4):594-605.

[79] Jaenisch R, Young R. Stem cells, the molecular circuitry of pluripotency and nuclear

Molecular Mechanisms of Embryonic Stem Cell Pluripotency

http://dx.doi.org/10.5772/54365

297

[80] Hollnagel A, Oehlmann V, Heymer J, Ruther U, Nordheim A. Id genes are direct tar‐ gets of bone morphogenetic protein induction in embryonic stem cells. Journal of Bi‐

[81] Ivanova NB, Dimos JT, Schaniel C, Hackney JA, Moore KA, Lemischka IR. A stem

[82] Ramalho-Santos M, Yoon S, Matsuzaki Y, Mulligan RC, Melton DA. "Stemness": tran‐ scriptional profiling of embryonic and adult stem cells. Science. 2002;298(5593):

[83] Sato N, Sanjuan IM, Heke M, Uchida M, Naef F, Brivanlou AH. Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev Biol.

[84] Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU

[85] Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, et al. Functional ex‐ pression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells.

[86] Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, et al. The ho‐ meoprotein Nanog is required for maintenance of pluripotency in mouse epiblast

[87] Liu Y, Labosky PA. Regulation of Embryonic Stem Cell Self-Renewal and Pluripoten‐

[88] Hanna LA, Foreman RK, Tarasenko IA, Kessler DS, Labosky PA. Requirement for Foxd3 in maintaining pluripotent cells of the early mouse embryo. Gene Dev.

[89] Rosner MH, Vigano MA, Ozato K, Timmons PM, Poirier F, Rigby PW, et al. A POUdomain transcription factor in early stem cells and germ cells of the mammalian em‐

[90] Scholer HR, Ruppert S, Suzuki N, Chowdhury K, Gruss P. New type of POU domain

[91] Okamoto K, Okazawa H, Okuda A, Sakai M, Muramatsu M, Hamada H. A Novel Octamer Binding Transcription Factor Is Differentially Expressed in Mouse Embry‐

[92] Yeom YI, Ha HS, Balling R, Scholer HR, Artzt K. Structure, expression and chromo‐

in germ line-specific protein Oct-4. Nature. 1990;344(6265):435-9.

somal location of the Oct-4 gene. Mech Dev. 1991;35(3):171-9.

reprogramming. Cell. 2008;132(4):567-82.

ological Chemistry. 1999;274(28):19838-45.

597-600.

2003;260(2):404-13.

Cell. 2003;113(5):643-55.

2002;16(20):2650-61.

and ES cells. Cell. 2003;113(5):631-42.

bryo. Nature. 1990;345(6277):686-92.

onic-Cells. Cell. 1990;60(3):461-72.

cy by Foxd3. Stem Cells. 2008;26(10):2475-84.

cell molecular signature. Science. 2002;298(5593):601-4.

transcription factor Oct4. Cell. 1998;95(3):379-91.


[79] Jaenisch R, Young R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell. 2008;132(4):567-82.

[64] Kielman MF, Rindapaa M, Gaspar C, van Poppel N, Breukel C, van Leeuwen S, et al. Apc modulates embryonic stem-cell differentiation by controlling the dosage of beta-

[65] Pereira L, Yi F, Merrill BJ. Repression of Nanog gene transcription by Tcf3 limits em‐

[66] Takao Y, Yokota T, Koide H. Beta-catenin up-regulates Nanog expression through in‐ teraction with Oct-3/4 in embryonic stem cells. Biochem Biophys Res Commun.

[67] Wray J, Kalkan T, Smith AG. The ground state of pluripotency. Biochem Soc Trans.

[68] Bao SQ, Tang FC, Li XH, Hayashi K, Gillich A, Lao KQ, et al. Epigenetic reversion of post-implantation epiblast to pluripotent embryonic stem cells. Nature.

[69] Bao S, Tang F, Li X, Hayashi K, Gillich A, Lao K, et al. Epigenetic reversion of post-im‐ plantation epiblast to pluripotent embryonic stem cells. Nature. 2009;461(7268):1292-5.

[70] Hanna J, Cheng AW, Saha K, Kim J, Lengner CJ, Soldner F, et al. Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse

[71] Nichols J, Smith A. Naive and primed pluripotent states. Cell Stem Cell. 2009;4(6):487-92.

[72] Silva J, Barrandon O, Nichols J, Kawaguchi J, Theunissen TW, Smith A. Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol.

[73] Silva J, Nichols J, Theunissen TW, Guo G, van Oosten AL, Barrandon O, et al. Nanog

[74] Tesar PJ, Chenoweth JG, Brook FA, Davies TJ, Evans EP, Mack DL, et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells.

[75] Pera MF, Tam PP. Extrinsic regulation of pluripotent stem cells. Nature.

[76] Tchieu J, Kuoy E, Chin MH, Trinh H, Patterson M, Sherman SP, et al. Female human

[77] Hanna JH, Saha K, Jaenisch R. Pluripotency and cellular reprogramming: facts, hy‐

[78] Lengner CJ, Gimelbrant AA, Erwin JA, Cheng AW, Guenther MG, Welstead GG, et al. Derivation of pre-X inactivation human embryonic stem cells under physiological

iPSCs retain an inactive X chromosome. Cell Stem Cell. 2010;7(3):329-42.

potheses, unresolved issues. Cell. 2010;143(4):508-25.

oxygen concentrations. Cell. 2010;141(5):872-83.

is the gateway to the pluripotent ground state. Cell. 2009;138(4):722-37.

bryonic stem cell self-renewal. Mol Cell Biol. 2006;26(20):7479-91.

catenin signaling. Nat Genet. 2002;32(4):594-605.

ESCs. Proc Natl Acad Sci U S A. 2010;107(20):9222-7.

2007;353(3):699-705.

296 Pluripotent Stem Cells

2010;38(4):1027-32.

2009;461(7268):1292-5.

2008;6(10):e253.

Nature. 2007;448(7150):196-9.

2010;465(7299):713-20.


[93] Nishimoto M, Fukushima A, Okuda A, Muramatsu M. The gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4 and Sox-2. Mol Cell Biol. 1999;19(8):5453-65.

[107] Kuroda T, Tada M, Kubota H, Kimura H, Hatano S, Suemori H, et al. Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expres‐

Molecular Mechanisms of Embryonic Stem Cell Pluripotency

http://dx.doi.org/10.5772/54365

299

[108] Nakatake Y, Fukui N, Iwamatsu Y, Masui S, Takahashi K, Yagi R, et al. Klf4 coopera‐ tes with Oct3/4 and Sox2 to activate the Lefty1 core promoter in embryonic stem

[109] Ivanova N, Dobrin R, Lu R, Kotenko I, Levorse J, DeCoste C, et al. Dissecting selfrenewal in stem cells with RNA interference. Nature. 2006;442(7102):533-8.

[110] Chambers I, Silva J, Colby D, Nichols J, Nijmeijer B, Robertson M, et al. Nanog safeguards pluripotency and mediates germline development. Nature. 2007;450(7173):1230-4.

[111] Suzuki A, Raya A, Kawakami Y, Morita M, Matsui T, Nakashima K, et al. Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiation of

[112] Vallier L, Mendjan S, Brown S, Chng Z, Teo A, Smithers LE, et al. Activin/Nodal sig‐ nalling maintains pluripotency by controlling Nanog expression. Development.

[113] Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, et al. Core transcrip‐ tional regulatory circuitry in human embryonic stem cells. Cell. 2005;122(6):947-56.

[114] Loh YH, Wu Q, Chew JL, Vega VB, Zhang W, Chen X, et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat

[115] Mathur D, Danford TW, Boyer LA, Young RA, Gifford DK, Jaenisch R. Analysis of the mouse embryonic stem cell regulatory networks obtained by ChIP-chip and

[116] Sharov AA, Masui S, Sharova LV, Piao Y, Aiba K, Matoba R, et al. Identification of Pou5f1, Sox2, and Nanog downstream target genes with statistical confidence by ap‐ plying a novel algorithm to time course microarray and genome-wide chromatin im‐

[117] Masui S, Nakatake Y, Toyooka Y, Shimosato D, Yagi R, Takahashi K, et al. Pluripo‐ tency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic

[118] Wang J, Rao S, Chu J, Shen X, Levasseur DN, Theunissen TW, et al. A protein interac‐ tion network for pluripotency of embryonic stem cells. Nature. 2006;444(7117):364-8.

[119] Goke J, Jung M, Behrens S, Chavez L, O'Keeffe S, Timmermann B, et al. Combinatori‐ al Binding in Human and Mouse Embryonic Stem Cells Identifies Conserved En‐ hancers Active in Early Embryonic Development. Plos Comput Biol. 2011;7(12).

embryonic stem cells. Proc Natl Acad Sci U S A. 2006;103(27):10294-9.

sion. Mol Cell Biol. 2005;25(6):2475-85.

cells. Mol Cell Biol. 2006;26(20):7772-82.

2009;136(8):1339-49.

Genet. 2006;38(4):431-40.

ChIP-PET. Genome Biol. 2008;9(8).

munoprecipitation data. BMC Genomics. 2008;9:269.

stem cells. Nat Cell Biol. 2007;9(6):625-35.


[107] Kuroda T, Tada M, Kubota H, Kimura H, Hatano S, Suemori H, et al. Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expres‐ sion. Mol Cell Biol. 2005;25(6):2475-85.

[93] Nishimoto M, Fukushima A, Okuda A, Muramatsu M. The gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4 and Sox-2. Mol Cell Biol. 1999;19(8):5453-65. [94] Tomioka M, Nishimoto M, Miyagi S, Katayanagi T, Fukui N, Niwa H, et al. Identifi‐ cation of Sox-2 regulatory region which is under the control of Oct-3/4-Sox-2 com‐

[95] Zeng XM, Miura T, Luo YQ, Bhattacharya B, Condie B, Chen J, et al. Properties of pluripotent human embryonic stem cells BG01 and BG02. Stem Cells. 2004;22(3):

[96] Niwa H, Toyooka T, Shimosato D, Strumpf D, Takahashi K, Yagi R, et al. Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. Cell.

[97] Ovitt CE, Scholer HR. The molecular biology of Oct-4 in the early mouse embryo.

[98] Pan GJ, Chang ZY, Scholer HR, Pei DQ. Stem cell pluripotency and transcription fac‐

[99] Yeom YI, Fuhrmann G, Ovitt CE, Brehm A, Ohbo K, Gross M, et al. Germline regula‐ tory element of Oct-4 specific for the totipotent cycle of embryonal cells. Develop‐

[100] Niwa H. Molecular mechanism to maintain stem cell renewal of ES cells. Cell Struct

[101] Li M, Pevny L, Lovell-Badge R, Smith A. Generation of purified neural precursors from embryonic stem cells by lineage selection. Curr Biol. 1998;8(17):971-4.

[102] Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R. Multipotent cell lineages in early mouse development depend on SOX2 function. Gene Dev.

[103] Zappone MV, Galli R, Catena R, Meani N, De Biasi S, Mattei E, et al. Sox2 regulatory sequences direct expression of a (beta)-geo transgene to telencephalic neural stem cells and precursors of the mouse embryo, revealing regionalization of gene expres‐

[104] Okumura-Nakanishi S, Saito M, Niwa H, Ishikawa F. Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem cells. Journal of Biological Chemistry. 2005;280(7):

[105] Tokuzawa Y, Kaiho E, Maruyama M, Takahashi K, Mitsui K, Maeda M, et al. Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and

[106] Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH, et al. Transcriptional regulation of Nanog by Oct4 and Sox2. Journal of Biological Chemistry. 2005;280(26):24731-7.

sion in CNS stem cells. Development. 2000;127(11):2367-82.

mouse development. Mol Cell Biol. 2003;23(8):2699-708.

plex. Nucleic Acids Res. 2002;30(14):3202-13.

Mol Hum Reprod. 1998;4(11):1021-31.

tor Oct4. Cell Res. 2002;12(5-6):321-9.

ment. 1996;122(3):881-94.

Funct. 2001;26(3):137-48.

2003;17(1):126-40.

5307-17.

292-312.

298 Pluripotent Stem Cells

2005;123(5):917-29.


**Chapter 14**

**Epigenetic Instability in Embryonic Stem Cells**

Embryonic stem (ES) cells constitute a very important tool for regenerative medicine today. Human ES cells, in particular, are almost all derived from embryos obtained by *in vitro* fer‐ tilization (IVF) followed by *in vitro* culture (IVC); however, such *in vitro* manipulated em‐ bryos often show epigenetic abnormalities in imprinted genes that can lead to the development of various diseases. We recently reported that epigenetic differences occurred between ES cells derived from *in vivo* developed embryos (Vivo ES) and ES cells derived from *in vitro* manipulated embryos (Vitro ES) [1]. In addition, we found that the DNA meth‐ ylation state of uniparental and somatic cell nuclear transfer (SCNT) ES cells exhibits epige‐ netic instability during *in vitro* culture [2]. In this chapter, we review studies that have examined the epigenetic instability of ES cells during generation and maintenance cultures, and discuss the candidate factors that may be responsible for this epigenetic instability.

In vertebrate genomic DNA, the 5' cytosine residues in CpG sequences are often methylated [3]. DNA methylation plays an essential role in the normal development of mammalian em‐ bryos by regulating gene expression through genomic imprinting and X chromosome inacti‐ vation, and confers genomic stability [4-7]. In this chapter, we focus primarily on genomic imprinting, which is the preferential silencing of one of the parental alleles of a gene by epi‐ genetic DNA methylation since epigenetic modifications to some imprinted genes cause dis‐ eases such as Beckwith-Wiedemann syndrome and Prader-Willie syndrome. For example, the expression level of the *H19* imprinted gene is regulated by an upstream differentially methylated region (DMR), and epigenetic alterations to the DMR result in Beckwith-Wiede‐ mann syndrome [8-10]. The *H19* mRNA is transcribed from the unmethylated maternal al‐

> © 2013 Horii and Hatada; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

> © 2013 Horii and Hatada; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Takuro Horii and Izuho Hatada

http://dx.doi.org/10.5772/54367

**1. Introduction**

Additional information is available at the end of the chapter

**2. Epigenetic regulation by DNA methylation**

## **Epigenetic Instability in Embryonic Stem Cells**

Takuro Horii and Izuho Hatada

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54367

## **1. Introduction**

Embryonic stem (ES) cells constitute a very important tool for regenerative medicine today. Human ES cells, in particular, are almost all derived from embryos obtained by *in vitro* fer‐ tilization (IVF) followed by *in vitro* culture (IVC); however, such *in vitro* manipulated em‐ bryos often show epigenetic abnormalities in imprinted genes that can lead to the development of various diseases. We recently reported that epigenetic differences occurred between ES cells derived from *in vivo* developed embryos (Vivo ES) and ES cells derived from *in vitro* manipulated embryos (Vitro ES) [1]. In addition, we found that the DNA meth‐ ylation state of uniparental and somatic cell nuclear transfer (SCNT) ES cells exhibits epige‐ netic instability during *in vitro* culture [2]. In this chapter, we review studies that have examined the epigenetic instability of ES cells during generation and maintenance cultures, and discuss the candidate factors that may be responsible for this epigenetic instability.

## **2. Epigenetic regulation by DNA methylation**

In vertebrate genomic DNA, the 5' cytosine residues in CpG sequences are often methylated [3]. DNA methylation plays an essential role in the normal development of mammalian em‐ bryos by regulating gene expression through genomic imprinting and X chromosome inacti‐ vation, and confers genomic stability [4-7]. In this chapter, we focus primarily on genomic imprinting, which is the preferential silencing of one of the parental alleles of a gene by epi‐ genetic DNA methylation since epigenetic modifications to some imprinted genes cause dis‐ eases such as Beckwith-Wiedemann syndrome and Prader-Willie syndrome. For example, the expression level of the *H19* imprinted gene is regulated by an upstream differentially methylated region (DMR), and epigenetic alterations to the DMR result in Beckwith-Wiede‐ mann syndrome [8-10]. The *H19* mRNA is transcribed from the unmethylated maternal al‐

© 2013 Horii and Hatada; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Horii and Hatada; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

lele but is not transcribed from the methylated paternal allele (Fig. 1). In contrast, DMRs of *Peg1* (Mest), *Snrpn* and *Igf2r* are methylated in the maternal allele and unmethylated in the paternal allele. Genomic imprinting is very stable except for the period when the reprogram‐ ming of genomic imprinting takes place in germline cells [11]. For the establishment and maintenance of DNA methylation, the cytosine-guanine (CpG) DNA methyltransferases (Dnmts), Dnmt1, Dnmt3a, and Dnmt3b, are the main factors that coordinately regulate CpG methylation in the genome [12-14]. Dnmt1 is involved in maintenance activity, while Dnmt3a and Dnmt3b are responsible primarily for the creation of new methylation patterns.

al applications. However, ES cells lose their pluripotency during prolonged *in vitro* culture [20]. Several studies indicate that the accumulation of epigenetic alterations over time is corre‐ lated with the loss of pluripotency in ES cells. Dean *et al*. reported that epigenetic alterations that occur in ES cells persist to later developmental stages and are associated with aberrant phenotypes in completely ES cell-derived mice [21]. Humpherys *et al*. show that variation in imprinted gene expression is observed in most cloned mice derived from ES cell donors, even those derived from ES cells of the same subclone [22]. Such epigenetic drift of imprinted genes was also observed in our experiments during prolonged culture of mouse ES cells (Fig. 2): DNA methylation of four imprinted genes, *Peg1*, *Snrpn*, *Igf2r* and *H19*, was unstable during cell cul‐ ture (P3-30), even in the same cell line, over time. Minoguchi and Iba reported that retroviral DNA that is introduced into mouse ES cells is progressively silenced by DNA methylation; however, a substantial amount of retroviral DNA is reversibly reactivated by DNA demethyla‐ tion [23]. Such epigenetic drift has also been observed in human ES cells, depending on the

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**Figure 2.** Epigenetic drift of imprinting methylations in fertilized embryo-derived ES cells. A. Combined bisulfite restriction analysis (COBRA) was conducted for three fertilized embryo-derived ES cell lines (B6-2, B6-6 and B6-8) during prolonged *in vitro* culture (P3, P10 and P30). The maternally methylated imprinted genes *Peg1*, *Snrpn* and *Igf2r*, and the paternally me‐ thylated imprinted gene, *H19*, were examined. B. Summary of imprinting methylations during prolonged culture of ES

cells. dig, digestion by restriction enzymes; u, unmethylated PCR products; m, methylated PCR products.

method of establishment and the culture conditions [24].

**Figure 1.** Regulation of gene expression in the *H19* imprinted gene.

## **3. Epigenetic instability in preimplantation embryos**

In general, ES cells, especially human ES cells, are generated from blastocyst stage embryos that are produced by *in vitro* manipulations such as IVF and IVC. However, *in vitro* manipulat‐ ed embryos may already possess epigenetic abnormalities because the culture conditions of fertilized embryos can influence the methylation state. For example, a sub-optimal culture me‐ dium (e.g., Whitten's medium) can cause aberrant genomic imprinting of the *H19* gene [15], and culture medium supplemented with fetal calf serum alters mRNA expression of imprinted genes [16]. Our recent study suggests that altered DNA methylation due to IVC conditions oc‐ curs not only in imprinted genes but also in genome-wide repetitive sequences, such as major and minor satellite sequences [17]. Thus, alteration of DNA methylation can occur in response to various factors, from the moment when embryos are collected from the oviducts or uterus.

## **4. Epigenetic instability in ES cells during prolonged culture**

ES cells are established from the inner cell mass (ICM) of blastocyst stage embryos [18,19]. Once ES cell lines are established, they can be maintained for long periods of time and used for sever‐ al applications. However, ES cells lose their pluripotency during prolonged *in vitro* culture [20]. Several studies indicate that the accumulation of epigenetic alterations over time is corre‐ lated with the loss of pluripotency in ES cells. Dean *et al*. reported that epigenetic alterations that occur in ES cells persist to later developmental stages and are associated with aberrant phenotypes in completely ES cell-derived mice [21]. Humpherys *et al*. show that variation in imprinted gene expression is observed in most cloned mice derived from ES cell donors, even those derived from ES cells of the same subclone [22]. Such epigenetic drift of imprinted genes was also observed in our experiments during prolonged culture of mouse ES cells (Fig. 2): DNA methylation of four imprinted genes, *Peg1*, *Snrpn*, *Igf2r* and *H19*, was unstable during cell cul‐ ture (P3-30), even in the same cell line, over time. Minoguchi and Iba reported that retroviral DNA that is introduced into mouse ES cells is progressively silenced by DNA methylation; however, a substantial amount of retroviral DNA is reversibly reactivated by DNA demethyla‐ tion [23]. Such epigenetic drift has also been observed in human ES cells, depending on the method of establishment and the culture conditions [24].

lele but is not transcribed from the methylated paternal allele (Fig. 1). In contrast, DMRs of *Peg1* (Mest), *Snrpn* and *Igf2r* are methylated in the maternal allele and unmethylated in the paternal allele. Genomic imprinting is very stable except for the period when the reprogram‐ ming of genomic imprinting takes place in germline cells [11]. For the establishment and maintenance of DNA methylation, the cytosine-guanine (CpG) DNA methyltransferases (Dnmts), Dnmt1, Dnmt3a, and Dnmt3b, are the main factors that coordinately regulate CpG methylation in the genome [12-14]. Dnmt1 is involved in maintenance activity, while Dnmt3a and Dnmt3b are responsible primarily for the creation of new methylation patterns.

**Figure 1.** Regulation of gene expression in the *H19* imprinted gene.

302 Pluripotent Stem Cells

**3. Epigenetic instability in preimplantation embryos**

**4. Epigenetic instability in ES cells during prolonged culture**

In general, ES cells, especially human ES cells, are generated from blastocyst stage embryos that are produced by *in vitro* manipulations such as IVF and IVC. However, *in vitro* manipulat‐ ed embryos may already possess epigenetic abnormalities because the culture conditions of fertilized embryos can influence the methylation state. For example, a sub-optimal culture me‐ dium (e.g., Whitten's medium) can cause aberrant genomic imprinting of the *H19* gene [15], and culture medium supplemented with fetal calf serum alters mRNA expression of imprinted genes [16]. Our recent study suggests that altered DNA methylation due to IVC conditions oc‐ curs not only in imprinted genes but also in genome-wide repetitive sequences, such as major and minor satellite sequences [17]. Thus, alteration of DNA methylation can occur in response to various factors, from the moment when embryos are collected from the oviducts or uterus.

ES cells are established from the inner cell mass (ICM) of blastocyst stage embryos [18,19]. Once ES cell lines are established, they can be maintained for long periods of time and used for sever‐

**Figure 2.** Epigenetic drift of imprinting methylations in fertilized embryo-derived ES cells. A. Combined bisulfite restriction analysis (COBRA) was conducted for three fertilized embryo-derived ES cell lines (B6-2, B6-6 and B6-8) during prolonged *in vitro* culture (P3, P10 and P30). The maternally methylated imprinted genes *Peg1*, *Snrpn* and *Igf2r*, and the paternally me‐ thylated imprinted gene, *H19*, were examined. B. Summary of imprinting methylations during prolonged culture of ES cells. dig, digestion by restriction enzymes; u, unmethylated PCR products; m, methylated PCR products.

## **5. Epigenetic differences between male and female ES cells**

Large differences in epigenetic drift have been observed between male (XY) and female (XX) mouse ES cells. Global demethylation, including imprinted genes and satellite re‐ peats, occurred more frequently in female ES cell lines compared to male ES cell lines [21, 25]. This global demethylation reflects the number and state of X chromosomes in ES cells. In general, both X chromosomes are active in female ES cells, whereas male ES cells have only one active X chromosome. The X chromosome state in female ES cells is thought to lead to downregulation of DNA methyltransferases (Dnmt3a and Dnmt3b) and, ultimately, to global hypomethylation [25]. Thus, DNA methylation of imprinted genes and repetitive sequences are gained or lost at high rates even in clonal popula‐ tions of ES cells, and these alterations may have deleterious effects on phenotypes of ES cell-derived animals or tissues.

**6.2. Gene expression of** *vivo* **and** *vitro* **ES cells**

We assessed gene expression patterns in ES cells at early and late passages by quantitative real-time RT-PCR. The expression of *Oct3/4* mRNA, a pluripotent cell marker, was signifi‐ cantly higher in early passage Vivo ES cells than in Vitro ES cells, whereas other pluripotent marker genes, *Nanog* and *Stella*, showed no significant differences in expression levels be‐ tween the two types of ES cells. Among the methylation-related genes, mRNA expression of the *de novo* DNA methyltransferase, Dnmt3b, was significantly higher in Vivo ES cells. Ex‐ pression of growth arrest and DNA damage-inducible protein 45 beta (Gadd45*b*), which is a putative demethylation factor [28,29], is higher in Vitro ES cells. Thus, mRNA expression patterns of several methylation-related genes tended to shift, resulting in the promotion of demethylation and the inhibition of methylation in Vitro ES cells. In contrast, at later passag‐ es, no significant differences between Vivo and Vitro ES cells were found with respect to the

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**Figure 3.** Epigenetic differences between Vivo and Vitro ES cells. DNA methylation status of imprinted genes, *H19*, *Snrpn* and *Igf2r*, and major satellite repeats were examined by COBRA in each ES cell line at an early passage (P2) and

a later passage (P5). These graphs summarize previously reported data [1]. \*, *P* < 0.05.

pluripotent marker genes and methylation-related genes that were examined.

## **6. Epigenetic differences between** *vivo* **and** *vitro* **ES cells**

#### **6.1. Methylation state of** *vivo* **and** *vitro* **ES cells**

In human ES cells, several studies have recently provided evidence for the efficient induc‐ tion of endoderm, mesoderm, and ectoderm, and many of their downstream derivatives [26], and these reports offer broad possibilities for regenerative medicine. However, all hu‐ man ES cell lines are established from *in vitro* manipulated embryos that often show abnor‐ mal genomic imprinting, which can lead to an increase in the frequency of diseases. Therefore, we have compared the methylation state of imprinted genes and the gene expres‐ sion patterns of both Vivo and Vitro ES cell lines in mice [1].

Although the genomic imprinting is maintained during preimplantation development, nor‐ mal imprinting can occasionally be disrupted in preimplantation embryos during IVC, re‐ sulting in biallelic expression of the *H19* gene [15,27]. To investigate whether Vitro ES cells take on abnormal imprinting from IVC blastocysts, we performed methylation analysis of the *H19* DMR for early passage (P2) cells (Fig. 3). COBRA analysis shows that the *H19* DMR is significantly demethylated in Vitro ES cells compared to Vivo ES cells. The *Igf2r* DMR2 also showed significant differences among Vitro vs. Vivo ES cells, but significant differences in the methylation of *Snrpn* and the major satellite repeats were not detected.

In additional experiments, both Vivo and Vitro ES cells were passaged several more times, and the methylation state of imprinted genes and satellite repeats was investigated at later passages (P5) (Fig. 3). Results from COBRA analysis at P5 showed no significant differences between Vivo and Vitro ES cells. Even Vivo ES cells exhibited highly demethylated alleles. In contrast, some Vitro ES cells had an almost normally methylated allele. This result indi‐ cates that the methylation state of ES cells at later passages depends more on the character of the individual cell lines than on the origin of the ES cells.

#### **6.2. Gene expression of** *vivo* **and** *vitro* **ES cells**

**5. Epigenetic differences between male and female ES cells**

**6. Epigenetic differences between** *vivo* **and** *vitro* **ES cells**

cell-derived animals or tissues.

304 Pluripotent Stem Cells

**6.1. Methylation state of** *vivo* **and** *vitro* **ES cells**

sion patterns of both Vivo and Vitro ES cell lines in mice [1].

the individual cell lines than on the origin of the ES cells.

Large differences in epigenetic drift have been observed between male (XY) and female (XX) mouse ES cells. Global demethylation, including imprinted genes and satellite re‐ peats, occurred more frequently in female ES cell lines compared to male ES cell lines [21, 25]. This global demethylation reflects the number and state of X chromosomes in ES cells. In general, both X chromosomes are active in female ES cells, whereas male ES cells have only one active X chromosome. The X chromosome state in female ES cells is thought to lead to downregulation of DNA methyltransferases (Dnmt3a and Dnmt3b) and, ultimately, to global hypomethylation [25]. Thus, DNA methylation of imprinted genes and repetitive sequences are gained or lost at high rates even in clonal popula‐ tions of ES cells, and these alterations may have deleterious effects on phenotypes of ES

In human ES cells, several studies have recently provided evidence for the efficient induc‐ tion of endoderm, mesoderm, and ectoderm, and many of their downstream derivatives [26], and these reports offer broad possibilities for regenerative medicine. However, all hu‐ man ES cell lines are established from *in vitro* manipulated embryos that often show abnor‐ mal genomic imprinting, which can lead to an increase in the frequency of diseases. Therefore, we have compared the methylation state of imprinted genes and the gene expres‐

Although the genomic imprinting is maintained during preimplantation development, nor‐ mal imprinting can occasionally be disrupted in preimplantation embryos during IVC, re‐ sulting in biallelic expression of the *H19* gene [15,27]. To investigate whether Vitro ES cells take on abnormal imprinting from IVC blastocysts, we performed methylation analysis of the *H19* DMR for early passage (P2) cells (Fig. 3). COBRA analysis shows that the *H19* DMR is significantly demethylated in Vitro ES cells compared to Vivo ES cells. The *Igf2r* DMR2 also showed significant differences among Vitro vs. Vivo ES cells, but significant differences

In additional experiments, both Vivo and Vitro ES cells were passaged several more times, and the methylation state of imprinted genes and satellite repeats was investigated at later passages (P5) (Fig. 3). Results from COBRA analysis at P5 showed no significant differences between Vivo and Vitro ES cells. Even Vivo ES cells exhibited highly demethylated alleles. In contrast, some Vitro ES cells had an almost normally methylated allele. This result indi‐ cates that the methylation state of ES cells at later passages depends more on the character of

in the methylation of *Snrpn* and the major satellite repeats were not detected.

We assessed gene expression patterns in ES cells at early and late passages by quantitative real-time RT-PCR. The expression of *Oct3/4* mRNA, a pluripotent cell marker, was signifi‐ cantly higher in early passage Vivo ES cells than in Vitro ES cells, whereas other pluripotent marker genes, *Nanog* and *Stella*, showed no significant differences in expression levels be‐ tween the two types of ES cells. Among the methylation-related genes, mRNA expression of the *de novo* DNA methyltransferase, Dnmt3b, was significantly higher in Vivo ES cells. Ex‐ pression of growth arrest and DNA damage-inducible protein 45 beta (Gadd45*b*), which is a putative demethylation factor [28,29], is higher in Vitro ES cells. Thus, mRNA expression patterns of several methylation-related genes tended to shift, resulting in the promotion of demethylation and the inhibition of methylation in Vitro ES cells. In contrast, at later passag‐ es, no significant differences between Vivo and Vitro ES cells were found with respect to the pluripotent marker genes and methylation-related genes that were examined.

**Figure 3.** Epigenetic differences between Vivo and Vitro ES cells. DNA methylation status of imprinted genes, *H19*, *Snrpn* and *Igf2r*, and major satellite repeats were examined by COBRA in each ES cell line at an early passage (P2) and a later passage (P5). These graphs summarize previously reported data [1]. \*, *P* < 0.05.

## **7. Epigenetic instability in SCNT and uniparental ES cells**

#### **7.1. SCNT ES cells**

Maintenance of the normal epigenetic state in SCNT-ES cells is crucial for their use in thera‐ peutic applications. We established two SCNT-ES cell lines from embryos that were pro‐ duced by introducing mouse embryonic fibroblast (MEF) donor cells into enucleated oocytes. Only two ES cell lines were generated by SCNT, which give a small sample size to examine, but the DNA methylation state of imprinted genes seems to be more severely al‐ tered compared to normal ES cell lines at early passages (Fig. 2 and Fig. 4). The abnormal DNA methylation in SCNT-ES cells undergoes further changes during prolonged culture (P10 and P30). For example, the imprinting methylation of the *Snrpn* gene has been com‐ pletely lost in both the Nt-1 and Nt-2 lines, and that of the *H19* gene has been completely lost in the Nt-1 line (Fig. 4). Chang *et al.* reported that the *H19* imprinted gene displays dis‐ tinct abnormalities both in SCNT-ES and fertilized embryo-derived ES cell lines after longterm culture *in vitro*, and both exhibit indistinguishable DNA methylation patterns of the imprinted gene [30]. Nevertheless, methylation imprints vary widely in cultured donor cells and their derivative cloned mice, even across the same subclone of donor cells [22]. In fact, results from previous studies indicate that the methylation state of imprinted genes is fre‐ quently disrupted in SCNT embryos and their derivative cloned animals [31,32]. In addition, the process of nuclear transfer itself could alter the DNA methylation and gene expression [33]. Thus, the epigenetic marks in SCNT-ES cells may potentially be varied and altered compared to normal ES cells, at least in early passages.

terms of gene expression pattern and pluripotency. Thus, reprogrammed PgES cells will provide a good tool for therapeutic applications. This is a case in which epigenetic insta‐ bility in ES cells resulted in a desirable outcome. However, epigenetic instability in ES

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**Figure 4.** Epigenetic instability in SCNT-ES cells during prolonged culture. Methylation in two SCNT ES cell lines (Nt-1

In humans, a growing number of reports suggest that children born following ART have an increased risk of developing epigenetic diseases such as Beckwith-Wiedemann syndrome [41,42] and Angelman Syndrome [43], which are caused by epigenetic modifications of im‐

**8. Effect of altered DNA methylation on pluripotency and disease**

and Nt-2) was examined by COBRA during prolonged *in vitro* culture (P3, P10 and P30).

cells most often leads to undesirable results.

#### **7.2. Uniparental (parthenogenetic) ES cells**

We and other groups have suggested that parthenogenetic ES (PgES) cells may be a plu‐ ripotent stem cell that could serve as a source of tissue for transplantation [34-36]. PgES cells do not require the destruction of viable biparental embryos as do normal ES cells. In addition, PgES cells do not need viruses or expression plasmids for the establishment of iPS cells. These are very powerful advantages for therapeutic applications. However, the biased epigenetic status and poor pluripotency of parthenogenetic cells are major is‐ sues to be overcome. PgES cells are established from parthenogenetic embryos that are produced by the artificial activation of the oocyte. Therefore, PgES cells that possess only maternal genomes could exhibit biallelic or silenced expression of imprinted genes, which causes poor pluripotency. Indeed, parthenogenetic embryos show poor growth and restricted tissue contribution in chimeras [37,38]. However, established PgES cells ex‐ hibit an improved contribution to chimeras, compared to chimeras derived from parthe‐ nogenetic embryos [39]. Recent reports have shown that loss of imprinting occurred in PgES cells and derivative somatic cells in chimeras and led to changes in the gene ex‐ pression of imprinted genes and improved pluripotency [2,40]. For example, *Peg1* and *Snrpn* genes are originally silenced in parthenogenetic cells, whereas expression of these genes is elevated in PgES cells by demethylation of the DMR of each gene. PgES cell lines that were reprogrammed by loss of imprinting are closest to normal ES cell lines in terms of gene expression pattern and pluripotency. Thus, reprogrammed PgES cells will provide a good tool for therapeutic applications. This is a case in which epigenetic insta‐ bility in ES cells resulted in a desirable outcome. However, epigenetic instability in ES cells most often leads to undesirable results.

**7. Epigenetic instability in SCNT and uniparental ES cells**

compared to normal ES cells, at least in early passages.

**7.2. Uniparental (parthenogenetic) ES cells**

Maintenance of the normal epigenetic state in SCNT-ES cells is crucial for their use in thera‐ peutic applications. We established two SCNT-ES cell lines from embryos that were pro‐ duced by introducing mouse embryonic fibroblast (MEF) donor cells into enucleated oocytes. Only two ES cell lines were generated by SCNT, which give a small sample size to examine, but the DNA methylation state of imprinted genes seems to be more severely al‐ tered compared to normal ES cell lines at early passages (Fig. 2 and Fig. 4). The abnormal DNA methylation in SCNT-ES cells undergoes further changes during prolonged culture (P10 and P30). For example, the imprinting methylation of the *Snrpn* gene has been com‐ pletely lost in both the Nt-1 and Nt-2 lines, and that of the *H19* gene has been completely lost in the Nt-1 line (Fig. 4). Chang *et al.* reported that the *H19* imprinted gene displays dis‐ tinct abnormalities both in SCNT-ES and fertilized embryo-derived ES cell lines after longterm culture *in vitro*, and both exhibit indistinguishable DNA methylation patterns of the imprinted gene [30]. Nevertheless, methylation imprints vary widely in cultured donor cells and their derivative cloned mice, even across the same subclone of donor cells [22]. In fact, results from previous studies indicate that the methylation state of imprinted genes is fre‐ quently disrupted in SCNT embryos and their derivative cloned animals [31,32]. In addition, the process of nuclear transfer itself could alter the DNA methylation and gene expression [33]. Thus, the epigenetic marks in SCNT-ES cells may potentially be varied and altered

We and other groups have suggested that parthenogenetic ES (PgES) cells may be a plu‐ ripotent stem cell that could serve as a source of tissue for transplantation [34-36]. PgES cells do not require the destruction of viable biparental embryos as do normal ES cells. In addition, PgES cells do not need viruses or expression plasmids for the establishment of iPS cells. These are very powerful advantages for therapeutic applications. However, the biased epigenetic status and poor pluripotency of parthenogenetic cells are major is‐ sues to be overcome. PgES cells are established from parthenogenetic embryos that are produced by the artificial activation of the oocyte. Therefore, PgES cells that possess only maternal genomes could exhibit biallelic or silenced expression of imprinted genes, which causes poor pluripotency. Indeed, parthenogenetic embryos show poor growth and restricted tissue contribution in chimeras [37,38]. However, established PgES cells ex‐ hibit an improved contribution to chimeras, compared to chimeras derived from parthe‐ nogenetic embryos [39]. Recent reports have shown that loss of imprinting occurred in PgES cells and derivative somatic cells in chimeras and led to changes in the gene ex‐ pression of imprinted genes and improved pluripotency [2,40]. For example, *Peg1* and *Snrpn* genes are originally silenced in parthenogenetic cells, whereas expression of these genes is elevated in PgES cells by demethylation of the DMR of each gene. PgES cell lines that were reprogrammed by loss of imprinting are closest to normal ES cell lines in

**7.1. SCNT ES cells**

306 Pluripotent Stem Cells

**Figure 4.** Epigenetic instability in SCNT-ES cells during prolonged culture. Methylation in two SCNT ES cell lines (Nt-1 and Nt-2) was examined by COBRA during prolonged *in vitro* culture (P3, P10 and P30).

#### **8. Effect of altered DNA methylation on pluripotency and disease**

In humans, a growing number of reports suggest that children born following ART have an increased risk of developing epigenetic diseases such as Beckwith-Wiedemann syndrome [41,42] and Angelman Syndrome [43], which are caused by epigenetic modifications of im‐ printed genes. In sheep, epigenetic changes in the *Igf2r* imprinted gene are associated with fetal overgrowth after IVC [44]. Genome-wide altered DNA methylation also causes epige‐ netic diseases. For example, genome-wide DNA hypomethylation is commonly observed in human cancers and schizophrenia, and occasionally induces tumors in mice [45-47]. More‐ over, hypomethylation in the classical DNA satellites II and III, which are major components of constitutive heterochromatin, is found in ICF (immunodeficiency, centromeric instability, facial anomalies) syndrome in humans [48].

**9.2. Other methylation factors**

**9.3. Active demethylation factors**

cause of epigenetic instability in ES cells.

**10. Conclusion**

**9.4. Chromatin structure specific to ES cells**

Other new methylation factors are Stella (PGC7) and Zfp57. Stella (PGC7), a primordial germ cell and ES cell marker, protects against DNA demethylation in early embryogenesis [58]. Zfp57, a putative KRAB zinc finger protein, is also required for the post-fertilization maintenance of maternal and paternal methylation at multiple imprinted domains [59]. Re‐ ductions of the levels of these factors could induce hypomethylation of DNA in ES cells.

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Active DNA demethylation via the base excision repair pathway has recently been proposed in mammals. In zebrafish, the coupling of a deaminase (activation-induced cytidine deami‐ nase, AID), a glycosylase (methyl-CpG binding domain protein 4, MBD4), and Gadd45 is in‐ volved in DNA demethylation [60]. In mammals, AID is indeed required for reprogramming of the somatic cell genome by demethylation of pluripotency genes in ESsomatic cell fusion [61]. Gadd45 also promotes epigenetic gene activation by repair-mediat‐ ed demethylation in mammals [28,29]. A *Gadd45b* gene is activated in Vitro ES cells that possess hypomethylated imprinted genes and repetitive sequences [1]. Another recently proposed demethylation pathway is the conversion of 5-methylcytosine (5-mC) to 5-hydrox‐ ymethylcytosine (5-hmC) mediated by the Ten-eleven translocation (TET) proteins, which ultimately results in DNA demethylation [62-63]. In fact, the TET proteins (Tet1 and Tet2) that regulate 5-hmC production [64] are abundantly expressed in ES cells and may be a

In ES cells, bivalent domains of chromatin, that regulate several key developmental genes, contain both repressive (histone H3 lysine 27 methylation) and activating (histone H3 lysine 4 methylation) histone modifications that are usually mutually exclusive [65]. Bivalent do‐ mains silence developmental genes in ES cells while preserving their potential to become ac‐ tivated upon initiation of specific differentiation programs. DNA methylation was thought to determine the chromatin structure; however, recent reports suggest that chromatin can af‐ fect DNA methylation and demethylation [66-67]. Therefore, bivalent chromatin modifica‐

ES cells exhibit instabilities in DNA methylation that are correlated with the origin of the blastocysts from which they were derived (*in vivo*, *in vitro*, SCNT and uniparental), the cul‐ ture conditions, sex, and prolonged culture. Epigenotyping of ES cells should be adopted as a prerequisite safety evaluation before their use in chimera production or therapeutic appli‐ cations. Furthermore, genes associated with aberrant DNA methylation should be moni‐ tored in ES cell lines to ensure that the cells do not accumulate epigenetic instabilities.

tions specific to ES cells could be associated with DNA methylation instability.

How do these abnormalities in ES cells affect chimeric mice or ES cell-derived tissues? Sev‐ eral studies have indicated that the accumulation of epigenetic alterations during prolonged culture causes a loss of pluripotency in ES cells [21,49]. In chimeras, prolonged culture of ES cells gives rise to abnormalities and frequently results in postnatal death of chimeras pos‐ sessing a high ES cell contribution [20]. One reason for these problems could be that a loss of imprinting enhances tumorigenesis. In fact, mice derived from ES cells that had a global loss of DNA methylation display widespread cancer formation [50].

## **9. Candidate genes that cause altered DNA methylation**

#### **9.1. DNA methyltransferases**

The most important factors for the maintenance of DNA methylation are the DNA meth‐ yltransferases. Three CpG DNA methyltransferases, Dnmt1, Dnmt3a and Dnmt3b, coor‐ dinately regulate CpG methylation in the genome [12-14]. Deletion of Dnmt1, Dnmt3a or Dnmt3b induces hypomethylation of genomic DNA [14,51], and forced expression of Dnmts causes genomic hypermethylation [52-54]. One of the Dnmt family members, Dnmt3L, is not expressed in differentiated somatic cells but is expressed in ES cells. Al‐ though Dnmt3L lacks the functional domains required for catalytic activity, overexpres‐ sion or downregulation of Dnmt3L results in changes in DNA methylation in ES cells [55]. Thus, the upregulation or downregulation of Dnmts could cause epigenetic instabili‐ ty in ES cells. Indeed, hypomethylation in XX ES cells is associated with reduced levels of Dnmt3a and Dnmt3b, which is the result of both X chromosomes being in the active state [25]. Among Dnmts, a number of alternative splicing variants that lack the regulato‐ ry and/or catalytic regions have been reported. In particular, Dnmt3b has nearly 40 dif‐ ferent isoforms generated by alternative splicing and/or alternative promoter usage. We recently reported that murine Dnmt3b lacking exon 6 (exon 5 in human) is highly ex‐ pressed in *in vitro* manipulated embryos and their derivative ES cells that exhibit CpG hypomethylation [17]. Gopalakrishnan *et al*. reported that this isoform is expressed in tu‐ mor and iPS cells, and that ectopic overexpression resulted in repetitive element hypo‐ methylation [56]. Similarly, forced expression of human specific DNMT3B4, which lacks a catalytic domain, induced DNA demethylation on satellite 2 in pericentromeric DNA [57]. These reports indicate that Dnmts have complex roles in the maintenance of the DNA methylation state. If this balance collapses, epigenetic instability will result.

#### **9.2. Other methylation factors**

printed genes. In sheep, epigenetic changes in the *Igf2r* imprinted gene are associated with fetal overgrowth after IVC [44]. Genome-wide altered DNA methylation also causes epige‐ netic diseases. For example, genome-wide DNA hypomethylation is commonly observed in human cancers and schizophrenia, and occasionally induces tumors in mice [45-47]. More‐ over, hypomethylation in the classical DNA satellites II and III, which are major components of constitutive heterochromatin, is found in ICF (immunodeficiency, centromeric instability,

How do these abnormalities in ES cells affect chimeric mice or ES cell-derived tissues? Sev‐ eral studies have indicated that the accumulation of epigenetic alterations during prolonged culture causes a loss of pluripotency in ES cells [21,49]. In chimeras, prolonged culture of ES cells gives rise to abnormalities and frequently results in postnatal death of chimeras pos‐ sessing a high ES cell contribution [20]. One reason for these problems could be that a loss of imprinting enhances tumorigenesis. In fact, mice derived from ES cells that had a global loss

The most important factors for the maintenance of DNA methylation are the DNA meth‐ yltransferases. Three CpG DNA methyltransferases, Dnmt1, Dnmt3a and Dnmt3b, coor‐ dinately regulate CpG methylation in the genome [12-14]. Deletion of Dnmt1, Dnmt3a or Dnmt3b induces hypomethylation of genomic DNA [14,51], and forced expression of Dnmts causes genomic hypermethylation [52-54]. One of the Dnmt family members, Dnmt3L, is not expressed in differentiated somatic cells but is expressed in ES cells. Al‐ though Dnmt3L lacks the functional domains required for catalytic activity, overexpres‐ sion or downregulation of Dnmt3L results in changes in DNA methylation in ES cells [55]. Thus, the upregulation or downregulation of Dnmts could cause epigenetic instabili‐ ty in ES cells. Indeed, hypomethylation in XX ES cells is associated with reduced levels of Dnmt3a and Dnmt3b, which is the result of both X chromosomes being in the active state [25]. Among Dnmts, a number of alternative splicing variants that lack the regulato‐ ry and/or catalytic regions have been reported. In particular, Dnmt3b has nearly 40 dif‐ ferent isoforms generated by alternative splicing and/or alternative promoter usage. We recently reported that murine Dnmt3b lacking exon 6 (exon 5 in human) is highly ex‐ pressed in *in vitro* manipulated embryos and their derivative ES cells that exhibit CpG hypomethylation [17]. Gopalakrishnan *et al*. reported that this isoform is expressed in tu‐ mor and iPS cells, and that ectopic overexpression resulted in repetitive element hypo‐ methylation [56]. Similarly, forced expression of human specific DNMT3B4, which lacks a catalytic domain, induced DNA demethylation on satellite 2 in pericentromeric DNA [57]. These reports indicate that Dnmts have complex roles in the maintenance of the

DNA methylation state. If this balance collapses, epigenetic instability will result.

facial anomalies) syndrome in humans [48].

308 Pluripotent Stem Cells

**9.1. DNA methyltransferases**

of DNA methylation display widespread cancer formation [50].

**9. Candidate genes that cause altered DNA methylation**

Other new methylation factors are Stella (PGC7) and Zfp57. Stella (PGC7), a primordial germ cell and ES cell marker, protects against DNA demethylation in early embryogenesis [58]. Zfp57, a putative KRAB zinc finger protein, is also required for the post-fertilization maintenance of maternal and paternal methylation at multiple imprinted domains [59]. Re‐ ductions of the levels of these factors could induce hypomethylation of DNA in ES cells.

#### **9.3. Active demethylation factors**

Active DNA demethylation via the base excision repair pathway has recently been proposed in mammals. In zebrafish, the coupling of a deaminase (activation-induced cytidine deami‐ nase, AID), a glycosylase (methyl-CpG binding domain protein 4, MBD4), and Gadd45 is in‐ volved in DNA demethylation [60]. In mammals, AID is indeed required for reprogramming of the somatic cell genome by demethylation of pluripotency genes in ESsomatic cell fusion [61]. Gadd45 also promotes epigenetic gene activation by repair-mediat‐ ed demethylation in mammals [28,29]. A *Gadd45b* gene is activated in Vitro ES cells that possess hypomethylated imprinted genes and repetitive sequences [1]. Another recently proposed demethylation pathway is the conversion of 5-methylcytosine (5-mC) to 5-hydrox‐ ymethylcytosine (5-hmC) mediated by the Ten-eleven translocation (TET) proteins, which ultimately results in DNA demethylation [62-63]. In fact, the TET proteins (Tet1 and Tet2) that regulate 5-hmC production [64] are abundantly expressed in ES cells and may be a cause of epigenetic instability in ES cells.

#### **9.4. Chromatin structure specific to ES cells**

In ES cells, bivalent domains of chromatin, that regulate several key developmental genes, contain both repressive (histone H3 lysine 27 methylation) and activating (histone H3 lysine 4 methylation) histone modifications that are usually mutually exclusive [65]. Bivalent do‐ mains silence developmental genes in ES cells while preserving their potential to become ac‐ tivated upon initiation of specific differentiation programs. DNA methylation was thought to determine the chromatin structure; however, recent reports suggest that chromatin can af‐ fect DNA methylation and demethylation [66-67]. Therefore, bivalent chromatin modifica‐ tions specific to ES cells could be associated with DNA methylation instability.

### **10. Conclusion**

ES cells exhibit instabilities in DNA methylation that are correlated with the origin of the blastocysts from which they were derived (*in vivo*, *in vitro*, SCNT and uniparental), the cul‐ ture conditions, sex, and prolonged culture. Epigenotyping of ES cells should be adopted as a prerequisite safety evaluation before their use in chimera production or therapeutic appli‐ cations. Furthermore, genes associated with aberrant DNA methylation should be moni‐ tored in ES cell lines to ensure that the cells do not accumulate epigenetic instabilities.

## **Nomenclature**

5-hmC, 5-hydroxymethylcytosine; 5-mC, 5-methylcytosine; AID, activation-induced cytidine deaminase; COBRA, Combined bisulfite restriction analysis; DMR, differentially methylated region; Dnmt, DNA methyltransferase; ES, embryonic stem; Gadd45, Growth arrest and DNA damage-inducible protein 45; ICM, inner cell mass; IVC, *in vitro* culture; IVF, *in vitro* fertilization; MBD4, methyl-CpG binding domain protein 4; PgES, parthenogenetic ES; SCNT, somatic cell nuclear transfer; TET, Ten-eleven translocation; Vitro ES, ES cells de‐ rived from in vitro manipulated embryos; Vivo ES, ES cells derived from embryos devel‐ oped in vivo.

[4] Jaenisch R. DNA methylation and imprinting: why bother? Trends in Genetics

Epigenetic Instability in Embryonic Stem Cells

http://dx.doi.org/10.5772/54367

311

[5] Jones P, Gonzalgo M. Altered DNA methylation and genome instability: a new path‐ way to cancer? Proceedings of National Academy Sciences of the United States of

[6] Robertson KD, Wolffe AP. DNA methylation in health and disease. Nature Reviews

[7] Surani MA. Imprinting and the initiation of gene silencing in the germline. Cell

[8] Bartolomei MS, Zemel S, Tilghman SM. Parental imprinting of the mouse H19 gene.

[9] Ferguson-Smith AC, Cattanach BM, Barton SC, Beechey CV, Surani MA. Embryolog‐ ical and molecular investigations of parental imprinting on mouse chromosome 7.

[10] Pfeifer K. Mechanisms of genomic imprinting. American Journal of Human Genetics.

[11] Szabo PE, and Mann JR. Biallelic expression of imprinted genes in the mouse germ line: implications for erasure, establishment, and mechanisms of genomic imprinting.

[12] Li E, Bestor TH, Jaenisch R. Targeted mutation of the DNA methyltransferase gene

[13] Okano M, Xie S, Li E. Cloning and characterization of a family of novel mammalian

[14] Okano, M., D. W. Bell, D. A. Haber, and E, Li. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell

[15] Doherty AS, Mann MR, Tremblay KD, Bartolomei MS, Schultz RM. Differential ef‐ fects of culture on imprinted H19 expression in the preimplantation mouse embryo.

[16] Fernández-Gonzalez R, Moreira P, Bilbao A, Jiménez A, Pérez-Crespo M, Ramírez MA, Rodríguez De Fonseca F, Pintado B, Gutiérrez-Adán A. Long-term effect of in vitro culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior. Proceedings of National Academy Sciences of the

[17] Horii T, Suetake I, Yanagisawa E, Morita S, Kimura M, Nagao Y, Imai H, Tajima S, Hatada I. The Dnmt3b splice variant is specifically expressed in in vitro-manipulated

DNA (cytosine-5) methyltransferases. Nature Genetics 1998;19(3) 219-20.

1997;13(8) 323-9.

America 1997;94(6) 2103-5.

Genetics 2000;1(1) 11–9.

Nature 1991;351(6322) 153-5.

Nature 1991;351(6328) 667-70.

Genes and Development 1995;9(15) 1857-68.

Biology of Reproduction 2000;62(6) 1526-35.

United States of America 2004;101(16) 5880-5.

results in embryonic lethality. Cell 1992;69(6) 915-26.

1998;93(3) 309-12.

2000;67(4) 777-87.

1999;99(3) 247-57.

## **Acknowledgements**

We thank Mr. Eikichi Yanagisawa, Dr. Sumiyo Morita, Ms. Mika Kimura, Mr. Daiki Tamura, Mr. Ryohei Kobayashi and Dr. Yasumitsu Nagao for technical assistance and helpful com‐ ments on the manuscript. This work was supported by grants from the Ministry of Educa‐ tion, Culture, Sports, Science and Technology of Japan; the Ministry of Health, Labour and Welfare of Japan; the National Institute of Biomedical Innovation; and the Takeda Science Foundation.

## **Author details**

Takuro Horii and Izuho Hatada\*

\*Address all correspondence to: hatada@gunma-u.ac.jp

Biosignal Genome Resource Center, Gunma University, Maebashi, Japan

## **References**


[4] Jaenisch R. DNA methylation and imprinting: why bother? Trends in Genetics 1997;13(8) 323-9.

**Nomenclature**

310 Pluripotent Stem Cells

oped in vivo.

Foundation.

**Author details**

**References**

p169-85.

Takuro Horii and Izuho Hatada\*

\*Address all correspondence to: hatada@gunma-u.ac.jp

Biosignal Genome Resource Center, Gunma University, Maebashi, Japan

vivo. Cellular Reprogramming 2010;12(5) 551-63.

**Acknowledgements**

5-hmC, 5-hydroxymethylcytosine; 5-mC, 5-methylcytosine; AID, activation-induced cytidine deaminase; COBRA, Combined bisulfite restriction analysis; DMR, differentially methylated region; Dnmt, DNA methyltransferase; ES, embryonic stem; Gadd45, Growth arrest and DNA damage-inducible protein 45; ICM, inner cell mass; IVC, *in vitro* culture; IVF, *in vitro* fertilization; MBD4, methyl-CpG binding domain protein 4; PgES, parthenogenetic ES; SCNT, somatic cell nuclear transfer; TET, Ten-eleven translocation; Vitro ES, ES cells de‐ rived from in vitro manipulated embryos; Vivo ES, ES cells derived from embryos devel‐

We thank Mr. Eikichi Yanagisawa, Dr. Sumiyo Morita, Ms. Mika Kimura, Mr. Daiki Tamura, Mr. Ryohei Kobayashi and Dr. Yasumitsu Nagao for technical assistance and helpful com‐ ments on the manuscript. This work was supported by grants from the Ministry of Educa‐ tion, Culture, Sports, Science and Technology of Japan; the Ministry of Health, Labour and Welfare of Japan; the National Institute of Biomedical Innovation; and the Takeda Science

[1] Horii T, Yanagisawa E, Kimura M, Morita S, Hatada I. Epigenetic differences be‐ tween embryonic stem cells generated from blastocysts developed in vitro and in

[2] Horii T, Kimura M, Morita S, Nagao Y, Hatada I. Loss of genomic imprinting in

[3] Antequera F, Bird A. CpG islands in DNA methylation. In: Jost JP, Saluz HP (eds.) Molecular Biology and Biological Significance. Basel: Birkhauser Verlag; 1993.

mouse parthenogenetic embryonic stem cells. Stem Cells 2008;26(1) 79-88.


blastocysts and their derivative ES cells. Journal of Reproduction and Development. 2011;57(5) 579-85.

[29] Ma DK, Jang MH, Guo JU, Kitabatake Y, Chang ML, Pow-Anpongkul N, Flavell RA, Lu B, Ming GL, Song H. Neuronal activity-induced Gadd45b promotes epigenetic

Epigenetic Instability in Embryonic Stem Cells

http://dx.doi.org/10.5772/54367

313

[30] Chang G, Liu S, Wang F, Zhang Y, Kou Z, Chen D, Gao S. Differential methylation status of imprinted genes in nuclear transfer derived ES (NT-ES) cells. Genomics

[31] Inoue K, Kohda T, Lee J, Ogonuki N, Mochida K, Noguchi Y, Tanemura K, Kaneko-Ishino T, Ishino F, Ogura A. Faithful expression of imprinted genes in cloned mice.

[32] Mann MR, Chung YG, Nolen LD, Verona RI, Latham KE, Bartolomei MS. Disruption of imprinted gene methylation and expression in cloned preimplantation stage

[33] Hikichi T, Kohda T, Wakayama S, Ishino F, Wakayama T. Nuclear transfer alters the DNA methylation status of specific genes in fertilized and parthenogenetically acti‐

[34] Cibelli JB, Grant KA, Chapman KB, Cunniff K, Worst T, Green HL, Walker SJ, Gutin PH, Vilner L, Tabar V, Dominko T, Kane J, Wettstein PJ, Lanza RP, Studer L, Vrana KE, West MD. Parthenogenetic stem cells in nonhuman primates. Science

[35] Kim K, Lerou P, Yabuuchi A, Lengerke C, Ng K, West J, Kirby A, Daly MJ, Daley GQ. Histocompatible embryonic stem cells by parthenogenesis. Science

[36] Horii T, Hatada I. Reprogrammed parthenogenetic ES cells - new choice for regener‐ ative medicine. In: Atwood C.S. (ed.) Methodological Advances in the Culture, Ma‐ nipulation and Utilization of Embryonic Stem Cells for Basic and Practical

[37] Surani, M. A. H. and Barton, S. C. Development of gynogenetic eggs in the mouse: implications for parthenogenetic embryos. Science 1983;222(4627) 1034-1036.

[38] Fundele RH, Norris ML, Barton SC, Fehlau M, Howlett SK, Mills WE, Surani MA. Temporal and spatial selection against parthenogenetic cells during development of

[39] Allen ND, Barton SC, Hilton K, Norris ML, Surani MA. A functional analysis of im‐ printing in parthenogenetic embryonic stem cells. Development 1994;120(6) 1473-82.

[40] Chen Z, Liu Z, Huang J, Amano T, Li C, Cao S, Wu C, Liu B, Zhou L, Carter MG, Keefe DL, Yang X, Liu L. Birth of parthenote mice directly from parthenogenetic em‐

[41] Maher ER, Brueton LA, Bowdin SC, Luharia A, Cooper W, Cole TR, Macdonald F, Sampson JR, Barratt CL, Reik W, Hawkins MM. Beckwith-Wiedemann syndrome

mouse embryos. Biology of Reproduction 2003;69(3) 902-14.

vated mouse embryonic stem cells. Stem Cells 2008;26(3) 783-8.

DNA demethylation and adult neurogenesis. Science 2009;323(5917) 1074-7.

2009;93(2) 112-9.

2002;295(5556) 819.

2007;315(5811) 482-6.

Applications. Rejeca: InTech; 2011. p221-36.

fetal chimeras. Development 1990;108(1) 203-11.

bryonic stem cells. Stem Cells 2009;27(9) 2136-45.

Science 2002;295(5553) 297.


[29] Ma DK, Jang MH, Guo JU, Kitabatake Y, Chang ML, Pow-Anpongkul N, Flavell RA, Lu B, Ming GL, Song H. Neuronal activity-induced Gadd45b promotes epigenetic DNA demethylation and adult neurogenesis. Science 2009;323(5917) 1074-7.

blastocysts and their derivative ES cells. Journal of Reproduction and Development.

[18] Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse

[19] Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proceedings of National Acade‐

[20] Nagy A, Gócza E, Diaz EM, Prideaux VR, Iványi E, Markkula M, Rossant J. Embry‐ onic stem cells alone are able to support fetal development in the mouse. Develop‐

[21] Dean W, Bowden L, Aitchison A, Klose J, Moore T, Meneses JJ, Reik W, Feil R. Al‐ tered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes. Development 1998;125(12)

[22] Humpherys D, Eggan K, Akutsu H, Hochedlinger K, Rideout WM 3rd, Biniszkiewicz D, Yanagimachi R, Jaenisch R. Epigenetic instability in ES cells and cloned mice. Sci‐

[23] Minoguchi S, Iba H. Instability of retroviral DNA methylation in embryonic stem

[24] Allegrucci C, Wu YZ, Thurston A, Denning CN, Priddle H, Mummery CL, Ward-van Oostwaard D, Andrews PW, Stojkovic M, Smith N, Parkin T, Jones ME, Warren G, Yu L, Brena RM, Plass C, Young LE. Restriction landmark genome scanning identi‐ fies culture-induced DNA methylation instability in the human embryonic stem cell

[25] Zvetkova I, Apedaile A, Ramsahoye B, Mermoud JE, Crompton LA, John R, Feil R, Brockdorff N. Global hypomethylation of the genome in XX embryonic stem cells.

[26] Murry CE, Keller G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 2008;132(4) 661-80.

[27] Mann MR, Lee SS, Doherty AS, Verona RI, Nolen LD, Schultz RM, Bartolomei MS. Selective loss of imprinting in the placenta following preimplantation development

[28] Barreto G, Schafer A, Marhold J, Stach D, Swaminathan SK, Handa V, Doderlein G, Maltry N, Wu W, Lyko F, Niehrs C. Gadd45a promotes epigenetic gene activation by

repair-mediated DNA demethylation. Nature 2007;445(7128) 671-5.

epigenome. Human Molecular Genetics 2007;16(10) 1253-68.

my Sciences of the United States of America 1981;78(12) 7634-8.

2011;57(5) 579-85.

312 Pluripotent Stem Cells

ment 1990;110(3) 815-21.

ence 2001;293(5527) 95-7.

cells. Stem Cells 2008;26(5) 1166-73.

Nature Genetics 2005;37(11), 1274-9.

in culture. Development 2004;131(15) 3727-35.

2273-82.

embryos. Nature 1981;292(5819) 154-6.


and assisted reproduction technology (ART). Journal of Medical Genetics 2003;40(1) 62-4.

[53] Hsieh CL. In vivo activity of murine de novo methyltransferases, Dnmt3a and

Epigenetic Instability in Embryonic Stem Cells

http://dx.doi.org/10.5772/54367

315

[54] Biniszkiewicz D, Gribnau J, Ramsahoye B, Gaudet F, Eggan K, Humpherys D, Mas‐ trangelo MA, Jun Z, Walter J, Jaenisch R. Dnmt1 overexpression causes genomic hy‐ permethylation, loss of imprinting, and embryonic lethality. Molecular and Cellular

[55] Ooi SK, Wolf D, Hartung O, Agarwal S, Daley GQ, Goff SP, Bestor TH. Dynamic in‐ stability of genomic methylation patterns in pluripotent stem cells. Epigenetics and

[56] Gopalakrishnan S, Van Emburgh BO, Shan J, Su Z, Fields CR, Vieweg J, Hamazaki T, Schwartz PH, Terada N, Robertson KD. A novel DNMT3B splice variant expressed in tumor and pluripotent cells modulates genomic DNA methylation patterns and dis‐

[57] Saito Y, Kanai Y, Sakamoto M, Saito H, Ishii H, Hirohashi S. Overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, associated with DNA hypo‐ methylation on pericentromeric satellite regions during human hepatocarcinogene‐ sis. Proceedings of National Academy Sciences of the United States of America

[58] Nakamura T, Arai Y, Umehara H, Masuhara M, Kimura T, Taniguchi H, Sekimoto T, Ikawa M, Yoneda Y, Okabe M, Tanaka S, Shiota K, Nakano T. PGC7/Stella protects against DNA demethylation in early embryogenesis. Nature Cell Biology 2007;9(1)

[59] Li X, Ito M, Zhou F, Youngson N, Zuo X, Leder P, Ferguson-Smith AC. A maternalzygotic effect gene, Zfp57, maintains both maternal and paternal imprints. Develop‐

[60] Rai K, Huggins IJ, James SR, Karpf AR, Jones DA, Cairns BR. DNA demethylation in zebrafish involves the coupling of a deaminase, a glycosylase, and gadd45. Cell

[61] Bhutani N, Brady JJ, Damian M, Sacco A, Corbel SY, Blau HM. Reprogramming to‐ wards pluripotency requires AID-dependent DNA demethylation. Nature

[62] Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L, Rao A. Conversion of 5-methylcytosine to 5-hydroxymethyl‐ cytosine in mammalian DNA by MLL partner TET1. Science 2009;324(5929) 930-5. [63] Ito S, D'Alessio AC, Taranova OV, Hong K, Sowers LC, Zhang Y. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification.

[64] Koh KP, Yabuuchi A, Rao S, Huang Y, Cunniff K, Nardone J, Laiho A, Tahiliani M, Sommer CA, Mostoslavsky G, Lahesmaa R, Orkin SH, Rodig SJ, Daley GQ, Rao A.

plays altered DNA binding. Molecular Cancer Research 2009;7(10) 1622-34.

Dnmt3b. Molecular and Cellular Biology 1999;19(12) 8211-8.

Biology 2002;22(7) 2124-35.

Chromatin 2010;3(1) 17.

2002;99(15) 10060-5.

mental Cell 2008;15(4) 547-57.

2008;135(7) 1201-12.

2010;463(7284) 1042-7.

Nature 2010;466(7310) 1129-33.

64-71.


[53] Hsieh CL. In vivo activity of murine de novo methyltransferases, Dnmt3a and Dnmt3b. Molecular and Cellular Biology 1999;19(12) 8211-8.

and assisted reproduction technology (ART). Journal of Medical Genetics 2003;40(1)

[42] DeBaun MR, Niemitz EL, Feinberg AP. Association of in vitro fertilization with Beck‐ with-Wiedemann syndrome and epigenetic alterations of LIT1 and H19. American

[43] Cox GF, Burger J, Lip V, Mau UA, Sperling K, Wu BL, Horsthemke B. Intracytoplas‐ mic sperm injection may increase the risk of imprinting defects. American Journal of

[44] Young LE, Fernandes K, McEvoy TG, Butterwith SC, Gutierrez CG, Carolan C, Broadbent PJ, Robinson JJ, Wilmut I, Sinclair KD. Epigenetic change in IGF2R is asso‐ ciated with fetal overgrowth after sheep embryo culture. Nature Genetics 2001;27(2)

[45] Eden A, Gaudet F, Waghmare A, Jaenisch R. Chromosomal instability and tumors

[46] Howard G, Eiges R, Gaudet F, Jaenisch R, Eden A. Activation and transposition of endogenous retroviral elements in hypomethylation induced tumors in mice. Onco‐

[47] Shimabukuro M, Sasaki T, Imamura A, Tsujita T, Fuke C, Umekage T, Tochigi M, Hiramatsu K, Miyazaki T, Oda T, Sugimoto J, Jinno Y, Okazaki Y. Global hypome‐ thylation of peripheral leukocyte DNA in male patients with schizophrenia: a poten‐ tial link between epigenetics and schizophrenia. Jounal of Psychiatric Research

[48] Jeanpierre M, Turleau C, Aurias A, Prieur M, Ledeist F, Fischer A, Viegas-Pequignot E. An embryonic-like methylation pattern of classical satellite DNA is observed in

[49] Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC. Derivation of com‐ pletely cell culture-derived mice from early-passage embryonic stem cells. Proceed‐ ings of National Academy Sciences of the United States of America 1993;90(18)

[50] Holm TM, Jackson-Grusby L, Brambrink T, Yamada Y, Rideout WM 3rd, Jaenisch R. Global loss of imprinting leads to widespread tumorigenesis in adult mice. Cancer

[51] Li E, Beard C, Jaenisch R. Role for DNA methylation in genomic imprinting. Nature

[52] Vertino PM, Yen RW, Gao J, Baylin SB. De novo methylation of CpG island sequen‐ ces in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase. Mo‐

promoted by DNA hypomethylation. Science 2003;300(5618) 455.

ICF syndrome. Human Molecular Genetics 1993;2(6) 731-5.

lecular and Cellular Biology 1996;16(8) 4555-65.

Journal of Human Genetics 2003;72(1) 156-60.

Human Genetics 2002;71(1) 162-164.

62-4.

314 Pluripotent Stem Cells

153-4.

gene 2008;27(3) 404-8.

2007;41(12) 1042-6.

Cell 2005;8(4) 275-85.

1993;366(6453) 362-5.

8424-8.


Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specifi‐ cation in mouse embryonic stem cells. Cell Stem Cell 2011;8(2) 200-13.

**Chapter 15**

**Function of KLF4 in Stem Cell Biology**

The Kruppel-like factor family is a group of zinc finger containing transcription factors, which are highly homologous with the Drosophila Kruppel protein. The feature that distin‐ guishes the KLF family from other zinc finger containing transcriptional factors is the pres‐ ence of three highly conserved C2H2 containing zinc finger motifs at the C-terminus [1-3]. These fingers enable KLFs to bind to the GC-box or CACCC-boxes on DNA with different affinities [4]. KLF4, as a member of KLF family, expresses in a wide range of tissues in mam‐ mals, and plays a critical role in regulating a diverse array of cellular processes including proliferation, differentiation, development, maintenance of normal tissue homeostasis and apoptosis. KLF4 can also acts either as a tumor suppressor or an oncogene depending on

The role that KLF4 plays in stem cell biology has attracted much more attention in recent years. For instance, in 2006, Takahashi K et al [5] reprogrammed somatic cells into pluripo‐ tent stem cells using KLF4 in combination with three other transcription factors: Oct4, Sox2 and c-Myc. Numerous recent literatures have further proved that KLF4 is essential for both embryonic stem (ES) cells self-renewal and maintenance, additionally our recent work re‐ vealed a critical role of KLF4 in maintenance of breast cancer stem cells [6]. Furthermore, we found that KLF4 is expressed in mouse skin hair follicle stem cells and such expression con‐ tributed to mouse cutaneous wound healing [7]. In this review, functions of KLF4 in stem cells, especially breast cancer stem cells and mouse hair follicle stem cells will be discussed,

Mouse KLF4 was first identified in 1996 independently by two groups and separately given two different names - GKLF (gut enriched Kruppel like factor):due to its high expression in the

> © 2013 Shi and Ai; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Shi and Ai; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

and the signaling pathways possibly involved will be addressed as well.

**2. Identification and characterization of KLF4**

Additional information is available at the end of the chapter

Ying Shi and Walden Ai

http://dx.doi.org/10.5772/54370

differing cellular context and cancer types.

**1. Introduction**


**Chapter 15**

## **Function of KLF4 in Stem Cell Biology**

Ying Shi and Walden Ai

Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specifi‐

[65] Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, Fry B, Meissner A, Wernig M, Plath K, Jaenisch R, Wagschal A, Feil R, Schreiber SL, Lander ES. A biva‐ lent chromatin structure marks key developmental genes in embryonic stem cells.

[66] Tamaru H, Selker EU. A histone H3 methyltransferase controls DNA methylation in

[67] Jackson JP, Lindroth AM, Cao X, Jacobsen SE. Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 2002;416(6880) 556-60.

[68] Bachman KE, Park BH, Rhee I, Rajagopalan H, Herman JG, Baylin SB, Kinzler KW, Vogelstein B. Histone modifications and silencing prior to DNA methylation of a tu‐

cation in mouse embryonic stem cells. Cell Stem Cell 2011;8(2) 200-13.

Neurospora crassa. Nature 2001;414(6861) 277-83.

mor suppressor gene. Cancer Cell 2003;3(1) 89-95.

Cell 2006;125(2) 315-26.

316 Pluripotent Stem Cells

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54370

## **1. Introduction**

The Kruppel-like factor family is a group of zinc finger containing transcription factors, which are highly homologous with the Drosophila Kruppel protein. The feature that distin‐ guishes the KLF family from other zinc finger containing transcriptional factors is the pres‐ ence of three highly conserved C2H2 containing zinc finger motifs at the C-terminus [1-3]. These fingers enable KLFs to bind to the GC-box or CACCC-boxes on DNA with different affinities [4]. KLF4, as a member of KLF family, expresses in a wide range of tissues in mam‐ mals, and plays a critical role in regulating a diverse array of cellular processes including proliferation, differentiation, development, maintenance of normal tissue homeostasis and apoptosis. KLF4 can also acts either as a tumor suppressor or an oncogene depending on differing cellular context and cancer types.

The role that KLF4 plays in stem cell biology has attracted much more attention in recent years. For instance, in 2006, Takahashi K et al [5] reprogrammed somatic cells into pluripo‐ tent stem cells using KLF4 in combination with three other transcription factors: Oct4, Sox2 and c-Myc. Numerous recent literatures have further proved that KLF4 is essential for both embryonic stem (ES) cells self-renewal and maintenance, additionally our recent work re‐ vealed a critical role of KLF4 in maintenance of breast cancer stem cells [6]. Furthermore, we found that KLF4 is expressed in mouse skin hair follicle stem cells and such expression con‐ tributed to mouse cutaneous wound healing [7]. In this review, functions of KLF4 in stem cells, especially breast cancer stem cells and mouse hair follicle stem cells will be discussed, and the signaling pathways possibly involved will be addressed as well.

## **2. Identification and characterization of KLF4**

Mouse KLF4 was first identified in 1996 independently by two groups and separately given two different names - GKLF (gut enriched Kruppel like factor):due to its high expression in the

© 2013 Shi and Ai; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Shi and Ai; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

gastrointestinal tract [8], and EZF (epithelial zinc finger) since it was highly expressed in differ‐ entiated epithelial cells of the skin [9]. Human KLF4 cDNA was cloned from human umbilical vein endothelial cell cDNA library [10] and later renamed as KLF4 to avoid confusion.

thelial barrier of the skin [32]. KLF4 null mice died one day after birth due to loss of barrier func‐ tion of the skin. It appears that KLF4 influences the formation of the cornified envelope in the late-stage differentiation process that was supported by upregulation of Sprr2a, a cornified en‐ velope gene, in KLF4 knockout mice. Two additional cornified envelope proteins: repetin (en‐ coded by Rptn) and plasminogen activating inhibitor 2 (encoded by Planh2) were found later. KLF4 may regulate these genes resulting in an imbalance in cornified envelope assembly or composition, thereby altering the structural scaffold on which the lipid lamellae are organized. A differential role of KLF4 has also been reported in smooth muscle cells [33], monocytes [34],

Function of KLF4 in Stem Cell Biology http://dx.doi.org/10.5772/54370 319

KLF4 is thought to be involved in chronic inflammatory disease since it has been shown to mediate proinflammatory signaling in human macrophages in vitro [37, 38] and regulate the expression of interleukin-10 in RAW264.7 macrophages [39]. KLF4 is also essential for differ‐ entiation of mouse inflammatory monocytes and involved in the differentiation of resident monocytes [34, 40]. The inflammation-selective effects of loss-of-KLF4 and gain-of-KLF4-in‐ duced monocytic differentiation in HL60 cells identify KLF4 as a key regulator of monocytic differentiation and a potential target for translational immune modulation [40]. KLF4 posi‐ tively regulates human ghrelin expression [41], which is expressed in the gastrointestinal tract. In addition, it was found that KLF4 is an immediate early gene for Nerve Growth Fac‐ tor [42]. A recent study showed that glutamatergic stimulation can trigger rapid elevation of KLF4 mRNA and protein levels, and that the over expression of KLF4 can regulate neuronal cell cycle proteins and sensitize neurons to NMDA-induced caspase-3 activity [43]. Another study demonstrated that KLF4 is involved in regulating the proliferation of CD8+ cells [44]. The transcription factor ELF4 directly activated the tumor suppressor KLF4 'downstream' of

T cell antigen receptor signaling to induce cell cycle arrest in naive CD8+ T cells [44].

essary for caspase inactivation and therefore acts as a negative regulator of apoptosis.

Embryonic stem (ES) cells are characterized by a self-renewal ability and pluripotency. Selfrenewal is the capability of ES cells to be maintained in a proliferative state for prolonged periods of time, whereas pluripotency is the ability of ES cells to differentiate into a diverse array of specialized cell types. It has been shown that self renewal and maintenance of pluri‐

KLF4 has been implicated in the regulation of apoptosis [45, 46]. During DNA damage, cells can take two routes - either pass into the next phase overcoming the checkpoint or get ar‐ rested at the checkpoint and activates the repair machinery. As discussed previously, over expression of KLF4 in RKO colon cancer cells, when subjected to UV radiation, reduced the percentage of apoptotic cells [47]. In esophageal cancer cell lines, KLF4 has been shown to bind to the promoter and repress the activity of the surviving gene *in vivo* [48], which is nec‐

testes [15], T cells [11, 35] and murine tooth development [36].

**3.3. Other functions**

**4. KLF4 in stem cell biology**

**4.1. KLF4 function in embryonic stem cells**

The human KLF4 gene locus is mapped on chromosome 9q31 whereas mouse KLF4 is on chromosome 4B3. Mouse KLF4 has a single ORF of 1449 bp that encodes a polypeptide of 483 amino acids with a predicted molecular weight of 53 Kd; while human KLF4 has an ORF of 1444 bp coding for a 470 amino acid protein with an estimated molecular mass 50 Kd. At the amino acid level the human and mouse KLF4 are shown to have 91% sequence similari‐ ty. The three tandem zinc finger motifs are conserved completely in the human and mouse sequences. Except skin and colon [8, 9], KLF4 is also found in lung, testis, small intestine [8, 9], thymus [11], cornea [12], cardiac myocytes [13] and lymphocytes [14]. In testis, four KLF4 transcripts with alternative polyadenylation were found and they generated different RNA species in various testicular cells, strongly suggesting translational regulation of KLF4 in spermatogenesis [15, 16].

## **3. General functions of KLF4**

#### **3.1. Inhibition of cell proliferation**

KLF4 is known to induce growth arrest, inhibiting cell proliferation by regulating the expres‐ sion of key cell cycle genes. Elevated expression of KLF4 in NIH3T3 subjected to serum starva‐ tion [8] has been shown to inhibit DNA synthesis. Microarray analysis confirms that a number of genes were up- or down-regulated upon KLF4 induction, most of which are involved in cell cycle control [17]. For example, the expression of cell cycle inhibitor p21/Cip1 was elevated [18], while cell cycle promoter Cyclin D1 was depressed [19]. KLF4 has been shown to inhibit cell proliferation by blocking G1/S progression of the cell cycle and to mediate p53 dependent G1/S cell cycle arrest in response to DNA damage [20, 21]. Furthermore, KLF4 plays an impor‐ tant role in maintaining the integrity of the G2/M checkpoint following DNA damage. While wild type HCT 116 colon cancer cells were arrested at the G2/M phase checkpoint upon γ-irra‐ diation, p53 -/- cells were able to enter M phase even after irradiation. It was observed that upon introduction of KLF4 into p53 -/- cells, the mitotic indices were considerably reduced and the Cyclin B1 levels were also risen [22]. These studies suggest that KLF4 is a critical factor in regu‐ lating entry of the cells into the mitotic phase. Finally, KLF4 was found both necessary and suf‐ ficient in preventing centrosome amplification following γ–irradiation-induced DNA damage by transcriptionally suppressing cyclin E expression [23].

#### **3.2. Promotion of cell differentiation**

Microarray analysis has shown that many keratin genes were upregulated on KLF4 induction, indicating its role in epithelial differentiation. Additionally, KLF4 has been reported to transac‐ tivate promoters of epithelial genes including CYP1A1 [24], laminin α 3A [25], laminin 1 [26], keratin 4 [27], keratin 19 [28]. Recent studies demonstrated that KLF4 plays a vital role in goblet cell differentiation in the intestine [29, 30], conjunctiva [31], and also in the formation of the epi‐ thelial barrier of the skin [32]. KLF4 null mice died one day after birth due to loss of barrier func‐ tion of the skin. It appears that KLF4 influences the formation of the cornified envelope in the late-stage differentiation process that was supported by upregulation of Sprr2a, a cornified en‐ velope gene, in KLF4 knockout mice. Two additional cornified envelope proteins: repetin (en‐ coded by Rptn) and plasminogen activating inhibitor 2 (encoded by Planh2) were found later. KLF4 may regulate these genes resulting in an imbalance in cornified envelope assembly or composition, thereby altering the structural scaffold on which the lipid lamellae are organized. A differential role of KLF4 has also been reported in smooth muscle cells [33], monocytes [34], testes [15], T cells [11, 35] and murine tooth development [36].

#### **3.3. Other functions**

gastrointestinal tract [8], and EZF (epithelial zinc finger) since it was highly expressed in differ‐ entiated epithelial cells of the skin [9]. Human KLF4 cDNA was cloned from human umbilical

The human KLF4 gene locus is mapped on chromosome 9q31 whereas mouse KLF4 is on chromosome 4B3. Mouse KLF4 has a single ORF of 1449 bp that encodes a polypeptide of 483 amino acids with a predicted molecular weight of 53 Kd; while human KLF4 has an ORF of 1444 bp coding for a 470 amino acid protein with an estimated molecular mass 50 Kd. At the amino acid level the human and mouse KLF4 are shown to have 91% sequence similari‐ ty. The three tandem zinc finger motifs are conserved completely in the human and mouse sequences. Except skin and colon [8, 9], KLF4 is also found in lung, testis, small intestine [8, 9], thymus [11], cornea [12], cardiac myocytes [13] and lymphocytes [14]. In testis, four KLF4 transcripts with alternative polyadenylation were found and they generated different RNA species in various testicular cells, strongly suggesting translational regulation of KLF4 in

KLF4 is known to induce growth arrest, inhibiting cell proliferation by regulating the expres‐ sion of key cell cycle genes. Elevated expression of KLF4 in NIH3T3 subjected to serum starva‐ tion [8] has been shown to inhibit DNA synthesis. Microarray analysis confirms that a number of genes were up- or down-regulated upon KLF4 induction, most of which are involved in cell cycle control [17]. For example, the expression of cell cycle inhibitor p21/Cip1 was elevated [18], while cell cycle promoter Cyclin D1 was depressed [19]. KLF4 has been shown to inhibit cell proliferation by blocking G1/S progression of the cell cycle and to mediate p53 dependent G1/S cell cycle arrest in response to DNA damage [20, 21]. Furthermore, KLF4 plays an impor‐ tant role in maintaining the integrity of the G2/M checkpoint following DNA damage. While wild type HCT 116 colon cancer cells were arrested at the G2/M phase checkpoint upon γ-irra‐ diation, p53 -/- cells were able to enter M phase even after irradiation. It was observed that upon introduction of KLF4 into p53 -/- cells, the mitotic indices were considerably reduced and the Cyclin B1 levels were also risen [22]. These studies suggest that KLF4 is a critical factor in regu‐ lating entry of the cells into the mitotic phase. Finally, KLF4 was found both necessary and suf‐ ficient in preventing centrosome amplification following γ–irradiation-induced DNA damage

Microarray analysis has shown that many keratin genes were upregulated on KLF4 induction, indicating its role in epithelial differentiation. Additionally, KLF4 has been reported to transac‐ tivate promoters of epithelial genes including CYP1A1 [24], laminin α 3A [25], laminin 1 [26], keratin 4 [27], keratin 19 [28]. Recent studies demonstrated that KLF4 plays a vital role in goblet cell differentiation in the intestine [29, 30], conjunctiva [31], and also in the formation of the epi‐

vein endothelial cell cDNA library [10] and later renamed as KLF4 to avoid confusion.

spermatogenesis [15, 16].

318 Pluripotent Stem Cells

**3. General functions of KLF4**

**3.1. Inhibition of cell proliferation**

by transcriptionally suppressing cyclin E expression [23].

**3.2. Promotion of cell differentiation**

KLF4 is thought to be involved in chronic inflammatory disease since it has been shown to mediate proinflammatory signaling in human macrophages in vitro [37, 38] and regulate the expression of interleukin-10 in RAW264.7 macrophages [39]. KLF4 is also essential for differ‐ entiation of mouse inflammatory monocytes and involved in the differentiation of resident monocytes [34, 40]. The inflammation-selective effects of loss-of-KLF4 and gain-of-KLF4-in‐ duced monocytic differentiation in HL60 cells identify KLF4 as a key regulator of monocytic differentiation and a potential target for translational immune modulation [40]. KLF4 posi‐ tively regulates human ghrelin expression [41], which is expressed in the gastrointestinal tract. In addition, it was found that KLF4 is an immediate early gene for Nerve Growth Fac‐ tor [42]. A recent study showed that glutamatergic stimulation can trigger rapid elevation of KLF4 mRNA and protein levels, and that the over expression of KLF4 can regulate neuronal cell cycle proteins and sensitize neurons to NMDA-induced caspase-3 activity [43]. Another study demonstrated that KLF4 is involved in regulating the proliferation of CD8+ cells [44]. The transcription factor ELF4 directly activated the tumor suppressor KLF4 'downstream' of T cell antigen receptor signaling to induce cell cycle arrest in naive CD8+ T cells [44].

KLF4 has been implicated in the regulation of apoptosis [45, 46]. During DNA damage, cells can take two routes - either pass into the next phase overcoming the checkpoint or get ar‐ rested at the checkpoint and activates the repair machinery. As discussed previously, over expression of KLF4 in RKO colon cancer cells, when subjected to UV radiation, reduced the percentage of apoptotic cells [47]. In esophageal cancer cell lines, KLF4 has been shown to bind to the promoter and repress the activity of the surviving gene *in vivo* [48], which is nec‐ essary for caspase inactivation and therefore acts as a negative regulator of apoptosis.

## **4. KLF4 in stem cell biology**

#### **4.1. KLF4 function in embryonic stem cells**

Embryonic stem (ES) cells are characterized by a self-renewal ability and pluripotency. Selfrenewal is the capability of ES cells to be maintained in a proliferative state for prolonged periods of time, whereas pluripotency is the ability of ES cells to differentiate into a diverse array of specialized cell types. It has been shown that self renewal and maintenance of pluri‐ potency in mouse ES cells requires leukemia inhibitory factor (LIF). LIF is a member of the IL6 cytokine family and is used to maintain ES cell cultures in an undifferentiated state through activation of the *Stat3* gene. Oct4, Sox2, and Nanog are all thought to be the master regulators of ES cell pluripotency. Although Oct4 and Sox2 are not direct targets of Stat3 [49], they have been identified as two essential transcription factors that form a heterodimer which binds to the Nanog promoter and regulates the expression of downstream genes that contribute to the maintenance of self-renewal [50]. KLF4 acts as a fast responding mediator to LIF-Stat3 signal changes, and directly binds to the promoter of Nanog to help Oct4 and Sox2 in regulating the expression of Nanog [51]. This observation confirms the critical role of KLF4 in ES cell self renewal as well as pluripotency.

Nevertheless, the iPS cell technology potentially can overcome two important obstacles as‐ sociated with human ES cells: immune rejection after transplantation and ethical concerns regarding the use of human embryos [61]. The advantage of iPS cell technology is that iPS cells can be generated using a few programming factors in any laboratory using standard techniques and equipment. Establishment of a stable and self-sustainable ES-specific tran‐ scriptional regulatory network is essential for reprogramming [62]. iPS cells still have the scope for clinical applications provided that proper ways are established to precisely evalu‐ ate each iPS cell clone and to select appropriate sub clones prior to clinical application.

Function of KLF4 in Stem Cell Biology http://dx.doi.org/10.5772/54370 321

Cancer stem cells (CSCs) are a subpopulation of tumor cells that possess the stem cell prop‐ erties of self renewal and differentiation, which allows them to generate the heterogeneous lineages of cancer cells that comprise the tumor. In 1997, a hierarchy in human acute mye‐ loid leukemia cells was first reported, which improved the understanding of tumorigenesis and cast new light on cancer therapy [63]. CSCs in other types of hematological malignan‐ cies were identified later, and then CSC research was expanded to solid tumors shortly after. The identification of CSCs in solid tumors depends on specific biomarker. Recently, CSCs have been identified in numerous solid tumors, including pancreas [64], colon [65], prostate

In breast cancer the first evidence of CSC was based on a combination of specific cellsurface antigen profile CD44+/CD24-/Lin- in 2003 [69]. More recently, aldehyde dehydro‐ genase (ALDH) was used as stem cell marker in a series of 577 breast carcinoma and 33 human breast cell lines [70]. ALDH is a detoxifying enzyme that oxidizes intracellular al‐ dehydes and is thought to play a role in the differentiation of stem cells via the metabo‐ lism of retinal to retinoic acid [71]. Side population (SP) was also defined as a characteristic of breast CSC, which indicated an inherently high resistance to chemothera‐ peutic agents [72]. Since the CSCs have the capacity for self-renewal, differentiation into multiple cancer cell lineages, extensive proliferation as normal stem cells, and are respon‐ sible for tumor recurrence and chemotherapeutic resistance, it is necessary to figure out the key regulators and related signaling pathways that regulate the CSC in the process

As discussed previously, KLF4 plays a critical role in ES self renewal and pluripotency, and is one of the four transcription factors creating iPS cells. Therefore, it's very worthy to ex‐ plore the relationship between KLF4 and breast CSCs along with underlying mechanisms. Our recent work provides evidence for the first time that KLF4 is essential for the mainte‐ nance of breast CSCs and cell migration and invasion [7]. This evidence may offer important

Earlier reports have shown that elevated KLF4 expression is detected in nearly 70% of breast carcinomas and that nuclear localization of KLF4 is associated with a more aggressive phe‐ notype in early-stage breast cancer [73, 74]. However, the ability of KLF4 to initiate aggres‐ sive tumors in vivo has not been examined yet. Our study showed that KLF4 was highly expressed in CSC-enriched populations in mouse primary mammary tumor and human

clues to understand how KLF4 promotes breast cancer development.

**4.3. KLF4 function in breast Cancer Stem Cells (CSC)**

[66], bladder [67], lung [68] and breast cancer [69].

of carcinogenesis and tumor metastasis.

#### **4.2. KLF4 function in generation of induced pluripotent stem cells**

ES cells are believed to hold great promise for regenerative medicine due to their unique ability to differentiate into any cell type. However, the application of human eggs or em‐ bryos encounters big ethical problems. This dilemma was broken in 2006 by Dr. Shinya Ya‐ manaka's group. They picked four transcription factors, including Oct4, Sox2, c-Myc, and KLF4, to introduce into mouse embryonic fibroblasts via retroviral transfection [5]. The modified embryonic fibroblasts were found to be reprogrammed to a pluripotent state simi‐ lar to that observed in ES cells. Later the finding was further confirmed by using either mouse or human adult fibroblasts [52-57]. The discovery of these "induced pluripotent stem cells" (iPS cells) was regarded as a great achievement in stem cell research and gave new insights into the feasibility of clinical application of stem cells.

A panel of assays has been performed to compare iPS cells with ES cells in morphology, surface marker expression, epigenetic status, formation of embryoid bodies *in vitro*, di‐ rected differentiation into neural cells and beating cardiomyocytes, teratoma formation *in vivo* and chimera contribution. The results indicated that iPS cells resemble ES cells by all measured criteria. Not only fibroblasts, but also other terminally differentiated cells can be reprogrammed to pluripotent cells [58]. After the introduction of pluripotency from terminally differentiated cells, the applications of the iPS cells have also been ex‐ plored. By using a humanized sickle cell anemia mouse model, mice can be rescued after transplantation with hematopoietic progenitors obtained from autologous iPS cells *in vi‐ tro*. Mechanistically, the rescue was due to the correction of the human sickle hemoglo‐ bin allele by gene specific targeting. This report provides the first proof of principle for using iPS cells for disease treatment in mice [59] and demonstrates the therapeutic poten‐ tial of iPS cells for human diseases.

Although iPS cells based on somatic cells avoid ethical issues, the use of oncogenes and ret‐ rovirus still raised safety concerns. For example, reactivation of the c-Myc retrovirus, in‐ creased tumorigenicity in the chimeras and progeny mice, hindering clinical applications [60]. Another problem is that iPS cells are refractory to differentiation and thereby increase the risk of immature teratoma formation after directed differentiation and transplantation into patients. Even if only a small portion of cells within each iPS cell clone shows impaired differentiation, then those cells might be sufficient to produce immature teratomas [61]. Nevertheless, the iPS cell technology potentially can overcome two important obstacles as‐ sociated with human ES cells: immune rejection after transplantation and ethical concerns regarding the use of human embryos [61]. The advantage of iPS cell technology is that iPS cells can be generated using a few programming factors in any laboratory using standard techniques and equipment. Establishment of a stable and self-sustainable ES-specific tran‐ scriptional regulatory network is essential for reprogramming [62]. iPS cells still have the scope for clinical applications provided that proper ways are established to precisely evalu‐ ate each iPS cell clone and to select appropriate sub clones prior to clinical application.

#### **4.3. KLF4 function in breast Cancer Stem Cells (CSC)**

potency in mouse ES cells requires leukemia inhibitory factor (LIF). LIF is a member of the IL6 cytokine family and is used to maintain ES cell cultures in an undifferentiated state through activation of the *Stat3* gene. Oct4, Sox2, and Nanog are all thought to be the master regulators of ES cell pluripotency. Although Oct4 and Sox2 are not direct targets of Stat3 [49], they have been identified as two essential transcription factors that form a heterodimer which binds to the Nanog promoter and regulates the expression of downstream genes that contribute to the maintenance of self-renewal [50]. KLF4 acts as a fast responding mediator to LIF-Stat3 signal changes, and directly binds to the promoter of Nanog to help Oct4 and Sox2 in regulating the expression of Nanog [51]. This observation confirms the critical role

ES cells are believed to hold great promise for regenerative medicine due to their unique ability to differentiate into any cell type. However, the application of human eggs or em‐ bryos encounters big ethical problems. This dilemma was broken in 2006 by Dr. Shinya Ya‐ manaka's group. They picked four transcription factors, including Oct4, Sox2, c-Myc, and KLF4, to introduce into mouse embryonic fibroblasts via retroviral transfection [5]. The modified embryonic fibroblasts were found to be reprogrammed to a pluripotent state simi‐ lar to that observed in ES cells. Later the finding was further confirmed by using either mouse or human adult fibroblasts [52-57]. The discovery of these "induced pluripotent stem cells" (iPS cells) was regarded as a great achievement in stem cell research and gave new

A panel of assays has been performed to compare iPS cells with ES cells in morphology, surface marker expression, epigenetic status, formation of embryoid bodies *in vitro*, di‐ rected differentiation into neural cells and beating cardiomyocytes, teratoma formation *in vivo* and chimera contribution. The results indicated that iPS cells resemble ES cells by all measured criteria. Not only fibroblasts, but also other terminally differentiated cells can be reprogrammed to pluripotent cells [58]. After the introduction of pluripotency from terminally differentiated cells, the applications of the iPS cells have also been ex‐ plored. By using a humanized sickle cell anemia mouse model, mice can be rescued after transplantation with hematopoietic progenitors obtained from autologous iPS cells *in vi‐ tro*. Mechanistically, the rescue was due to the correction of the human sickle hemoglo‐ bin allele by gene specific targeting. This report provides the first proof of principle for using iPS cells for disease treatment in mice [59] and demonstrates the therapeutic poten‐

Although iPS cells based on somatic cells avoid ethical issues, the use of oncogenes and ret‐ rovirus still raised safety concerns. For example, reactivation of the c-Myc retrovirus, in‐ creased tumorigenicity in the chimeras and progeny mice, hindering clinical applications [60]. Another problem is that iPS cells are refractory to differentiation and thereby increase the risk of immature teratoma formation after directed differentiation and transplantation into patients. Even if only a small portion of cells within each iPS cell clone shows impaired differentiation, then those cells might be sufficient to produce immature teratomas [61].

of KLF4 in ES cell self renewal as well as pluripotency.

320 Pluripotent Stem Cells

**4.2. KLF4 function in generation of induced pluripotent stem cells**

insights into the feasibility of clinical application of stem cells.

tial of iPS cells for human diseases.

Cancer stem cells (CSCs) are a subpopulation of tumor cells that possess the stem cell prop‐ erties of self renewal and differentiation, which allows them to generate the heterogeneous lineages of cancer cells that comprise the tumor. In 1997, a hierarchy in human acute mye‐ loid leukemia cells was first reported, which improved the understanding of tumorigenesis and cast new light on cancer therapy [63]. CSCs in other types of hematological malignan‐ cies were identified later, and then CSC research was expanded to solid tumors shortly after. The identification of CSCs in solid tumors depends on specific biomarker. Recently, CSCs have been identified in numerous solid tumors, including pancreas [64], colon [65], prostate [66], bladder [67], lung [68] and breast cancer [69].

In breast cancer the first evidence of CSC was based on a combination of specific cellsurface antigen profile CD44+/CD24-/Lin- in 2003 [69]. More recently, aldehyde dehydro‐ genase (ALDH) was used as stem cell marker in a series of 577 breast carcinoma and 33 human breast cell lines [70]. ALDH is a detoxifying enzyme that oxidizes intracellular al‐ dehydes and is thought to play a role in the differentiation of stem cells via the metabo‐ lism of retinal to retinoic acid [71]. Side population (SP) was also defined as a characteristic of breast CSC, which indicated an inherently high resistance to chemothera‐ peutic agents [72]. Since the CSCs have the capacity for self-renewal, differentiation into multiple cancer cell lineages, extensive proliferation as normal stem cells, and are respon‐ sible for tumor recurrence and chemotherapeutic resistance, it is necessary to figure out the key regulators and related signaling pathways that regulate the CSC in the process of carcinogenesis and tumor metastasis.

As discussed previously, KLF4 plays a critical role in ES self renewal and pluripotency, and is one of the four transcription factors creating iPS cells. Therefore, it's very worthy to ex‐ plore the relationship between KLF4 and breast CSCs along with underlying mechanisms. Our recent work provides evidence for the first time that KLF4 is essential for the mainte‐ nance of breast CSCs and cell migration and invasion [7]. This evidence may offer important clues to understand how KLF4 promotes breast cancer development.

Earlier reports have shown that elevated KLF4 expression is detected in nearly 70% of breast carcinomas and that nuclear localization of KLF4 is associated with a more aggressive phe‐ notype in early-stage breast cancer [73, 74]. However, the ability of KLF4 to initiate aggres‐ sive tumors in vivo has not been examined yet. Our study showed that KLF4 was highly expressed in CSC-enriched populations in mouse primary mammary tumor and human breast cancer cell lines (Figure 1). Knockdown of KLF4 in breast cancer cell MCF-7 and MDA-MB-231 inhibits cell migration, invasion and adhesion *in vitro*, and the self-renewal of breast CSCs (Figure 2). Tumor growth in mouse xenograft mode was suppressed as well (Figure 3), suggesting that KLF4 could act as an oncogenic protein in breast cancers.

of KLF4 knockdown cells was comparable to that of the control cells would argue against this possibility. We have not performed limiting-dilution assays to determine the tumor-initiating capacities of CSC cells in non-obese diabetic/severe combined immu‐ nodeficiency mice yet, which is a traditional method in CSC studies. Nevertheless, our results not only provide additional experimental support for the important function of KLF4 in stem cell biology, but also are important for breast cancer studies. CSCs have been shown to foster blood vessel formation and promote cell motility. They are also re‐ sistant to chemotherapy and radiotherapy [76] and have been implicated in breast cancer metastasis that remains the number one cause of cancer-related mortality in women [77]. Our study suggested that overexpression of KLF4 was sufficient to drive cell migration and invasion. Additional studies on the mechanisms by which KLF4 maintains cancer stem cell phenotype will be very helpful to develop novel therapeutic strategies targeting KLF4 or the related signaling pathway to treat malignant breast cancer and metastasis.

Function of KLF4 in Stem Cell Biology http://dx.doi.org/10.5772/54370 323

**Figure 2.** Knockdown of KLF4 resulted in a reduced stem cell population and decreased self-renewal of breast cancer stem cells. (a) Freshly isolated siCon and siKLF4 MCF-7 cells were labeled with CD24 (fluorescein isothiocyanate (FITC)) and CD44 (phycoerythrin (PE)) antibodies to identify CD44+/CD24-population using a FACSCalibur flow cytometer. (b) SP popula‐ tion in MCF-7 stable cells was determined by Hoechst 33342 efflux assays. (c) Left, MCF-7 cells (siCon and siKLF4) were grown in ultra-low attachment surface plates at a density of 1000, 500, 200, and 100 per well. Assays were conducted after 10 days (left). The symbol \* indicates *P*<0.05 vs siCon group. Right, primary (P1) and secondary (P2) mammosphere forma‐

tion under suspension culture conditions were evaluated in MCF-7 mammary tumor cell lines.

**Figure 1.** KLF4 was highly expressed in CSC-enriched population. (a) KLF4 expression was examined in adherent cells and mammospheres of primary tumors originated from MMTV-Neu transgenic mice. Oct4 and Nanog were used as positive and negative controls, respectively. (b) KLF4 expression was examined in SP and non-SP cells of MCF-7. The symbol \* indicates *P*<0.05 vs non-SP cells group. (c) KLF4 expression was determined in CD44+/CD24- and CD44-/ CD24- populations isolated by flow cytometry. The symbol \* indicates *P*<0.05 vs CD44-/CD24- group.

The anti-proliferative function of KLF4 is associated with inhibition of cell cycle promot‐ er cyclin-D1 [19] and activation of the cell-cycle inhibitor p21/Cip1 [18]. Since inactiva‐ tion of either protein not only neutralizes the cytostatic effect of KLF4 but also collaborates with KLF4 in oncogenic transformation [75], thus further highlighting the importance of p21/Cip1. Although p21/Cip1 status might be a switch that determines the tumor suppressor or oncoprotein function of KLF4, the exact mechanism has not been elucidated yet. Moreover, a cellular mechanism by which KLF4 contributes to the aggres‐ sive characteristics of breast cancers remains unknown. Our current studies indicate that KLF4 is required for the maintenance of breast CSCs and the knockdown of KLF4 sig‐ nificantly decrease the self-renewal of breast CSCs by examining several different CSC markers. Notably KLF4 exerted an anti-apoptotic function in many cancer cell lines, so it is possible that the decreased CSC population upon KLF4 knockdown may be a result of the increased apoptosis mediated by KLF4 reduction. However, the fact that cell viability of KLF4 knockdown cells was comparable to that of the control cells would argue against this possibility. We have not performed limiting-dilution assays to determine the tumor-initiating capacities of CSC cells in non-obese diabetic/severe combined immu‐ nodeficiency mice yet, which is a traditional method in CSC studies. Nevertheless, our results not only provide additional experimental support for the important function of KLF4 in stem cell biology, but also are important for breast cancer studies. CSCs have been shown to foster blood vessel formation and promote cell motility. They are also re‐ sistant to chemotherapy and radiotherapy [76] and have been implicated in breast cancer metastasis that remains the number one cause of cancer-related mortality in women [77]. Our study suggested that overexpression of KLF4 was sufficient to drive cell migration and invasion. Additional studies on the mechanisms by which KLF4 maintains cancer stem cell phenotype will be very helpful to develop novel therapeutic strategies targeting KLF4 or the related signaling pathway to treat malignant breast cancer and metastasis.

breast cancer cell lines (Figure 1). Knockdown of KLF4 in breast cancer cell MCF-7 and MDA-MB-231 inhibits cell migration, invasion and adhesion *in vitro*, and the self-renewal of breast CSCs (Figure 2). Tumor growth in mouse xenograft mode was suppressed as well

**Figure 1.** KLF4 was highly expressed in CSC-enriched population. (a) KLF4 expression was examined in adherent cells and mammospheres of primary tumors originated from MMTV-Neu transgenic mice. Oct4 and Nanog were used as positive and negative controls, respectively. (b) KLF4 expression was examined in SP and non-SP cells of MCF-7. The symbol \* indicates *P*<0.05 vs non-SP cells group. (c) KLF4 expression was determined in CD44+/CD24- and CD44-/

The anti-proliferative function of KLF4 is associated with inhibition of cell cycle promot‐ er cyclin-D1 [19] and activation of the cell-cycle inhibitor p21/Cip1 [18]. Since inactiva‐ tion of either protein not only neutralizes the cytostatic effect of KLF4 but also collaborates with KLF4 in oncogenic transformation [75], thus further highlighting the importance of p21/Cip1. Although p21/Cip1 status might be a switch that determines the tumor suppressor or oncoprotein function of KLF4, the exact mechanism has not been elucidated yet. Moreover, a cellular mechanism by which KLF4 contributes to the aggres‐ sive characteristics of breast cancers remains unknown. Our current studies indicate that KLF4 is required for the maintenance of breast CSCs and the knockdown of KLF4 sig‐ nificantly decrease the self-renewal of breast CSCs by examining several different CSC markers. Notably KLF4 exerted an anti-apoptotic function in many cancer cell lines, so it is possible that the decreased CSC population upon KLF4 knockdown may be a result of the increased apoptosis mediated by KLF4 reduction. However, the fact that cell viability

CD24- populations isolated by flow cytometry. The symbol \* indicates *P*<0.05 vs CD44-/CD24- group.

(Figure 3), suggesting that KLF4 could act as an oncogenic protein in breast cancers.

322 Pluripotent Stem Cells

**Figure 2.** Knockdown of KLF4 resulted in a reduced stem cell population and decreased self-renewal of breast cancer stem cells. (a) Freshly isolated siCon and siKLF4 MCF-7 cells were labeled with CD24 (fluorescein isothiocyanate (FITC)) and CD44 (phycoerythrin (PE)) antibodies to identify CD44+/CD24-population using a FACSCalibur flow cytometer. (b) SP popula‐ tion in MCF-7 stable cells was determined by Hoechst 33342 efflux assays. (c) Left, MCF-7 cells (siCon and siKLF4) were grown in ultra-low attachment surface plates at a density of 1000, 500, 200, and 100 per well. Assays were conducted after 10 days (left). The symbol \* indicates *P*<0.05 vs siCon group. Right, primary (P1) and secondary (P2) mammosphere forma‐ tion under suspension culture conditions were evaluated in MCF-7 mammary tumor cell lines.

expression of KLF4 gradually recovered. Kenpaullone-treated cells possessed phenotypes similar to KLF4 knockdown cells in our studies, which, from another point of view, confirmed the indispensable role of KLF4 in CSCs and extended a function of Kenpaullone

Function of KLF4 in Stem Cell Biology http://dx.doi.org/10.5772/54370 325

Our research also indicates that KLF4 might promote epithelial-mesenchymal transition (EMT) in breast cancers. EMT is a unique process by which epithelial cells undergo remarka‐ ble morphological changes (leading to increased motility and invasion) and believed to be reminiscent of 'cancer stem-like cells', showing characteristics similar to many cancer sys‐ tems [81, 82]. It has been reported that KLF4 interacts with transforming growth factor-β, a well established regulator of EMT [83], and β-catenin, one of the most important mesenchy‐ mal markers. Based on the pivotal role of KLF4 in CSCs, in combination with its links to the transforming growth factor-β signaling pathway, we highly suspected that KLF4 improved EMT in breast cancers. In our studies, KLF4 knockdown MCF-7 cells exhibited a well-spread morphology, with the majority of cells forming a rounded, epithelial-like form and aggre‐ gating together in groups, a typical characteristic of mesenchymal to epithelial transition [84] and a reversal of EMT. Fibronectin and vimentin, two critical mesenchymal-associated markers, were both decreased in KLF4 downregulated cells, which were consistent with re‐ duced ability of migration and invasion of these cells. However, E-cadherin expression and localization, a hallmark of the EMT phenotype, showed no significant difference after KLF4 was knocked down. Contrary to our results though, KLF4 was reported to inhibit EMT in non-transformed MCF-10A cells by another group [85]. Our major argument is that MCF-10A cells are spontaneously transformed cells with no potential of tumorigenesis. Therefore, the results from MCF-10A cells may not be readily applicable to other mammary tumor cells. In their study, MDA-MB-231 tumor cells with KLF4 overexpression had also been used. However, results from our studies, using KLF4 knockdown and overexpression stable cells, supported a positive connection between KLF4 and EMT. Clearly, more studies are necessary to examine whether the difference of the two systems or the genetic back‐ ground of specific MDA-MB-231 clones contributes to the discrepancies between the previ‐

Skin is renewed throughout life by proliferation of a multipotential stem cell population and terminal differentiation of stem cell progeny. Epidermal renewal is thought to be controlled by stem cells located either in the basal layer of the interfollicular epidermis (IFE) or in the deepest portion of permanent hair follicle called bulge [86]. Mouse hair follicle stem cells which reside in the hair follicle bulge are characterized by expression of CD34 and CD49 [87-89], retention of either DNA or histone labels over long periods [90, 91], and expression of Leucine-rich repeats and immunoglobin-like domain protein 1 (Lrig1) [92, 93]. Wound healing is an important response of skin in order that it might repair itself after an injury. Regeneration of epidermis after wounding involves activation, migration and proliferation of keratinocytes from both the surrounding epidermis and the adnexal structures such as hair follicles [94-96]. The discovery of properties of epidermal stem cells led to the hypothe‐

from the induction of iPS cells to the maintenance of mammary CSCs.

ously reported results and our current results.

**4.4. KLF4 function in mouse hair follicle stem cells**

**Figure 3.** Knockdown of KLF4 reduced tumorigenesis in vitro and in vivo. (a) Colony-forming abilities of siCon and siKLF4 cells were assessed. The symbol \* indicates *P*<0.05 vs siCon group. (b) Tumor growth curves were plotted for immunocompromised non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice injected with KLFknockdown (siKLF4, solid line) and control cells (siCon, dashed line). Data are shown as mean size ± s.e.m. of tumors in five mice per cell line.

The function of KLF4 in maintenance of CSCs has been further confirmed in our study by using Kenpaullone, a small molecule inhibitor of KLF4. Previous work has demonstrated that Kenpaullone is able to replace KLF4 in the reprogramming of primary and secondary fibroblasts, and that Kenpaullone-induced iPS cells display characteristics of pluripotent ES cells [78]. We tested KLF4 expression in Kenpaullone-treated breast cancer cell lines and found that it decreased at both of the mRNA and protein levels. Additional reporter assays showed that KLF4 promoter activity was significantly inhibited by Kenpaullone treatment, suggesting that Kenpaullone-mediated downregulation of KLF4 occurred at a transcription‐ al level. KLF4 downregulation was also accompanied by decreased expression of two previ‐ ously reported down-stream targets [79, 80]: p53 and intestinal alkaline phosphatase. This further validates the regulation of KLF4 by Kenpaullone. Since a maximal downregulation of KLF4 was observed at a 4 h time point after Kenpaullone treatment, we postulate that KLF4 may be an early responsive gene after Kenpaullone treatment, and after this point, the expression of KLF4 gradually recovered. Kenpaullone-treated cells possessed phenotypes similar to KLF4 knockdown cells in our studies, which, from another point of view, confirmed the indispensable role of KLF4 in CSCs and extended a function of Kenpaullone from the induction of iPS cells to the maintenance of mammary CSCs.

Our research also indicates that KLF4 might promote epithelial-mesenchymal transition (EMT) in breast cancers. EMT is a unique process by which epithelial cells undergo remarka‐ ble morphological changes (leading to increased motility and invasion) and believed to be reminiscent of 'cancer stem-like cells', showing characteristics similar to many cancer sys‐ tems [81, 82]. It has been reported that KLF4 interacts with transforming growth factor-β, a well established regulator of EMT [83], and β-catenin, one of the most important mesenchy‐ mal markers. Based on the pivotal role of KLF4 in CSCs, in combination with its links to the transforming growth factor-β signaling pathway, we highly suspected that KLF4 improved EMT in breast cancers. In our studies, KLF4 knockdown MCF-7 cells exhibited a well-spread morphology, with the majority of cells forming a rounded, epithelial-like form and aggre‐ gating together in groups, a typical characteristic of mesenchymal to epithelial transition [84] and a reversal of EMT. Fibronectin and vimentin, two critical mesenchymal-associated markers, were both decreased in KLF4 downregulated cells, which were consistent with re‐ duced ability of migration and invasion of these cells. However, E-cadherin expression and localization, a hallmark of the EMT phenotype, showed no significant difference after KLF4 was knocked down. Contrary to our results though, KLF4 was reported to inhibit EMT in non-transformed MCF-10A cells by another group [85]. Our major argument is that MCF-10A cells are spontaneously transformed cells with no potential of tumorigenesis. Therefore, the results from MCF-10A cells may not be readily applicable to other mammary tumor cells. In their study, MDA-MB-231 tumor cells with KLF4 overexpression had also been used. However, results from our studies, using KLF4 knockdown and overexpression stable cells, supported a positive connection between KLF4 and EMT. Clearly, more studies are necessary to examine whether the difference of the two systems or the genetic back‐ ground of specific MDA-MB-231 clones contributes to the discrepancies between the previ‐ ously reported results and our current results.

#### **4.4. KLF4 function in mouse hair follicle stem cells**

**Figure 3.** Knockdown of KLF4 reduced tumorigenesis in vitro and in vivo. (a) Colony-forming abilities of siCon and siKLF4 cells were assessed. The symbol \* indicates *P*<0.05 vs siCon group. (b) Tumor growth curves were plotted for immunocompromised non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice injected with KLFknockdown (siKLF4, solid line) and control cells (siCon, dashed line). Data are shown as mean size ± s.e.m. of tumors in

The function of KLF4 in maintenance of CSCs has been further confirmed in our study by using Kenpaullone, a small molecule inhibitor of KLF4. Previous work has demonstrated that Kenpaullone is able to replace KLF4 in the reprogramming of primary and secondary fibroblasts, and that Kenpaullone-induced iPS cells display characteristics of pluripotent ES cells [78]. We tested KLF4 expression in Kenpaullone-treated breast cancer cell lines and found that it decreased at both of the mRNA and protein levels. Additional reporter assays showed that KLF4 promoter activity was significantly inhibited by Kenpaullone treatment, suggesting that Kenpaullone-mediated downregulation of KLF4 occurred at a transcription‐ al level. KLF4 downregulation was also accompanied by decreased expression of two previ‐ ously reported down-stream targets [79, 80]: p53 and intestinal alkaline phosphatase. This further validates the regulation of KLF4 by Kenpaullone. Since a maximal downregulation of KLF4 was observed at a 4 h time point after Kenpaullone treatment, we postulate that KLF4 may be an early responsive gene after Kenpaullone treatment, and after this point, the

five mice per cell line.

324 Pluripotent Stem Cells

Skin is renewed throughout life by proliferation of a multipotential stem cell population and terminal differentiation of stem cell progeny. Epidermal renewal is thought to be controlled by stem cells located either in the basal layer of the interfollicular epidermis (IFE) or in the deepest portion of permanent hair follicle called bulge [86]. Mouse hair follicle stem cells which reside in the hair follicle bulge are characterized by expression of CD34 and CD49 [87-89], retention of either DNA or histone labels over long periods [90, 91], and expression of Leucine-rich repeats and immunoglobin-like domain protein 1 (Lrig1) [92, 93]. Wound healing is an important response of skin in order that it might repair itself after an injury. Regeneration of epidermis after wounding involves activation, migration and proliferation of keratinocytes from both the surrounding epidermis and the adnexal structures such as hair follicles [94-96]. The discovery of properties of epidermal stem cells led to the hypothe‐ sis that these stem cells play a critical role in epidermal repair after wounding. Previous work has reported that bulge stem cells rapidly respond to wounding and migrate towards the IFE to help with the rapid hair-follicle regeneration, and that bulge-derived cells are transient amplifying cells committed to differentiation [93, 95, 97]. However, the role and contribution of keratinocytes derived from hair follicle bulge stem cells to cutaneous wound healing needs further elucidation.

It has been proven that KLF4 is essential for establishing the barrier function of skin. How‐ ever, KLF4 expression and potential function in epidermal stem cells has not been studied before. In our current study, we have shown that KLF4 is likely expressed in mouse epider‐ mal stem cells. A decreased number of hair bulge stem cells was observed in KLF4 knockout mice, which was accompanied by a decreased ability of colony formation from these cells when compared to those from control mice, suggesting that KLF4 may be required for the maintenance of skin hair follicle stem cells. Notably, KLF4 deficiency delayed the process of mouse cutaneous wound healing, during which KLF4-expressing multipotent cells migrated towards the wound area [6].

**Figure 4.** KLF4-expressing cells possessed label retaining property. 3-day-old KLF4/EGFP mice were injected with BrdU (75mg/kg) for 5 consecutive days. BrdU-positive cells were examined 3 months later by immunohistochemical staining. Anti-KLF4, anti-BrdU, and anti-Ki67 antibodies were used to stain consecutive slides. Insets show enlarged portion of the staining indicating co-localization of KLF4 and BrdU positive cells with no Ki67 signals (red arrows). Scale bars, 50 mm.

Function of KLF4 in Stem Cell Biology http://dx.doi.org/10.5772/54370 327

**Figure 5.** KLF4-expressing hair follicle stem cells were examined by lineage tracing. KLF4/CreERTM/Rosa26RLacZ mice were induced by tamoxifen (100mg/kg) for 5 consecutive days at 6-week-old (a).4 weeks later X-gal staining was per‐ formed. Potential KLF4 expression in interfollicular epidermis (shown by red arrows in c, d) and bulge area (b, and black arrows in c, d) was shown. A typical epithelial proliferation unit was shown in e (inset). Note that fixation was

performed without xylene in a and b. Scale bars, 80 mm.

Using the wild type mice and KLF4/EGFP mouse model, we found that KLF4 was expressed in CD34+/CD49f+ bulge stem cell-enriched populations. However, KLF4 gene expression in CD34+/CD49f+/Lrig1+ cells was about 2.2 fold higher than in CD34+/CD49f-/Lrig1- cells sort‐ ed from wild-type mice. High levels of KLF4 expression in most differentiated, post mitotic skin epithelial cells [98] and low percentage of skin epidermal stem cells may be reasons why a difference has not been observed. Nevertheless, our studies collectively provide the first evidence that KLF4 was likely expressed in mouse hair follicle stem cells, especially in bulge stem cells.

The label retention cell (LRC) assay was used to confirm the quiescent nature of KLF4-ex‐ pressing cells (Figure 4). Three-day-old KLF4/EGFP mice were injected with BrdU and left for an extended period. Twelve weeks later, the proportion of KLF4-positive cells in LRCs was 4.1%, suggesting that only a subset of these LRCs expressed KLF4. These results reveal a heterogeneous nature of LRCs. However, the difference between KLF4-expressing and KLF4-non-expressing LRCs and the related functional influence in wound healing still re‐ main unknown. By lineage tracing to the KLF4/CreERTM/ Rosa26RLacZ mouse model, a multipotent and clonal nature of KLF4 expressing cells was identified as well (Figure 5). Our studies have also shown that KLF4 knockout decreased the population of CD34+/CD49f+ cells accompanied by reduced self-renewal ability of these cells. Together with the label re‐ taining ability of KLF4 expressing cells, our results indicated KLF4 plays an important role in the homeostasis of skin bulge stem cells. In addition, expression of KLF4 in rare skin stem cells and in the bulk of differentiated keratinocytes may suggest that the functions of KLF4 in these populations are different. It has been reported that different KLF4 isoforms may ex‐ ist and exhibit different functions in pancreatic cancer cell [99]. Characterization of different KLF4 isoforms and/or separation of distinct KLF4 expressing cells will be necessary for dis‐ secting specific functions of KLF4 in skin homeostasis as well as pathogenesis including wound healing.

sis that these stem cells play a critical role in epidermal repair after wounding. Previous work has reported that bulge stem cells rapidly respond to wounding and migrate towards the IFE to help with the rapid hair-follicle regeneration, and that bulge-derived cells are transient amplifying cells committed to differentiation [93, 95, 97]. However, the role and contribution of keratinocytes derived from hair follicle bulge stem cells to cutaneous wound

It has been proven that KLF4 is essential for establishing the barrier function of skin. How‐ ever, KLF4 expression and potential function in epidermal stem cells has not been studied before. In our current study, we have shown that KLF4 is likely expressed in mouse epider‐ mal stem cells. A decreased number of hair bulge stem cells was observed in KLF4 knockout mice, which was accompanied by a decreased ability of colony formation from these cells when compared to those from control mice, suggesting that KLF4 may be required for the maintenance of skin hair follicle stem cells. Notably, KLF4 deficiency delayed the process of mouse cutaneous wound healing, during which KLF4-expressing multipotent cells migrated

Using the wild type mice and KLF4/EGFP mouse model, we found that KLF4 was expressed in CD34+/CD49f+ bulge stem cell-enriched populations. However, KLF4 gene expression in CD34+/CD49f+/Lrig1+ cells was about 2.2 fold higher than in CD34+/CD49f-/Lrig1- cells sort‐ ed from wild-type mice. High levels of KLF4 expression in most differentiated, post mitotic skin epithelial cells [98] and low percentage of skin epidermal stem cells may be reasons why a difference has not been observed. Nevertheless, our studies collectively provide the first evidence that KLF4 was likely expressed in mouse hair follicle stem cells, especially in

The label retention cell (LRC) assay was used to confirm the quiescent nature of KLF4-ex‐ pressing cells (Figure 4). Three-day-old KLF4/EGFP mice were injected with BrdU and left for an extended period. Twelve weeks later, the proportion of KLF4-positive cells in LRCs was 4.1%, suggesting that only a subset of these LRCs expressed KLF4. These results reveal a heterogeneous nature of LRCs. However, the difference between KLF4-expressing and KLF4-non-expressing LRCs and the related functional influence in wound healing still re‐ main unknown. By lineage tracing to the KLF4/CreERTM/ Rosa26RLacZ mouse model, a multipotent and clonal nature of KLF4 expressing cells was identified as well (Figure 5). Our studies have also shown that KLF4 knockout decreased the population of CD34+/CD49f+ cells accompanied by reduced self-renewal ability of these cells. Together with the label re‐ taining ability of KLF4 expressing cells, our results indicated KLF4 plays an important role in the homeostasis of skin bulge stem cells. In addition, expression of KLF4 in rare skin stem cells and in the bulk of differentiated keratinocytes may suggest that the functions of KLF4 in these populations are different. It has been reported that different KLF4 isoforms may ex‐ ist and exhibit different functions in pancreatic cancer cell [99]. Characterization of different KLF4 isoforms and/or separation of distinct KLF4 expressing cells will be necessary for dis‐ secting specific functions of KLF4 in skin homeostasis as well as pathogenesis including

healing needs further elucidation.

326 Pluripotent Stem Cells

towards the wound area [6].

bulge stem cells.

wound healing.

**Figure 4.** KLF4-expressing cells possessed label retaining property. 3-day-old KLF4/EGFP mice were injected with BrdU (75mg/kg) for 5 consecutive days. BrdU-positive cells were examined 3 months later by immunohistochemical staining. Anti-KLF4, anti-BrdU, and anti-Ki67 antibodies were used to stain consecutive slides. Insets show enlarged portion of the staining indicating co-localization of KLF4 and BrdU positive cells with no Ki67 signals (red arrows). Scale bars, 50 mm.

**Figure 5.** KLF4-expressing hair follicle stem cells were examined by lineage tracing. KLF4/CreERTM/Rosa26RLacZ mice were induced by tamoxifen (100mg/kg) for 5 consecutive days at 6-week-old (a).4 weeks later X-gal staining was per‐ formed. Potential KLF4 expression in interfollicular epidermis (shown by red arrows in c, d) and bulge area (b, and black arrows in c, d) was shown. A typical epithelial proliferation unit was shown in e (inset). Note that fixation was performed without xylene in a and b. Scale bars, 80 mm.

even after acute, selective loss of mature neurons; meanwhile previously identified progeni‐ tors are largely responsible for tissue maintenance. Surprisingly after extensive injuries that deplete resident neuronal precursors, these quiescent stem cells transiently proliferate and reconstitute the neuroepithelium to maintain homeostasis [102]. Moreover, KLF4 deficiency delayed the process of wound healing and cell migration. It has been proven that KLF4 is essential for establishing skin barrier function because KLF4 deficiency selectively perturbed the late-stage differentiation structures including the cornified envelope [32]. It is not clear though, whether the role of KLF4 in barrier function is also involved in wound healing in our setting. Finally, our wound healing model did not limit for contraction. Although this simple method allowed us to observe an obvious phenotype, more rigorous models should be used in the future in order to define the role of KLF4 in the complex wound healing proc‐ ess. Nonetheless, our results suggest a critical function of KLF4-expressing epidermal multi‐

Function of KLF4 in Stem Cell Biology http://dx.doi.org/10.5772/54370 329

Stem cells often reside in locations called stem cell niches. Specifically, stem cell niches are defined as particular locations or microenvironments that maintain the combined properties of stem cell self-renewal and multipotency [103]. A combination of genetic and molecular analyses has identified many factors that support stem cell niches that also control stem cell identity. These factors include components of Notch, Wnt, and Hedgehog signaling path‐

Notch signaling is involved in cell proliferation and apoptosis, which affects the develop‐ ment and function of many organs. The signal is initiated by interaction of a Notch receptor with a Notch ligand on an adjacent cell. Upon activation, Notch is cleaved, releasing intra‐ cellular domain of the Notch (ICN) through a cascade of proteolytic cleavages by the metal‐ loprotease tumor necrosis factor-α-converting enzyme (TACE) and γ-secretase. ICN then translocates to the nucleus where it displaces corepressor complexes that are prebound with CSL. The following recruitment of coactivators, including Mastermind-like proteins and

It has been reported that altered Notch signaling affects the function of a variety of mamma‐ lian stem cells such as hematopoietic, intestinal, and skin stem cells, and intestinal stem cells in Drosophila and germ stem cells in C. elegans [103, 105, 108]. KLF4 is proposed as the downstream target of Notch signaling pathway and KLF4 promoter activity is inhibited by Notch, but the relationship between the Notch signaling pathway and KLF4 appears de‐ pendent on different cellular contexts. Our early work and that of others suggest that KLF4 is inhibited by Notch in the gastrointestinal tract [107, 109, 110]. Recently, downregulation of Notch1 gene expression in keratinocytes by KLF4 has also been reported [111].In our current study on breast CSCs, we found that the expression of Notch1, Notch2 and Jagged1 were

CBP/p300, then activates gene expression of downstream target genes [107].

potent stem cells in cutaneous wound healing.

**5. Notch signaling and KLF4**

**4.5. Signaling pathways regulating KLF4 and stem cell biology**

ways, all of which KLF4 is thought to be involved in [104-106].

**Figure 6.** Knockout of KLF4 decreased hair follicle stem cell population and self-renewal potential *in vitro* and retard‐ ed would healing *in vivo*. (a) Dorsal skin keratinocytes isolatedfrom control (KLF4+/+) and KLF4 knockout (KLF4-/-) mice were analyzed by flow cytometry using mouse epidermal stem cell markers CD34 and CD49f. (b) Quantitation of the colony numbers from 2000 seeded keratinocytes. Data shown were the mean ± SM of three separate experiments. \**P* <0.05 vs. control. 5mm wounds were introduced into the backs of KLF4/CreERTM/Rosa26RLacZ mice 5 (c, d) or 10 days (e–h) after using control (c, f) or tamoxifen (d, e, g, h) induction and X-gal staining was performed. Blue strips on epidermis were shown in d (inset 1) and h. Blue cells was indicated by black arrows outside (d) and by green arrows inside (e) the conjunction of the wound (separated by dashed green lines). Inset 2 in d showed blue cells around hair follicles. Migration of KLF4 expressing multipotent cells from hair follicles (g) and interfollicular epidermis towards the wound area was detected similarly.. Scale bars, 80 mm.

Previous work has demonstrated that stem cells located in the bulge area [95] and isthmus [100] contribute to wound healing. Our work has shown that KLF4-expressing multipotent cells participate in re-epithelialization during cutaneous wound healing. It known that cuta‐ neous wounds heal with an acute delay in re-epithelialization in the absence of hair follicles [101]. From our study we learned that KLF4 expression in possible hair follicle stem cells may contribute to the wound healing (Figure 6). We also observed that KLF4-expressing stem cells remained quiescent as evidenced by rarely detectable blue cells eight months after the cells were labeled. However, they were readily activated and detectable when the cuta‐ neous wound occurred. This observation is consistent with a recent proposal for olfactory neural stem cells. In this pattern, stem cells within the LRC population serve as a reservoir of long-lived progenitors that remain largely quiescent during normal neuronal turnover or even after acute, selective loss of mature neurons; meanwhile previously identified progeni‐ tors are largely responsible for tissue maintenance. Surprisingly after extensive injuries that deplete resident neuronal precursors, these quiescent stem cells transiently proliferate and reconstitute the neuroepithelium to maintain homeostasis [102]. Moreover, KLF4 deficiency delayed the process of wound healing and cell migration. It has been proven that KLF4 is essential for establishing skin barrier function because KLF4 deficiency selectively perturbed the late-stage differentiation structures including the cornified envelope [32]. It is not clear though, whether the role of KLF4 in barrier function is also involved in wound healing in our setting. Finally, our wound healing model did not limit for contraction. Although this simple method allowed us to observe an obvious phenotype, more rigorous models should be used in the future in order to define the role of KLF4 in the complex wound healing proc‐ ess. Nonetheless, our results suggest a critical function of KLF4-expressing epidermal multi‐ potent stem cells in cutaneous wound healing.

#### **4.5. Signaling pathways regulating KLF4 and stem cell biology**

Stem cells often reside in locations called stem cell niches. Specifically, stem cell niches are defined as particular locations or microenvironments that maintain the combined properties of stem cell self-renewal and multipotency [103]. A combination of genetic and molecular analyses has identified many factors that support stem cell niches that also control stem cell identity. These factors include components of Notch, Wnt, and Hedgehog signaling path‐ ways, all of which KLF4 is thought to be involved in [104-106].

## **5. Notch signaling and KLF4**

**Figure 6.** Knockout of KLF4 decreased hair follicle stem cell population and self-renewal potential *in vitro* and retard‐ ed would healing *in vivo*. (a) Dorsal skin keratinocytes isolatedfrom control (KLF4+/+) and KLF4 knockout (KLF4-/-) mice were analyzed by flow cytometry using mouse epidermal stem cell markers CD34 and CD49f. (b) Quantitation of the colony numbers from 2000 seeded keratinocytes. Data shown were the mean ± SM of three separate experiments. \**P* <0.05 vs. control. 5mm wounds were introduced into the backs of KLF4/CreERTM/Rosa26RLacZ mice 5 (c, d) or 10 days (e–h) after using control (c, f) or tamoxifen (d, e, g, h) induction and X-gal staining was performed. Blue strips on epidermis were shown in d (inset 1) and h. Blue cells was indicated by black arrows outside (d) and by green arrows inside (e) the conjunction of the wound (separated by dashed green lines). Inset 2 in d showed blue cells around hair follicles. Migration of KLF4 expressing multipotent cells from hair follicles (g) and interfollicular epidermis towards the

Previous work has demonstrated that stem cells located in the bulge area [95] and isthmus [100] contribute to wound healing. Our work has shown that KLF4-expressing multipotent cells participate in re-epithelialization during cutaneous wound healing. It known that cuta‐ neous wounds heal with an acute delay in re-epithelialization in the absence of hair follicles [101]. From our study we learned that KLF4 expression in possible hair follicle stem cells may contribute to the wound healing (Figure 6). We also observed that KLF4-expressing stem cells remained quiescent as evidenced by rarely detectable blue cells eight months after the cells were labeled. However, they were readily activated and detectable when the cuta‐ neous wound occurred. This observation is consistent with a recent proposal for olfactory neural stem cells. In this pattern, stem cells within the LRC population serve as a reservoir of long-lived progenitors that remain largely quiescent during normal neuronal turnover or

wound area was detected similarly.. Scale bars, 80 mm.

328 Pluripotent Stem Cells

Notch signaling is involved in cell proliferation and apoptosis, which affects the develop‐ ment and function of many organs. The signal is initiated by interaction of a Notch receptor with a Notch ligand on an adjacent cell. Upon activation, Notch is cleaved, releasing intra‐ cellular domain of the Notch (ICN) through a cascade of proteolytic cleavages by the metal‐ loprotease tumor necrosis factor-α-converting enzyme (TACE) and γ-secretase. ICN then translocates to the nucleus where it displaces corepressor complexes that are prebound with CSL. The following recruitment of coactivators, including Mastermind-like proteins and CBP/p300, then activates gene expression of downstream target genes [107].

It has been reported that altered Notch signaling affects the function of a variety of mamma‐ lian stem cells such as hematopoietic, intestinal, and skin stem cells, and intestinal stem cells in Drosophila and germ stem cells in C. elegans [103, 105, 108]. KLF4 is proposed as the downstream target of Notch signaling pathway and KLF4 promoter activity is inhibited by Notch, but the relationship between the Notch signaling pathway and KLF4 appears de‐ pendent on different cellular contexts. Our early work and that of others suggest that KLF4 is inhibited by Notch in the gastrointestinal tract [107, 109, 110]. Recently, downregulation of Notch1 gene expression in keratinocytes by KLF4 has also been reported [111].In our current study on breast CSCs, we found that the expression of Notch1, Notch2 and Jagged1 were significantly decreased in KLF4 knockdown cells, and upregulated by overexpression of KLF4. Unexpectedly, inhibition of the Notch pathway by CompE, a γ-secretase inhibitor, had no effect on stem cell numbers and self-renewal potential of breast cancer cells. This re‐ sult suggested that the Notch signaling pathway is not required for KLF4-mediated mainte‐ nance of stem cells in breast cancer cells (Figure 7). On the other hand, inhibition of Notch signaling by CompE in KLF4-overexpressing cells led to decreased migration and invasion ability, which indicated that the Notch signaling pathway was responsible for KLF4-mediat‐ ed mobility characteristics of breast cancer cells. These results are consistent with the role of Notch signaling as potent drivers during tumor progression and in converting polarized ep‐ ithelial cells into motile, invasive cells [112]. However, in breast cancer cells, inhibitors of canonical Notch1 signaling suppressed the transformation induced by Notch1 whereas it had no effect on the transformation by KLF4, indicating KLF4-induced transformation re‐ quires Notch1, canonical Notch1 signaling is not required, and Notch1 may signal through a distinct pathway in cells with increased KLF4 activity. These results suggest that KLF4 could contribute to breast tumor progression by activating synthesis of Notch1 and by promoting signaling through a non-canonical Notch1 pathway [113].

naling cascades have traditionally fallen into two categories: canonical and noncanonical, differentiated by their dependence on β-catenin. Canonical Wnt signaling is initiated when a Wnt ligand engages co-receptors of the Frizzled (Fzd) and low-density lipoprotein (LDL)-related protein (either Lrp5 or Lrp6), ultimately leading to β-catenin stabilization, nuclear translocation and activation of target genes. The canonical Wnt/βcatenin pathway plays a crucial role in stem and cancer stem cells' self-renewal and/or

Function of KLF4 in Stem Cell Biology http://dx.doi.org/10.5772/54370 331

In the absence of Wnt stimulus, β-catenin is held in an inactive state by a multimeric "de‐ struction" complex comprised of adenomatous polyposis coli (APC), Axin, glycogen syn‐ thase kinase 3β (GSK3β) and casein kinase1α (CK1α) [118]. Nearly 90% of colon cancer harbors Wnt/β-catenin signaling mutations that result in β-catenin mutation. The most com‐ mon type of mutation in colon cancer results in the inactivation of APC, thus driving constit‐ utive activation of β-catenin [119-121]. KLF4 binds the transcriptional activation domain of β-catenin and inhibits β-catenin-mediated transcription in colorectal cancer cells, suggesting that the cross talk between KLF4 and β-catenin plays an important role in intestinal homeo‐ stasis and colorectal carcinogenesis [122]. A growing body of evidence illustrates a critical role of β-catenin in CSCs. For example, stem-like colon cells with a high level of β-catenin signaling have a much greater tumorigenic potential than counterpart cells with low β-cate‐ nin signaling [123]. The latest report shows that in stem cells and cancer cells, TERT, the en‐ zymatic subunit of telomerase complex controlling telomere length, is directly regulated by

β-catenin, and klf4 is required for β-catenin to localize to the *Tert* promoter [124].

and the relationship between them needs further investigation as well.

**7. Hedgehog signaling and KLF4**

In over 50% of clinical breast cancer cases a stabilization of β-catenin has been demonstrat‐ ed. Inhibition of Wnt/β-catenin signaling in the mouse mammary gland blocks organ devel‐ opment and pregnancy-induced proliferation and heavily reduces the numbers of alveolar progenitor cells [125]. Wnt/β-catenin has also been implicated in mediating the radiation re‐ sistance of mouse mammary gland progenitor cells. Our recent study shows that KLF4 is re‐ quired for maintenance of breast CSCs and for cell migration and invasion along with Notch signaling pathway [7]. However, the reaction of KLF4 and Wnt/β-catenin signaling in this setting still remains unknown and needs further investigation. Our other work showed that KLF4 contributes to cutaneous wound healing [6]. Additionally, the canonical Wnt signals are required in the normal skin to instruct bulge stem cells toward the hair cell fate [126], while in epidermal tumors, they control the maintenance of skin CSCs [84]. Therefore it is speculated that both of KLF4 and Wnt/β-catenin signaling are implicated in this process,

Under normal conditions, HH signaling plays important roles in embryonic development and is also involved in tissue regeneration in adults [127, 128]. Activating events in the HH pathway are involved in numerous human cancers, including melanoma [129], glioma [130], and basal cell carcinoma (BCC) [131]. Mammalian HH signaling is initiated when one of

differentiation of skin, intestine and mammary gland [117].

**Figure 7.** Notch signaling pathway is activated but not required for KLF4-mediated maintenance of stem cells in breast cancer cells (a) Levels of Notch1, Notch2 and Jagged1 expression in siCon and siKLF4 MCF-7 cells were detected by real-time PCR. The symbol \* indicates *P*<0.05 vs siCon group. (b) Similar to (a) except that control and KLF4-N (KLF4 overexpression) MCF-7 cells were used. (c) MCF-7 cells (siCon and siKLF4) were seeded into ultra-low attachment sur‐ face plates and incubated with CompE at a concentration of 1 mM.

#### **6. Wnt signaling and KLF4**

Wnt signaling is an ancient and highly conserved system that is involved in embryogen‐ esis, development, cell polarization, differentiation and proliferation [114-116]. Wnt sig‐ naling cascades have traditionally fallen into two categories: canonical and noncanonical, differentiated by their dependence on β-catenin. Canonical Wnt signaling is initiated when a Wnt ligand engages co-receptors of the Frizzled (Fzd) and low-density lipoprotein (LDL)-related protein (either Lrp5 or Lrp6), ultimately leading to β-catenin stabilization, nuclear translocation and activation of target genes. The canonical Wnt/βcatenin pathway plays a crucial role in stem and cancer stem cells' self-renewal and/or differentiation of skin, intestine and mammary gland [117].

In the absence of Wnt stimulus, β-catenin is held in an inactive state by a multimeric "de‐ struction" complex comprised of adenomatous polyposis coli (APC), Axin, glycogen syn‐ thase kinase 3β (GSK3β) and casein kinase1α (CK1α) [118]. Nearly 90% of colon cancer harbors Wnt/β-catenin signaling mutations that result in β-catenin mutation. The most com‐ mon type of mutation in colon cancer results in the inactivation of APC, thus driving constit‐ utive activation of β-catenin [119-121]. KLF4 binds the transcriptional activation domain of β-catenin and inhibits β-catenin-mediated transcription in colorectal cancer cells, suggesting that the cross talk between KLF4 and β-catenin plays an important role in intestinal homeo‐ stasis and colorectal carcinogenesis [122]. A growing body of evidence illustrates a critical role of β-catenin in CSCs. For example, stem-like colon cells with a high level of β-catenin signaling have a much greater tumorigenic potential than counterpart cells with low β-cate‐ nin signaling [123]. The latest report shows that in stem cells and cancer cells, TERT, the en‐ zymatic subunit of telomerase complex controlling telomere length, is directly regulated by β-catenin, and klf4 is required for β-catenin to localize to the *Tert* promoter [124].

In over 50% of clinical breast cancer cases a stabilization of β-catenin has been demonstrat‐ ed. Inhibition of Wnt/β-catenin signaling in the mouse mammary gland blocks organ devel‐ opment and pregnancy-induced proliferation and heavily reduces the numbers of alveolar progenitor cells [125]. Wnt/β-catenin has also been implicated in mediating the radiation re‐ sistance of mouse mammary gland progenitor cells. Our recent study shows that KLF4 is re‐ quired for maintenance of breast CSCs and for cell migration and invasion along with Notch signaling pathway [7]. However, the reaction of KLF4 and Wnt/β-catenin signaling in this setting still remains unknown and needs further investigation. Our other work showed that KLF4 contributes to cutaneous wound healing [6]. Additionally, the canonical Wnt signals are required in the normal skin to instruct bulge stem cells toward the hair cell fate [126], while in epidermal tumors, they control the maintenance of skin CSCs [84]. Therefore it is speculated that both of KLF4 and Wnt/β-catenin signaling are implicated in this process, and the relationship between them needs further investigation as well.

### **7. Hedgehog signaling and KLF4**

significantly decreased in KLF4 knockdown cells, and upregulated by overexpression of KLF4. Unexpectedly, inhibition of the Notch pathway by CompE, a γ-secretase inhibitor, had no effect on stem cell numbers and self-renewal potential of breast cancer cells. This re‐ sult suggested that the Notch signaling pathway is not required for KLF4-mediated mainte‐ nance of stem cells in breast cancer cells (Figure 7). On the other hand, inhibition of Notch signaling by CompE in KLF4-overexpressing cells led to decreased migration and invasion ability, which indicated that the Notch signaling pathway was responsible for KLF4-mediat‐ ed mobility characteristics of breast cancer cells. These results are consistent with the role of Notch signaling as potent drivers during tumor progression and in converting polarized ep‐ ithelial cells into motile, invasive cells [112]. However, in breast cancer cells, inhibitors of canonical Notch1 signaling suppressed the transformation induced by Notch1 whereas it had no effect on the transformation by KLF4, indicating KLF4-induced transformation re‐ quires Notch1, canonical Notch1 signaling is not required, and Notch1 may signal through a distinct pathway in cells with increased KLF4 activity. These results suggest that KLF4 could contribute to breast tumor progression by activating synthesis of Notch1 and by promoting

**Figure 7.** Notch signaling pathway is activated but not required for KLF4-mediated maintenance of stem cells in breast cancer cells (a) Levels of Notch1, Notch2 and Jagged1 expression in siCon and siKLF4 MCF-7 cells were detected by real-time PCR. The symbol \* indicates *P*<0.05 vs siCon group. (b) Similar to (a) except that control and KLF4-N (KLF4 overexpression) MCF-7 cells were used. (c) MCF-7 cells (siCon and siKLF4) were seeded into ultra-low attachment sur‐

Wnt signaling is an ancient and highly conserved system that is involved in embryogen‐ esis, development, cell polarization, differentiation and proliferation [114-116]. Wnt sig‐

signaling through a non-canonical Notch1 pathway [113].

330 Pluripotent Stem Cells

face plates and incubated with CompE at a concentration of 1 mM.

**6. Wnt signaling and KLF4**

Under normal conditions, HH signaling plays important roles in embryonic development and is also involved in tissue regeneration in adults [127, 128]. Activating events in the HH pathway are involved in numerous human cancers, including melanoma [129], glioma [130], and basal cell carcinoma (BCC) [131]. Mammalian HH signaling is initiated when one of three HH ligands (Sonic, Indian, and Desert HH) binds the dodecatransmembrane receptor Patched (Ptch1). Ligand/receptor interactions occur through an autocrine or paracrine man‐ ner, depending on the context. Receptor engagement results in activation of the heptatrans‐ membrane Smoothened (Smo), which is held in an inactive state in the absence of a ligand. Smo activation in turn regulates the activity of transcription factors Gli1, Gli2 and Gli3. Gli1/2/3 function to regulate transcription of genes involved in HH signaling such as Gli1 and Ptch1, and importantly genes involved in epithelial-mesenchymaltransition (EMT), such as SNAIL1[127, 128].

regulate TERT expression in stem cells and cancer cells. As to HH signaling and KLF4, the research is still just beginning, but considering the crosstalk between Wnt/β-catenin and HH, it is very important to discern the communication between them. Nevertheless, under‐ standing the signaling circuitries regulating stem cell fate decisions might provide impor‐

Function of KLF4 in Stem Cell Biology http://dx.doi.org/10.5772/54370 333

The authors gratefully acknowledge Mr. Andrew Vaughan and Mr. Matthew Riester for critical reading and editing of the manuscript. This work was supported by NIH grants to

1 Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of

2 Department of Pathology, Microbiology, and Immunology, University of South Carolina

[1] Bieker JJ.Kruppel-like factors: three fingers in many pies. Journal of Biological Chem‐

[2] Philipsen S, Suske G.A tale of three fingers: the family of mammalian Sp/XKLF tran‐

[3] Turner J, Crossley M.Mammalian Kruppel-like transcription factors: more than just a

[4] Kaczynski J, Cook T, Urrutia R.Sp1- and Kruppel-like transcription factors. Genome

[5] Takahashi K, Yamanaka S.Induction of pluripotent stem cells from mouse embryonic

and adult fibroblast cultures by defined factors. Cell 2006;126(4)663-676.

scription factors. Nucleic Acids Research 1999;27(15)2991-3000.

pretty finger. Trends In Biochemical Sciences 1999;24(6)236-240.

tant insights into novel therapeutic strategies for cancer and regeneration medicine.

**Acknowledgements**

**Author details**

Ying Shi1

**References**

W. A. (KO1DK069489 and RO3AR060987).

and Walden Ai2\*

\*Address all correspondence to: Walden.Ai@uscmed.sc.edu

Science and Technology, Wuhan, Hubai, China

School of Medicine, Columbia, SC, USA

istry 2001;276(37)34355-34358.

Biology 2003;4(2)206.

HH-GLI signaling was found to modulate normal dorsal brain growth by controlling pre‐ cursor proliferation [132]; it was also found to have an essential role in controlling the be‐ havior of CD133+ glioma cancer stem cells [130]. However, HH pathway-driven tumorigenesis depends on canonical Wnt/β-catenin signaling in BCC [131]. Recently, CSC/ tumor initiating cells (TIC) in human melanomas were found in a collection of human mela‐ nomas obtained from a broad spectrum of sites and stages by using non-adherent spheres and ALDH enzymatic activity. Both pharmacological inhibition of HH signaling by the SMO antagonist cyclopamine and GLI antagonist GANT61, and stable expression of shRNA tar‐ geting either SMO or GLI1 result in a significant decrease in melanoma stem cell self-renew‐ al *in vitro* and a reduction in the number of ALDH high melanoma stem cells, indicating an essential role of the HH-GLI1 signaling in of melanoma CSC/TIC. Notably, melanoma‐ spheres express not only high levels of Hedgehog pathway components, but also high levels of embryonic pluripotent stem cell factors Sox2, Nanog, Oct4 and KLF4 [129]. This is the first report that reveals a possible correlation of HH signaling and KLF4 in CSC, though the un‐ derlying mechanism appears entirely unknown.

## **8. Concluding remarks**

Since the identification and characterization of KLF4 over 10 years ago, significant progres‐ sion has been made to understand its biological function, including its role in cell prolifera‐ tion, differentiation, apoptosis and maintenance of normal tissue homeostasis. However, a novel role of KLF4 in stem cell biology further opens a window to study KLF4 in a different area. KLF4 is believed to play a significant role in ES cell self-renewal and pluripotency. No‐ tably, KLF4 collaborating with other transcription factors including Oct4, Sox2 and c-Myc, drives somatic cells into iPS cells. CSCs have been identified in various tumors, and KLF4 can be speculated to have similar functions in CSCs based on its function in ES cell [133]. Our work provides evidence for the first time that KLF4 is essential for the maintenance of breast CSC and cell migration and invasion, which may be helpful to develop new therapeu‐ tic strategies for breast cancer. Apart from just breast CSCs, our work also demonstrates that KLF4 is highly expressed in skin hair follicle stem cells and facilitates the process of cutane‐ ous wound healing. Many papers have confirmed the underlying molecular mechanism that KLF4 exerts its action in stem cell biology by integration of different signaling pathways, in‐ cluding Notch, Wnt and HH. Notch signaling pathway is responsible for KLF4-mediated mobility characteristics of breast cancer cells, while Wnt/β-catenin signaling recruits KLF4 to regulate TERT expression in stem cells and cancer cells. As to HH signaling and KLF4, the research is still just beginning, but considering the crosstalk between Wnt/β-catenin and HH, it is very important to discern the communication between them. Nevertheless, under‐ standing the signaling circuitries regulating stem cell fate decisions might provide impor‐ tant insights into novel therapeutic strategies for cancer and regeneration medicine.

## **Acknowledgements**

three HH ligands (Sonic, Indian, and Desert HH) binds the dodecatransmembrane receptor Patched (Ptch1). Ligand/receptor interactions occur through an autocrine or paracrine man‐ ner, depending on the context. Receptor engagement results in activation of the heptatrans‐ membrane Smoothened (Smo), which is held in an inactive state in the absence of a ligand. Smo activation in turn regulates the activity of transcription factors Gli1, Gli2 and Gli3. Gli1/2/3 function to regulate transcription of genes involved in HH signaling such as Gli1 and Ptch1, and importantly genes involved in epithelial-mesenchymaltransition (EMT), such

HH-GLI signaling was found to modulate normal dorsal brain growth by controlling pre‐ cursor proliferation [132]; it was also found to have an essential role in controlling the be‐ havior of CD133+ glioma cancer stem cells [130]. However, HH pathway-driven tumorigenesis depends on canonical Wnt/β-catenin signaling in BCC [131]. Recently, CSC/ tumor initiating cells (TIC) in human melanomas were found in a collection of human mela‐ nomas obtained from a broad spectrum of sites and stages by using non-adherent spheres and ALDH enzymatic activity. Both pharmacological inhibition of HH signaling by the SMO antagonist cyclopamine and GLI antagonist GANT61, and stable expression of shRNA tar‐ geting either SMO or GLI1 result in a significant decrease in melanoma stem cell self-renew‐ al *in vitro* and a reduction in the number of ALDH high melanoma stem cells, indicating an essential role of the HH-GLI1 signaling in of melanoma CSC/TIC. Notably, melanoma‐ spheres express not only high levels of Hedgehog pathway components, but also high levels of embryonic pluripotent stem cell factors Sox2, Nanog, Oct4 and KLF4 [129]. This is the first report that reveals a possible correlation of HH signaling and KLF4 in CSC, though the un‐

Since the identification and characterization of KLF4 over 10 years ago, significant progres‐ sion has been made to understand its biological function, including its role in cell prolifera‐ tion, differentiation, apoptosis and maintenance of normal tissue homeostasis. However, a novel role of KLF4 in stem cell biology further opens a window to study KLF4 in a different area. KLF4 is believed to play a significant role in ES cell self-renewal and pluripotency. No‐ tably, KLF4 collaborating with other transcription factors including Oct4, Sox2 and c-Myc, drives somatic cells into iPS cells. CSCs have been identified in various tumors, and KLF4 can be speculated to have similar functions in CSCs based on its function in ES cell [133]. Our work provides evidence for the first time that KLF4 is essential for the maintenance of breast CSC and cell migration and invasion, which may be helpful to develop new therapeu‐ tic strategies for breast cancer. Apart from just breast CSCs, our work also demonstrates that KLF4 is highly expressed in skin hair follicle stem cells and facilitates the process of cutane‐ ous wound healing. Many papers have confirmed the underlying molecular mechanism that KLF4 exerts its action in stem cell biology by integration of different signaling pathways, in‐ cluding Notch, Wnt and HH. Notch signaling pathway is responsible for KLF4-mediated mobility characteristics of breast cancer cells, while Wnt/β-catenin signaling recruits KLF4 to

as SNAIL1[127, 128].

332 Pluripotent Stem Cells

derlying mechanism appears entirely unknown.

**8. Concluding remarks**

The authors gratefully acknowledge Mr. Andrew Vaughan and Mr. Matthew Riester for critical reading and editing of the manuscript. This work was supported by NIH grants to W. A. (KO1DK069489 and RO3AR060987).

## **Author details**

Ying Shi1 and Walden Ai2\*

\*Address all correspondence to: Walden.Ai@uscmed.sc.edu

1 Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubai, China

2 Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, SC, USA

## **References**


[6] Li J, Zheng H, Wang J, Yu F, Morris RJ, et al.Expression of Kruppel-Like Factor KLF4 in Mouse Hair Follicle Stem Cells Contributes to Cutaneous Wound Healing. PLoS One 2012;7(6)e39663.

[17] Chen X, Whitney EM, Gao SY, Yang VW.Transcriptional profiling of Kruppel-like factor 4 reveals a function in cell cycle regulation and epithelial differentiation. Jour‐

Function of KLF4 in Stem Cell Biology http://dx.doi.org/10.5772/54370 335

[18] Zhang W, Geiman DE, Shields JM, Dang DT, Mahatan CS, et al.The gut-enriched Kruppel-like factor (Kruppel-like factor 4) mediates the transactivating effect of p53 on the p21WAF1/Cip1 promoter. Journal of Biological Chemistry

[19] Shie JL, Chen ZY, Fu M, Pestell RG, Tseng CC.Gut-enriched Kruppel-like factor re‐ presses cyclin D1 promoter activity through Sp1 motif. Nucleic Acids Research

[20] Chen X, Johns DC, Geiman DE, Marban E, Dang DT, et al.Kruppel-like factor 4 (gutenriched Kruppel-like factor) inhibits cell proliferation by blocking G1/S progression

[21] Yoon HS, Chen X, Yang VW.Kruppel-like factor 4 mediates p53-dependent G1/S cell cycle arrest in response to DNA damage. Journal of Biological Chemistry

[22] Yoon HS, Yang VW.Requirement of Kruppel-like factor 4 in preventing entry into mitosis following DNA damage. Journal of Biological Chemistry

[23] Yoon HS, Ghaleb AM, Nandan MO, Hisamuddin IM, Dalton WB, et al.Kruppel-like factor 4 prevents centrosome amplification following gamma-irradiation-induced

[24] Zhang W, Shields JM, Sogawa K, Fujii-Kuriyama Y, Yang VW.The gut-enriched Kruppel-like factor suppresses the activity of the CYP1A1 promoter in an Sp1-de‐

[25] Miller KA, Eklund EA, Peddinghaus ML, Cao Z, Fernandes N, et al.Kruppel-like fac‐ tor 4 regulates laminin alpha 3A expression in mammary epithelial cells. Journal of

[26] Higaki Y, Schullery D, Kawata Y, Shnyreva M, Abrass C, et al.Synergistic activation of the rat laminin gamma1 chain promoter by the gut-enriched Kruppel-like factor

[27] Okano J, Opitz OG, Nakagawa H, Jenkins TD, Friedman SL, et al.The Kruppel-like transcriptional factors Zf9 and GKLF coactivate the human keratin 4 promoter and

[28] Brembeck FH, Rustgi AK.The tissue-dependent keratin 19 gene transcription is regu‐ lated by GKLF/KLF4 and Sp1. Journal of Biological Chemistry

(GKLF/KLF4) and Sp1. Nucleic Acids Research 2002;30(11)2270-2279.

pendent fashion. Journal of Biological Chemistry 1998;273(28)17917-17925.

of the cell cycle. Journal of Biological Chemistry 2001;276(32)30423-30428.

nal of Molecular Biology 2003;326(3)665-677.

2000;275(24)18391-18398.

2000;28(15)2969-2976.

2003;278(4)2101-2105.

2004;279(6)5035-5041.

DNA damage. Oncogene 2005;24(25)4017-4025.

Biological Chemistry 2001;276(46)42863-42868.

physically interact. Febs Letters 2000;473(1)95-100.

2000;275(36)28230-28239.


[17] Chen X, Whitney EM, Gao SY, Yang VW.Transcriptional profiling of Kruppel-like factor 4 reveals a function in cell cycle regulation and epithelial differentiation. Jour‐ nal of Molecular Biology 2003;326(3)665-677.

[6] Li J, Zheng H, Wang J, Yu F, Morris RJ, et al.Expression of Kruppel-Like Factor KLF4 in Mouse Hair Follicle Stem Cells Contributes to Cutaneous Wound Healing. PLoS

[7] Yu F, Li J, Chen H, Fu J, Ray S, et al.Kruppel-like factor 4 (KLF4) is required for main‐ tenance of breast cancer stem cells and for cell migration and invasion. Oncogene

[8] Shields JM, Christy RJ, Yang VW.Identification and characterization of a gene encod‐ ing a gut-enriched Kruppel-like factor expressed during growth arrest. Journal of Bi‐

[9] Garrett-Sinha LA, Eberspaecher H, Seldin MF, de Crombrugghe B.A gene for a novel zinc-finger protein expressed in differentiated epithelial cells and transiently in cer‐ tain mesenchymal cells. Journal of Biological Chemistry 1996;271(49)31384-31390.

[10] Yet SF, McA'Nulty MM, Folta SC, Yen HW, Yoshizumi M, et al.Human EZF, a Krup‐ pel-like zinc finger protein, is expressed in vascular endothelial cells and contains transcriptional activation and repression domains. Journal of Biological Chemistry

[11] Panigada M, Porcellini S, Sutti F, Doneda L, Pozzoli O, et al.GKLF in thymus epithe‐ lium as a developmentally regulated element of thymocyte-stroma cross-talk. Mecha‐

[12] Chiambaretta F, De Graeve F, Turet G, Marceau G, Gain P, et al.Cell and tissue spe‐ cific expression of human Kruppel-like transcription factors in human ocular surface.

[13] Cullingford TE, Butler MJ, Marshall AK, Tham el L, Sugden PH, et al.Differential regulation of Kruppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: effects of endothelin-1, oxidative stress and cytokines. Biochi‐

[14] Fruman DA, Ferl GZ, An SS, Donahue AC, Satterthwaite AB, et al.Phosphoinositide 3-kinase and Bruton's tyrosine kinase regulate overlapping sets of genes in B lym‐ phocytes. Proceedings of the National Academy of Sci ence of the United States of

[15] Behr R, Kaestner KH.Developmental and cell type-specific expression of the zinc fin‐ ger transcription factor Kruppel-like factor 4 (Klf4) in postnatal mouse testis. Mecha‐

[16] Godmann M, Kromberg I, Mayer J, Behr R.The mouse Kruppel-like Factor 4 (Klf4) gene: four functional polyadenylation sites which are used in a cell-specific manner as revealed by testicular transcript analysis and multiple processed pseudogenes.

One 2012;7(6)e39663.

334 Pluripotent Stem Cells

2011;30(18)2161-2172.

1998;273(2)1026-1031.

ological Chemistry 1996;271(33)20009-20017.

niams of Development 1999;81(1-2)103-113.

mica et Biophysica Acta 2008;1783(6)1229-1236.

niams of Development 2002;115(1-2)167-169.

Molecular Vision 2004;10 901-909.

America 2002;99(1)359-364.

Gene 2005;361(149-156.


[29] Katz JP, Perreault N, Goldstein BG, Lee CS, Labosky PA, et al.The zinc-finger tran‐ scription factor Klf4 is required for terminal differentiation of goblet cells in the co‐ lon. Development 2002;129(11)2619-2628.

[41] Lee HJ, Kang YM, Moon CS, Joe MK, Lim JH, et al.KLF4 positively regulates human

Function of KLF4 in Stem Cell Biology http://dx.doi.org/10.5772/54370 337

[42] Dijkmans TF, van Hooijdonk LW, Schouten TG, Kamphorst JT, Fitzsimons CP, et al.Identification of new Nerve Growth Factor-responsive immediate-early genes.

[43] Zhu S, Tai C, MacVicar BA, Jia W, Cynader MS.Glutamatergic stimulation triggers rapid Krupple-like factor 4 expression in neurons and the overexpression of KLF4 sensitizes neurons to NMDA-induced caspase-3 activity. Brain Research

[44] Yamada T, Park CS, Mamonkin M, Lacorazza HD.Transcription factor ELF4 controls the proliferation and homing of CD8+ T cells via the Kruppel-like factors KLF4 and

[45] Wei D, Gong W, Kanai M, Schlunk C, Wang L, et al.Drastic down-regulation of Kruppel-like factor 4 expression is critical in human gastric cancer development and

[46] Ghaleb AM, Katz JP, Kaestner KH, Du JX, Yang VW.Kruppel-like factor 4 exhibits antiapoptotic activity following gamma-radiation-induced DNA damage. Oncogene

[47] Dang DT, Chen X, Feng J, Torbenson M, Dang LH, et al.Overexpression of Kruppellike factor 4 in the human colon cancer cell line RKO leads to reduced tumorigeneci‐

[48] Zhang G, Zhu H, Wang Y, Yang S, Liu M, et al.Kruppel-like factor 4 represses tran‐ scription of the survivin gene in esophageal cancer cell lines. Biological Chemistry

[49] Kidder BL, Yang J, Palmer S.Stat3 and c-Myc genome-wide promoter occupancy in

[50] Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, et al.Transcriptional regulation of nanog by OCT4 and SOX2. Journal of Biological Chemistry 2005;280(26)24731-24737.

[51] Zhang P, Andrianakos R, Yang Y, Liu C, Lu W.Kruppel-like factor 4 (Klf4) prevents embryonic stem (ES) cell differentiation by regulating Nanog gene expression. Jour‐

[52] Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, et al.In vitro reprogram‐ ming of fibroblasts into a pluripotent ES-cell-like state. Nature 2007;448(7151)318-324.

[53] Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, et al.Directly reprogrammed fi‐ broblasts show global epigenetic remodeling and widespread tissue contribution.

ghrelin expression. Biochemical Journal 2009;420(3)403-411.

Brain Research 2009;1249 19-33.

KLF2. Nature Immunology 2009;10(6)618-626.

progression. Cancer Research 2005;65(7)2746-2754.

embryonic stem cells. PLoS One 2008;3(12)e3932.

nal of Biological Chemistry 2010;285(12)9180-9189.

2009;1250(49-62.

2007;26(16)2365-2373.

2009;390(5-6)463-469.

Cell Stem Cell 2007;1(1)55-70.

ty. Oncogene 2003;22(22)3424-3430.


[41] Lee HJ, Kang YM, Moon CS, Joe MK, Lim JH, et al.KLF4 positively regulates human ghrelin expression. Biochemical Journal 2009;420(3)403-411.

[29] Katz JP, Perreault N, Goldstein BG, Lee CS, Labosky PA, et al.The zinc-finger tran‐ scription factor Klf4 is required for terminal differentiation of goblet cells in the co‐

[30] Katz JP, Perreault N, Goldstein BG, Actman L, McNally SR, et al.Loss of Klf4 in mice causes altered proliferation and differentiation and precancerous changes in the

[31] Swamynathan SK, Katz JP, Kaestner KH, Ashery-Padan R, Crawford MA, et al.Con‐ ditional deletion of the mouse Klf4 gene results in corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells. Molecular and Cellular Biology

[32] Segre JA, Bauer C, Fuchs E.Klf4 is a transcription factor required for establishing the

[33] Adam PJ, Regan CP, Hautmann MB, Owens GK.Positive- and negative-acting Krup‐ pel-like transcription factors bind a transforming growth factor beta control element required for expression of the smooth muscle cell differentiation marker SM22alpha

[34] Feinberg MW, Wara AK, Cao Z, Lebedeva MA, Rosenbauer F, et al.The Kruppel-like factor KLF4 is a critical regulator of monocyte differentiation. Embo Journal

[35] Dik WA, Pike-Overzet K, Weerkamp F, de Ridder D, de Haas EF, et al.New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling. Journal of Experimental Medicine

[36] Chen Z, Couble ML, Mouterfi N, Magloire H, Bleicher F.Spatial and temporal expres‐ sion of KLF4 and KLF5 during murine tooth development. Archives of Oral Biology

[37] Feinberg MW, Cao Z, Wara AK, Lebedeva MA, Senbanerjee S, et al.Kruppel-like fac‐ tor 4 is a mediator of proinflammatory signaling in macrophages. Journal of Biologi‐

[38] Liu J, Liu Y, Zhang H, Chen G, Wang K, et al.KLF4 promotes the expression, translo‐ cation, and releas eof HMGB1 in RAW264.7 macrophages in response to LPS. Shock

[39] Liu J, Zhang H, Liu Y, Wang K, Feng Y, et al.KLF4 regulates the expression of inter‐ leukin-10 in RAW264.7 macrophages. Biochemical and Biophysical Research Com‐

[40] Alder JK, Georgantas RW, 3rd, Hildreth RL, Kaplan IM, Morisot S, et al.Kruppel-like factor 4 is essential for inflammatory monocyte differentiation in vivo. Journal of Im‐

barrier function of the skin. Nature Genetics 1999;22(4)356-360.

in vivo. Journal of Biological Chemistry 2000;275(48)37798-37806.

lon. Development 2002;129(11)2619-2628.

2007;27(1)182-194.

336 Pluripotent Stem Cells

2007;26(18)4138-4148.

2005;201(11)1715-1723.

cal Chemistry 2005;280(46)38247-38258.

munications 2007;362(3)575-581.

munology 2008;180(8)5645-5652.

2009;54(5)403-411.

2008;30(3)260-266.

adult stomach. Gastroenterology 2005;128(4)935-945.


[54] Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, et al.Induced pluripotent stem cell lines derived from human somatic cells. Science 2007;318(5858)1917-1920.

[69] Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF.Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Acade‐

Function of KLF4 in Stem Cell Biology http://dx.doi.org/10.5772/54370 339

[70] Charafe-Jauffret E, Ginestier C, Iovino F, Wicinski J, Cervera N, et al.Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct

[71] Chute JP, Muramoto GG, Whitesides J, Colvin M, Safi R, et al.Inhibition of aldehyde dehydrogenase and retinoid signaling induces the expansion of human hematopoiet‐ ic stem cells. Proceedings of the National Academy of Sci ence of the United States of

[72] Hirschmann-Jax C, Foster AE, Wulf GG, Nuchtern JG, Jax TW, et al.A distinct "side population" of cells with high drug efflux capacity in human tumor cells. Proceed‐ ings of the National Academy of Sci ence of the United States of America

[73] Foster KW, Frost AR, McKie-Bell P, Lin CY, Engler JA, et al.Increase of GKLF mes‐ senger RNA and protein expression during progression of breast cancer. Cancer Re‐

[74] Pandya AY, Talley LI, Frost AR, Fitzgerald TJ, Trivedi V, et al.Nuclear localization of KLF4 is associated with an aggressive phenotype in early-stage breast cancer. Clini‐

[75] Rowland BD, Bernards R, Peeper DS.The KLF4 tumour suppressor is a transcription‐ al repressor of p53 that acts as a context-dependent oncogene. Nature Cell Biology

[76] Charafe-Jauffret E, Monville F, Ginestier C, Dontu G, Birnbaum D, et al.Cancer stem cells in breast: current opinion and future challenges. Pathobiology 2008;75(2)75-84.

[77] Lawson JC, Blatch GL, Edkins AL.Cancer stem cells in breast cancer and metastasis.

[78] Lyssiotis CA, Foreman RK, Staerk J, Garcia M, Mathur D, et al.Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4. Proceedings of the National Academy of Sci ence of the United States of

[79] Hinnebusch BF, Siddique A, Henderson JW, Malo MS, Zhang W, et al.Enterocyte dif‐ ferentiation marker intestinal alkaline phosphatase is a target gene of the gut-en‐ riched Kruppel-like factor. American Journal of Physiology. Gastrointestinal and

[80] Wassmann S, Wassmann K, Jung A, Velten M, Knuefermann P, et al.Induction of p53 by GKLF is essential for inhibition of proliferation of vascular smooth muscle cells.

Journal of Molecular and Cellular Cardiology 2007;43(3)301-307.

Breast Cancer Research and Treatment 2009;118(2)241-254.

my of Sci ence of the United States of America 2003;100(7)3983-3988.

molecular signature. Cancer Research 2009;69(4)1302-1313.

America 2006;103(31)11707-11712.

2004;101(39)14228-14233.

search 2000;60(22)6488-6495.

2005;7(11)1074-1082.

America 2009;106(22)8912-8917.

Liver Physiology 2004;286(1)G23-30.

cal Cancer Research 2004;10(8)2709-2719.


[69] Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF.Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Acade‐ my of Sci ence of the United States of America 2003;100(7)3983-3988.

[54] Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, et al.Induced pluripotent stem cell lines derived from human somatic cells. Science

[55] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, et al.Induction of pluripo‐ tent stem cells from adult human fibroblasts by defined factors. Cell

[56] Park IH, Zhao R, West JA, Yabuuchi A, Huo H, et al.Reprogramming of human so‐ matic cells to pluripotency with defined factors. Nature 2008;451(7175)141-146.

[57] Lowry WE, Richter L, Yachechko R, Pyle AD, Tchieu J, et al.Generation of human in‐ duced pluripotent stem cells from dermal fibroblasts. Proceedings of the National

[58] Hanna J, Markoulaki S, Schorderet P, Carey BW, Beard C, et al.Direct reprogram‐ ming of terminally differentiated mature B lymphocytes to pluripotency. Cell

[59] Hanna J, Wernig M, Markoulaki S, Sun CW, Meissner A, et al.Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science

[60] Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, et al.Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Na‐

[62] Zhao R, Daley GQ.From fibroblasts to iPS cells: induced pluripotency by defined fac‐

[63] Bonnet D, Dick JE.Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nature Medicine 1997;3(7)730-737.

[64] Schatton T, Frank NY, Frank MH.Identification and targeting of cancer stem cells. Bi‐

[65] O'Brien CA, Pollett A, Gallinger S, Dick JE.A human colon cancer cell capable of ini‐ tiating tumour growth in immunodeficient mice. Nature 2007;445(7123)106-110.

[66] Visvader JE, Lindeman GJ.Cancer stem cells in solid tumours: accumulating evidence

[67] Yang YM, Chang JW.Bladder cancer initiating cells (BCICs) are among EMA-CD44v6+ subset: novel methods for isolating undetermined cancer stem (initiating)

[68] Eramo A, Haas TL, De Maria R.Lung cancer stem cells: tools and targets to fight lung

and unresolved questions. Nature Reviews. Cancer 2008;8(10)755-768.

Academy of Sci ence of the United States of America 2008;105(8)2883-2888.

2007;318(5858)1917-1920.

2007;131(5)861-872.

338 Pluripotent Stem Cells

2008;133(2)250-264.

2007;318(5858)1920-1923.

ture Biotechnology 2008;26(1)101-106.

oessays 2009;31(10)1038-1049.

cells. Cancer Investigation 2008;26(7)725-733.

cancer. Oncogene 2010;29(33)4625-4635.

[61] Yamanaka S.A fresh look at iPS cells. Cell 2009;137(1)13-17.

tors. Journal of Cellular Biochemistry 2008;105(4)949-955.


[81] Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, et al.The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 2008;133(4)704-715.

[94] Taylor G, Lehrer MS, Jensen PJ, Sun TT, Lavker RM.Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 2000;102(4)451-461.

Function of KLF4 in Stem Cell Biology http://dx.doi.org/10.5772/54370 341

[95] Ito M, Liu Y, Yang Z, Nguyen J, Liang F, et al.Stem cells in the hair follicle bulge con‐ tribute to wound repair but not to homeostasis of the epidermis. Nature Medicine

[96] Levy V, Lindon C, Zheng Y, Harfe BD, Morgan BA.Epidermal stem cells arise from

[97] Nowak JA, Polak L, Pasolli HA, Fuchs E.Hair follicle stem cells are specified and

[98] Dang DT, Pevsner J, Yang VW.The biology of the mammalian Kruppel-like family of transcription factors. The International Journal of Biochemistry & Cell Biology

[99] Wei D, Wang L, Kanai M, Jia Z, Le X, et al.KLF4alpha up-regulation promotes cell cycle progression and reduces survival time of patients with pancreatic cancer. Gas‐

[100] Snippert HJ, Haegebarth A, Kasper M, Jaks V, van Es JH, et al.Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin. Science

[101] Langton AK, Herrick SE, Headon DJ.An extended epidermal response heals cutane‐ ous wounds in the absence of a hair follicle stem cell contribution. Journal of Investi‐

[102] Leung CT, Coulombe PA, Reed RR.Contribution of olfactory neural stem cells to tis‐ sue maintenance and regeneration. Nature Neuroscience 2007;10(6)720-726.

[103] Ohlstein B, Kai T, Decotto E, Spradling A.The stem cell niche: theme and variations.

[104] Bray SJ.Notch signalling: a simple pathway becomes complex. Nature Reviews. Mo‐

[105] Joseph NM, Morrison SJ.Toward an understanding of the physiological function of

[106] Keith B, Simon MC.Hypoxia-inducible factors, stem cells, and cancer. Cell

[107] Zheng H, Pritchard DM, Yang X, Bennett E, Liu G, et al.KLF4 gene expression is in‐ hibited by the notch signaling pathway that controls goblet cell differentiation in mouse gastrointestinal tract. American Journal of Physiology. Gastrointestinal and

the hair follicle after wounding. Faseb Journal 2007;21(7)1358-1366.

function in early skin morphogenesis. Cell Stem Cell 2008;3(1)33-43.

2005;11(12)1351-1354.

2000;32(11-12)1103-1121.

2010;327(5971)1385-1389.

troenterology 2010;139(6)2135-2145.

gative Dermatology 2008;128(5)1311-1318.

lecular Cell Biology 2006;7(9)678-689.

Liver Physiology 2009;296(3)G490-498.

2007;129(3)465-472.

Current Opinion In Cell Biology 2004;16(6)693-699.

Mammalian stem cells. Developmental Cell 2005;9(2)173-183.


[94] Taylor G, Lehrer MS, Jensen PJ, Sun TT, Lavker RM.Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 2000;102(4)451-461.

[81] Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, et al.The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 2008;133(4)704-715.

[82] Yang J, Weinberg RA.Epithelial-mesenchymal transition: at the crossroads of devel‐

[83] Miettinen PJ, Ebner R, Lopez AR, Derynck R.TGF-beta induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors.

[84] Malanchi I, Peinado H, Kassen D, Hussenet T, Metzger D, et al.Cutaneous cancer stem cell maintenance is dependent on beta-catenin signalling. Nature

[85] Yori JL, Johnson E, Zhou G, Jain MK, Keri RA.Kruppel-like factor 4 inhibits epithe‐ lial-to-mesenchymal transition through regulation of E-cadherin gene expression.

[86] Cotsarelis G, Sun TT, Lavker RM.Label-retaining cells reside in the bulge area of pilo‐ sebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogene‐

[87] Trempus CS, Morris RJ, Bortner CD, Cotsarelis G, Faircloth RS, et al.Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker

[88] Morris RJ, Liu Y, Marles L, Yang Z, Trempus C, et al.Capturing and profiling adult

[89] Blanpain C, Lowry WE, Geoghegan A, Polak L, Fuchs E.Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell

[90] Braun KM, Niemann C, Jensen UB, Sundberg JP, Silva-Vargas V, et al.Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis. Development 2003;130(21)5241-5255.

[91] Tumbar T, Guasch G, Greco V, Blanpain C, Lowry WE, et al.Defining the epithelial

[92] Jensen KB, Watt FM.Single-cell expression profiling of human epidermal stem and transit-amplifying cells: Lrig1 is a regulator of stem cell quiescence. Proceedings of the National Academy of Sci ence of the United States of America

[93] Jensen KB, Collins CA, Nascimento E, Tan DW, Frye M, et al.Lrig1 expression de‐ fines a distinct multipotent stem cell population in mammalian epidermis. Cell Stem

opment and tumor metastasis. Developmental Cell 2008;14(6)818-829.

Journal of Cell Biology 1994;127(6 Pt 2)2021-2036.

Journal of Biological Chemistry 2010;285(22)16854-16863.

CD34. Journal of Investigative Dermatology 2003;120(4)501-511.

hair follicle stem cells. Nature Biotechnology 2004;22(4)411-417.

stem cell niche in skin. Science 2004;303(5656)359-363.

2008;452(7187)650-653.

340 Pluripotent Stem Cells

sis. Cell 1990;61(7)1329-1337.

2004;118(5)635-648.

2006;103(32)11958-11963.

Cell 2009;4(5)427-439.


[108] Ohlstein B, Spradling A.The adult Drosophila posterior midgut is maintained by plu‐ ripotent stem cells. Nature 2006;439(7075)470-474.

[122] Zhang W, Chen X, Kato Y, Evans PM, Yuan S, et al.Novel cross talk of Kruppel-like factor 4 and beta-catenin regulates normal intestinal homeostasis and tumor repres‐

Function of KLF4 in Stem Cell Biology http://dx.doi.org/10.5772/54370 343

[123] Vermeulen L, De Sousa EMF, van der Heijden M, Cameron K, de Jong JH, et al.Wnt activity defines colon cancer stem cells and is regulated by the microenvironment.

[124] Hoffmeyer K, Raggioli A, Rudloff S, Anton R, Hierholzer A, et al.Wnt/beta-catenin signaling regulates telomerase in stem cells and cancer cells. Science

[125] Brennan KR, Brown AM.Wnt proteins in mammary development and cancer. Jour‐

[126] Grigoryan T, Wend P, Klaus A, Birchmeier W.Deciphering the function of canonical Wnt signals in development and disease: conditional loss- and gain-of-function mu‐

[127] Ingham PW, McMahon AP.Hedgehog signaling in animal development: paradigms

[128] Varjosalo M, Taipale J.Hedgehog: functions and mechanisms. Genes & Development

[129] Santini R, Vinci MC, Pandolfi S, Penachioni JY, Montagnani V, et al.HEDGEHOG-GLI Signaling Drives Self-Renewal and Tumorigenicity of Human Melanoma-Initiat‐

[130] Clement V, Sanchez P, de Tribolet N, Radovanovic I, Ruiz i Altaba A.HEDGEHOG-GLI1 signaling regulates human glioma growth, cancer stem cell self-renewal, and

[131] Yang SH, Andl T, Grachtchouk V, Wang A, Liu J, et al.Pathological responses to on‐ cogenic Hedgehog signaling in skin are dependent on canonical Wnt/beta3-catenin

[132] Dahmane N, Sanchez P, Gitton Y, Palma V, Sun T, et al.The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis. Development

[133] Schoenhals M, Kassambara A, De Vos J, Hose D, Moreaux J, et al.Embryonic stem cell markers expression in cancers. Biochemical and Biophysical Research Communi‐

tations of beta-catenin in mice. Genes & Development 2008;22(17)2308-2341.

nalof Mammary Gland Biology and Neoplasia 2004;9(2)119-131.

and principles. Genes & Development 2001;15(23)3059-3087.

tumorigenicity. Current Biology 2007;17(2)165-172.

signaling. Nature Genetics 2008;40(9)1130-1135.

sion. Molecular and Cellular Biology 2006;26(6)2055-2064.

Nature Cell Biology 2010;12(5)468-476.

2012;336(6088)1549-1554.

2008;22(18)2454-2472.

ing Cells. Stem Cells 2012;

2001;128(24)5201-5212.

cations 2009;383(2)157-162.


[122] Zhang W, Chen X, Kato Y, Evans PM, Yuan S, et al.Novel cross talk of Kruppel-like factor 4 and beta-catenin regulates normal intestinal homeostasis and tumor repres‐ sion. Molecular and Cellular Biology 2006;26(6)2055-2064.

[108] Ohlstein B, Spradling A.The adult Drosophila posterior midgut is maintained by plu‐

[109] Ghaleb AM, Aggarwal G, Bialkowska AB, Nandan MO, Yang VW.Notch inhibits ex‐ pression of the Kruppel-like factor 4 tumor suppressor in the intestinal epithelium.

[110] Real PJ, Tosello V, Palomero T, Castillo M, Hernando E, et al.Gamma-secretase inhib‐ itors reverse glucocorticoid resistance in T cell acute lymphoblastic leukemia. Nature

[111] Lambertini C, Pantano S, Dotto GP.Differential control of Notch1 gene transcription by Klf4 and Sp3 transcription factors in normal versus cancer-derived keratinocytes.

[112] Sahlgren C, Gustafsson MV, Jin S, Poellinger L, Lendahl U.Notch signaling mediates hypoxia-induced tumor cell migration and invasion. Proceedings of the National

[113] Liu Z, Teng L, Bailey SK, Frost AR, Bland KI, et al.Epithelial transformation by KLF4 requires Notch1 but not canonical Notch1 signaling. Cancer Biology & Therapy

[114] Cadigan KM, Nusse R.Wnt signaling: a common theme in animal development.

[115] Clevers H.Wnt/beta-catenin signaling in development and disease. Cell

[116] van de Wetering M, Sancho E, Verweij C, de Lau W, Oving I, et al.The beta-catenin/ TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell

[117] Curtin JC, Lorenzi MV.Drug discovery approaches to target Wnt signaling in cancer

[118] MacDonald BT, Tamai K, He X.Wnt/beta-catenin signaling: components, mecha‐

[119] Groden J, Thliveris A, Samowitz W, Carlson M, Gelbert L, et al.Identification and characterization of the familial adenomatous polyposis coli gene. Cell

[120] Kinzler KW, Nilbert MC, Su LK, Vogelstein B, Bryan TM, et al.Identification of FAP

[121] Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, et al.Activation of beta-cate‐ nin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science

locus genes from chromosome 5q21. Science 1991;253(5020)661-665.

Academy of Sci ence of the United States of America 2008;105(17)6392-6397.

ripotent stem cells. Nature 2006;439(7075)470-474.

Medicine 2009;15(1)50-58.

342 Pluripotent Stem Cells

PLoS One 2010;5(4)e10369.

2009;8(19)1840-1851.

2006;127(3)469-480.

2002;111(2)241-250.

1991;66(3)589-600.

1997;275(5307)1787-1790.

Genes & Development 1997;11(24)3286-3305.

stem cells. Oncotarget 2010;1(7)563-577.

nisms, and diseases. Developmental Cell 2009;17(1)9-26.

Moecularl Cancer Research: MCR 2008;6(12)1920-1927.


**Chapter 16**

**β1,4-Galactosyltransferases, Potential Modifiers of**

The ability of embryonic stem cells to self renew and, at a given signal, give rise to the multi‐ faceted cell types normally observed in the body, is highly dependent on the complex inter‐ play between both intrinsic (inside the cell) and extrinsic (outside of the cell) factors. Despite progress in analyzing the genome, proteome, and the transcriptome, challenges still exists to find the most efficient and specific conditions in which human embryonic stem cells (hESC) can maintain pluripotency and or/can be efficiently directed to differentiate towards a ho‐ mogenous cell type. In a stem cell niche, the integrity of the cell matrix and the manifold of different cell-cell interactions and the ability of the cells to respond to a variety of cytokine cues from both interstitial fluids and from extracellular matrices, are crucial factors in giving the right signal signals to the cells internal machinery, in a space (spatio) and time (tempo‐ ral) manner during different developmental stages. One of these molecules is the glycan. A glycan is a polysaccharide or oligosaccharide, that is attached to a glucoconjugate such as glycoprotein, glycolipid, and proteoglycan. Cell surface glycoproteins are abundant and constitute approximately 50% of all proteins in nature. For many years, the biological func‐ tion of glycosylation in stem cell behavior/homeostasis was overlooked and thought of as a more or less redundant process with applications only limited to the identification and sort‐ ing of cells at different stages of pluripotency and during formation of induced pluripotent stem cells (iPSC). Markers such as stage specific embryonic antigen (SSEA1 and -3/4) and the tumor rejection antigens (TRA-1-60 and TRA-1-81) have been used to analyze the pluripo‐ tency and differentiation stages of embryonic stem cells and induced pluripotent stem cell (iPSC).The research of how glycosylation can impact stem cells has long been hampered by the structural complexities of glycosylation and the difficulties to identify and purify the en‐ zymes, glycosyltransferases, responsible for these processes. This problem is partly due to

> © 2013 Wassler; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Wassler; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

**Stem Cell Pluripotency and Differentiation**

Additional information is available at the end of the chapter

Michael Wassler

**1. Introduction**

http://dx.doi.org/10.5772/54376

## **β1,4-Galactosyltransferases, Potential Modifiers of Stem Cell Pluripotency and Differentiation**

Michael Wassler

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54376

## **1. Introduction**

The ability of embryonic stem cells to self renew and, at a given signal, give rise to the multi‐ faceted cell types normally observed in the body, is highly dependent on the complex inter‐ play between both intrinsic (inside the cell) and extrinsic (outside of the cell) factors. Despite progress in analyzing the genome, proteome, and the transcriptome, challenges still exists to find the most efficient and specific conditions in which human embryonic stem cells (hESC) can maintain pluripotency and or/can be efficiently directed to differentiate towards a ho‐ mogenous cell type. In a stem cell niche, the integrity of the cell matrix and the manifold of different cell-cell interactions and the ability of the cells to respond to a variety of cytokine cues from both interstitial fluids and from extracellular matrices, are crucial factors in giving the right signal signals to the cells internal machinery, in a space (spatio) and time (tempo‐ ral) manner during different developmental stages. One of these molecules is the glycan. A glycan is a polysaccharide or oligosaccharide, that is attached to a glucoconjugate such as glycoprotein, glycolipid, and proteoglycan. Cell surface glycoproteins are abundant and constitute approximately 50% of all proteins in nature. For many years, the biological func‐ tion of glycosylation in stem cell behavior/homeostasis was overlooked and thought of as a more or less redundant process with applications only limited to the identification and sort‐ ing of cells at different stages of pluripotency and during formation of induced pluripotent stem cells (iPSC). Markers such as stage specific embryonic antigen (SSEA1 and -3/4) and the tumor rejection antigens (TRA-1-60 and TRA-1-81) have been used to analyze the pluripo‐ tency and differentiation stages of embryonic stem cells and induced pluripotent stem cell (iPSC).The research of how glycosylation can impact stem cells has long been hampered by the structural complexities of glycosylation and the difficulties to identify and purify the en‐ zymes, glycosyltransferases, responsible for these processes. This problem is partly due to

© 2013 Wassler; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Wassler; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the fact that glycans are not encoded directly from the genome but rather depends on the collaboration of a limited number of both glycosyltransferases and glycosidases, whose ex‐ pression are reliant upon both intracellular as well as extracellular changes. Furthermore, glycosyltransferases are expressed differentially between many cell types and disease states in a spatio- temporal manner during development. In this review, I will summarize research on what is known for glycosyltransferases in stem cell pluripotency and differentiation. I will specifically focus on one glycosyltransferase, N-acetylglucosamin β1,4- Galactosyltrans‐ ferase 1 (β4Gal-T1), a unique galactosyltrasferase implicated in a variety of cellular process‐ es such as cell-cell and cell-matrix adhesion, apoptosis, proliferation and differentiation, to mention a few. I will discuss its regulation and potential mechanism(s) in cell-cell, cell-ma‐ trix and cytokine signaling pathways. Finally, in the last section, I will talk about some dis‐ eases related to galactosyltransferase deficiency. All in all, this chapter is intended to evoke more interest in the field of stem cell glycobiology, both for the layman as well as for the bench scientist. Ultimately, the goal of this review is to encourage future research to find al‐ ternative therapeutic modalities for glycoprotein related diseases, such as cancer, congenital disease and even Alzheimer's.

#### **2. What is glycosyltransferases?**

Glycosyltransferases (GTs; EC 2.4.x.y) constitute a large protein family of about 200-300 en‐ zymes that are involved in the biosynthesis of glycans. GTs are type II transmembrane pro‐ teins with large carboxy-terminal globular catalytic domains, that face the luminal side of the Golgi complex, and a short cytoplasmic domain. The sequential action of GTs results in the formation of both linear as well as highly branched glycan structures that are present in both prokaryotes and eukaryotes. Mammalian GTs utilize a variety of uridine diphosphate activated (UDP) sugars as donors: UDP-glucose, UDP-galactose, UDP-GlcNAc, UDP-Gal‐ NAc, UDP-xylose, UDP-glucuronic acid, GDP-mannose, GDP-fucose, and CMP-sialic acid. Glycosyl transfer can occur on protein residues, usually to asparagine, to give N-linked gly‐ coproteins and on tyrosine, serine, or threonine to give O-linked glycoproteins [1]. The first step in N-linked glycosylation occurs in the endoplamic reticulum (ER) in which a "high mannose" oligosaccharide branch is added to an Asparagine (Asn) residue in the protein backbone (N-linkage). Another type of glycan linkage is the *O*-linked glycosylation, which occurs through serine/threonin residues in the protein back bone during transport within the Golgie complex [2]. Other GTs are responsible for extensive branching of glycan struc‐ tures such as the galactosyltransferase family (GalTs) [3] which together with glycosidases give rise to more "complex" type sugar chains (Figure 1). These processes creates oligosac‐ charide structures of enormous diversity and whose functions spans from cell adhesion, in‐ flammation, cancer metastasis, stem cell proliferation and development [4]. This exciting area of biology has resulted in an intensive research to unveil the function of individual GTs in during stem cell pluripotency and differentiation. Several studies have implicated a varie‐ ty of GTs in stem cell biology, some of which are presented below:

**Figure 1.** General view of an O-linked (A), and a (B) "complex" N-linked cell surface glycoprotein. A lactosylceramide‐ glycolipid (LacCer) (C) is also shown, located at the upper leaflet of the plasma membrane (PM). Ser; Serine, Thr;Threo‐ nine, Asn;Asparagine Sial;Sialic acid, Gal;Galactose, Glc;Glucose,Man;Mannose, GlcNac; N-Acetylglucoseamine,

β1,4-Galactosyltransferases, Potential Modifiers of Stem Cell Pluripotency and Differentiation

http://dx.doi.org/10.5772/54376

347

**1.** N-acetylglucosaminyl-1 phosphate transferase (GPT): The first steps in N-linked glycan synthesis begins on both the cytosolic and luminal side of the endoplasmic reticulum where nine mannosyl residues are sequentially added to a poly-isoprenoid lipid, doly‐ chylmonophosphate by the activity of N-acetylglucosaminyl-1 phosphate transferase (GPT) and a number of mannosyltransferases. One inhibitor to GPT, tunicamycin (TM), inhibits N-linked glycosylation and has been reported affect cell proliferation, neu-vas‐

cularization and cancer progression, due to induced cell death from ER stress [4].

**2.** βGalNAc-T3: The cell surface glycan epitope LacdiNac (GalNac-β4GlcNAc) has been shown to be an important glycosylation modification of leukemia inhibitor factor recep‐ tor (LIFR) and its co-receptor, gp130.The addition of LacdiNac epitopes to LIFR was de‐ pendent on a specific transferase, β-3-N-acetyl-Galactosyl transferase 3 (βGalNac-T3). This modification is crucial for the localization of LIF to lipid rafts/ calveolar compo‐ nents, such as caveolin-1, in order to enhance its activity. Mouse and human stem cells (mESC, hESC) differ from each other in some aspects on how they respond to cytokines necessary for pluripotency. hESCs seem to be at a later developmental stage than mESCs, because of their independency of the LIF pathway for self renewal. Interesting‐ ly, the level of βGalNac-T3 was much lower in human versus mouse embryonic stem

GalTNAc; N-Acetylglucoseamine, Fuc;Fucose

the fact that glycans are not encoded directly from the genome but rather depends on the collaboration of a limited number of both glycosyltransferases and glycosidases, whose ex‐ pression are reliant upon both intracellular as well as extracellular changes. Furthermore, glycosyltransferases are expressed differentially between many cell types and disease states in a spatio- temporal manner during development. In this review, I will summarize research on what is known for glycosyltransferases in stem cell pluripotency and differentiation. I will specifically focus on one glycosyltransferase, N-acetylglucosamin β1,4- Galactosyltrans‐ ferase 1 (β4Gal-T1), a unique galactosyltrasferase implicated in a variety of cellular process‐ es such as cell-cell and cell-matrix adhesion, apoptosis, proliferation and differentiation, to mention a few. I will discuss its regulation and potential mechanism(s) in cell-cell, cell-ma‐ trix and cytokine signaling pathways. Finally, in the last section, I will talk about some dis‐ eases related to galactosyltransferase deficiency. All in all, this chapter is intended to evoke more interest in the field of stem cell glycobiology, both for the layman as well as for the bench scientist. Ultimately, the goal of this review is to encourage future research to find al‐ ternative therapeutic modalities for glycoprotein related diseases, such as cancer, congenital

Glycosyltransferases (GTs; EC 2.4.x.y) constitute a large protein family of about 200-300 en‐ zymes that are involved in the biosynthesis of glycans. GTs are type II transmembrane pro‐ teins with large carboxy-terminal globular catalytic domains, that face the luminal side of the Golgi complex, and a short cytoplasmic domain. The sequential action of GTs results in the formation of both linear as well as highly branched glycan structures that are present in both prokaryotes and eukaryotes. Mammalian GTs utilize a variety of uridine diphosphate activated (UDP) sugars as donors: UDP-glucose, UDP-galactose, UDP-GlcNAc, UDP-Gal‐ NAc, UDP-xylose, UDP-glucuronic acid, GDP-mannose, GDP-fucose, and CMP-sialic acid. Glycosyl transfer can occur on protein residues, usually to asparagine, to give N-linked gly‐ coproteins and on tyrosine, serine, or threonine to give O-linked glycoproteins [1]. The first step in N-linked glycosylation occurs in the endoplamic reticulum (ER) in which a "high mannose" oligosaccharide branch is added to an Asparagine (Asn) residue in the protein backbone (N-linkage). Another type of glycan linkage is the *O*-linked glycosylation, which occurs through serine/threonin residues in the protein back bone during transport within the Golgie complex [2]. Other GTs are responsible for extensive branching of glycan struc‐ tures such as the galactosyltransferase family (GalTs) [3] which together with glycosidases give rise to more "complex" type sugar chains (Figure 1). These processes creates oligosac‐ charide structures of enormous diversity and whose functions spans from cell adhesion, in‐ flammation, cancer metastasis, stem cell proliferation and development [4]. This exciting area of biology has resulted in an intensive research to unveil the function of individual GTs in during stem cell pluripotency and differentiation. Several studies have implicated a varie‐

ty of GTs in stem cell biology, some of which are presented below:

disease and even Alzheimer's.

346 Pluripotent Stem Cells

**2. What is glycosyltransferases?**

**Figure 1.** General view of an O-linked (A), and a (B) "complex" N-linked cell surface glycoprotein. A lactosylceramide‐ glycolipid (LacCer) (C) is also shown, located at the upper leaflet of the plasma membrane (PM). Ser; Serine, Thr;Threo‐ nine, Asn;Asparagine Sial;Sialic acid, Gal;Galactose, Glc;Glucose,Man;Mannose, GlcNac; N-Acetylglucoseamine, GalTNAc; N-Acetylglucoseamine, Fuc;Fucose


indicating that LacdNac play an important role for adopting stem cells from a primed state (already programmed for germ line specification) to a more naïve state, e g fully pluripotent cells[5]

β4Gal-T2 preferentially transfer galactose to the GlcNacβ1-2Manα and the GlcNAcβ1-4Man1-3 branch. β4Gal-T4 and β4Gal-T5 catalyzes the addition of galactose to GlcNAcβ1-6Man and the GlcNAcβ1-4 Man, respectively (Figure 2). The β4Gal-T1, β4Gal-T2, and β4Gal-T3 can also transfer galactose residues to tetra-antenna oligosaccharides. In addi‐ tion being involved in glycoconjugate synthesis, β4Gal-T2, -3, -4 and -6, are also important catalysts for glycolipid biosynthesis. β4Gal-T2 and -3 prefers a glycolipid intermediate, Lc3Cer, as a substrate and β4Gal-T4 uses GlcNac-6-sulphate, a common constituent of kera‐ tin sulphate, as a substrate [14]. β4Gal-T6 has been shown to have Lactosyl Ceramide syn‐ thase activity. Finally, β4Gal-T7, transfers a Galactose to an O-linked Xylose on a serine residue to start the synthesis of the linker region between glycosaminoglycans (GAG) and proteoglycans [15]. A general summary or the chromosomal location, tissue expression, gly‐ cosidic linkage and potential biological function of currently known β, 4-GalTs is summar‐

β1,4-Galactosyltransferases, Potential Modifiers of Stem Cell Pluripotency and Differentiation

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349

**Figure 2.** An example of a tetra antenna structure in a complex-type N-glycan. The numbers indicate the glycosidic linkages. The arrows and the boxed areas represent the bonds catalyzed by β1,4-galactosyltransferase (β4GaIT, blue area) and βGlcNAc Transferases (βGlcNAcT, green area), respectively. Gal; Galactose, Man: Mannose, GlcNAc; N-Acetyl‐

One member of the β4galactosyltransferase family, that has got increased attention in stem cell biology, is the β4Gal-T1. β4Gal-T1 catalyze the transfer of galactose (Gal) from uridine diphosphate-galactose (UDP-Gal) to terminal N-Acetylglucosamine (GlcNac) residues of oli‐ gosaccharide chains in a β1,4 linkage, to form N-acetyllactosamine. β4Gal-T1 and βal-T2 are unique among the β4galactosyltransferases (β4GalTs) genes that they form a heterodimer with alpha-lactalbumin and changes substrate specificity from GlcNac towards Glucose (Glc) as a substrate, forming lactose, a very common protein in the mammary glands. Inter‐ estingly, β4Gal-T1 is constitutively expressed. However, apart from β4Gal-T1, β4Gal-T2 is only expressed in fetal brain. β4GalT-2 is a key regulator of glycosylation of the proteins in‐

ized in Table 1.

glucosamin, R; glycoprotein back bone.

**4. β-1,4-Galactosyltransferase 1 (β4Gal-T1 )**


## **3. β-1,4-Galactosyltransferases**

β-1,4-Galactosyltransferases (β4GalTs) are type II membrane proteins of the glycosyltrans‐ ferase family that have the exclusive specificity to transfer an active UDP-galactose in a β1,4 linkage to acceptor sugars such as N-acetylglucosamine (GlcNAc), Glucose (Glc), Galactose (Gal) and even Xylose (Xyl). Each β4-GalTs have a distinct function in the biosynthesis of different glycoconjugates and disaccharide structures.The most common structure, the Galβ1-β4GlcNAc, or N-Acetyllactosamine, exists as disaccharide repeats within linear or branched poly-N-acetyllactoseamine chains, but also at the terminal ends of oligosaccharide chains where they become sialyllated. These structures are formed by a combined action of UDP-GlcNac:Mannosyl N-acetylglucosaminyltransferases and β-1,4-galactosyltransferases (β4GalTs) [10]. The first galactosyltransferase, β4GalT-1, was cloned in 1986 due to its func‐ tion of transfer galactosyl residues to β-1,4-linked GlcNac found in glycoconjugates [11]. Targeted inactivation of mouse β4Gal-T1 gene, however, revealed that both tissue and se‐ rum glycoproteins still contained residual β4GalT-1 activity towards glycoprotein acceptors [10]. To date there are currently seven members of the β4GalT gene family designated β4Gal-T1-T7. Even though, β4Gal-T1 to -T6 shares various homologies (30-50%) to β4GalT-1 at the amino acid level, their substrate affinities and end products appear to be slightly dif‐ ferent, depending on nature of the branched oligosaccharide structure tissue expression and the cellular milieu for the enzymes, e.g. lipid -rich environment [12, 13]. Both β4Gal-T1 and β4Gal-T2 preferentially transfer galactose to the GlcNacβ1-2Manα and the GlcNAcβ1-4Man1-3 branch. β4Gal-T4 and β4Gal-T5 catalyzes the addition of galactose to GlcNAcβ1-6Man and the GlcNAcβ1-4 Man, respectively (Figure 2). The β4Gal-T1, β4Gal-T2, and β4Gal-T3 can also transfer galactose residues to tetra-antenna oligosaccharides. In addi‐ tion being involved in glycoconjugate synthesis, β4Gal-T2, -3, -4 and -6, are also important catalysts for glycolipid biosynthesis. β4Gal-T2 and -3 prefers a glycolipid intermediate, Lc3Cer, as a substrate and β4Gal-T4 uses GlcNac-6-sulphate, a common constituent of kera‐ tin sulphate, as a substrate [14]. β4Gal-T6 has been shown to have Lactosyl Ceramide syn‐ thase activity. Finally, β4Gal-T7, transfers a Galactose to an O-linked Xylose on a serine residue to start the synthesis of the linker region between glycosaminoglycans (GAG) and proteoglycans [15]. A general summary or the chromosomal location, tissue expression, gly‐ cosidic linkage and potential biological function of currently known β, 4-GalTs is summar‐ ized in Table 1.

**Figure 2.** An example of a tetra antenna structure in a complex-type N-glycan. The numbers indicate the glycosidic linkages. The arrows and the boxed areas represent the bonds catalyzed by β1,4-galactosyltransferase (β4GaIT, blue area) and βGlcNAc Transferases (βGlcNAcT, green area), respectively. Gal; Galactose, Man: Mannose, GlcNAc; N-Acetyl‐ glucosamin, R; glycoprotein back bone.

## **4. β-1,4-Galactosyltransferase 1 (β4Gal-T1 )**

indicating that LacdNac play an important role for adopting stem cells from a primed state (already programmed for germ line specification) to a more naïve state, e g fully

**3.** Ext1 and Ext2: Heparan sulphate is a large sulphated oligosaccharide chain located on proteoglycans impacting both the stability of pluripotency and differentiation into neu‐ ral stem cell lineage. Ext1 and Ext2 encodes two bifunctional endoplasmic reticulumresident type II transmembrane glycosyltransferase that are involved in the chain elongation and modification of HS biosynthesis. HS on embryonic stem cells has been shown to exhibit a lower amount of sulfated glycosaminoglycans relative to differenti‐ ated cells indicating that the ratio between nonsulphated versus sulphated HS is impor‐

**4.** O-GlcNac Transferase (OGT): O-GlcNAcylation is a O-β-glycosidic attachment of a sin‐ gle N-acetyl glucosamine to a serine or threonin residue in nucleoplasmic proteins. Some of these proteins are represented by the transcription factors Oct4, Klf4, Sox and Nanog, which are involved in the pluripotency network in stem cell self renewal and in the core proteins responsible for the production of induced pluripotent stem cells (iPSCs). Recently it was discovered that this specific O-linked modification of Oct4 and Sox was crucial for their transcriptional activities. Two enzymes are responsible for O-GlcNAcylation: O-GlcNac Transferase (OGT) adds the modification and O-glucNAcase

β-1,4-Galactosyltransferases (β4GalTs) are type II membrane proteins of the glycosyltrans‐ ferase family that have the exclusive specificity to transfer an active UDP-galactose in a β1,4 linkage to acceptor sugars such as N-acetylglucosamine (GlcNAc), Glucose (Glc), Galactose (Gal) and even Xylose (Xyl). Each β4-GalTs have a distinct function in the biosynthesis of different glycoconjugates and disaccharide structures.The most common structure, the Galβ1-β4GlcNAc, or N-Acetyllactosamine, exists as disaccharide repeats within linear or branched poly-N-acetyllactoseamine chains, but also at the terminal ends of oligosaccharide chains where they become sialyllated. These structures are formed by a combined action of UDP-GlcNac:Mannosyl N-acetylglucosaminyltransferases and β-1,4-galactosyltransferases (β4GalTs) [10]. The first galactosyltransferase, β4GalT-1, was cloned in 1986 due to its func‐ tion of transfer galactosyl residues to β-1,4-linked GlcNac found in glycoconjugates [11]. Targeted inactivation of mouse β4Gal-T1 gene, however, revealed that both tissue and se‐ rum glycoproteins still contained residual β4GalT-1 activity towards glycoprotein acceptors [10]. To date there are currently seven members of the β4GalT gene family designated β4Gal-T1-T7. Even though, β4Gal-T1 to -T6 shares various homologies (30-50%) to β4GalT-1 at the amino acid level, their substrate affinities and end products appear to be slightly dif‐ ferent, depending on nature of the branched oligosaccharide structure tissue expression and the cellular milieu for the enzymes, e.g. lipid -rich environment [12, 13]. Both β4Gal-T1 and

pluripotent cells[5]

348 Pluripotent Stem Cells

removes it [9].

**3. β-1,4-Galactosyltransferases**

tant in stem cell pluripotency [6-8]

One member of the β4galactosyltransferase family, that has got increased attention in stem cell biology, is the β4Gal-T1. β4Gal-T1 catalyze the transfer of galactose (Gal) from uridine diphosphate-galactose (UDP-Gal) to terminal N-Acetylglucosamine (GlcNac) residues of oli‐ gosaccharide chains in a β1,4 linkage, to form N-acetyllactosamine. β4Gal-T1 and βal-T2 are unique among the β4galactosyltransferases (β4GalTs) genes that they form a heterodimer with alpha-lactalbumin and changes substrate specificity from GlcNac towards Glucose (Glc) as a substrate, forming lactose, a very common protein in the mammary glands. Inter‐ estingly, β4Gal-T1 is constitutively expressed. However, apart from β4Gal-T1, β4Gal-T2 is only expressed in fetal brain. β4GalT-2 is a key regulator of glycosylation of the proteins in‐ volved in neuronal development [16] and is responsible for the synthesis of complex-type N-linked oligosaccharides in many glycoproteins, as well as the carbohydrate moieties of glycolipids. Like the β4Gal-T1 enzyme, its substrate specificity is affected by alpha-lactalbu‐ min but is not expressed in lactating mammary tissue Apart from the other βGalTs, βGal-T1 encodes two protein isoforms produced by differential translation initiation at the 5' end of the mRNA transcript: a long isoform, containing a 24 amino acid cytoplasmic domain, and a short isoform with only an 11 amino acid domain [24]. Both isoforms are localized to trans-Golgi network and are able to function as glycoprotein processing enzymes (Fig.3). Howev‐ er, a small fraction of the long isoform of βGaT-1, preferentially targets the cell surface of various cells [25]. The specific signal sequence in β4GalTs that regulate the differential local‐ ization between cell surface and the Golgi complex, has been shown to consist of a short Nterminal hydrophobic sequence in the cytoplasmic domain, adjacent to the plasma membrane. This observation was further extended by the findings that the 13 amino acid se‐ quence in the cytoplasmic domain of long Gal-T1, could be phophorylated by p58 (CDK11), a GalT1 associated and cell cycle related Serine/Threonin kinase and, hence, could act as a retention signal for β4Gal-T1 in the Golgi complex [26, 27, 28, 38, 55] (Fig.3). Apart from be‐ ing involved in a variety of physiological activities, such as, for example mouse gamete in‐ teraction, neurite extension, epithelial mesenchymal transition and neural crest cell migration [29], cell surface GalT1 is also responsible for late morula compaction during de‐ velopment [30]. For more than a decade ago, β4Gal-T1 was found to facilitate cell migration on laminin 1, an important constituent of the extra cellular matrix (ECM) and during devel‐ opment [31, 32]. Furthermore, addition of β4Gal-T1 perturbants to F9 embryonic carcinoma led to an arrest in cell growth and morphological changes of embryoid bodies (EB) during differentiation [33]. Eckstein et. al., showed that cell surface β4Gal-T1 needed to associate with intact actin cytoskeleton in order for its cell surface activity [34] Interestingly, the intra‐ cellular domain of long form of β4Gal-T1 has been shown to bind to an array of signal trans‐ duction molecules such as a trimeric G-proteins (Gi) [35], Src Suppressed C-kinase Substrate (SSECKs) [36, 37], CDK11 (p58) [26, 38] and a novel ubiquitin conjugating enzyme [39]. The β4Gal-T1 interaction with SSeCKS was detected using the two hybrid system with the ami‐ no terminal 13 amino acid long cytoplasmic domain of β4GalT-1 [37]. The β4Gal-T1 associa‐ tion with SSeCKS is interesting since both proteins show similar subcellular distributions and share important cellular functions, such as cell proliferation, actin dynamics, and cell migration during development [36, 40]. For example, ectopic expression of both cell surface β4Gal-T1 and SSeCKS has been reported to induce a transient tyrosine phosphorylation of focal adhesion kinase (FAK) and rearrangement of filamentous actin [41]. Furthermore, SSeCKs also control the G1 to S phase progression through regulation of cyclin D1 expres‐ sion and localization. Since SSeCKS is a scaffolding molecule that can binds to several sig‐ naling proteins, such as PKC, Rho family members, and FAK, to mention a few, it is possible that most effects attributed to cell surface GalT1 in stem cell growh and differentiation may be mediated through SSeCKS. However it is unclear if cell surface β4GalT-1 performs in a similar manner as a lectin for its biological function [42, 105] or whether it utilizes its enzy‐ matic activity to modify and release its galactosylated product [31].

**(β4GalTs) (Chrom. #)**

β4Gal-T1 (9p13)

β4Gal-T2 (1p32-33)

β4Gal-T3 (1q23)

β4Gal-T4 (3q13.3)

β4Gal-T5 (11)

β4Gal-T6 (18q)

β4Gal-T7 (5q35.1-35.3)

**5. β4GalTs in cancer**

**Expression Glycosidic linkage and**

Restricted in brain, testis Galβ1-4GlcNac-R

Restricted to adult brain Galβ1-4GlcNac-R

Heart, liver, lung, testis, ovary, placenta, fetal brain

Constitutively expressed, high in fetal brain.

Testis, ovary, placenta, pancreas

heart, lung, liver, kidney, testis, Restricted in brain

Heart, Brain, Placenta, Liver, kidney, pancreas

β4Galactosyltransferase family, related to stem cells and development.

**Acceptor substrates**

Glycolipid

Galβ1-4GlcNac-R Glycolipid

Galβ1-4GlcNac-R GlcNac-6-sulphate LacCer

> Galβ1-4GlcNac-R LacCer

> > LacCer

GlcAβ1-3Galβ1-3Galβ1-4 Xylβ1-R

Glycosylation of cell surface glycoproteins and glycolipids changes dramatically upon the malignant transformation of cells [43]. β4GalTs have been reported to be increased in a fair amount of cancer. However, is not currently known if the elevated expression of β4GalTs contributes to the induction of cancer/malignancy, by affecting the cell surface landscape of glycans, or is an indirect effect of cancer progression or metasisis. β4Gal-T1 has been detect‐ ed in highly metastatic lung cancer by transcription factor E1AF activation of the β4Gal-T1 promoter [17, 51]. Furthermore, siRNA interference of surface β4Gal-T1 function, inhibited cell adhesion on laminin, the invasive potential in vitro,and tyrosine phosphorylation of fo‐ cal adhesion kinase [17]. The relative level of β4Gal-T1 has been reported to be important in melanoma invasiveness. For example, increasing cell surface β4Gal-T1 expression in cells of low metastatic potential promoted their invasive potential [44]. Other β4GalTs such as β4Gal-T5, function as an important growth regulator in glioma cells using both the E1AF and Sp1 transcription factors for its metastatic potential [17, 45]. Furthermore, clinically over expressed β4Gal-T4 and β4Gal-T6 have been shown to increase E2F1 and cyclin D3 tran‐ scription in colorectal cancer, respectively [18, 19]. Moreover, β4Gal-T1, -T2 and -T5 levels

**Table 1.** Table depicting the chromosomal region, the glycosidic linkage, substrate, and the function for the

**Function in stem cell, cancer and/or development**

growth, laminin dependent migration

Neuronal development, spermatogenic differentiation

Testicular development, tumor metastasis, keratin sulfate synthesis.

Self renewal of glioma cells, astrocytoma, extraembryonic development

> Extra embryonic development

Glycosaminoglycan (GAG) biosynthesis

N/A [14]

Galβ1-4GlcNac-R Morula compaction, cell

β1,4-Galactosyltransferases, Potential Modifiers of Stem Cell Pluripotency and Differentiation

**References**

351

http://dx.doi.org/10.5772/54376

[17], [18]

[16], [18]

[19], [14], [20, 21]

[18], [22]

[18, 19]

[15, 23]


**Table 1.** Table depicting the chromosomal region, the glycosidic linkage, substrate, and the function for the β4Galactosyltransferase family, related to stem cells and development.

## **5. β4GalTs in cancer**

volved in neuronal development [16] and is responsible for the synthesis of complex-type N-linked oligosaccharides in many glycoproteins, as well as the carbohydrate moieties of glycolipids. Like the β4Gal-T1 enzyme, its substrate specificity is affected by alpha-lactalbu‐ min but is not expressed in lactating mammary tissue Apart from the other βGalTs, βGal-T1 encodes two protein isoforms produced by differential translation initiation at the 5' end of the mRNA transcript: a long isoform, containing a 24 amino acid cytoplasmic domain, and a short isoform with only an 11 amino acid domain [24]. Both isoforms are localized to trans-Golgi network and are able to function as glycoprotein processing enzymes (Fig.3). Howev‐ er, a small fraction of the long isoform of βGaT-1, preferentially targets the cell surface of various cells [25]. The specific signal sequence in β4GalTs that regulate the differential local‐ ization between cell surface and the Golgi complex, has been shown to consist of a short Nterminal hydrophobic sequence in the cytoplasmic domain, adjacent to the plasma membrane. This observation was further extended by the findings that the 13 amino acid se‐ quence in the cytoplasmic domain of long Gal-T1, could be phophorylated by p58 (CDK11), a GalT1 associated and cell cycle related Serine/Threonin kinase and, hence, could act as a retention signal for β4Gal-T1 in the Golgi complex [26, 27, 28, 38, 55] (Fig.3). Apart from be‐ ing involved in a variety of physiological activities, such as, for example mouse gamete in‐ teraction, neurite extension, epithelial mesenchymal transition and neural crest cell migration [29], cell surface GalT1 is also responsible for late morula compaction during de‐ velopment [30]. For more than a decade ago, β4Gal-T1 was found to facilitate cell migration on laminin 1, an important constituent of the extra cellular matrix (ECM) and during devel‐ opment [31, 32]. Furthermore, addition of β4Gal-T1 perturbants to F9 embryonic carcinoma led to an arrest in cell growth and morphological changes of embryoid bodies (EB) during differentiation [33]. Eckstein et. al., showed that cell surface β4Gal-T1 needed to associate with intact actin cytoskeleton in order for its cell surface activity [34] Interestingly, the intra‐ cellular domain of long form of β4Gal-T1 has been shown to bind to an array of signal trans‐ duction molecules such as a trimeric G-proteins (Gi) [35], Src Suppressed C-kinase Substrate (SSECKs) [36, 37], CDK11 (p58) [26, 38] and a novel ubiquitin conjugating enzyme [39]. The β4Gal-T1 interaction with SSeCKS was detected using the two hybrid system with the ami‐ no terminal 13 amino acid long cytoplasmic domain of β4GalT-1 [37]. The β4Gal-T1 associa‐ tion with SSeCKS is interesting since both proteins show similar subcellular distributions and share important cellular functions, such as cell proliferation, actin dynamics, and cell migration during development [36, 40]. For example, ectopic expression of both cell surface β4Gal-T1 and SSeCKS has been reported to induce a transient tyrosine phosphorylation of focal adhesion kinase (FAK) and rearrangement of filamentous actin [41]. Furthermore, SSeCKs also control the G1 to S phase progression through regulation of cyclin D1 expres‐ sion and localization. Since SSeCKS is a scaffolding molecule that can binds to several sig‐ naling proteins, such as PKC, Rho family members, and FAK, to mention a few, it is possible that most effects attributed to cell surface GalT1 in stem cell growh and differentiation may be mediated through SSeCKS. However it is unclear if cell surface β4GalT-1 performs in a similar manner as a lectin for its biological function [42, 105] or whether it utilizes its enzy‐

350 Pluripotent Stem Cells

matic activity to modify and release its galactosylated product [31].

Glycosylation of cell surface glycoproteins and glycolipids changes dramatically upon the malignant transformation of cells [43]. β4GalTs have been reported to be increased in a fair amount of cancer. However, is not currently known if the elevated expression of β4GalTs contributes to the induction of cancer/malignancy, by affecting the cell surface landscape of glycans, or is an indirect effect of cancer progression or metasisis. β4Gal-T1 has been detect‐ ed in highly metastatic lung cancer by transcription factor E1AF activation of the β4Gal-T1 promoter [17, 51]. Furthermore, siRNA interference of surface β4Gal-T1 function, inhibited cell adhesion on laminin, the invasive potential in vitro,and tyrosine phosphorylation of fo‐ cal adhesion kinase [17]. The relative level of β4Gal-T1 has been reported to be important in melanoma invasiveness. For example, increasing cell surface β4Gal-T1 expression in cells of low metastatic potential promoted their invasive potential [44]. Other β4GalTs such as β4Gal-T5, function as an important growth regulator in glioma cells using both the E1AF and Sp1 transcription factors for its metastatic potential [17, 45]. Furthermore, clinically over expressed β4Gal-T4 and β4Gal-T6 have been shown to increase E2F1 and cyclin D3 tran‐ scription in colorectal cancer, respectively [18, 19]. Moreover, β4Gal-T1, -T2 and -T5 levels are higher in astrocytoma [18]. The expression of the β4Gal-T5 gene has also been shown to be regulated by transcription factors Sp1 and Ets-1 in cancer cells. Both these transcription factors regulate the gene expression levels of not only glycosyltransferases, but also key molecules involved in tumor growth, invasion and metastisis. Finally, small molecules that increase expression of GalTs could have beneficial effects during treatment of various cancer forms [45].

sitivity associated with G1 phase. Changes in the length of G1 phase are understood to ac‐ company the differentiation of human embryonic stem cells (hESCs), but the timing and extent of such changes are poorly defined. Terminally differentiated cells usually have a lon‐ ger G1 phase than those of stem cell and progenitor cells. Understanding the early steps governing the differentiation of hESCs will facilitate better control over differentiation for regenerative medicine and drug discovery applications. To avoid that cells with genetic aberrations are expanded in the population, stem cells have adapted to their harsh environ‐ ment by shutting off specific checkpoints normally activated in somatic cells. This will result in cell death as a default pathway for stem cells exhibiting chromosomal deveations, with‐ out slowing down proliferation of otherwise healthy cells. Since the upstream promoter re‐ gion of the 4.1 kb β-GalT1 transcript is mainly occupied by the Sp1 transcriptional factor, GalT1 was long believed to be another "house keeping" gene. However, several laboratories have shown that β4GalT-1 is regulated during cell cycle [28, 48, 49]. Interestingly, experi‐ ments in F9 embryonic carcinoma cells and in 3T3 cells have indicated that the cell surface bound and the Golgi related forms of β4GalT-1 are regulated differently, in which the long form is induced much earlier than the short and Golgie bound form. β4Gal-T1 showed the highest activity during G1-S phase and during interphase of the cell cycle [50]. There are many transcription factors important during the G1-S transition. The E2F family members of transcription factors serve as key regulators of the cell cycle progression by inducing activa‐ tors of S-phase related genes. Normally, during the onset of G1/S transition in cell cycle, the cyclic dependent kinases (CDKs) phosphorylate the retina blastoma (Rb) protein, resulting in a conformational change in Rb and subsequent release of active E2F from the Rb-E2F complex. This event results in transcription of G1-phase activating proteins such as e.g Cy‐ clin D3. Interestingly, E2F1, one of the best characterized members of this family, also binds to a promoter element in β4Gal-T1 transcript and positively regulates its activity. Moreover, cells subjected to a short hairpin RNA (shRNA) to β4Gal-T1 became less responsive for E2F1 activation [51].The effect of E2F1 on the expression of the other family members of β4GalTs, however, (β4Gal-T1, -T7) has not been exclusively determined. Another cell cycle related protein that has been found to regulate β4GalT1 expression is the p16 protein. This protein is a product of a tumor suppressor gene called CDKN2A that inhibits the cyclin-dependent kinases (CDK)-4 and 6 which are responsible for the G1 checkpoint in cell cycle. Transfec‐ tion of A549 human lung cancer with p16 led to down regulation of βGalT1 activity [53]. Thus, inactivation of either p16 or pRb function allows the cells to enter the S-phase only after a brief pause at the G1 checkpoint, leading to accelerated cell proliferation. Similar re‐ sults for GalT1 expression was obtained in hepatocarcinoma SMMC-7721 cells after blocking endogenous activity of TGFβ, a known regulator of the G1 to S-phase transition of cell cycle, by arresting cells in G1 phase [54]. Over expressing β4Gal-T1 has also been shown to exas‐ perate cyclohexamid induced apoptosis of [45]. This process is partly dependent on the ac‐ tivity of the CDK11(p58), a CDK11 family Ser/Thr kinase, a G2/M specific protein that contributes to regulation of cell cycle [55]. Recently GalT1 has been shown to interact with CDK11(p58) [26, 38] where it has an important function during cell cycle in stem cells pro‐ gression [28, 56]. Furhtermore, β4Gal-T1 contributes to HBx-induced cell cycle progression In hepatoma cells [57]. All these findings have led to the conclusion that β4Gal-T1 may be

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**Figure 3.** The long isomer of β1,4Galactosyltransferase 1 (β4GalT-1). β4GalT-1 catalyzes the transfer of UDP-galactose (red circle) to a terminal N-Acetylglycosamine (GlcNAc) residue in a newly synthesized glycoprotein in the golgie lu‐ men. The cytosolic domain of the long β4Gal-T1 consists of 11 amino acids (a.a) together with a 13 a.a extension (24 a.a in total). Phosphorylation of Serine 11 (S11) and/or Theonine 18 (T18) in the cytoplasmic domain negatively regu‐ late the localization and function GalT-1 as a cell surface receptor. The figure is not in scale.

## **6. β4Gal-T1 in cell cycle**

The observation that some, or maybe all, of the β4GalTs have relevancy in cancer progres‐ sion and/or metastasis, has highlighted the idea that stem cell pluripotency and differentia‐ tion may also depend on N-glycan structures [46]. One decisive factor in pluripotency and stem cell differentiation is the speed by which cells goes through the G1 phase in the cell cycle [47]. The cell cycle in pluripotent stem cells is remarkable for the shortness of the G1 phase, permitting rapid proliferation and reducing the duration of differentiation signal sen‐ sitivity associated with G1 phase. Changes in the length of G1 phase are understood to ac‐ company the differentiation of human embryonic stem cells (hESCs), but the timing and extent of such changes are poorly defined. Terminally differentiated cells usually have a lon‐ ger G1 phase than those of stem cell and progenitor cells. Understanding the early steps governing the differentiation of hESCs will facilitate better control over differentiation for regenerative medicine and drug discovery applications. To avoid that cells with genetic aberrations are expanded in the population, stem cells have adapted to their harsh environ‐ ment by shutting off specific checkpoints normally activated in somatic cells. This will result in cell death as a default pathway for stem cells exhibiting chromosomal deveations, with‐ out slowing down proliferation of otherwise healthy cells. Since the upstream promoter re‐ gion of the 4.1 kb β-GalT1 transcript is mainly occupied by the Sp1 transcriptional factor, GalT1 was long believed to be another "house keeping" gene. However, several laboratories have shown that β4GalT-1 is regulated during cell cycle [28, 48, 49]. Interestingly, experi‐ ments in F9 embryonic carcinoma cells and in 3T3 cells have indicated that the cell surface bound and the Golgi related forms of β4GalT-1 are regulated differently, in which the long form is induced much earlier than the short and Golgie bound form. β4Gal-T1 showed the highest activity during G1-S phase and during interphase of the cell cycle [50]. There are many transcription factors important during the G1-S transition. The E2F family members of transcription factors serve as key regulators of the cell cycle progression by inducing activa‐ tors of S-phase related genes. Normally, during the onset of G1/S transition in cell cycle, the cyclic dependent kinases (CDKs) phosphorylate the retina blastoma (Rb) protein, resulting in a conformational change in Rb and subsequent release of active E2F from the Rb-E2F complex. This event results in transcription of G1-phase activating proteins such as e.g Cy‐ clin D3. Interestingly, E2F1, one of the best characterized members of this family, also binds to a promoter element in β4Gal-T1 transcript and positively regulates its activity. Moreover, cells subjected to a short hairpin RNA (shRNA) to β4Gal-T1 became less responsive for E2F1 activation [51].The effect of E2F1 on the expression of the other family members of β4GalTs, however, (β4Gal-T1, -T7) has not been exclusively determined. Another cell cycle related protein that has been found to regulate β4GalT1 expression is the p16 protein. This protein is a product of a tumor suppressor gene called CDKN2A that inhibits the cyclin-dependent kinases (CDK)-4 and 6 which are responsible for the G1 checkpoint in cell cycle. Transfec‐ tion of A549 human lung cancer with p16 led to down regulation of βGalT1 activity [53]. Thus, inactivation of either p16 or pRb function allows the cells to enter the S-phase only after a brief pause at the G1 checkpoint, leading to accelerated cell proliferation. Similar re‐ sults for GalT1 expression was obtained in hepatocarcinoma SMMC-7721 cells after blocking endogenous activity of TGFβ, a known regulator of the G1 to S-phase transition of cell cycle, by arresting cells in G1 phase [54]. Over expressing β4Gal-T1 has also been shown to exas‐ perate cyclohexamid induced apoptosis of [45]. This process is partly dependent on the ac‐ tivity of the CDK11(p58), a CDK11 family Ser/Thr kinase, a G2/M specific protein that contributes to regulation of cell cycle [55]. Recently GalT1 has been shown to interact with CDK11(p58) [26, 38] where it has an important function during cell cycle in stem cells pro‐ gression [28, 56]. Furhtermore, β4Gal-T1 contributes to HBx-induced cell cycle progression In hepatoma cells [57]. All these findings have led to the conclusion that β4Gal-T1 may be

are higher in astrocytoma [18]. The expression of the β4Gal-T5 gene has also been shown to be regulated by transcription factors Sp1 and Ets-1 in cancer cells. Both these transcription factors regulate the gene expression levels of not only glycosyltransferases, but also key molecules involved in tumor growth, invasion and metastisis. Finally, small molecules that increase expression of GalTs could have beneficial effects during treatment of various cancer

**Figure 3.** The long isomer of β1,4Galactosyltransferase 1 (β4GalT-1). β4GalT-1 catalyzes the transfer of UDP-galactose (red circle) to a terminal N-Acetylglycosamine (GlcNAc) residue in a newly synthesized glycoprotein in the golgie lu‐ men. The cytosolic domain of the long β4Gal-T1 consists of 11 amino acids (a.a) together with a 13 a.a extension (24 a.a in total). Phosphorylation of Serine 11 (S11) and/or Theonine 18 (T18) in the cytoplasmic domain negatively regu‐

The observation that some, or maybe all, of the β4GalTs have relevancy in cancer progres‐ sion and/or metastasis, has highlighted the idea that stem cell pluripotency and differentia‐ tion may also depend on N-glycan structures [46]. One decisive factor in pluripotency and stem cell differentiation is the speed by which cells goes through the G1 phase in the cell cycle [47]. The cell cycle in pluripotent stem cells is remarkable for the shortness of the G1 phase, permitting rapid proliferation and reducing the duration of differentiation signal sen‐

late the localization and function GalT-1 as a cell surface receptor. The figure is not in scale.

**6. β4Gal-T1 in cell cycle**

forms [45].

352 Pluripotent Stem Cells

directly or indirectly connected to cell cycle progression and could be a potential reason for the growth impeded phenotype observed earlier in knock out β4Gal-T1 mice [52]

sources. Several members of β4GalT family seem to be important enzymes in the synthesis of GSL [62]. The basic structure for GLS is a monosaccharide, usually glucose, attached di‐ rectly to a ceramide molecule, mediated through the action of ceramide glucosyltransferase (Ugcg), resulting in a glycosylceramide (glucocerebroside;GlcCer) (Figure 5). βGalT-2 then transfer a UDP-Galactose to the GlcCer moiety, forming Lactosylceramide (LacCer) [62] (Figure 5). A variety of structural subclasses of GLS may then be synthesized from LacCer by the addition of other mono and disaccharides, resulting in the synthesis of structural sub‐ classes of GLS such as ganglio-, lacto/neolacto-, globo,- -isoglo, and ganglioseries-series [63]. Many of these structures are important for various biological functions, such as for example cell growth, myocardial differentiation cell migration and during development of the nerv‐ ous system[60, 61, 64]. When the Lex epitope is attached to a lactosylceramid it is identical to stage specific antigen (SSEA-1). This antigen is highly regulated during embryogenesis, ex‐ pressed at the morula stage in embryos and is considered to function as a cell-cell interac‐

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**Figure 5.** Core 2 structure of the glycoplipid, Lactosylceramide (LacCer) synthesized by UDP-glucose ceramide glucosyl transferase (Ugcg) and by β1,4Ga|act0sy|transferases (β4Ga|Ts) forming the β1,4-glycosidic linkage to ceramide.

Embryoglycans: Most developmentally regulated epitopes identified on embryonal carcino‐ ma cells and murine preimplantation embryos are associated with a glycoprotein-bound and large glycans, called embryoglycans. Embryoglycans consists of linear of branched poly-N-acetyllactoseamines with high molecular weight that carries a number of different

6). Apart from the mouse, where SSEA-1 is abundant from the 8-cell morula stage, SSEA-1 in human is not expressed until the germ cell line and in neural stem cells. Interestingly, β4Gal-T1 is expressed during the morula stage and has been shown to affect the compaction process [30]. Furthermore, human ES cells express SSEA-3 and -4 SSEA-1. SSEA-1 is also ex‐ pressed in undifferentiated F9 teratocarcinoma cells. After induction of differentiation the expression of SSEA-1 decreases. This is caused by the upregulation of alpha-1,3-galactosyl‐

, described above (Figure

structure [66, 67]. The stage specific

developmentally regulated carbohydrate epitopes, such as e. g. Lex

transferase that is responsible for masking of the Lex

tion ligand in the compaction process [65].

**Figure 4.** Biosynthesis of a core 2 O-glycan with Lewis X, (LeX,SSEA-1), Sialyl-Lewis (SLex) or 6-sulpho Sialyl (6-Sulpho-Lex synthesized at the terminus of poly-N-Acetyllactosamine chains. The action of β4Ga|Ts and β3GnT are indicated with arrows. Sia|;Sia|ic acid, Gal;Ga|act0se, G|c;G|uc0se, Man;Mann0se, G|cNac; N-Acetylglucoseamine, Ga|TNAc; N-Acetylgalactoseamine, β4Ga|act0syltransferases; β4GalT, β1,3-N- Acetylglucosaminyltransferase; β3GnT

## **7. β4GalTs involvement in Lewis X, glycosphingolipids and embryoglycans**

Lewis X: As mentioned in the beginning of this chapter, β4GalTs are Important for the syn‐ thesis of linear or branched poly-N-acetyllactoseamines chains. They are attached to N-gly‐ can, O-Glycans or glycolipids and are synthesized by the repeating and alternate action of N-acetylglucosaminyltransferases (β3GnT or β4GnT) and β4Gal-T1 [58]. These structures of‐ ten carry various functional epitopes important in stem cell homeostasis and inflammation [59]. One of these antigen is called the Lewis X antigen (Le<sup>x</sup> ) and constitutes the core struc‐ ture from which other antigens are synthesized. Le<sup>x</sup> epitope consists of a trisaccharide, Galβ1-4(Fucα1-3) GlcNAcβ1 which is produced by the action of β4Gal-T1 and α-1,3-Fuco‐ syltransferase (FUT). Other examples of epitopes formed from this core, are the Sialyl-Lewis (SLe<sup>x</sup> ) and 6-sulpho Sialyl (6-Sulpho-Lex) epitopes (Figure 3), in which the latter involve the activity of β4Gal-T4 (Table 1). These epitopes are implicated in biospecific interactions with selectins and other glycan-binding proteins during inflammatory processes [59] as well as in important regulatory functions during development [60]. Also, Le<sup>x</sup> structures has been im‐ plicated in specific differentiation, such as myocardial differentiation from embryonic stem cells [60, 61].

Glycosphingolipids: Glycosphingolipids, or sometimes called glycolipids (GLS) have been found in the upper leaflet of the plasma membrane in both lower and higher eukaryotic sources. Several members of β4GalT family seem to be important enzymes in the synthesis of GSL [62]. The basic structure for GLS is a monosaccharide, usually glucose, attached di‐ rectly to a ceramide molecule, mediated through the action of ceramide glucosyltransferase (Ugcg), resulting in a glycosylceramide (glucocerebroside;GlcCer) (Figure 5). βGalT-2 then transfer a UDP-Galactose to the GlcCer moiety, forming Lactosylceramide (LacCer) [62] (Figure 5). A variety of structural subclasses of GLS may then be synthesized from LacCer by the addition of other mono and disaccharides, resulting in the synthesis of structural sub‐ classes of GLS such as ganglio-, lacto/neolacto-, globo,- -isoglo, and ganglioseries-series [63]. Many of these structures are important for various biological functions, such as for example cell growth, myocardial differentiation cell migration and during development of the nerv‐ ous system[60, 61, 64]. When the Lex epitope is attached to a lactosylceramid it is identical to stage specific antigen (SSEA-1). This antigen is highly regulated during embryogenesis, ex‐ pressed at the morula stage in embryos and is considered to function as a cell-cell interac‐ tion ligand in the compaction process [65].

directly or indirectly connected to cell cycle progression and could be a potential reason for

**Figure 4.** Biosynthesis of a core 2 O-glycan with Lewis X, (LeX,SSEA-1), Sialyl-Lewis (SLex) or 6-sulpho Sialyl (6-Sulpho-Lex synthesized at the terminus of poly-N-Acetyllactosamine chains. The action of β4Ga|Ts and β3GnT are indicated with arrows. Sia|;Sia|ic acid, Gal;Ga|act0se, G|c;G|uc0se, Man;Mann0se, G|cNac; N-Acetylglucoseamine, Ga|TNAc; N-

Lewis X: As mentioned in the beginning of this chapter, β4GalTs are Important for the syn‐ thesis of linear or branched poly-N-acetyllactoseamines chains. They are attached to N-gly‐ can, O-Glycans or glycolipids and are synthesized by the repeating and alternate action of N-acetylglucosaminyltransferases (β3GnT or β4GnT) and β4Gal-T1 [58]. These structures of‐ ten carry various functional epitopes important in stem cell homeostasis and inflammation

Galβ1-4(Fucα1-3) GlcNAcβ1 which is produced by the action of β4Gal-T1 and α-1,3-Fuco‐ syltransferase (FUT). Other examples of epitopes formed from this core, are the Sialyl-Lewis (SLe<sup>x</sup> ) and 6-sulpho Sialyl (6-Sulpho-Lex) epitopes (Figure 3), in which the latter involve the activity of β4Gal-T4 (Table 1). These epitopes are implicated in biospecific interactions with selectins and other glycan-binding proteins during inflammatory processes [59] as well as in important regulatory functions during development [60]. Also, Le<sup>x</sup> structures has been im‐ plicated in specific differentiation, such as myocardial differentiation from embryonic stem

Glycosphingolipids: Glycosphingolipids, or sometimes called glycolipids (GLS) have been found in the upper leaflet of the plasma membrane in both lower and higher eukaryotic

) and constitutes the core struc‐

epitope consists of a trisaccharide,

Acetylgalactoseamine, β4Ga|act0syltransferases; β4GalT, β1,3-N- Acetylglucosaminyltransferase; β3GnT

**7. β4GalTs involvement in Lewis X, glycosphingolipids and**

[59]. One of these antigen is called the Lewis X antigen (Le<sup>x</sup>

ture from which other antigens are synthesized. Le<sup>x</sup>

**embryoglycans**

354 Pluripotent Stem Cells

cells [60, 61].

the growth impeded phenotype observed earlier in knock out β4Gal-T1 mice [52]

**Figure 5.** Core 2 structure of the glycoplipid, Lactosylceramide (LacCer) synthesized by UDP-glucose ceramide glucosyl transferase (Ugcg) and by β1,4Ga|act0sy|transferases (β4Ga|Ts) forming the β1,4-glycosidic linkage to ceramide.

Embryoglycans: Most developmentally regulated epitopes identified on embryonal carcino‐ ma cells and murine preimplantation embryos are associated with a glycoprotein-bound and large glycans, called embryoglycans. Embryoglycans consists of linear of branched poly-N-acetyllactoseamines with high molecular weight that carries a number of different developmentally regulated carbohydrate epitopes, such as e. g. Lex , described above (Figure 6). Apart from the mouse, where SSEA-1 is abundant from the 8-cell morula stage, SSEA-1 in human is not expressed until the germ cell line and in neural stem cells. Interestingly, β4Gal-T1 is expressed during the morula stage and has been shown to affect the compaction process [30]. Furthermore, human ES cells express SSEA-3 and -4 SSEA-1. SSEA-1 is also ex‐ pressed in undifferentiated F9 teratocarcinoma cells. After induction of differentiation the expression of SSEA-1 decreases. This is caused by the upregulation of alpha-1,3-galactosyl‐ transferase that is responsible for masking of the Lex structure [66, 67]. The stage specific embryonic antigens 3 and 4, (SSEA-3,-4) are from the globo-series of glycosphingolipids (GL-5 and GL-7) and have not been found on linear poly-N-lactosamines [68].

inhibition of cell surface β4GalT-1 resulted in increased tyrosine phophorylation of the EGF receptor and attenuated cell proliferation, while the shorter form of βGal-T1 did not have any effect [48]. These results implies that cell surface β4Gal-T1 has an inhibitory ef‐ fect on EGF activity. Later, several groups substantiated this observation by showing that knock-down of β4GalT1 activity in SMMC7721 hepatocarcinoma cells, elevated the auto‐ phosporylation of EGFR. Reversibly, the level of tyrosine phosphorylation was attenuated if cell surface βGal-T1 was over expressed [74]. Interestingly, EGF treatment of HeLa cells has been shown to increase the β4Gal-T1 mRNA level, suggesting that β4GalT1 also act in a negative feedback loop on EGF activity [17]. In another elegant experiment, using mutant Chinese hamster ovary cells (CHO), where the levels of six beta β4Galactosyl‐ transferases (βGalT1-6) were reduced, the protein level of active and surface-located EGFR was greatly attenuated without affecting the transcriptional level and activity of EGF receptor [75]. β4Gal-T1 has also been shown to positively affect EGFR activity. Iso‐ prenaline, a β-adrenergic receptor has a dramatic growth stimulating activation on the salivary glands of rat and mice, eventually leading to hyperplastic and hypertrophic gland enlargement. This effect has been suggested to be mediated in part by cell surface β4Gal-T1 by mimicking EGF receptor mediated receptor ligand binding and activation [76]. In any case, the specific β4GalT1 binding site on the EGF receptor has not, as yet, been investigated but it is possible that the recently discovered extracellular location of O-linked GlcNac moieties on the EGF receptor, could act as a recognition signal, as has been observed for other membrane anchored extracellular proteins, such as Notch and Dumpy receptor [77, 78]. In this scenario, β4GalT1 could act as a lectin like molecule, us‐ ing its substrate, GlcNAc [79, 80].There are also possibilities for other, more indirect and β4Gal-T1 dependent effects on EGF receptor function, such as the ganglioside GM3. The synthesis of this glycolipid is dependent on β4Gal-T2 activity, and has been shown to in‐ hibit ligand-induced tyrosine phophorylation of EGF receptor through its sialyllactose

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carbohydrate moiety by interacting with the GlcNAc termini [72, 81].

Fibroblast growth factor (FGF) functions as a natural inducer of mesoderm, regulator of cell differentiation and autocrine modulator of cell growth and transformation of various cell types. FGF is activated by ligand-receptor interaction that results in tyrosine phosphoryla‐ tion of the intracellular domain of the FGF receptor [82]. FGF-2 is often used as a key player in regulating self renewal and proliferation of human embryonic stem cells. Recently FGF-2 has been shown to regulate the transition from one pluripotent state to another. It has been speculated that human embryonic stem cells, due to their precautious ability to differentiate in culture, are identical to a later or "primed" developmental stage of mouse embryonic stem cells, EpiESC. LIF signaling is dispensable for this state, but instead relies on FGF sig‐ naling. Inhibition of FGF signaling with inhibitors in the presence of human LIF can "res‐ cue" human embryonic stem cells from a primed state to a more naïve state, e g full pluripotency [83].This difference is still unclear but there are indication that extracellular proteoglycans, such as heparin sulphate (HSPG) acts as key co-activators of FGF receptors. Furthermore, during development, oligosaccharides from embryoglycans are often shed in‐

**8.2. FGF-2**

Glycoseaminoglycans (GAG): GAGs are long unbranched polysaccharides containing a re‐ peating disaccharide unit. The disaccharide units contain either one of two modified sugars, *N*-acetylgalactosamine (GalNAc) or *N*-acetylglucosamine (GlcNAc), and a uronic acid such as glucuronate or iduronate, forming heparin sulphate and hondroitin sulphate, respectively [69]. GAGs are highly negatively charged molecules, and are located primarily on the sur‐ face of cells or in the extracellular matrix (ECM). GAGs are normally attached to soluble or membranes bound core proteins to form proteoglycans which carries various carbohydrate markers expressed on early embryonic cells [60]. In the few past years it has become clear that many growth factor such as EGF and FGF has been shown to bind specific pentasac‐ charides within GAGs efficiently affect signaling during development [70]. The integrity of proteoglycans is important. One of the β4galactosyltransferase, β4Gal-T4, is one has recently been shown to be involved in the biosynthesis of keratin sulphate (KS), in which TRA-1-60 and TRA-1-80 epitopes are found, [14]. Furthermore, β4GalT-7 is involved in the synthesis of the GAG linkage region to proteoglycans, by catalyzing the transfer UDP-Gal to an *O*linked Xylose/Ser residue in the sequence, GlcAcβ1-3Galβ1-3Galβ1-4Xylβ1-O-ser [23].

## **8. βGalTs and ESC signaling pathways**

A number of reports have suggested β4GalTs to be direct or indirect mediators and regula‐ tors of cytokine signaling during stem cell and/or cancer development. As discusses below, many signal transduction pathways, such as EGF, FGF, Wnt and the Notch pathway, that utilize Lex -containing carbohydrates are potential targets for aberrations in β4GalTs activi‐ ties:

#### **8.1. Epidermal Growth Factor (EGF)**

EGF is involved in the regulation of cell proliferation and exerts its effects in the target cells by binding to the plasma membrane located EGF receptor. The EGF receptor is a transmembrane protein tyrosine kinase. Binding of EGF to the receptor causes activation of receptor autophosphorylation, which is essential for the interaction of the receptor with its cytosolic substrates. In mouse embryonic stem cells (mESC), EGF has been shown to stimulate proliferation of mouse ES cells via PLC/PKC, Ca2+influx and p44/42 MAPK signal pathway through EGF tyrosine kinase phosphorylation [71]. Altering the core components of *N*-linked glycans will change the EGF binding, the transport and the receptor endocytosis meanwhile substitution of the outer chain or terminal glucosides have been shown to affect the phosphorylation state and the dimerization of the receptor [72, 73]. Cell surface βGalT1 has been suggested to associate with and disrupt autopho‐ phorylation of EGF receptor Hinton et. al, showed that when a dominant negative form of long β4GalT-1 was over expressed in F9 embryonic carcinoma cells, the endogenous and active cell surface GalT-1 is displaced from its association to actin cytoskeleton. This inhibition of cell surface β4GalT-1 resulted in increased tyrosine phophorylation of the EGF receptor and attenuated cell proliferation, while the shorter form of βGal-T1 did not have any effect [48]. These results implies that cell surface β4Gal-T1 has an inhibitory ef‐ fect on EGF activity. Later, several groups substantiated this observation by showing that knock-down of β4GalT1 activity in SMMC7721 hepatocarcinoma cells, elevated the auto‐ phosporylation of EGFR. Reversibly, the level of tyrosine phosphorylation was attenuated if cell surface βGal-T1 was over expressed [74]. Interestingly, EGF treatment of HeLa cells has been shown to increase the β4Gal-T1 mRNA level, suggesting that β4GalT1 also act in a negative feedback loop on EGF activity [17]. In another elegant experiment, using mutant Chinese hamster ovary cells (CHO), where the levels of six beta β4Galactosyl‐ transferases (βGalT1-6) were reduced, the protein level of active and surface-located EGFR was greatly attenuated without affecting the transcriptional level and activity of EGF receptor [75]. β4Gal-T1 has also been shown to positively affect EGFR activity. Iso‐ prenaline, a β-adrenergic receptor has a dramatic growth stimulating activation on the salivary glands of rat and mice, eventually leading to hyperplastic and hypertrophic gland enlargement. This effect has been suggested to be mediated in part by cell surface β4Gal-T1 by mimicking EGF receptor mediated receptor ligand binding and activation [76]. In any case, the specific β4GalT1 binding site on the EGF receptor has not, as yet, been investigated but it is possible that the recently discovered extracellular location of O-linked GlcNac moieties on the EGF receptor, could act as a recognition signal, as has been observed for other membrane anchored extracellular proteins, such as Notch and Dumpy receptor [77, 78]. In this scenario, β4GalT1 could act as a lectin like molecule, us‐ ing its substrate, GlcNAc [79, 80].There are also possibilities for other, more indirect and β4Gal-T1 dependent effects on EGF receptor function, such as the ganglioside GM3. The synthesis of this glycolipid is dependent on β4Gal-T2 activity, and has been shown to in‐ hibit ligand-induced tyrosine phophorylation of EGF receptor through its sialyllactose carbohydrate moiety by interacting with the GlcNAc termini [72, 81].

#### **8.2. FGF-2**

embryonic antigens 3 and 4, (SSEA-3,-4) are from the globo-series of glycosphingolipids

Glycoseaminoglycans (GAG): GAGs are long unbranched polysaccharides containing a re‐ peating disaccharide unit. The disaccharide units contain either one of two modified sugars, *N*-acetylgalactosamine (GalNAc) or *N*-acetylglucosamine (GlcNAc), and a uronic acid such as glucuronate or iduronate, forming heparin sulphate and hondroitin sulphate, respectively [69]. GAGs are highly negatively charged molecules, and are located primarily on the sur‐ face of cells or in the extracellular matrix (ECM). GAGs are normally attached to soluble or membranes bound core proteins to form proteoglycans which carries various carbohydrate markers expressed on early embryonic cells [60]. In the few past years it has become clear that many growth factor such as EGF and FGF has been shown to bind specific pentasac‐ charides within GAGs efficiently affect signaling during development [70]. The integrity of proteoglycans is important. One of the β4galactosyltransferase, β4Gal-T4, is one has recently been shown to be involved in the biosynthesis of keratin sulphate (KS), in which TRA-1-60 and TRA-1-80 epitopes are found, [14]. Furthermore, β4GalT-7 is involved in the synthesis of the GAG linkage region to proteoglycans, by catalyzing the transfer UDP-Gal to an *O*linked Xylose/Ser residue in the sequence, GlcAcβ1-3Galβ1-3Galβ1-4Xylβ1-O-ser [23].

A number of reports have suggested β4GalTs to be direct or indirect mediators and regula‐ tors of cytokine signaling during stem cell and/or cancer development. As discusses below, many signal transduction pathways, such as EGF, FGF, Wnt and the Notch pathway, that

EGF is involved in the regulation of cell proliferation and exerts its effects in the target cells by binding to the plasma membrane located EGF receptor. The EGF receptor is a transmembrane protein tyrosine kinase. Binding of EGF to the receptor causes activation of receptor autophosphorylation, which is essential for the interaction of the receptor with its cytosolic substrates. In mouse embryonic stem cells (mESC), EGF has been shown to stimulate proliferation of mouse ES cells via PLC/PKC, Ca2+influx and p44/42 MAPK signal pathway through EGF tyrosine kinase phosphorylation [71]. Altering the core components of *N*-linked glycans will change the EGF binding, the transport and the receptor endocytosis meanwhile substitution of the outer chain or terminal glucosides have been shown to affect the phosphorylation state and the dimerization of the receptor [72, 73]. Cell surface βGalT1 has been suggested to associate with and disrupt autopho‐ phorylation of EGF receptor Hinton et. al, showed that when a dominant negative form of long β4GalT-1 was over expressed in F9 embryonic carcinoma cells, the endogenous and active cell surface GalT-1 is displaced from its association to actin cytoskeleton. This


(GL-5 and GL-7) and have not been found on linear poly-N-lactosamines [68].

**8. βGalTs and ESC signaling pathways**

**8.1. Epidermal Growth Factor (EGF)**

utilize Lex

356 Pluripotent Stem Cells

ties:

Fibroblast growth factor (FGF) functions as a natural inducer of mesoderm, regulator of cell differentiation and autocrine modulator of cell growth and transformation of various cell types. FGF is activated by ligand-receptor interaction that results in tyrosine phosphoryla‐ tion of the intracellular domain of the FGF receptor [82]. FGF-2 is often used as a key player in regulating self renewal and proliferation of human embryonic stem cells. Recently FGF-2 has been shown to regulate the transition from one pluripotent state to another. It has been speculated that human embryonic stem cells, due to their precautious ability to differentiate in culture, are identical to a later or "primed" developmental stage of mouse embryonic stem cells, EpiESC. LIF signaling is dispensable for this state, but instead relies on FGF sig‐ naling. Inhibition of FGF signaling with inhibitors in the presence of human LIF can "res‐ cue" human embryonic stem cells from a primed state to a more naïve state, e g full pluripotency [83].This difference is still unclear but there are indication that extracellular proteoglycans, such as heparin sulphate (HSPG) acts as key co-activators of FGF receptors. Furthermore, during development, oligosaccharides from embryoglycans are often shed in‐ to the extra cellular environment where they can influence cytokine and mitogen signaling. Lewis x epitopes on embryoglycans acts as a recognition molecule for FGF2 and plays an active role in the formation of FGF ligand receptor complexes. Free and soluble sulphated Lewis X was most prominent to activate the FGF-2 mitogenic acitivity [84, 85]. Also exoge‐ nous and free glycolipids in the form of gangliosides, can interact with the FGF-2. Ganglio‐ sides are derivatives of LacCer with a neuraminic acid (NeuAc) attached to the core, and seem to have dual roles in affecting both EGF and FGF proliferative action; soluble ganglio‐ sides and sulphated heparin act in a negative manner meanwhile membrane bound ganglio‐ sides increase the receptor activity. It seems clear that the close interplay between Lex epitopes, adhesion molecules and cytokines has an important impact on the efficiency by which ligands are presented, and ultimately results in receptor oligomerization of the recep‐ tors and signalling [70]. It is therefore possible that β4GalTs could mediate some aspect of FGF receptor signalling, as described below.

syltransferase (O-FucT1). An N-acetyl glucosamine (GlcNac) and a Galactose (Gal) residue are then sequentially added to the fucosyl residue by the action of Fringe, a O-fucose β1,3- N-Acetyl glucosaminyl transferase and β4Gal-T1, respectively. The addition of Gal is neces‐ sary for the enhancement of Delta dependent signaling but not sufficient for the inhibition of Jagged induced Notch activation [89]. Recently, another layer of regulation of Delta induced Notch signaling was discovered in which the two Fringe genes, Lunatic Fringe (LFNG) and Manic Fringe (MFNG), seem to exhibit differential activity toward Delta dependent Notch activation. Gal was required for enhancement of Notch activation through LFNG and inhib‐ ited the enhancement of Delta induced signaling [90, 91]. Apart from O-linked Fucosylation, an O-linked GlcNAc modification of Notch EGF repeats was recently discovered [77]. Al‐ though the O-GlcNac modification is known to regulate a wide range of cellular processes, the list of known modified proteins has previously been limited to intracellular proteins in animals. Thus, this novel finding predicts a distinct glycosylation process associated with a novel regulatory mechanism for Notch receptor activity that may include a variety of βGalTs [77]. Furthermore, continuous hypoxic culturing conditions have been shown to acti‐ vate Notch signaling to allow long-term propagation of human embryonic stem cells with‐ out spontaneous differentiation. Stem cells isolated and cultured under low oxygen tension (hypoxia) condition have been shown to maintain a stable pluripotency potential because of Notch activation [92]. Recently, it was also shown that β4GalT1 derived Lewis X epitopes on N-linked glycans was necessary for Notch activity and in the propagation of neural stem

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359

It has been a challenge to get a consensus of the mechanisms by which complex carbohy‐ drates control aspects of mammalian development and early differentiation. Some of the information has been available from knock-down experiment of individual galactosyl‐ transferases. However, since many carbohydrate functions during early development in mammals are confined to " *in utero"*, further analysis of the physiological effects of galac‐ tosyltransferases has not been possible. An attractive model using a more efficient "highthroughput " a assay system, is the zebrafish system. **β4Gal-T1:** The zebrafish β4Gal-T1 has the highest sequence homology to β4Gal-T1 among the human β4GalT family. β4Gal-T1 morpholino treated embryos had a truncated anterior-posterior axis phenotype that was a result of a defect in convergent extension [94]. Convergent extension is a develop‐ mental process that relies on coordinated cell migration to elongate and narrow a field of cells. Laminin is an extracellular substrate for cell surface β4Gal-T1 and constitutes one of the major components of the basement membrane upon which cell adhesion and migra‐ tion occur during development [29]. Interestingly, in the mopholino treated embryos, laminin was hypo-galactosylated and hence could explain the decreased in ectodermal cell migration of [94]. **β4Gal-T2:** Tonoyama, et al. showed that β4Gal-T2 was indispensa‐ ble for mediolateral cell intercalation and thus extension movement during gastrulation [95]. The specific substrates for β4Gal-T2 activity in glycoproteins responsible for these ef‐

cells (NSC) [93].

**9. β4GalTs deficiency in fish**

#### **8.3. Wnt pathway**

The Wnt family of growth/differentiation factors has important developmental roles in em‐ bryonic stem cells. They act through the complex of Frizzled receptor and LPR co-receptor with effect on β-catenin transcriptional activity [86]. Similarly to EGF, the activity of Wnt al‐ so depends on association with HSPG for activity. HSPG is a rich source for developmental‐ ly regulated Lex epitopes. Furthermore, Wnt-1 has been shown to interact directly with Lex epitopes [87]. These observations suggests that surface bound and secreted Lex have a regu‐ latory function in stabilizing the stem cell niche, where they binds to and present appropri‐ ate factors, important for cell proliferation and self renewal.

#### **8.4. Notch pathway**

In a stem cell niche, stem cells and a variety of progenitor cells have to receive both temporal and spatial signals in order to differentiate or stay pluripotent. Also, during development and differentiation, cells have to decipher their precise localization in the dorso-ventral plane in order to form distinct and proper boundaries with other cell types in the tissue. These processes are governed by the Notch/ Delta system [88]. Notch is an essential devel‐ opmental glycoprotein that plays key roles in both growth control and cell fate decisions. It is a transmembrane glycoprotein with a large extracellular domain made up of 29-36 EGF repeats, which can contain both N-linked and O-linked EGF repeats [90]. When Notch re‐ ceptor is activated by a ligand on adjacent cells it is proteolytically cleaved, disposing the extracellular domain, followed by a second cleavage resulting in the released of the intracel‐ lular domain into the cytosol where it translocates to the nucleus and activates the transcrip‐ tion of numerous developmental genes. There are two ligands to Notch receptor, Delta and Jagged. Even though Notch receptor is ubiquitously expressed, Delta and Jagged are not usually located in the same cells but rather in different parts of the tissue during develop‐ ment where they exert their effect dependent on cell type and/or the environment. To avoid ubiquitous activation, Notch undergoes a post translational modification in which Fucose is first attached to certain EGF repeats on the extracellular domain of the receptor by *O*-Fuco‐ syltransferase (O-FucT1). An N-acetyl glucosamine (GlcNac) and a Galactose (Gal) residue are then sequentially added to the fucosyl residue by the action of Fringe, a O-fucose β1,3- N-Acetyl glucosaminyl transferase and β4Gal-T1, respectively. The addition of Gal is neces‐ sary for the enhancement of Delta dependent signaling but not sufficient for the inhibition of Jagged induced Notch activation [89]. Recently, another layer of regulation of Delta induced Notch signaling was discovered in which the two Fringe genes, Lunatic Fringe (LFNG) and Manic Fringe (MFNG), seem to exhibit differential activity toward Delta dependent Notch activation. Gal was required for enhancement of Notch activation through LFNG and inhib‐ ited the enhancement of Delta induced signaling [90, 91]. Apart from O-linked Fucosylation, an O-linked GlcNAc modification of Notch EGF repeats was recently discovered [77]. Al‐ though the O-GlcNac modification is known to regulate a wide range of cellular processes, the list of known modified proteins has previously been limited to intracellular proteins in animals. Thus, this novel finding predicts a distinct glycosylation process associated with a novel regulatory mechanism for Notch receptor activity that may include a variety of βGalTs [77]. Furthermore, continuous hypoxic culturing conditions have been shown to acti‐ vate Notch signaling to allow long-term propagation of human embryonic stem cells with‐ out spontaneous differentiation. Stem cells isolated and cultured under low oxygen tension (hypoxia) condition have been shown to maintain a stable pluripotency potential because of Notch activation [92]. Recently, it was also shown that β4GalT1 derived Lewis X epitopes on N-linked glycans was necessary for Notch activity and in the propagation of neural stem cells (NSC) [93].

## **9. β4GalTs deficiency in fish**

to the extra cellular environment where they can influence cytokine and mitogen signaling. Lewis x epitopes on embryoglycans acts as a recognition molecule for FGF2 and plays an active role in the formation of FGF ligand receptor complexes. Free and soluble sulphated Lewis X was most prominent to activate the FGF-2 mitogenic acitivity [84, 85]. Also exoge‐ nous and free glycolipids in the form of gangliosides, can interact with the FGF-2. Ganglio‐ sides are derivatives of LacCer with a neuraminic acid (NeuAc) attached to the core, and seem to have dual roles in affecting both EGF and FGF proliferative action; soluble ganglio‐ sides and sulphated heparin act in a negative manner meanwhile membrane bound ganglio‐ sides increase the receptor activity. It seems clear that the close interplay between Lex epitopes, adhesion molecules and cytokines has an important impact on the efficiency by which ligands are presented, and ultimately results in receptor oligomerization of the recep‐ tors and signalling [70]. It is therefore possible that β4GalTs could mediate some aspect of

The Wnt family of growth/differentiation factors has important developmental roles in em‐ bryonic stem cells. They act through the complex of Frizzled receptor and LPR co-receptor with effect on β-catenin transcriptional activity [86]. Similarly to EGF, the activity of Wnt al‐ so depends on association with HSPG for activity. HSPG is a rich source for developmental‐

epitopes [87]. These observations suggests that surface bound and secreted Lex have a regu‐ latory function in stabilizing the stem cell niche, where they binds to and present appropri‐

In a stem cell niche, stem cells and a variety of progenitor cells have to receive both temporal and spatial signals in order to differentiate or stay pluripotent. Also, during development and differentiation, cells have to decipher their precise localization in the dorso-ventral plane in order to form distinct and proper boundaries with other cell types in the tissue. These processes are governed by the Notch/ Delta system [88]. Notch is an essential devel‐ opmental glycoprotein that plays key roles in both growth control and cell fate decisions. It is a transmembrane glycoprotein with a large extracellular domain made up of 29-36 EGF repeats, which can contain both N-linked and O-linked EGF repeats [90]. When Notch re‐ ceptor is activated by a ligand on adjacent cells it is proteolytically cleaved, disposing the extracellular domain, followed by a second cleavage resulting in the released of the intracel‐ lular domain into the cytosol where it translocates to the nucleus and activates the transcrip‐ tion of numerous developmental genes. There are two ligands to Notch receptor, Delta and Jagged. Even though Notch receptor is ubiquitously expressed, Delta and Jagged are not usually located in the same cells but rather in different parts of the tissue during develop‐ ment where they exert their effect dependent on cell type and/or the environment. To avoid ubiquitous activation, Notch undergoes a post translational modification in which Fucose is first attached to certain EGF repeats on the extracellular domain of the receptor by *O*-Fuco‐

epitopes. Furthermore, Wnt-1 has been shown to interact directly with Lex

FGF receptor signalling, as described below.

ate factors, important for cell proliferation and self renewal.

**8.3. Wnt pathway**

358 Pluripotent Stem Cells

ly regulated Lex

**8.4. Notch pathway**

It has been a challenge to get a consensus of the mechanisms by which complex carbohy‐ drates control aspects of mammalian development and early differentiation. Some of the information has been available from knock-down experiment of individual galactosyl‐ transferases. However, since many carbohydrate functions during early development in mammals are confined to " *in utero"*, further analysis of the physiological effects of galac‐ tosyltransferases has not been possible. An attractive model using a more efficient "highthroughput " a assay system, is the zebrafish system. **β4Gal-T1:** The zebrafish β4Gal-T1 has the highest sequence homology to β4Gal-T1 among the human β4GalT family. β4Gal-T1 morpholino treated embryos had a truncated anterior-posterior axis phenotype that was a result of a defect in convergent extension [94]. Convergent extension is a develop‐ mental process that relies on coordinated cell migration to elongate and narrow a field of cells. Laminin is an extracellular substrate for cell surface β4Gal-T1 and constitutes one of the major components of the basement membrane upon which cell adhesion and migra‐ tion occur during development [29]. Interestingly, in the mopholino treated embryos, laminin was hypo-galactosylated and hence could explain the decreased in ectodermal cell migration of [94]. **β4Gal-T2:** Tonoyama, et al. showed that β4Gal-T2 was indispensa‐ ble for mediolateral cell intercalation and thus extension movement during gastrulation [95]. The specific substrates for β4Gal-T2 activity in glycoproteins responsible for these ef‐ fects are currently not known but has been speculated to be related with N-glycosylated FGF receptor signaling. FGF signaling pathway is dependent on its N-glycans in the in‐ teraction with heparin co-receptor, regulating the efficiency of signaling [96]. **β4Gal-T5:** Transforming Growth factor (TGFβ) and bone morphogenic protein (BMP) are polypep‐ tide members of the transforming growth factor beta (TGF) super family of cytokines. They are both secreted protein that performs many cellular functions, including the con‐ trol of cell growth, cell proliferation, cell differentiation and apoptosis. In this context, knock-down of β4Gal-T5 using morpholino-injected zebrafish resulted in embryos with an elongated dorso-ventral axis and a defective tail bud [97]. This effect was suggested to be mediated through a decreased BMP-2 (a TGFβ family member) binding to proteogly‐ can due to defective glycosylation, and subsequent attenuation of SMAD signaling.

ance, developmental delay, dwarfism, craniofacial disproportion, delayed wound healing,

β1,4-Galactosyltransferases, Potential Modifiers of Stem Cell Pluripotency and Differentiation

http://dx.doi.org/10.5772/54376

361

The involvement of β4GalTs in cancer, inflammation and during development / stem cell homeostasis has encouraged research to come up with new modalities that can either boost or inhibit the expression/activity of endogenous glycosyltransferases. I will briefly discuss potential therapeutic models for treatment that will inhibit or activate specific gal‐

A potential regulator of a galactosyltransferase, GTAP, was discovered 2008 in a two hybrid screen of a mouse embryonic library, using the cytoplasmic domain of cell surface Gal-T1 as bait. Ectopically expressed GTAP down regulated the expression of cell surface bound GalT-1 and negatively affected both laminin dependent stem cell migration and embryonic body formation during differentiation. GTAP is an ubiqutin conjugating enzyme that is ex‐ pressed during early development of the inner cell mass and in embryonic stem cells but al‐ so in highly proliferative tissues, such as, such as kidney, lung and testis. This effect was not due to a proteasome dependent degradation of βGal-T1 but an increase of ubiquitin depend‐ ent lysosomal activity. So far this is the only report on ubiquitin related regulation of a cell surface galactosyltranferase and may be important for the development of more effective and specific inhibitors of various glycosyltransferases in glycan related diseases. The only known ubiquitin/proteaseome regulated system of glycans so far, is the endoplasmic reticu‐ lum assisted degradation (ERAD).This system helps cells to avoid stress and cell death by

A limited number of GalT-1 inhibitors have been described. Most of them have been ana‐ logues of either the donor substrate (e.g Gal) or the acceptor (GlcNac) molecules to galac‐ tosyltransferases. E. g. a modified GlcNac acceptor, called compound 612, was recently discovered showing differential affinities for β4Gal-T1 and β4Gal-T5, two galactosyl transferases with similar acceptor specificities [102]. Also, in contrast to other β4galacto‐ syltransferases, β4Gal-T7 has the ability to bind, but not actively transfer Mannose or GalNAc to an acceptor substrate, implying that these donors can be used as potential in‐

During recent years, several laboratories, using specific cell lines that either over express or lack different glycosyltransferases in combination with high density lectin microarrays.

loose skin, and general ostopenia [15, 100].

degradation of missfolded proteins in the ER [101]

**11.2. Analogues to GalT donor and acceptor**

hibitors to GAG synthesis [103]

**11.3. Lectins**

**11. Potential treatments**

actosyltransferases.

**11.1. Protein ubiquitination**

#### **10. β4GalTs deficiency in mouse and human**

Many diseases such as disorders of blood clotting, congenital disorder of glycosylation, diseases of blood vessels, cancer, angiogenesis essential for breast and other solid tumor progression and metastasis, are all associated with a dysfunctional *N*-glycan expression. The expression of many galactosyltransferases is under control of cytokines and could therefore become altered in various disease states. In order to find physiological func‐ tions for each galactosyl transferases, researchers have used both mouse and rat knockout models. **β4Ga-T1:** β4Ga-T1 was the first galactosyltransferase that indicated potential relevance in physiology. About 50% of β4Gal-T1, knock-out mice died prematurely be‐ cause of pituitary deficiency [10].The surviving animals showed growth retardation, ele‐ vated proliferation of skin epidermis, and delayed wound healing due to attenuated leukocyte recruitment and infiltration [59]. Recently, some diseases in humans due to aberrations in β4Gal-T1 have emerged. For example, congenital disorders of glycosyla‐ tion (CDGs) comprise a group of inherited disorders associated with psychomotor and mental disorders. One of these groups, CDGII, comprises all defects in trimming and elongation of N-linked oligosaccharides. CDGIId fall into a group in which β4Gal-T1 is mutated in its catalytic domain. This resulted in an aberrant translation product that was 15 kDa shorter than normal. Since β4GalT-1 has been shown to be is important during the early development of the brain, the phenotype from this mutation is mental retarda‐ tion [98]. **β4Gal-T5:** Furthermore, knock-out β4Gal-T5 in mouse resulted in growth retar‐ dation and early lethality of embryos due to hematopoietic and/or placental defects [99]. Also the expression of β4Gal-T5 strongly increased during embryonic stem (ES) cell dif‐ ferentiation [22]. Both β4Gal-T5 and β4Gal-T6 are lactosylceramid synthases. However, β4Gal-T5 is more restricted to the early embryogenisis than β4Gal-T6, which is more lim‐ ited to adult brain. β4GalT-5 deficient animals showed abnormal extra embryonic struc‐ tures that led to embryonic lethal phenotype at day E10.5. **β4Gal-T7:** A rare genetic mutation of β4Gal-T7, believed to be the consequence of two missense mutations in the active domain resulted defective GAG chain formation [15] gives rise to Ehlers-Danlos disease. This is a disorder in which patients exhibit phenotypes such as aged appear‐ ance, developmental delay, dwarfism, craniofacial disproportion, delayed wound healing, loose skin, and general ostopenia [15, 100].

## **11. Potential treatments**

fects are currently not known but has been speculated to be related with N-glycosylated FGF receptor signaling. FGF signaling pathway is dependent on its N-glycans in the in‐ teraction with heparin co-receptor, regulating the efficiency of signaling [96]. **β4Gal-T5:** Transforming Growth factor (TGFβ) and bone morphogenic protein (BMP) are polypep‐ tide members of the transforming growth factor beta (TGF) super family of cytokines. They are both secreted protein that performs many cellular functions, including the con‐ trol of cell growth, cell proliferation, cell differentiation and apoptosis. In this context, knock-down of β4Gal-T5 using morpholino-injected zebrafish resulted in embryos with an elongated dorso-ventral axis and a defective tail bud [97]. This effect was suggested to be mediated through a decreased BMP-2 (a TGFβ family member) binding to proteogly‐ can due to defective glycosylation, and subsequent attenuation of SMAD signaling.

Many diseases such as disorders of blood clotting, congenital disorder of glycosylation, diseases of blood vessels, cancer, angiogenesis essential for breast and other solid tumor progression and metastasis, are all associated with a dysfunctional *N*-glycan expression. The expression of many galactosyltransferases is under control of cytokines and could therefore become altered in various disease states. In order to find physiological func‐ tions for each galactosyl transferases, researchers have used both mouse and rat knockout models. **β4Ga-T1:** β4Ga-T1 was the first galactosyltransferase that indicated potential relevance in physiology. About 50% of β4Gal-T1, knock-out mice died prematurely be‐ cause of pituitary deficiency [10].The surviving animals showed growth retardation, ele‐ vated proliferation of skin epidermis, and delayed wound healing due to attenuated leukocyte recruitment and infiltration [59]. Recently, some diseases in humans due to aberrations in β4Gal-T1 have emerged. For example, congenital disorders of glycosyla‐ tion (CDGs) comprise a group of inherited disorders associated with psychomotor and mental disorders. One of these groups, CDGII, comprises all defects in trimming and elongation of N-linked oligosaccharides. CDGIId fall into a group in which β4Gal-T1 is mutated in its catalytic domain. This resulted in an aberrant translation product that was 15 kDa shorter than normal. Since β4GalT-1 has been shown to be is important during the early development of the brain, the phenotype from this mutation is mental retarda‐ tion [98]. **β4Gal-T5:** Furthermore, knock-out β4Gal-T5 in mouse resulted in growth retar‐ dation and early lethality of embryos due to hematopoietic and/or placental defects [99]. Also the expression of β4Gal-T5 strongly increased during embryonic stem (ES) cell dif‐ ferentiation [22]. Both β4Gal-T5 and β4Gal-T6 are lactosylceramid synthases. However, β4Gal-T5 is more restricted to the early embryogenisis than β4Gal-T6, which is more lim‐ ited to adult brain. β4GalT-5 deficient animals showed abnormal extra embryonic struc‐ tures that led to embryonic lethal phenotype at day E10.5. **β4Gal-T7:** A rare genetic mutation of β4Gal-T7, believed to be the consequence of two missense mutations in the active domain resulted defective GAG chain formation [15] gives rise to Ehlers-Danlos disease. This is a disorder in which patients exhibit phenotypes such as aged appear‐

**10. β4GalTs deficiency in mouse and human**

360 Pluripotent Stem Cells

The involvement of β4GalTs in cancer, inflammation and during development / stem cell homeostasis has encouraged research to come up with new modalities that can either boost or inhibit the expression/activity of endogenous glycosyltransferases. I will briefly discuss potential therapeutic models for treatment that will inhibit or activate specific gal‐ actosyltransferases.

#### **11.1. Protein ubiquitination**

A potential regulator of a galactosyltransferase, GTAP, was discovered 2008 in a two hybrid screen of a mouse embryonic library, using the cytoplasmic domain of cell surface Gal-T1 as bait. Ectopically expressed GTAP down regulated the expression of cell surface bound GalT-1 and negatively affected both laminin dependent stem cell migration and embryonic body formation during differentiation. GTAP is an ubiqutin conjugating enzyme that is ex‐ pressed during early development of the inner cell mass and in embryonic stem cells but al‐ so in highly proliferative tissues, such as, such as kidney, lung and testis. This effect was not due to a proteasome dependent degradation of βGal-T1 but an increase of ubiquitin depend‐ ent lysosomal activity. So far this is the only report on ubiquitin related regulation of a cell surface galactosyltranferase and may be important for the development of more effective and specific inhibitors of various glycosyltransferases in glycan related diseases. The only known ubiquitin/proteaseome regulated system of glycans so far, is the endoplasmic reticu‐ lum assisted degradation (ERAD).This system helps cells to avoid stress and cell death by degradation of missfolded proteins in the ER [101]

#### **11.2. Analogues to GalT donor and acceptor**

A limited number of GalT-1 inhibitors have been described. Most of them have been ana‐ logues of either the donor substrate (e.g Gal) or the acceptor (GlcNac) molecules to galac‐ tosyltransferases. E. g. a modified GlcNac acceptor, called compound 612, was recently discovered showing differential affinities for β4Gal-T1 and β4Gal-T5, two galactosyl transferases with similar acceptor specificities [102]. Also, in contrast to other β4galacto‐ syltransferases, β4Gal-T7 has the ability to bind, but not actively transfer Mannose or GalNAc to an acceptor substrate, implying that these donors can be used as potential in‐ hibitors to GAG synthesis [103]

#### **11.3. Lectins**

During recent years, several laboratories, using specific cell lines that either over express or lack different glycosyltransferases in combination with high density lectin microarrays. In order to entangle the mechanism by which the cellular glycome can influence stem cell pluripotency and differentiation. Lectins are proteins that bind to particular carbohydrate epitopes in a similar manner as an antibody. Glycans are located at the cell surface where many signal transduction pathways, cell-cell interaction and cell-to cell recognition are constantly active. Interactions between glycans and endogenous lectins may influence self renewal, maintenance of pluripotency and differentiaon of iPS/ESC. Such an approach has already been tested in which synthetic substrates, mimicking endogenous lectins, can facilitate the formation of induced pluripotent cell( iPSC) and help sustain long term cul‐ ture of human ESCs [104]

#### **12. Conclusion and perspectives**

It is clear that both N-linked and O-linked glycans are implicated in many intricate and com‐ plex processes during development, differentiation and in many diseases. For many years glycosyltransferases were thought of as just redundant enzymes acting solely in the ER and Golgie, creating oligosaccharide structure mostly important for transport and solubility of secreted proteins. However, in the last decades, the functions of glycosyl transferases have been expanded to involve receptor oligomerization, antigen presentation, endocytosis, li‐ gand-receptor binding, and even signal transduction. These observations have attracted at‐ tention in the stem cell biology field. Several markers for pluripotency, such as Lewis X antigen, e.g. SSEA-1, -3 and -4, and the keratin sulphate related markers, TRA-1-60 and TRA-1-80, are all dependent on functional galactosylation for their synthesis and functional‐ ity. The levels and modifications of these embryonic derived antigens are changing upon differentiation. These markers have mainly been used, and are still used, as markers for iso‐ lation and propagation of different stem cell populations. With recent technological advan‐ ces and the development of more efficient lectin microarrays and HPLC systems, more and more details of the functional and structural requirements of early epitopes during stem cell self renewal and differentiation, are emerging. These techniques, combined with specific knock- down models and ectopical expression of individual galactosyltransferases, would eventually reveal the molecular mechanisms by which glycans influence stem cell and can‐ cer progression. The complex interplay between members of the galactosyltransferase fami‐ ly, does not only affect the core structures of glycans but are also extensively involved in the synthesis of other bioactive compounds, such as glycolipids and the Lexis X antigens that affect a variety of biological systems spanning from cell migration to signal transduction. The presence of the long form of β4Gal-T1 at the cell surface raises many interesting ques‐ tions on how this receptor, or maybe other glycosyltranferases as well, can influence so many different signal transduction pathways in the regulation of cell cycle, cell death, prolif‐ eration and differentiation. Apart from being located to the Golgi complex, where it is re‐ sponsible for creating complex oligosaccharide structures on proteoglycans and glycolipid, the cell surface β4Gal-Ts also affect intracellular signal transduction pathways. As seen in Figure 6, cell surface β4GalTs can indirectly affect many cell specific functions because of its involvement in the synthesis of glycolipids, embryoglycans and many embryonal epitopes,

such Lewis X antigens. These complexes will either stabilize growth factor or cytokine-re‐ ceptor complexes or, after shedded into the extracellular matrix during differentiation, in‐ hibit receptor function. A change in galactosyltransferase activity could therefore indirectly affect the stem cell nitch by hinder effective glycolipid, proteoglycan/GAG synthesis and signal transduction through tyrosin kinase (TK) receptors. Secondly, apart from binding to the extracellular matrix, such as laminin, the cell surface β4Gal-T1 could also act directly as a lectin-like molecule that bind to tyrosine receptors (EGF, FGF or Notch), either on the same cells, or on adjacent cells, as long as a terminal GlcNAc are presented. This could ei‐

cose.

**Figure 6.** Schematic view of cell surface β4GalTs potential functions. Cell surface as well as Golgi bound long β34Gal‐ actosyltransferase (GalTs) can influence stem cell homeostatis. TK; Tyrosin kinase. AC; actin, GC; Golgi complex, GL;Gly‐ colipid, PM; Plasma membrane, Ptyr; Tyrosine phosphorylation, PG; Proteoglycan, S04; sulphate, Neu;Neuramic acid, Gal;Galactose, Glc;Glucose, Man;Mannose, GalNAc; N-Acetylgalactosamine, GalNAc; N-Acetylgalactoseamine, Fuc;Fu‐

β1,4-Galactosyltransferases, Potential Modifiers of Stem Cell Pluripotency and Differentiation

http://dx.doi.org/10.5772/54376

363

β1,4-Galactosyltransferases, Potential Modifiers of Stem Cell Pluripotency and Differentiation http://dx.doi.org/10.5772/54376 363

In order to entangle the mechanism by which the cellular glycome can influence stem cell pluripotency and differentiation. Lectins are proteins that bind to particular carbohydrate epitopes in a similar manner as an antibody. Glycans are located at the cell surface where many signal transduction pathways, cell-cell interaction and cell-to cell recognition are constantly active. Interactions between glycans and endogenous lectins may influence self renewal, maintenance of pluripotency and differentiaon of iPS/ESC. Such an approach has already been tested in which synthetic substrates, mimicking endogenous lectins, can facilitate the formation of induced pluripotent cell( iPSC) and help sustain long term cul‐

It is clear that both N-linked and O-linked glycans are implicated in many intricate and com‐ plex processes during development, differentiation and in many diseases. For many years glycosyltransferases were thought of as just redundant enzymes acting solely in the ER and Golgie, creating oligosaccharide structure mostly important for transport and solubility of secreted proteins. However, in the last decades, the functions of glycosyl transferases have been expanded to involve receptor oligomerization, antigen presentation, endocytosis, li‐ gand-receptor binding, and even signal transduction. These observations have attracted at‐ tention in the stem cell biology field. Several markers for pluripotency, such as Lewis X antigen, e.g. SSEA-1, -3 and -4, and the keratin sulphate related markers, TRA-1-60 and TRA-1-80, are all dependent on functional galactosylation for their synthesis and functional‐ ity. The levels and modifications of these embryonic derived antigens are changing upon differentiation. These markers have mainly been used, and are still used, as markers for iso‐ lation and propagation of different stem cell populations. With recent technological advan‐ ces and the development of more efficient lectin microarrays and HPLC systems, more and more details of the functional and structural requirements of early epitopes during stem cell self renewal and differentiation, are emerging. These techniques, combined with specific knock- down models and ectopical expression of individual galactosyltransferases, would eventually reveal the molecular mechanisms by which glycans influence stem cell and can‐ cer progression. The complex interplay between members of the galactosyltransferase fami‐ ly, does not only affect the core structures of glycans but are also extensively involved in the synthesis of other bioactive compounds, such as glycolipids and the Lexis X antigens that affect a variety of biological systems spanning from cell migration to signal transduction. The presence of the long form of β4Gal-T1 at the cell surface raises many interesting ques‐ tions on how this receptor, or maybe other glycosyltranferases as well, can influence so many different signal transduction pathways in the regulation of cell cycle, cell death, prolif‐ eration and differentiation. Apart from being located to the Golgi complex, where it is re‐ sponsible for creating complex oligosaccharide structures on proteoglycans and glycolipid, the cell surface β4Gal-Ts also affect intracellular signal transduction pathways. As seen in Figure 6, cell surface β4GalTs can indirectly affect many cell specific functions because of its involvement in the synthesis of glycolipids, embryoglycans and many embryonal epitopes,

ture of human ESCs [104]

362 Pluripotent Stem Cells

**12. Conclusion and perspectives**

**Figure 6.** Schematic view of cell surface β4GalTs potential functions. Cell surface as well as Golgi bound long β34Gal‐ actosyltransferase (GalTs) can influence stem cell homeostatis. TK; Tyrosin kinase. AC; actin, GC; Golgi complex, GL;Gly‐ colipid, PM; Plasma membrane, Ptyr; Tyrosine phosphorylation, PG; Proteoglycan, S04; sulphate, Neu;Neuramic acid, Gal;Galactose, Glc;Glucose, Man;Mannose, GalNAc; N-Acetylgalactosamine, GalNAc; N-Acetylgalactoseamine, Fuc;Fu‐ cose.

such Lewis X antigens. These complexes will either stabilize growth factor or cytokine-re‐ ceptor complexes or, after shedded into the extracellular matrix during differentiation, in‐ hibit receptor function. A change in galactosyltransferase activity could therefore indirectly affect the stem cell nitch by hinder effective glycolipid, proteoglycan/GAG synthesis and signal transduction through tyrosin kinase (TK) receptors. Secondly, apart from binding to the extracellular matrix, such as laminin, the cell surface β4Gal-T1 could also act directly as a lectin-like molecule that bind to tyrosine receptors (EGF, FGF or Notch), either on the same cells, or on adjacent cells, as long as a terminal GlcNAc are presented. This could ei‐ ther create a block or enhancement of the TK receptor- ligand complexes, or even hinder di‐ merization and activation of the receptors. Furthermore, the β4GalT-receptor binding could lead to aggregation of cell surface β4Gal-T1, increasing its association to actin, and subse‐ quently lead to increase in intracellular signal transduction through FAK, SSeCKS and other signalling molecules. In this scenario, it is plausible that β4GalTs, control a myriad regulato‐ ry feedback loops. It is clear that so much more of the biological function of GalTs has to be understood in order to unravel attractive and potential therapies for cancer and in regenera‐ tive medicine.

[8] Smith, R.A., et al., Glycosaminoglycans as regulators of stem cell differentiation. Bio‐

β1,4-Galactosyltransferases, Potential Modifiers of Stem Cell Pluripotency and Differentiation

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365

[9] Jang, H., et al., O-GlcNAc Regulates Pluripotency and Reprogramming by Directly Acting on Core Components of the Pluripotency Network. Cell Stem Cell, 2012.

[10] Furukawa, K. and T. Sato, Beta-1,4-galactosylation of N-glycans is a complex process.

[11] Shaper, N.L., et al., Bovine galactosyltransferase: identification of a clone by direct immunological screening of a cDNA expression library. Proc Natl Acad Sci U S A,

[12] Guo, S., et al., Galactosylation of N-linked oligosaccharides by human beta-1,4-galac‐ tosyltransferases I, II, III, IV, V, and VI expressed in Sf-9 cells. Glycobiology, 2001.

[13] Lee, J., et al., Chinese hamster ovary (CHO) cells may express six beta 4-galactosyl‐ transferases (beta 4GalTs). Consequences of the loss of functional beta 4GalT-1, beta 4GalT-6, or both in CHO glycosylation mutants. J Biol Chem, 2001. 276(17): p.

[14] Seko, A., et al., Beta 1,4-galactosyltransferase (beta 4GalT)-IV is specific for GlcNAc 6-O-sulfate. Beta 4GalT-IV acts on keratan sulfate-related glycans and a precursor

[15] Bui, C., et al., Molecular characterization of beta1,4-galactosyltransferase 7 genetic mutations linked to the progeroid form of Ehlers-Danlos syndrome (EDS). FEBS Lett,

[16] Sasaki, N., et al., beta4GalT-II is a key regulator of glycosylation of the proteins in‐ volved in neuronal development. Biochem Biophys Res Commun, 2005. 333(1): p.

[17] Zhu, X., et al., Elevated beta1,4-galactosyltransferase I in highly metastatic human lung cancer cells. Identification of E1AF as important transcription activator. J Biol

[18] Xu, S., et al., Over-expression of beta-1,4-galactosyltransferase I, II, and V in human

[19] Chen, W.S., et al., Tumor beta-1,4-galactosyltransferase IV overexpression is closely associated with colorectal cancer metastasis and poor prognosis. Clin Cancer Res,

[20] Chatterjee, S., A. Kolmakova, and M. Rajesh, Regulation of lactosylceramide syn‐ thase (glucosylceramide beta1-->4 galactosyltransferase); implication as a drug tar‐

astrocytoma. J Cancer Res Clin Oncol, 2001. 127(8): p. 502-6.

glycan of 6-sulfosialyl-Lewis X. J Biol Chem, 2003. 278(11): p. 9150-8.

chem Soc Trans, 2011. 39(1): p. 383-7.

Biochim Biophys Acta, 1999. 1473(1): p. 54-66.

11(1): p. 62-74.

1986. 83(6): p. 1573-7.

2010. 584(18): p. 3962-8.

Chem, 2005. 280(13): p. 12503-16.

2005. 11(24 Pt 1): p. 8615-22.

get. Curr Drug Targets, 2008. 9(4): p. 272-81.

11(10): p. 813-20.

13924-34.

131-7.

## **Author details**

Michael Wassler\*

Address all correspondence to: Michael.Wassler@uth.tmc.edu

The center for cardiovascular Biology and Atherosclerosis Research, Department of Internal Medicine, The University of Texas Medical School at Houston, Houston, TX, USA

## **References**


[8] Smith, R.A., et al., Glycosaminoglycans as regulators of stem cell differentiation. Bio‐ chem Soc Trans, 2011. 39(1): p. 383-7.

ther create a block or enhancement of the TK receptor- ligand complexes, or even hinder di‐ merization and activation of the receptors. Furthermore, the β4GalT-receptor binding could lead to aggregation of cell surface β4Gal-T1, increasing its association to actin, and subse‐ quently lead to increase in intracellular signal transduction through FAK, SSeCKS and other signalling molecules. In this scenario, it is plausible that β4GalTs, control a myriad regulato‐ ry feedback loops. It is clear that so much more of the biological function of GalTs has to be understood in order to unravel attractive and potential therapies for cancer and in regenera‐

The center for cardiovascular Biology and Atherosclerosis Research, Department of Internal

[1] Taniguchi, N. and H. Korekane, Branched N-glycans and their implications for cell adhesion, signaling and clinical applications for cancer biomarkers and in therapeu‐

[2] Weerapana, E. and B. Imperiali, Asparagine-linked protein glycosylation: from eu‐

[3] Amado, M., et al., Identification and characterization of large galactosyltransferase gene families: galactosyltransferases for all functions. Biochim Biophys Acta, 1999.

[4] Banerjee, D.K., N-glycans in cell survival and death: Cross-talk between glycosyl‐

[5] Sasaki, N., et al., LacdiNAc (GalNAcbeta1-4GlcNAc) contributes to self-renewal of mouse embryonic stem cells by regulating leukemia inhibitory factor/STAT3 signal‐

[6] Kraushaar, D.C., Y. Yamaguchi, and L. Wang, Heparan sulfate is required for embry‐ onic stem cells to exit from self-renewal. J Biol Chem, 2010. 285(8): p. 5907-16.

[7] Sasaki, N., et al., Heparan sulfate regulates self-renewal and pluripotency of embry‐

karyotic to prokaryotic systems. Glycobiology, 2006. 16(6): p. 91R-101R.

transferases. Biochim Biophys Acta, 2012. 1820(9): p. 1338-46.

onic stem cells. J Biol Chem, 2008. 283(6): p. 3594-606.

Medicine, The University of Texas Medical School at Houston, Houston, TX, USA

tive medicine.

364 Pluripotent Stem Cells

**Author details**

Michael Wassler\*

**References**

Address all correspondence to: Michael.Wassler@uth.tmc.edu

tics. BMB Rep., 2011. 44 (12) p. 772-81.

ing. Stem Cells, 2011. 29(4): p. 641-50.

1473(1): p. 35-53.


[21] Schwientek, T., et al., Cloning of a novel member of the UDP-galactose:beta-N-acetyl‐ glucosamine beta1,4-galactosyltransferase family, beta4Gal-T4, involved in glyco‐ sphingolipid biosynthesis. J Biol Chem, 1998. 273(45): p. 29331-40.

[34] Eckstein, D.J. and B.D. Shur, Cell surface beta-1,4-galactosyltransferase is associated with the detergent-insoluble cytoskeleton on migrating mesenchymal cells. Exp Cell

β1,4-Galactosyltransferases, Potential Modifiers of Stem Cell Pluripotency and Differentiation

http://dx.doi.org/10.5772/54376

367

[35] Gong, X., et al., Activation of a G protein complex by aggregation of beta-1,4-galacto‐ syltransferase on the surface of sperm. Science, 1995. 269(5231): p. 1718-21.

[36] Lin, X., P. Nelson, and I.H. Gelman, SSeCKS, a major protein kinase C substrate with tumor suppressor activity, regulates G(1)-->S progression by controlling the expres‐ sion and cellular compartmentalization of cyclin D. Mol Cell Biol, 2000. 20(19): p.

[37] Wassler, M.J., et al., Functional interaction between the SSeCKS scaffolding protein and the cytoplasmic domain of beta1,4-galactosyltransferase. J Cell Sci, 2001. 114(Pt

[38] Bunnell, B.A., et al., Increased expression of a 58-kDa protein kinase leads to changes

[39] Wassler, M.J., et al., Characterization of a novel ubiquitin-conjugating enzyme that regulates beta1,4-galactosyltransferase-1 in embryonic stem cells. Stem Cells, 2008.

[40] Gelman, I.H., E. Tombler, and J. Vargas, Jr., A role for SSeCKS, a major protein kin‐ ase C substrate with tumour suppressor activity, in cytoskeletal architecture, forma‐ tion of migratory processes, and cell migration during embryogenesis. Histochem J,

[41] Wassler, M.J. and B.D. Shur, Clustering of cell surface (beta)1,4-galactosyltransferase I induces transient tyrosine phosphorylation of focal adhesion kinase and loss of

[42] Zeng, F.Y., et al., Differential response of the epidermal growth factor receptor tyro‐ sine kinase activity to several plant and mammalian lectins. Mol Cell Biochem, 1995.

[43] Dennis, J.W., M. Granovsky, and C.E. Warren, Glycoprotein glycosylation and cancer

[44] Johnson, F.M. and B.D. Shur, The level of cell surface beta1,4-galactosyltransferase I influences the invasive potential of murine melanoma cells. J Cell Sci, 1999. 112 ( Pt

[45] Shen, J., et al., Two specific inhibitors of the phosphatidylinositol 3-kinase LY294002 and wortmannin up-regulate beta1,4-galactosyltransferase I and thus sensitize SMMC-7721 human hepatocarcinoma cells to cycloheximide-induced apoptosis. Mol

in the CHO cell cycle. Proc Natl Acad Sci U S A, 1990. 87(19): p. 7467-71.

Res, 1992. 201(1): p. 83-90.

7259-72.

12): p. 2291-300.

26(8): p. 2006-18.

2000. 32(1): p. 13-26.

142(2): p. 117-24.

16): p. 2785-95.

stress fibers. J Cell Sci, 2000. 113 Pt 2: p. 237-45.

Cell Biochem, 2007. 304(1-2): p. 361-7.

progression. Biochim Biophys Acta, 1999. 1473(1): p. 21-34.


[34] Eckstein, D.J. and B.D. Shur, Cell surface beta-1,4-galactosyltransferase is associated with the detergent-insoluble cytoskeleton on migrating mesenchymal cells. Exp Cell Res, 1992. 201(1): p. 83-90.

[21] Schwientek, T., et al., Cloning of a novel member of the UDP-galactose:beta-N-acetyl‐ glucosamine beta1,4-galactosyltransferase family, beta4Gal-T4, involved in glyco‐

[22] Nishie, T., et al., Beta4-galactosyltransferase-5 is a lactosylceramide synthase essen‐ tial for mouse extra-embryonic development. Glycobiology, 2010. 20(10): p. 1311-22.

[23] Almeida, R., et al., Cloning and expression of a proteoglycan UDP-galactose:beta-xy‐ lose beta1,4-galactosyltransferase I. A seventh member of the human beta4-galacto‐

[24] Shur, B.D., S. Evans, and Q. Lu, Cell surface galactosyltransferase: current issues.

[25] Shur, B.D., Cell surface beta 1,4 galactosyltransferase: twenty years later. Glycobiolo‐

[26] Bunnell, B.A., D.E. Adams, and V.J. Kidd, Transient expression of a p58 protein kin‐ ase cDNA enhances mammalian glycosyltransferase activity. Biochem Biophys Res

[27] Hathaway, H.J., et al., Mutational analysis of the cytoplasmic domain of beta1,4-gal‐ actosyltransferase I: influence of phosphorylation on cell surface expression. J Cell

[28] Zhang, S.W., et al., Effect of p58GTA on beta-1,4-galactosyltransferase 1 activity and cell-cycle in human hepatocarcinoma cells. Mol Cell Biochem, 2001. 221(1-2): p. 161-8.

[29] Shur, B.D., Glycosyltransferases as cell adhesion molecules. Curr Opin Cell Biol,

[30] Bayna, E.M., J.H. Shaper, and B.D. Shur, Temporally specific involvement of cell sur‐ face beta-1,4 galactosyltransferase during mouse embryo morula compaction. Cell,

[31] Begovac, P.C., et al., Evidence that cell surface beta 1,4-galactosyltransferase sponta‐ neously galactosylates an underlying laminin substrate during fibroblast migration. J

[32] Maillet, C.M. and B.D. Shur, Uvomorulin, LAMP-1, and laminin are substrates for cell surface beta-1,4-galactosyltransferase on F9 embryonal carcinoma cells: compari‐ sons between wild-type and mutant 5.51 att- cells. Exp Cell Res, 1993. 208(1): p.

[33] Maillet, C.M. and B.D. Shur, Perturbing cell surface beta-(1,4)-galactosyltransferase on F9 embryonal carcinoma cells arrests cell growth and induces laminin synthesis. J

sphingolipid biosynthesis. J Biol Chem, 1998. 273(45): p. 29331-40.

syltransferase gene family. J Biol Chem, 1999. 274(37): p. 26165-71.

Glycoconj J, 1998. 15(6): p. 537-48.

Commun, 1990. 171(1): p. 196-203.

Sci, 2003. 116(Pt 21): p. 4319-30.

1993. 5(5): p. 854-63.

1988. 53(1): p. 145-57.

282-95.

Biol Chem, 1994. 269(50): p. 31793-9.

Cell Sci, 1994. 107 ( Pt 6): p. 1713-24.

gy, 1991. 1(6): p. 563-75.

366 Pluripotent Stem Cells


[46] Satomaa, T., et al., The N-glycome of human embryonic stem cells. BMC Cell Biol, 2009. 10: p. 42.

[60] Muramatsu, T. and H. Muramatsu, Carbohydrate antigens expressed on stem cells

β1,4-Galactosyltransferases, Potential Modifiers of Stem Cell Pluripotency and Differentiation

http://dx.doi.org/10.5772/54376

369

[61] Sudou, A., et al., Le(X) structure enhances myocardial differentiation from embryon‐

[62] Ma, R., et al., Post-translational and transcriptional regulation of glycolipid glycosyl‐ transferase genes in apoptotic breast carcinoma cells: VII. Studied by DNA-microar‐

[63] Biellmann, F., et al., The Lc3-synthase gene B3gnt5 is essential to pre-implantation

[64] Yamashita, T., et al., A vital role for glycosphingolipid synthesis during development

[65] Rastan, S., et al., Cell interactions in preimplantation embryos: evidence for involve‐ ment of saccharides of the poly-N-acetyllactosamine series. J Embryol Exp Morphol,

[66] Cho, S.K., et al., Transcriptional regulation of alpha1,3-galactosyltransferase in em‐ bryonal carcinoma cells by retinoic acid. Masking of Lewis X antigens by alpha-gal‐

[67] Patil, S.A., et al., Scaling down the size and increasing the throughput of glycosyl‐ transferase assays: activity changes on stem cell differentiation. Anal Biochem, 2012.

[68] Liang, Y.J., et al., Changes in glycosphingolipid composition during differentiation of human embryonic stem cells to ectodermal or endodermal lineages. Stem Cells, 2011.

[69] Hacker, U., K. Nybakken, and N. Perrimon, Heparan sulphate proteoglycans: the

[70] Raman, R., V. Sasisekharan, and R. Sasisekharan, Structural insights into biological roles of protein-glycosaminoglycan interactions. Chem Biol, 2005. 12(3): p. 267-77.

[71] Heo, J.S., Y.J. Lee, and H.J. Han, EGF stimulates proliferation of mouse embryonic stem cells: involvement of Ca2+ influx and p44/42 MAPKs. Am J Physiol Cell Physi‐

[72] Kawashima, N., et al., Tyrosine kinase activity of epidermal growth factor receptor is regulated by GM3 binding through carbohydrate to carbohydrate interactions. J Biol

[73] Takahashi, M., et al., Role of N-glycans in growth factor signaling. Glycoconj J, 2004.

sweet side of development. Nat Rev Mol Cell Biol, 2005. 6(7): p. 530-41.

ray after treatment with L-PPMP. Glycoconj J, 2009. 26(6): p. 647-61.

development of the murine embryo. BMC Dev Biol, 2008. 8: p. 109.

and differentiation. Proc Natl Acad Sci U S A, 1999. 96(16): p. 9142-7.

and early embryonic cells. Glycoconj J, 2004. 21(1-2): p. 41-5.

ic stem cells. Cell Struct Funct, 1997. 22(2): p. 247-51.

actosylation. J Biol Chem, 1996. 271(6): p. 3238-46.

1985. 87: p. 115-28.

425(2): p. 135-44.

29(12): p. 1995-2004.

ol, 2006. 290(1): p. C123-33.

20(3): p. 207-12.

Chem, 2009. 284(10): p. 6147-55.


[60] Muramatsu, T. and H. Muramatsu, Carbohydrate antigens expressed on stem cells and early embryonic cells. Glycoconj J, 2004. 21(1-2): p. 41-5.

[46] Satomaa, T., et al., The N-glycome of human embryonic stem cells. BMC Cell Biol,

[47] Calder, A., et al., Lengthened G1 Phase Indicates Differentiation Status In Human

[48] Hinton, D.A., S.C. Evans, and B.D. Shur, Altering the expression of cell surface beta 1,4-galactosyltransferase modulates cell growth. Exp Cell Res, 1995. 219(2): p. 640-9.

[49] Pouncey, L., et al., Beta 1-4-galactosyltransferase gene expression is regulated during entry into the cell cycle and during the cell cycle. Somat Cell Mol Genet, 1991. 17(5):

[50] Wu, G.Q., S.M. Jiang, and J.X. Gu, Studies on the beta(l-4)Galactosyltransferase of Cell Surface in Induced HL60 Cells. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue

[51] Wei, Y., et al., Regulation of the beta1,4-Galactosyltransferase I promoter by E2F1. J

[52] Asano, M., et al., Growth retardation and early death of beta-1,4-galactosyltransfer‐ ase knockout mice with augmented proliferation and abnormal differentiation of epi‐

[53] Zhang, S.W., et al., Down-regulation of beta1,4-galactosyltransferase gene expression by cell-cycle suppressor gene p16. Biochim Biophys Acta, 1999. 1444(1): p. 49-54.

[54] Zhang, S.W., et al., Effect of suppression of TGF-beta1 expression on cell-cycle and gene expression of beta-1,4-galactosyltransferase 1 in human hepatocarcinoma cells.

[55] Li, Z., et al., Downregulation of beta1,4-galactosyltransferase 1 inhibits CDK11(p58) mediated apoptosis induced by cycloheximide. Biochem Biophys Res Commun,

[56] Ohno, S., et al., Polypyrimidine tract-binding protein regulates the cell cycle through IRES-dependent translation of CDK11(p58) in mouse embryonic stem cells. Cell Cy‐

[57] Wei, Y., et al., Identification of beta-1,4-galactosyltransferase I as a target gene of HBx-induced cell cycle progression of hepatoma cell. J Hepatol, 2008. 49(6): p.

[58] Ujita, M., et al., Poly-N-acetyllactosamine synthesis in branched N-glycans is control‐ led by complemental branch specificity of I-extension enzyme and beta1,4-galactosyl‐

[59] Asano, M., et al., Impaired selectin-ligand biosynthesis and reduced inflammatory responses in beta-1,4-galactosyltransferase-I-deficient mice. Blood, 2003. 102(5): p.

2009. 10: p. 42.

368 Pluripotent Stem Cells

p. 435-43.

Embryonic Stem Cells. Stem Cells Dev, 2012.

Bao (Shanghai), 1997. 29(4): p. 402-408.

thelial cells. EMBO J, 1997. 16(8): p. 1850-7.

Biochem Biophys Res Commun, 2000. 273(3): p. 833-8.

transferase I. J Biol Chem, 1999. 274(24): p. 16717-26.

Biochem, 2010. 148(3): p. 263-71.

2005. 327(2): p. 628-36.

1029-37.

1678-85.

cle, 2011. 10(21): p. 3706-13.


[74] Li, Z., et al., Cell surface beta 1, 4-galactosyltransferase 1 promotes apoptosis by in‐ hibiting epidermal growth factor receptor pathway. Mol Cell Biochem, 2006. 291(1-2): p. 69-76.

[89] Chen, J., D.J. Moloney, and P. Stanley, Fringe modulation of Jagged1-induced Notch signaling requires the action of beta 4galactosyltransferase-1. Proc Natl Acad Sci U S

β1,4-Galactosyltransferases, Potential Modifiers of Stem Cell Pluripotency and Differentiation

http://dx.doi.org/10.5772/54376

371

[90] Haltiwanger, R.S. and P. Stanley, Modulation of receptor signaling by glycosylation: fringe is an O-fucose-beta1,3-N-acetylglucosaminyltransferase. Biochim Biophys Ac‐

[91] Hou, X., Y. Tashima, and P. Stanley, Galactose differentially modulates lunatic and manic fringe effects on Delta1-induced NOTCH signaling. J Biol Chem, 2012. 287(1):

[92] Prasad, S.M., et al., Continuous hypoxic culturing maintains activation of Notch and allows long-term propagation of human embryonic stem cells without spontaneous

[93] Yagi, H., et al., Lewis X-carrying N-Glycans Regulate the Proliferation of Mouse Em‐ bryonic Neural Stem Cells via the Notch Signaling Pathway. J Biol Chem, 2012.

[94] Machingo, Q.J., A. Fritz, and B.D. Shur, A beta1,4-galactosyltransferase is required for convergent extension movements in zebrafish. Dev Biol, 2006. 297(2): p. 471-82.

[95] Tonoyama, Y., et al., Essential role of beta-1,4-galactosyltransferase 2 during medaka

[96] Duchesne, L., et al., N-glycosylation of fibroblast growth factor receptor 1 regulates ligand and heparan sulfate co-receptor binding. J Biol Chem, 2006. 281(37): p.

[97] Machingo, Q.J., A. Fritz, and B.D. Shur, A beta1,4-galactosyltransferase is required for Bmp2-dependent patterning of the dorsoventral axis during zebrafish embryo‐

[98] Hansske, B., et al., Deficiency of UDP-galactose:N-acetylglucosamine beta-1,4-galac‐ tosyltransferase I causes the congenital disorder of glycosylation type IId. J Clin In‐

[99] Kumagai, T., et al., Early lethality of beta-1,4-galactosyltransferase V-mutant mice by growth retardation. Biochem Biophys Res Commun, 2009. 379(2): p. 456-9.

[100] Talhaoui, I., et al., Identification of key functional residues in the active site of human {beta}1,4-galactosyltransferase 7: a major enzyme in the glycosaminoglycan synthesis

[101] Mallinger, A., et al., Using a ubiquitin ligase as an unfolded protein sensor. Biochem

(Oryzias latipes) gastrulation. Mech Dev, 2009. 126(7): p. 580-94.

A, 2001. 98(24): p. 13716-21.

ta, 2002. 1573(3): p. 328-35.

differentiation. Cell Prolif, 2009. 42(1): p. 63-74.

genesis. Development, 2006. 133(11): p. 2233-41.

pathway. J Biol Chem, 2010. 285(48): p. 37342-58.

Biophys Res Commun, 2012. 418(1): p. 44-8.

vest, 2002. 109(6): p. 725-33.

p. 474-83.

27178-89.

287(29): p. 24356-64.


[89] Chen, J., D.J. Moloney, and P. Stanley, Fringe modulation of Jagged1-induced Notch signaling requires the action of beta 4galactosyltransferase-1. Proc Natl Acad Sci U S A, 2001. 98(24): p. 13716-21.

[74] Li, Z., et al., Cell surface beta 1, 4-galactosyltransferase 1 promotes apoptosis by in‐ hibiting epidermal growth factor receptor pathway. Mol Cell Biochem, 2006. 291(1-2):

[75] Gabius, H.J., et al., Down-regulation of the epidermal growth factor receptor by alter‐ ing N-glycosylation: emerging role of beta1,4-galactosyltransferases. Anticancer Res,

[76] Purushotham, K.R., et al., A novel mechanism for isoprenaline-stimulated prolifera‐ tion of rat parotid acinar cells involving the epidermal growth factor receptor and

[77] Matsuura, A., et al., O-linked N-acetylglucosamine is present on the extracellular do‐

[78] Sakaidani, Y., et al., O-linked-N-acetylglucosamine on extracellular protein domains

[79] Hazan, R., L. Krushel, and K.L. Crossin, EGF receptor-mediated signals are differen‐

[80] Hebert, E., Endogenous lectins as cell surface transducers. Biosci Rep, 2000. 20(4): p.

[81] Rusnati, M., et al., Interaction of fibroblast growth factor-2 (FGF-2) with free ganglio‐ sides: biochemical characterization and biological consequences in endothelial cell

[82] Basilico, C. and D. Moscatelli, The FGF family of growth factors and oncogenes. Adv

[83] Lanner, F. and J. Rossant, The role of FGF/Erk signaling in pluripotent cells. Develop‐

[84] Dvorak, P., et al., Embryoglycan ectodomains regulate biological activity of FGF-2 to

[85] Jirmanova, L., et al., O-linked carbohydrates are required for FGF-2-mediated prolif‐

[86] Katoh, M., WNT signaling pathway and stem cell signaling network. Clin Cancer

[87] Capela, A. and S. Temple, LeX is expressed by principle progenitor cells in the em‐ bryonic nervous system, is secreted into their environment and binds Wnt-1. Dev Bi‐

[88] Haltiwanger, R.S., Regulation of signal transduction pathways in development by

eration of mouse embryonic cells. Int J Dev Biol, 1999. 43(6): p. 555-62.

embryonic stem cells. J Cell Sci, 1998. 111 ( Pt 19): p. 2945-52.

glycosylation. Curr Opin Struct Biol, 2002. 12(5): p. 593-8.

mediates epithelial cell-matrix interactions. Nat Commun, 2011. 2: p. 583.

tially modulated by concanavalin A. J Cell Physiol, 1995. 162(1): p. 74-85.

cell surface galactosyltransferase. Biochem J, 1992. 284 ( Pt 3): p. 767-76.

main of notch receptors. J Biol Chem, 2008. 283(51): p. 35486-95.

cultures. Mol Biol Cell, 1999. 10(2): p. 313-27.

Cancer Res, 1992. 59: p. 115-65.

ment. 2010.137 (20) p. 3351-60.

Res, 2007. 13(14): p. 4042-5.

ol, 2006. 291(2): p. 300-13.

p. 69-76.

370 Pluripotent Stem Cells

213-37.

2012. 32(5): p. 1565-72.


[102] Gao, Y., et al., Specificity of beta1,4-galactosyltransferase inhibition by 2-naphthyl 2 butanamido-2-deoxy-1-thio-beta-D-glucopyranoside. Glycoconj J, 2010. 27(7-9): p. 673-84.

**Section 3**

**Therapeutic Implications and Ethical Concerns**


**Therapeutic Implications and Ethical Concerns**

[102] Gao, Y., et al., Specificity of beta1,4-galactosyltransferase inhibition by 2-naphthyl 2 butanamido-2-deoxy-1-thio-beta-D-glucopyranoside. Glycoconj J, 2010. 27(7-9): p.

[103] Daligault, F., et al., Thermodynamic insights into the structural basis governing the donor substrate recognition by human beta1,4-galactosyltransferase 7. Biochem J,

[104] Tateno, H., et al., Glycome diagnosis of human induced pluripotent stem cells using

[105] Roth, S., E.J. McGuire, and S. Roseman, Evidence for cell-surface glycosyltransferas‐ es. Their potential role in cellular recognition. J Cell Biol, 1971. 51(21): p. 536-47.

lectin microarray. J Biol Chem, 2011. 286(23): p. 20345-53.

673-84.

372 Pluripotent Stem Cells

2009. 418(3): p. 605-14.

**Chapter 17**

**Advances in Stem Cell Therapies**

Additional information is available at the end of the chapter

diseases while minimizing unwanted side effects.

Four key milestones have to be realized for the ideal customized stem cell therapy to be successful. First, stem cells utilized in these therapies have to be genetically stable and epigenetically regulated to ensure the safety of stem cells employed in any future therapies. This is essential to ensure that patients undergoing stem cell therapy are not exposed to increased risks of tumorigenesis and other mutagenic diseases. Second, stem cells should be able to evade the innate immune response of patients, possibly via the secretion of immuno‐ suppressive molecules that inhibit immune responses or by displaying host cellular recogni‐ tion markers. The survival of transplanted stem cells is crucial for the design of an effective therapy. Additionally the ability of transplanted stem cells to evade immune detection and inflammatory responses will prevent undesired symptoms such as graft-versus-host-disease in patients. Third, stem cells employed in these therapies should be location specific. These stem cells should possess specific homing cell surface markers that will allow them to locate and migrate to specific localities. This will ensure that stem cells used in therapies will only accumulate in diseased tissues for targeted therapeutic effect, and not in other healthy regions where detrimental non-specific interactions might occur. Finally, the stem cells used in these therapies should be functionally specific and disease relevant. Transplanted stem cells should be designed to restore a healthy phenotype in patients. These cells should be able to restore organ and tissue function in regenerative therapies, either directly by replicating to replace damaged portions of these organs and tissues and/or indirectly by secreting therapeutic molecules to mediate their functional restoration. These stem cells should also be epigenetically primed for specific functions to ensure that they are able to reverse the effects of treated

Stem cells are commonly classified into three broad categories based on how they were derived. Embryonic stem cells (ESCs) are stem cells that are isolated from the inner cell mass of the early developing embryo. Adult stem cells assist in the natural regeneration and repair

> © 2013 Sng and Lufkin; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Sng and Lufkin; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Joel Sng and Thomas Lufkin

http://dx.doi.org/10.5772/55061

**1. Introduction**

## **Chapter 17**

## **Advances in Stem Cell Therapies**

Joel Sng and Thomas Lufkin

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55061

## **1. Introduction**

Four key milestones have to be realized for the ideal customized stem cell therapy to be successful. First, stem cells utilized in these therapies have to be genetically stable and epigenetically regulated to ensure the safety of stem cells employed in any future therapies. This is essential to ensure that patients undergoing stem cell therapy are not exposed to increased risks of tumorigenesis and other mutagenic diseases. Second, stem cells should be able to evade the innate immune response of patients, possibly via the secretion of immuno‐ suppressive molecules that inhibit immune responses or by displaying host cellular recogni‐ tion markers. The survival of transplanted stem cells is crucial for the design of an effective therapy. Additionally the ability of transplanted stem cells to evade immune detection and inflammatory responses will prevent undesired symptoms such as graft-versus-host-disease in patients. Third, stem cells employed in these therapies should be location specific. These stem cells should possess specific homing cell surface markers that will allow them to locate and migrate to specific localities. This will ensure that stem cells used in therapies will only accumulate in diseased tissues for targeted therapeutic effect, and not in other healthy regions where detrimental non-specific interactions might occur. Finally, the stem cells used in these therapies should be functionally specific and disease relevant. Transplanted stem cells should be designed to restore a healthy phenotype in patients. These cells should be able to restore organ and tissue function in regenerative therapies, either directly by replicating to replace damaged portions of these organs and tissues and/or indirectly by secreting therapeutic molecules to mediate their functional restoration. These stem cells should also be epigenetically primed for specific functions to ensure that they are able to reverse the effects of treated diseases while minimizing unwanted side effects.

Stem cells are commonly classified into three broad categories based on how they were derived. Embryonic stem cells (ESCs) are stem cells that are isolated from the inner cell mass of the early developing embryo. Adult stem cells assist in the natural regeneration and repair

© 2013 Sng and Lufkin; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Sng and Lufkin; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

in developed organisms and can be purified from their tissues. Induced pluripotent stem cells (iPSCs) are artificially derived stem cells that are formed via various genetic and epigenetic reprogramming procedures. Of the three broad categories of stem cells, adult stem cells are most widely utilized in clinical trials and experimental therapies worldwide. Most adult stem cells are multipotent and differentiate to form only a limited subset of cell types. Hence these stem cells are commonly classified according to their developmental commitment or tissue source. Examples of adult stem cells include mesenchymal stem cells (MSCs), neural stem cells (NSCs), hematopoietic stem cells, inner ear stem cells, mammary stem cells, endothelial stem cells, intestinal stem cells, and testicular stem cells.

phenotypes unlike umbilical cord blood MSC that display a lower capacity to form adipocytes [3, 6]. MSCs also express different cellular markers. For example A-MSCs express CD34, CD49d, and CD54 at higher levels than M-MSCs while M-MSCs and U-MSCs express higher levels of CD 106 than A-MSCs [7]. M-MSCs and A-MSCs also have higher levels of CD90 and CD105 expression when compared to U-MSCs [3]. The morphology of MSCs can also differ significantly and even MSCs from the same source display heterogeneous morphologies. Various descriptions of MSCs in the literature include spindle shaped, round, fibroblastoid cells, flattened cells, and blanket cells [8, 9]. Further studies to understand these inherent differences in various subpopulations of MSCs could lead to an improved understanding of how epigenetic differences regulate stem cell differentiation fates, homing to specific recog‐

Advances in Stem Cell Therapies http://dx.doi.org/10.5772/55061 377

M-MSCs are currently the most widely used stem cells in clinical trials and therapies. Both autograft and allograft M-MSCs have been extensively tested for their therapeutic safety and effectiveness in alleviating the symptoms of several diseases. One of the key reasons for the success of M-MSCs therapy is because these cells possess intrinsic immunomodulatory properties that enable M-MSCs to inhibit and evade potential immune rejection when transplanted [10]. M-MSCs are able to inhibit the maturation and function of various immune cells including dendritic cells, natural killer cells, B cells, and T lymphocytes [11]. Additionally M-MSCs have reduced immunogenicity due to their minimal expression of surface MHC ІІ proteins and the lack of T cell stimulatory proteins like CD80 and CD86 [12]. Another important reason for the early success of M-MSCs based therapies is that MSCs have low tumorigenic potential and are safer than therapies based on ESCs or iPSCs which display robust tumori‐

Due to these intrinsic advantages of MSCs, clinical trials can be conducted to evaluate their safety and effectiveness in treating various diseases. For example, the safety and effectiveness of M-MSC transplantation for joint cartilage repair has been evaluated in several studies. In a clinical trial involving 41 patients studied over a period of between 5 to 137 months, M-MSC transplantation did not contribute to increased risk of tumors or infection [14]. Another study has reported the potential for M-MSC regenerative knee therapy to induce cartilage and meniscus growth and increase range of motion [15]. These results are supported in a larger scale M-MSC transplantation study involving 339 patients which reported no increased risk of tumor formation and a significant improvement of knee function in transplant patients [16]. While further clinical trials have to be conducted to verify these preliminary results, the successes of these initial clinical trials indicate that M-MSC therapy is likely to be safe and can

The effectiveness of M-MSC and other MSC therapies for various autoimmune diseases has also been studied in several small clinical trials. These autoimmune diseases include multiple sclerosis, Crohn's disease, scleroderma, and systemic lupus erythematosus [17]. The causes of many of these autoimmune diseases are not well understood and it is likely that while the patients suffer from similar symptoms, contributing disease factors may vary significantly between patients. However the application of a generic MSC transplantation therapy was

successful in alleviating the symptoms of these patients in several clinical trials.

nition sites, proliferation rates, and senescence.

genicity [13].

catalyze cartilage repair.

Adult stem cells present the first success of human experimental stem cell therapy. There are several reasons why adult stem cells therapies are currently more successful than ESC and iPSC therapies. Firstly, stem cell therapies involving adult stem cells are often autotransplants with minimal potential for immune rejection. These adult stem cells can be harvested directly from individual patients before being utilized as transplants. Hence these adult stem cells will exhibit host cell recognition molecules unlike ESCs and iPSCs that may provoke an immune response when used in therapies. Secondly since most adult stem cells therapies involve minimally processed cells, there a reduced possibility of genetic mutation or chromosome aberration occurring compared to ESCs and iPSCs that have to be cultured extensively in vitro before their use in therapies. Thirdly, adult stem cells do not readily form tumors when introduced into patients and are considered to be safer than ESCs and iPSCs that display greater carcinogenic potential. Finally, the use of adult stem cells in therapies is not considered to be controversial as they can be readily extracted from patient tissues and do not require the destruction of embryos to derive stable cell lines unlike ESCs. These key advantages of adult stem cells have led to their wider utilization in research and various clinical trials compared to ESCs and iPSCs.

## **2. Mesenchymal stem cell therapy**

Mesenchymal stem cells (MSCs) are one of the first multipotent adult stem cells to be utilized in stem cell therapies. These stem cells have the ability to differentiate and form bone, cartilage, and adipose tissues. While the bone marrow is the most common source of MSCs for thera‐ peutic purposes, they can also be found in adipose and synovial tissue, skeletal muscles, peripheral blood, breast milk, and the umbilical cord [1, 2].

While these stem cells are commonly referred to as MSCs, they actually form a heterogeneous population of cells as evidenced by differences in proliferative capacity, differentiation potential, cellular markers, and morphology. For example, MSCs derived from the bone marrow (M-MSC) have lower proliferative capacity, followed by adipose tissue MSCs (A-MSC) and umbilical cord blood MSCs (U-MSC) which have the highest proliferative capacity [3]. MSCs also have differing differential potentials. For example, bone marrows MSCs have a higher chondrogenic potential while adipose MSCs have a lower chondrogenic potential [4, 5]. In addition both bone marrow and adipose MSCs readily form cells with adipogenic phenotypes unlike umbilical cord blood MSC that display a lower capacity to form adipocytes [3, 6]. MSCs also express different cellular markers. For example A-MSCs express CD34, CD49d, and CD54 at higher levels than M-MSCs while M-MSCs and U-MSCs express higher levels of CD 106 than A-MSCs [7]. M-MSCs and A-MSCs also have higher levels of CD90 and CD105 expression when compared to U-MSCs [3]. The morphology of MSCs can also differ significantly and even MSCs from the same source display heterogeneous morphologies. Various descriptions of MSCs in the literature include spindle shaped, round, fibroblastoid cells, flattened cells, and blanket cells [8, 9]. Further studies to understand these inherent differences in various subpopulations of MSCs could lead to an improved understanding of how epigenetic differences regulate stem cell differentiation fates, homing to specific recog‐ nition sites, proliferation rates, and senescence.

in developed organisms and can be purified from their tissues. Induced pluripotent stem cells (iPSCs) are artificially derived stem cells that are formed via various genetic and epigenetic reprogramming procedures. Of the three broad categories of stem cells, adult stem cells are most widely utilized in clinical trials and experimental therapies worldwide. Most adult stem cells are multipotent and differentiate to form only a limited subset of cell types. Hence these stem cells are commonly classified according to their developmental commitment or tissue source. Examples of adult stem cells include mesenchymal stem cells (MSCs), neural stem cells (NSCs), hematopoietic stem cells, inner ear stem cells, mammary stem cells, endothelial stem

Adult stem cells present the first success of human experimental stem cell therapy. There are several reasons why adult stem cells therapies are currently more successful than ESC and iPSC therapies. Firstly, stem cell therapies involving adult stem cells are often autotransplants with minimal potential for immune rejection. These adult stem cells can be harvested directly from individual patients before being utilized as transplants. Hence these adult stem cells will exhibit host cell recognition molecules unlike ESCs and iPSCs that may provoke an immune response when used in therapies. Secondly since most adult stem cells therapies involve minimally processed cells, there a reduced possibility of genetic mutation or chromosome aberration occurring compared to ESCs and iPSCs that have to be cultured extensively in vitro before their use in therapies. Thirdly, adult stem cells do not readily form tumors when introduced into patients and are considered to be safer than ESCs and iPSCs that display greater carcinogenic potential. Finally, the use of adult stem cells in therapies is not considered to be controversial as they can be readily extracted from patient tissues and do not require the destruction of embryos to derive stable cell lines unlike ESCs. These key advantages of adult stem cells have led to their wider utilization in research and various clinical trials compared

Mesenchymal stem cells (MSCs) are one of the first multipotent adult stem cells to be utilized in stem cell therapies. These stem cells have the ability to differentiate and form bone, cartilage, and adipose tissues. While the bone marrow is the most common source of MSCs for thera‐ peutic purposes, they can also be found in adipose and synovial tissue, skeletal muscles,

While these stem cells are commonly referred to as MSCs, they actually form a heterogeneous population of cells as evidenced by differences in proliferative capacity, differentiation potential, cellular markers, and morphology. For example, MSCs derived from the bone marrow (M-MSC) have lower proliferative capacity, followed by adipose tissue MSCs (A-MSC) and umbilical cord blood MSCs (U-MSC) which have the highest proliferative capacity [3]. MSCs also have differing differential potentials. For example, bone marrows MSCs have a higher chondrogenic potential while adipose MSCs have a lower chondrogenic potential [4, 5]. In addition both bone marrow and adipose MSCs readily form cells with adipogenic

cells, intestinal stem cells, and testicular stem cells.

to ESCs and iPSCs.

376 Pluripotent Stem Cells

**2. Mesenchymal stem cell therapy**

peripheral blood, breast milk, and the umbilical cord [1, 2].

M-MSCs are currently the most widely used stem cells in clinical trials and therapies. Both autograft and allograft M-MSCs have been extensively tested for their therapeutic safety and effectiveness in alleviating the symptoms of several diseases. One of the key reasons for the success of M-MSCs therapy is because these cells possess intrinsic immunomodulatory properties that enable M-MSCs to inhibit and evade potential immune rejection when transplanted [10]. M-MSCs are able to inhibit the maturation and function of various immune cells including dendritic cells, natural killer cells, B cells, and T lymphocytes [11]. Additionally M-MSCs have reduced immunogenicity due to their minimal expression of surface MHC ІІ proteins and the lack of T cell stimulatory proteins like CD80 and CD86 [12]. Another important reason for the early success of M-MSCs based therapies is that MSCs have low tumorigenic potential and are safer than therapies based on ESCs or iPSCs which display robust tumori‐ genicity [13].

Due to these intrinsic advantages of MSCs, clinical trials can be conducted to evaluate their safety and effectiveness in treating various diseases. For example, the safety and effectiveness of M-MSC transplantation for joint cartilage repair has been evaluated in several studies. In a clinical trial involving 41 patients studied over a period of between 5 to 137 months, M-MSC transplantation did not contribute to increased risk of tumors or infection [14]. Another study has reported the potential for M-MSC regenerative knee therapy to induce cartilage and meniscus growth and increase range of motion [15]. These results are supported in a larger scale M-MSC transplantation study involving 339 patients which reported no increased risk of tumor formation and a significant improvement of knee function in transplant patients [16]. While further clinical trials have to be conducted to verify these preliminary results, the successes of these initial clinical trials indicate that M-MSC therapy is likely to be safe and can catalyze cartilage repair.

The effectiveness of M-MSC and other MSC therapies for various autoimmune diseases has also been studied in several small clinical trials. These autoimmune diseases include multiple sclerosis, Crohn's disease, scleroderma, and systemic lupus erythematosus [17]. The causes of many of these autoimmune diseases are not well understood and it is likely that while the patients suffer from similar symptoms, contributing disease factors may vary significantly between patients. However the application of a generic MSC transplantation therapy was successful in alleviating the symptoms of these patients in several clinical trials.

Multiple sclerosis is a debilitating autoimmune disease caused by immune mediated damage of neural myelin sheath. Progressive neural damage results in many disabling symptoms including the loss of balance, vision and memory. M-MSC clinical trials for multiple sclerosis therapy have provided limited preliminary data indicating that M-MSC transplantation is safe, inhibits the progress of multiple sclerosis through immune regulated neuroprotection, and can repair limited damage to the CNS [18, 19]. For example, in a preliminary phase 2 clinical trial involving 10 patients diagnosed with progressive multiple sclerosis, autologous infusion of externally expanded M-MSCs was shown to improve visual acuity and increase optic nerve area without any major side-effects [18].

In the first clinical trial SLE patients were treated with allogeneic M-MSC infusion [24]. Treatment with donor M-MSCs was shown to restore kidney function and reverse the progression of SLE. The second clinical trial involved a larger group of patients and provided additional evidence that M-MSC therapy could mitigate the symptoms of SLE in patients [25].

Advances in Stem Cell Therapies http://dx.doi.org/10.5772/55061 379

Other studies have also attempted to verify the effectiveness of M-MSC therapies for various diseases. M-MSC therapy has been shown to improve liver function in patients suffering from liver cirrhosis by encouraging hepatocyte proliferation [26]. The co-transplantation of M-MSCs and kidney transplants for patients with kidney failure can reduce the risk of acute transplant rejection and improve transplant function in treated patients [27]. M-MSC therapy can also catalyze functional recovery and improve survival rates in ischemic stroke patients [28, 29]. These clinical studies provide preliminary evidence that M-MSC therapy is safe and the regenerative properties of these stem cells can be harnessed to treat a wide variety of diseases.

To develop the ideal next generation stem cell therapy, it is necessary to evaluate currently available therapies to identify their current limitations and suggest areas for improvement. Next generation stem cell therapies will have to fulfill the four key milestones (safety, immune evasion, location specificity, and disease relevancy) of customized stem cell therapy. Human M-MSC therapy has been extensively studied in multiple experiments and clinical trials and

Firstly the safety of M-MSC therapies must be considered. Multiple clinical trials mentioned previously involving the infusion and injection of both autologous and allogeneic M-MSCs for therapeutic purposes stated that patients were generally not exposed to increased risks of cancer or other serious side-effects. However, a study stated that the infusion of M-MSC may lead to pericardial calcification and increased risk of cardiac impairment in some patients [22]. As currently completed clinical trials often only involve a relatively small patient population or are only conducted over a brief period of time, the risks of M-MSC therapies may not be fully understood and more studies have to be conducted to ensure that the benefits of these therapies outweigh their potential risks. Another source of concern is the use of in vitro cultured M-MSCs in therapies. M-MSCs exist naturally in low concentrations in the human bone marrow, and often have to be concentrated and expanded in vitro media to provide sufficient numbers of stem cells for therapeutic purposes. This process may expose M-MSCs to xenogeneic antigens such as in fetal calf serum in the media. Culture of M-MSCs in vitro also exposes cells to an atmospheric oxygen concentration of 21% that is radically different from physiological conditions of 1-7% [30]. These in vitro culture conditions may affect the genetic and epigenetic stability of these stem cells resulting in an increased chance of muta‐ genesis. In an effort to resolve these potential issues, several studies have attempted to identify the ideal M-MSC culture media. From these studies, it has been proposed that human platelet lysate can be used as a viable substitute to fetal calf serum to reduce unnecessary exposure to xenogeneic antigens [31]. M-MSCs should also be cultured in low oxygen concentrations of approximately 3% to reduce oxidative stress and telomere shortening and increase the proliferative lifespan and genetic stability of in vitro M-MSCs [32]. The implementation of these protocols will provide M-MSCs with culture conditions that are more similar to the M-MSC native environment and minimize the impact of in vitro expansion on the genetic and

is an ideal candidate for evaluation against these key milestones.

Crohn's disease is a chronic autoimmune bowel disease characterized by inflammation of the gastrointestinal tract. In severe cases, this uncontrolled immune response may result in infection, hemorrhage, and intestinal fistulas. M-MSC clinical trials involving patients suffering from Crohn's disease have sought to harness the innate immunomodulatory capacity of MSCs to mitigate abnormal immune response in these patients and determine the safety of any potential therapies. In two phase one clinical trials a total of 22 adult Crohn's disease patients were enrolled to investigate the effects of M-MSC therapy. In the first trial it was determined that while autologous M-MSC infusion therapy did not result in adverse side effects, it only had a modest impact in alleviating the autoimmune response in these patients [20]. In the second trial in vitro expanded M-MSCs were directly injected into the intestinal wall and lumen [21]. When M-MSCs were directly injected, they were able to inhibit inflam‐ mation locally and mediate healing of intestinal tissue in these regions.

Scleroderma is an autoimmune connective tissue disorder characterized by accumulation of collagen in the skin, heart, kidneys or lungs. This buildup of collagen may lead to skin ulcers, pulmonary fibrosis, heart and kidney failure. Exploratory M-MSC clinical trials involving patients suffering from Scleroderma have sought to harness the regenerative and immuno‐ modulatory capacity of MSCs to initiate ulcer healing and prevent organ failure while evaluating the safety of these therapies. In two separate phase one clinical trials a total of 7 adult scleroderma patients were enrolled to determine the effects of M-MSC therapy. In the first trial allogeneic transplantation of donor M-MSC was performed via intravenous infusion and was associated with possible pericardial calcification and increased risk of cardiac impairment. While patients in this trial displayed a slight improvement in MRSS score and healing of skin ulcers, the effects were on occasion only temporary and the disease regressed in some patients [22]. The second trial involved autologous transplantation of either M-MSC or peripheral stem cells in patients via intramuscular injection [23]. This local stem cell therapy was able to induce healing of skin ulcers in these patients and improved endothelial function of blood vessels.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder that can affect the kidney, lung, brain, and other organs. Severe SLE may result in kidney failure, stroke, and inflammation of blood vessels. M-MSC clinical trials in SLE patients have attempted to treat progression of SLE symptoms by harnessing the immunomodulatory properties of MSCs. In two clinical trials a total of 19 patients suffering from SLE were treated with M-MSC transplants to determine if MSC therapy is safe and effective in reversing the symptoms of SLE patients. In the first clinical trial SLE patients were treated with allogeneic M-MSC infusion [24]. Treatment with donor M-MSCs was shown to restore kidney function and reverse the progression of SLE. The second clinical trial involved a larger group of patients and provided additional evidence that M-MSC therapy could mitigate the symptoms of SLE in patients [25].

Multiple sclerosis is a debilitating autoimmune disease caused by immune mediated damage of neural myelin sheath. Progressive neural damage results in many disabling symptoms including the loss of balance, vision and memory. M-MSC clinical trials for multiple sclerosis therapy have provided limited preliminary data indicating that M-MSC transplantation is safe, inhibits the progress of multiple sclerosis through immune regulated neuroprotection, and can repair limited damage to the CNS [18, 19]. For example, in a preliminary phase 2 clinical trial involving 10 patients diagnosed with progressive multiple sclerosis, autologous infusion of externally expanded M-MSCs was shown to improve visual acuity and increase optic nerve

Crohn's disease is a chronic autoimmune bowel disease characterized by inflammation of the gastrointestinal tract. In severe cases, this uncontrolled immune response may result in infection, hemorrhage, and intestinal fistulas. M-MSC clinical trials involving patients suffering from Crohn's disease have sought to harness the innate immunomodulatory capacity of MSCs to mitigate abnormal immune response in these patients and determine the safety of any potential therapies. In two phase one clinical trials a total of 22 adult Crohn's disease patients were enrolled to investigate the effects of M-MSC therapy. In the first trial it was determined that while autologous M-MSC infusion therapy did not result in adverse side effects, it only had a modest impact in alleviating the autoimmune response in these patients [20]. In the second trial in vitro expanded M-MSCs were directly injected into the intestinal wall and lumen [21]. When M-MSCs were directly injected, they were able to inhibit inflam‐

Scleroderma is an autoimmune connective tissue disorder characterized by accumulation of collagen in the skin, heart, kidneys or lungs. This buildup of collagen may lead to skin ulcers, pulmonary fibrosis, heart and kidney failure. Exploratory M-MSC clinical trials involving patients suffering from Scleroderma have sought to harness the regenerative and immuno‐ modulatory capacity of MSCs to initiate ulcer healing and prevent organ failure while evaluating the safety of these therapies. In two separate phase one clinical trials a total of 7 adult scleroderma patients were enrolled to determine the effects of M-MSC therapy. In the first trial allogeneic transplantation of donor M-MSC was performed via intravenous infusion and was associated with possible pericardial calcification and increased risk of cardiac impairment. While patients in this trial displayed a slight improvement in MRSS score and healing of skin ulcers, the effects were on occasion only temporary and the disease regressed in some patients [22]. The second trial involved autologous transplantation of either M-MSC or peripheral stem cells in patients via intramuscular injection [23]. This local stem cell therapy was able to induce healing of skin ulcers in these patients and improved endothelial function

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder that can affect the kidney, lung, brain, and other organs. Severe SLE may result in kidney failure, stroke, and inflammation of blood vessels. M-MSC clinical trials in SLE patients have attempted to treat progression of SLE symptoms by harnessing the immunomodulatory properties of MSCs. In two clinical trials a total of 19 patients suffering from SLE were treated with M-MSC transplants to determine if MSC therapy is safe and effective in reversing the symptoms of SLE patients.

mation locally and mediate healing of intestinal tissue in these regions.

area without any major side-effects [18].

378 Pluripotent Stem Cells

of blood vessels.

Other studies have also attempted to verify the effectiveness of M-MSC therapies for various diseases. M-MSC therapy has been shown to improve liver function in patients suffering from liver cirrhosis by encouraging hepatocyte proliferation [26]. The co-transplantation of M-MSCs and kidney transplants for patients with kidney failure can reduce the risk of acute transplant rejection and improve transplant function in treated patients [27]. M-MSC therapy can also catalyze functional recovery and improve survival rates in ischemic stroke patients [28, 29]. These clinical studies provide preliminary evidence that M-MSC therapy is safe and the regenerative properties of these stem cells can be harnessed to treat a wide variety of diseases.

To develop the ideal next generation stem cell therapy, it is necessary to evaluate currently available therapies to identify their current limitations and suggest areas for improvement. Next generation stem cell therapies will have to fulfill the four key milestones (safety, immune evasion, location specificity, and disease relevancy) of customized stem cell therapy. Human M-MSC therapy has been extensively studied in multiple experiments and clinical trials and is an ideal candidate for evaluation against these key milestones.

Firstly the safety of M-MSC therapies must be considered. Multiple clinical trials mentioned previously involving the infusion and injection of both autologous and allogeneic M-MSCs for therapeutic purposes stated that patients were generally not exposed to increased risks of cancer or other serious side-effects. However, a study stated that the infusion of M-MSC may lead to pericardial calcification and increased risk of cardiac impairment in some patients [22]. As currently completed clinical trials often only involve a relatively small patient population or are only conducted over a brief period of time, the risks of M-MSC therapies may not be fully understood and more studies have to be conducted to ensure that the benefits of these therapies outweigh their potential risks. Another source of concern is the use of in vitro cultured M-MSCs in therapies. M-MSCs exist naturally in low concentrations in the human bone marrow, and often have to be concentrated and expanded in vitro media to provide sufficient numbers of stem cells for therapeutic purposes. This process may expose M-MSCs to xenogeneic antigens such as in fetal calf serum in the media. Culture of M-MSCs in vitro also exposes cells to an atmospheric oxygen concentration of 21% that is radically different from physiological conditions of 1-7% [30]. These in vitro culture conditions may affect the genetic and epigenetic stability of these stem cells resulting in an increased chance of muta‐ genesis. In an effort to resolve these potential issues, several studies have attempted to identify the ideal M-MSC culture media. From these studies, it has been proposed that human platelet lysate can be used as a viable substitute to fetal calf serum to reduce unnecessary exposure to xenogeneic antigens [31]. M-MSCs should also be cultured in low oxygen concentrations of approximately 3% to reduce oxidative stress and telomere shortening and increase the proliferative lifespan and genetic stability of in vitro M-MSCs [32]. The implementation of these protocols will provide M-MSCs with culture conditions that are more similar to the M-MSC native environment and minimize the impact of in vitro expansion on the genetic and epigenetic stability of M-MSCs. In conclusion, while some doubts about the safety of M-MSC based therapies remain, various clinical trials and experiments have indicated that the use of minimally expanded M-MSCs is relatively safe for patients, especially when coupled with the latest M-MSC expansion protocols.

cure or alleviate various disease symptoms through their general immunomodulatory and regenerative properties. While non-specific M-MSCs may still be a viable therapy for a wide range of diseases, the lack of specificity in these therapies may result in potentially lethal consequences. For example, the general immunosuppressive properties of M-MSCs can increase the severity of breast cancer by increasing the concentration of regulatory T cells and inhibiting the innate immune response against cancer cells [35, 40]. M-MSCs could also secrete soluble factors that accelerate tumor growth, such as through the activation of the phosphati‐ dylinositol-3-kinase/Akt signaling pathway which can prevent apoptosis and induce prolif‐ eration of cancer cells [41]. Hence additional studies have to be performed to understand how stem cells can be epigenetically reprogrammed to enhance their specificity for disease treat‐

Advances in Stem Cell Therapies http://dx.doi.org/10.5772/55061 381

In conclusion, it can be seen that while current clinical data demonstrates that M-MSC based therapies are relatively safe and M-MSC transplants can evade immune detection and survive in patients, these therapies rely on the general immunosuppressive and regenerative proper‐ ties of M-MSCs and are neither specific nor disease relevant. Hence although the utilization of M-MSC based therapies may potentially result in cures for various diseases, more research is

While the bone marrow is the most commonly mentioned source of MSCs, MSCs can also be extracted from other sources including adipose and synovial tissue, skeletal muscles, periph‐ eral blood, breast milk, and the umbilical cord [1, 2]. In particular, adipose MSCs (A-MSCs) have been increasingly studied because these cells can be readily purified from adipose tissue via liposuction and is a relatively non-invasive procedure compared to bone marrow extraction of M-MSCs [42, 43]. A-MSCs have similar immunomodulatory effects compared to M-MSCs and can be utilized for treatment of similar diseases such as scleroderma [44, 45]. A-MSCs also possess a similar capacity to regenerate cartilage and bone tissues and mediate some symptoms in patients with osteonecrosis and osteoarthritis [46]. Hence the discovery of A-MSCs provides patients with an alternative source of MSCs in the event that they are unable to undergo M-

MSCs are also present in human and animal synovial fluid. These synovial MSCs (S-MSCs) have a greater ability to proliferate and differentiate compared to other MSCs and can form osteoblasts, adipocytes, chondrocytes, and neurons [47, 48]. S-MSCs also possess greater cartilage regenerative potential than other MSCs with 60% of S-MSCs placed on cartilage defects attaching to the defect within 10 minutes [49]. S-MSCs posses similar regenerative potential as M-MSCs and can also initiate regeneration of the nucleus pulposus in the damaged rabbit intervertebral disc by suppressing inflammation and inducing the synthesis of type ІІ collagen which acts as a supportive framework for nucleus pulposus repair [50]. In addition S-MSCs can be readily harvested via punch biopsy [9]. The greater innate proliferative ability of S-MSCs and the relative ease of obtaining S-MSCs indicate that it may be an excellent source

ment and reduce undesirable side effects.

MSC extraction.

of MSCs for future regenerative therapies

necessary for developing the ideal stem cell therapy.

**3. Other MSC and adult stem cell therapies**

Secondly the ability of M-MSCs to evade immune detection must be accessed. No incidents of acute immune rejection were reported in the various clinical trials involving autologous and allogeneic transplants of M-MSCs. This could possibly be attributed to the fact that autologous M-MSCs are extracted from the treated patients and present host cellular recognition markers. Additionally M-MSCs have reduced immunogenicity due to the naturally low expression levels of surface MHC ІІ proteins and the lack of other T cell stimulatory proteins like CD80 and CD 86 in M-MSCs. The multifaceted immunomodulatory capacity of M-MSCs must also be considered. Various studies have indicated that M-MSCs are able to inhibit the proliferation of T lymphocytes possibly via the activation of regulatory T cells and secretion of immuno‐ suppressive factors like transforming growth factor beta1 and hepatocyte growth factor [33-35]. The inhibition of T lymphocytes that are essential for the recognition and destruction of foreign transplants contributes to the ability of M-MSCs to evade immune detection. M-MSCs can also interfere with the development and function of antigen-presenting dendritic cells. Soluble factors secreted by M-MSCs can inhibit differentiation of monocytes to dendritic cells and suppress the production of cytokines [36]. M-MSCs can also affect the function of mature dendritic cells by suppressing the expression of various presentation and co-stimula‐ tory molecules like CD1a, CD80, CD83, and CD86 [37]. This impedes dendritic cells from inducing T cells and B cells and prevents resistance of foreign transplants from developing. Finally M-MSCs can also inhibit the proliferation of B cells stimulated with anti-CD40 monoclonal antibody and IL-4 by halting the G0/G1 cell cycle phase [38, 39]. The data from these studies indicate that M-MSCs are able to efficiently evade the innate immune response of patients via various mechanisms of cellular recognition and immunosuppression.

Thirdly the location specificity of M-MSCs employed in various therapies should be consid‐ ered. While M-MSC clinical trials discussed previously indicate that M-MSC therapy is able to alleviate the conditions of various autoimmune diseases and induce cartilage repair, infusion of M-MSCs resulted in non-specific distribution of these cells within the patient. Nonspecific infusion of M-MSCs resulted in a distribution of these cells in various organs including the heart muscle, liver, kidney, skin, and lung. This may result in undesirable side-effects such as pericardial calcification and increased risk of cardiac impairment in patients as described in a clinical study [22]. Hence further research is required to design a stem cell therapy that is more specific to the injury location. In the ideal therapy, stem cells could be engineered with receptors for mobilization to the location of injury. Alternatively, stem cells could be integrated within a scaffold that would then be implanted into patients to improve the specificity of these therapies. The direct injection of M-MSCs near sites of injury may also provide increased specificity to these therapies.

The fourth consideration is stem cells utilized in these therapies should be disease relevant. Disease relevant stem cells should be epigenetically primed to treat specific underlying causes of disease in each patient. M-MSCs utilized in these clinical trials are not disease relevant and cure or alleviate various disease symptoms through their general immunomodulatory and regenerative properties. While non-specific M-MSCs may still be a viable therapy for a wide range of diseases, the lack of specificity in these therapies may result in potentially lethal consequences. For example, the general immunosuppressive properties of M-MSCs can increase the severity of breast cancer by increasing the concentration of regulatory T cells and inhibiting the innate immune response against cancer cells [35, 40]. M-MSCs could also secrete soluble factors that accelerate tumor growth, such as through the activation of the phosphati‐ dylinositol-3-kinase/Akt signaling pathway which can prevent apoptosis and induce prolif‐ eration of cancer cells [41]. Hence additional studies have to be performed to understand how stem cells can be epigenetically reprogrammed to enhance their specificity for disease treat‐ ment and reduce undesirable side effects.

In conclusion, it can be seen that while current clinical data demonstrates that M-MSC based therapies are relatively safe and M-MSC transplants can evade immune detection and survive in patients, these therapies rely on the general immunosuppressive and regenerative proper‐ ties of M-MSCs and are neither specific nor disease relevant. Hence although the utilization of M-MSC based therapies may potentially result in cures for various diseases, more research is necessary for developing the ideal stem cell therapy.

## **3. Other MSC and adult stem cell therapies**

epigenetic stability of M-MSCs. In conclusion, while some doubts about the safety of M-MSC based therapies remain, various clinical trials and experiments have indicated that the use of minimally expanded M-MSCs is relatively safe for patients, especially when coupled with the

Secondly the ability of M-MSCs to evade immune detection must be accessed. No incidents of acute immune rejection were reported in the various clinical trials involving autologous and allogeneic transplants of M-MSCs. This could possibly be attributed to the fact that autologous M-MSCs are extracted from the treated patients and present host cellular recognition markers. Additionally M-MSCs have reduced immunogenicity due to the naturally low expression levels of surface MHC ІІ proteins and the lack of other T cell stimulatory proteins like CD80 and CD 86 in M-MSCs. The multifaceted immunomodulatory capacity of M-MSCs must also be considered. Various studies have indicated that M-MSCs are able to inhibit the proliferation of T lymphocytes possibly via the activation of regulatory T cells and secretion of immuno‐ suppressive factors like transforming growth factor beta1 and hepatocyte growth factor [33-35]. The inhibition of T lymphocytes that are essential for the recognition and destruction of foreign transplants contributes to the ability of M-MSCs to evade immune detection. M-MSCs can also interfere with the development and function of antigen-presenting dendritic cells. Soluble factors secreted by M-MSCs can inhibit differentiation of monocytes to dendritic cells and suppress the production of cytokines [36]. M-MSCs can also affect the function of mature dendritic cells by suppressing the expression of various presentation and co-stimula‐ tory molecules like CD1a, CD80, CD83, and CD86 [37]. This impedes dendritic cells from inducing T cells and B cells and prevents resistance of foreign transplants from developing. Finally M-MSCs can also inhibit the proliferation of B cells stimulated with anti-CD40 monoclonal antibody and IL-4 by halting the G0/G1 cell cycle phase [38, 39]. The data from these studies indicate that M-MSCs are able to efficiently evade the innate immune response

of patients via various mechanisms of cellular recognition and immunosuppression.

Thirdly the location specificity of M-MSCs employed in various therapies should be consid‐ ered. While M-MSC clinical trials discussed previously indicate that M-MSC therapy is able to alleviate the conditions of various autoimmune diseases and induce cartilage repair, infusion of M-MSCs resulted in non-specific distribution of these cells within the patient. Nonspecific infusion of M-MSCs resulted in a distribution of these cells in various organs including the heart muscle, liver, kidney, skin, and lung. This may result in undesirable side-effects such as pericardial calcification and increased risk of cardiac impairment in patients as described in a clinical study [22]. Hence further research is required to design a stem cell therapy that is more specific to the injury location. In the ideal therapy, stem cells could be engineered with receptors for mobilization to the location of injury. Alternatively, stem cells could be integrated within a scaffold that would then be implanted into patients to improve the specificity of these therapies. The direct injection of M-MSCs near sites of injury may also provide increased

The fourth consideration is stem cells utilized in these therapies should be disease relevant. Disease relevant stem cells should be epigenetically primed to treat specific underlying causes of disease in each patient. M-MSCs utilized in these clinical trials are not disease relevant and

latest M-MSC expansion protocols.

380 Pluripotent Stem Cells

specificity to these therapies.

While the bone marrow is the most commonly mentioned source of MSCs, MSCs can also be extracted from other sources including adipose and synovial tissue, skeletal muscles, periph‐ eral blood, breast milk, and the umbilical cord [1, 2]. In particular, adipose MSCs (A-MSCs) have been increasingly studied because these cells can be readily purified from adipose tissue via liposuction and is a relatively non-invasive procedure compared to bone marrow extraction of M-MSCs [42, 43]. A-MSCs have similar immunomodulatory effects compared to M-MSCs and can be utilized for treatment of similar diseases such as scleroderma [44, 45]. A-MSCs also possess a similar capacity to regenerate cartilage and bone tissues and mediate some symptoms in patients with osteonecrosis and osteoarthritis [46]. Hence the discovery of A-MSCs provides patients with an alternative source of MSCs in the event that they are unable to undergo M-MSC extraction.

MSCs are also present in human and animal synovial fluid. These synovial MSCs (S-MSCs) have a greater ability to proliferate and differentiate compared to other MSCs and can form osteoblasts, adipocytes, chondrocytes, and neurons [47, 48]. S-MSCs also possess greater cartilage regenerative potential than other MSCs with 60% of S-MSCs placed on cartilage defects attaching to the defect within 10 minutes [49]. S-MSCs posses similar regenerative potential as M-MSCs and can also initiate regeneration of the nucleus pulposus in the damaged rabbit intervertebral disc by suppressing inflammation and inducing the synthesis of type ІІ collagen which acts as a supportive framework for nucleus pulposus repair [50]. In addition S-MSCs can be readily harvested via punch biopsy [9]. The greater innate proliferative ability of S-MSCs and the relative ease of obtaining S-MSCs indicate that it may be an excellent source of MSCs for future regenerative therapies

Neural Stem Cells (NSCs) have also been studied in an attempt to harness their regenerative potential for therapeutic purposes. NSCs can be found in various tissues including the bone marrow and striatum [51, 52] and their regenerative properties have been assessed by both NSC transplantation and endogenous NSC functional studies. NSCs can initiate axon remye‐ lination, neuroprotection, proliferation of oligodendrocyte progenitors, and functional recovery when transplanted into mice experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis [53]. NSCs also possess similar immunomodulatory properties as MSCs. For example, NSCs can inhibit dendritic cell and antigen-specific T cell maturation through the release of morphogens such as bone morphogenetic protein 4 [54]. Additionally, NSCs can suppress T-cell proliferation through the release of prostaglandin E2 and nitric oxide [55]. This innate immunomodulatory property of NSCs has been harnessed to induce stable pancreatic islet graft function in mice, without the need for long-term immunosuppression [56]. The immunosuppressive potential of NSC can also be enhanced by engineering NSCs to produce anti-inflammatory cytokines such as IL-10 [57]. Engineered NSC transplants have greater therapeutic potential than ordinary NSCs and give rise to enhanced functional recovery of EAE mice.

studies have revealed that the over-expression of *SKP2* can induce proliferation of non-sensory cells that can differentiate to form hair cells through the co-expression of *Atoh1* [67]. Develop‐ mental studies have also provided insight into the *Notch* signaling pathway, and its influence on the lateral-inhibition mediation differentiation of hair cells [68, 69]. Further studies could lead to the development of a viable stem cell therapy for regenerating auditory hair cells and

Advances in Stem Cell Therapies http://dx.doi.org/10.5772/55061 383

Mammary stem cells are indispensible in the formation of mammary glands and can possess the capacity to form myoepithelial cells, alveolar epithelial cells, and ductal epithelial cells [70, 71]. The deregulation of various signaling pathways including the Notch, Wnt, and Hedgehog pathways in mammary stem cells has been implicated in breast cancer development [71, 72]. These studies could lead to the development of anti-cancer drugs that target specific signaling

Intestinal stem cells are multipotent progenitors of the intestinal epithelial cell lineages. Studies of intestinal stem cells have revealed the role of the Notch and Wnt signaling pathways in intestinal stem cell maintenance, differentiation, and proliferation and how deregulation of these pathways can promote intestinal carcinogenesis [73, 74]. An impaired differential capacity of intestinal stem cells has also been linked to inflammatory bowel diseases like Crohn's disease and ulcerative colitis [75]. Future studies based on these discoveries could lead

Adult germline stem cells are essential for gamete generation and can be derived from testis spermatogonial cells. These stem cells are pluripotent and share characteristics similar to ESCs [76]. The pluripotent nature of these stem cells may allow the development of regenerative therapies not possible with other multipotent adult stem cells. Adult germline stem cell transplantation can also be utilized for fertility restoration in animals [77-79]. This regenerative ability could be utilized for maintaining the fertility of patients undergoing radiotherapy,

A comparison to determine how epigenetic differences inherent to these different classes of adult stem cells lead to a wide variation in differentiation, homing, proliferation, and immu‐ nomodulation capacities will enable the design of novel stem cell therapies for specific diseases. The differentiation potentials of adult stem cells can vary widely, for instance hematopoietic stem cells tend to form cells from the lymphoid and myeloid lineages, while neural stem cells tend to form neural cells like neurons, oligodendrocytes and astrocytes. Differences in differential predisposition also exist within a similar class of stem cells. A-MSCs tend to form adipocytes and cardiomyocytes, while M-MSCs form chondrocytes more readily [4, 5]. Further studies to map epigenetic differences between these stem cell populations will reveal how differentiation is regulated. This will lead to an improved ability to prime and select optimal stem cell transplants for disease therapy. For example, a better understanding of underlying pro-chondrogenic factors will enable the engineering of stem cells specialized in cartilage regeneration. Detailed studies of other intra-population epigenetic variations will also lead to better understanding of how these differences lead to differences in other prop‐ erties of stem cells and augment the safety, effectiveness, and specificity of stem cell therapies. In conclusion, it can be seen that while the safety of adult stem cell therapies remains a key

a cure for hearing impairment.

to more effective cures for these diseases.

chemotherapy and other therapies that may cause infertility.

pathways.

Functional studies of endogenous NSCs have revealed the complex regulatory pathways governing in vivo neuronal regeneration. While neural stem cell niches exist in the subven‐ tricular zone and the subgranular zone of the hippocampal dentate gyrus [58], NSCs in these niches are unable to initiate spontaneous neural regeneration in many diseases. Hence recent research has been concerned with elucidating the regulators of neurogenesis and repair. For example neurogenesis can be initiated by suppressing Olig2 resulting in increased neurogen‐ esis for brain injury repair [59]. Other molecular regulators of neurogenesis include morpho‐ gens like Shh and Wnt, transcription factors like Sox2, growth factors like Fibroblast Growth Factor family, and cell surface molecules like Notch1 [60]. An improved understanding of the molecular pathways that regulate the differentiation, mobilization, and proliferation of endogenous NSCs and the development of molecular tools to manipulate these pathways may lead to the development of novel minimally invasive regenerative therapies.

Other adult stem cells that have been evaluated for therapeutic use include hematopoietic stem cells, inner ear stem cells, mammary stem cells, intestinal stem cells, and adult germline stem cells. Hematopoietic stem cells are multipotent and can form various blood cells such as those from the lymphoid and myeloid lineages. Allogeneic hematopoietic stem cell transplantation (HSCT) therapy in leukemia patients can lead to remission by inducing an immune antitumor response [61]. HSCT has also been utilized to cure other diseases including sickle cell anemia, acquired aplastic anemia and thalassemia [62, 63]. HSCT can also halt neurological deteriora‐ tion in X-linked adrenoleukodystrophy patients [64]. HSCT is also useful for alleviating symptoms of Hurler Syndrome and other lysosomal storage diseases and these grafts can replace metabolic enzymes that are lacking in host cells.

Inner ear stem cells are important progenitors of auditory hair cells and exist endogenously in both the utricular sensory epithelium and the dorsal epithelium of the cochlear canal [65, 66]. An improved understanding of molecular regulatory pathways in these stem cells could lead to the development of regenerative therapies for treating hearing impairment. Ongoing studies have revealed that the over-expression of *SKP2* can induce proliferation of non-sensory cells that can differentiate to form hair cells through the co-expression of *Atoh1* [67]. Develop‐ mental studies have also provided insight into the *Notch* signaling pathway, and its influence on the lateral-inhibition mediation differentiation of hair cells [68, 69]. Further studies could lead to the development of a viable stem cell therapy for regenerating auditory hair cells and a cure for hearing impairment.

Neural Stem Cells (NSCs) have also been studied in an attempt to harness their regenerative potential for therapeutic purposes. NSCs can be found in various tissues including the bone marrow and striatum [51, 52] and their regenerative properties have been assessed by both NSC transplantation and endogenous NSC functional studies. NSCs can initiate axon remye‐ lination, neuroprotection, proliferation of oligodendrocyte progenitors, and functional recovery when transplanted into mice experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis [53]. NSCs also possess similar immunomodulatory properties as MSCs. For example, NSCs can inhibit dendritic cell and antigen-specific T cell maturation through the release of morphogens such as bone morphogenetic protein 4 [54]. Additionally, NSCs can suppress T-cell proliferation through the release of prostaglandin E2 and nitric oxide [55]. This innate immunomodulatory property of NSCs has been harnessed to induce stable pancreatic islet graft function in mice, without the need for long-term immunosuppression [56]. The immunosuppressive potential of NSC can also be enhanced by engineering NSCs to produce anti-inflammatory cytokines such as IL-10 [57]. Engineered NSC transplants have greater therapeutic potential than ordinary NSCs and give rise to enhanced functional recovery

Functional studies of endogenous NSCs have revealed the complex regulatory pathways governing in vivo neuronal regeneration. While neural stem cell niches exist in the subven‐ tricular zone and the subgranular zone of the hippocampal dentate gyrus [58], NSCs in these niches are unable to initiate spontaneous neural regeneration in many diseases. Hence recent research has been concerned with elucidating the regulators of neurogenesis and repair. For example neurogenesis can be initiated by suppressing Olig2 resulting in increased neurogen‐ esis for brain injury repair [59]. Other molecular regulators of neurogenesis include morpho‐ gens like Shh and Wnt, transcription factors like Sox2, growth factors like Fibroblast Growth Factor family, and cell surface molecules like Notch1 [60]. An improved understanding of the molecular pathways that regulate the differentiation, mobilization, and proliferation of endogenous NSCs and the development of molecular tools to manipulate these pathways may

Other adult stem cells that have been evaluated for therapeutic use include hematopoietic stem cells, inner ear stem cells, mammary stem cells, intestinal stem cells, and adult germline stem cells. Hematopoietic stem cells are multipotent and can form various blood cells such as those from the lymphoid and myeloid lineages. Allogeneic hematopoietic stem cell transplantation (HSCT) therapy in leukemia patients can lead to remission by inducing an immune antitumor response [61]. HSCT has also been utilized to cure other diseases including sickle cell anemia, acquired aplastic anemia and thalassemia [62, 63]. HSCT can also halt neurological deteriora‐ tion in X-linked adrenoleukodystrophy patients [64]. HSCT is also useful for alleviating symptoms of Hurler Syndrome and other lysosomal storage diseases and these grafts can

Inner ear stem cells are important progenitors of auditory hair cells and exist endogenously in both the utricular sensory epithelium and the dorsal epithelium of the cochlear canal [65, 66]. An improved understanding of molecular regulatory pathways in these stem cells could lead to the development of regenerative therapies for treating hearing impairment. Ongoing

lead to the development of novel minimally invasive regenerative therapies.

replace metabolic enzymes that are lacking in host cells.

of EAE mice.

382 Pluripotent Stem Cells

Mammary stem cells are indispensible in the formation of mammary glands and can possess the capacity to form myoepithelial cells, alveolar epithelial cells, and ductal epithelial cells [70, 71]. The deregulation of various signaling pathways including the Notch, Wnt, and Hedgehog pathways in mammary stem cells has been implicated in breast cancer development [71, 72]. These studies could lead to the development of anti-cancer drugs that target specific signaling pathways.

Intestinal stem cells are multipotent progenitors of the intestinal epithelial cell lineages. Studies of intestinal stem cells have revealed the role of the Notch and Wnt signaling pathways in intestinal stem cell maintenance, differentiation, and proliferation and how deregulation of these pathways can promote intestinal carcinogenesis [73, 74]. An impaired differential capacity of intestinal stem cells has also been linked to inflammatory bowel diseases like Crohn's disease and ulcerative colitis [75]. Future studies based on these discoveries could lead to more effective cures for these diseases.

Adult germline stem cells are essential for gamete generation and can be derived from testis spermatogonial cells. These stem cells are pluripotent and share characteristics similar to ESCs [76]. The pluripotent nature of these stem cells may allow the development of regenerative therapies not possible with other multipotent adult stem cells. Adult germline stem cell transplantation can also be utilized for fertility restoration in animals [77-79]. This regenerative ability could be utilized for maintaining the fertility of patients undergoing radiotherapy, chemotherapy and other therapies that may cause infertility.

A comparison to determine how epigenetic differences inherent to these different classes of adult stem cells lead to a wide variation in differentiation, homing, proliferation, and immu‐ nomodulation capacities will enable the design of novel stem cell therapies for specific diseases. The differentiation potentials of adult stem cells can vary widely, for instance hematopoietic stem cells tend to form cells from the lymphoid and myeloid lineages, while neural stem cells tend to form neural cells like neurons, oligodendrocytes and astrocytes. Differences in differential predisposition also exist within a similar class of stem cells. A-MSCs tend to form adipocytes and cardiomyocytes, while M-MSCs form chondrocytes more readily [4, 5]. Further studies to map epigenetic differences between these stem cell populations will reveal how differentiation is regulated. This will lead to an improved ability to prime and select optimal stem cell transplants for disease therapy. For example, a better understanding of underlying pro-chondrogenic factors will enable the engineering of stem cells specialized in cartilage regeneration. Detailed studies of other intra-population epigenetic variations will also lead to better understanding of how these differences lead to differences in other prop‐ erties of stem cells and augment the safety, effectiveness, and specificity of stem cell therapies. In conclusion, it can be seen that while the safety of adult stem cell therapies remains a key concern especially in the less studied stem cells, the innate immunomodulatory and regener‐ ative capacity of adult stem cells can be exploited for curing a wide range of diseases.

efficiency. High atmospheric oxygen concentration (~21%) exposes stem cells to increased oxidative stress and DNA damage [32]. Lowering the oxygen concentration to 5% can improve iPSC generation efficiency and genetic stability of stem cells [96]. The addition of vitamin C and other antioxidants can also improve the efficiency of iPSC generation by preventing the accumulation of reactive oxygen species and promoting epigenetic modifications required forreprogramming to occur[97]. Reprogramming and iPSC culture maintenance also requires precise manipulation of other medium conditions. iPSC progenitors have to be cultivated in conditions that facilitate their survival but these original conditions may have to be modi‐ fied to enhance reprogramming efficiency and maintain iPSC populations [98, 99]. The search for improving the efficiency of iPSC generation has also led to the use of miRNA sequen‐ ces in reprogramming. Viral aided *miR302/367* cell reprogramming can reprogram fibro‐

Advances in Stem Cell Therapies http://dx.doi.org/10.5772/55061 385

The reprogramming of adult somatic cells to ideal iPSCs will involve a complex epigenomic transformation of the cellular epigenome to resemble ESC epigenetics. However iPSCs derived with current procedures retain unique epigenetic signatures that differ from the ESC epige‐ nome. Some common epigenetic differences include variations in DNA methylation at CpG islands and histone modifications [101, 102]. The epigenetic memory of iPSCs is an artifact from the reprogramming process and parental cell epigenetics and can affect the differentiation predisposition of iPSCs [90]. This iPSC epigenetic signature can also be transmitted to successive generations of iPSCs and their differentiated progeny [102]. A failure to reset the epigenetic memory of iPSCs to more closely resemble the epigenetic ground state of ESCs could

affect the function and safety of differentiated cells derived from these iPSC lines.

adult somatic cells to mirror this ideal epigenomic ground state.

The issue of residual iPSC epigenetic memory can be partially addressed. For example, somatic cell nuclear transfer has been proposed as a viable method for resetting epigenetic memory [91]. Sodium butyrate, a short-chain fatty acid, could also assist in programming iPSCs closer to the epigenetic ground state by inhibiting histone deacetylase, directing the acetylation of specific genes, and encouraging stem cell renewal [103, 104]. DNA methyltransferase inhibitors can also be used to direct DNA methylation at specific sites for more complete epigenetic reprogramming [105]. The iPSC culture environment can also be manipulated to achieve a desired epigenetic state. For example, reducing culture oxygen concentration to 2% can induce epigenetic modifications that increase the expression of the retinal genes *Six3* and *Lhx2* in iPSCs while an oxygen concentration of 5% can increase the efficiency of iPSC regeneration [96, 106]. However despite the increased availability of tools for epigenetic modification, more studies are required to determine the ideal epigenomic ground state for therapeutic stem cells. Comprehensive epigenetic mapping of ESCs and adult stem cells could provide important clues and enable the development of improved experimental procedures for reprogramming

When evaluated against the four key milestones, iPSCs are clearly inferior to M-MSCs and other adult stem cells currently being evaluated in experimental therapies. First, iPSCs are neither genetically nor epigenetically stable, this inherent property of iPSCs, along with the integration of proto-oncogenes as a byproduct of some iPSC reprogramming procedures results in increased propensity for tumorigenesis in vivo. Hence the safety of iPSC based

blasts to iPSCs with up to 10% efficiency [100].

## **4. Induced pluripotent stem cells**

The discovery of iPSCs has led to a revolution in stem cell research. The ability to reprogram adult somatic cells to iPSCs using an increasing array of novel vectors and strategies has opened up a myriad of possibilities for therapeutic stem cell development. iPSC based therapies possess several advantages over adult stem cell and ESC based therapies. First, since iPSCs can be derived from patients like adult stem cells, they will exhibit host cellular recognition markers and can evade immune rejection more readily than ESCs. Additionally since iPSC lines can be derived from patients, they do not face the ethical concerns associated with ESC derivation. The use of iPSCs is also advantageous because iPSCs can be modified to produce desired cell phenotypes that may not be naturally available in adult stem cell and ESC populations. Hence iPSCs can be customized for treating specific diseases unlike other stem cells whose curative properties tend to be more general.

However currently available iPSCs face several limitations that prevent their use in patient therapies. First the iPSC derivation process commonly involves the use of viral vectors such as lentiviral and retroviral vectors which results in the integration of viral DNA in iPSCs [80, 81]. Second, many iPSC derivation processes involve the over-expression or integration of proto-oncogenes such as *Oct4*, *c-Myc*, and *Sox2* [82-86]. Third, iPSC cultures are genetically unstable and contain numerous genetic abnormalities including protein coding mutations, copy number variations, and chromosomal aberrations [87-89]. Fourth, the iPSC reprogram‐ ming process may be incomplete and iPSCs can retain epigenetic memory from parental somatic cells [90, 91]. Finally, the transformation efficiency of adult somatic cells to iPSCs is inefficient (0.001% - 4.4%) and only a small fraction of adult somatic cells can be transformed to iPSCs via existing methods.

These current shortcomings hinder the development of iPSCs suitable for patient therapy. The use of viral reprogramming vectors, over-expression of proto-oncogenes, and suboptimal culture conditions contribute to widespread genetic mutation and increased tumorigenic potential in iPSCs. Consequently, iPSCs can readily form tumors in immune deficient mice and mice derived from iPSCs have a high chance of developing tumors [82, 92]. To overcome these limitations, new methods for iPSC reprogramming were developed to enhance the genetic integrity of iPSCs. Advances in reprogramming enabled the genera‐ tion of iPSCs without *c-Myc* and mice without tumors could be derived from these iPSCs [93]. Additionally, non-integrating viral vectors like adenoviruses were used to prevent the introduction of foreign viral DNA into iPSCs [94]. More recently iPSCs have been generat‐ ed via transfection of modified mRNA, this DNA free method results in a higher efficiency of iPSC generation compared to previous methods and does not introduce any exogenous DNA into reprogrammed cells [95]. Optimized iPSC culture and reprogramming condi‐ tions will also be essential for maintaining genetic stability and increasing transformation efficiency. High atmospheric oxygen concentration (~21%) exposes stem cells to increased oxidative stress and DNA damage [32]. Lowering the oxygen concentration to 5% can improve iPSC generation efficiency and genetic stability of stem cells [96]. The addition of vitamin C and other antioxidants can also improve the efficiency of iPSC generation by preventing the accumulation of reactive oxygen species and promoting epigenetic modifications required forreprogramming to occur[97]. Reprogramming and iPSC culture maintenance also requires precise manipulation of other medium conditions. iPSC progenitors have to be cultivated in conditions that facilitate their survival but these original conditions may have to be modi‐ fied to enhance reprogramming efficiency and maintain iPSC populations [98, 99]. The search for improving the efficiency of iPSC generation has also led to the use of miRNA sequen‐ ces in reprogramming. Viral aided *miR302/367* cell reprogramming can reprogram fibro‐ blasts to iPSCs with up to 10% efficiency [100].

concern especially in the less studied stem cells, the innate immunomodulatory and regener‐

The discovery of iPSCs has led to a revolution in stem cell research. The ability to reprogram adult somatic cells to iPSCs using an increasing array of novel vectors and strategies has opened up a myriad of possibilities for therapeutic stem cell development. iPSC based therapies possess several advantages over adult stem cell and ESC based therapies. First, since iPSCs can be derived from patients like adult stem cells, they will exhibit host cellular recognition markers and can evade immune rejection more readily than ESCs. Additionally since iPSC lines can be derived from patients, they do not face the ethical concerns associated with ESC derivation. The use of iPSCs is also advantageous because iPSCs can be modified to produce desired cell phenotypes that may not be naturally available in adult stem cell and ESC populations. Hence iPSCs can be customized for treating specific diseases unlike other stem

However currently available iPSCs face several limitations that prevent their use in patient therapies. First the iPSC derivation process commonly involves the use of viral vectors such as lentiviral and retroviral vectors which results in the integration of viral DNA in iPSCs [80, 81]. Second, many iPSC derivation processes involve the over-expression or integration of proto-oncogenes such as *Oct4*, *c-Myc*, and *Sox2* [82-86]. Third, iPSC cultures are genetically unstable and contain numerous genetic abnormalities including protein coding mutations, copy number variations, and chromosomal aberrations [87-89]. Fourth, the iPSC reprogram‐ ming process may be incomplete and iPSCs can retain epigenetic memory from parental somatic cells [90, 91]. Finally, the transformation efficiency of adult somatic cells to iPSCs is inefficient (0.001% - 4.4%) and only a small fraction of adult somatic cells can be transformed

These current shortcomings hinder the development of iPSCs suitable for patient therapy. The use of viral reprogramming vectors, over-expression of proto-oncogenes, and suboptimal culture conditions contribute to widespread genetic mutation and increased tumorigenic potential in iPSCs. Consequently, iPSCs can readily form tumors in immune deficient mice and mice derived from iPSCs have a high chance of developing tumors [82, 92]. To overcome these limitations, new methods for iPSC reprogramming were developed to enhance the genetic integrity of iPSCs. Advances in reprogramming enabled the genera‐ tion of iPSCs without *c-Myc* and mice without tumors could be derived from these iPSCs [93]. Additionally, non-integrating viral vectors like adenoviruses were used to prevent the introduction of foreign viral DNA into iPSCs [94]. More recently iPSCs have been generat‐ ed via transfection of modified mRNA, this DNA free method results in a higher efficiency of iPSC generation compared to previous methods and does not introduce any exogenous DNA into reprogrammed cells [95]. Optimized iPSC culture and reprogramming condi‐ tions will also be essential for maintaining genetic stability and increasing transformation

ative capacity of adult stem cells can be exploited for curing a wide range of diseases.

**4. Induced pluripotent stem cells**

384 Pluripotent Stem Cells

cells whose curative properties tend to be more general.

to iPSCs via existing methods.

The reprogramming of adult somatic cells to ideal iPSCs will involve a complex epigenomic transformation of the cellular epigenome to resemble ESC epigenetics. However iPSCs derived with current procedures retain unique epigenetic signatures that differ from the ESC epige‐ nome. Some common epigenetic differences include variations in DNA methylation at CpG islands and histone modifications [101, 102]. The epigenetic memory of iPSCs is an artifact from the reprogramming process and parental cell epigenetics and can affect the differentiation predisposition of iPSCs [90]. This iPSC epigenetic signature can also be transmitted to successive generations of iPSCs and their differentiated progeny [102]. A failure to reset the epigenetic memory of iPSCs to more closely resemble the epigenetic ground state of ESCs could affect the function and safety of differentiated cells derived from these iPSC lines.

The issue of residual iPSC epigenetic memory can be partially addressed. For example, somatic cell nuclear transfer has been proposed as a viable method for resetting epigenetic memory [91]. Sodium butyrate, a short-chain fatty acid, could also assist in programming iPSCs closer to the epigenetic ground state by inhibiting histone deacetylase, directing the acetylation of specific genes, and encouraging stem cell renewal [103, 104]. DNA methyltransferase inhibitors can also be used to direct DNA methylation at specific sites for more complete epigenetic reprogramming [105]. The iPSC culture environment can also be manipulated to achieve a desired epigenetic state. For example, reducing culture oxygen concentration to 2% can induce epigenetic modifications that increase the expression of the retinal genes *Six3* and *Lhx2* in iPSCs while an oxygen concentration of 5% can increase the efficiency of iPSC regeneration [96, 106]. However despite the increased availability of tools for epigenetic modification, more studies are required to determine the ideal epigenomic ground state for therapeutic stem cells. Comprehensive epigenetic mapping of ESCs and adult stem cells could provide important clues and enable the development of improved experimental procedures for reprogramming adult somatic cells to mirror this ideal epigenomic ground state.

When evaluated against the four key milestones, iPSCs are clearly inferior to M-MSCs and other adult stem cells currently being evaluated in experimental therapies. First, iPSCs are neither genetically nor epigenetically stable, this inherent property of iPSCs, along with the integration of proto-oncogenes as a byproduct of some iPSC reprogramming procedures results in increased propensity for tumorigenesis in vivo. Hence the safety of iPSC based therapies remains a key concern and must be resolved before they can be tested in clinical therapies. Second, while iPSCs can be derived from patients and should be able to evade patient immune response, abnormal expression of genes in iPSCs and their differentiated progeny has been shown to induce immune responses in recipients [107]. A possible consequence of the genetic and epigenetic instability, the inability of iPSCs to evade the innate immune response of patients could lead to the rejection of iPSC grafts. Similarly, genomic and epigenetic instability of iPSCs will frustrate efforts for developing iPSCs with specific function and homing abilities. Hence more research is required before iPSCs suitable for use in patient therapies will be available.

that human ESC transplantation can rescue animal models of retinal degeneration, Stargardt, and spinal cord injury, and catalyze limited visual improvement in human macular degener‐

Advances in Stem Cell Therapies http://dx.doi.org/10.5772/55061 387

Despite these apparent advantages of human ESCs, its use in research and medical therapy has been fairly controversial historically as the derivation of human ESC lines requires the destruction of human embryos. Pro-life advocates have strongly opposed the destruction of embryos for research on the basis that human life begins when a human egg cell is fertilized, and the belief that human life is inviolable. More recently, these objections have been partially overcome through the development of human ESC derivation procedures that do not require embryo destruction [116] and the use of surplus frozen embryos from in vitro fertilization

When evaluated against the four key milestones, it can be determined that while ESCs are potentially safer than iPSCs, several key concerns continue to forestall their wider use in human clinical trials. First while some studies of human ESC based transplants in animals and humans have suggested that there is minimal risk of teratoma formation or uncontrolled proliferation of transplanted cells, other studies contend that *in vitro* culture conditions can result in potentially hazardous epigenetic modifications [117]. Second since ESC transplants are allogeneic, there is a higher likelihood of immune rejection compared to autographs of adult stem cells. Third ESC transplants potentially share similar location specificity limitations as adult stem cell transplants. This limitation has to be addressed for the development of a viable next generation stem cell therapy. Fourth since ESCs have a higher innate pluripotent capacity than adult stem cells and iPSCs, it may be easier to obtain pure populations of functionally specific and disease relevant transplant cells from ESC lines. In conclusion, while more clinical trials will be required to assess the viability of ESC therapy, studies have indicated that ESC therapy seems to offer a promising alternative for treating currently incurable

Transdifferentiation is the direct conversion of one cell type to another without the involve‐ ment of an intermediate pluripotent state. Transdifferentiation could be a viable alternative therapy to stem cell therapies and relatively abundant adult somatic cells like fibroblasts and adipocytes could be harvested from patients and directly converted by transdifferentiation to neurons or cardiomyocytes before being used as autologous grafts in regenerative therapies. Transdifferentiation is advantageous compared to adult stem cell therapy because cell grafts could be designed specifically for each disease therapy resulting in improved functional and positional specificity. Transdifferentiation is also advantageous compared to iPSC based therapy because conversion to a desired cell type is a one step process requiring lesser epigenetic modification, and is a more rapid and direct process than dedifferentiation to form

ation patients.

clinics.

diseases.

**6. The promise of transdifferentiation therapy**

iPSCs followed by controlled differentiation into the desired cell type.

While current limitations of iPSC technology forestalls their direct use in patient therapy, the versatility of iPSCs and their ease of derivation from patients has enabled their use in disease modeling and in vitro drug screening. iPSCs can be derived from patients affected by various diseases including LEOPARD syndrome, Schizophrenia, and X-linked adrenoleukodystrophy and used in drug and functional tests [108, 109]. This has enabled the molecular pathways and genetic mutations that cause these diseases to be studied in greater detail and led to the development of new therapies for patients. Hence the discovery of iPSCs continues to contribute to an improved knowledge of the underlying molecular mechanisms of various diseases and catalyze the development of novel drugs for their treatment.

## **5. Embryonic stem cells**

The first breakthrough technique for isolating and growing human ESCs *in vitro* was devel‐ oped at the University of Wisconsin-Madison in 1998 [110]. Since then, interest in developing more efficient methods for deriving ESCs and research into potential therapies involving ESCs has increased exponentially. ESC based therapies possess several natural advantages over other stem cell therapies. Since ESCs are directly derived from the developing embryo, they possess greater innate pluripotent capacity compared to most adult stem cells and could potentially be used in a wider range of therapies. Additionally, while the pluripotent potential and number of autogenic adult stem cells available may decline as patients age, ESC based therapies do not share the same limitation and a potentially limitless source of stem cells can be derived and cultured from blastocysts. Finally ESCs occur naturally in the inner cell mass of blastocysts and can be easily derived with minimal genetic or epigenetic manipulation unlike iPSCs.

The effectiveness and safety of ESCs therapies for treating various medical conditions includ‐ ing spinal cord injury, Stargardt's disease, and macular degeneration have been tested in animal and human clinical trials [111-115]. These studies demonstrate that it is relatively easy to obtain high quality and pathogen free human ESC cells, stimulate hESCs to form pure populations of differentiated cells for transplantation, and obtain sufficient quantities of cells for transplantation. Some studies of human ESC based transplants have also demonstrated that ESC is potentially safe and can be conducted with minimal risk of teratoma formation and graft rejection. The results of these animal and human ESC based clinical trials also indicate that human ESC transplantation can rescue animal models of retinal degeneration, Stargardt, and spinal cord injury, and catalyze limited visual improvement in human macular degener‐ ation patients.

therapies remains a key concern and must be resolved before they can be tested in clinical therapies. Second, while iPSCs can be derived from patients and should be able to evade patient immune response, abnormal expression of genes in iPSCs and their differentiated progeny has been shown to induce immune responses in recipients [107]. A possible consequence of the genetic and epigenetic instability, the inability of iPSCs to evade the innate immune response of patients could lead to the rejection of iPSC grafts. Similarly, genomic and epigenetic instability of iPSCs will frustrate efforts for developing iPSCs with specific function and homing abilities. Hence more research is required before iPSCs suitable for use in patient

While current limitations of iPSC technology forestalls their direct use in patient therapy, the versatility of iPSCs and their ease of derivation from patients has enabled their use in disease modeling and in vitro drug screening. iPSCs can be derived from patients affected by various diseases including LEOPARD syndrome, Schizophrenia, and X-linked adrenoleukodystrophy and used in drug and functional tests [108, 109]. This has enabled the molecular pathways and genetic mutations that cause these diseases to be studied in greater detail and led to the development of new therapies for patients. Hence the discovery of iPSCs continues to contribute to an improved knowledge of the underlying molecular mechanisms of various

The first breakthrough technique for isolating and growing human ESCs *in vitro* was devel‐ oped at the University of Wisconsin-Madison in 1998 [110]. Since then, interest in developing more efficient methods for deriving ESCs and research into potential therapies involving ESCs has increased exponentially. ESC based therapies possess several natural advantages over other stem cell therapies. Since ESCs are directly derived from the developing embryo, they possess greater innate pluripotent capacity compared to most adult stem cells and could potentially be used in a wider range of therapies. Additionally, while the pluripotent potential and number of autogenic adult stem cells available may decline as patients age, ESC based therapies do not share the same limitation and a potentially limitless source of stem cells can be derived and cultured from blastocysts. Finally ESCs occur naturally in the inner cell mass of blastocysts and can be easily derived with minimal genetic or epigenetic manipulation

The effectiveness and safety of ESCs therapies for treating various medical conditions includ‐ ing spinal cord injury, Stargardt's disease, and macular degeneration have been tested in animal and human clinical trials [111-115]. These studies demonstrate that it is relatively easy to obtain high quality and pathogen free human ESC cells, stimulate hESCs to form pure populations of differentiated cells for transplantation, and obtain sufficient quantities of cells for transplantation. Some studies of human ESC based transplants have also demonstrated that ESC is potentially safe and can be conducted with minimal risk of teratoma formation and graft rejection. The results of these animal and human ESC based clinical trials also indicate

diseases and catalyze the development of novel drugs for their treatment.

therapies will be available.

386 Pluripotent Stem Cells

**5. Embryonic stem cells**

unlike iPSCs.

Despite these apparent advantages of human ESCs, its use in research and medical therapy has been fairly controversial historically as the derivation of human ESC lines requires the destruction of human embryos. Pro-life advocates have strongly opposed the destruction of embryos for research on the basis that human life begins when a human egg cell is fertilized, and the belief that human life is inviolable. More recently, these objections have been partially overcome through the development of human ESC derivation procedures that do not require embryo destruction [116] and the use of surplus frozen embryos from in vitro fertilization clinics.

When evaluated against the four key milestones, it can be determined that while ESCs are potentially safer than iPSCs, several key concerns continue to forestall their wider use in human clinical trials. First while some studies of human ESC based transplants in animals and humans have suggested that there is minimal risk of teratoma formation or uncontrolled proliferation of transplanted cells, other studies contend that *in vitro* culture conditions can result in potentially hazardous epigenetic modifications [117]. Second since ESC transplants are allogeneic, there is a higher likelihood of immune rejection compared to autographs of adult stem cells. Third ESC transplants potentially share similar location specificity limitations as adult stem cell transplants. This limitation has to be addressed for the development of a viable next generation stem cell therapy. Fourth since ESCs have a higher innate pluripotent capacity than adult stem cells and iPSCs, it may be easier to obtain pure populations of functionally specific and disease relevant transplant cells from ESC lines. In conclusion, while more clinical trials will be required to assess the viability of ESC therapy, studies have indicated that ESC therapy seems to offer a promising alternative for treating currently incurable diseases.

## **6. The promise of transdifferentiation therapy**

Transdifferentiation is the direct conversion of one cell type to another without the involve‐ ment of an intermediate pluripotent state. Transdifferentiation could be a viable alternative therapy to stem cell therapies and relatively abundant adult somatic cells like fibroblasts and adipocytes could be harvested from patients and directly converted by transdifferentiation to neurons or cardiomyocytes before being used as autologous grafts in regenerative therapies. Transdifferentiation is advantageous compared to adult stem cell therapy because cell grafts could be designed specifically for each disease therapy resulting in improved functional and positional specificity. Transdifferentiation is also advantageous compared to iPSC based therapy because conversion to a desired cell type is a one step process requiring lesser epigenetic modification, and is a more rapid and direct process than dedifferentiation to form iPSCs followed by controlled differentiation into the desired cell type.

Transdifferentiation can be a relatively spontaneous process such as the in vitro transdiffer‐ entiation of chick retinal cells to lens cells [118]. It can also be induced via the guided expression of various molecular factors and genes. For example overexpression of *Atoh1* can induce the transdifferentiation of non-sensory supporting cells in the cochlea to auditory hair cells [67]. The expression of the microRNAs *miR-9/9\** and *miR-124* can also induce the transdifferentia‐ tion of human fibroblasts to functional neurons [119]. However despite initial successes, currently available methods for inducing transdifferentiation remain too inefficient in vivo therapeutic purposes and further research is required to improve the process before it can be considered as a viable therapeutic alternative.

[3] Kern, S, et al. Comparative analysis of mesenchymal stem cells from bone marrow,

Advances in Stem Cell Therapies http://dx.doi.org/10.5772/55061 389

[4] Yang, J, et al. Differentiation potential of human mesenchymal stem cells derived from adipose tissue and bone marrow to sinus node-like cells. Mol Med Report,

[5] Huang, J. I, et al. Chondrogenic potential of progenitor cells derived from human bone marrow and adipose tissue: a patient-matched comparison. J Orthop Res,

[6] Chang, Y. J, et al. Disparate mesenchyme-lineage tendencies in mesenchymal stem cells from human bone marrow and umbilical cord blood. Stem Cells, (2006). ,

[7] De Ugarte, D. A, et al. Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow. Immunol

[8] Pevsner-fischer, M, Levin, S, & Zipori, D. The origins of mesenchymal stromal cell

[9] Sakaguchi, Y, et al. Comparison of human stem cells derived from various mesen‐ chymal tissues: superiority of synovium as a cell source. Arthritis Rheum, (2005). ,

[10] Nauta, A. J, & Fibbe, W. E. Immunomodulatory properties of mesenchymal stromal

[11] Zhao, S, et al. Immunomodulatory properties of mesenchymal stromal cells and their therapeutic consequences for immune-mediated disorders. Stem Cells Dev, (2010). ,

[12] Barry, F. P, et al. Immunogenicity of adult mesenchymal stem cells: lessons from the

[13] Knoepfler, P. S. Deconstructing stem cell tumorigenicity: a roadmap to safe regenera‐

[14] Wakitani, S, et al. Safety of autologous bone marrow-derived mesenchymal stem cell transplantation for cartilage repair in 41 patients with 45 joints followed for up to 11

[15] Centeno, C. J, et al. Increased knee cartilage volume in degenerative joint disease us‐ ing percutaneously implanted, autologous mesenchymal stem cells. Pain Physician,

[16] Centeno, C. J, et al. Safety and complications reporting update on the re-implantation of culture-expanded mesenchymal stem cells using autologous platelet lysate techni‐

years and 5 months. J Tissue Eng Regen Med, (2011). , 146-150.

umbilical cord blood, or adipose tissue. Stem Cells, (2006). , 1294-1301.

(2012). , 108-113.

(2005). , 1383-1389.

Lett, (2003). , 267-270.

cells. Blood, (2007). , 3499-3506.

heterogeneity. Stem Cell Rev, (2011). , 560-568.

fetal allograft. Stem Cells Dev, (2005). , 252-265.

tive medicine. Stem Cells, (2009). , 1050-1056.

que. Curr Stem Cell Res Ther, (2011). , 368-378.

679-685.

2521-2529.

607-614.

(2008). , 343-353.

### **7. Summary**

The use of stem cells for therapeutic purposes will be increasingly widespread as improved knowledge leads to the development of safer and more effective therapies. Stem cells derived from patients have also been successfully used in disease modeling and therapy evaluation. Further studies will enable the innate regenerative and immunomodulatory properties of stem cells to be harnessed more effectively for treating a larger variety of diseases and injury. The study and use of adult stem cells will continue to play a pivotal role in the ongoing search for novel therapies due to their availability and safety, while further developments in iPSC and ESC derivation and cultivation processes will be required before they can be used in therapies. An improved understanding of genetic and epigenetic control continues to be a prerequisite for the development of an ideal stem cell therapy. Finally further improvements in inducing direct transdifferentiation of adult non-stem cells to the desired cell types may be an alternative regenerative therapy that may circumvent the use of stem cells entirely.

## **Author details**

Joel Sng and Thomas Lufkin\*

\*Address all correspondence to: lufkin@gis.a-star.edu.sg

Stem Cell and Developmental Biology Genome Institute of Singapore, Singapore

### **References**


[3] Kern, S, et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells, (2006). , 1294-1301.

Transdifferentiation can be a relatively spontaneous process such as the in vitro transdiffer‐ entiation of chick retinal cells to lens cells [118]. It can also be induced via the guided expression of various molecular factors and genes. For example overexpression of *Atoh1* can induce the transdifferentiation of non-sensory supporting cells in the cochlea to auditory hair cells [67]. The expression of the microRNAs *miR-9/9\** and *miR-124* can also induce the transdifferentia‐ tion of human fibroblasts to functional neurons [119]. However despite initial successes, currently available methods for inducing transdifferentiation remain too inefficient in vivo therapeutic purposes and further research is required to improve the process before it can be

The use of stem cells for therapeutic purposes will be increasingly widespread as improved knowledge leads to the development of safer and more effective therapies. Stem cells derived from patients have also been successfully used in disease modeling and therapy evaluation. Further studies will enable the innate regenerative and immunomodulatory properties of stem cells to be harnessed more effectively for treating a larger variety of diseases and injury. The study and use of adult stem cells will continue to play a pivotal role in the ongoing search for novel therapies due to their availability and safety, while further developments in iPSC and ESC derivation and cultivation processes will be required before they can be used in therapies. An improved understanding of genetic and epigenetic control continues to be a prerequisite for the development of an ideal stem cell therapy. Finally further improvements in inducing direct transdifferentiation of adult non-stem cells to the desired cell types may be an alternative

regenerative therapy that may circumvent the use of stem cells entirely.

Stem Cell and Developmental Biology Genome Institute of Singapore, Singapore

man mesenchymal stem cells. ScientificWorldJournal, (2012). , 793823.

[1] Zhang, Y, et al. Mechanisms underlying the osteo- and adipo-differentiation of hu‐

[2] Patki, S, et al. Human breast milk is a rich source of multipotent mesenchymal stem

\*Address all correspondence to: lufkin@gis.a-star.edu.sg

cells. Hum Cell, (2010). , 35-40.

considered as a viable therapeutic alternative.

**7. Summary**

388 Pluripotent Stem Cells

**Author details**

**References**

Joel Sng and Thomas Lufkin\*


[17] Tyndall, A. Successes and failures of stem cell transplantation in autoimmune diseas‐ es. Hematology Am Soc Hematol Educ Program, (2011). , 280-284.

[31] Perez-ilzarbe, M, et al. Comparison of ex vivo expansion culture conditions of mes‐ enchymal stem cells for human cell therapy. Transfusion, (2009). , 1901-1910.

Advances in Stem Cell Therapies http://dx.doi.org/10.5772/55061 391

[32] Estrada, J. C, et al. Culture of human mesenchymal stem cells at low oxygen tension improves growth and genetic stability by activating glycolysis. Cell Death Differ,

[33] Rasmusson, I, et al. Mesenchymal stem cells inhibit the formation of cytotoxic T lym‐ phocytes, but not activated cytotoxic T lymphocytes or natural killer cells. Transplan‐

[34] Di NicolaM., et al., Human bone marrow stromal cells suppress T-lymphocyte prolif‐ eration induced by cellular or nonspecific mitogenic stimuli. Blood, (2002). ,

[35] Djouad, F, et al. Immunosuppressive effect of mesenchymal stem cells favors tumor

[36] Nauta, A. J, et al. Mesenchymal stem cells inhibit generation and function of both CD34+-derived and monocyte-derived dendritic cells. J Immunol, (2006). , 2080-2087.

[37] Jiang, X. X, et al. Human mesenchymal stem cells inhibit differentiation and function

[38] Corcione, A, et al. Human mesenchymal stem cells modulate B-cell functions. Blood,

[39] Glennie, S, et al. Bone marrow mesenchymal stem cells induce division arrest anergy

[40] Patel, S. A, et al. Mesenchymal stem cells protect breast cancer cells through regulato‐ ry T cells: role of mesenchymal stem cell-derived TGF-beta. J Immunol, (2010). ,

[41] Torsvik, A, & Bjerkvig, R. Mesenchymal stem cell signaling in cancer progression.

[42] Schreml, S, et al. Harvesting human adipose tissue-derived adult stem cells: resection

[43] Dubois, S. G, et al. Isolation of human adipose-derived stem cells from biopsies and

[44] Lee, J. M, et al. Comparison of immunomodulatory effects of placenta mesenchymal stem cells with bone marrow and adipose mesenchymal stem cells. Int Immunophar‐

[45] Scuderi, N, et al. Human Adipose-Derived Stem Cells for Cell-Based Therapies in the

growth in allogeneic animals. Blood, (2003). , 3837-3844.

of activated T cells. Blood, (2005). , 2821-2827.

versus liposuction. Cytotherapy, (2009). , 947-957.

liposuction specimens. Methods Mol Biol, (2008). , 69-79.

Treatment of Systemic Sclerosis. Cell Transplant, (2012).

of monocyte-derived dendritic cells. Blood, (2005). , 4120-4126.

(2012). , 743-755.

3838-3843.

(2006). , 367-372.

5885-5894.

Cancer Treat Rev, (2012).

macol, (2012). , 219-224.

tation, (2003). , 1208-1213.


[31] Perez-ilzarbe, M, et al. Comparison of ex vivo expansion culture conditions of mes‐ enchymal stem cells for human cell therapy. Transfusion, (2009). , 1901-1910.

[17] Tyndall, A. Successes and failures of stem cell transplantation in autoimmune diseas‐

[18] Connick, P, et al. Autologous mesenchymal stem cells for the treatment of secondary progressive multiple sclerosis: an open-label phase 2a proof-of-concept study. Lancet

[19] Freedman, M. S, et al. The therapeutic potential of mesenchymal stem cell transplan‐ tation as a treatment for multiple sclerosis: consensus report of the International

[20] Duijvestein, M, et al. Autologous bone marrow-derived mesenchymal stromal cell treatment for refractory luminal Crohn's disease: results of a phase I study. Gut,

[21] Ciccocioppo, R, et al. Autologous bone marrow-derived mesenchymal stromal cells

[22] Keyszer, G, et al. Treatment of severe progressive systemic sclerosis with transplan‐ tation of mesenchymal stromal cells from allogeneic related donors: report of five

[23] Nevskaya, T, et al. Autologous progenitor cell implantation as a novel therapeutic in‐ tervention for ischaemic digits in systemic sclerosis. Rheumatology (Oxford), (2009). ,

[24] Sun, L, et al. Mesenchymal stem cell transplantation reverses multiorgan dysfunction in systemic lupus erythematosus mice and humans. Stem Cells, (2009). , 1421-1432.

[25] Liang, J, et al. Allogenic mesenchymal stem cells transplantation in refractory sys‐ temic lupus erythematosus: a pilot clinical study. Ann Rheum Dis, (2010). ,

[26] Terai, S, et al. Timeline for development of autologous bone marrow infusion (ABMi) therapy and perspective for future stem cell therapy. J Gastroenterol, (2012).

[27] Tan, J, et al. Induction therapy with autologous mesenchymal stem cells in living-re‐ lated kidney transplants: a randomized controlled trial. JAMA, (2012). , 1169-1177.

[28] Lee, J. S, et al. A long-term follow-up study of intravenous autologous mesenchymal stem cell transplantation in patients with ischemic stroke. Stem Cells, (2010). ,

[29] Bang, O. Y, et al. Autologous mesenchymal stem cell transplantation in stroke pa‐

[30] Fehrer, C, et al. Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan. Aging Cell, (2007). , 745-757.

in the treatment of fistulising Crohn's disease. Gut, (2011). , 788-798.

es. Hematology Am Soc Hematol Educ Program, (2011). , 280-284.

MSCT Study Group. Mult Scler, (2010). , 503-510.

cases. Arthritis Rheum, (2011). , 2540-2542.

Neurol, (2012). , 150-156.

390 Pluripotent Stem Cells

(2010). , 1662-1669.

61-64.

1423-1429.

1099-1106.

tients. Ann Neurol, (2005). , 874-882.


[46] Pak, J. Regeneration of human bones in hip osteonecrosis and human cartilage in knee osteoarthritis with autologous adipose-tissue-derived stem cells: a case series. J Med Case Reports, (2011). , 296.

[60] Saha, B, Jaber, M, & Gaillard, A. Potentials of endogenous neural stem cells in corti‐

Advances in Stem Cell Therapies http://dx.doi.org/10.5772/55061 393

[61] Nishida, T, et al. Development of tumor-reactive T cells after nonmyeloablative allo‐ geneic hematopoietic stem cell transplant for chronic lymphocytic leukemia. Clin

[62] Lucarelli, G, et al. Hematopoietic stem cell transplantation in thalassemia and sickle

[63] Kim, H, et al. Allogeneic Hematopoietic Stem Cell Transplant for Adults over 40 Years Old with Acquired Aplastic Anemia. Biol Blood Marrow Transplant, (2012).

[64] Shapiro, E, et al. Long-term effect of bone-marrow transplantation for childhood-on‐

[65] Breuskin, I, et al. Strategies to regenerate hair cells: identification of progenitors and

[66] Li, H, Liu, H, & Heller, S. Pluripotent stem cells from the adult mouse inner ear. Nat

[67] Minoda, R, et al. Manipulating cell cycle regulation in the mature cochlea. Hear Res,

[68] Dabdoub, A, et al. Sox2 signaling in prosensory domain specification and subsequent hair cell differentiation in the developing cochlea. Proc Natl Acad Sci U S A, (2008). ,

[69] Kiernan, A. E, et al. The Notch ligands DLL1 and JAG2 act synergistically to regulate hair cell development in the mammalian inner ear. Development, (2005). , 4353-4362.

[70] Shackleton, M, et al. Generation of a functional mammary gland from a single stem

[71] Liu, S, Dontu, G, & Wicha, M. S. Mammary stem cells, self-renewal pathways, and

[72] Ercan, C, Van Diest, P. J, & Vooijs, M. Mammary development and breast cancer: the

[73] Katoh, M. Notch signaling in gastrointestinal tract (review). Int J Oncol, (2007). ,

[74] Crosnier, C, Stamataki, D, & Lewis, J. Organizing cell renewal in the intestine: stem

[75] Gersemann, M, Stange, E. F, & Wehkamp, J. From intestinal stem cells to inflammato‐

cells, signals and combinatorial control. Nat Rev Genet, (2006). , 349-359.

ry bowel diseases. World J Gastroenterol, (2011). , 3198-3203.

set cerebral X-linked adrenoleukodystrophy. Lancet, (2000). , 713-718.

cell anemia. Cold Spring Harb Perspect Med, (2012). , a011825.

cal repair. Front Cell Neurosci, (2012). , 14.

Cancer Res, (2009). , 4759-4768.

critical genes. Hear Res, (2008). , 1-10.

Med, (2003). , 1293-1299.

cell. Nature, (2006). , 84-88.

carcinogenesis. Breast Cancer Res, (2005). , 86-95.

role of stem cells. Curr Mol Med, (2011). , 270-285.

(2007). , 44-51.

18396-18401.

247-251.


[60] Saha, B, Jaber, M, & Gaillard, A. Potentials of endogenous neural stem cells in corti‐ cal repair. Front Cell Neurosci, (2012). , 14.

[46] Pak, J. Regeneration of human bones in hip osteonecrosis and human cartilage in knee osteoarthritis with autologous adipose-tissue-derived stem cells: a case series. J

[47] Koyama, N, et al. Pluripotency of mesenchymal cells derived from synovial fluid in patients with temporomandibular joint disorder. Life Sci, (2011). , 741-747.

[48] Ju, Y. J, et al. Synovial mesenchymal stem cells accelerate early remodeling of ten‐

[49] Sekiya, I, et al. Articular cartilage regeneration with synovial mesenchymal stem

[50] Miyamoto, T, et al. Intradiscal transplantation of synovial mesenchymal stem cells prevents intervertebral disc degeneration through suppression of matrix metallopro‐ teinase-related genes in nucleus pulposus cells in rabbits. Arthritis Res Ther, (2010). ,

[51] Yang, J, et al. Evaluation of bone marrow- and brain-derived neural stem cells in therapy of central nervous system autoimmunity. Am J Pathol, (2010). , 1989-2001.

[52] Reynolds, B. A, & Weiss, S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science, (1992). , 1707-1710.

[53] Pluchino, S, et al. Injection of adult neurospheres induces recovery in a chronic mod‐

[54] Pluchino, S, et al. Immune regulatory neural stem/precursor cells protect from cen‐ tral nervous system autoimmunity by restraining dendritic cell function. PLoS One,

[55] Wang, L, et al. Neural stem/progenitor cells modulate immune responses by sup‐ pressing T lymphocytes with nitric oxide and prostaglandin E2. Exp Neurol, (2009). ,

[56] Melzi, R, et al. Co-graft of allogeneic immune regulatory neural stem cells (NPC) and pancreatic islets mediates tolerance, while inducing NPC-derived tumors in mice.

[57] Yang, J, et al. Adult neural stem cells expressing IL-10 confer potent immunomodula‐ tion and remyelination in experimental autoimmune encephalitis. J Clin Invest,

[58] Conover, J. C, & Notti, R. Q. The neural stem cell niche. Cell Tissue Res, (2008). ,

[59] Buffo, A, et al. Expression pattern of the transcription factor Olig2 in response to brain injuries: implications for neuronal repair. Proc Natl Acad Sci U S A, (2005). ,

Med Case Reports, (2011). , 296.

cells]. Clin Calcium, (2011). , 879-889.

R206.

392 Pluripotent Stem Cells

(2009). , e5959.

PLoS One, (2010). , e10357.

(2009). , 3678-3691.

177-183.

211-224.

18183-18188.

don-bone healing. Cell Tissue Res, (2008). , 469-478.

el of multiple sclerosis. Nature, (2003). , 688-694.


[76] Conrad, S, et al. Generation of pluripotent stem cells from adult human testis. Na‐ ture, (2008). , 344-349.

[92] Okita, K, Ichisaka, T, & Yamanaka, S. Generation of germline-competent induced

Advances in Stem Cell Therapies http://dx.doi.org/10.5772/55061 395

[93] Nakagawa, M, et al. Generation of induced pluripotent stem cells without Myc from

[94] Stadtfeld, M, et al. Induced pluripotent stem cells generated without viral integra‐

[95] Warren, L, et al. Highly efficient reprogramming to pluripotency and directed differ‐ entiation of human cells with synthetic modified mRNA. Cell Stem Cell, (2010). ,

[96] Yoshida, Y, et al. Hypoxia enhances the generation of induced pluripotent stem cells.

[97] Esteban, M. A, et al. Vitamin C enhances the generation of mouse and human in‐

[98] Maherali, N, & Hochedlinger, K. Guidelines and techniques for the generation of in‐

[99] Wernig, M, et al. A drug-inducible transgenic system for direct reprogramming of

[100] Anokye-danso, F, et al. Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell, (2011). , 376-388.

[101] Doi, A, et al. Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibro‐

[102] Lister, R, et al. Hotspots of aberrant epigenomic reprogramming in human induced

[103] Govindarajan, N, et al. Sodium butyrate improves memory function in an Alzheim‐ er's disease mouse model when administered at an advanced stage of disease pro‐

[104] Ware, C. B, et al. Histone deacetylase inhibition elicits an evolutionarily conserved self-renewal program in embryonic stem cells. Cell Stem Cell, (2009). , 359-369.

[105] Hobley, G, et al. Development of rationally designed DNA N6 adenine methyltrans‐

[106] Bae, D, et al. Hypoxia enhances the generation of retinal progenitor cells from human induced pluripotent and embryonic stem cells. Stem Cells Dev, (2012). , 1344-1355.

[107] Zhao, T, et al. Immunogenicity of induced pluripotent stem cells. Nature, (2011). ,

ferase inhibitors. Bioorg Med Chem Lett, (2012). , 3079-3082.

mouse and human fibroblasts. Nat Biotechnol, (2008). , 101-106.

duced pluripotent stem cells. Cell Stem Cell, (2010). , 71-79.

duced pluripotent stem cells. Cell Stem Cell, (2008). , 595-605.

multiple somatic cell types. Nat Biotechnol, (2008). , 916-924.

pluripotent stem cells. Nature, (2007). , 313-317.

tion. Science, (2008). , 945-949.

Cell Stem Cell, (2009). , 237-241.

blasts. Nat Genet, (2009). , 1350-1353.

pluripotent stem cells. Nature, (2011). , 68-73.

gression. J Alzheimers Dis, (2011). , 187-197.

618-630.

212-215.


[92] Okita, K, Ichisaka, T, & Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature, (2007). , 313-317.

[76] Conrad, S, et al. Generation of pluripotent stem cells from adult human testis. Na‐

[77] Dobrinski, I. Germ cell transplantation and testis tissue xenografting in domestic ani‐

[78] Honaramooz, A, et al. Fertility and germline transmission of donor haplotype follow‐ ing germ cell transplantation in immunocompetent goats. Biol Reprod, (2003). ,

[79] Brinster, R. L, & Avarbock, M. R. Germline transmission of donor haplotype follow‐ ing spermatogonial transplantation. Proc Natl Acad Sci U S A, (1994). , 11303-11307.

[80] Stadtfeld, M, Brennand, K, & Hochedlinger, K. Reprogramming of pancreatic beta

[81] Takahashi, K, & Yamanaka, S. Induction of pluripotent stem cells from mouse em‐ bryonic and adult fibroblast cultures by defined factors. Cell, (2006). , 663-676.

[82] Takahashi, K, et al. Induction of pluripotent stem cells from adult human fibroblasts

[83] Yu, J, et al. Induced pluripotent stem cell lines derived from human somatic cells.

[84] Neumann, J, et al. SOX2 expression correlates with lymph-node metastases and dis‐

[85] De Nigris, F, et al. c-Myc oncoprotein: cell cycle-related events and new therapeutic challenges in cancer and cardiovascular diseases. Cell Cycle, (2003). , 325-328.

[86] Wang, P, et al. The POU homeodomain protein OCT3 as a potential transcriptional activator for fibroblast growth factor-4 (FGF-4) in human breast cancer cells. Biochem

[87] Gore, A, et al. Somatic coding mutations in human induced pluripotent stem cells.

[88] Hussein, S. M, et al. Copy number variation and selection during reprogramming to

[89] Mayshar, Y, et al. Identification and classification of chromosomal aberrations in hu‐

[90] Bar-nur, O, et al. Epigenetic memory and preferential lineage-specific differentiation in induced pluripotent stem cells derived from human pancreatic islet beta cells. Cell

[91] Kim, K, et al. Epigenetic memory in induced pluripotent stem cells. Nature, (2010). ,

man induced pluripotent stem cells. Cell Stem Cell, (2010). , 521-531.

cells into induced pluripotent stem cells. Curr Biol, (2008). , 890-894.

tant spread in right-sided colon cancer. BMC Cancer, (2011). , 518.

ture, (2008). , 344-349.

1260-1264.

394 Pluripotent Stem Cells

mals. Anim Reprod Sci, (2005). , 137-145.

by defined factors. Cell, (2007). , 861-872.

Science, (2007). , 1917-1920.

J, (2003). Pt 1): , 199-205.

Nature, (2011). , 63-67.

Stem Cell, (2011). , 17-23.

285-290.

pluripotency. Nature, (2011). , 58-62.


[108] Robinton, D. A, & Daley, G. Q. The promise of induced pluripotent stem cells in re‐ search and therapy. Nature, (2012). , 295-305.

**Chapter 18**

**Embryonic Stem Cell Therapy – From Bench to Bed**

The term stem cell includes a large class of cells defined by their ability to give rise to vari‐ ous mature progeny while maintaining the capacity to self-renew. Embryonic stem cells (ESCs) were first isolated from the inner mass of late blastocysts in mice by Sir Martin J. Evans and Matthew Kaufman (Evans & Kaufman, 1981) and independently by Gail R. Mar‐ tin (Martin, 1981). Later, it became possible to obtain ESCs from non-human primates and humans. In 1998, James Thomson and his team reported the first successful derivation of hu‐ man ESC lines (Thomson *et al.*, 1998), thus extending the great potential of ESCs by provid‐

Embryonic stem cells are pluripotent, a term that defines the ability of a cell to differentiate into cells of all three germ layers. There are different types of mammalian pluripotent stem cells: embryonic stem cells derived from pre-implantation embryos (Evans & Kaufman, 1981), embryonic carcinoma (EC) cells, the stem cells of testicular tumors (Stevens, 1966; Evans, 1972), epiblast stem cells (EpiSCs) derived from the late epiblast layer of post-implan‐ tation embryos (Brons *et al.*, 2007), and embryonic germ (EG) cells derived from primordial germ cells (PGCs) of the post-implantation embryo (Matsui *et al.*, 1992; Stewart *et al.*, 1994).

Besides isolating pluripotent cells from different embryonic tissues, various experimental methods are available nowadays for inducing pluripotency *in vitro*. These methods include cloning by somatic cell nuclear transfer (SCNT), cellular fusion with embryonic stem cells, the induction of parthenogenesis, and direct reprogramming by addition of reprogramming transcription factors. SCNT is done by replacing the oocyte genome at metaphase II of meio‐ sis with a somatic cell nucleus. Although somatic cell reprogramming has been achieved in several mammalian species (Wilmut *et al.*, 1997), this seems to be very difficult to achieve in humans. Only in 2011 Noggle et al. (Noggle *et al.*, 2011) succeeded to generate human pluri‐ potent cells by using SCNT. However, their study revealed that the classical SCNT consis‐

> © 2013 Sperling; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Sperling; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

ing the opportunity to develop stem cell-based therapies for human disease.

Laura E. Sperling

**1. Introduction**

http://dx.doi.org/10.5772/54368

Additional information is available at the end of the chapter


## **Embryonic Stem Cell Therapy – From Bench to Bed**

Laura E. Sperling

[108] Robinton, D. A, & Daley, G. Q. The promise of induced pluripotent stem cells in re‐

[109] Carvajal-vergara, X, et al. Patient-specific induced pluripotent stem-cell-derived

[110] Thomson, J. A, et al. Embryonic stem cell lines derived from human blastocysts. Sci‐

[111] Schwartz, S. D, et al. Embryonic stem cell trials for macular degeneration: a prelimi‐

[112] Lu, B, et al. Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration. Stem Cells, (2009). , 2126-2135.

[113] Lukovic, D, et al. Concise review: human pluripotent stem cells in the treatment of

[114] Wichterle, H, et al. Directed differentiation of embryonic stem cells into motor neu‐

[115] All, A. H, et al. Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitors Aid in Functional Recovery of Sensory Pathways following Contusive Spinal Cord

[116] Klimanskaya, I, et al. Human embryonic stem cell lines derived from single blasto‐

[117] Tompkins, J. D, et al. Epigenetic stability, adaptability, and reversibility in human

[118] Eguchi, G, & Okada, T. S. Differentiation of lens tissue from the progeny of chick reti‐ nal pigment cells cultured in vitro: a demonstration of a switch of cell types in clonal

[119] Yoo, A. S, et al. MicroRNA-mediated conversion of human fibroblasts to neurons.

embryonic stem cells. Proc Natl Acad Sci U S A, (2012). , 12544-12549.

cell culture. Proc Natl Acad Sci U S A, (1973). , 1495-1499.

search and therapy. Nature, (2012). , 295-305.

ence, (1998). , 1145-1147.

396 Pluripotent Stem Cells

rons. Cell, (2002). , 385-397.

Injury. PLoS One, (2012). , e47645.

meres. Nature, (2006). , 481-485.

Nature, (2011). , 228-231.

nary report. Lancet, (2012). , 713-720.

spinal cord injury. Stem Cells, (2012). , 1787-1792.

models of LEOPARD syndrome. Nature, (2010). , 808-812.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54368

## **1. Introduction**

The term stem cell includes a large class of cells defined by their ability to give rise to vari‐ ous mature progeny while maintaining the capacity to self-renew. Embryonic stem cells (ESCs) were first isolated from the inner mass of late blastocysts in mice by Sir Martin J. Evans and Matthew Kaufman (Evans & Kaufman, 1981) and independently by Gail R. Mar‐ tin (Martin, 1981). Later, it became possible to obtain ESCs from non-human primates and humans. In 1998, James Thomson and his team reported the first successful derivation of hu‐ man ESC lines (Thomson *et al.*, 1998), thus extending the great potential of ESCs by provid‐ ing the opportunity to develop stem cell-based therapies for human disease.

Embryonic stem cells are pluripotent, a term that defines the ability of a cell to differentiate into cells of all three germ layers. There are different types of mammalian pluripotent stem cells: embryonic stem cells derived from pre-implantation embryos (Evans & Kaufman, 1981), embryonic carcinoma (EC) cells, the stem cells of testicular tumors (Stevens, 1966; Evans, 1972), epiblast stem cells (EpiSCs) derived from the late epiblast layer of post-implan‐ tation embryos (Brons *et al.*, 2007), and embryonic germ (EG) cells derived from primordial germ cells (PGCs) of the post-implantation embryo (Matsui *et al.*, 1992; Stewart *et al.*, 1994).

Besides isolating pluripotent cells from different embryonic tissues, various experimental methods are available nowadays for inducing pluripotency *in vitro*. These methods include cloning by somatic cell nuclear transfer (SCNT), cellular fusion with embryonic stem cells, the induction of parthenogenesis, and direct reprogramming by addition of reprogramming transcription factors. SCNT is done by replacing the oocyte genome at metaphase II of meio‐ sis with a somatic cell nucleus. Although somatic cell reprogramming has been achieved in several mammalian species (Wilmut *et al.*, 1997), this seems to be very difficult to achieve in humans. Only in 2011 Noggle et al. (Noggle *et al.*, 2011) succeeded to generate human pluri‐ potent cells by using SCNT. However, their study revealed that the classical SCNT consis‐

© 2013 Sperling; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Sperling; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

tently leads to developmental arrest. The activated human oocytes develop to the blastocyst stage only when the somatic cell genome is merely added and the oocyte genome is not re‐ moved. Human stem cells derived from these blastocysts contain both a haploid genome de‐ rived from the oocyte and a diploid somatic cell genome reprogrammed to a pluripotent state (Noggle *et al.*, 2011). However, the SCNT raises some ethical concerns regarding the use of human eggs. It has also been reported that somatic cells could be reprogrammed by fusion with ES cells (Do *et al.*, 2006). These cells offer a good alternative to SCNT, especially for studying the mechanisms of reprogramming, but are thought to be less interesting for therapies due to the presence of the nuclei of stem cells in the hybrids and their instability. Human ESC lines derived from parthenogenetic blastocysts obtained by artificial activation of an oocyte have been obtained (Turovets *et al.*, 2011). Their immune-matching advantage, combined with the advantage of derivation from nonviable human embryos makes these cells a good source for cell-based transplantation therapy. However, one of the most exciting reports in reprogramming was the generation of iPSCs from terminally differentiated so‐ matic cells by transduction of four transcription factors (*OCT4*, *SOX2*, *KLF4* and *c-MYC*) into fibroblasts (Takahashi & Yamanaka, 2006).

choriocapillaries (Katta *et al.*, 2009). Other retinal diseases with limited conventional treat‐ ments include Stargardt's macular dystrophy (SMD) and retinitis pigmentosa (RP). SMD is the most common early-onset macular degeneration disease, usually manifesting in people between ages 10 to 20. Initially there is an abnormal deposit of lipofuscin (yellow–brown granules of pigment that manifest with age) in the RPE. The RPE eventually degrades, which leads to photoreceptor loss, causing a decrease in central vision (Rowland *et al.*, 2012). In attempts to develop cell-based therapies for blinding diseases, two different approaches have to be distinguished. The first is a more direct approach of implanting appropriate reti‐ nal or RPE precursor cells, with the hope that they may integrate autonomously into the re‐ maining (and diseased) target tissue. The second strategy counts on a lesser degree of cell autonomy within the diseased environment. Therefore, in this case, the bioengineer will first reconstruct a piece of retina or RPE tissue *in vitro*, which then can be implanted into the le‐ sioned or diseased location (Layer *et al.,* 2010). This approach is called tissue engineering.

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Restoration of vision has focused up to now on transplantation of neural progenitor cells (NPCs) and retinal pigmented epithelium (RPE) to the retina. The retinal pigment epitheli‐ um (RPE) is a monolayer of pigmented cells forming a part of the blood/retina barrier and plays crucial roles in the maintenance and function of the retina and its photoreceptors (Strauss, 2005). The apical membrane of the RPE is associated with the rod and cone photo‐ receptors of the retina. The basal side of the RPE faces Bruch's membrane, thereby separat‐ ing the NR from the blood. The RPE absorbs light energy to increase visual sensitivity and protect against photooxidation, transports nutrients and ions between the photoreceptors at its apical surface and the choriocapillaries at its basal surface, phagocytoses photoreceptor outer segments, according to a daily circadian cycle, to relieve the photoreceptors of lightinduced free radicals. The RPE secretes a variety of growth factors, such as the neuroprotec‐ tive-antiangiogenic pigment epithelium-derived factor (PEDF) which is released to the neural retina, and the vasoprotective-angiogenic vascular endothelial growth factor (VEGF) that is secreted to the choroid (Layer *et al.,* 2010). With these diverse functions of the RPE it is not surprising that dysfunction and loss of RPE leads to degeneration of photoreceptors several diseases such as age-related macular degeneration (AMD), retinitis pigmentosa and

Cell transplantation is a novel therapeutic strategy to restore visual responses. Human em‐ bryonic stem cells (hESCs) may serve as an unlimited source of RPE cells and photorecep‐

hESC studies have focused on the derivation of subsets of retinal cell populations (Meyer *et al.*, 2009), with emphasis on the production of either retinal progenitors (Banin *et al.*, 2006; Lamba *et al.*, 2006), or more mature cells such as retinal pigment epithelium (RPE) (Kliman‐

Several groups have demonstrated that differentiating hESCs mimic the stepwise develop‐ ment of retinal cells *in vivo* (Meyer *et al.*, 2009). Furthermore, hESCs appear to respond to secreted morphogens in a manner predicted by studies of vertebrate neural induction and

Stargardt´s disease.

**2.1. Preclinical work**

tors for transplantation in different blinding conditions.

skaya *et al.*, 2004) or photoreceptors (Osakada *et al.*, 2008).

By using various biological reagents (e.g. growth factors) (Schuldiner *et al.*, 2000), ESCs can be differentiated in the laboratory into a range of different cell types, including neurons, glia, cardiomyocytes, islet beta cells, hepatocytes, hematopoietic progenitors and retinal pig‐ ment epithelium. The ESC ability to give rise to many different cell types is the reason that makes them very good candidates for cellular therapies. Many of the diseases that place the greatest burden on society are, at their root, diseases of cellular deficiency. Diabetes, stroke, heart diseases, hematological and neurodegenerative disorders, blindness, spinal cord in‐ jury, osteoarthritis, and kidney failure all result from the absence of one or more populations of cells that the body is unable to replace. Three basic methods have been developed to pro‐ mote differentiation of ESCs: (1) the formation of three-dimensional aggregates known as embryoid bodies (EBs) (Itskovitz-Eldor *et al.*, 2000), (2) the culture of ESCs as monolayers on extracellular matrix proteins, and (3) the culture of ESCs directly on supportive stromal lay‐ ers (Kawasaki *et al.*, 2000; Murry & Keller, 2008). However, the controlled differentiation of ESCs is rather difficult to optimize due to the use of serum in the culture media and difficul‐ ty to select differentiated cells. In this chapter I will focus on the differentiation of ESCs into the ectodermal lineage and on the two in 2012 ongoing clinical trials involving transplanta‐ tion of ESCs derivates into eye and spinal cord.

#### **2. Treatment of eye diseases**

Retinal degenerative diseases that target photoreceptors or the adjacent retinal pigment epi‐ thelium (RPE) affect millions of people worldwide. Age-related macular degeneration (AMD) is a late-onset, complex disorder of the eye with a multi-factorial etiology in elderly (Katta *et al.*, 2009). Being the third leading cause of blindness worldwide, it accounts for 8.7% of blind persons globally. AMD results in progressive and irreversible loss of central vision affecting the macula of the eye and involves the RPE, Bruch's membrane (BM) and choriocapillaries (Katta *et al.*, 2009). Other retinal diseases with limited conventional treat‐ ments include Stargardt's macular dystrophy (SMD) and retinitis pigmentosa (RP). SMD is the most common early-onset macular degeneration disease, usually manifesting in people between ages 10 to 20. Initially there is an abnormal deposit of lipofuscin (yellow–brown granules of pigment that manifest with age) in the RPE. The RPE eventually degrades, which leads to photoreceptor loss, causing a decrease in central vision (Rowland *et al.*, 2012). In attempts to develop cell-based therapies for blinding diseases, two different approaches have to be distinguished. The first is a more direct approach of implanting appropriate reti‐ nal or RPE precursor cells, with the hope that they may integrate autonomously into the re‐ maining (and diseased) target tissue. The second strategy counts on a lesser degree of cell autonomy within the diseased environment. Therefore, in this case, the bioengineer will first reconstruct a piece of retina or RPE tissue *in vitro*, which then can be implanted into the le‐ sioned or diseased location (Layer *et al.,* 2010). This approach is called tissue engineering.

Restoration of vision has focused up to now on transplantation of neural progenitor cells (NPCs) and retinal pigmented epithelium (RPE) to the retina. The retinal pigment epitheli‐ um (RPE) is a monolayer of pigmented cells forming a part of the blood/retina barrier and plays crucial roles in the maintenance and function of the retina and its photoreceptors (Strauss, 2005). The apical membrane of the RPE is associated with the rod and cone photo‐ receptors of the retina. The basal side of the RPE faces Bruch's membrane, thereby separat‐ ing the NR from the blood. The RPE absorbs light energy to increase visual sensitivity and protect against photooxidation, transports nutrients and ions between the photoreceptors at its apical surface and the choriocapillaries at its basal surface, phagocytoses photoreceptor outer segments, according to a daily circadian cycle, to relieve the photoreceptors of lightinduced free radicals. The RPE secretes a variety of growth factors, such as the neuroprotec‐ tive-antiangiogenic pigment epithelium-derived factor (PEDF) which is released to the neural retina, and the vasoprotective-angiogenic vascular endothelial growth factor (VEGF) that is secreted to the choroid (Layer *et al.,* 2010). With these diverse functions of the RPE it is not surprising that dysfunction and loss of RPE leads to degeneration of photoreceptors several diseases such as age-related macular degeneration (AMD), retinitis pigmentosa and Stargardt´s disease.

#### **2.1. Preclinical work**

tently leads to developmental arrest. The activated human oocytes develop to the blastocyst stage only when the somatic cell genome is merely added and the oocyte genome is not re‐ moved. Human stem cells derived from these blastocysts contain both a haploid genome de‐ rived from the oocyte and a diploid somatic cell genome reprogrammed to a pluripotent state (Noggle *et al.*, 2011). However, the SCNT raises some ethical concerns regarding the use of human eggs. It has also been reported that somatic cells could be reprogrammed by fusion with ES cells (Do *et al.*, 2006). These cells offer a good alternative to SCNT, especially for studying the mechanisms of reprogramming, but are thought to be less interesting for therapies due to the presence of the nuclei of stem cells in the hybrids and their instability. Human ESC lines derived from parthenogenetic blastocysts obtained by artificial activation of an oocyte have been obtained (Turovets *et al.*, 2011). Their immune-matching advantage, combined with the advantage of derivation from nonviable human embryos makes these cells a good source for cell-based transplantation therapy. However, one of the most exciting reports in reprogramming was the generation of iPSCs from terminally differentiated so‐ matic cells by transduction of four transcription factors (*OCT4*, *SOX2*, *KLF4* and *c-MYC*) into

By using various biological reagents (e.g. growth factors) (Schuldiner *et al.*, 2000), ESCs can be differentiated in the laboratory into a range of different cell types, including neurons, glia, cardiomyocytes, islet beta cells, hepatocytes, hematopoietic progenitors and retinal pig‐ ment epithelium. The ESC ability to give rise to many different cell types is the reason that makes them very good candidates for cellular therapies. Many of the diseases that place the greatest burden on society are, at their root, diseases of cellular deficiency. Diabetes, stroke, heart diseases, hematological and neurodegenerative disorders, blindness, spinal cord in‐ jury, osteoarthritis, and kidney failure all result from the absence of one or more populations of cells that the body is unable to replace. Three basic methods have been developed to pro‐ mote differentiation of ESCs: (1) the formation of three-dimensional aggregates known as embryoid bodies (EBs) (Itskovitz-Eldor *et al.*, 2000), (2) the culture of ESCs as monolayers on extracellular matrix proteins, and (3) the culture of ESCs directly on supportive stromal lay‐ ers (Kawasaki *et al.*, 2000; Murry & Keller, 2008). However, the controlled differentiation of ESCs is rather difficult to optimize due to the use of serum in the culture media and difficul‐ ty to select differentiated cells. In this chapter I will focus on the differentiation of ESCs into the ectodermal lineage and on the two in 2012 ongoing clinical trials involving transplanta‐

Retinal degenerative diseases that target photoreceptors or the adjacent retinal pigment epi‐ thelium (RPE) affect millions of people worldwide. Age-related macular degeneration (AMD) is a late-onset, complex disorder of the eye with a multi-factorial etiology in elderly (Katta *et al.*, 2009). Being the third leading cause of blindness worldwide, it accounts for 8.7% of blind persons globally. AMD results in progressive and irreversible loss of central vision affecting the macula of the eye and involves the RPE, Bruch's membrane (BM) and

fibroblasts (Takahashi & Yamanaka, 2006).

398 Pluripotent Stem Cells

tion of ESCs derivates into eye and spinal cord.

**2. Treatment of eye diseases**

Cell transplantation is a novel therapeutic strategy to restore visual responses. Human em‐ bryonic stem cells (hESCs) may serve as an unlimited source of RPE cells and photorecep‐ tors for transplantation in different blinding conditions.

hESC studies have focused on the derivation of subsets of retinal cell populations (Meyer *et al.*, 2009), with emphasis on the production of either retinal progenitors (Banin *et al.*, 2006; Lamba *et al.*, 2006), or more mature cells such as retinal pigment epithelium (RPE) (Kliman‐ skaya *et al.*, 2004) or photoreceptors (Osakada *et al.*, 2008).

Several groups have demonstrated that differentiating hESCs mimic the stepwise develop‐ ment of retinal cells *in vivo* (Meyer *et al.*, 2009). Furthermore, hESCs appear to respond to secreted morphogens in a manner predicted by studies of vertebrate neural induction and retinogenesis. In particular, blockade of bone morphogenetic protein and canonical Wnt sig‐ naling is known to be important for neural and retinal patterning, and many retinal differ‐ entiation protocols call for antagonists of one or both of these pathways to be included in the culture medium (Gamm & Meyer, 2010). Furthermore, the differentiation toward neural and further toward RPE fate is augmented by nicotinamide and Activin A (Idelson *et al.*, 2009). Several hESC lines actually generate neuroectodermal progenitors by spontaneous differen‐ tiation, without the addition of specific factors. RPE cells for example, were being isolated from several spontaneously differentiating human ES cell lines (Klimanskaya *et al.*, 2004). In their hands (Klimanskaya *et al.*, 2004), RPE-like differentiation occurred independently of the presence of serum. RPE cells reliably appeared in cultures grown in the presence or ab‐ sence of FBS without significant variations in RPE number or time of appearance. The inde‐ pendence of this differentiation pathway on either coculture or extracellular matrix suggests the involvement of other differentiation cues, such as potential autocrine factors produced by differentiating hES cells. The hES-derived RPE-like cells expressed the same makers as RPE cells, e.g. RPE65 protein and CRALBP (Alge *et al.*, 2003; Klimanskaya *et al.*, 2004).

One of the rationales behind using the eye for cell therapy is that the eye represents an immuno‐ privileged site. The failure of the immune system to elicit an immune response in this and other such sites constitutes the hallmark of the immune privilege status (Hori *et al.*, 2010). The re‐ markably successful field of corneal transplantation in clinical practice is undoubtedly associ‐ ated with corneal immune privilege. The subretinal space is protected by a blood–ocular barrier and the ocular fluids contain a potpourri of immunosuppressive and immunoregulato‐ ry factors that suppress T-cell proliferation and secretion of proinflammatory cytokines and in‐

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401

hibiting of both the cellular and humoral immune responses (Niederkorn, 2002).

**Figure 1.** Scheme of procedure for replacing damaged retinal pigment epithelium cells.

So far, it has been shown that transplanted postmitotic photoreceptor precursors are able to functionally integrate into the adult mouse neural retina. However, photoreceptors are neu‐ rons and they need to form synaptic connections in order to be functional. This makes the cell therapy with photoreceptors more challenging when compared to RPE cells. Interesting‐ ly, a group from Japan (Eiraku *et al.*, 2011) could obtain formation of a fully stratified neural retina from by using a three dimensional ESCs culture system. The 3D organoids would open up new avenues for the transplantation of artificial retinal tissue sheets, rather than simple cell grafting.

#### **2.2. Clinical trial**

Until shortly, the most relevant clinical studies currently being conducted in patients with reti‐ nal degeneration were fetal retinal sheet transplants (Radtke *et al.*, 2008). This strategy has its basis on the fact that immature retinal sheet extends cell processes and forms synaptic connec‐ tions with the degenerate host retina. The underlying principle is that the inner retinal neurons of the host remain intact and therefore only require synaptic connections with photoreceptors for visual function to be restored. One big problem for the application of photoreceptor cell transplantation is that an appropriate source of the precursor cells is required.

Advanced Cell Technology and Jules Stein Eye Institute at UCLA started two prospective clinical studies to establish the safety and tolerability of subretinal transplantation of human ESC-derived retinal pigment epithelium (RPE) in patients with Stargardt's macular dystro‐ phy (clinical trial identifier-NCT01469832) and dry age-related macular degeneration (clini‐ cal trial identifier-NCT01344993) — the leading cause of blindness in the developed world (Schwartz *et al.*, 2012). The studies are in phase I/II, where only the safety and tolerability of human ESC-derived RPE cells is assessed. The team of researchers from ACT and UCLA re‐ ported their preliminary work in two patients, one with AMD, the other with Stargardt's macular dystrophy, being the first to publish data on the use of human ESC-derived cells in the clinic (Schwartz *et al.*, 2012).

One of the rationales behind using the eye for cell therapy is that the eye represents an immuno‐ privileged site. The failure of the immune system to elicit an immune response in this and other such sites constitutes the hallmark of the immune privilege status (Hori *et al.*, 2010). The re‐ markably successful field of corneal transplantation in clinical practice is undoubtedly associ‐ ated with corneal immune privilege. The subretinal space is protected by a blood–ocular barrier and the ocular fluids contain a potpourri of immunosuppressive and immunoregulato‐ ry factors that suppress T-cell proliferation and secretion of proinflammatory cytokines and in‐ hibiting of both the cellular and humoral immune responses (Niederkorn, 2002).

retinogenesis. In particular, blockade of bone morphogenetic protein and canonical Wnt sig‐ naling is known to be important for neural and retinal patterning, and many retinal differ‐ entiation protocols call for antagonists of one or both of these pathways to be included in the culture medium (Gamm & Meyer, 2010). Furthermore, the differentiation toward neural and further toward RPE fate is augmented by nicotinamide and Activin A (Idelson *et al.*, 2009). Several hESC lines actually generate neuroectodermal progenitors by spontaneous differen‐ tiation, without the addition of specific factors. RPE cells for example, were being isolated from several spontaneously differentiating human ES cell lines (Klimanskaya *et al.*, 2004). In their hands (Klimanskaya *et al.*, 2004), RPE-like differentiation occurred independently of the presence of serum. RPE cells reliably appeared in cultures grown in the presence or ab‐ sence of FBS without significant variations in RPE number or time of appearance. The inde‐ pendence of this differentiation pathway on either coculture or extracellular matrix suggests the involvement of other differentiation cues, such as potential autocrine factors produced by differentiating hES cells. The hES-derived RPE-like cells expressed the same makers as RPE cells, e.g. RPE65 protein and CRALBP (Alge *et al.*, 2003; Klimanskaya *et al.*, 2004).

So far, it has been shown that transplanted postmitotic photoreceptor precursors are able to functionally integrate into the adult mouse neural retina. However, photoreceptors are neu‐ rons and they need to form synaptic connections in order to be functional. This makes the cell therapy with photoreceptors more challenging when compared to RPE cells. Interesting‐ ly, a group from Japan (Eiraku *et al.*, 2011) could obtain formation of a fully stratified neural retina from by using a three dimensional ESCs culture system. The 3D organoids would open up new avenues for the transplantation of artificial retinal tissue sheets, rather than

Until shortly, the most relevant clinical studies currently being conducted in patients with reti‐ nal degeneration were fetal retinal sheet transplants (Radtke *et al.*, 2008). This strategy has its basis on the fact that immature retinal sheet extends cell processes and forms synaptic connec‐ tions with the degenerate host retina. The underlying principle is that the inner retinal neurons of the host remain intact and therefore only require synaptic connections with photoreceptors for visual function to be restored. One big problem for the application of photoreceptor cell

Advanced Cell Technology and Jules Stein Eye Institute at UCLA started two prospective clinical studies to establish the safety and tolerability of subretinal transplantation of human ESC-derived retinal pigment epithelium (RPE) in patients with Stargardt's macular dystro‐ phy (clinical trial identifier-NCT01469832) and dry age-related macular degeneration (clini‐ cal trial identifier-NCT01344993) — the leading cause of blindness in the developed world (Schwartz *et al.*, 2012). The studies are in phase I/II, where only the safety and tolerability of human ESC-derived RPE cells is assessed. The team of researchers from ACT and UCLA re‐ ported their preliminary work in two patients, one with AMD, the other with Stargardt's macular dystrophy, being the first to publish data on the use of human ESC-derived cells in

transplantation is that an appropriate source of the precursor cells is required.

simple cell grafting.

the clinic (Schwartz *et al.*, 2012).

**2.2. Clinical trial**

400 Pluripotent Stem Cells

**Figure 1.** Scheme of procedure for replacing damaged retinal pigment epithelium cells.

Two patients enrolled in the clinical trial in order to test the safety of such cell trans‐ plantations. 50 000 viable RPE cells differentiated from the hESC line MA09 (Kliman‐ skaya *et al.*, 2006) by embryoid body formation were injected into the subretinal space of each patient's eye (see Fig. 1 for schematic overview). The cells were resuspended in phosphate buffered saline (PBS) and delivered in a region of pericentral macula that was not completely lost to the disease. The authors thought that engraftment of the cells into a completely atrophic macula was unlikely due to the loss of Bruch's membrane. The primary outcome was positive: none of the concerns related to stem cell transplantations as teratomas, rejection, or inflammation were observed. The transplanted cells attached to Bruch's membrane and persisted for the duration of the observation period. This was however possible only in one of the two patients. Moreover, clear functional visual im‐ provement was noted in the patient with Stargardt's macular dystrophy.

There are many repair strategies in spinal cord injury, as prevention of cell death by antiglutamatergic drugs, promotion of axonal regeneration, compensation of the lost myelina‐ tion or cell replacement therapy (McDonald *et al.*, 2002; McDonald & Sadowsky, 2002). Different sources and types of cells, including stem/progenitor cells (embryonic stem cells, neural progenitor cells, bone marrow mesenchymal cells) and non-stem cells (olfactory en‐ sheathing cells [OECs] and Schwann cells) have been, and/or are being tested in clinical tri‐

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As mentioned before in the case of spinal cord injury, diseases of the nervous system in‐ volve proliferation of astrocytes and loss of oligodendrocytes (OLN) and the protective myelin sheath they produce. Transplantation of oligodendrocyte precursors in different animals systems show that these precursors can myelinate axons (Groves *et al.*, 1993). Thus, derivation of oligodendrocytes from ESCs has been an important goal for cell re‐ placement therapy. The most common protocols involve an initial differentiation step to neural progenitors (Reubinoff *et al.*, 2001), followed by expansion, further differentiation, and selection. These protocols follow the differentiation steps that take place *in vivo*. Dur‐ ing development, oligodendrocytes differentiate from precursors, which migrate and pro‐ liferate, through immature oligodendrocytes, which send out processes seeking axons to myelinate, to mature myelinating oligodendrocytes that form myelin sheaths. The precur‐ sor cells are morphologically bipolar (when migrating) or stellate (after migration). These initially differentiate into immature cells that put out processes seeking axons to myeli‐ nate, and eventually form mature cells with parallel processes myelinating up to 30 dif‐

Oligodendrocytes were first efficiently derived from mouse ESCs (Brustle *et al.*, 1999), where ESCs were aggregated to embryoid bodies and plated in a defined medium that favors the survival of ES cell–derived neural precursors, followed by the expansion of progenitors in culture medium containing FGF2 and EGF, and a switch to FGF2 and PDGF to yield bipo‐ tential glial progenitors (Brustle *et al.*, 1999). These glial progenitors were transplanted into the spinal cords of rats with a genetic deficiency in myelin production, yielding myelinated fibers in the majority of animals (Learish *et al.*, 1999). Human ESCs were first shown to dif‐ ferentiate into oligodendrocytes by Zhang et al., 2001, who used a similar strategy involving FGF treatment followed by growth as neurospheres (Zhang *et al.*, 2001). They reported the differentiation of neural precursors into neurons, astrocytes and oligodendrocytes. Howev‐ er, no human oligodendrocytes were detected after transplantation of neural precursors into the brains of newborn mice, although human neurons and some astrocytes were found to

The first detailed protocol for directed differentiation of oligodendrocytes from human ESCs was published in 2005 and involved the induction of neural lineage by retinoic acid treat‐ ment, followed by expansion and selection in various media containing the differentiation factors triiodothyroidin hormone, FGF2, EGF, and insulin (Nistor *et al.*, 2005). After 42 days of culture, the desired cells were found in yellow spheroids, which upon differentiation as

als for spinal cord injury (Fehlings & Vawda, 2011).

**3.1. Differentiation to oligodendrocytes**

ferent axons (Karadottir & Attwell, 2007).

have engrafted (Zhang *et al.*, 2001).

This is the first peer reviewed study that uses human ESCs for cell therapy. Although their report is preliminary, in only two patients, and with a short-term follow-up, the results are impressive - especially considering the progressive nature of both diseases (Atala, 2012).

## **3. Treatment of spinal cord injury**

More than a decade ago, spinal-cord injury meant confinement to a wheelchair and a lifetime of medical care. Published incidence rates for traumatic spinal-cord injury in the USA range between 28 and 55 per million people, with about 10 000 new cases reported every year. Causes include motor vehicle accidents (36–48%), violence (5–29%), falls (17– 21%), and recreational activities (7–16%) (McDonald & Sadowsky, 2002). The primary in‐ jury (the initial insult) is usually due to the mechanical trauma and includes traction and compression forces. Neural elements are compressed by fractured and displaced bone fragments, disc material, and ligaments and leads to injuries on both the central and pe‐ ripheral nervous systems. Blood vessels are damaged, axons disrupted and cell mem‐ branes broken. Micro-haemorrhages occur within minutes in the central grey matter and spread out over the next few hours. Within minutes, the spinal cord swells to occupy the entire diameter of the spinal canal at the injury level. Secondary ischaemia results when cord swelling exceeds venous blood pressure. The more destructive phase of secondary injury is, however, more responsible for cell death and functional deficits. Hemorrhage, edema, ischaemia, release of toxic chemicals from disrupted neural membranes, and elec‐ trolyte shifts trigger a secondary injury cascade that substantially compounds initial me‐ chanical damage by harming or killing neighbouring cells (McDonald & Sadowsky, 2002). Glutamate plays a key part in a highly disruptive process known as excitotoxicity. It was demonstrated that glutamate, released during injury, damages oligodendocytes (Domercq *et al.*, 2005). Oligodendrocytes express glutamate receptors as NMDA (Karadot‐ tir *et al.*, 2005) and AMPA/kainate receptors (Domercq *et al.*, 1999). Up to now, the pri‐ mary approach in treatment is limitation of secondary injury by removal of damaging bone, disc, and ligament fragments to decompress the swollen cord, followed by the ad‐ ministration of the steroid methyl-prednisolone (Bracken *et al.*, 1990).

There are many repair strategies in spinal cord injury, as prevention of cell death by antiglutamatergic drugs, promotion of axonal regeneration, compensation of the lost myelina‐ tion or cell replacement therapy (McDonald *et al.*, 2002; McDonald & Sadowsky, 2002). Different sources and types of cells, including stem/progenitor cells (embryonic stem cells, neural progenitor cells, bone marrow mesenchymal cells) and non-stem cells (olfactory en‐ sheathing cells [OECs] and Schwann cells) have been, and/or are being tested in clinical tri‐ als for spinal cord injury (Fehlings & Vawda, 2011).

#### **3.1. Differentiation to oligodendrocytes**

Two patients enrolled in the clinical trial in order to test the safety of such cell trans‐ plantations. 50 000 viable RPE cells differentiated from the hESC line MA09 (Kliman‐ skaya *et al.*, 2006) by embryoid body formation were injected into the subretinal space of each patient's eye (see Fig. 1 for schematic overview). The cells were resuspended in phosphate buffered saline (PBS) and delivered in a region of pericentral macula that was not completely lost to the disease. The authors thought that engraftment of the cells into a completely atrophic macula was unlikely due to the loss of Bruch's membrane. The primary outcome was positive: none of the concerns related to stem cell transplantations as teratomas, rejection, or inflammation were observed. The transplanted cells attached to Bruch's membrane and persisted for the duration of the observation period. This was however possible only in one of the two patients. Moreover, clear functional visual im‐

This is the first peer reviewed study that uses human ESCs for cell therapy. Although their report is preliminary, in only two patients, and with a short-term follow-up, the results are impressive - especially considering the progressive nature of both diseases (Atala, 2012).

More than a decade ago, spinal-cord injury meant confinement to a wheelchair and a lifetime of medical care. Published incidence rates for traumatic spinal-cord injury in the USA range between 28 and 55 per million people, with about 10 000 new cases reported every year. Causes include motor vehicle accidents (36–48%), violence (5–29%), falls (17– 21%), and recreational activities (7–16%) (McDonald & Sadowsky, 2002). The primary in‐ jury (the initial insult) is usually due to the mechanical trauma and includes traction and compression forces. Neural elements are compressed by fractured and displaced bone fragments, disc material, and ligaments and leads to injuries on both the central and pe‐ ripheral nervous systems. Blood vessels are damaged, axons disrupted and cell mem‐ branes broken. Micro-haemorrhages occur within minutes in the central grey matter and spread out over the next few hours. Within minutes, the spinal cord swells to occupy the entire diameter of the spinal canal at the injury level. Secondary ischaemia results when cord swelling exceeds venous blood pressure. The more destructive phase of secondary injury is, however, more responsible for cell death and functional deficits. Hemorrhage, edema, ischaemia, release of toxic chemicals from disrupted neural membranes, and elec‐ trolyte shifts trigger a secondary injury cascade that substantially compounds initial me‐ chanical damage by harming or killing neighbouring cells (McDonald & Sadowsky, 2002). Glutamate plays a key part in a highly disruptive process known as excitotoxicity. It was demonstrated that glutamate, released during injury, damages oligodendocytes (Domercq *et al.*, 2005). Oligodendrocytes express glutamate receptors as NMDA (Karadot‐ tir *et al.*, 2005) and AMPA/kainate receptors (Domercq *et al.*, 1999). Up to now, the pri‐ mary approach in treatment is limitation of secondary injury by removal of damaging bone, disc, and ligament fragments to decompress the swollen cord, followed by the ad‐

provement was noted in the patient with Stargardt's macular dystrophy.

ministration of the steroid methyl-prednisolone (Bracken *et al.*, 1990).

**3. Treatment of spinal cord injury**

402 Pluripotent Stem Cells

As mentioned before in the case of spinal cord injury, diseases of the nervous system in‐ volve proliferation of astrocytes and loss of oligodendrocytes (OLN) and the protective myelin sheath they produce. Transplantation of oligodendrocyte precursors in different animals systems show that these precursors can myelinate axons (Groves *et al.*, 1993). Thus, derivation of oligodendrocytes from ESCs has been an important goal for cell re‐ placement therapy. The most common protocols involve an initial differentiation step to neural progenitors (Reubinoff *et al.*, 2001), followed by expansion, further differentiation, and selection. These protocols follow the differentiation steps that take place *in vivo*. Dur‐ ing development, oligodendrocytes differentiate from precursors, which migrate and pro‐ liferate, through immature oligodendrocytes, which send out processes seeking axons to myelinate, to mature myelinating oligodendrocytes that form myelin sheaths. The precur‐ sor cells are morphologically bipolar (when migrating) or stellate (after migration). These initially differentiate into immature cells that put out processes seeking axons to myeli‐ nate, and eventually form mature cells with parallel processes myelinating up to 30 dif‐ ferent axons (Karadottir & Attwell, 2007).

Oligodendrocytes were first efficiently derived from mouse ESCs (Brustle *et al.*, 1999), where ESCs were aggregated to embryoid bodies and plated in a defined medium that favors the survival of ES cell–derived neural precursors, followed by the expansion of progenitors in culture medium containing FGF2 and EGF, and a switch to FGF2 and PDGF to yield bipo‐ tential glial progenitors (Brustle *et al.*, 1999). These glial progenitors were transplanted into the spinal cords of rats with a genetic deficiency in myelin production, yielding myelinated fibers in the majority of animals (Learish *et al.*, 1999). Human ESCs were first shown to dif‐ ferentiate into oligodendrocytes by Zhang et al., 2001, who used a similar strategy involving FGF treatment followed by growth as neurospheres (Zhang *et al.*, 2001). They reported the differentiation of neural precursors into neurons, astrocytes and oligodendrocytes. Howev‐ er, no human oligodendrocytes were detected after transplantation of neural precursors into the brains of newborn mice, although human neurons and some astrocytes were found to have engrafted (Zhang *et al.*, 2001).

The first detailed protocol for directed differentiation of oligodendrocytes from human ESCs was published in 2005 and involved the induction of neural lineage by retinoic acid treat‐ ment, followed by expansion and selection in various media containing the differentiation factors triiodothyroidin hormone, FGF2, EGF, and insulin (Nistor *et al.*, 2005). After 42 days of culture, the desired cells were found in yellow spheroids, which upon differentiation as low-density monolayers formed 85%–95% oligodendrocytes expressing typical markers as GalC, RIP, and O4. Human embryonic stem cell (hESC)-derived oligodendrocytes were able to integrate, differentiate and display a functional myelinating phenotype following trans‐ plantation into the shiverer mutant mouse (Nistor *et al.*, 2005). Recently, other protocols were developed for generation of oligodendrocytes from ESCs. The Neman and de Vellis (Neman & de Vellis, 2012) laboratory has reported usage of defined serum-free media to‐ gether with morphogens, as retinoic acid and sonic hedgehog, to devise a new method to derive a pure population of OLN from ESCs. These experiments show that human oligoden‐ drocytes can be generated in large numbers and used to restore myelination under some cir‐ cumstances in mice.

#### **3.2. Clinical trial**

In October 2010 the world's first clinical trial using human embryonic stem cells began, using ESCs converted into OLN precursor cells. The feasibility of the treatment was proofed by a wide range of pre-clinical studies that have shown that human oligoden‐ drocyte progenitor cells implanted after spinal cord injury in rodent models show func‐ tional improvement (Keirstead, 2005; Keirstead *et al.*, 2005; Sharp *et al.*, 2010). Geron of Menlo Park, California, is the biotech company that received FDA approval to proceed with clinical trials that transplant cells derived from embryonic stem cells into the spinal cord (Alper, 2009). This company has pioneered translational research into human ESC therapies. The Geron trial (trial identification number NCT01217008), which was original‐ ly approved by the FDA, but then halted due to concerns of abnormal cyst formation, was reinitiated and approved for phase I clinical trials in the U.S. in October 2010. The trial was suspended following news that animals in a dose-escalation study developed microscopic cysts in regenerating tissue sites. In november 2011 Geron announced that it is dropping its entire program owing to financial concerns and started looking for part‐ ners for stem cell treatments and decided to not further invest in the clinical trials in‐ volving treatments with ESCs.

**Figure 2.** Scheme of procedure for treating spinal cord injury with human ESCs derived oligodendrocyte precursor cells.

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http://dx.doi.org/10.5772/54368

405

The major safety concerns for the use of hESCs are related to the achievement of xenobi‐ otic-free culture conditions, avoidance of genetic abnormalities, development of good dif‐ ferentiation and selection protocols, and the avoidance of the immune rejection. Moreover, the unlimited proliferative capacity of ESCs is a disadvantage in clinical appli‐ cations because this could cause tumor formation upon transplantation. When implanted in an undifferentiated state, ESCs cause teratoma, a tumor type that consists of different kinds of differentiated cells. Teratomas are encapsulated, usually benign tumors that can occur naturally, but there is the fear, based on some animal studies, that some propor‐ tion of the cells derived from ESCs injected into the body could drift from their intended developmental pathway. Teratoma formation was reported in various cases when mouse

**4. Embryonic stem cells and tumorigenesis**

The trial was planned to involve treating ten patients who have suffered a complete thora‐ cic-level spinal cord injury in a phase 1 multicenter trial. The pioneering therapy is Geron's 'GRNOPC1 product', which contains hES cell–derived oligodendrocyte progenitor cells that have demonstrated remyelinating and nerve growth–stimulating properties. In the human SCI lesion site, it is hoped that OLN precursors will work as a "combination therapy" - phe‐ notypically replacing lost oligodendrocytes and hence remyelinating axons that have be‐ come demyelinated during injury, as well as secreting neurotrophic factors to establish a repair environment in the lesion (Hatch *et al.*, 2009). The ESCs were differentiated into OLN precursors (Hatch *et al.*, 2009) and one injection of 2 million GRNOPC1 cells was adminis‐ tered within 2 weeks in patients with thoracic spinal cord injury (Fig. 2). No serious adverse effects were observed in the 2 patients enrolled, only one of the patients experienced some side effects due to the immunosupression (Watson & Yeung, 2011). However, the data gen‐ erated by Geron for the FDA are not published and no preliminary report on the safety of their product is available up to now.

**Figure 2.** Scheme of procedure for treating spinal cord injury with human ESCs derived oligodendrocyte precursor cells.

## **4. Embryonic stem cells and tumorigenesis**

low-density monolayers formed 85%–95% oligodendrocytes expressing typical markers as GalC, RIP, and O4. Human embryonic stem cell (hESC)-derived oligodendrocytes were able to integrate, differentiate and display a functional myelinating phenotype following trans‐ plantation into the shiverer mutant mouse (Nistor *et al.*, 2005). Recently, other protocols were developed for generation of oligodendrocytes from ESCs. The Neman and de Vellis (Neman & de Vellis, 2012) laboratory has reported usage of defined serum-free media to‐ gether with morphogens, as retinoic acid and sonic hedgehog, to devise a new method to derive a pure population of OLN from ESCs. These experiments show that human oligoden‐ drocytes can be generated in large numbers and used to restore myelination under some cir‐

In October 2010 the world's first clinical trial using human embryonic stem cells began, using ESCs converted into OLN precursor cells. The feasibility of the treatment was proofed by a wide range of pre-clinical studies that have shown that human oligoden‐ drocyte progenitor cells implanted after spinal cord injury in rodent models show func‐ tional improvement (Keirstead, 2005; Keirstead *et al.*, 2005; Sharp *et al.*, 2010). Geron of Menlo Park, California, is the biotech company that received FDA approval to proceed with clinical trials that transplant cells derived from embryonic stem cells into the spinal cord (Alper, 2009). This company has pioneered translational research into human ESC therapies. The Geron trial (trial identification number NCT01217008), which was original‐ ly approved by the FDA, but then halted due to concerns of abnormal cyst formation, was reinitiated and approved for phase I clinical trials in the U.S. in October 2010. The trial was suspended following news that animals in a dose-escalation study developed microscopic cysts in regenerating tissue sites. In november 2011 Geron announced that it is dropping its entire program owing to financial concerns and started looking for part‐ ners for stem cell treatments and decided to not further invest in the clinical trials in‐

The trial was planned to involve treating ten patients who have suffered a complete thora‐ cic-level spinal cord injury in a phase 1 multicenter trial. The pioneering therapy is Geron's 'GRNOPC1 product', which contains hES cell–derived oligodendrocyte progenitor cells that have demonstrated remyelinating and nerve growth–stimulating properties. In the human SCI lesion site, it is hoped that OLN precursors will work as a "combination therapy" - phe‐ notypically replacing lost oligodendrocytes and hence remyelinating axons that have be‐ come demyelinated during injury, as well as secreting neurotrophic factors to establish a repair environment in the lesion (Hatch *et al.*, 2009). The ESCs were differentiated into OLN precursors (Hatch *et al.*, 2009) and one injection of 2 million GRNOPC1 cells was adminis‐ tered within 2 weeks in patients with thoracic spinal cord injury (Fig. 2). No serious adverse effects were observed in the 2 patients enrolled, only one of the patients experienced some side effects due to the immunosupression (Watson & Yeung, 2011). However, the data gen‐ erated by Geron for the FDA are not published and no preliminary report on the safety of

cumstances in mice.

volving treatments with ESCs.

their product is available up to now.

**3.2. Clinical trial**

404 Pluripotent Stem Cells

The major safety concerns for the use of hESCs are related to the achievement of xenobi‐ otic-free culture conditions, avoidance of genetic abnormalities, development of good dif‐ ferentiation and selection protocols, and the avoidance of the immune rejection. Moreover, the unlimited proliferative capacity of ESCs is a disadvantage in clinical appli‐ cations because this could cause tumor formation upon transplantation. When implanted in an undifferentiated state, ESCs cause teratoma, a tumor type that consists of different kinds of differentiated cells. Teratomas are encapsulated, usually benign tumors that can occur naturally, but there is the fear, based on some animal studies, that some propor‐ tion of the cells derived from ESCs injected into the body could drift from their intended developmental pathway. Teratoma formation was reported in various cases when mouse ESCs-derived cells like insulin producing islets (Fujikawa *et al.*, 2005), ESC-derived cardi‐ omyocytes (Cao *et al.*, 2006), and ESC-derived neurons (Schuldiner *et al.*, 2001) were transplanted into immunosuppressed mice even though there was successful engraftment and functional improvement. When undifferentiated human ESCs were injected into the hind limb muscles or under the kidney capsule of SCID mice, teratomas were readily formed after 8–12 weeks (Richards *et al.*, 2002). Evidence of tumor formation has also been observed in differentiated hESC derivatives transplanted *in vivo* (Roy *et al.,* 2006). In another study, successful hESC-derived neuronal engraftment in a Parkinsonian rat model did not yield teratomas after 12 weeks (Ben-Hur *et al.*, 2004). When hESC-derived osteocytes or cardiomyocytes were transplanted into the bone or heart of severe com‐ bined immunodeficient mice (SCID), there was also no teratoma production within 1 month after injection (Bielby *et al.*, 2004; Laflamme *et al.*, 2007). It seems that the longer hESCs are differentiated *in vitro*, the risk of teratoma formation appears to be reduced. Certain sites appear to favor the growth of teratomas, while others do not, confirming a phenomenon already described that tumorigenesis of ESCs is site dependent. For exam‐ ple the rate of teratoma formation with hESCs in immunodeficient mice was subcutane‐ ously 25–100%, intratesticularly 60%, intramuscularly 12.5% and under the kidney capsule 100% (Prokhorova *et al.*, 2009). Furthermore, tumor formation in the lung and thymus had the highest probability of teratoma formation while the pancreas was parti‐ ally site-privileged (Shih *et al.*, 2007). Shih et al. observed an aggressive growth of tu‐ mors when human ESCs were injected into engrafted human fetal tissues in SCID mice (Shih *et al.*, 2007).

hESCs (Bongso *et al.*, 2008). It is also very important to develop very good and reliable methods to detect residual ESCs contamination in ESCs derived cells prior to clinical ap‐ plication. In their review, Fong et al. (Fong *et al.*, 2010) presented some available meth‐ ods for the elimination of undifferentiated ESCs. These included single cell propagation with encapsulation, usage of density gradients, MACS and FACS, usage of tumor privi‐ leged sites, usage of antibodies against undifferentiated ESCs, prolonged differentiation in vitro before transplantation or destruction of teratoma after engraftment. However, be‐ cause differentiation is not an on/off process, it is probably the best to use a combination

Embryonic Stem Cell Therapy – From Bench to Bed

http://dx.doi.org/10.5772/54368

407

**5. Embryonic stem cells versus induced pluripotent stem cells in clinics**

Induced pluripotent cells (iPS) are generated by re-engineering mature, fully differentiat‐ ed cells (e.g. human skin fibroblasts) by modifying the cells with a set of transgenes (Ta‐ kahashi & Yamanaka, 2006; Takahashi *et al.*, 2007). Induced pluripotent stem cells, created by turning back the developmental clock on adult tissues, display similar geneexpression patterns to ESCs, and can produce various tissues in the human body. How‐ ever, iPS cells have a major advantage over ESCs; they can be obtained directly from the individual that has to be treated. Thus, as a source of cells for therapy, they are able to avoid the immunocompatibility issues. Furthermore, the utilization of these stem cells in both clinical and basic research studies does not face ethical and political issues that oth‐

During the last years various studies reported the differentiation of iPS cells to various types of cells in vitro and these cells were used for cellular therapy in various mouse models (Wer‐

However, before bringing these cells into the clinics, their safety should be tested. For example, the initial enthusiasm related to bringing iPS cells into clinics dampened when it was shown that these cells develop teratoma more efficiently than ESCs (Gutierrez-Aranda *et al.*, 2010). It was also shown that iPS retain the epigenetic memory of the cells from which they are derived; this fact makes them to preferentially differentiate into the cell lineage from which they came from. Future clinical applications will demand new techniques for generating factor-free iPS cells such as virus-free or DNA-free approaches at acceptable efficiencies. There are also other disadvantages in using iPS cells in the clinics. Usually, they are made by integrating retroviruses into the cells as shuttle for the reprogramming factors. This problem may be solved by transient gene transfer or by de‐ livering the pluripotency factors in protein form (Murry & Keller, 2008). The second is that iPS cells are not an ''off-the-shelf'' product and would likely only be produced after the patient becomes ill, precluding their use in the acute phase of the disease (Murry & Keller, 2008). Quality control is will also be difficult and expensive, because a separate

of these methods in order to do safe cell therapy.

erwise surround the use of embryonic stem cells.

batch of iPS cells would have to be made for each patient.

nig *et al.*, 2008; Saha & Jaenisch, 2009).

The simplest way to slow or even eliminate the tumorigenicity of normal stem cells prior to transplantation may be to take advantage of pluripotency by partially differentiating them into progenitors. Therefore, a promising proposed method for making stem cell-based re‐ generative medicine therapies safer may seem paradoxical: to not transplant stem cells at all into patients. The idea is to use the stem cells to produce progenitor or precursor cells of the desired lineage and then transplant progenitors purified by sorting (Knoepfler, 2009). This approach was presented in this chapter and is actually used in the clinical trial with oligo‐ dendrocyte progenitor cells. However, not only the embryonic stem cells, but also the im‐ planted precursor cells seem to form teratoma in some cases. A group of Israeli researchers reported that a boy with ataxia telangiectasia who had received several fetal neural stem cell transplants developed teratomas in his brain and spinal cord four years after treatment (Amariglio *et al.*, 2009). For this reason is very important to achieve a 100% pure population of differentiated cells when using ESCs for cell therapy.

Currently, the only way to ensure that teratomas do not form is to differentiate the ESCs in advance, enrich for the desired cell type, and screen for the presence of undifferentiat‐ ed cells. The elimination of undifferentiated hESCs may best be achieved by (1) destroy‐ ing the remaining undifferentiated hESCs in the differentiated tissue population with specific agents or antibodies, (2) separating or removing the undifferentiated hESCs from the differentiated cell population, (3) eliminating pluripotent cells during the differentia‐ tion process, and (4) inducing further differentiation of left-over rogue undifferentiated hESCs (Bongso *et al.*, 2008). It is also very important to develop very good and reliable methods to detect residual ESCs contamination in ESCs derived cells prior to clinical ap‐ plication. In their review, Fong et al. (Fong *et al.*, 2010) presented some available meth‐ ods for the elimination of undifferentiated ESCs. These included single cell propagation with encapsulation, usage of density gradients, MACS and FACS, usage of tumor privi‐ leged sites, usage of antibodies against undifferentiated ESCs, prolonged differentiation in vitro before transplantation or destruction of teratoma after engraftment. However, be‐ cause differentiation is not an on/off process, it is probably the best to use a combination of these methods in order to do safe cell therapy.

ESCs-derived cells like insulin producing islets (Fujikawa *et al.*, 2005), ESC-derived cardi‐ omyocytes (Cao *et al.*, 2006), and ESC-derived neurons (Schuldiner *et al.*, 2001) were transplanted into immunosuppressed mice even though there was successful engraftment and functional improvement. When undifferentiated human ESCs were injected into the hind limb muscles or under the kidney capsule of SCID mice, teratomas were readily formed after 8–12 weeks (Richards *et al.*, 2002). Evidence of tumor formation has also been observed in differentiated hESC derivatives transplanted *in vivo* (Roy *et al.,* 2006). In another study, successful hESC-derived neuronal engraftment in a Parkinsonian rat model did not yield teratomas after 12 weeks (Ben-Hur *et al.*, 2004). When hESC-derived osteocytes or cardiomyocytes were transplanted into the bone or heart of severe com‐ bined immunodeficient mice (SCID), there was also no teratoma production within 1 month after injection (Bielby *et al.*, 2004; Laflamme *et al.*, 2007). It seems that the longer hESCs are differentiated *in vitro*, the risk of teratoma formation appears to be reduced. Certain sites appear to favor the growth of teratomas, while others do not, confirming a phenomenon already described that tumorigenesis of ESCs is site dependent. For exam‐ ple the rate of teratoma formation with hESCs in immunodeficient mice was subcutane‐ ously 25–100%, intratesticularly 60%, intramuscularly 12.5% and under the kidney capsule 100% (Prokhorova *et al.*, 2009). Furthermore, tumor formation in the lung and thymus had the highest probability of teratoma formation while the pancreas was parti‐ ally site-privileged (Shih *et al.*, 2007). Shih et al. observed an aggressive growth of tu‐ mors when human ESCs were injected into engrafted human fetal tissues in SCID mice

The simplest way to slow or even eliminate the tumorigenicity of normal stem cells prior to transplantation may be to take advantage of pluripotency by partially differentiating them into progenitors. Therefore, a promising proposed method for making stem cell-based re‐ generative medicine therapies safer may seem paradoxical: to not transplant stem cells at all into patients. The idea is to use the stem cells to produce progenitor or precursor cells of the desired lineage and then transplant progenitors purified by sorting (Knoepfler, 2009). This approach was presented in this chapter and is actually used in the clinical trial with oligo‐ dendrocyte progenitor cells. However, not only the embryonic stem cells, but also the im‐ planted precursor cells seem to form teratoma in some cases. A group of Israeli researchers reported that a boy with ataxia telangiectasia who had received several fetal neural stem cell transplants developed teratomas in his brain and spinal cord four years after treatment (Amariglio *et al.*, 2009). For this reason is very important to achieve a 100% pure population

Currently, the only way to ensure that teratomas do not form is to differentiate the ESCs in advance, enrich for the desired cell type, and screen for the presence of undifferentiat‐ ed cells. The elimination of undifferentiated hESCs may best be achieved by (1) destroy‐ ing the remaining undifferentiated hESCs in the differentiated tissue population with specific agents or antibodies, (2) separating or removing the undifferentiated hESCs from the differentiated cell population, (3) eliminating pluripotent cells during the differentia‐ tion process, and (4) inducing further differentiation of left-over rogue undifferentiated

of differentiated cells when using ESCs for cell therapy.

(Shih *et al.*, 2007).

406 Pluripotent Stem Cells

## **5. Embryonic stem cells versus induced pluripotent stem cells in clinics**

Induced pluripotent cells (iPS) are generated by re-engineering mature, fully differentiat‐ ed cells (e.g. human skin fibroblasts) by modifying the cells with a set of transgenes (Ta‐ kahashi & Yamanaka, 2006; Takahashi *et al.*, 2007). Induced pluripotent stem cells, created by turning back the developmental clock on adult tissues, display similar geneexpression patterns to ESCs, and can produce various tissues in the human body. How‐ ever, iPS cells have a major advantage over ESCs; they can be obtained directly from the individual that has to be treated. Thus, as a source of cells for therapy, they are able to avoid the immunocompatibility issues. Furthermore, the utilization of these stem cells in both clinical and basic research studies does not face ethical and political issues that oth‐ erwise surround the use of embryonic stem cells.

During the last years various studies reported the differentiation of iPS cells to various types of cells in vitro and these cells were used for cellular therapy in various mouse models (Wer‐ nig *et al.*, 2008; Saha & Jaenisch, 2009).

However, before bringing these cells into the clinics, their safety should be tested. For example, the initial enthusiasm related to bringing iPS cells into clinics dampened when it was shown that these cells develop teratoma more efficiently than ESCs (Gutierrez-Aranda *et al.*, 2010). It was also shown that iPS retain the epigenetic memory of the cells from which they are derived; this fact makes them to preferentially differentiate into the cell lineage from which they came from. Future clinical applications will demand new techniques for generating factor-free iPS cells such as virus-free or DNA-free approaches at acceptable efficiencies. There are also other disadvantages in using iPS cells in the clinics. Usually, they are made by integrating retroviruses into the cells as shuttle for the reprogramming factors. This problem may be solved by transient gene transfer or by de‐ livering the pluripotency factors in protein form (Murry & Keller, 2008). The second is that iPS cells are not an ''off-the-shelf'' product and would likely only be produced after the patient becomes ill, precluding their use in the acute phase of the disease (Murry & Keller, 2008). Quality control is will also be difficult and expensive, because a separate batch of iPS cells would have to be made for each patient.

## **6. Conclusion**

There is no doubt that after the hurdles are overcome, hESC-derived cells have a promising future for transplantation therapy given the versatility of these cells. It is very encouraging to see that clinical trials involving the use of hESCs have begun, and that extensive efforts are underway to efficiently, and safely differentiate hESCs into specific cell types.

[6] Ben-Hur, T., Idelson, M., Khaner, H., Pera, M., Reinhartz, E., Itzik, A. & Reubinoff, B.E. (2004) Transplantation of human embryonic stem cell-derived neural progeni‐ tors improves behavioral deficit in Parkinsonian rats. Stem Cells, 22, 1246-1255.

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http://dx.doi.org/10.5772/54368

409

[7] Bielby, R.C., Boccaccini, A.R., Polak, J.M. & Buttery, L.D. (2004) In vitro differentia‐ tion and in vivo mineralization of osteogenic cells derived from human embryonic

[8] Bongso, A., Fong, C.Y. & Gauthaman, K. (2008) Taking stem cells to the clinic: Major

[9] Bracken, M.B., Shepard, M.J., Collins, W.F., Holford, T.R., Young, W., Baskin, D.S., Eisenberg, H.M., Flamm, E., Leo-Summers, L., Maroon, J. & et al. (1990) A random‐ ized, controlled trial of methylprednisolone or naloxone in the treatment of acute spi‐ nal-cord injury. Results of the Second National Acute Spinal Cord Injury Study. The

[10] Brons, I.G., Smithers, L.E., Trotter, M.W., Rugg-Gunn, P., Sun, B., Chuva de Sousa Lopes, S.M., Howlett, S.K., Clarkson, A., Ahrlund-Richter, L., Pedersen, R.A. & Valli‐ er, L. (2007) Derivation of pluripotent epiblast stem cells from mammalian embryos.

[11] Brustle, O., Jones, K.N., Learish, R.D., Karram, K., Choudhary, K., Wiestler, O.D., Duncan, I.D. & McKay, R.D. (1999) Embryonic stem cell-derived glial precursors: a

[12] Cao, F., Lin, S., Xie, X., Ray, P., Patel, M., Zhang, X., Drukker, M., Dylla, S.J., Connol‐ ly, A.J., Chen, X., Weissman, I.L., Gambhir, S.S. & Wu, J.C. (2006) In vivo visualiza‐ tion of embryonic stem cell survival, proliferation, and migration after cardiac

[13] Do, J.T., Han, D.W. & Scholer, H.R. (2006) Reprogramming somatic gene activity by

[14] Domercq, M., Etxebarria, E., Perez-Samartin, A. & Matute, C. (2005) Excitotoxic oli‐ godendrocyte death and axonal damage induced by glutamate transporter inhibi‐

[15] Domercq, M., Sanchez-Gomez, M.V., Areso, P. & Matute, C. (1999) Expression of glu‐ tamate transporters in rat optic nerve oligodendrocytes. The European journal of

[16] Eiraku, M., Takata, N., Ishibashi, H., Kawada, M., Sakakura, E., Okuda, S., Sekiguchi, K., Adachi, T. & Sasai, Y. (2011) Self-organizing optic-cup morphogenesis in three-di‐

[17] Evans, M.J. (1972) The isolation and properties of a clonal tissue culture strain of plu‐ ripotent mouse teratoma cells. Journal of embryology and experimental morphology,

stem cells. Tissue engineering, 10, 1518-1525.

New England journal of medicine, 322, 1405-1411.

source of myelinating transplants. Science, 285, 754-756.

fusion with pluripotent cells. Stem cell reviews, 2, 257-264.

delivery. Circulation, 113, 1005-1014.

Nature, 448, 191-195.

tion. Glia, 52, 36-46.

28, 163-176.

neuroscience, 11, 2226-2236.

mensional culture. Nature, 472, 51-56.

challenges. Journal of cellular biochemistry, 105, 1352-1360.

## **Acknowledgements**

I would like to thank Paul G. Layer for carefully reading the manuscript.

## **Author details**

Laura E. Sperling

Technische Universität Darmstadt, Fachbereich Biologie, Entwicklungsbiologie & Neuroge‐ netik, Darmstadt, Germany

### **References**


[6] Ben-Hur, T., Idelson, M., Khaner, H., Pera, M., Reinhartz, E., Itzik, A. & Reubinoff, B.E. (2004) Transplantation of human embryonic stem cell-derived neural progeni‐ tors improves behavioral deficit in Parkinsonian rats. Stem Cells, 22, 1246-1255.

**6. Conclusion**

408 Pluripotent Stem Cells

**Acknowledgements**

**Author details**

Laura E. Sperling

**References**

netik, Darmstadt, Germany

e1000029.

cet, 379, 689-690.

There is no doubt that after the hurdles are overcome, hESC-derived cells have a promising future for transplantation therapy given the versatility of these cells. It is very encouraging to see that clinical trials involving the use of hESCs have begun, and that extensive efforts

Technische Universität Darmstadt, Fachbereich Biologie, Entwicklungsbiologie & Neuroge‐

[1] Alge, C.S., Suppmann, S., Priglinger, S.G., Neubauer, A.S., May, C.A., Hauck, S., Welge-Lussen, U., Ueffing, M. & Kampik, A. (2003) Comparative proteome analysis of native differentiated and cultured dedifferentiated human RPE cells. Investigative

[2] Alper, J. (2009) Geron gets green light for human trial of ES cell-derived product. Na‐

[3] Amariglio, N., Hirshberg, A., Scheithauer, B.W., Cohen, Y., Loewenthal, R., Trakh‐ tenbrot, L., Paz, N., Koren-Michowitz, M., Waldman, D., Leider-Trejo, L., Toren, A., Constantini, S. & Rechavi, G. (2009) Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient. PLoS medicine, 6,

[4] Atala, A. (2012) Human embryonic stem cells: early hints on safety and efficacy. Lan‐

[5] Banin, E., Obolensky, A., Idelson, M., Hemo, I., Reinhardtz, E., Pikarsky, E., Ben-Hur, T. & Reubinoff, B. (2006) Retinal incorporation and differentiation of neural precur‐

sors derived from human embryonic stem cells. Stem Cells, 24, 246-257.

are underway to efficiently, and safely differentiate hESCs into specific cell types.

I would like to thank Paul G. Layer for carefully reading the manuscript.

ophthalmology & visual science, 44, 3629-3641.

ture biotechnology, 27, 213-214.


[18] Evans, M.J. & Kaufman, M.H. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature, 292, 154-156.

[31] Katta, S., Kaur, I. & Chakrabarti, S. (2009) The molecular genetic basis of age-related

Embryonic Stem Cell Therapy – From Bench to Bed

http://dx.doi.org/10.5772/54368

411

[32] Kawasaki, H., Mizuseki, K., Nishikawa, S., Kaneko, S., Kuwana, Y., Nakanishi, S., Nishikawa, S.I. & Sasai, Y. (2000) Induction of midbrain dopaminergic neurons from

[33] Keirstead, H.S. (2005) Stem cells for the treatment of myelin loss. Trends in neuro‐

[34] Keirstead, H.S., Nistor, G., Bernal, G., Totoiu, M., Cloutier, F., Sharp, K. & Steward, O. (2005) Human embryonic stem cell-derived oligodendrocyte progenitor cell trans‐ plants remyelinate and restore locomotion after spinal cord injury. The Journal of neuroscience : the official journal of the Society for Neuroscience, 25, 4694-4705.

[35] Klimanskaya, I., Chung, Y., Becker, S., Lu, S.J. & Lanza, R. (2006) Human embryonic

[36] Klimanskaya, I., Hipp, J., Rezai, K.A., West, M., Atala, A. & Lanza, R. (2004) Deriva‐ tion and comparative assessment of retinal pigment epithelium from human embry‐

[37] Knoepfler, P.S. (2009) Deconstructing stem cell tumorigenicity: a roadmap to safe re‐

[38] Laflamme, M.A., Zbinden, S., Epstein, S.E. & Murry, C.E. (2007) Cell-based therapy for myocardial ischemia and infarction: pathophysiological mechanisms. Annual re‐

[39] Layer P.G., Araki, M. & Vogel-Höpker, A. (2010) New concepts for reconstruction of retinal and pigment epithelial tissues. *Expert Review of Ophthalmology*, 5, No. 4,

[40] Lamba, D.A., Karl, M.O., Ware, C.B. & Reh, T.A. (2006) Efficient generation of retinal progenitor cells from human embryonic stem cells. Proceedings of the National

[41] Learish, R.D., Brustle, O., Zhang, S.C. & Duncan, I.D. (1999) Intraventricular trans‐ plantation of oligodendrocyte progenitors into a fetal myelin mutant results in wide‐

[42] Martin, G.R. (1981) Isolation of a pluripotent cell line from early mouse embryos cul‐ tured in medium conditioned by teratocarcinoma stem cells. Proceedings of the Na‐

[43] Matsui, Y., Zsebo, K. & Hogan, B.L. (1992) Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell, 70, 841-847.

tional Academy of Sciences of the United States of America, 78, 7634-7638.

Academy of Sciences of the United States of America, 103, 12769-12774.

spread formation of myelin. Annals of neurology, 46, 716-722.

stem cell lines derived from single blastomeres. Nature, 444, 481-485.

onic stem cells using transcriptomics. Cloning and stem cells, 6, 217-245.

generative medicine. Stem Cells, 27, 1050-1056.

view of pathology, 2, 307-339.

523-543.

ES cells by stromal cell-derived inducing activity. Neuron, 28, 31-40.

macular degeneration: an overview. J Genet, 88, 425-449.

sciences, 28, 677-683.


[31] Katta, S., Kaur, I. & Chakrabarti, S. (2009) The molecular genetic basis of age-related macular degeneration: an overview. J Genet, 88, 425-449.

[18] Evans, M.J. & Kaufman, M.H. (1981) Establishment in culture of pluripotential cells

[19] Fehlings, M.G. & Vawda, R. (2011) Cellular treatments for spinal cord injury: the time is right for clinical trials. Neurotherapeutics : the journal of the American Soci‐

[20] Fong, C.Y., Gauthaman, K. & Bongso, A. (2010) Teratomas from pluripotent stem

[21] Fujikawa, T., Oh, S.H., Pi, L., Hatch, H.M., Shupe, T. & Petersen, B.E. (2005) Teratoma formation leads to failure of treatment for type I diabetes using embryonic stem cellderived insulin-producing cells. The American journal of pathology, 166, 1781-1791.

[22] Gamm, D.M. & Meyer, J.S. (2010) Directed differentiation of human induced pluripo‐

[23] Groves, A.K., Barnett, S.C., Franklin, R.J., Crang, A.J., Mayer, M., Blakemore, W.F. & Noble, M. (1993) Repair of demyelinated lesions by transplantation of purified O-2A

[24] Gutierrez-Aranda, I., Ramos-Mejia, V., Bueno, C., Munoz-Lopez, M., Real, P.J., Ma‐ cia, A., Sanchez, L., Ligero, G., Garcia-Parez, J.L. & Menendez, P. (2010) Human in‐ duced pluripotent stem cells develop teratoma more efficiently and faster than human embryonic stem cells regardless the site of injection. Stem Cells, 28,

[25] Hatch, M.N., Nistor, G. & Keirstead, H.S. (2009) Derivation of high-purity oligoden‐

[26] Hori, J., Vega, J.L. & Masli, S. (2010) Review of ocular immune privilege in the year 2010: modifying the immune privilege of the eye. Ocular immunology and inflam‐

[27] Idelson, M., Alper, R., Obolensky, A., Ben-Shushan, E., Hemo, I., Yachimovich-Co‐ hen, N., Khaner, H., Smith, Y., Wiser, O., Gropp, M., Cohen, M.A., Even-Ram, S., Ber‐ man-Zaken, Y., Matzrafi, L., Rechavi, G., Banin, E. & Reubinoff, B. (2009) Directed differentiation of human embryonic stem cells into functional retinal pigment epithe‐

[28] Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O., Amit, M., Sor‐ eq, H. & Benvenisty, N. (2000) Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med, 6, 88-95.

[29] Karadottir, R. & Attwell, D. (2007) Neurotransmitter receptors in the life and death of

[30] Karadottir, R., Cavelier, P., Bergersen, L.H. & Attwell, D. (2005) NMDA receptors are expressed in oligodendrocytes and activated in ischaemia. Nature, 438, 1162-1166.

cells: A clinical hurdle. Journal of cellular biochemistry, 111, 769-781.

tent stem cells: a retina perspective. Regenerative medicine, 5, 315-317.

from mouse embryos. Nature, 292, 154-156.

progenitor cells. Nature, 362, 453-455.

droglial progenitors. Methods Mol Biol, 549, 59-75.

1568-1570.

410 Pluripotent Stem Cells

mation, 18, 325-333.

lium cells. Cell stem cell, 5, 396-408.

oligodendrocytes. Neuroscience, 145, 1426-1438.

ety for Experimental NeuroTherapeutics, 8, 704-720.


[44] McDonald, J.W., Becker, D., Sadowsky, C.L., Jane, J.A., Sr., Conturo, T.E. & Schultz, L.M. (2002) Late recovery following spinal cord injury. Case report and review of the literature. Journal of neurosurgery, 97, 252-265.

[56] Richards, M., Fong, C.Y., Chan, W.K., Wong, P.C. & Bongso, A. (2002) Human feed‐ ers support prolonged undifferentiated growth of human inner cell masses and em‐

Embryonic Stem Cell Therapy – From Bench to Bed

http://dx.doi.org/10.5772/54368

413

[57] Rowland, T.J., Buchholz, D.E. & Clegg, D.O. (2012) Pluripotent human stem cells for the treatment of retinal disease. Journal of cellular physiology, 227, 457-466.

[58] Roy, N.S., Cleren, C., Singh, S.K., Yang, L., Beal, M.F. & Goldman, S.A. (2006) Func‐ tional engraftment of human ES cell-derived dopaminergic neurons enriched by co‐ culture with telomerase-immortalized midbrain astrocytes. Nature medicine, 12,

[59] Saha, K. & Jaenisch, R. (2009) Technical challenges in using human induced pluripo‐

[60] Schuldiner, M., Eiges, R., Eden, A., Yanuka, O., Itskovitz-Eldor, J., Goldstein, R.S. & Benvenisty, N. (2001) Induced neuronal differentiation of human embryonic stem

[61] Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D.A. & Benvenisty, N. (2000) Effects of eight growth factors on the differentiation of cells derived from human em‐ bryonic stem cells. Proceedings of the National Academy of Sciences of the United

[62] Schwartz, S.D., Hubschman, J.P., Heilwell, G., Franco-Cardenas, V., Pan, C.K., Os‐ trick, R.M., Mickunas, E., Gay, R., Klimanskaya, I. & Lanza, R. (2012) Embryonic stem

cell trials for macular degeneration: a preliminary report. Lancet, 379, 713-720.

ery after cervical spinal cord injury. Stem Cells, 28, 152-163.

reenter the germ line. Developmental biology, 161, 626-628.

[63] Sharp, J., Frame, J., Siegenthaler, M., Nistor, G. & Keirstead, H.S. (2010) Human em‐ bryonic stem cell-derived oligodendrocyte progenitor cell transplants improve recov‐

[64] Shih, C.C., Forman, S.J., Chu, P. & Slovak, M. (2007) Human embryonic stem cells are prone to generate primitive, undifferentiated tumors in engrafted human fetal tissues in severe combined immunodeficient mice. Stem cells and development, 16, 893-902.

[65] Stevens, L.C. (1966) Development of resistance to teratocarcinogenesis by primordial

[66] Stewart, C.L., Gadi, I. & Bhatt, H. (1994) Stem cells from primordial germ cells can

[67] Strauss, O. (2005) The retinal pigment epithelium in visual function. Physiological re‐

[68] Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K. & Yama‐ naka, S. (2007) Induction of pluripotent stem cells from adult human fibroblasts by

germ cells in mice. Journal of the National Cancer Institute, 37, 859-867.

bryonic stem cells. Nature biotechnology, 20, 933-936.

tent scells to model disease. Cell stem cell, 5, 584-595.

cells. Brain research, 913, 201-205.

States of America, 97, 11307-11312.

views, 85, 845-881.

defined factors. Cell, 131, 861-872.

1259-1268.


[56] Richards, M., Fong, C.Y., Chan, W.K., Wong, P.C. & Bongso, A. (2002) Human feed‐ ers support prolonged undifferentiated growth of human inner cell masses and em‐ bryonic stem cells. Nature biotechnology, 20, 933-936.

[44] McDonald, J.W., Becker, D., Sadowsky, C.L., Jane, J.A., Sr., Conturo, T.E. & Schultz, L.M. (2002) Late recovery following spinal cord injury. Case report and review of the

[46] Meyer, J.S., Shearer, R.L., Capowski, E.E., Wright, L.S., Wallace, K.A., McMillan, E.L., Zhang, S.C. & Gamm, D.M. (2009) Modeling early retinal development with human embryonic and induced pluripotent stem cells. Proceedings of the National Academy

[47] Murry, C.E. & Keller, G. (2008) Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell, 132, 661-680.

[48] Neman, J. & de Vellis, J. (2012) A method for deriving homogenous population of oli‐ godendrocytes from mouse embryonic stem cells. Developmental neurobiology, 72,

[49] Niederkorn, J.Y. (2002) Immune privilege in the anterior chamber of the eye. Critical

[50] Nistor, G.I., Totoiu, M.O., Haque, N., Carpenter, M.K. & Keirstead, H.S. (2005) Hu‐ man embryonic stem cells differentiate into oligodendrocytes in high purity and

[51] Noggle, S., Fung, H.L., Gore, A., Martinez, H., Satriani, K.C., Prosser, R., Oum, K., Paull, D., Druckenmiller, S., Freeby, M., Greenberg, E., Zhang, K., Goland, R., Sauer, M.V., Leibel, R.L. & Egli, D. (2011) Human oocytes reprogram somatic cells to a plu‐

[52] Osakada, F., Ikeda, H., Mandai, M., Wataya, T., Watanabe, K., Yoshimura, N., Akaike, A., Sasai, Y. & Takahashi, M. (2008) Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells. Nature bio‐

[53] Prokhorova, T.A., Harkness, L.M., Frandsen, U., Ditzel, N., Schroder, H.D., Burns, J.S. & Kassem, M. (2009) Teratoma formation by human embryonic stem cells is site dependent and enhanced by the presence of Matrigel. Stem cells and development,

[54] Radtke, N.D., Aramant, R.B., Petry, H.M., Green, P.T., Pidwell, D.J. & Seiler, M.J. (2008) Vision improvement in retinal degeneration patients by implantation of retina together with retinal pigment epithelium. American journal of ophthalmology, 146,

[55] Reubinoff, B.E., Itsykson, P., Turetsky, T., Pera, M.F., Reinhartz, E., Itzik, A. & Ben-Hur, T. (2001) Neural progenitors from human embryonic stem cells. Nature biotech‐

[45] McDonald, J.W. & Sadowsky, C. (2002) Spinal-cord injury. Lancet, 359, 417-425.

of Sciences of the United States of America, 106, 16698-16703.

myelinate after spinal cord transplantation. Glia, 49, 385-396.

literature. Journal of neurosurgery, 97, 252-265.

777-788.

412 Pluripotent Stem Cells

reviews in immunology, 22, 13-46.

ripotent state. Nature, 478, 70-75.

technology, 26, 215-224.

nology, 19, 1134-1140.

18, 47-54.

172-182.


[69] Takahashi, K. & Yamanaka, S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126, 663-676.

**Chapter 19**

**Disease Models for the Genetic Cardiac Diseases**

The ability to reprogram somatic cells into pluripotent stem cells has presented a significant advancement in stem cell research. This technique enables derivation of induced pluripotent stem (iPS) cells from any individual having a unique genotype. iPS cells can be derived from human somatic cells such as fibroblasts, keratinocytes or blood cells. Since, the production of iPS cell lines does not require the destruction of human embryos as in the production of the human embryonic stem cells (hESCs), the legal and ethical issues associated with hESCs can be at least partly avoided. The characteristics of iPS cells are very similar to those of pluripotent hESCs in many respects, including cell morphology, immortal growth characteristics in culture, expression of pluripotent markers, and differentiation potential. The iPS cells com‐ bined with the various differentiation protocols developed enable the production of genotype specific cell types. This feature enables also to produce disease-specific iPS cell lines from patients bearing defined genetic mutations. Traditionally, it has been challenging to study genetic cardiac diseases because cardiomyocytes from the heart biopsies of patients are difficult to obtain and the procedure carries a high risk. Additionally these cardiomyocytes do not survive long in culture. Animal models, mostly developed in rodent, have aided in elucidating the basic mechanisms of several genetic cardiac diseases. The disadvantages of small animal models are marked differences in anatomy and physiology of the cardiovascular system in comparison to humans. Rodent models are far from ideal when used in the identi‐ fication of contractile deficits and signals that initiate pathological growth [1]. Furthermore, the results obtained from neonatal rat cell experiments can be problematic because these cells possess different relative receptor subtypes and cell-signaling mechanisms. It will thus be especially important to investigate functional consequences of genetic cardiac diseases in human cardiomyocytes in which the functional effects of specific proteins have been adjusted to optimize electrical properties, contractile efficiency and power output of larger hearts [2].

> © 2013 Pekkanen-Mattila et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Pekkanen-Mattila et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Mari Pekkanen-Mattila, Kristiina Rajala and

Additional information is available at the end of the chapter

Katriina Aalto-Setälä

http://dx.doi.org/10.5772/55773

**1. Introduction**


## **Disease Models for the Genetic Cardiac Diseases**

[69] Takahashi, K. & Yamanaka, S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126, 663-676.

[70] Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Mar‐ shall, V.S. & Jones, J.M. (1998) Embryonic stem cell lines derived from human blasto‐

[71] Turovets, N., Semechkin, A., Kuzmichev, L., Janus, J., Agapova, L. & Revazova, E. (2011) Derivation of human parthenogenetic stem cell lines. Methods Mol Biol, 767,

[72] Watson, R.A. & Yeung, T.M. (2011) What is the potential of oligodendrocyte progeni‐ tor cells to successfully treat human spinal cord injury? BMC neurology, 11, 113.

[73] Wernig, M., Zhao, J.P., Pruszak, J., Hedlund, E., Fu, D., Soldner, F., Broccoli, V., Con‐ stantine-Paton, M., Isacson, O. & Jaenisch, R. (2008) Neurons derived from reprog‐ rammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Proceedings of the National Academy of Sciences of

[74] Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J. & Campbell, K.H. (1997) Viable off‐ spring derived from fetal and adult mammalian cells. Nature, 385, 810-813.

[75] Zhang, S.C., Wernig, M., Duncan, I.D., Brustle, O. & Thomson, J.A. (2001) In vitro dif‐ ferentiation of transplantable neural precursors from human embryonic stem cells.

cysts. Science, 282, 1145-1147.

the United States of America, 105, 5856-5861.

Nature biotechnology, 19, 1129-1133.

37-54.

414 Pluripotent Stem Cells

Mari Pekkanen-Mattila, Kristiina Rajala and Katriina Aalto-Setälä

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55773

## **1. Introduction**

The ability to reprogram somatic cells into pluripotent stem cells has presented a significant advancement in stem cell research. This technique enables derivation of induced pluripotent stem (iPS) cells from any individual having a unique genotype. iPS cells can be derived from human somatic cells such as fibroblasts, keratinocytes or blood cells. Since, the production of iPS cell lines does not require the destruction of human embryos as in the production of the human embryonic stem cells (hESCs), the legal and ethical issues associated with hESCs can be at least partly avoided. The characteristics of iPS cells are very similar to those of pluripotent hESCs in many respects, including cell morphology, immortal growth characteristics in culture, expression of pluripotent markers, and differentiation potential. The iPS cells com‐ bined with the various differentiation protocols developed enable the production of genotype specific cell types. This feature enables also to produce disease-specific iPS cell lines from patients bearing defined genetic mutations. Traditionally, it has been challenging to study genetic cardiac diseases because cardiomyocytes from the heart biopsies of patients are difficult to obtain and the procedure carries a high risk. Additionally these cardiomyocytes do not survive long in culture. Animal models, mostly developed in rodent, have aided in elucidating the basic mechanisms of several genetic cardiac diseases. The disadvantages of small animal models are marked differences in anatomy and physiology of the cardiovascular system in comparison to humans. Rodent models are far from ideal when used in the identi‐ fication of contractile deficits and signals that initiate pathological growth [1]. Furthermore, the results obtained from neonatal rat cell experiments can be problematic because these cells possess different relative receptor subtypes and cell-signaling mechanisms. It will thus be especially important to investigate functional consequences of genetic cardiac diseases in human cardiomyocytes in which the functional effects of specific proteins have been adjusted to optimize electrical properties, contractile efficiency and power output of larger hearts [2].

© 2013 Pekkanen-Mattila et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Pekkanen-Mattila et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Genetic cardiac diseases, such as long QT syndrome, belong to a severe class of diseases which are unpredictable, have variable clinical picture ranging from asymptomatic to sudden cardiac death and lack specific medication. These inherited arrhythmic diseases are caused by single mutations which are relatively common in population. Earlier we did not have *in vitro* models for these diseases, but with the aid of iPS cell derived cardiomyocytes genetic cardiac diseases can now be modeled in cell culture. The patient specific iPS cell derived cardiomyocytes have been demonstrated to manifest the disease-associated electrophysiological abnormalities in a dish [3-6]. Therefore, these cells allow researchers to study and understand disease mecha‐ nisms more readily as well as to investigate the effects of different chemical compounds on the electrophysiology of the cardiomyocytes. In addition to basic research, iPS cell derived cardiomyocytes would provide an effective tool for novel drug or treatment discovery. However, before iPS cell derived cardiomyocytes are ready to be considered for use as disease models, the cells produced need to be confirmed to exhibit the essential functional character‐ istics of human cardiomyocytes.

efficiency and kinetics as well as to generate iPS cells without viral integration in the genome (Table 1). The nonintegrating reprogramming methods developed thus far include adeno- and sendai viruses, plasmid- and episomal vector-based approaches, excision systems of integrat‐ ed transgenes such as Cre/loxP recombination or PiggyBac transposition, and delivery of reprogramming factors directly as RNAs, proteins and chemicals. However, most of these nonintegrating approaches are still highly inefficient when compared to the original retro- or lentiviral reprogramming systems with the exception of nowadays widely used sendai virus

**Methods Efficiency % Details References**

tumorigenicity possible

tumorigenicity possible

vector-fragment possible

fragment possible, T sensitive Sendai vector allowing removal of the virus

site excision, screening needed, tumorigenicity possible

treatment required, genetic abnormality

treatment required, abnormal signaling

pathway possible, virus used

[7]

Disease Models for the Genetic Cardiac Diseases

http://dx.doi.org/10.5772/55773

417

[9]

[14]

[15]

[17]

[18, 19]

[20]

[21, 22]

[23, 24]

[25]

[26]

Retroviral vectors Medium, 0.01-0.5 Multiple integration, incomplete silencing,

Lentiviral vectors Medium, 0.1-1 Multiple integration, incomplete silencing,

Adenoviral vectors Low, 0.001 Non-integrating, however integrated

Sendaiviral vectors Medium, 0.01-1 Non-integrating, integrated vector-

Plasmids Low, 0.001 Occasional integration, simple transfection [16]

Cre/loxP system Medium, 0.01-1 Integration but excisable, inefficient loxP

PiggyBac system Medium, 0.1 Precise excision possible, screening

RNAs High, 1 Non-integrating, DNA-free, multiple

Protein Low, 0.001 Non-integrating, DNA-free, long-term

**Table 1.** Overview of the reprogramming methods for the generation of iPS cells.

Low, 0.0003 Non-integrating, long-term persistent transcription

Low, 0.005 Non-integrating, multiple transductions needed

needed

possible

High, >1 Non-integrating, DNA-free, long-term

transfection needed

reprogramming method.

OriP/EBNA-1 episomal vectors

Minicircle DNA episomal vectors

Factors + small molecules

In this chapter, the production and the characterization of patient specific iPS cell derived cardiomyocytes is described. In addition, we discuss the genetic cardiac disease models so far developed based on iPS technique, their demands, advantages and disadvantages. Further‐ more, the future applications for iPS cell derived cardiomyocytes are discussed.

## **2. Production of disease specific iPS cell lines**

The discovery of cellular reprogramming as a technology to generate iPS cells offers a potential solution to the challenge when studying genetic cardiac diseases. In this approach, human adult somatic cells are reprogrammed into stem cells offering comparable function to human pluripotent ESCs in their ability to develop differentiated progeny from all developmental lineages of the human being. When somatic cells are reprogrammed to iPS cells, they shut down the expression of genes specific for that somatic cell type and activate genes that maintain pluripotency. Once reprogramming has occurred, endogenous counterparts of the exoge‐ nously supplied reprogramming factors are activated, indicating that exogenous factors are only required for the induction, not for the maintenance of pluripotency [7]. Up to date, various human somatic cell types, including fibroblasts, keratinocytes, and different blood cells have been reprogrammed to iPS cells [7-11].

The initial methods used to generate iPS cells involved the retroviral overexpression of four transcription factors Oct4, Sox2, Klf4, and c-myc observed to be essential in maintaining pluripotency of hESCs [7, 12]. Another set of four transcription factors Oct4, Sox2, Nanog, and Lin-28 was also found to induce pluripotency [9]. Efficient retro- and lentiviral vector systems that have been most widely used to generate iPS cells have several drawbacks including the possibility of proviral genomic integration, which may cause both the reactivation of silenced exogenous genes and the alteration of genomic integrity, thereby increasing the risk for tumorigenesis [12, 13]. Since the seminal discovery the development in this field has been rapid and numerous alternative strategies have been applied to improve the reprogramming safety, efficiency and kinetics as well as to generate iPS cells without viral integration in the genome (Table 1). The nonintegrating reprogramming methods developed thus far include adeno- and sendai viruses, plasmid- and episomal vector-based approaches, excision systems of integrat‐ ed transgenes such as Cre/loxP recombination or PiggyBac transposition, and delivery of reprogramming factors directly as RNAs, proteins and chemicals. However, most of these nonintegrating approaches are still highly inefficient when compared to the original retro- or lentiviral reprogramming systems with the exception of nowadays widely used sendai virus reprogramming method.

Genetic cardiac diseases, such as long QT syndrome, belong to a severe class of diseases which are unpredictable, have variable clinical picture ranging from asymptomatic to sudden cardiac death and lack specific medication. These inherited arrhythmic diseases are caused by single mutations which are relatively common in population. Earlier we did not have *in vitro* models for these diseases, but with the aid of iPS cell derived cardiomyocytes genetic cardiac diseases can now be modeled in cell culture. The patient specific iPS cell derived cardiomyocytes have been demonstrated to manifest the disease-associated electrophysiological abnormalities in a dish [3-6]. Therefore, these cells allow researchers to study and understand disease mecha‐ nisms more readily as well as to investigate the effects of different chemical compounds on the electrophysiology of the cardiomyocytes. In addition to basic research, iPS cell derived cardiomyocytes would provide an effective tool for novel drug or treatment discovery. However, before iPS cell derived cardiomyocytes are ready to be considered for use as disease models, the cells produced need to be confirmed to exhibit the essential functional character‐

In this chapter, the production and the characterization of patient specific iPS cell derived cardiomyocytes is described. In addition, we discuss the genetic cardiac disease models so far developed based on iPS technique, their demands, advantages and disadvantages. Further‐

The discovery of cellular reprogramming as a technology to generate iPS cells offers a potential solution to the challenge when studying genetic cardiac diseases. In this approach, human adult somatic cells are reprogrammed into stem cells offering comparable function to human pluripotent ESCs in their ability to develop differentiated progeny from all developmental lineages of the human being. When somatic cells are reprogrammed to iPS cells, they shut down the expression of genes specific for that somatic cell type and activate genes that maintain pluripotency. Once reprogramming has occurred, endogenous counterparts of the exoge‐ nously supplied reprogramming factors are activated, indicating that exogenous factors are only required for the induction, not for the maintenance of pluripotency [7]. Up to date, various human somatic cell types, including fibroblasts, keratinocytes, and different blood cells have

The initial methods used to generate iPS cells involved the retroviral overexpression of four transcription factors Oct4, Sox2, Klf4, and c-myc observed to be essential in maintaining pluripotency of hESCs [7, 12]. Another set of four transcription factors Oct4, Sox2, Nanog, and Lin-28 was also found to induce pluripotency [9]. Efficient retro- and lentiviral vector systems that have been most widely used to generate iPS cells have several drawbacks including the possibility of proviral genomic integration, which may cause both the reactivation of silenced exogenous genes and the alteration of genomic integrity, thereby increasing the risk for tumorigenesis [12, 13]. Since the seminal discovery the development in this field has been rapid and numerous alternative strategies have been applied to improve the reprogramming safety,

more, the future applications for iPS cell derived cardiomyocytes are discussed.

**2. Production of disease specific iPS cell lines**

istics of human cardiomyocytes.

416 Pluripotent Stem Cells

been reprogrammed to iPS cells [7-11].


**Table 1.** Overview of the reprogramming methods for the generation of iPS cells.

## **3. Cardiomyocyte differentiation**

Cardiomyocytes have been differentiated from the hESCs over a decade [27, 28] and multiple cardiac differentiation methods have been developed. The differentiation methods developed for hESC derived cardiomyocytes have been proven to be applicable also for cardiac differ‐ entiation of iPS cells.

Overall the differentiation event of hESC and iPS cell derived cardiomyocytes is quite rapid, 10-20 days regardless of the differentiation method used. However, all the differentiation methods share common problems, including uncontrolled differentiation and low differen‐ tiation rates. With common differentiation methods the cardiomyocyte yield is between ~1-25 % of the total cell number [28-30]. In addition, the cardiomyocyte differentiation efficiency has been shown to vary markedly between different stem cell lines [31].

All differentiation methods end up with a heterogeneous cell population. In addition to the other cell types, the differentiated population includes all cardiomyocyte subtypes; ventricular, atrial and nodal –like cells [32]. The ventricular cells form usually the majori‐ ty of differentiated cells (60-80%), atrial cells form usually 10-40 % of the population and only <5% of cells are nodal-like cells [32, 33]. However, these numbers can differ depend‐ ing on the cell line used [34].

The cardiac differentiation methods are lately reviewed [35] and described in Figure 1.

## **4. Transdifferentiation of fibroblasts into cardiac cells**

Murine fibroblasts can be reprogrammed directly into cardiomyocytes by overexpression of Gata4, Mef2c and Tbx5 (GMT) [36]. This combination of factors has been reported to convert murine cardiac fibroblasts and tail tip fibroblasts into spontaneous beating cells having cardiomyocyte expression profiles. In addition, epigenetic status is typical for cardiomyocytes in these cells. However, Chen and co-workers have shown this method to be inefficient [37]. Overexpression of GMT factors resulted in an increase in cardiac troponin expression but spontaneous action potentials were lacking even though 22% of the cells exhibited voltagedependent calcium currents.

**Figure 1.** Generation of disease –specific iPS cell lines and cardiomyocytes. Cardiac differentiation methods can be div‐ ided into three classes; (1) Embryoid body (EB) based, (2) END-2 coculture based or (3) directed differentiation meth‐ ods. Traditionally EB method has been based on spontaneous aggregation of EBs and spontaneous differentiation [28]. However, lately multiple methods controlling the EB formation has been developed [41] enhancing the reprodu‐ cibility and productivity of the cardiac differentiation. END-2 method can be performed in two ways, either co-cultur‐ ing the hESC or iPS cells in contact with END-2 cells [42] or by using END-2 conditioned media [43]. A lot of effort has been made in enhancing and defining the cardiac differentiation and this has led to the development of directed dif‐ ferentiation methods with growth factors or small molecules. Activin A and BMP-4 has been used in combination with monolayer cultures on matrigel to differentiate cardiomyocytes [44]. A temporal modulation of Wnt signaling by us‐ ing small molecules has been proven to an even more robust and, in addition, rather inexpensive method for cardio‐ myocyte differentiation [45]. Directed reprogramming of fibroblasts to cardiomyocytes has been successful with

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The first characterization step for the differentiated hESC or iPS cell derived cardiomyocytes is the observation of spontaneously beating cells. In addition, cardiomyocyte phenotype can

mouse cells. However, this method has not yet been proven to work with human cells.

**5. The assesment of the cardiomyocyte functionality**

**5.1. Cardiomyocyte characterization at gene and protein level**

A lot of effort has been done to transdifferentiate human fibroblasts into cardiomyocytes. So far spontaneously beating human cells have not been obtained. However, with transcription factors mesoderm posterior (MESP) homolog and mammalian v-ets erythoblastosis virus E26 oncogene homolog ETS2 cardiomyocyte progenitors expressing cardiac mesoderm marker KDR have been obtained [38]. It seems that the GMT method alone is not robust enough for direct reprogramming of human cardiomyocytes. Therefore it has been suggested that combination of GMT with other transcription factors, mRNAs or small molecules could provide more efficient reprogramming procedure [39]. In addition, based on animal experi‐ ments it can be concluded that cardiac microenvironment has also important role in reprog‐ ramming [40].

**3. Cardiomyocyte differentiation**

entiation of iPS cells.

418 Pluripotent Stem Cells

ing on the cell line used [34].

dependent calcium currents.

ramming [40].

Cardiomyocytes have been differentiated from the hESCs over a decade [27, 28] and multiple cardiac differentiation methods have been developed. The differentiation methods developed for hESC derived cardiomyocytes have been proven to be applicable also for cardiac differ‐

Overall the differentiation event of hESC and iPS cell derived cardiomyocytes is quite rapid, 10-20 days regardless of the differentiation method used. However, all the differentiation methods share common problems, including uncontrolled differentiation and low differen‐ tiation rates. With common differentiation methods the cardiomyocyte yield is between ~1-25 % of the total cell number [28-30]. In addition, the cardiomyocyte differentiation efficiency has

All differentiation methods end up with a heterogeneous cell population. In addition to the other cell types, the differentiated population includes all cardiomyocyte subtypes; ventricular, atrial and nodal –like cells [32]. The ventricular cells form usually the majori‐ ty of differentiated cells (60-80%), atrial cells form usually 10-40 % of the population and only <5% of cells are nodal-like cells [32, 33]. However, these numbers can differ depend‐

The cardiac differentiation methods are lately reviewed [35] and described in Figure 1.

Murine fibroblasts can be reprogrammed directly into cardiomyocytes by overexpression of Gata4, Mef2c and Tbx5 (GMT) [36]. This combination of factors has been reported to convert murine cardiac fibroblasts and tail tip fibroblasts into spontaneous beating cells having cardiomyocyte expression profiles. In addition, epigenetic status is typical for cardiomyocytes in these cells. However, Chen and co-workers have shown this method to be inefficient [37]. Overexpression of GMT factors resulted in an increase in cardiac troponin expression but spontaneous action potentials were lacking even though 22% of the cells exhibited voltage-

A lot of effort has been done to transdifferentiate human fibroblasts into cardiomyocytes. So far spontaneously beating human cells have not been obtained. However, with transcription factors mesoderm posterior (MESP) homolog and mammalian v-ets erythoblastosis virus E26 oncogene homolog ETS2 cardiomyocyte progenitors expressing cardiac mesoderm marker KDR have been obtained [38]. It seems that the GMT method alone is not robust enough for direct reprogramming of human cardiomyocytes. Therefore it has been suggested that combination of GMT with other transcription factors, mRNAs or small molecules could provide more efficient reprogramming procedure [39]. In addition, based on animal experi‐ ments it can be concluded that cardiac microenvironment has also important role in reprog‐

been shown to vary markedly between different stem cell lines [31].

**4. Transdifferentiation of fibroblasts into cardiac cells**

**Figure 1.** Generation of disease –specific iPS cell lines and cardiomyocytes. Cardiac differentiation methods can be div‐ ided into three classes; (1) Embryoid body (EB) based, (2) END-2 coculture based or (3) directed differentiation meth‐ ods. Traditionally EB method has been based on spontaneous aggregation of EBs and spontaneous differentiation [28]. However, lately multiple methods controlling the EB formation has been developed [41] enhancing the reprodu‐ cibility and productivity of the cardiac differentiation. END-2 method can be performed in two ways, either co-cultur‐ ing the hESC or iPS cells in contact with END-2 cells [42] or by using END-2 conditioned media [43]. A lot of effort has been made in enhancing and defining the cardiac differentiation and this has led to the development of directed dif‐ ferentiation methods with growth factors or small molecules. Activin A and BMP-4 has been used in combination with monolayer cultures on matrigel to differentiate cardiomyocytes [44]. A temporal modulation of Wnt signaling by us‐ ing small molecules has been proven to an even more robust and, in addition, rather inexpensive method for cardio‐ myocyte differentiation [45]. Directed reprogramming of fibroblasts to cardiomyocytes has been successful with mouse cells. However, this method has not yet been proven to work with human cells.

## **5. The assesment of the cardiomyocyte functionality**

#### **5.1. Cardiomyocyte characterization at gene and protein level**

The first characterization step for the differentiated hESC or iPS cell derived cardiomyocytes is the observation of spontaneously beating cells. In addition, cardiomyocyte phenotype can be assessed at the gene or protein level with cardiomyocyte specific markers such as structural proteins troponin, alpha-actinin or myosins. The commonly used markers in monitoring the cardiac differentiation are listed in Table 2.

cardiac action potential is composed of co-operation of these channels and the action potential curve can be divided into five different phases (Figure 2). Phase 0 of the action potential is the depolarization phase of the cardiomyocytes from the negative membrane potential to positive, called the upstroke. This is followed by phase 1, the short transient repolarization that is followed by the plateau phase 2. Phase 2 is followed by phase 3, which is the repolarization back to the resting membrane potential. The resting state of the membrane potential is called

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As mentioned, cardiac action potential results from the chain reaction of multiple ion channels. Therefore a malfunction of a single ion channel can be observed from the action potential curve. Figure 2 presents the parameters which are used in analyzing the action potential. In regard to analyzing cardiac disease specific cells, the action potential duration plays an important role

As a method, patch clamp is very informative and provides invaluable data for example for pharmacological and safety pharmacological studies. However, it is very laborious, needs highly specialized machinery and, most importantly, dedicated and specialized users. For these reasons, semi-automated and automated patch clamp machinery are being developed

**Figure 2.** The phases of the cardiac action potential. ADP50 and ADP90 represent the action potential duration at 50% and 90% of the repolarization and these parameters are used in determining the duration of the action potential. The dV/dtmax represents the maximal upstroke velocity and can be used in assessing the electrophysiological phenotype

because the lengthening of the action potential may lead to severe arrhythmias.

and would be valuable for cardiomyocyte applications [50, 51].

as the phase 4 [49].

and maturity stage of the cardiomyocytes.


**Table 2.** Markers used in monitoring the cardiac differentiation.

#### **5.2. Electrophysiological methods**

#### *5.2.1. Patch clamp*

Traditional way to study the functionality and the electrical activity of the cardiomyocytes is the patch clamp technique [46]. Originally patch clamp method has been developed to study ion channels in excitable membranes [47]. In this technique micropipette is attached to the cell membrane by a giga seal and this can be exploited to measure current changes and voltage across the membrane. Due to the unique nature of the cardiomyocyte action potential curve, the ion channel composition and the maturation stage of the cardiomyocyte can be assessed and therefore the method has been widely used with stem cell derived cardiomyocyte studies.

Key cardiac ion channels (and respective current) involved in the human action potential are NaV1.5 (INa), KV4.3 (Ito), CaV1.2 (ICa,L) KV11.1 (IKr), KV7.1 (IKs), and Kir2.X (IK1) [48]. The cardiac action potential is composed of co-operation of these channels and the action potential curve can be divided into five different phases (Figure 2). Phase 0 of the action potential is the depolarization phase of the cardiomyocytes from the negative membrane potential to positive, called the upstroke. This is followed by phase 1, the short transient repolarization that is followed by the plateau phase 2. Phase 2 is followed by phase 3, which is the repolarization back to the resting membrane potential. The resting state of the membrane potential is called as the phase 4 [49].

be assessed at the gene or protein level with cardiomyocyte specific markers such as structural proteins troponin, alpha-actinin or myosins. The commonly used markers in monitoring the

> Nanog SOX2 Tra-1-60 SSEA-4

FoxC1 Dkk-1 Mesp1 Flk-1

Nkx2.5 GATA4 Tbx5 Isl-1 Mef2c Hand1/2

Sarcomeric α-actinin Myosin heavy- and lightchain (MHC and MLC)

cardiac differentiation are listed in Table 2.

420 Pluripotent Stem Cells

**Cell stage Markers** Pluripotent cells OCT4

Precardiac/cardiac mesodermal cells Brachyury T

Cardiac precursor cells KDR

**Table 2.** Markers used in monitoring the cardiac differentiation.

**5.2. Electrophysiological methods**

*5.2.1. Patch clamp*

Cardiac cells Troponin I and T

Traditional way to study the functionality and the electrical activity of the cardiomyocytes is the patch clamp technique [46]. Originally patch clamp method has been developed to study ion channels in excitable membranes [47]. In this technique micropipette is attached to the cell membrane by a giga seal and this can be exploited to measure current changes and voltage across the membrane. Due to the unique nature of the cardiomyocyte action potential curve, the ion channel composition and the maturation stage of the cardiomyocyte can be assessed and therefore the method has been widely used with stem cell derived cardiomyocyte studies.

Key cardiac ion channels (and respective current) involved in the human action potential are NaV1.5 (INa), KV4.3 (Ito), CaV1.2 (ICa,L) KV11.1 (IKr), KV7.1 (IKs), and Kir2.X (IK1) [48]. The

As mentioned, cardiac action potential results from the chain reaction of multiple ion channels. Therefore a malfunction of a single ion channel can be observed from the action potential curve. Figure 2 presents the parameters which are used in analyzing the action potential. In regard to analyzing cardiac disease specific cells, the action potential duration plays an important role because the lengthening of the action potential may lead to severe arrhythmias.

As a method, patch clamp is very informative and provides invaluable data for example for pharmacological and safety pharmacological studies. However, it is very laborious, needs highly specialized machinery and, most importantly, dedicated and specialized users. For these reasons, semi-automated and automated patch clamp machinery are being developed and would be valuable for cardiomyocyte applications [50, 51].

**Figure 2.** The phases of the cardiac action potential. ADP50 and ADP90 represent the action potential duration at 50% and 90% of the repolarization and these parameters are used in determining the duration of the action potential. The dV/dtmax represents the maximal upstroke velocity and can be used in assessing the electrophysiological phenotype and maturity stage of the cardiomyocytes.

#### *5.2.2. Micro electrode array*

In addition to the traditional patch clamp technique [46] the micro electrode array (MEA) – platform [52] offers practical, relative easy and non-invasive technique to assess the electrical properties of the differentiated cardiomyocytes [53]. Contrary to the patch clamp, the MEA system measures the electrical activity of a cell population. Therefore the signal resembles electrocardiogram (ECG) and is called field potential instead of action potential. Even though the ion channel function cannot be studied in the similar accuracy as with patch clamp, it allows examination field potential properties, such as cardiac repolarization, and therefore enables drug effect investigation [53]. During the last years, MEA has been widely used in character‐ ization of hESC- and iPS cell derived cardiomyocytes [31, 54]. MEA has been become a basic electrophysiological tool and in addition to cardiomyocytes, it has been successfully used also with other cell types, such as neurons [55].

*5.2.4. Force measurement*

with custom made platforms [61, 62].

**6.1. Long QT-syndrome**

seizures and even sudden cardiac death.

**6. Diseases modeled with iPS cell technique**

edly provide groundbreaking innovations in the future.

Recently a lot of effort has been applied to develop measuring systems to understand the mechanobiology of cardiomyocytes. Force measurement technique can be applied to measure isometric cardiomyocyte force contraction. A number of parameters can be determined by using the cardiomyocyte force measurement such as determination of Ca-sensitivity, cooper‐ ativity of force production and maximal Ca-activated force. Kinetics of the contractile re‐ sponses can also be measured such as the actin-myosin turnover kinetics. These parameters can be useful in the characterization of myofibrillar pathologies of various origin and drug effects. Most of the currently existing systems are only suitable for the study of cardiac tissue slices and therefore inappropriate to be used for iPS cell derived cardiomyocytes. Recently, however, cardiomyocyte force measurement system based on atomic force microscopy (AFM) was developed which can also be used to study single cardiomyocytes and small clusters of cardiomyocytes [60]. With the AFM system they were able to measure contractile forces, beat frequencies and durations of single cardiomyocytes and small cardiomyocyte clusters. The AFM-based method is also applicable for the screening of cardiac-active pharmacological agents. Cardiac microtissues have also been constructed using human pluripotent stem cell derived cardiomyocytes and the contraction force of the beating tissues has been analyzed

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Since the revolutionary discovery of iPS cells, multiple genetic diseases including cardiac and neuronal diseases have been modeled with patient specific iPS cell derived cells. Since primary human cardiomyocytes are not available for research in vitro, iPS cell derived cardiomyocytes are invaluable tool to study the pathophysiology of severe cardiac diseases and will undoubt‐

Long QT-syndrome (LQTS) appears as a genetic or a drug-induced form. It is characterized by a prolonged cardiac repolarization phase resulting in a prolonged QT interval in the surface electrocardiogram (ECG). The clinical symptoms of LQTS include palpitations, syncope and

More than 700 mutations in 12 different genes (LQT1–12) have been found to affect genetic forms of LQTS [63]. However, two of these subtypes account the majority (>90%) of all the genetically identified LQTSs. Both of these mutations affect potassium channels altering their proper function. LQTS type 1 (LQT1) is the most common LQTS subtype, resulting from mutations in the KCNQ1 gene. This gene encodes the α-subunit of the slow component of the delayed rectifier potassium current (IKs) channel [64]. Individuals with LQT1 typically have

LQTS type 2 (LQT2) is due to non-proper functioning of the α-subunit of the rapid delayed potassium channel (IKr), which is encoded by the human ether-a-go-go-related gene (HERG),

symptoms when the heart rate is elevated e.g. during exercise [65, 66].

The MEA system is also applicable in studying cardiac cell responses to pharmaceutical agents [54]. It also enables cells to be measured repeatedly for longer periods of times e.g. multiple days or weeks. However, the analysis of MEA measurement data is laborious. Therefore, semiautomated and automated systems for data analysis have been developed, which makes MEA system more reliable and efficient tool in research [56].

#### *5.2.3. The assessment of calcium homeostasis*

In addition to the unique co-operation of cardiac ion channels, the interaction of calcium-ions with cardiac structure proteins is another crucial feature in cardiomyocytes that is essential for the proper function of the heart. In human cardiomyocytes, calcium ion (Ca2+) influx through L-type calcium channels during the plateau phase triggers the Ca2+-release from the sarcoplasmic reticulum (SR) which is mediated by the ryanodine receptors (RyR2). The Ca2+ influx together with the release raises the free calcium concentration inside the cardiomyo‐ cytes. In sytosol, free calcium binds to troponin C in the myofilaments and triggers the machinery which induces the cell contraction. For the cell relaxation to occur, the calcium has to be rapidly removed from the cytosol. The removal is efficient with the aid of four separate pathways; sarcoplasmic reticulum Ca2+-ATPase (Serca2a), sarcolemmal Ca2+-ATPase, sarcolemmal Na+/Ca2+ exchanger and mitochondrial Ca2+ uniport [57].

Similarly as the regular and synchronous chain of action potentials, calcium concentration fluctuates in the cardiomyocytes. Therefore, with the aid of calcium binding dyes and modern fluorescence microscope systems, the function and response to pharmaceutical agents of cardiomyocytes can be monitored. This method is called calcium imaging [58, 59]. The calcium binding dyes, such as Fura-2 and Fluo-4, can be loaded inside the cardiomyocyte cytosol and when the calcium ions are released to the cytosol, the ions bind to the dyes and a fluorescence signal can be detected. When the fluorescence intensity is measured from the single cell, the calcium handling of the single cardiomyocyte can be monitored and analyzed. From the calcium imaging data, the beating rate and the function of the calcium handling machinery in the cardiomyocytes can be assessed. If the calcium is not released or withdrawn from the cardiomyocyte cytosol in a proper way, irregularity or multiple peaks can be seen in the calcium imaging curve.

#### *5.2.4. Force measurement*

*5.2.2. Micro electrode array*

422 Pluripotent Stem Cells

with other cell types, such as neurons [55].

*5.2.3. The assessment of calcium homeostasis*

calcium imaging curve.

system more reliable and efficient tool in research [56].

In addition to the traditional patch clamp technique [46] the micro electrode array (MEA) – platform [52] offers practical, relative easy and non-invasive technique to assess the electrical properties of the differentiated cardiomyocytes [53]. Contrary to the patch clamp, the MEA system measures the electrical activity of a cell population. Therefore the signal resembles electrocardiogram (ECG) and is called field potential instead of action potential. Even though the ion channel function cannot be studied in the similar accuracy as with patch clamp, it allows examination field potential properties, such as cardiac repolarization, and therefore enables drug effect investigation [53]. During the last years, MEA has been widely used in character‐ ization of hESC- and iPS cell derived cardiomyocytes [31, 54]. MEA has been become a basic electrophysiological tool and in addition to cardiomyocytes, it has been successfully used also

The MEA system is also applicable in studying cardiac cell responses to pharmaceutical agents [54]. It also enables cells to be measured repeatedly for longer periods of times e.g. multiple days or weeks. However, the analysis of MEA measurement data is laborious. Therefore, semiautomated and automated systems for data analysis have been developed, which makes MEA

In addition to the unique co-operation of cardiac ion channels, the interaction of calcium-ions with cardiac structure proteins is another crucial feature in cardiomyocytes that is essential for the proper function of the heart. In human cardiomyocytes, calcium ion (Ca2+) influx through L-type calcium channels during the plateau phase triggers the Ca2+-release from the sarcoplasmic reticulum (SR) which is mediated by the ryanodine receptors (RyR2). The Ca2+ influx together with the release raises the free calcium concentration inside the cardiomyo‐ cytes. In sytosol, free calcium binds to troponin C in the myofilaments and triggers the machinery which induces the cell contraction. For the cell relaxation to occur, the calcium has to be rapidly removed from the cytosol. The removal is efficient with the aid of four separate pathways; sarcoplasmic reticulum Ca2+-ATPase (Serca2a), sarcolemmal Ca2+-ATPase,

Similarly as the regular and synchronous chain of action potentials, calcium concentration fluctuates in the cardiomyocytes. Therefore, with the aid of calcium binding dyes and modern fluorescence microscope systems, the function and response to pharmaceutical agents of cardiomyocytes can be monitored. This method is called calcium imaging [58, 59]. The calcium binding dyes, such as Fura-2 and Fluo-4, can be loaded inside the cardiomyocyte cytosol and when the calcium ions are released to the cytosol, the ions bind to the dyes and a fluorescence signal can be detected. When the fluorescence intensity is measured from the single cell, the calcium handling of the single cardiomyocyte can be monitored and analyzed. From the calcium imaging data, the beating rate and the function of the calcium handling machinery in the cardiomyocytes can be assessed. If the calcium is not released or withdrawn from the cardiomyocyte cytosol in a proper way, irregularity or multiple peaks can be seen in the

sarcolemmal Na+/Ca2+ exchanger and mitochondrial Ca2+ uniport [57].

Recently a lot of effort has been applied to develop measuring systems to understand the mechanobiology of cardiomyocytes. Force measurement technique can be applied to measure isometric cardiomyocyte force contraction. A number of parameters can be determined by using the cardiomyocyte force measurement such as determination of Ca-sensitivity, cooper‐ ativity of force production and maximal Ca-activated force. Kinetics of the contractile re‐ sponses can also be measured such as the actin-myosin turnover kinetics. These parameters can be useful in the characterization of myofibrillar pathologies of various origin and drug effects. Most of the currently existing systems are only suitable for the study of cardiac tissue slices and therefore inappropriate to be used for iPS cell derived cardiomyocytes. Recently, however, cardiomyocyte force measurement system based on atomic force microscopy (AFM) was developed which can also be used to study single cardiomyocytes and small clusters of cardiomyocytes [60]. With the AFM system they were able to measure contractile forces, beat frequencies and durations of single cardiomyocytes and small cardiomyocyte clusters. The AFM-based method is also applicable for the screening of cardiac-active pharmacological agents. Cardiac microtissues have also been constructed using human pluripotent stem cell derived cardiomyocytes and the contraction force of the beating tissues has been analyzed with custom made platforms [61, 62].

## **6. Diseases modeled with iPS cell technique**

Since the revolutionary discovery of iPS cells, multiple genetic diseases including cardiac and neuronal diseases have been modeled with patient specific iPS cell derived cells. Since primary human cardiomyocytes are not available for research in vitro, iPS cell derived cardiomyocytes are invaluable tool to study the pathophysiology of severe cardiac diseases and will undoubt‐ edly provide groundbreaking innovations in the future.

#### **6.1. Long QT-syndrome**

Long QT-syndrome (LQTS) appears as a genetic or a drug-induced form. It is characterized by a prolonged cardiac repolarization phase resulting in a prolonged QT interval in the surface electrocardiogram (ECG). The clinical symptoms of LQTS include palpitations, syncope and seizures and even sudden cardiac death.

More than 700 mutations in 12 different genes (LQT1–12) have been found to affect genetic forms of LQTS [63]. However, two of these subtypes account the majority (>90%) of all the genetically identified LQTSs. Both of these mutations affect potassium channels altering their proper function. LQTS type 1 (LQT1) is the most common LQTS subtype, resulting from mutations in the KCNQ1 gene. This gene encodes the α-subunit of the slow component of the delayed rectifier potassium current (IKs) channel [64]. Individuals with LQT1 typically have symptoms when the heart rate is elevated e.g. during exercise [65, 66].

LQTS type 2 (LQT2) is due to non-proper functioning of the α-subunit of the rapid delayed potassium channel (IKr), which is encoded by the human ether-a-go-go-related gene (HERG), also known as KCNH2-gene [67]. Contrary to type 1, individuals with LQT2 have clinical symptoms when the heart rate is slow [65, 66] and syptoms can be triggered e.g. by an alarm clock during sleep. The drug induced form of LQTS is due to altered function of the HERGchannel by the drug, therefore this channel has a significant importance during drug devel‐ opment and in safety studies.

A human cell model for LQT3 has also been produced and its function and characteristics were compared with a mouse models which were based on both mouse ESCs and mouse iPS cells affected with the same disease specific mutation [71]. LQT3 syndrome is due to mutations in the SCN5A gene, which encodes for the α-subunit of the cardiac sodium (Na+) channel. These mutations disrupt the inactivation of the Na+ channel during the action potential plateu phase and this irruption leads to the delay in repolarization and further prolonged QT interval [72]. In addition to LQT3, another kind of cardiac arrhythmia syndromes such as Brugada syndrome and cardiac conduction disease are associated with mutations in the SCN5A gene. In these syndromes the mutations are loss-of-function-type whereas LQT3 syndrome they are gain-offunction-type mutations [72, 73]. The comparison of multiple types of pluripotent stem cell derived cardiomyocytes showed that all of these models manifest the symptoms of the disease and, furthermore, the characteristics are similar within both species [71]. iPS cell models for

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Timothy syndrome is caused by a single mutation in the CACNA1C-gene. This gene encodes the main L-type calcium channel, Cav1.2, in the mammalian heart which is essential for the cardiac action potential and also for cardiomyocyte contraction [74-76]. Timothy sydrome characterized by LQTS, syndactyly (webbing of fingers and toes), immune deficiency and autism [77] iPS cell derived cardiomyocytes originating from Timothy syndrome patients exhibited irregular functional properties typical for the disease [78]. Interestingly, these irregularities were restored by roscovitine, a compound which increases the voltage-depend‐

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disorder characterized by stress-induced polymorphic ventricular tachycardia in a structurally normal heart. CPVT is a very severe disease and 30-35% of mutation carriers have had symptoms (stress-related syncope, seizures or sudden death) by the age of 30. This disease is caused by mutations in the genes of RyR2 or calsequestrin (CASQ2) which is a regulatory

Multiple iPS-based CPVT disease models have been published, most of them having the disease specific mutation in the RyR2 gene while [83-85] and one having the mutation in the CASQ2 gene [86]. The congruent result from these CPVT model studies was the occurrence of delayed after depolarizations (DADs) and arrhythmias which are caused by the aberrant diastolic Ca2+ from the SR. Notably the model with RyR2-P2328S mutation also exhibited early after depolarizations (EADs) in addition to DADs suggesting suggest‐ ing another pathophysiological mechanism for CPVT [85]. Intriguing finding was also the

these loss-of-function diseases have not yet been described.

*6.1.2. Catecholaminergic Polymorphic Ventricular Tachycardia*

calcium-buffering protein associated with RyR2 in the SR [79-82].

effect of dantrolene in rescuing the arrhythmogenic phenotype [84].

*6.1.1. Timothy syndrome*

ent inactivation of Cav1.2 [78].

The prevalence of the genetic form of LQTS is 1:2,000 in the general population [63]. However, the penetrance of the clinical symptoms of LQTS is low and there is considerable variation in phenotypic expression even within families carrying the same mutation [68]. It has also been suggested, that the population prevalence of milder LQTS mutations might be higher. Therefore the prevalence of latent or concealed LQTS, i.e. relatively asymptomatic individuals, would be higher than currently anticipated [69]. Due to this challenging and complex nature of LQTS, in addition, to the great interest of pharmaceutical industry towards this disease, multiple reports of iPS cell- based LQTS cell models have been published since 2007 when the iPS technology was invented.

Moretti and co-workers produced iPS cell derived cardiomycotyes from two patients carrying a KCNQ1 (R190Q) mutation [6]. In this study, the cardiomyocytes possessed the LQT1 genotype and exhibited prolonged action potential duration. The action potential prolongation was determined to be caused by the ion-channel trafficking defect resulting in a 70-80% IKs current density reduction. A β-adrenergic agonist isoproterenol altered the activation and deactivation kinetics of the IKs and this effect was rescued by the β-blockade [6]. Egashira and co-workers also produced a disease model for LQTS type 1 [70]. In their study, the iPS cells were derived from a sporadic patient who did not have a family history of significant QT interval abnormality. The mutation of the patient in the KCNQ1 was novel (1893delC) and the cells exhibited prolonged action potential duration in addition to arrhythmogenity.

Similarly results were found with iPS-CM derived from a patient suffering from the severe LQT type 2 syndrome. The patient had hERG (A614V) mutation and previously presented episodes of torsade de pointes (TdP), a special type of polymorphic ventricular tachycardia which is associated with LQTS [4]. The LQT2-cardiomyocytes derived from the patient's iPS cells demonstrated increased arrhythmogenicity associated with early after depolarizations (EADs) [5]. In addition, significant APD prolongation due to a reduced IKr current density was observed [4]. Arrhytmia and EADs were also induced by a specific HERG-channel blocker E-4031 to iPS cell derived CM having a hERG (G1681A) mutation. In addition, these cells exhibited EADs caused by the isoproterenol treatment and these EADs were rescued by βblockade [5].

All the aforementioned studies were done with iPS cells derived from the symptomatic LQTS patients. Nevertheless, similar results have been obtained from patients without severe symptoms. In the study made in our institute, iPS cell lines were derived from a patient having a KCNH2 (R176W) mutation and a family history of LQTS. However, this individual was asymptomatic except for occasional palpitations. iPS cell derived cardiomyocytes from this patient manifest the phenotype characteristics to LQT2, such as a prolonged repolarization time and increased arrhythmogenicity [3].

A human cell model for LQT3 has also been produced and its function and characteristics were compared with a mouse models which were based on both mouse ESCs and mouse iPS cells affected with the same disease specific mutation [71]. LQT3 syndrome is due to mutations in the SCN5A gene, which encodes for the α-subunit of the cardiac sodium (Na+) channel. These mutations disrupt the inactivation of the Na+ channel during the action potential plateu phase and this irruption leads to the delay in repolarization and further prolonged QT interval [72]. In addition to LQT3, another kind of cardiac arrhythmia syndromes such as Brugada syndrome and cardiac conduction disease are associated with mutations in the SCN5A gene. In these syndromes the mutations are loss-of-function-type whereas LQT3 syndrome they are gain-offunction-type mutations [72, 73]. The comparison of multiple types of pluripotent stem cell derived cardiomyocytes showed that all of these models manifest the symptoms of the disease and, furthermore, the characteristics are similar within both species [71]. iPS cell models for these loss-of-function diseases have not yet been described.

#### *6.1.1. Timothy syndrome*

also known as KCNH2-gene [67]. Contrary to type 1, individuals with LQT2 have clinical symptoms when the heart rate is slow [65, 66] and syptoms can be triggered e.g. by an alarm clock during sleep. The drug induced form of LQTS is due to altered function of the HERGchannel by the drug, therefore this channel has a significant importance during drug devel‐

The prevalence of the genetic form of LQTS is 1:2,000 in the general population [63]. However, the penetrance of the clinical symptoms of LQTS is low and there is considerable variation in phenotypic expression even within families carrying the same mutation [68]. It has also been suggested, that the population prevalence of milder LQTS mutations might be higher. Therefore the prevalence of latent or concealed LQTS, i.e. relatively asymptomatic individuals, would be higher than currently anticipated [69]. Due to this challenging and complex nature of LQTS, in addition, to the great interest of pharmaceutical industry towards this disease, multiple reports of iPS cell- based LQTS cell models have been published since 2007 when the

Moretti and co-workers produced iPS cell derived cardiomycotyes from two patients carrying a KCNQ1 (R190Q) mutation [6]. In this study, the cardiomyocytes possessed the LQT1 genotype and exhibited prolonged action potential duration. The action potential prolongation was determined to be caused by the ion-channel trafficking defect resulting in a 70-80% IKs current density reduction. A β-adrenergic agonist isoproterenol altered the activation and deactivation kinetics of the IKs and this effect was rescued by the β-blockade [6]. Egashira and co-workers also produced a disease model for LQTS type 1 [70]. In their study, the iPS cells were derived from a sporadic patient who did not have a family history of significant QT interval abnormality. The mutation of the patient in the KCNQ1 was novel (1893delC) and the

cells exhibited prolonged action potential duration in addition to arrhythmogenity.

Similarly results were found with iPS-CM derived from a patient suffering from the severe LQT type 2 syndrome. The patient had hERG (A614V) mutation and previously presented episodes of torsade de pointes (TdP), a special type of polymorphic ventricular tachycardia which is associated with LQTS [4]. The LQT2-cardiomyocytes derived from the patient's iPS cells demonstrated increased arrhythmogenicity associated with early after depolarizations (EADs) [5]. In addition, significant APD prolongation due to a reduced IKr current density was observed [4]. Arrhytmia and EADs were also induced by a specific HERG-channel blocker E-4031 to iPS cell derived CM having a hERG (G1681A) mutation. In addition, these cells exhibited EADs caused by the isoproterenol treatment and these EADs were rescued by β-

All the aforementioned studies were done with iPS cells derived from the symptomatic LQTS patients. Nevertheless, similar results have been obtained from patients without severe symptoms. In the study made in our institute, iPS cell lines were derived from a patient having a KCNH2 (R176W) mutation and a family history of LQTS. However, this individual was asymptomatic except for occasional palpitations. iPS cell derived cardiomyocytes from this patient manifest the phenotype characteristics to LQT2, such as a prolonged repolarization

opment and in safety studies.

424 Pluripotent Stem Cells

iPS technology was invented.

blockade [5].

time and increased arrhythmogenicity [3].

Timothy syndrome is caused by a single mutation in the CACNA1C-gene. This gene encodes the main L-type calcium channel, Cav1.2, in the mammalian heart which is essential for the cardiac action potential and also for cardiomyocyte contraction [74-76]. Timothy sydrome characterized by LQTS, syndactyly (webbing of fingers and toes), immune deficiency and autism [77] iPS cell derived cardiomyocytes originating from Timothy syndrome patients exhibited irregular functional properties typical for the disease [78]. Interestingly, these irregularities were restored by roscovitine, a compound which increases the voltage-depend‐ ent inactivation of Cav1.2 [78].

#### *6.1.2. Catecholaminergic Polymorphic Ventricular Tachycardia*

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disorder characterized by stress-induced polymorphic ventricular tachycardia in a structurally normal heart. CPVT is a very severe disease and 30-35% of mutation carriers have had symptoms (stress-related syncope, seizures or sudden death) by the age of 30. This disease is caused by mutations in the genes of RyR2 or calsequestrin (CASQ2) which is a regulatory calcium-buffering protein associated with RyR2 in the SR [79-82].

Multiple iPS-based CPVT disease models have been published, most of them having the disease specific mutation in the RyR2 gene while [83-85] and one having the mutation in the CASQ2 gene [86]. The congruent result from these CPVT model studies was the occurrence of delayed after depolarizations (DADs) and arrhythmias which are caused by the aberrant diastolic Ca2+ from the SR. Notably the model with RyR2-P2328S mutation also exhibited early after depolarizations (EADs) in addition to DADs suggesting suggest‐ ing another pathophysiological mechanism for CPVT [85]. Intriguing finding was also the effect of dantrolene in rescuing the arrhythmogenic phenotype [84].

#### *6.1.3. Cardiomyopathies*

Mutations in the genes expressed in the cardiomyocytes can cause heart diseases known as cardiomyopathies. Cardiomyopathies are currently categorized into the following four classes: arrythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, and restrictive cardiomyopathy [87]. Cardiomyopathies that are associated with mutations in genes encoding for sarcomeric proteins are a frequent cause of heart failure. changes including increase in protein synthesis, enhanced sarcomere reorganization as well

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427

iPS cell technology has not yet been reported to model HCM. However, iPS cells were used to generate cardiomyocytes from two LEOPARD syndrome patients carrying mutation in the PTPN11 gene encoding for the SHP2 phosphatase [101]. LEOPARD syndrome is an autosomaldominant developmental disorder belonging to inherited RAS-mitogen-activated protein kinase signalling diseases. A major disease phenotype of the LEOPARD syndrome patients is HCM [102]. The iPS cell derived cardiomyocytes from LEOPARD syndrome were larger, had a higher degree of sarcomeric organization as well as preferential localization of NFATC4 in the nucleus when compared to iPS cell derived cardiomyocytes from healthy sibling of the LEOPARD syndrome patient thus presenting some indications of hypertrophy in patient

There are still several challenges that need to be carefully considered when designing disease modeling studies with specialized cell types derived from iPS cells. One potential challenge relates to the reactivation of silenced exogenous transgenes in the iPS cells or in their differ‐ entiated derivatives leading to the altered genomic integrity which may have unknown effects on the differentiation potential and characteristics of differentiated cell types. Efforts to improve the reprogramming methods have led to the technical development of nonintegrating approaches for iPS cell generation which will eliminate this risk in the future iPS cell lines and their differentiated derivatives. The nonintegrating sendai virus tehnique is already widely used in the generation of iPS cells. Regular monitoring of exogenous genes in iPS cells lines

Many genetic cardiac diseases are complex demonstrating huge clinical heterogeneity even within families and patients having the same mutation. In addition, reprogrammed cells carry genetic alterations that have accumulated through life, thus there is a risk that the variance overwhelms the ability to detect the authentic mechanisms in the pathophysiology of the disease. Thus, it will be essential to investigate adequate number of iPSC lines and patients to be able to demonstrate the common features of the cardiac disease phenotype. Further, it may be advantageous to initially compare the characteristics of cardiomyocytes from patients

Most likely in many genetic cardiac diseases various cell types in the heart contribute to the pathophysiological responses of the disease, thus there is a risk that it is impossible to recapitulate the features of the disorder by using solely cardiomyocytes. A 3D human heart tissue model with proper composition of cardiomyocytes, endothelial cells, fibroblasts, smooth muscle cells as well as neurons has not been developed but in recent years the advancement in this field of research has been rapid and hopefully in future we have besides cell models

**7. Challenges with iPS cell technology and disease modeling**

generated by using the integrating techniques is advisable.

authentic tissue models to study genetic cardiac diseases.

as activation of specific cardiac genes [98-100].

specific cardiomyocytes.

having severe symptoms.

iPS cells have been used to generate cardiomyocytes from patients in a family with inherited dilated cardiomyopathy (DCM) [88]. The researchers generated a large number of individualspecific cardiomyocytes from a family carrying a deleterious point mutation (R173W) in TNNT2, a gene encoding for a sarcomeric protein cardiac troponin T, which regulates cardiomyocyte contraction. When compared to cardiomyocytes derived from iPS cells of healthy controls within the same family, the researchers showed that cardiomyocytes derived from iPS cells of DCM patients exhibited an increased heterogenous myofilament organization due to abnormal distribution of α-actinin, compromised ability to regulate calcium flux, and decreased contraction force. When DCM specific cardiomyocytes were stimulated with a βadrenergic agonist, the cells showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric αactinin distribution. The authors also showed that the function of DCM-specific cardiomyo‐ cytes was improved with the treatment with β-adrenergic blockers or overexpression of Serca2a.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is another genetic cardiomyop‐ athy characterized by replacement of cardiomyocytes by adipose and fibrous tissue leading to right ventricular failure, arrhythmias and even sudden death [89]. Twelve different genes have been linked to ARVC and all these encode cardiac cell adhesion proteins resulting in dysfunc‐ tional cardiac desmosomes. Cell adhesion proteins resulting in ARVC include plakoglobin (JUP), desmoplakin (DSP) and plakophilin 2 (PKP2). Patient specific iPS cells have been generated from an ARCV patient carrying a PKP2 mutation and having clinical manifestations of the disease [90]. ARVC specific cardiomyocytes revealed reduced amount of desmosomal proteins and more lipid droplets in the cardiomyocytes compared to control cardiomyocytes thus presenting the abnormalities observed in ARCV patients.

The third form of cardiomyopathy, hypertrophic cardiomyopathy (HCM), is a complex autosomal-dominant disease and the affected individuals acquire cardiac hypertrophy without external stimuli. Cardiac hypertrophy can be induced by different exogenous factors such as hypertension and valvular disease and even by severe exercise [91]. Affecting in 1 in 500 individuals within the general population, genetic HCM is the most common inherited cardiovascular disorder and the leading cause of sudden cardiac death in adolescents and young adults, especially in atheletes [92-94]. The majority of gene mutations associated with HCM occur in 13 sarcomere-related genes where several hundred mutations have been identified [94-97]. Typically cardiac hypertrophy affects the left ventricle and the interventric‐ ular septum and may eventually lead to left ventricular outflow tract obstruction, arrythmias, diastolic dysfunction, and sudden death. Other hallmark features are myocyte disarray and fibrosis [94-97]. The hypertrophic process in cardiomyocytes is characterized by morphological changes including increase in protein synthesis, enhanced sarcomere reorganization as well as activation of specific cardiac genes [98-100].

*6.1.3. Cardiomyopathies*

426 Pluripotent Stem Cells

Serca2a.

Mutations in the genes expressed in the cardiomyocytes can cause heart diseases known as cardiomyopathies. Cardiomyopathies are currently categorized into the following four classes: arrythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, and restrictive cardiomyopathy [87]. Cardiomyopathies that are associated with mutations in genes encoding for sarcomeric proteins are a frequent cause of heart failure. iPS cells have been used to generate cardiomyocytes from patients in a family with inherited dilated cardiomyopathy (DCM) [88]. The researchers generated a large number of individualspecific cardiomyocytes from a family carrying a deleterious point mutation (R173W) in TNNT2, a gene encoding for a sarcomeric protein cardiac troponin T, which regulates cardiomyocyte contraction. When compared to cardiomyocytes derived from iPS cells of healthy controls within the same family, the researchers showed that cardiomyocytes derived from iPS cells of DCM patients exhibited an increased heterogenous myofilament organization due to abnormal distribution of α-actinin, compromised ability to regulate calcium flux, and decreased contraction force. When DCM specific cardiomyocytes were stimulated with a βadrenergic agonist, the cells showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric αactinin distribution. The authors also showed that the function of DCM-specific cardiomyo‐ cytes was improved with the treatment with β-adrenergic blockers or overexpression of

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is another genetic cardiomyop‐ athy characterized by replacement of cardiomyocytes by adipose and fibrous tissue leading to right ventricular failure, arrhythmias and even sudden death [89]. Twelve different genes have been linked to ARVC and all these encode cardiac cell adhesion proteins resulting in dysfunc‐ tional cardiac desmosomes. Cell adhesion proteins resulting in ARVC include plakoglobin (JUP), desmoplakin (DSP) and plakophilin 2 (PKP2). Patient specific iPS cells have been generated from an ARCV patient carrying a PKP2 mutation and having clinical manifestations of the disease [90]. ARVC specific cardiomyocytes revealed reduced amount of desmosomal proteins and more lipid droplets in the cardiomyocytes compared to control cardiomyocytes

The third form of cardiomyopathy, hypertrophic cardiomyopathy (HCM), is a complex autosomal-dominant disease and the affected individuals acquire cardiac hypertrophy without external stimuli. Cardiac hypertrophy can be induced by different exogenous factors such as hypertension and valvular disease and even by severe exercise [91]. Affecting in 1 in 500 individuals within the general population, genetic HCM is the most common inherited cardiovascular disorder and the leading cause of sudden cardiac death in adolescents and young adults, especially in atheletes [92-94]. The majority of gene mutations associated with HCM occur in 13 sarcomere-related genes where several hundred mutations have been identified [94-97]. Typically cardiac hypertrophy affects the left ventricle and the interventric‐ ular septum and may eventually lead to left ventricular outflow tract obstruction, arrythmias, diastolic dysfunction, and sudden death. Other hallmark features are myocyte disarray and fibrosis [94-97]. The hypertrophic process in cardiomyocytes is characterized by morphological

thus presenting the abnormalities observed in ARCV patients.

iPS cell technology has not yet been reported to model HCM. However, iPS cells were used to generate cardiomyocytes from two LEOPARD syndrome patients carrying mutation in the PTPN11 gene encoding for the SHP2 phosphatase [101]. LEOPARD syndrome is an autosomaldominant developmental disorder belonging to inherited RAS-mitogen-activated protein kinase signalling diseases. A major disease phenotype of the LEOPARD syndrome patients is HCM [102]. The iPS cell derived cardiomyocytes from LEOPARD syndrome were larger, had a higher degree of sarcomeric organization as well as preferential localization of NFATC4 in the nucleus when compared to iPS cell derived cardiomyocytes from healthy sibling of the LEOPARD syndrome patient thus presenting some indications of hypertrophy in patient specific cardiomyocytes.

## **7. Challenges with iPS cell technology and disease modeling**

There are still several challenges that need to be carefully considered when designing disease modeling studies with specialized cell types derived from iPS cells. One potential challenge relates to the reactivation of silenced exogenous transgenes in the iPS cells or in their differ‐ entiated derivatives leading to the altered genomic integrity which may have unknown effects on the differentiation potential and characteristics of differentiated cell types. Efforts to improve the reprogramming methods have led to the technical development of nonintegrating approaches for iPS cell generation which will eliminate this risk in the future iPS cell lines and their differentiated derivatives. The nonintegrating sendai virus tehnique is already widely used in the generation of iPS cells. Regular monitoring of exogenous genes in iPS cells lines generated by using the integrating techniques is advisable.

Many genetic cardiac diseases are complex demonstrating huge clinical heterogeneity even within families and patients having the same mutation. In addition, reprogrammed cells carry genetic alterations that have accumulated through life, thus there is a risk that the variance overwhelms the ability to detect the authentic mechanisms in the pathophysiology of the disease. Thus, it will be essential to investigate adequate number of iPSC lines and patients to be able to demonstrate the common features of the cardiac disease phenotype. Further, it may be advantageous to initially compare the characteristics of cardiomyocytes from patients having severe symptoms.

Most likely in many genetic cardiac diseases various cell types in the heart contribute to the pathophysiological responses of the disease, thus there is a risk that it is impossible to recapitulate the features of the disorder by using solely cardiomyocytes. A 3D human heart tissue model with proper composition of cardiomyocytes, endothelial cells, fibroblasts, smooth muscle cells as well as neurons has not been developed but in recent years the advancement in this field of research has been rapid and hopefully in future we have besides cell models authentic tissue models to study genetic cardiac diseases.

The current cardiomyocyte differentiation protocols generate cells lacking full maturity when compared to human adult cardiomyocytes. This may lead to a situation where it is impossible to detect some molecular or functional basis of the cardiac disease. To reduce this risk it will be advisable to use control cells to compare diseased cardiomyocytes to healthy cardiomyo‐ cytes. For reliable and reproducible modeling of cardiac diseases it is necessary to have preferable multiple iPS lines from healthy controls. For monogenic diseases the use of iPS cells derived from the healthy family members would be favorable for minimizing the effect of genetic variation. However, iPS cells from family members are not always available. On possibility to overcome this challenge is to use genome editing techniques such as zinc finger nuclease technology and transcription activator–like effectors (TALEs) in modifying the iPS cells [103, 104]. With these methods, it is possible to correct a targeted point mutation in human iPS cells and produce control cells for disease specific iPS cells.

**References**

471(7337), 225-9.

(2010). , 363(15), 1397-409.

(2007). , 131(5), 861-72.

(2009). , 5(4), 434-41.

(2007). , 318(5858), 1917-20.

Nat Biotechnol (2008). , 26(11), 1276-84.

and SOX2. Cell Stem Cell (2009). , 5(4), 353-7.

[1] Dixon, J. A, & Spinale, F. G. Large animal models of heart failure: a critical link in the translation of basic science to clinical practice. Circ Heart Fail (2009). , 2(3), 262-71.

Disease Models for the Genetic Cardiac Diseases

http://dx.doi.org/10.5772/55773

429

[2] Cingolani, H. E, & Ennis, I. L. Sodium-hydrogen exchanger, cardiac overload, and

[3] Lahti, A. L, Kujala, V. J, Chapman, H, Koivisto, A. P, Pekkanen-mattila, M, Kerkela, E, et al. Model for long QT syndrome type 2 using human iPS cells demonstrates ar‐ rhythmogenic characteristics in cell culture. Dis Model Mech (2012). , 5(2), 220-30.

[4] Itzhaki, I, Maizels, L, Huber, I, Zwi-dantsis, L, Caspi, O, Winterstern, A, et al. Model‐ ling the long QT syndrome with induced pluripotent stem cells. Nature (2011). ,

[5] Matsa, E, Rajamohan, D, Dick, E, Young, L, Mellor, I, Staniforth, A, et al. Drug evalu‐ ation in cardiomyocytes derived from human induced pluripotent stem cells carry‐

[6] Moretti, A, Bellin, M, Welling, A, Jung, C. B, Lam, J. T, Bott-flugel, L, et al. Patientspecific induced pluripotent stem-cell models for long-QT syndrome. N Engl J Med

[7] Takahashi, K, Tanabe, K, Ohnuki, M, Narita, M, Ichisaka, T, Tomoda, K, et al. Induc‐ tion of pluripotent stem cells from adult human fibroblasts by defined factors. Cell

[8] Haase, A, Olmer, R, Schwanke, K, Wunderlich, S, Merkert, S, Hess, C, et al. Genera‐ tion of induced pluripotent stem cells from human cord blood. Cell Stem Cell

[9] Yu, J, Vodyanik, M. A, Smuga-otto, K, Antosiewicz-bourget, J, Frane, J. L, Tian, S, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science

[10] Aasen, T, Raya, A, Barrero, M. J, Garreta, E, Consiglio, A, Gonzalez, F, et al. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.

[11] Giorgetti, A, Montserrat, N, Aasen, T, Gonzalez, F, Rodriguez-piza, I, Vassena, R, et al. Generation of induced pluripotent stem cells from human cord blood using OCT4

[12] Takahashi, K, & Yamanaka, S. Induction of pluripotent stem cells from mouse em‐ bryonic and adult fibroblast cultures by defined factors. Cell (2006). , 126(4), 663-76.

ing a long QT syndrome type 2 mutation. Eur Heart J (2011). , 32(8), 952-62.

myocardial hypertrophy. Circulation (2007). , 115(9), 1090-100.

## **8. Conclusions**

The most relevant human disease model uses cells of human origin, of the appropriate cell type, and with the identical genetic background as the patients. Traditionally, this approach in cardiac diseases has been out of reach as human cardiomyocytes are not easily procured and their propagation in vitro is extremely problematic. The revolutionary discovery of cellular reprogramming as a technology to generate iPS cells enables the production of patient specific cell types such as cardiomyocytes which can be used as authentic and relevant human cell models to study the pathophysiology of genetic cardiac diseases as well as in drug discovery and safety assays. The most relevant aspects in disease modeling are to show that the produced disease specific cell type bears the disease causing mutation and further to present the functional consequences of the mutant protein. Here we have reviewed the genetic cardiac diseases modeled thus far by using the iPS cell technology. Worthwhile of noticing is that the era of iPS cells in disease modeling is just in the very beginning. As the production of iPS cells and cardiomyocytes with more mature phenotype and the methods available for the functional characterization of cardiomyocytes continue to develop the future looks bright for modeling genetic cardiac diseases. Importantly these models will be extremely valuable for drug discovery and toxicology in the future.

### **Author details**


## **References**

The current cardiomyocyte differentiation protocols generate cells lacking full maturity when compared to human adult cardiomyocytes. This may lead to a situation where it is impossible to detect some molecular or functional basis of the cardiac disease. To reduce this risk it will be advisable to use control cells to compare diseased cardiomyocytes to healthy cardiomyo‐ cytes. For reliable and reproducible modeling of cardiac diseases it is necessary to have preferable multiple iPS lines from healthy controls. For monogenic diseases the use of iPS cells derived from the healthy family members would be favorable for minimizing the effect of genetic variation. However, iPS cells from family members are not always available. On possibility to overcome this challenge is to use genome editing techniques such as zinc finger nuclease technology and transcription activator–like effectors (TALEs) in modifying the iPS cells [103, 104]. With these methods, it is possible to correct a targeted point mutation in human

The most relevant human disease model uses cells of human origin, of the appropriate cell type, and with the identical genetic background as the patients. Traditionally, this approach in cardiac diseases has been out of reach as human cardiomyocytes are not easily procured and their propagation in vitro is extremely problematic. The revolutionary discovery of cellular reprogramming as a technology to generate iPS cells enables the production of patient specific cell types such as cardiomyocytes which can be used as authentic and relevant human cell models to study the pathophysiology of genetic cardiac diseases as well as in drug discovery and safety assays. The most relevant aspects in disease modeling are to show that the produced disease specific cell type bears the disease causing mutation and further to present the functional consequences of the mutant protein. Here we have reviewed the genetic cardiac diseases modeled thus far by using the iPS cell technology. Worthwhile of noticing is that the era of iPS cells in disease modeling is just in the very beginning. As the production of iPS cells and cardiomyocytes with more mature phenotype and the methods available for the functional characterization of cardiomyocytes continue to develop the future looks bright for modeling genetic cardiac diseases. Importantly these models will be extremely valuable for drug

iPS cells and produce control cells for disease specific iPS cells.

**8. Conclusions**

428 Pluripotent Stem Cells

discovery and toxicology in the future.

Mari Pekkanen-Mattila1,2, Kristiina Rajala1,2 and Katriina Aalto-Setälä1,2,3

2 BioMediTech, University of Tampere, Tampere, Finland

3 Heart Center, Tampere University Hospital, Tampere, Finland

1 Institute of Biomedical Technology, University of Tampere, Tampere, Finland

**Author details**


[13] Hussein, S. M, Batada, N. N, Vuoristo, S, Ching, R. W, Autio, R, Narva, E, et al. Copy number variation and selection during reprogramming to pluripotency. Nature (2011). , 471(7336), 58-62.

[25] Kim, D, Kim, C. H, Moon, J. I, Chung, Y. G, Chang, M. Y, Han, B. S, et al. Generation of human induced pluripotent stem cells by direct delivery of reprogramming pro‐

Disease Models for the Genetic Cardiac Diseases

http://dx.doi.org/10.5772/55773

431

[26] Lin, T, Ambasudhan, R, Yuan, X, Li, W, Hilcove, S, Abujarour, R, et al. A chemical platform for improved induction of human iPSCs. Nat Methods (2009). , 6(11), 805-8.

[27] Mummery, C, & Ward, D. van den Brink CE, Bird SD, Doevendans PA, Opthof T, et al. Cardiomyocyte differentiation of mouse and human embryonic stem cells. J Anat

[28] Kehat, I, Kenyagin-karsenti, D, Snir, M, Segev, H, Amit, M, Gepstein, A, et al. Hu‐ man embryonic stem cells can differentiate into myocytes with structural and func‐

[29] Passier, R, Oostwaard, D. W, Snapper, J, Kloots, J, Hassink, R. J, Kuijk, E, et al. In‐ creased cardiomyocyte differentiation from human embryonic stem cells in serum-

[30] Zhang, J, Wilson, G. F, Soerens, A. G, Koonce, C. H, Yu, J, Palecek, S. P, et al. Func‐ tional cardiomyocytes derived from human induced pluripotent stem cells. Circ Res

[31] Pekkanen-mattila, M, Kerkela, E, Tanskanen, J. M, Pietila, M, Pelto-huikko, M, Hytti‐ nen, J, et al. Substantial variation in the cardiac differentiation of human embryonic stem cell lines derived and propagated under the same conditions--a comparison of

[32] He, J-Q, Ma, Y, Lee, Y, Thomson, J. A, & Kamp, T. J. Human Embryonic Stem Cells Develop Into Multiple Types of Cardiac Myocytes: Action Potential Characterization.

[33] Pekkanen-mattila, M, Chapman, H, Kerkela, E, Suuronen, R, Skottman, H, Koivisto, A. P, et al. Human embryonic stem cell-derived cardiomyocytes: demonstration of a portion of cardiac cells with fairly mature electrical phenotype. Exp Biol Med (May‐

[34] Moore, J. C, Fu, J, Chan, Y. C, Lin, D, Tran, H, Tse, H. F, et al. Distinct cardiogenic preferences of two human embryonic stem cell (hESC) lines are imprinted in their proteomes in the pluripotent state. Biochem Biophys Res Commun (2008). , 372(4),

[35] Mummery, C. L, Zhang, J, Ng, E. S, Elliott, D. A, Elefanty, A. G, & Kamp, T. J. Differ‐ entiation of human embryonic stem cells and induced pluripotent stem cells to cardi‐

[36] Ieda, M, Fu, J. D, Delgado-olguin, P, Vedantham, V, Hayashi, Y, Bruneau, B. G, et al. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined fac‐

omyocytes: a methods overview. Circ Res (2012). , 111(3), 344-58.

tional properties of cardiomyocytes. J Clin Invest (2001). , 108(3), 407-14.

teins. Cell Stem Cell (2009). , 4(6), 472-6.

free cultures. Stem Cells (2005). , 23(6), 772-80.

multiple cell lines. Ann Med (2009). , 41(5), 360-70.

(2002). Pt 3):233-42.

(2009). e, 30-41.

Circ Res (2003). , 93(1), 32-39.

wood) (2010). , 235(4), 522-30.

tors. Cell (2010). , 142(3), 375-86.

553-8.


[25] Kim, D, Kim, C. H, Moon, J. I, Chung, Y. G, Chang, M. Y, Han, B. S, et al. Generation of human induced pluripotent stem cells by direct delivery of reprogramming pro‐ teins. Cell Stem Cell (2009). , 4(6), 472-6.

[13] Hussein, S. M, Batada, N. N, Vuoristo, S, Ching, R. W, Autio, R, Narva, E, et al. Copy number variation and selection during reprogramming to pluripotency. Nature

[14] Stadtfeld, M, Nagaya, M, Utikal, J, Weir, G, & Hochedlinger, K. Induced pluripotent stem cells generated without viral integration. Science (2008). , 322(5903), 945-9.

[15] Fusaki, N, Ban, H, Nishiyama, A, Saeki, K, & Hasegawa, M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys

[16] Okita, K, Nakagawa, M, Hyenjong, H, Ichisaka, T, & Yamanaka, S. Generation of mouse induced pluripotent stem cells without viral vectors. Science (2008). ,

[17] Yu, J, Hu, K, Smuga-otto, K, Tian, S, Stewart, R, Slukvin, I. I, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science (2009). ,

[18] Ebert, A. D, Yu, J, & Rose, F. F. Jr., Mattis VB, Lorson CL, Thomson JA, et al. Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature (2009). ,

[19] Jia, F, Wilson, K. D, Sun, N, Gupta, D. M, Huang, M, Li, Z, et al. A nonviral minicircle

[20] Lee, C. H, Kim, J. H, Lee, H. J, Jeon, K, Lim, H, Choi, H, et al. The generation of iPS cells using non-viral magnetic nanoparticle based transfection. Biomaterials (2011). ,

[21] Shao, L, Feng, W, Sun, Y, Bai, H, Liu, J, Currie, C, et al. Generation of iPS cells using defined factors linked via the self-cleaving 2A sequences in a single open reading

[22] Yusa, K, Rad, R, Takeda, J, & Bradley, A. Generation of transgene-free induced pluri‐ potent mouse stem cells by the piggyBac transposon. Nat Methods (2009). , 6(5),

[23] Yakubov, E, Rechavi, G, Rozenblatt, S, & Givol, D. Reprogramming of human fibro‐ blasts to pluripotent stem cells using mRNA of four transcription factors. Biochem

[24] Warren, L, Manos, P. D, Ahfeldt, T, Loh, Y. H, Li, H, Lau, F, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with syn‐

vector for deriving human iPS cells. Nat Methods (2010). , 7(3), 197-9.

(2011). , 471(7336), 58-62.

430 Pluripotent Stem Cells

Biol Sci (2009). , 85(8), 348-62.

322(5903), 949-53.

324(5928), 797-801.

457(7227), 277-80.

32(28), 6683-91.

363-9.

frame. Cell Res (2009). , 19(3), 296-306.

Biophys Res Commun (2010). , 394(1), 189-93.

thetic modified mRNA. Cell Stem Cell (2010). , 7(5), 618-30.


[37] Chen, J. X, Krane, M, Deutsch, M. A, Wang, L, Rav-acha, M, Gregoire, S, et al. Ineffi‐ cient reprogramming of fibroblasts into cardiomyocytes using Gata4, Mef2c, and Tbx5. Circ Res (2012). , 111(1), 50-5.

[50] Rajamohan, D, Matsa, E, Kalra, S, Crutchley, J, Patel, A, George, V, et al. Current sta‐ tus of drug screening and disease modelling in human pluripotent stem cells. Bioes‐

Disease Models for the Genetic Cardiac Diseases

http://dx.doi.org/10.5772/55773

433

[51] Ma, J, Guo, L, Fiene, S. J, Anson, B. D, Thomson, J. A, Kamp, T. J, et al. High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents. Am J Physiol Heart Circ Physiol

[52] Reppel, M, Pillekamp, F, Lu, Z. J, Halbach, M, Brockmeier, K, Fleischmann, B. K, et al. Microelectrode arrays: a new tool to measure embryonic heart activity. J Electro‐

[53] Reppel, M, Pillekamp, F, Brockmeier, K, Matzkies, M, Bekcioglu, A, Lipke, T, et al. The electrocardiogram of human embryonic stem cell-derived cardiomyocytes. J

[54] Caspi, O, Itzhaki, I, Kehat, I, Gepstein, A, Arbel, G, Huber, I, et al. In vitro electro‐ physiological drug testing using human embryonic stem cell derived cardiomyo‐

[55] Heikkila, T. J, Yla-outinen, L, Tanskanen, J. M, Lappalainen, R. S, Skottman, H, Suur‐ onen, R, et al. Human embryonic stem cell-derived neuronal cells form spontaneous‐

[56] Kujala, V. J, Jimenez, Z. C, Vaisanen, J, Tanskanen, J. M, Kerkela, E, Hyttinen, J, et al. Averaging in vitro cardiac field potential recordings obtained with microelectrode

[57] Bers, D. M. Cardiac excitation-contraction coupling. Nature (2002). , 415(6868),

[58] Wier, W. G, Beuckelmann, D. J, & Barcenas-ruiz, L. Ca2+]i in single isolated cardiac cells: a review of recent results obtained with digital imaging microscopy and fura-2.

[59] Herron, T. J, Lee, P, & Jalife, J. Optical imaging of voltage and calcium in cardiac cells

[60] Liu, J, Sun, N, Bruce, M. A, Wu, J. C, & Butte, M. J. Atomic force mechanobiology of

[61] Boudou, T, Legant, W. R, Mu, A, Borochin, M. A, Thavandiran, N, Radisic, M, et al. A microfabricated platform to measure and manipulate the mechanics of engineered

[62] Schaaf, S, Shibamiya, A, Mewe, M, Eder, A, Stohr, A, Hirt, M. N, et al. Human engi‐ neered heart tissue as a versatile tool in basic research and preclinical toxicology.

pluripotent stem cell-derived cardiomyocytes. PLoS One (2012). e37559.

ly active neuronal networks in vitro. Exp Neurol (2009). , 218(1), 109-16.

arrays. Comput Methods Programs Biomed (2011). , 104(2), 199-205.

says (2012).

198-205.

(2011). H, 2006-17.

cardiol (2004). Suppl:, 104-9.

Electrocardiol (2005). Suppl):, 166-70.

cytes. Stem Cells Dev (2009). , 18(1), 161-72.

Can J Physiol Pharmacol (1988). , 66(9), 1224-31.

cardiac microtissues. Tissue Eng Part A (2012).

& tissues. Circ Res;, 110(4), 609-23.

PLoS One (2011). e26397.


[50] Rajamohan, D, Matsa, E, Kalra, S, Crutchley, J, Patel, A, George, V, et al. Current sta‐ tus of drug screening and disease modelling in human pluripotent stem cells. Bioes‐ says (2012).

[37] Chen, J. X, Krane, M, Deutsch, M. A, Wang, L, Rav-acha, M, Gregoire, S, et al. Ineffi‐ cient reprogramming of fibroblasts into cardiomyocytes using Gata4, Mef2c, and

[38] Islas, J. F, Liu, Y, Weng, K. C, Robertson, M. J, Zhang, S, Prejusa, A, et al. Transcrip‐ tion factors ETS2 and MESP1 transdifferentiate human dermal fibroblasts into car‐

[39] Yoshida, Y, & Yamanaka, S. Labor pains of new technology: direct cardiac reprog‐

[40] Qian, L, Huang, Y, Spencer, C. I, Foley, A, Vedantham, V, Liu, L, et al. In vivo re‐ programming of murine cardiac fibroblasts into induced cardiomyocytes. Nature

[41] Burridge, P. W, Thompson, S, Millrod, M. A, Weinberg, S, Yuan, X, Peters, A, et al. A universal system for highly efficient cardiac differentiation of human induced pluri‐

potent stem cells that eliminates interline variability. PLoS One (2011). e18293.

[42] Mummery, C. Ward-van Oostwaard D, Doevendans P, Spijker R, van den Brink S, Hassink R, et al. Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation (2003). , 107(21),

[43] Graichen, R, Xu, X, Braam, S. R, Balakrishnan, T, Norfiza, S, Sieh, S, et al. Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of

[44] Laflamme, M. A, Chen, K. Y, Naumova, A. V, Muskheli, V, Fugate, J. A, Dupras, S. K, et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival fac‐ tors enhance function of infarcted rat hearts. Nat Biotechnol (2007). , 25(9), 1015-24.

[45] Lian, X, Hsiao, C, Wilson, G, Zhu, K, Hazeltine, L. B, Azarin, S. M, et al. Robust cardi‐ omyocyte differentiation from human pluripotent stem cells via temporal modula‐

[46] Hamill, O. P, Marty, A, Neher, E, Sakmann, B, & Sigworth, F. J. Improved patchclamp techniques for high-resolution current recording from cells and cell-free mem‐

[47] Sakmann, B, & Neher, E. Patch clamp techniques for studying ionic channels in excit‐

[48] Pollard, C. E. Abi Gerges N, Bridgland-Taylor MH, Easter A, Hammond TG, Valen‐ tin JP. An introduction to QT interval prolongation and non-clinical approaches to

[49] Nerbonne, J. M, & Kass, R. S. Molecular physiology of cardiac repolarization. Physiol

tion of canonical Wnt signaling. Proc Natl Acad Sci U S A (2012). E, 1848-57.

brane patches. Pflugers Arch (1981). , 391(2), 85-100.

able membranes. Annu Rev Physiol (1984). , 46, 455-72.

assessing and reducing risk. Br J Pharmacol (2010). , 159(1), 12-21.

diac progenitors. Proc Natl Acad Sci U S A (2012). , 109(32), 13016-21.

Tbx5. Circ Res (2012). , 111(1), 50-5.

ramming. Circ Res (2012). , 111(1), 3-4.

MAPK. Differentiation (2008). , 38.

Rev (2005). , 85(4), 1205-53.

(2012). , 485(7400), 593-8.

2733-40.

432 Pluripotent Stem Cells


[63] Hedley, P. L, Jorgensen, P, Schlamowitz, S, Wangari, R, Moolman-smook, J, Brink, P. A, et al. The genetic basis of long QT and short QT syndromes: a mutation update. Hum Mutat (2009). , 30(11), 1486-511.

[77] Splawski, I, Timothy, K. W, Sharpe, L. M, Decher, N, Kumar, P, Bloise, R, et al. Ca(calcium channel dysfunction causes a multisystem disorder including arrhythmia

Disease Models for the Genetic Cardiac Diseases

http://dx.doi.org/10.5772/55773

435

[78] Yazawa, M, Hsueh, B, Jia, X, Pasca, A. M, Bernstein, J. A, Hallmayer, J, et al. Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syn‐

[79] Hayashi, M, Denjoy, I, Extramiana, F, Maltret, A, Buisson, N. R, Lupoglazoff, J. M, et al. Incidence and risk factors of arrhythmic events in catecholaminergic polymorphic

[80] Leenhardt, A, Lucet, V, Denjoy, I, Grau, F, Ngoc, D. D, & Coumel, P. Catecholami‐ nergic polymorphic ventricular tachycardia in children. A 7-year follow-up of 21 pa‐

[81] Laitinen, P. J, Brown, K. M, Piippo, K, Swan, H, Devaney, J. M, Brahmbhatt, B, et al. Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic

[82] Priori, S. G, Napolitano, C, Tiso, N, Memmi, M, Vignati, G, Bloise, R, et al. Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymor‐

[83] Itzhaki, I, Maizels, L, Huber, I, Gepstein, A, Arbel, G, Caspi, O, et al. Modeling of cat‐ echolaminergic polymorphic ventricular tachycardia with patient-specific human-in‐

[84] Jung, C. B, & Moretti, A. Mederos y Schnitzler M, Iop L, Storch U, Bellin M, et al. Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia. EMBO Mol Med (2012). ,

[85] Kujala, K, Paavola, J, Lahti, A, Larsson, K, Pekkanen-mattila, M, Viitasalo, M, et al. Cell model of catecholaminergic polymorphic ventricular tachycardia reveals early

[86] Novak, A, Barad, L, Zeevi-levin, N, Shick, R, Shtrichman, R, Lorber, A, et al. Cardio‐ myocytes generated from CPVTD307H patients are arrhythmogenic in response to

[87] Richardson, P, Mckenna, W, Bristow, M, Maisch, B, Mautner, B, & Connell, O. J, et al. Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the Definition and Classification of cardiomyopathies.

[88] Sun, R. H, Hu, B. C, & Li, Q. Stress-induced cardiomyopathy complicated by multi‐ ple organ failure following cephalosporin-induced anaphylaxis. Intern Med (2012). ,

duced pluripotent stem cells. J Am Coll Cardiol (2012). , 60(11), 990-1000.

ventricular tachycardia. Circulation (2009). , 119(18), 2426-34.

ventricular tachycardia. Circulation (2001). , 103(4), 485-90.

and delayed afterdepolarizations. PLoS One (2012). e44660.

beta-adrenergic stimulation. J Cell Mol Med (2012). , 16(3), 468-82.

phic ventricular tachycardia. Circulation (2001). , 103(2), 196-200.

and autism. Cell (2004). , 1

drome. Nature (2011). , 471(7337), 230-4.

tients. Circulation (1995). , 91(5), 1512-9.

4(3), 180-91.

51(8), 895-9.

Circulation (1996). , 93(5), 841-2.


[77] Splawski, I, Timothy, K. W, Sharpe, L. M, Decher, N, Kumar, P, Bloise, R, et al. Ca(calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. Cell (2004). , 1

[63] Hedley, P. L, Jorgensen, P, Schlamowitz, S, Wangari, R, Moolman-smook, J, Brink, P. A, et al. The genetic basis of long QT and short QT syndromes: a mutation update.

[64] Chiang, C. E, & Roden, D. M. The long QT syndromes: genetic basis and clinical im‐

[65] Roden, D. M. Clinical practice. Long-QT syndrome. N Engl J Med (2008). , 358(2),

[66] Schwartz, P. J, Priori, S. G, Spazzolini, C, Moss, A. J, Vincent, G. M, Napolitano, C, et al. Genotype-phenotype correlation in the long-QT syndrome: gene-specific triggers

[67] Curran, M. E, Splawski, I, Timothy, K. W, Vincent, G. M, Green, E. D, & Keating, M. T. A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syn‐

[68] Priori, S. G, Napolitano, C, & Schwartz, P. J. Low penetrance in the long-QT syn‐

[69] Marjamaa, A, Salomaa, V, Newton-cheh, C, Porthan, K, Reunanen, A, Karanko, H, et al. High prevalence of four long QT syndrome founder mutations in the Finnish pop‐

[70] Egashira, T, Yuasa, S, Suzuki, T, Aizawa, Y, Yamakawa, H, Matsuhashi, T, et al. Dis‐ ease characterization using LQTS-specific induced pluripotent stem cells. Cardiovasc

[71] Davis, R. P, & Casini, S. van den Berg CW, Hoekstra M, Remme CA, Dambrot C, et al. Cardiomyocytes derived from pluripotent stem cells recapitulate electrophysio‐ logical characteristics of an overlap syndrome of cardiac sodium channel disease.

[72] Bennett, P. B, Yazawa, K, Makita, N, & George, A. L. Jr. Molecular mechanism for an

[73] Remme, C. A, & Bezzina, C. R. Sodium channel (dys)function and cardiac arrhyth‐

[74] Reuter, H. Ion channels in cardiac cell membranes. Annu Rev Physiol (1984). , 46,

[75] Flucher, B. E, & Franzini-armstrong, C. Formation of junctions involved in excitationcontraction coupling in skeletal and cardiac muscle. Proc Natl Acad Sci U S A

[76] Seisenberger, C, Specht, V, Welling, A, Platzer, J, Pfeifer, A, Kuhbandner, S, et al. Functional embryonic cardiomyocytes after disruption of the L-type alpha1C (Cav1.2) calcium channel gene in the mouse. J Biol Chem (2000). , 275(50), 39193-9.

inherited cardiac arrhythmia. Nature (1995). , 376(6542), 683-5.

for life-threatening arrhythmias. Circulation (2001). , 103(1), 89-95.

drome: clinical impact. Circulation (1999). , 99(4), 529-33.

Hum Mutat (2009). , 30(11), 1486-511.

drome. Cell (1995). , 80(5), 795-803.

ulation. Ann Med (2009). , 41(3), 234-40.

Circulation (2012). , 125(25), 3079-91.

mias. Cardiovasc Ther (2010). , 28(5), 287-94.

Res (2012). , 95(4), 419-29.

473-84.

(1996). , 93(15), 8101-6.

169-76.

434 Pluripotent Stem Cells

plications. J Am Coll Cardiol (2000). , 36(1), 1-12.


[89] Azaouagh, A, Churzidse, S, Konorza, T, & Erbel, R. Arrhythmogenic right ventricu‐ lar cardiomyopathy/dysplasia: a review and update. Clin Res Cardiol (2012). , 100(5), 383-94.

[103] Soldner, F, Laganiere, J, Cheng, A. W, Hockemeyer, D, Gao, Q, Alagappan, R, et al. Generation of isogenic pluripotent stem cells differing exclusively at two early onset

Disease Models for the Genetic Cardiac Diseases

http://dx.doi.org/10.5772/55773

437

[104] Hockemeyer, D, Wang, H, Kiani, S, Lai, C. S, Gao, Q, Cassady, J. P, et al. Genetic en‐ gineering of human pluripotent cells using TALE nucleases. Nat Biotechnol (2011). ,

Parkinson point mutations. Cell (2011). , 146(2), 318-31.

29(8), 731-4.


[103] Soldner, F, Laganiere, J, Cheng, A. W, Hockemeyer, D, Gao, Q, Alagappan, R, et al. Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell (2011). , 146(2), 318-31.

[89] Azaouagh, A, Churzidse, S, Konorza, T, & Erbel, R. Arrhythmogenic right ventricu‐ lar cardiomyopathy/dysplasia: a review and update. Clin Res Cardiol (2012). , 100(5),

[90] Ma, D, Wei, H, Lu, J, Ho, S, Zhang, G, Sun, X, et al. Generation of patient-specific in‐ duced pluripotent stem cell-derived cardiomyocytes as a cellular model of arrhyth‐

[91] Rohini, A, Agrawal, N, Koyani, C. N, & Singh, R. Molecular targets and regulators of

[92] Soor, G. S, Luk, A, Ahn, E, Abraham, J. R, Woo, A, Ralph-edwards, A, et al. Hyper‐ trophic cardiomyopathy: current understanding and treatment objectives. J Clin

[93] Maron, B. J, Gardin, J. M, Flack, J. M, Gidding, S. S, Kurosaki, T. T, & Bild, D. E. Prev‐ alence of hypertrophic cardiomyopathy in a general population of young adults. Echocardiographic analysis of 4111 subjects in the CARDIA Study. Coronary Artery

[94] Poliac, L. C, Barron, M. E, & Maron, B. J. Hypertrophic cardiomyopathy. Anesthesiol‐

[95] Richard, P, Charron, P, Carrier, L, Ledeuil, C, Cheav, T, Pichereau, C, et al. Hypertro‐ phic cardiomyopathy: distribution of disease genes, spectrum of mutations, and im‐ plications for a molecular diagnosis strategy. Circulation (2003). , 107(17), 2227-32.

[96] Lind, J. M, Chiu, C, & Semsarian, C. Genetic basis of hypertrophic cardiomyopathy.

[97] Maron, M. S, Olivotto, I, Betocchi, S, Casey, S. A, Lesser, J. R, Losi, M. A, et al. Effect of left ventricular outflow tract obstruction on clinical outcome in hypertrophic car‐

[98] Lorell, B. H, & Carabello, B. A. Left ventricular hypertrophy: pathogenesis, detection,

[99] Frey, N, Katus, H. A, Olson, E. N, & Hill, J. A. Hypertrophy of the heart: a new thera‐

[100] Wolf, C. M, Moskowitz, I. P, Arno, S, Branco, D. M, Semsarian, C, Bernstein, S. A, et al. Somatic events modify hypertrophic cardiomyopathy pathology and link hyper‐

[101] Carvajal-vergara, X, Sevilla, A, Souza, D, Ang, S. L, Schaniel, Y. S, & Lee, C. DF, et al. Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syn‐

[102] Sarkozy, A, Digilio, M. C, & Dallapiccola, B. Leopard syndrome. Orphanet J Rare Dis

trophy to arrhythmia. Proc Natl Acad Sci U S A (2005). , 102(50), 18123-8.

Risk Development in (Young) Adults. Circulation (1995). , 92(4), 785-9.

mogenic right ventricular cardiomyopathy. Eur Heart J (2012).

cardiac hypertrophy. Pharmacol Res (2010). , 61(4), 269-80.

383-94.

436 Pluripotent Stem Cells

Pathol (2009). , 62(3), 226-35.

ogy (2006). , 104(1), 183-92.

Expert Rev Cardiovasc Ther (2006). , 4(6), 927-34.

diomyopathy. N Engl J Med (2003). , 348(4), 295-303.

and prognosis. Circulation (2000). , 102(4), 470-9.

peutic target? Circulation (2004). , 109(13), 1580-9.

drome. Nature (2010). , 465(7299), 808-12.

(2008).

[104] Hockemeyer, D, Wang, H, Kiani, S, Lai, C. S, Gao, Q, Cassady, J. P, et al. Genetic en‐ gineering of human pluripotent cells using TALE nucleases. Nat Biotechnol (2011). , 29(8), 731-4.

**Chapter 20**

**Pluripotent Stem Cells to**

Calvin C. Sheng and Charles C. Hong

**2. Human cardiovascular diseases**

http://dx.doi.org/10.5772/54373

**1. Introduction**

**Model Human Cardiac Diseases**

Additional information is available at the end of the chapter

For past several decades, laboratory animal models have been the prevailing paradigm for studying human diseases. A classic approach is to study the impact of specific genes through the use of gain- or loss-of-function mutant animals. While the animal models have greatly contributed to our understanding of the etiology and mechanisms of disease, they often fall short of fully recapitulating human pathophysiology and translating to clinical ap‐ plications due to interspecies physiologic differences. In a review of preclinical studies of animal models published in high-impact scientific journals, approximately one-third trans‐ lated to the level of human randomized trials and only one-tenth were subsequently ap‐ proved clinically for patient use [1]. This attrition rate would have been even higher if less frequently cited animal research had been included. These unresolved issues with animal models have set the stage for the emergence of human embryonic stem cell (hESC) and hu‐

Laid out in this chapter, we will discuss the development of various stem cell paradigms includ‐ ing mESC, hESC, and hiPSC (Figure 1); examine the utilization of these models via studies of cardiac diseases; assess the current limitations and future challenges; and finally conclude with the prospective outlook and viability of the field holistically in the scope of disease modeling.

According to the American Heart Association, cardiovascular diseases (CVD) remains the leading cause of deaths in United States, accounting for 32.8% of all deaths or roughly one of every three deaths [2]. To put into perspective, that is an average of 1 death every 39 sec‐

> © 2013 Sheng and Hong; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

> © 2013 Sheng and Hong; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

man induced pluripotent stem cell (hiPSC) for modeling human diseases.

## **Chapter 20**

## **Pluripotent Stem Cells to Model Human Cardiac Diseases**

Calvin C. Sheng and Charles C. Hong

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54373

## **1. Introduction**

For past several decades, laboratory animal models have been the prevailing paradigm for studying human diseases. A classic approach is to study the impact of specific genes through the use of gain- or loss-of-function mutant animals. While the animal models have greatly contributed to our understanding of the etiology and mechanisms of disease, they often fall short of fully recapitulating human pathophysiology and translating to clinical ap‐ plications due to interspecies physiologic differences. In a review of preclinical studies of animal models published in high-impact scientific journals, approximately one-third trans‐ lated to the level of human randomized trials and only one-tenth were subsequently ap‐ proved clinically for patient use [1]. This attrition rate would have been even higher if less frequently cited animal research had been included. These unresolved issues with animal models have set the stage for the emergence of human embryonic stem cell (hESC) and hu‐ man induced pluripotent stem cell (hiPSC) for modeling human diseases.

Laid out in this chapter, we will discuss the development of various stem cell paradigms includ‐ ing mESC, hESC, and hiPSC (Figure 1); examine the utilization of these models via studies of cardiac diseases; assess the current limitations and future challenges; and finally conclude with the prospective outlook and viability of the field holistically in the scope of disease modeling.

### **2. Human cardiovascular diseases**

According to the American Heart Association, cardiovascular diseases (CVD) remains the leading cause of deaths in United States, accounting for 32.8% of all deaths or roughly one of every three deaths [2]. To put into perspective, that is an average of 1 death every 39 sec‐

onds. CVD is a generic term that encompasses conditions that affect the circulatory system, including myocardial infarction, angina pectoris, heart failure, stroke, and congenital cardio‐ vascular defects. Both genetic and environmental factors are implicated in the pathogenesis of CVDs. While some risk factors such as lifestyle habits and family history have been iden‐ tified for CVDs, much more remains to be learned about the pathophysiology, optimal man‐ agement, and proper prevention. Moreover, genetic predispositions like abnormalities in specific ion channels and sarcomere proteins pose special diagnostic and therapeutic chal‐ lenges. In fact, for most heritable forms of heart diseases, current treatment options leave much to be desired.

**Figure 1. Timeline of stem cell modeling progress.** Stem cell platforms are a new technology that was only intro‐ duced within the last two decades. The most recent breakthrough in hiPSC occurred just six years ago.

#### **3. Stem cell disease modeling**

Despite much progress in the past couple decades in the discovery of the molecular and ge‐ netic causes of many heart diseases, a detailed mechanic understanding of failing heart at the cellular level remains rudimentary. The main reason for this situation is the lack of ac‐ cess to live human tissues and unproven human cardiomyocyte cell culture models. Post‐ partum, cardiomyocytes become terminally differentiated and cease to proliferate, thus making isolation and culture of human myocardial cells extremely challenging. One surro‐ gate for human cardiomyocyte culture is the use of rat neonatal cardiomyocytes, which has been shown to yield 8.4x106 cells per heart [3]. However, with both human and rat neonatal cardiomyocytes, the inability to continuously passage cells and scarcity of resource make them unsustainable candidates for disease modeling.

**Figure 2. Overview of the stem cell disease modeling process.** The blue and yellow lightning bolts indicate the

Pluripotent Stem Cells to Model Human Cardiac Diseases

http://dx.doi.org/10.5772/54373

441

Furthermore, special considerations must be taken into account for critical differences be‐ tween animal and human cardiomyocytes, in terms of cell biological, mechanical and elec‐ trophysiological properties. The lack of appropriate human heart disease models have hindered development of rational therapies, and the prospects for new therapies to treat heart diseases remain dim despite tremendous advances in various animal models. An alter‐ native human biology based approach for heart disease modeling is to use human stem cells

addition of reprogramming and directed differentiation factors, respectively.

onds. CVD is a generic term that encompasses conditions that affect the circulatory system, including myocardial infarction, angina pectoris, heart failure, stroke, and congenital cardio‐ vascular defects. Both genetic and environmental factors are implicated in the pathogenesis of CVDs. While some risk factors such as lifestyle habits and family history have been iden‐ tified for CVDs, much more remains to be learned about the pathophysiology, optimal man‐ agement, and proper prevention. Moreover, genetic predispositions like abnormalities in specific ion channels and sarcomere proteins pose special diagnostic and therapeutic chal‐ lenges. In fact, for most heritable forms of heart diseases, current treatment options leave

**Figure 1. Timeline of stem cell modeling progress.** Stem cell platforms are a new technology that was only intro‐

Despite much progress in the past couple decades in the discovery of the molecular and ge‐ netic causes of many heart diseases, a detailed mechanic understanding of failing heart at the cellular level remains rudimentary. The main reason for this situation is the lack of ac‐ cess to live human tissues and unproven human cardiomyocyte cell culture models. Post‐ partum, cardiomyocytes become terminally differentiated and cease to proliferate, thus making isolation and culture of human myocardial cells extremely challenging. One surro‐ gate for human cardiomyocyte culture is the use of rat neonatal cardiomyocytes, which has

cardiomyocytes, the inability to continuously passage cells and scarcity of resource make

cells per heart [3]. However, with both human and rat neonatal

duced within the last two decades. The most recent breakthrough in hiPSC occurred just six years ago.

much to be desired.

440 Pluripotent Stem Cells

**3. Stem cell disease modeling**

been shown to yield 8.4x106

them unsustainable candidates for disease modeling.

**Figure 2. Overview of the stem cell disease modeling process.** The blue and yellow lightning bolts indicate the addition of reprogramming and directed differentiation factors, respectively.

Furthermore, special considerations must be taken into account for critical differences be‐ tween animal and human cardiomyocytes, in terms of cell biological, mechanical and elec‐ trophysiological properties. The lack of appropriate human heart disease models have hindered development of rational therapies, and the prospects for new therapies to treat heart diseases remain dim despite tremendous advances in various animal models. An alter‐ native human biology based approach for heart disease modeling is to use human stem cells as a renewable source of cells for cardiomyocytes. In the following section, we will discuss the various stem cell platforms (mESC, hESC, & hiPSC) for disease modeling, with specific focus on cardiovascular diseases (Figure 2).

cell lines [11]. Nonetheless, this discovery paved the way for modeling diseases directly on a

Pluripotent Stem Cells to Model Human Cardiac Diseases

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443

In a study in 2009, Lu et al. evaluated long-term safety and function of retinal pigment epitheli‐ um (RPE) as preclinical models of macular degeneration using hESCs [10]. When hESC-in‐ duced RPE were subsequently transplanted into mutant mice, they demonstrated long-term functional rescue, though progressively deteriorating function was noted due to the immuno‐ genic response elicited by the xenografts. The initial data showed promise for future elucidation of macular degenerative disease pathophysiology. However, there were important obstacles to widespread clinical translation. First, transplantation of hESC requires immunosuppression, since the cells are allogeneic. In addition, a well known risk of this technology is the formation of teratomas, tumor-like formations containing tissues belonging to all three germ layers, if some undifferentiated pluripotent cells are transplanted [12]. Finally, perhaps the biggest obstacle to a widespread acceptance of human ESC transplantation is ethical and religious, as derivation of

Given these obstacles to a widespread use of the human ESCs, a new stem cell technology, hu‐ man induced pluripotent stem cells (hiPSCs), has rapidly overtaken hESC research. Introduced in 2006 by Takahashi and Yamanaka, hiPSCs have been hailed as "the molecular equivalent of the discovery of antibiotics or vaccines in the last century [13]." The technology revolutionized the stem cell field, and for his achievement, Yamanaka received the 2012 Nobel Prize in Medi‐ cine." In a span of just six years, the field has rapidly expanded the repertoire of reprogramma‐ ble terminally differentiated tissue into hiPSC (keratinocytes [14, 15], hepatocytes [16], adiposederived stem cells [17, 18], neural stem cells [19], astrocytes [20], cord blood [21, 22], amniotic cells [23], peripheral blood [24, 25], mesenchymal stromal cells [26], oral mucosa fibroblasts [27], and T-cells [28]). Most recently, the ability to generate hiPSC from Epstein-Barr virus (EBV)-im‐ mortalized B cell lines (lymphoblastoid B-cell lines) provides the opportunity to obtain samples from disease cohort repositories such as the Coriell Institute for Medical Research or the UK Bio‐

In parallel, tremendous progress has been made towards the directing differentiation of these hiPSCs into various cell fates (neural progenitors [31], [32] motor neurons [33] [34], dopaminergic neurons [35], retinal cells [36], hepatocytes [37], blood cells [25, 38], adipo‐ cytes [39], endothelial cells [37, 38], fibroblasts [40, 41], and cardiomyocytes [42]). In theory, these patient-derived hiPSCs should be capable of differentiating into all of the >210 adult cell lineages. Nonetheless, our current growing repertoire sets the stage for studying various disease mechanisms in the laboratory, with the caveat that monogenic diseases such as long-

As alluded to above, the somatic cell reprogramming offers several distinct advantages over embryonic stem cells. In the U.S. particularly, funding may be scarce at times due to the government's political stance regarding stem cell research. Importantly, somatic cells can be obtained from individual patients, enabling the development of truly personalized diagnos‐

QT syndrome will be much easier to model than complex diseases like Parkinson's.

human ESCs typically involves the consumption of a human embryo.

human-based paradigm.

**3.3. hiPSC paradigm**

bank [29, 30].

tics and therapeutics.

#### **3.1. mESC paradigm**

In 1981, the first pluripotent mouse embryonic stem cells (mESCs) were isolated *in vitro* by culturing the inner cell mass of pre-implantation mouse blastocysts [4, 5]. These cells were capable of self-renewal and pluripotent differentiation into all three germ layers (ectoderm, mesoderm, and endoderm) [6]. The initial studies demonstrated a proof of concept, showing the feasibility of isolating pluripotent cells directly from early embryos. The unique capabili‐ ty of culturing pluripotent cells *in vitro* provided the means for genetic manipulation via se‐ lection or transformation of specific DNA fragments, and importantly to develop genetic mouse models of human disease. This platform allowed researchers to begin exploring path‐ ways in cardiac development to dissect underlying molecular and cellular mechanisms causing congenital defects and other abnormalities.

While the general use of mESCs was promising, inherent problems with using animal mod‐ els remained in the context of studying disease pathogenesis and pathophysiology. One of the crucial points of divergence is the shear difference in size and complexity between hu‐ mans and mice both macroscopically and genomically [7]. Consequently, disease suscepti‐ bility may vary drastically. For instance, a mouse heart is ten thousand times smaller but beats roughly seven times faster than that of a human. The two organisms also differ in their expression of myosin heavy chain (MHC) isoforms. βMHC is the predominant isoform in fetal mouse hearts, whereas mainly αMHC is expressed in adults; conversely, the vice versa is true for humans [8]. Furthermore, mice are resistant to the development of coronary athe‐ rosclerosis even on a high-fat, high-cholesterol diet, because they lack cholesteryl ester trans‐ fer protein (CETP), an enzyme responsible for the transfer of cholesterol from high-density lipoprotein to low-density lipoprotein [9].

#### **3.2. hESC paradigm**

Building on the initial discovery of mESC technologies, increased research focus has been directed towards developing a human-based stem cell approach in anticipation of creating a more accurate disease model. It would be another seventeen years before human embryonic stem cells (hESCs) derived from the inner cell mass of the human blastocyst (stage 4-5 days post-fertilization) were isolated by Thomson *et al.* in 1998 [10]. Many factors hindered the transition from mESC to hESC, such as the limited availability of surplus human embryos, stringent growth requirements for culturing hESC, and the shroud of ethical controversies. Generating hESCs require the destruction of the donor embryo that is considered a potential human life by some ethical and religious groups. The debate revolving around hESC has de‐ terred many researchers, mainly in the United States, from pursuit of this technology. In Au‐ gust of 2001, President Bush became the first President to provide federal funding for embryonic stem cell research, albeit limited to experimenting with only the 15 existing stem cell lines [11]. Nonetheless, this discovery paved the way for modeling diseases directly on a human-based paradigm.

In a study in 2009, Lu et al. evaluated long-term safety and function of retinal pigment epitheli‐ um (RPE) as preclinical models of macular degeneration using hESCs [10]. When hESC-in‐ duced RPE were subsequently transplanted into mutant mice, they demonstrated long-term functional rescue, though progressively deteriorating function was noted due to the immuno‐ genic response elicited by the xenografts. The initial data showed promise for future elucidation of macular degenerative disease pathophysiology. However, there were important obstacles to widespread clinical translation. First, transplantation of hESC requires immunosuppression, since the cells are allogeneic. In addition, a well known risk of this technology is the formation of teratomas, tumor-like formations containing tissues belonging to all three germ layers, if some undifferentiated pluripotent cells are transplanted [12]. Finally, perhaps the biggest obstacle to a widespread acceptance of human ESC transplantation is ethical and religious, as derivation of human ESCs typically involves the consumption of a human embryo.

#### **3.3. hiPSC paradigm**

as a renewable source of cells for cardiomyocytes. In the following section, we will discuss the various stem cell platforms (mESC, hESC, & hiPSC) for disease modeling, with specific

In 1981, the first pluripotent mouse embryonic stem cells (mESCs) were isolated *in vitro* by culturing the inner cell mass of pre-implantation mouse blastocysts [4, 5]. These cells were capable of self-renewal and pluripotent differentiation into all three germ layers (ectoderm, mesoderm, and endoderm) [6]. The initial studies demonstrated a proof of concept, showing the feasibility of isolating pluripotent cells directly from early embryos. The unique capabili‐ ty of culturing pluripotent cells *in vitro* provided the means for genetic manipulation via se‐ lection or transformation of specific DNA fragments, and importantly to develop genetic mouse models of human disease. This platform allowed researchers to begin exploring path‐ ways in cardiac development to dissect underlying molecular and cellular mechanisms

While the general use of mESCs was promising, inherent problems with using animal mod‐ els remained in the context of studying disease pathogenesis and pathophysiology. One of the crucial points of divergence is the shear difference in size and complexity between hu‐ mans and mice both macroscopically and genomically [7]. Consequently, disease suscepti‐ bility may vary drastically. For instance, a mouse heart is ten thousand times smaller but beats roughly seven times faster than that of a human. The two organisms also differ in their expression of myosin heavy chain (MHC) isoforms. βMHC is the predominant isoform in fetal mouse hearts, whereas mainly αMHC is expressed in adults; conversely, the vice versa is true for humans [8]. Furthermore, mice are resistant to the development of coronary athe‐ rosclerosis even on a high-fat, high-cholesterol diet, because they lack cholesteryl ester trans‐ fer protein (CETP), an enzyme responsible for the transfer of cholesterol from high-density

Building on the initial discovery of mESC technologies, increased research focus has been directed towards developing a human-based stem cell approach in anticipation of creating a more accurate disease model. It would be another seventeen years before human embryonic stem cells (hESCs) derived from the inner cell mass of the human blastocyst (stage 4-5 days post-fertilization) were isolated by Thomson *et al.* in 1998 [10]. Many factors hindered the transition from mESC to hESC, such as the limited availability of surplus human embryos, stringent growth requirements for culturing hESC, and the shroud of ethical controversies. Generating hESCs require the destruction of the donor embryo that is considered a potential human life by some ethical and religious groups. The debate revolving around hESC has de‐ terred many researchers, mainly in the United States, from pursuit of this technology. In Au‐ gust of 2001, President Bush became the first President to provide federal funding for embryonic stem cell research, albeit limited to experimenting with only the 15 existing stem

focus on cardiovascular diseases (Figure 2).

causing congenital defects and other abnormalities.

lipoprotein to low-density lipoprotein [9].

**3.2. hESC paradigm**

**3.1. mESC paradigm**

442 Pluripotent Stem Cells

Given these obstacles to a widespread use of the human ESCs, a new stem cell technology, hu‐ man induced pluripotent stem cells (hiPSCs), has rapidly overtaken hESC research. Introduced in 2006 by Takahashi and Yamanaka, hiPSCs have been hailed as "the molecular equivalent of the discovery of antibiotics or vaccines in the last century [13]." The technology revolutionized the stem cell field, and for his achievement, Yamanaka received the 2012 Nobel Prize in Medi‐ cine." In a span of just six years, the field has rapidly expanded the repertoire of reprogramma‐ ble terminally differentiated tissue into hiPSC (keratinocytes [14, 15], hepatocytes [16], adiposederived stem cells [17, 18], neural stem cells [19], astrocytes [20], cord blood [21, 22], amniotic cells [23], peripheral blood [24, 25], mesenchymal stromal cells [26], oral mucosa fibroblasts [27], and T-cells [28]). Most recently, the ability to generate hiPSC from Epstein-Barr virus (EBV)-im‐ mortalized B cell lines (lymphoblastoid B-cell lines) provides the opportunity to obtain samples from disease cohort repositories such as the Coriell Institute for Medical Research or the UK Bio‐ bank [29, 30].

In parallel, tremendous progress has been made towards the directing differentiation of these hiPSCs into various cell fates (neural progenitors [31], [32] motor neurons [33] [34], dopaminergic neurons [35], retinal cells [36], hepatocytes [37], blood cells [25, 38], adipo‐ cytes [39], endothelial cells [37, 38], fibroblasts [40, 41], and cardiomyocytes [42]). In theory, these patient-derived hiPSCs should be capable of differentiating into all of the >210 adult cell lineages. Nonetheless, our current growing repertoire sets the stage for studying various disease mechanisms in the laboratory, with the caveat that monogenic diseases such as long-QT syndrome will be much easier to model than complex diseases like Parkinson's.

As alluded to above, the somatic cell reprogramming offers several distinct advantages over embryonic stem cells. In the U.S. particularly, funding may be scarce at times due to the government's political stance regarding stem cell research. Importantly, somatic cells can be obtained from individual patients, enabling the development of truly personalized diagnos‐ tics and therapeutics.

## **4. Modeling cardiovascular diseases**

While there is a wide array of cardiovascular diseases, we chose to focus on several with welldefined clinical presentation, strong genetic component, and significant research progress (Long QT syndrome types 1 and 2, Timothy syndrome, LEOPARD syndrome, & dilated cardio‐ myopathy; see Table 1). As discussed below, the paradigm of using stem cells to model inherit‐ ed cardiovascular diseases is rapidly being established and validated. Moreover, these advances with the rare inherited conditions may lead to new paradigms to study the much more prevalent acquired heart and vascular diseases at the cellular and molecular levels.

**4.1. Long QT syndrome**

b) Surface EKG

a) Cardiac Action Potential

indication is the prolongation of that interval on an EKG.

Long-QT syndrome (LQTS) is a rare congenital channelopathy disease that is characterized by an abnormally prolonged ventricular repolarization phase, inherited primarily in an au‐ tosomal dominant manner but sometimes autosomal recessively. It was first described in 1957 in a family with normal parents and two healthy children but also in which three chil‐ dren experienced recurrent syncope and sudden death [43]. Electrocardiography (EKG) studies showed prolonged QT interval due to increased ventricular action potential, hence the name of the disease (Figure 3). The prevalence of LQTS in the U.S. is approximately 1 in 7,000 individuals, causing 2,000 to 3,000 sudden deaths annually in children or adolescents [44]. This abnormality can lead to an increased risk of such reported incidence of sudden death, usually triggered by the resulting ventricular fibrillation or torsade de pointes (poly‐ morphic ventricular tachycardia). Depending on the specific gene mutation, long-QT syn‐ drome can be classified into 12 genetic subtypes [45]. Together, LQT1, LQT2, and LQT3

**Long QT Syndrome**

**0**

I Na

P

**QT<\$%&?>interval**

Q

**Figure 3. Long QT Syndrome. a)** a visual representation of the cardiac action potential during depolarization and repolarization of the cell. There are 4 phases of the cycle in which various ion channels open and close, causing the flux of charged ions (red: into the cell & blue: out of the cell) and reflecting the change in overall action potential. **b)** an illustration of a normal surface EKG plot, highlighting the QT interval in particular. In long QT syndrome, a clear

S

R

**<sup>1</sup> <sup>2</sup>**

I to1

I Kr

QT Interval

I ca (L)

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**3**

I Ks

> I K1

> > T

**4**

genotypes account for 97% of the mutations identified to date [46].


**Table 1.** hiPSC studies modeling cardiovascular diseases.

#### **4.1. Long QT syndrome**

**4. Modeling cardiovascular diseases**

*KCNQ1* R190Q

*KCNH2* R176W

*KCNH2* A614V

*KCNH2* G1681A

*CACNA1C* G1216A

> *PTPN11* T468M

*TNNT2* R173W

**Table 1.** hiPSC studies modeling cardiovascular diseases.

Long QT syndrome Type 1

444 Pluripotent Stem Cells

Moretti et al. (2010) [48]

Long QT syndrome Type 2

Lahti et al. (2012) [83]

Itzhaki et al. (2011) [50]

Matsa et al. (2011) [84]

Timothy syndrome

Yazawa et al. (2011) [52]

LEOPARD syndrome Carvajal-Vergara et al. (2010) [57]

> Dilated cardiomyopathy

Sun et al. (2012) [67]

While there is a wide array of cardiovascular diseases, we chose to focus on several with welldefined clinical presentation, strong genetic component, and significant research progress (Long QT syndrome types 1 and 2, Timothy syndrome, LEOPARD syndrome, & dilated cardio‐ myopathy; see Table 1). As discussed below, the paradigm of using stem cells to model inherit‐ ed cardiovascular diseases is rapidly being established and validated. Moreover, these advances with the rare inherited conditions may lead to new paradigms to study the much more

> marked prolonged action potentials; dominant negative trafficking defect associated with a 70 to 80% reduction in Iks current; altered channel activation and deactivation properties; increased susceptibility to catecholamine-induced tachyarrhythmia attenuated by β-blockage

prolonged action potential; reduced Ikr density; more sensitive to potentially arrhythmogenic drugs; more pronounced inverse correlation between the beating rate and repolarization time

significant reduction of potassium current IKr; marked arrhythmogenecity; evaluated potency of existing & novel pharmacological agents

prolonged field/action potential duration; Ikr blocker & isoprenaline induced arrhythmias presenting as early after depolarizations; attenuated by β-blockers propranolol & nadolol

irregular contraction; excess Ca2+ influx; prolonged action potentials; irregular electrical activity; abnormal calcium transients in ventricular-like cells; roscovitine restored electrical and Ca2+ signaling properties

hypertrophic cardiomyopathy; higher degree of sarcomeric organization; preferential localization of NFATC4 in the nucleus

altered regulation of Ca2+; decreased contractility; abnormal distribution of sarcomeric α-actinin; β-drenergic agonist induced cellular stress; β-adrenergic blockers or overexpression of Serca2a improved function

prevalent acquired heart and vascular diseases at the cellular and molecular levels.

**Genetic Disorder Mutation Main findings**

Long-QT syndrome (LQTS) is a rare congenital channelopathy disease that is characterized by an abnormally prolonged ventricular repolarization phase, inherited primarily in an au‐ tosomal dominant manner but sometimes autosomal recessively. It was first described in 1957 in a family with normal parents and two healthy children but also in which three chil‐ dren experienced recurrent syncope and sudden death [43]. Electrocardiography (EKG) studies showed prolonged QT interval due to increased ventricular action potential, hence the name of the disease (Figure 3). The prevalence of LQTS in the U.S. is approximately 1 in 7,000 individuals, causing 2,000 to 3,000 sudden deaths annually in children or adolescents [44]. This abnormality can lead to an increased risk of such reported incidence of sudden death, usually triggered by the resulting ventricular fibrillation or torsade de pointes (poly‐ morphic ventricular tachycardia). Depending on the specific gene mutation, long-QT syn‐ drome can be classified into 12 genetic subtypes [45]. Together, LQT1, LQT2, and LQT3 genotypes account for 97% of the mutations identified to date [46].

## **Long QT Syndrome**

**Figure 3. Long QT Syndrome. a)** a visual representation of the cardiac action potential during depolarization and repolarization of the cell. There are 4 phases of the cycle in which various ion channels open and close, causing the flux of charged ions (red: into the cell & blue: out of the cell) and reflecting the change in overall action potential. **b)** an illustration of a normal surface EKG plot, highlighting the QT interval in particular. In long QT syndrome, a clear indication is the prolongation of that interval on an EKG.

Our current understanding of how mutations in ion channels cause disease can only be extrapolated from, at best, mammalian cell lines such as immortalized human em‐ bryonic kidney 293 cells or *Xenopus* oocytes using heterologous expression systems de‐ signed with the mutant channel of interest [47]. Commonly used mouse models are not apt for studying LQTS because the IKr current is essentially absent in the mouse heart. With the advent of patient-derived iPSC technology, cardiac induction of these cell lines may recapitulate their respective disease pathophysiology *in vitro*, providing a unique platform for studying cellular and molecular mechanisms and assessing the efficacy of various therapies.

tudes of IKr activation and tail currents in the cells derived from the LQTS patient compared to those generated from a healthy individual, both hallmark signatures of LQTS. They also reported observing early-after depolarizations in 66% of the iPSC-CMs on both cellular and multicellular levels, a key finding suggestive of arrhythmogenicity that explains sudden death in LQTS patients clinically. With the amount of clinical evidence extracted from these patient-derived cardiomocytes, this novel technology can serve as an excellent *in vitro* dis‐ ease model for understanding cellular & molecular pathogenesis and becomes a very viable

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In contrast to the previously detailed potassium channel defects that lead to LQTS, Timothy syndrome is a form of LQTS caused by a missense mutation in the L-type calcium channel Cav1.2, encoded by the CACNA1C gene. This is the predominant L-type channel in the mammalian heart, which is essential for normal heart development and excitation-contrac‐ tion coupling [51]. Mutations in this Ca2+ channel cause delayed channel closing and conse‐

Concurrent with Itzhaki et al.'s publication LQTS type 2, Yazawa et al. reported their findings on Timothy syndrome using a patient-derived iPSC-CM disease model [52]. To summarize, using a similar cardiac induction protocol, they successfully reproduced *in vitro* cardiomyocytes exhibiting clinical Timothy syndrome phenotypes. Electrophysiology and calcium imaging studies showed irregular contraction, excess Ca2+ influx, prolonged action potentials, irregular electrical activity, and abnormal calcium transients in ventric‐

One of the key findings in their study was the functional difference between Timothy syn‐ drome and LQTS type 1 cardiomyocytes. Unlike the latter where both ventricular- and at‐ rial-like cells had prolonged action potentials, only ventricular Timothy syndrome cardiomyocytes exhibited this phenotype. Additionally, drug-induced triggering of arrhyth‐ mias and delayed depolarizations in LQTS type 1 cells were not necessary, because they were observed spontaneously in Timothy syndrome cells. While a direct correlation has yet to be established to the clinical outcomes (i.e. torsades de points and ventricular fibrillation), this study is another proof-of-concept that iPSC-CMs are invaluable for examining detailed

LEOPARD syndrome is an autosomal-dominant developmental disorder with clinical mani‐ festations described by its acronym: lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitalia, retardation of growth, and deafness [53]. It is caused by a mutation in the PTPN11 gene, which impairs the catalytic re‐ gion of the encoded SHP2 phosphatase [54]. Currently, drosophila [55] and zebrafish [56] models of LEOPARD syndrome have been described in literature, but the molecular basis of

option for personalized medicine in the future.

quently, increased cellular excitability.

pathogenesis of human diseases.

pathogenesis remains to be addressed.

**4.3. LEOPARD syndrome**

**4.2. Timothy syndrome**

ular-like cells.

#### *4.1.1. Long QT syndrome type 1*

The most common type LQT1, accounting for roughly 45% of genotyped patients, results from mutations of the alpha subunit of the slow delayed rectifier potassium channel KvLQT1, encoded by gene KCNQ1 on chromosome 11 [48]. In a recent study aimed at reca‐ pitulating disease phenotype using patient-derived iPSCs, Moretti et al. initially screened a family affected by LQTS type 1 through genotyping and electrophysiology studies, identify‐ ing an autosomal dominant missense mutation R190Q of KCNQ1 [48]. Then, they reprog‐ rammed skin fibroblast from two affected family members into iPSCs and directed cardiac induction to yield spontaneously beating cardiomyocytes. Finally, they characterized these heart cells through whole-cell patch clamp, observing reduced Iks, a slow delayed rectifier potassium current, by 70-80%, altered Iks activation and deactivation properties, and an ab‐ normal response to catecholamine stimulation.

Not only were Moretti et al. able to capture characteristics of LQTS type 1 *in vitro*, they were also able to demonstrate physiologically how beta-blockers, clinically administered as a pro‐ phylactic therapy for asymptomatic LQTS type 1 patients, had protective effects against cat‐ echolamine-induced tachyarrhythmia by reducing early afterdepolarizations [49]. This ability to mimic LQTS type 1 in an *in vitro* human model paved way for similar studies in‐ volving other genetic diseases.

#### *4.1.2. Long QT syndrome type 2*

Similar to LQTS type 1, LQTS type 2 is another mutation arising from the alpha subunit of a potassium channel, but one with different properties: a KCNH2 (also known as hERG)-en‐ coded rapid delayed rectifier potassium channel [50]. A diagnostic finding in patients is the onset of clinical symptoms such as syncope triggered by sudden loud noises [45].

In a study by Itzhaki et al., A614V missense mutation was identified in the KCNH2 gene in a 28 year old patient with clinically diagnosed type 2 LQTS [50]. Dermal fibroblast samples were obtained, reprogrammed to generate patient-specific human iPSCs, and through retro‐ viral transduction, differentiated into embryoid bodies of spontaneously beating cardiomyo‐ cytes. Through the use of these iPSC-generated heart cells, they were able to conduct electrophysiology studies and test the effects of pharmacological intervention. Itzhaki et al. found marked prolonged action potential duration and significantly reduced peak ampli‐ tudes of IKr activation and tail currents in the cells derived from the LQTS patient compared to those generated from a healthy individual, both hallmark signatures of LQTS. They also reported observing early-after depolarizations in 66% of the iPSC-CMs on both cellular and multicellular levels, a key finding suggestive of arrhythmogenicity that explains sudden death in LQTS patients clinically. With the amount of clinical evidence extracted from these patient-derived cardiomocytes, this novel technology can serve as an excellent *in vitro* dis‐ ease model for understanding cellular & molecular pathogenesis and becomes a very viable option for personalized medicine in the future.

#### **4.2. Timothy syndrome**

Our current understanding of how mutations in ion channels cause disease can only be extrapolated from, at best, mammalian cell lines such as immortalized human em‐ bryonic kidney 293 cells or *Xenopus* oocytes using heterologous expression systems de‐ signed with the mutant channel of interest [47]. Commonly used mouse models are not apt for studying LQTS because the IKr current is essentially absent in the mouse heart. With the advent of patient-derived iPSC technology, cardiac induction of these cell lines may recapitulate their respective disease pathophysiology *in vitro*, providing a unique platform for studying cellular and molecular mechanisms and assessing the

The most common type LQT1, accounting for roughly 45% of genotyped patients, results from mutations of the alpha subunit of the slow delayed rectifier potassium channel KvLQT1, encoded by gene KCNQ1 on chromosome 11 [48]. In a recent study aimed at reca‐ pitulating disease phenotype using patient-derived iPSCs, Moretti et al. initially screened a family affected by LQTS type 1 through genotyping and electrophysiology studies, identify‐ ing an autosomal dominant missense mutation R190Q of KCNQ1 [48]. Then, they reprog‐ rammed skin fibroblast from two affected family members into iPSCs and directed cardiac induction to yield spontaneously beating cardiomyocytes. Finally, they characterized these heart cells through whole-cell patch clamp, observing reduced Iks, a slow delayed rectifier potassium current, by 70-80%, altered Iks activation and deactivation properties, and an ab‐

Not only were Moretti et al. able to capture characteristics of LQTS type 1 *in vitro*, they were also able to demonstrate physiologically how beta-blockers, clinically administered as a pro‐ phylactic therapy for asymptomatic LQTS type 1 patients, had protective effects against cat‐ echolamine-induced tachyarrhythmia by reducing early afterdepolarizations [49]. This ability to mimic LQTS type 1 in an *in vitro* human model paved way for similar studies in‐

Similar to LQTS type 1, LQTS type 2 is another mutation arising from the alpha subunit of a potassium channel, but one with different properties: a KCNH2 (also known as hERG)-en‐ coded rapid delayed rectifier potassium channel [50]. A diagnostic finding in patients is the

In a study by Itzhaki et al., A614V missense mutation was identified in the KCNH2 gene in a 28 year old patient with clinically diagnosed type 2 LQTS [50]. Dermal fibroblast samples were obtained, reprogrammed to generate patient-specific human iPSCs, and through retro‐ viral transduction, differentiated into embryoid bodies of spontaneously beating cardiomyo‐ cytes. Through the use of these iPSC-generated heart cells, they were able to conduct electrophysiology studies and test the effects of pharmacological intervention. Itzhaki et al. found marked prolonged action potential duration and significantly reduced peak ampli‐

onset of clinical symptoms such as syncope triggered by sudden loud noises [45].

efficacy of various therapies.

446 Pluripotent Stem Cells

*4.1.1. Long QT syndrome type 1*

volving other genetic diseases.

*4.1.2. Long QT syndrome type 2*

normal response to catecholamine stimulation.

In contrast to the previously detailed potassium channel defects that lead to LQTS, Timothy syndrome is a form of LQTS caused by a missense mutation in the L-type calcium channel Cav1.2, encoded by the CACNA1C gene. This is the predominant L-type channel in the mammalian heart, which is essential for normal heart development and excitation-contrac‐ tion coupling [51]. Mutations in this Ca2+ channel cause delayed channel closing and conse‐ quently, increased cellular excitability.

Concurrent with Itzhaki et al.'s publication LQTS type 2, Yazawa et al. reported their findings on Timothy syndrome using a patient-derived iPSC-CM disease model [52]. To summarize, using a similar cardiac induction protocol, they successfully reproduced *in vitro* cardiomyocytes exhibiting clinical Timothy syndrome phenotypes. Electrophysiology and calcium imaging studies showed irregular contraction, excess Ca2+ influx, prolonged action potentials, irregular electrical activity, and abnormal calcium transients in ventric‐ ular-like cells.

One of the key findings in their study was the functional difference between Timothy syn‐ drome and LQTS type 1 cardiomyocytes. Unlike the latter where both ventricular- and at‐ rial-like cells had prolonged action potentials, only ventricular Timothy syndrome cardiomyocytes exhibited this phenotype. Additionally, drug-induced triggering of arrhyth‐ mias and delayed depolarizations in LQTS type 1 cells were not necessary, because they were observed spontaneously in Timothy syndrome cells. While a direct correlation has yet to be established to the clinical outcomes (i.e. torsades de points and ventricular fibrillation), this study is another proof-of-concept that iPSC-CMs are invaluable for examining detailed pathogenesis of human diseases.

#### **4.3. LEOPARD syndrome**

LEOPARD syndrome is an autosomal-dominant developmental disorder with clinical mani‐ festations described by its acronym: lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitalia, retardation of growth, and deafness [53]. It is caused by a mutation in the PTPN11 gene, which impairs the catalytic re‐ gion of the encoded SHP2 phosphatase [54]. Currently, drosophila [55] and zebrafish [56] models of LEOPARD syndrome have been described in literature, but the molecular basis of pathogenesis remains to be addressed.

In 2010, Carvajal-Vergara et al. successfully demonstrated the use of iPSC technology to characterize LEOPARD syndrome *in vitro* [57]. One of the clinical hallmarks of the dis‐ ease is hypertrophic cardiomyopathy. In this study, iPSC-CMs derived from a 25-year old female patient with the condition were compared to those differentiated a healthy brother as a control. Carvajal-Vergara et al. showed, by comparison to the wild-type, larger patient-derived iPSC-CMs with a higher degree of sarcomeric organization and preferential localization of NFATC4 (calcineurin-NFAT pathway is an important regula‐ tor of cardiac hypertrophy [58]) in the nucleus [57]. Using antibody microarrays on pa‐ tient-specific iPSCs, they also noted increased phosphorylation of certain proteins such as MEK1 leading to perturbations in the RAS-MAPK signaling cascade, which can begin to provide some preliminary understanding and elucidation of LEOPARD syndrome's pathogenesis on a molecular level [57].

**5. Stem cell disease modeling challenges**

nant approach for disease modeling over hESCs.

ting to the disease such as oxidative stress [73].

ery and development.

In the framework of disease modeling, both hESC and hiPSC technologies still have unre‐ solved issues to address. For instance, hESCs display chromosomal instability with longterm *in vitro* culture [69], and iPSCs undergo dynamic changes in copy number variations during reprogramming, especially in the early passages [70]. In the U.S., research funding for hESC often fluctuates, subjecting to restrictions imposed by Congress and its current stance on the destruction of fertilized human embryos. The advantage of hiPSC over hESC is that it bypasses this controversy and generates autologous cells while maintaining key char‐ acteristics: morphology, feeder dependency, surface markers, gene expression, promoter methylation status, telomerase activities, *in vitro* differentiation potential, and *in vivo* terato‐ ma forming capacity [71]. These features heavily favor hiPSC technology as the predomi‐

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In the near future, the hiPSC model faces several main challenges. One of the concerns is developing a robust and efficient methodology for yielding large quantities of differenti‐ ated and functional cells of a designated lineage. Depending on the protocol and cell lines used, efficiencies can range anywhere from <0.0001% to >50%. Specifically in the case of cardiac induction, the hiPSC-induced cardiomyocytes resemble immature fetal cardiomyocytes in their gene expression profile (key marker is β-tubulin) as well as elec‐ trophysiologic and structural properties [72]. Resolving this hindrance will also have great impact on facilitating *in vivo* studies and widespread applications in drug discov‐

The practicality of studying disease pathogenesis *in vitro*, especially those with systemic involvement, raises another question. This intrinsic lack of an *in vivo* environment pre‐ vents a global understanding of how a disease may impact the body and simplifies inter‐ actions of basic signaling pathways. For more complex diseases, it may also be difficult to replicate conditions in a petri dish with a single lineage cell type, even if done via cocultures. Furthermore, the current designation of a control line is arbitrary since it is mainly a criterion of exclusions. In diseases such as Alzheimer's or Parkinson's, there is a long latency period, which would be hard to mimic *in vitro* due to the dynamics of re‐ al-time disease progression. Studies are currently underway to assess the possibility of accelerating disease progression *in vitro* via exposure to environmental factors contribu‐

Finally, not all diseases can be readily modeled using hiPSCs. For example, patients with Fanconi anemia have a defective DNA repair mechanism, and therefore cannot be reprog‐ rammed without antecedent gene correction [74]. For other conditions, some may exhibit low penetrance or do not follow a simple Mendelian form of inheritance and are affected by a multitude of factors ranging from the environment to epigenetics. The latter in diseased state may become an inevitable confounding factor working with iPSCs, because of its con‐ tribution to the low efficiency of reprogramming and its stochastic nature. In a study by Meissner et al., sub-clone lineages transfected with an Oct4-GFP reporter were obtained from early appearing iPSC colonies and displayed temporally different expression patterns

#### **4.4. Dilated cardiomyopathy**

As previously mentioned, cardiovascular disease is the leading cause of morbidity and mor‐ tality worldwide, projected to represent 30% of all deaths in 2015 [59]. In the United States alone, heart disease accounts for roughly one-third of all deaths [60]. Of those, dilated cardi‐ omyopathy (DCM) is one of the leading causes of heart failure and is associated with sub‐ stantial mortality [61]. It leads to progressive cardiac remodeling, characterized by ventricular dilatation, hypertrophy, and systolic dysfunction [62]. In an estimated 20% to 48% of cases depending on the study, DCM is identified as a familial disorder with strong heritability [63]. Mutations in over 30 genes have been shown to be disease causing or dis‐ ease associated [64].

One of the more common genetic defects causing DCM is a mutation in the cardiac troponin T gene (TNNT2) [65]. Mouse models have already provided invaluable insight to the disease mechanism. For instance, mice still displayed normal phenotype after knockout of one TNNT2 allele, because they only lead to a mild deficit in transcript but not protein [66]. Fur‐ thermore, the severity of DCM depends on the ratio of mutant to wild-type TNNT2 tran‐ script, since mutant protein is associated with cardiomyocyte Ca2+ desensitization [66]. However, given the differences in electrophysiological and developmental properties, *in vi‐ tro* human models of DCM would conceivably provide a more precise platform for under‐ standing molecular basis of pathogenesis.

In Sun et al.'s study published in 2012, they characterized iPSC-CMs from a family carry‐ ing a point mutation (R173W) in the TNNT2 gene by comparing to healthy individuals in the same cohort [67]. These patient-specific cardiomyocytes from diseased individuals exhibited dysregulated calcium handling, decreased contractility, and abnormal heteroge‐ nous distribution of sarcomeric α-actinin. The overexpression of Serca2a, a gene therapy treatment for heart failure currently in clinical trials [68], significantly improved the con‐ tractility force generated by iPSC-CMs derived from DCM patients [67]. Much like the use of hiPSC technology for other cardiovascular diseases discussed previously, it ap‐ pears to be a robust system for describing pathogenesis of disease that has yielded pre‐ liminary positive results.

## **5. Stem cell disease modeling challenges**

In 2010, Carvajal-Vergara et al. successfully demonstrated the use of iPSC technology to characterize LEOPARD syndrome *in vitro* [57]. One of the clinical hallmarks of the dis‐ ease is hypertrophic cardiomyopathy. In this study, iPSC-CMs derived from a 25-year old female patient with the condition were compared to those differentiated a healthy brother as a control. Carvajal-Vergara et al. showed, by comparison to the wild-type, larger patient-derived iPSC-CMs with a higher degree of sarcomeric organization and preferential localization of NFATC4 (calcineurin-NFAT pathway is an important regula‐ tor of cardiac hypertrophy [58]) in the nucleus [57]. Using antibody microarrays on pa‐ tient-specific iPSCs, they also noted increased phosphorylation of certain proteins such as MEK1 leading to perturbations in the RAS-MAPK signaling cascade, which can begin to provide some preliminary understanding and elucidation of LEOPARD syndrome's

As previously mentioned, cardiovascular disease is the leading cause of morbidity and mor‐ tality worldwide, projected to represent 30% of all deaths in 2015 [59]. In the United States alone, heart disease accounts for roughly one-third of all deaths [60]. Of those, dilated cardi‐ omyopathy (DCM) is one of the leading causes of heart failure and is associated with sub‐ stantial mortality [61]. It leads to progressive cardiac remodeling, characterized by ventricular dilatation, hypertrophy, and systolic dysfunction [62]. In an estimated 20% to 48% of cases depending on the study, DCM is identified as a familial disorder with strong heritability [63]. Mutations in over 30 genes have been shown to be disease causing or dis‐

One of the more common genetic defects causing DCM is a mutation in the cardiac troponin T gene (TNNT2) [65]. Mouse models have already provided invaluable insight to the disease mechanism. For instance, mice still displayed normal phenotype after knockout of one TNNT2 allele, because they only lead to a mild deficit in transcript but not protein [66]. Fur‐ thermore, the severity of DCM depends on the ratio of mutant to wild-type TNNT2 tran‐ script, since mutant protein is associated with cardiomyocyte Ca2+ desensitization [66]. However, given the differences in electrophysiological and developmental properties, *in vi‐ tro* human models of DCM would conceivably provide a more precise platform for under‐

In Sun et al.'s study published in 2012, they characterized iPSC-CMs from a family carry‐ ing a point mutation (R173W) in the TNNT2 gene by comparing to healthy individuals in the same cohort [67]. These patient-specific cardiomyocytes from diseased individuals exhibited dysregulated calcium handling, decreased contractility, and abnormal heteroge‐ nous distribution of sarcomeric α-actinin. The overexpression of Serca2a, a gene therapy treatment for heart failure currently in clinical trials [68], significantly improved the con‐ tractility force generated by iPSC-CMs derived from DCM patients [67]. Much like the use of hiPSC technology for other cardiovascular diseases discussed previously, it ap‐ pears to be a robust system for describing pathogenesis of disease that has yielded pre‐

pathogenesis on a molecular level [57].

standing molecular basis of pathogenesis.

liminary positive results.

**4.4. Dilated cardiomyopathy**

448 Pluripotent Stem Cells

ease associated [64].

In the framework of disease modeling, both hESC and hiPSC technologies still have unre‐ solved issues to address. For instance, hESCs display chromosomal instability with longterm *in vitro* culture [69], and iPSCs undergo dynamic changes in copy number variations during reprogramming, especially in the early passages [70]. In the U.S., research funding for hESC often fluctuates, subjecting to restrictions imposed by Congress and its current stance on the destruction of fertilized human embryos. The advantage of hiPSC over hESC is that it bypasses this controversy and generates autologous cells while maintaining key char‐ acteristics: morphology, feeder dependency, surface markers, gene expression, promoter methylation status, telomerase activities, *in vitro* differentiation potential, and *in vivo* terato‐ ma forming capacity [71]. These features heavily favor hiPSC technology as the predomi‐ nant approach for disease modeling over hESCs.

In the near future, the hiPSC model faces several main challenges. One of the concerns is developing a robust and efficient methodology for yielding large quantities of differenti‐ ated and functional cells of a designated lineage. Depending on the protocol and cell lines used, efficiencies can range anywhere from <0.0001% to >50%. Specifically in the case of cardiac induction, the hiPSC-induced cardiomyocytes resemble immature fetal cardiomyocytes in their gene expression profile (key marker is β-tubulin) as well as elec‐ trophysiologic and structural properties [72]. Resolving this hindrance will also have great impact on facilitating *in vivo* studies and widespread applications in drug discov‐ ery and development.

The practicality of studying disease pathogenesis *in vitro*, especially those with systemic involvement, raises another question. This intrinsic lack of an *in vivo* environment pre‐ vents a global understanding of how a disease may impact the body and simplifies inter‐ actions of basic signaling pathways. For more complex diseases, it may also be difficult to replicate conditions in a petri dish with a single lineage cell type, even if done via cocultures. Furthermore, the current designation of a control line is arbitrary since it is mainly a criterion of exclusions. In diseases such as Alzheimer's or Parkinson's, there is a long latency period, which would be hard to mimic *in vitro* due to the dynamics of re‐ al-time disease progression. Studies are currently underway to assess the possibility of accelerating disease progression *in vitro* via exposure to environmental factors contribu‐ ting to the disease such as oxidative stress [73].

Finally, not all diseases can be readily modeled using hiPSCs. For example, patients with Fanconi anemia have a defective DNA repair mechanism, and therefore cannot be reprog‐ rammed without antecedent gene correction [74]. For other conditions, some may exhibit low penetrance or do not follow a simple Mendelian form of inheritance and are affected by a multitude of factors ranging from the environment to epigenetics. The latter in diseased state may become an inevitable confounding factor working with iPSCs, because of its con‐ tribution to the low efficiency of reprogramming and its stochastic nature. In a study by Meissner et al., sub-clone lineages transfected with an Oct4-GFP reporter were obtained from early appearing iPSC colonies and displayed temporally different expression patterns of GFP, some never expressing it at all [75]. Because of the sensitivity to epigenetic events, the use of histone deacetylase (HDAC) inhibitors may help promote self-renewal and/or di‐ rected differentiation of stem cells [76].

Further studies are needed to evaluate the optimal cardiac induction protocol. Once a ro‐ bust, universal, and scalable system for directed differentiation of iPSCs into cardiomyo‐ cytes is established, we can provide an inexhaustible supply of patient-derived cells for

Pluripotent Stem Cells to Model Human Cardiac Diseases

http://dx.doi.org/10.5772/54373

451

With some host-specific modifications, currently available technologies such as zinc fin‐ ger nucleases can be applied as the next step in disease modeling after understanding the pathogenesis, developing a cure. Zinc finger nucleases are enzymes that manipulate specific sites of the host genome, generating transgenic lines via knocking-in and knock‐ ing-out of genes. The homologous recombination pathway, naturally occurring at DNA replication forks and repairing double stranded breaks, can be exploited to selectively target a locus for modification while leaving the rest of the genome in tact [82]. Through this method, we have been able to identify new gene function in mouse and other ho‐ mologous mammalian models. The same concept can be applied to gene therapy for hu‐ mans. For example, with patient-specific cardiomyocytes, constructs can be created and

High-throughput screening is another means of advancing disease therapy, but it hinges on its scalability; in other words, whether or not cells of the disease model can be massproduced. With current protocols for directed cardiac differentiation, every round of ex‐ periments would take at least 2 weeks [81]. If hiPSC-derived cardiomyocytes could be consistently generated in 96-well plates, then these high-throughput screenings that could propel translational research from a cellular and molecular level of disease directly

In the new era of personalized medicine, the stem cell platform for disease modeling ap‐ pears very viable, especially given the rapid advancements in the field over the past several decades. We have thoroughly discussed the advantages and disadvantages of using mESC, hESC, and hiPSC, all of which have the common end goal of best recapitulating disease phe‐ notype *in vitro*. Of those, we strongly believe that hiPSC-derived cells can eventually be the gold standard for personalized medicine. Using a heritable cardiovascular class of diseases as an example, we endeavored to convey the potential benefits of harnessing iPSC technolo‐ gy to study the pathogenesis of various disorders. One of the most difficult challenges cur‐ rently is establishing a robust, universal, and scalable cardiac induction protocol. Combined with the genetic tools available, we will be able to break the barriers to disease modeling

research and therapeutic purposes.

tested *in vitro* to restore wild-type function.

with the limitless supply of human cells *in vitro*.

**6.2. High-throughput screening**

to therapeutic applications.

**7. Conclusion**

**6.1. Zinc finger nucleases**

## **6. Future outlook & research direction**

The intent of stem cell technology was to recapitulate, as closely as possible, disease pheno‐ type in the human body for three primary outcomes: disease modeling, drug discovery & development, and regenerative medicine. The first of which will provide the initial platform from which drugs and therapeutic applications can be derived. In some cases, a treatment could be discovered before the underlying disease mechanism is understood, because pa‐ tient-derived hiPSCs can be differentiated without any genetic modifications *in vitro* into the desired cell type and characterized in drug screenings.

In the context of patient-derived cardiomyocytes, while not a perfect *in vivo* surrogate, they will still be one of the better models currently available due to their identical genomes and phenotype. The complex interactions of normal human physiology is incredibly difficult to mimic outside the host, let alone recapitulating a diseased phenotype. The mouse model is currently the most common mammalian system used to study human physiology for several reasons: 90% genetic homology with comparable genomic sizes, relatively easy mainte‐ nance, rapid cost-effective breeding under laboratory settings, and capability for genetic ma‐ nipulation. It is great for initial studies and insight into basic understanding and elucidation of the mechanisms underlying the disease.

Building on the gradual advancement from mESC to hESC to the current hiPSC technology, one of the technical goals remains to be removing all extrinsic factors with the goal of mim‐ icking *in vivo* conditions. Most established mESC and hESC protocols relied on a fibroblast feeder-cell layer for culture and proliferation, which secrete undefined substrates into the medium and cause batch-to-batch variation [77]. Similarly, initial hiPSC protocols used a mouse embryonic fibroblast (MEF) feeder-cell layer that had similar problems [78]. In 2011, Yu et al. developed a feeder-free system with chemically defined medium and also replaced conventional transfection of somatic cells with footprint-less episomal reprogramming using small molecules to generate hiPSCs [79].

Furthermore, mESC and hESC-directed differentiation formed embryoid bodies (EBs), which are spheroids with an inner layer of ectoderm and a single outer layer of endoderm. These EBs differentiate to derivatives of all 3 primary germ layers, leaving a very low yield of spontaneously contracting cardiomyocytes. While this was sufficient for initial studies, larger quantities of pure cardiomyocytes are necessary to establish a scalable system for dis‐ ease modeling and drug development. In 2007, Laflamme et al. reported the use of a mono‐ layer cardiac induction system based on activin A and BMP4 with a 30-fold higher yield of pure cardiomyocytes than through the formation of EBs [80]. Most recently, Lian et al. of the Wisconsin stem cell group identified temporal modulation of canonical Wnt signaling as a key step for robust cardiomyocyte differentiation reporting efficient yields of up to 98% [81]. Further studies are needed to evaluate the optimal cardiac induction protocol. Once a ro‐ bust, universal, and scalable system for directed differentiation of iPSCs into cardiomyo‐ cytes is established, we can provide an inexhaustible supply of patient-derived cells for research and therapeutic purposes.

#### **6.1. Zinc finger nucleases**

of GFP, some never expressing it at all [75]. Because of the sensitivity to epigenetic events, the use of histone deacetylase (HDAC) inhibitors may help promote self-renewal and/or di‐

The intent of stem cell technology was to recapitulate, as closely as possible, disease pheno‐ type in the human body for three primary outcomes: disease modeling, drug discovery & development, and regenerative medicine. The first of which will provide the initial platform from which drugs and therapeutic applications can be derived. In some cases, a treatment could be discovered before the underlying disease mechanism is understood, because pa‐ tient-derived hiPSCs can be differentiated without any genetic modifications *in vitro* into the

In the context of patient-derived cardiomyocytes, while not a perfect *in vivo* surrogate, they will still be one of the better models currently available due to their identical genomes and phenotype. The complex interactions of normal human physiology is incredibly difficult to mimic outside the host, let alone recapitulating a diseased phenotype. The mouse model is currently the most common mammalian system used to study human physiology for several reasons: 90% genetic homology with comparable genomic sizes, relatively easy mainte‐ nance, rapid cost-effective breeding under laboratory settings, and capability for genetic ma‐ nipulation. It is great for initial studies and insight into basic understanding and elucidation

Building on the gradual advancement from mESC to hESC to the current hiPSC technology, one of the technical goals remains to be removing all extrinsic factors with the goal of mim‐ icking *in vivo* conditions. Most established mESC and hESC protocols relied on a fibroblast feeder-cell layer for culture and proliferation, which secrete undefined substrates into the medium and cause batch-to-batch variation [77]. Similarly, initial hiPSC protocols used a mouse embryonic fibroblast (MEF) feeder-cell layer that had similar problems [78]. In 2011, Yu et al. developed a feeder-free system with chemically defined medium and also replaced conventional transfection of somatic cells with footprint-less episomal reprogramming using

Furthermore, mESC and hESC-directed differentiation formed embryoid bodies (EBs), which are spheroids with an inner layer of ectoderm and a single outer layer of endoderm. These EBs differentiate to derivatives of all 3 primary germ layers, leaving a very low yield of spontaneously contracting cardiomyocytes. While this was sufficient for initial studies, larger quantities of pure cardiomyocytes are necessary to establish a scalable system for dis‐ ease modeling and drug development. In 2007, Laflamme et al. reported the use of a mono‐ layer cardiac induction system based on activin A and BMP4 with a 30-fold higher yield of pure cardiomyocytes than through the formation of EBs [80]. Most recently, Lian et al. of the Wisconsin stem cell group identified temporal modulation of canonical Wnt signaling as a key step for robust cardiomyocyte differentiation reporting efficient yields of up to 98% [81].

rected differentiation of stem cells [76].

450 Pluripotent Stem Cells

**6. Future outlook & research direction**

desired cell type and characterized in drug screenings.

of the mechanisms underlying the disease.

small molecules to generate hiPSCs [79].

With some host-specific modifications, currently available technologies such as zinc fin‐ ger nucleases can be applied as the next step in disease modeling after understanding the pathogenesis, developing a cure. Zinc finger nucleases are enzymes that manipulate specific sites of the host genome, generating transgenic lines via knocking-in and knock‐ ing-out of genes. The homologous recombination pathway, naturally occurring at DNA replication forks and repairing double stranded breaks, can be exploited to selectively target a locus for modification while leaving the rest of the genome in tact [82]. Through this method, we have been able to identify new gene function in mouse and other ho‐ mologous mammalian models. The same concept can be applied to gene therapy for hu‐ mans. For example, with patient-specific cardiomyocytes, constructs can be created and tested *in vitro* to restore wild-type function.

#### **6.2. High-throughput screening**

High-throughput screening is another means of advancing disease therapy, but it hinges on its scalability; in other words, whether or not cells of the disease model can be massproduced. With current protocols for directed cardiac differentiation, every round of ex‐ periments would take at least 2 weeks [81]. If hiPSC-derived cardiomyocytes could be consistently generated in 96-well plates, then these high-throughput screenings that could propel translational research from a cellular and molecular level of disease directly to therapeutic applications.

## **7. Conclusion**

In the new era of personalized medicine, the stem cell platform for disease modeling ap‐ pears very viable, especially given the rapid advancements in the field over the past several decades. We have thoroughly discussed the advantages and disadvantages of using mESC, hESC, and hiPSC, all of which have the common end goal of best recapitulating disease phe‐ notype *in vitro*. Of those, we strongly believe that hiPSC-derived cells can eventually be the gold standard for personalized medicine. Using a heritable cardiovascular class of diseases as an example, we endeavored to convey the potential benefits of harnessing iPSC technolo‐ gy to study the pathogenesis of various disorders. One of the most difficult challenges cur‐ rently is establishing a robust, universal, and scalable cardiac induction protocol. Combined with the genetic tools available, we will be able to break the barriers to disease modeling with the limitless supply of human cells *in vitro*.

## **Acknowledgements**

CCH was funded by the Veterans Affairs VA Merit Award BX000771, NIH grants 5U01HL100398 and 1R01HL104040. CCS was funded by NIH NRSA 5T35HL090555.

[9] Thomson, J.A., et al., *Embryonic stem cell lines derived from human blastocysts.* science,

Pluripotent Stem Cells to Model Human Cardiac Diseases

http://dx.doi.org/10.5772/54373

453

[10] Lu, B., et al., *Long'ÄêTerm Safety and Function of RPE from Human Embryonic Stem Cells in Preclinical Models of Macular Degeneration.* Stem Cells, 2009. 27(9): p. 2126-2135. [11] Walters, L.R., *Human embryonic stem cell research: an intercultural perspective.* Kennedy

[12] Zhang, Q., et al., *Stem cells and cardiovascular tissue repair: Mechanism, methods, and clinical applications.* Journal of Cardiothoracic Renal Research, 2006. 1(1): p. 3-14. [13] Wu, S.M. and K. Hochedlinger, *Harnessing the potential of induced pluripotent stem cells*

[14] Aasen, T., et al., *Efficient and rapid generation of induced pluripotent stem cells from hu‐*

[15] Carey, B.W., et al., *Reprogramming of murine and human somatic cells using a single poly‐ cistronic vector.* Proceedings of the National Academy of Sciences, 2009. 106(1): p.

[16] Liu, H., et al., *Generation of endoderm'Äêderived human induced pluripotent stem cells*

[17] Sun, N., et al., *Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells.* Proceedings of the National Academy of Sciences, 2009. 106(37): p.

[18] Aoki, T., et al., *Generation of induced pluripotent stem cells from human adipose-derived stem cells without c-MYC.* Tissue Engineering Part A, 2010. 16(7): p. 2197-2206.

[19] Kim, J.B., et al., *Direct reprogramming of human neural stem cells by OCT4.* Nature, 2009.

[20] Ruiz, S., et al., *High-efficient generation of induced pluripotent stem cells from human astro‐*

[21] Haase, A., et al., *Generation of induced pluripotent stem cells from human cord blood.* Cell

[22] Giorgetti, A., et al., *Generation of induced pluripotent stem cells from human cord blood us‐*

[23] Li, C., et al., *Pluripotency can be rapidly and efficiently induced in human amniotic fluid-*

[24] Loh, Y.H., et al., *Generation of induced pluripotent stem cells from human blood.* Blood,

[25] Ye, Z., et al., *Human-induced pluripotent stem cells from blood cells of healthy donors and*

*derived cells.* Human molecular genetics, 2009. 18(22): p. 4340-4349.

*patients with acquired blood disorders.* Blood, 2009. 114(27): p. 5473-5480.

*for regenerative medicine.* Nature cell biology, 2011. 13(5): p. 497-505.

*man keratinocytes.* Nature biotechnology, 2008. 26(11): p. 1276-1284.

*from primary hepatocytes.* Hepatology, 2010. 51(5): p. 1810-1819.

1998. 282(5391): p. 1145-1147.

157-162.

15720-15725.

461(7264): p. 649-643.

*cytes.* PLoS One, 2010. 5(12): p. e15526.

*ing OCT4 and SOX2.* Cell Stem Cell, 2009. 5(4): p. 353.

Stem Cell, 2009. 5(4): p. 434-441.

2009. 113(22): p. 5476-5479.

Institute of Ethics Journal, 2004. 14(1): p. 3-38.

## **Author details**

Calvin C. Sheng1 and Charles C. Hong1,2\*

\*Address all correspondence to: charles.c.hong@vanderbilt.edu

1 Division of Cardiovascular Medicine, Center for Inherited Heart Disease, Department of Cell and Developmental Biology, Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, USA

2 Research Medicine, Veterans Affairs TVHS, Nashville, USA

## **References**


[9] Thomson, J.A., et al., *Embryonic stem cell lines derived from human blastocysts.* science, 1998. 282(5391): p. 1145-1147.

**Acknowledgements**

452 Pluripotent Stem Cells

**Author details**

Calvin C. Sheng1

**References**

1731-1732.

2390-2397.

2012. 125(1): p. e2-e220.

technology, 2005. 49(2): p. 109-116.

*mouse embryos.* Nature, 1981. 292(5819): p. 154-156.

Sciences, 1981. 78(12): p. 7634.

School of Medicine, Nashville, USA

CCH was funded by the Veterans Affairs VA Merit Award BX000771, NIH grants

1 Division of Cardiovascular Medicine, Center for Inherited Heart Disease, Department of Cell and Developmental Biology, Department of Pharmacology, Vanderbilt University

[1] Hackam, D.G. and D.A. Redelmeier, *Translation of research evidence from animals to hu‐ mans.* JAMA: the journal of the American Medical Association, 2006. 296(14): p.

[2] Members, W.G., et al., *Heart Disease and Stroke Statistics—2012 Update.* Circulation,

[3] Fu, J., et al., *An optimized protocol for culture of cardiomyocyte from neonatal rat.* Cyto‐

[4] Martin, G.R., *Isolation of a pluripotent cell line from early mouse embryos cultured in medi‐ um conditioned by teratocarcinoma stem cells.* Proceedings of the National Academy of

[5] Evans, M.J. and M.H. Kaufman, *Establishment in culture of pluripotential cells from*

[6] Urbach, A., M. Schuldiner, and N. Benvenisty, *Modeling for Lesch'ÄêNyhan Disease by Gene Targeting in Human Embryonic Stem Cells.* Stem Cells, 2004. 22(4): p. 635-641. [7] Musunuru, K., I.J. Domian, and K.R. Chien, *Stem cell models of cardiac development and disease.* Annual review of cell and developmental biology, 2010. 26: p. 667-687.

[8] Krenz, M. and J. Robbins, *Impact of beta-myosin heavy chain expression on cardiac func‐ tion during stress.* Journal of the American College of Cardiology, 2004. 44(12): p.

5U01HL100398 and 1R01HL104040. CCS was funded by NIH NRSA 5T35HL090555.

and Charles C. Hong1,2\*

\*Address all correspondence to: charles.c.hong@vanderbilt.edu

2 Research Medicine, Veterans Affairs TVHS, Nashville, USA


[26] Oda, Y., et al., *Induction of pluripotent stem cells from human third molar mesenchymal stromal cells.* Journal of Biological Chemistry, 2010. 285(38): p. 29270-29278.

[43] Jervell, A. and F. Lange-Nielsen, *Congenital deaf-mutism, functional heart disease with prolongation of the QT interval, and sudden death.* American heart journal, 1957. 54(1): p.

Pluripotent Stem Cells to Model Human Cardiac Diseases

http://dx.doi.org/10.5772/54373

455

[44] Vincent, G.M., *The long QT syndrome.* Indian pacing and electrophysiology journal,

[45] Roden, D.M., *Long-QT Syndrome.* New England Journal of Medicine, 2008. 358(2): p.

[46] Goldenberg, I., W. Zareba, and A.J. Moss, *Long QT syndrome.* Current problems in

[47] Kamp, T.J., *An electrifying iPSC disease model: long QT syndrome type 2 and heart cells in*

[48] Moretti, A., et al., *Patient-specific induced pluripotent stem-cell models for long-QT syn‐*

[49] Vincent, G.M., et al., *High Efficacy of β-Blockers in Long-QT Syndrome Type 1.* Circula‐

[50] Itzhaki, I., et al., *Modelling the long QT syndrome with induced pluripotent stem cells.* Na‐

[51] Xu, M., et al., *Enhanced expression of L-type Cav1. 3 calcium channels in murine embryonic hearts from Cav1. 2-deficient mice.* Journal of Biological Chemistry, 2003. 278(42): p.

[52] Yazawa, M., et al., *Using induced pluripotent stem cells to investigate cardiac phenotypes*

[53] Legius, E., et al., *PTPN11 mutations in LEOPARD syndrome.* Journal of medical genet‐

[54] Kontaridis, M.I., et al., *PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects.* Journal of Biological Chemistry, 2006. 281(10): p.

[55] Oishi, K., et al., *Phosphatase-defective LEOPARD syndrome mutations in PTPN11 gene have gain-of-function effects during Drosophila development.* Human molecular genetics,

[56] Jopling, C., D. Van Geemen, and J. Den Hertog, *Shp2 knockdown and Noonan/LEOP‐ ARD mutant Shp2–induced gastrulation defects.* PLoS genetics, 2007. 3(12): p. e225.

[57] Carvajal-Vergara, X., et al., *Patient-specific induced pluripotent stem-cell-derived models of*

[58] Heineke, J. and J.D. Molkentin, *Regulation of cardiac hypertrophy by intracellular signal‐ ling pathways.* Nature Reviews Molecular Cell Biology, 2006. 7(8): p. 589-600.

*in Timothy syndrome.* Nature, 2011. 471(7337): p. 230-234.

*LEOPARD syndrome.* Nature, 2010. 465(7299): p. 808-812.

*drome.* New England Journal of Medicine, 2010. 363(15): p. 1397-1409.

59-68.

169-176.

2002. 2(4): p. 127.

cardiology, 2008. 33(11): p. 629-694.

tion, 2009. 119(2): p. 215-221.

ics, 2002. 39(8): p. 571-574.

2009. 18(1): p. 193-201.

40837-40841.

6785-6792.

ture, 2011. 471(7337): p. 225-229.

*a dish.* Cell Stem Cell, 2011. 8(2): p. 130-131.


[43] Jervell, A. and F. Lange-Nielsen, *Congenital deaf-mutism, functional heart disease with prolongation of the QT interval, and sudden death.* American heart journal, 1957. 54(1): p. 59-68.

[26] Oda, Y., et al., *Induction of pluripotent stem cells from human third molar mesenchymal stromal cells.* Journal of Biological Chemistry, 2010. 285(38): p. 29270-29278.

[27] Miyoshi, K., et al., *Generation of human induced pluripotent stem cells from oral mucosa.*

[28] Seki, T., et al., *Generation of induced pluripotent stem cells from human terminally differen‐*

[29] Rajesh, D., et al., *Human lymphoblastoid B-cell lines reprogrammed to EBV-free induced*

[30] Choi, S.M., et al., *Reprogramming of EBV-immortalized B-lymphocyte cell lines into in‐*

[31] Chambers, S.M., et al., *Highly efficient neural conversion of human ES and iPS cells by du‐ al inhibition of SMAD signaling.* Nature biotechnology, 2009. 27(3): p. 275-280.

[32] Neely, M.D., et al., *DMH1, a highly selective small molecule BMP inhibitor promotes neu‐ rogenesis of hiPSCs: Comparison of PAX6 and SOX1 expression during neural induction.*

[33] Dimos, J.T., et al., *Induced pluripotent stem cells generated from patients with ALS can be*

[34] Ebert, A.D., et al., *Induced pluripotent stem cells from a spinal muscular atrophy patient.*

[35] Soldner, F., et al., *Parkinson's disease patient-derived induced pluripotent stem cells free of*

[36] Osakada, F., et al., *In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction.* Journal of cell science, 2009. 122(17): p. 3169-3179.

[37] Sullivan, G.J., et al., *Generation of functional human hepatic endoderm from human induced*

[38] Choi, K.D., et al., *Hematopoietic and endothelial differentiation of human induced pluripo‐*

[39] Taura, D., et al., *Adipogenic differentiation of human induced pluripotent stem cells: com‐ parison with that of human embryonic stem cells.* FEBS letters, 2009. 583(6): p. 1029-1033.

[40] Hockemeyer, D., et al., *A drug-inducible system for direct reprogramming of human so‐*

[41] Maherali, N., et al., *A high-efficiency system for the generation and study of human induced*

[42] Uosaki, H., et al., *Efficient and scalable purification of cardiomyocytes from human embry‐ onic and induced pluripotent stem cells by VCAM1 surface expression.* PLoS One, 2011.

*differentiated into motor neurons.* science, 2008. 321(5893): p. 1218-1221.

*viral reprogramming factors.* Cell, 2009. 136(5): p. 964-977.

*pluripotent stem cells.* Hepatology, 2010. 51(1): p. 329-335.

*matic cells to pluripotency.* Cell Stem Cell, 2008. 3(3): p. 346-353.

*pluripotent stem cells.* Cell Stem Cell, 2008. 3(3): p. 340-345.

6(8): p. e23657.

*tent stem cells.* Stem Cells, 2009. 27(3): p. 559-567.

Journal of bioscience and bioengineering, 2010. 110(3): p. 345-350.

*tiated circulating T cells.* Cell Stem Cell, 2010. 7(1): p. 11.

*pluripotent stem cells.* Blood, 2011. 118(7): p. 1797-1800.

Nature, 2008. 457(7227): p. 277-280.

454 Pluripotent Stem Cells

*duced pluripotent stem cells.* Blood, 2011. 118(7): p. 1801-1805.


[74] Raya, Å., et al., *Disease-corrected haematopoietic progenitors from Fanconi anaemia induced*

Pluripotent Stem Cells to Model Human Cardiac Diseases

http://dx.doi.org/10.5772/54373

457

[75] Meissner, A., M. Wernig, and R. Jaenisch, *Direct reprogramming of genetically unmodi‐ fied fibroblasts into pluripotent stem cells.* Nature biotechnology, 2007. 25(10): p.

[76] Kretsovali, A., C. Hadjimichael, and N. Charmpilas, *Histone deacetylase inhibitors in cell pluripotency, differentiation, and reprogramming.* Stem Cells International, 2012.

[77] Boheler, K.R., et al., *Differentiation of pluripotent embryonic stem cells into cardiomyo‐*

[78] Zhang, J., et al., *Functional cardiomyocytes derived from human induced pluripotent stem*

[79] Yu, J., et al., *Efficient feeder-free episomal reprogramming with small molecules.* PLoS One,

[80] Laflamme, M.A., et al., *Cardiomyocytes derived from human embryonic stem cells in prosurvival factors enhance function of infarcted rat hearts.* Nature biotechnology, 2007.

[81] Lian, X., et al., *Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.* Proceedings of the National Academy

[82] Lombardo, A., et al., *Gene editing in human stem cells using zinc finger nucleases and inte‐ grase-defective lentiviral vector delivery.* Nature biotechnology, 2007. 25(11): p.

[83] Lahti, A.L., et al., *Model for long QT syndrome type 2 using human iPS cells demonstrates arrhythmogenic characteristics in cell culture.* Disease Models & Mechanisms, 2012. 5(2):

[84] Matsa, E., et al., *Drug evaluation in cardiomyocytes derived from human induced pluripo‐ tent stem cells carrying a long QT syndrome type 2 mutation.* European heart journal,

*pluripotent stem cells.* Nature, 2009. 460(7251): p. 53-59.

*cytes.* Circulation research, 2002. 91(3): p. 189-201.

*cells.* Circulation research, 2009. 104(4): p. e30-e41.

of Sciences, 2012. 109(27): p. E1848-E1857.

1177-1181.

2011. 6(3): p. e17557.

25(9): p. 1015-1024.

1298-1306.

p. 220.

2011. 32(8): p. 952-962.


[74] Raya, Å., et al., *Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells.* Nature, 2009. 460(7251): p. 53-59.

[59] Fuster, V. and B.B. Kelly, *Epidemiology of Cardiovascular Disease.* 2010.

*sociated with Dilated Cardiomyopathy.* Heart Rhythm, 2011.

*demiology and prevention.* Circulation, 2006. 113(14): p. 1807-1816.

*opathy.* Clinical and translational science, 2010. 3(3): p. 90-97.

*ment and dilated cardiomyopathy.* PLoS One, 2008. 3(7): p. e2642.

*American Heart Association.* Circulation, 2011.

930-941.

456 Pluripotent Stem Cells

2010. 48(5): p. 882-892.

130ra47-130ra47.

Circulation, 2011. 124(3): p. 304-313.

Apmis, 2005. 113(11'Äê12): p. 751-755.

*potency.* Nature, 2011. 471(7336): p. 58-62.

*defined factors.* Cell, 2007. 131(5): p. 861-872.

*cells to model disease.* Cell Stem Cell, 2009. 5(6): p. 584.

[60] Roger, V.L., et al., *Heart Disease and Stroke Statistics--2012 Update: A Report From the*

[61] Refaat, M.M., et al., *Genetic Variation in the Alternative Splicing Regulator, RBM20, is as‐*

[62] Maron, B.J., et al., *Contemporary definitions and classification of the cardiomyopathies an American heart association scientific statement from the council on clinical cardiology, heart failure and transplantation committee; quality of care and outcomes research and functional genomics and translational biology interdisciplinary working groups; and council on epi‐*

[63] Brauch, K.M., et al., *Mutations in ribonucleic acid binding protein gene cause familial dilat‐ ed cardiomyopathy.* Journal of the American College of Cardiology, 2009. 54(10): p.

[64] Li, D., et al., *Identification of novel mutations in RBM20 in patients with dilated cardiomy‐*

[65] Willott, R.H., et al., *Mutations in Troponin that cause HCM, DCM AND RCM: What can we learn about thin filament function?* Journal of molecular and cellular cardiology,

[66] Ahmad, F., et al., *The role of cardiac troponin T quantity and function in cardiac develop‐*

[67] Sun, N., et al., *Patient-Specific Induced Pluripotent Stem Cells as a Model for Familial Di‐ lated Cardiomyopathy.* Science Translational Medicine, 2012. 4(130): p.

[68] Jessup, M., et al., *Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) Clinical Perspective A Phase 2 Trial of Intracoronary Gene Therapy of Sarcoplasmic Reticulum Ca2+-ATPase in Patients With Advanced Heart Failure.*

[69] Hanson, C. and G. Caisander, *Human embryonic stem cells and chromosome stability.*

[70] Hussein, S.M., et al., *Copy number variation and selection during reprogramming to pluri‐*

[71] Takahashi, K., et al., *Induction of pluripotent stem cells from adult human fibroblasts by*

[72] Josowitz, R., et al., *Induced pluripotent stem cell-derived cardiomyocytes as models for ge‐ netic cardiovascular disorders.* Current Opinion in Cardiology, 2011. 26(3): p. 223. [73] Saha, K. and R. Jaenisch, *Technical challenges in using human induced pluripotent stem*


**Chapter 21**

**Pluripotent Stem Cells for Cardiac Cell Therapy:**

Cardiovascular disease remains the leading cause of death worldwide despite many years of declining mortality rates in the Western world [1,2]. Myocardial infarction carries a short term mortality rate of about 7% even with aggressive therapy, and congestive heart failure with even more distressing 20% one-year mortality [3]. Despite significant advances in therapeutic modalities and risk-reduction strategies, the substantial burden remains. This continued health problem has prompted research into new therapeutic strategies including cardiac regenerative therapy as a new approach for severe cardiac diseases resistant to conventional therapies [4,5]. Acute ischemic injury and chronic cardiomyopathies lead to permanent loss of cardiac tissue, leading to heart failure. For pathologic situations, cell transplantation is thought to be an ideal therapeutic method for supplying *de novo* myocardium [6]. Of the available cell sources for cardiac cell therapy, stem cells (e.g. pluripotent stem cells, bone-marrow derived stem cells, skeletal myoblasts and cardiac stem cells) are now being prioritized for basic research and clinical trials [4,7]. The discoveries of various stem cell populations possessing cardiogenic potential and the advance of methods to isolate and expand these cells have shaped the notion of cell-based restorative therapy [8-11]. Despite much knowledge gained through numerous basic researches, significant challenges for true cardiac regeneration remain, and the field lacks sufficient results conclusive to support full-scale implementation of such treatments. Further‐ more, results of clinical researches in cardiac stem cell therapy with a relatively small cohort

scale were marginal, thus only showing little clinical advantages so far [12].

Among the stem cell types, pluripotent stem cells (PSCs) [Embryonic stem cells (ESCs) / induced pluripotent stem cells (iPSCs)] possess great capacity for cardiac regeneration mainly due to the prominent potential to expand and differentiate into most somatic cell lineages [13,14]. To date, no human trials using PSCs for cardiac repair have been attempted. Intensive

> © 2013 Masumoto and Yamashita; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Masumoto and Yamashita; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**The Application of Cell Sheet Technology**

Hidetoshi Masumoto and Jun K. Yamashita

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56326

**1. Introduction**

## **Pluripotent Stem Cells for Cardiac Cell Therapy: The Application of Cell Sheet Technology**

Hidetoshi Masumoto and Jun K. Yamashita

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56326

## **1. Introduction**

Cardiovascular disease remains the leading cause of death worldwide despite many years of declining mortality rates in the Western world [1,2]. Myocardial infarction carries a short term mortality rate of about 7% even with aggressive therapy, and congestive heart failure with even more distressing 20% one-year mortality [3]. Despite significant advances in therapeutic modalities and risk-reduction strategies, the substantial burden remains. This continued health problem has prompted research into new therapeutic strategies including cardiac regenerative therapy as a new approach for severe cardiac diseases resistant to conventional therapies [4,5].

Acute ischemic injury and chronic cardiomyopathies lead to permanent loss of cardiac tissue, leading to heart failure. For pathologic situations, cell transplantation is thought to be an ideal therapeutic method for supplying *de novo* myocardium [6]. Of the available cell sources for cardiac cell therapy, stem cells (e.g. pluripotent stem cells, bone-marrow derived stem cells, skeletal myoblasts and cardiac stem cells) are now being prioritized for basic research and clinical trials [4,7]. The discoveries of various stem cell populations possessing cardiogenic potential and the advance of methods to isolate and expand these cells have shaped the notion of cell-based restorative therapy [8-11]. Despite much knowledge gained through numerous basic researches, significant challenges for true cardiac regeneration remain, and the field lacks sufficient results conclusive to support full-scale implementation of such treatments. Further‐ more, results of clinical researches in cardiac stem cell therapy with a relatively small cohort scale were marginal, thus only showing little clinical advantages so far [12].

Among the stem cell types, pluripotent stem cells (PSCs) [Embryonic stem cells (ESCs) / induced pluripotent stem cells (iPSCs)] possess great capacity for cardiac regeneration mainly due to the prominent potential to expand and differentiate into most somatic cell lineages [13,14]. To date, no human trials using PSCs for cardiac repair have been attempted. Intensive

translational researches, including the demonstration of effectiveness and safety, are needed to realize clinical application of PSCs.

disadvantage of MSCs for this clinical application is the broad differentiation capacity; MSC populations remain highly heterogeneous and are less predictable after transplantation. Some studies have shown that MSCs differentiated into osteoblasts inside ventricular tissue after

Pluripotent Stem Cells for Cardiac Cell Therapy: The Application of Cell Sheet Technology

http://dx.doi.org/10.5772/56326

461

Endothelial progenitor cells (EPCs) are another promising stem cell subset which accumulate to vascular injury sites from bone marrow and incorporate into the microvasculature (vascu‐ logenesis) [9]. EPCs can be identified by the ability to acquire the expression of endothelial cell surface makers, such as cluster of differentiation molecule 133 (CD133), CD34 and so on, both in vitro and in vivo [25]. The research into their therapeutic use began with attempts to enhance their mobilization or incorporate EPCs directly into the vasculature of injured sites [26]. Preclinical studies of the injection of EPCs to infarct myocardium improved left ventricular function [15]. Although EPCs remain promising as a potential therapeutic material, they have several disadvantages for cell therapy: 1) Their heterogeneity. EPCs circulating in the periph‐ eral blood span the full range of differentiation from angioblasts to mature endothelial cells. 2) Limited stem cell pool. Ex vivo expansion would be the only way to obtain a sufficient amount of EPCs for the treatment of an ischemic injury [27]. 3) The pool of EPCs is reduced in patients with common comorbidities of cardiac ischemia (e.g. diabetes mellitus, hypertension,

Skeletal myoblasts (SMs) are a stem cell population derived from the satellite cells which exists beneath the basal membrane of adult skeletal muscle tissue [29]. SMs have been considered as an attractive source for cardiac restoration because of the small potential for teratoma forma‐ tion, availability for autologous transplantation, resistance to ischemic condition and so on [10]. Most transplantations in animal disease models improved left ventricular function and decreased ventricular remodeling [10,30]. There are however, two main limitations; the first is the arrhythmogenic potential of the engrafted SMs. It is reported that only a fraction of skeletal myoblasts differentiate into cardiomyocytes after transplantation, and the generated myotubules may not synchronically work with the native myocardium [31]. A large scale clinical trial, Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) trial, showed a higher number of arrhythmic events in myoblast-treated patients [32]. The second limitation is the relatively poor engraftment of the transplanted cells into the host myocardium. It is reported that less than 10% of transplanted cells could survive within the first few days

Several populations of cardiac progenitor / stem cells derived from mature cardiac tissue have been reported, which may hold the natural and endogenous cardiac regenerative mechanisms. Traditionally, the heart has been considered to be a post-mitotic organ, and withdrawn from the proliferative cell cycle. However, some contradictory data have reported, as cardiomyocyte proliferation and cell cycling have been observed under pathological conditions (e.g. hyper‐ tension or myocardial infarction) [34, 35] and even in the healthy heart [36]. These evidences prompted further research for such resident cardiac cells. The first cell population with stem cell properties is called the side population (SP) cells. Isolated cardiac SP cells represent cardiac and vascular progenitor cells and can differentiate into cardiomyocytes, endothelial cells, or smooth muscle cells [37]. The second progenitor population is the cells expressing the stem

transplantation [24].

and hyperlipidemia) [28].

after injection in mice [33].

Another concern is the actual phenomena which are taking place in the niche of transplanted site: does cardiac stem cell therapy bring *de novo* functional myocardium, or some indirect mechanisms mediate cardiac repair? It is reported that very few of the transplanted tissue stem cells seem to differentiate into mature cardiovascular cell types, suggesting that transplanted cells exert indirect paracrine effects by which humoral factors induce or support favorable processes, including angiogenesis, prevention of apoptosis, and promotion of healing, in the injured myocardium rather than differentiating into *de novo* myocardium [4,15]. PSCs might possess advantages in this context; defined cell populations differentiated from PSCs might be effective to elucidate underlying paracrine mechanisms in cardiac restoration compared to bulk cell mixture derived from somatic stem cells with various cell lineages and differentiation stages [16].

Concerning stem cell transplantation, as well as the transplanted cell type, the method for transplantation is also important to overcome the poor efficiency of engraftment with needle injection. A promising approach is the creation of cell sheets that better support effective engraftment of the transplants. We have shown the effectiveness of temperature-responsive cell sheet technology in basic studies [16].

In this chapter, we introduce the clarification for the progress and drawbacks of current cardiac stem cell therapy, and finally indicate the future directions of cardiac cell therapy through our recent researches combining PSCs and cell sheet technology.

## **2. Various somatic stem cell populations for cardiac stem cell therapy**

To date, various somatic stem cells have been investigated for their feasibility to cardiac regenerative therapy with many basic studies.

Bone marrow hematopoietic stem cells (or circulating peripheral-blood progenitor cells) are an abundant and well characterized source of progenitor cells. A number of studies have shown that direct transplantation of bone marrow-derived cells or mobilization from endog‐ enous reservoirs of the cell population significantly improves cardiac function [17,18]. However, other investigations found limited differentiation of bone marrow cells into cardiovascular cell types [19]. This suggests that beneficial results were mainly due to indirect paracrine effects such as neovascularization, independent of direct tissue regeneration.

Mesenchymal stem cells (MSCs) are a subset of stem cells found in the stroma of the bone marrow, adipose tissue, fetal membrane and many other tissues that can differentiate into osteoblasts, chondrocytes, and adipocytes [20,21] and also into small numbers of cardiomyo‐ cytes [8]. MSCs are thought to be either less immunogenic than other stem cell populations or inherently immunomodulatory [22], alleviating the need for immunosuppression prior to transplantation. Transplantation of MSCs into infarct animal models demonstrated improved left ventricular function, reduced infarct size, and increased survival rate [8,22,23]. The major disadvantage of MSCs for this clinical application is the broad differentiation capacity; MSC populations remain highly heterogeneous and are less predictable after transplantation. Some studies have shown that MSCs differentiated into osteoblasts inside ventricular tissue after transplantation [24].

translational researches, including the demonstration of effectiveness and safety, are needed

Another concern is the actual phenomena which are taking place in the niche of transplanted site: does cardiac stem cell therapy bring *de novo* functional myocardium, or some indirect mechanisms mediate cardiac repair? It is reported that very few of the transplanted tissue stem cells seem to differentiate into mature cardiovascular cell types, suggesting that transplanted cells exert indirect paracrine effects by which humoral factors induce or support favorable processes, including angiogenesis, prevention of apoptosis, and promotion of healing, in the injured myocardium rather than differentiating into *de novo* myocardium [4,15]. PSCs might possess advantages in this context; defined cell populations differentiated from PSCs might be effective to elucidate underlying paracrine mechanisms in cardiac restoration compared to bulk cell mixture derived from somatic stem cells with various cell lineages and differentiation

Concerning stem cell transplantation, as well as the transplanted cell type, the method for transplantation is also important to overcome the poor efficiency of engraftment with needle injection. A promising approach is the creation of cell sheets that better support effective engraftment of the transplants. We have shown the effectiveness of temperature-responsive

In this chapter, we introduce the clarification for the progress and drawbacks of current cardiac stem cell therapy, and finally indicate the future directions of cardiac cell therapy through our

**2. Various somatic stem cell populations for cardiac stem cell therapy**

To date, various somatic stem cells have been investigated for their feasibility to cardiac

Bone marrow hematopoietic stem cells (or circulating peripheral-blood progenitor cells) are an abundant and well characterized source of progenitor cells. A number of studies have shown that direct transplantation of bone marrow-derived cells or mobilization from endog‐ enous reservoirs of the cell population significantly improves cardiac function [17,18]. However, other investigations found limited differentiation of bone marrow cells into cardiovascular cell types [19]. This suggests that beneficial results were mainly due to indirect paracrine effects such as neovascularization, independent of direct tissue regeneration.

Mesenchymal stem cells (MSCs) are a subset of stem cells found in the stroma of the bone marrow, adipose tissue, fetal membrane and many other tissues that can differentiate into osteoblasts, chondrocytes, and adipocytes [20,21] and also into small numbers of cardiomyo‐ cytes [8]. MSCs are thought to be either less immunogenic than other stem cell populations or inherently immunomodulatory [22], alleviating the need for immunosuppression prior to transplantation. Transplantation of MSCs into infarct animal models demonstrated improved left ventricular function, reduced infarct size, and increased survival rate [8,22,23]. The major

to realize clinical application of PSCs.

cell sheet technology in basic studies [16].

regenerative therapy with many basic studies.

recent researches combining PSCs and cell sheet technology.

stages [16].

460 Pluripotent Stem Cells

Endothelial progenitor cells (EPCs) are another promising stem cell subset which accumulate to vascular injury sites from bone marrow and incorporate into the microvasculature (vascu‐ logenesis) [9]. EPCs can be identified by the ability to acquire the expression of endothelial cell surface makers, such as cluster of differentiation molecule 133 (CD133), CD34 and so on, both in vitro and in vivo [25]. The research into their therapeutic use began with attempts to enhance their mobilization or incorporate EPCs directly into the vasculature of injured sites [26]. Preclinical studies of the injection of EPCs to infarct myocardium improved left ventricular function [15]. Although EPCs remain promising as a potential therapeutic material, they have several disadvantages for cell therapy: 1) Their heterogeneity. EPCs circulating in the periph‐ eral blood span the full range of differentiation from angioblasts to mature endothelial cells. 2) Limited stem cell pool. Ex vivo expansion would be the only way to obtain a sufficient amount of EPCs for the treatment of an ischemic injury [27]. 3) The pool of EPCs is reduced in patients with common comorbidities of cardiac ischemia (e.g. diabetes mellitus, hypertension, and hyperlipidemia) [28].

Skeletal myoblasts (SMs) are a stem cell population derived from the satellite cells which exists beneath the basal membrane of adult skeletal muscle tissue [29]. SMs have been considered as an attractive source for cardiac restoration because of the small potential for teratoma forma‐ tion, availability for autologous transplantation, resistance to ischemic condition and so on [10]. Most transplantations in animal disease models improved left ventricular function and decreased ventricular remodeling [10,30]. There are however, two main limitations; the first is the arrhythmogenic potential of the engrafted SMs. It is reported that only a fraction of skeletal myoblasts differentiate into cardiomyocytes after transplantation, and the generated myotubules may not synchronically work with the native myocardium [31]. A large scale clinical trial, Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) trial, showed a higher number of arrhythmic events in myoblast-treated patients [32]. The second limitation is the relatively poor engraftment of the transplanted cells into the host myocardium. It is reported that less than 10% of transplanted cells could survive within the first few days after injection in mice [33].

Several populations of cardiac progenitor / stem cells derived from mature cardiac tissue have been reported, which may hold the natural and endogenous cardiac regenerative mechanisms. Traditionally, the heart has been considered to be a post-mitotic organ, and withdrawn from the proliferative cell cycle. However, some contradictory data have reported, as cardiomyocyte proliferation and cell cycling have been observed under pathological conditions (e.g. hyper‐ tension or myocardial infarction) [34, 35] and even in the healthy heart [36]. These evidences prompted further research for such resident cardiac cells. The first cell population with stem cell properties is called the side population (SP) cells. Isolated cardiac SP cells represent cardiac and vascular progenitor cells and can differentiate into cardiomyocytes, endothelial cells, or smooth muscle cells [37]. The second progenitor population is the cells expressing the stem cell factor receptor c-Kit (also designed as CD117), which are located in small clusters within the adult cardiac tissue. c-Kit+ cells hold regenerative potential after transplantation and give rise to cardiomyocytes, endothelial cells, and smooth muscle cells [38]. The third cell type expresses stem cell antigen 1 (Sca-1). Sca-1+ cells migrate to infarcted myocardium and differentiate into cardiomyocytes around the injured area [39]. Finally, enzymatic digestion of heart tissue obtained via endomyocardial biopsy or during cardiac surgery yields cardiac progenitor cells that form what is called cardiospheres. Cardiosphere derived cells (CDCs) can also differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells, exhibiting prominent capacities for proliferation and differentiation [11]. This population can be differ‐ entiated into aggregates of cardiomyocytes that when transplanted into injured myocardium produced functional improvement in preclinical studies [40]. It is unclear whether the various cardiac stem cells shown here are different populations, or represent various stages of a single cell lineage. A major limitation of cardiac progenitor / stem cell populations is that the cardiac stem cell pool appears to diminish along with age, which may limit the efficacy of regeneration in elderly people [41]. Considering that it is mostly the elderly who suffer increased mortality from cardiac ischemia, intensive research aiming to rejuvenate this senescent stem cell population is required.

The second reason is that the capacity for the differentiation towards a desired cell type, such as cardiomyocytes or other vascular cell types is the highest among various stem cell popula‐ tions known to possess cardiogenic potential. The differentiation of PSCs can be driven towards cardiomyocytes or others by culture conditions as monolayers or embryoid bodies in various growth media [52–55]. Previously, we have developed a novel monolayer culturebased ESC / iPSC differentiation system that recapitulates early cardiovascular developmental processes using Flk1 (also designed as vascular endothelial cell growth factor [VEGF] recep‐ tor-2)-positive cells as common cardiovascular progenitors. Cardiovascular cell types, namely cardiomyocytes [53], endothelial cells, and vascular mural cells [52], can be systematically induced and purified with this system (Figure 1A). In fact, of the various stem cell populations studied so far, PSCs have demonstrated probably the greatest capacity for cardiac cell

Pluripotent Stem Cells for Cardiac Cell Therapy: The Application of Cell Sheet Technology

http://dx.doi.org/10.5772/56326

463

The third reason is that PSCs might be advantageous for further elucidation of regenerative mechanisms. In the field of cardiac restoration with stem cell therapy, it has been widely believed that transplanted cells act as an inducer of indirect paracrine effects such as angio‐ genesis, prevention of apoptosis, and so on rather than regeneration of *de novo* myocardium [4,15]. Considering this point, the transplantation of somatic stem cells, which are largely performed thus far as mentioned above, may raise a question, "which cells are really effec‐ tive?", because the transplanted cells from somatic stem cells might consist of heterogeneous cell populations. In this regard, the transplantation of defined cardiovascular cell populations systematically derived from PSCs might be much more superior to that of somatic stem cellderived populations for the sake of the elucidation of regenerative mechanisms (Figure 1B).

The final reason is the discovery of iPSCs. The generation of iPSCs by reprogramming autologous somatic cells with genes regulating pluripotency may resolve the ethical and immunogenic issues associated with the use of ESCs. Furthermore, we have reported that cardiovascular cell types can be differentiated respectively from mouse iPSCs almost identi‐ cally with those from mouse ESCs [57]. This indicates that iPSCs possess almost the same regenerative capacity as that of ESCs. A potent differentiation protocol based on high-density monolayer culture and chemically defined factors, and modifications thereof, have been reported to induce cardiomyocytes from human iPSCs with a robust efficiency of 40–70 % [54, 58]. The application of this method would strongly promote cardiac regeneration using human

The transplantation of cardiac cells derived from PSCs has been tested in animal studies with encouraging results [16, 54]. However, no human studies using PSCs for cardiac repair have been attempted so far. A major concern regarding iPSC transplantation as a treatment modality is related to the potential tumor formation. The differentiating cells from PSCs contain derivatives from three germ layers (ectoderm, mesoderm and endoderm), possessing the capacity to differentiate along any or all of these three lineages. This increases the risk of teratoma formation at the transplantation site. Although such teratomas are believed to be largely benign, some teratoma cells have been reported to express markers similar to those seen in malignant tumors [59]. Recently, protocols for generating human iPSCs without genomic integration by utilizing episomal vectors [60] or human artificial chromosome vectors

differentiation and long-term cell survival [56].

iPSCs.

Many clinical studies have been conducted using these somatic stem cells so far: TOPCARE-AMI [42], BOOST [43], REPAIR-AMI [44], LateTime [45] (Bone marrow hematopoietic stem cells), REGENT [46] (EPCs), MAGIC [32], CAuSMIC [47] (SMs), CADUCEUS [48], SCIPIO [49] (cardiac progenitor / stem cells) and so on. However, most of these clinical studies have shown relatively limited clinical benefits in general. These marginal results indicate that more efficient approaches for stem cell therapy are needed to realize full-scale stem cell-based therapy.

## **3. Advantages of pluripotent stem cells in cardiac regeneration**

Embryonic stem cells (ESCs) are one of the stem cell populations which can be removed from the inner cell mass of the blastocyst and expanded in vitro with practically no limitations [13]. Yamanaka and colleagues have discovered that reprogramming of adult somatic cells with transcription factor genes that confer pluripotency generates ESC-like cells, called induced pluripotent stem cells (iPSCs) [14,50]. Among the stem cell types, these pluripotent stem cells (PSCs) [ESCs / iPSCs] possess great capacity especially for cardiac regeneration due to several reasons.

The first reason is that PSCs can be expanded practically indefinitely in vitro remaining pluripotent in an undifferentiated state in culture, and can give rise to most somatic cell lineages once allowed to differentiate. In this regard, the regenerative capacity is theoretically limitless [51]. The merit of PSCs is larger especially for the heart compared to other organs, such as endocrine or sensory organs, as the heart functions as an assembly of a large number of cells including cardiomyocytes and other cell types (e.g. vascular cells, cardiac fibroblasts), and numerous (>108 ) heart-composing cells might be required to fully compensate for the damaged human heart [5].

The second reason is that the capacity for the differentiation towards a desired cell type, such as cardiomyocytes or other vascular cell types is the highest among various stem cell popula‐ tions known to possess cardiogenic potential. The differentiation of PSCs can be driven towards cardiomyocytes or others by culture conditions as monolayers or embryoid bodies in various growth media [52–55]. Previously, we have developed a novel monolayer culturebased ESC / iPSC differentiation system that recapitulates early cardiovascular developmental processes using Flk1 (also designed as vascular endothelial cell growth factor [VEGF] recep‐ tor-2)-positive cells as common cardiovascular progenitors. Cardiovascular cell types, namely cardiomyocytes [53], endothelial cells, and vascular mural cells [52], can be systematically induced and purified with this system (Figure 1A). In fact, of the various stem cell populations studied so far, PSCs have demonstrated probably the greatest capacity for cardiac cell differentiation and long-term cell survival [56].

cell factor receptor c-Kit (also designed as CD117), which are located in small clusters within

rise to cardiomyocytes, endothelial cells, and smooth muscle cells [38]. The third cell type

differentiate into cardiomyocytes around the injured area [39]. Finally, enzymatic digestion of heart tissue obtained via endomyocardial biopsy or during cardiac surgery yields cardiac progenitor cells that form what is called cardiospheres. Cardiosphere derived cells (CDCs) can also differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells, exhibiting prominent capacities for proliferation and differentiation [11]. This population can be differ‐ entiated into aggregates of cardiomyocytes that when transplanted into injured myocardium produced functional improvement in preclinical studies [40]. It is unclear whether the various cardiac stem cells shown here are different populations, or represent various stages of a single cell lineage. A major limitation of cardiac progenitor / stem cell populations is that the cardiac stem cell pool appears to diminish along with age, which may limit the efficacy of regeneration in elderly people [41]. Considering that it is mostly the elderly who suffer increased mortality from cardiac ischemia, intensive research aiming to rejuvenate this senescent stem cell

Many clinical studies have been conducted using these somatic stem cells so far: TOPCARE-AMI [42], BOOST [43], REPAIR-AMI [44], LateTime [45] (Bone marrow hematopoietic stem cells), REGENT [46] (EPCs), MAGIC [32], CAuSMIC [47] (SMs), CADUCEUS [48], SCIPIO [49] (cardiac progenitor / stem cells) and so on. However, most of these clinical studies have shown relatively limited clinical benefits in general. These marginal results indicate that more efficient approaches for stem cell therapy are needed to realize full-scale stem cell-based therapy.

Embryonic stem cells (ESCs) are one of the stem cell populations which can be removed from the inner cell mass of the blastocyst and expanded in vitro with practically no limitations [13]. Yamanaka and colleagues have discovered that reprogramming of adult somatic cells with transcription factor genes that confer pluripotency generates ESC-like cells, called induced pluripotent stem cells (iPSCs) [14,50]. Among the stem cell types, these pluripotent stem cells (PSCs) [ESCs / iPSCs] possess great capacity especially for cardiac regeneration due to several

The first reason is that PSCs can be expanded practically indefinitely in vitro remaining pluripotent in an undifferentiated state in culture, and can give rise to most somatic cell lineages once allowed to differentiate. In this regard, the regenerative capacity is theoretically limitless [51]. The merit of PSCs is larger especially for the heart compared to other organs, such as endocrine or sensory organs, as the heart functions as an assembly of a large number of cells including cardiomyocytes and other cell types (e.g. vascular cells, cardiac fibroblasts),

) heart-composing cells might be required to fully compensate for the

**3. Advantages of pluripotent stem cells in cardiac regeneration**

cells hold regenerative potential after transplantation and give

cells migrate to infarcted myocardium and

the adult cardiac tissue. c-Kit+

462 Pluripotent Stem Cells

population is required.

reasons.

and numerous (>108

damaged human heart [5].

expresses stem cell antigen 1 (Sca-1). Sca-1+

The third reason is that PSCs might be advantageous for further elucidation of regenerative mechanisms. In the field of cardiac restoration with stem cell therapy, it has been widely believed that transplanted cells act as an inducer of indirect paracrine effects such as angio‐ genesis, prevention of apoptosis, and so on rather than regeneration of *de novo* myocardium [4,15]. Considering this point, the transplantation of somatic stem cells, which are largely performed thus far as mentioned above, may raise a question, "which cells are really effec‐ tive?", because the transplanted cells from somatic stem cells might consist of heterogeneous cell populations. In this regard, the transplantation of defined cardiovascular cell populations systematically derived from PSCs might be much more superior to that of somatic stem cellderived populations for the sake of the elucidation of regenerative mechanisms (Figure 1B).

The final reason is the discovery of iPSCs. The generation of iPSCs by reprogramming autologous somatic cells with genes regulating pluripotency may resolve the ethical and immunogenic issues associated with the use of ESCs. Furthermore, we have reported that cardiovascular cell types can be differentiated respectively from mouse iPSCs almost identi‐ cally with those from mouse ESCs [57]. This indicates that iPSCs possess almost the same regenerative capacity as that of ESCs. A potent differentiation protocol based on high-density monolayer culture and chemically defined factors, and modifications thereof, have been reported to induce cardiomyocytes from human iPSCs with a robust efficiency of 40–70 % [54, 58]. The application of this method would strongly promote cardiac regeneration using human iPSCs.

The transplantation of cardiac cells derived from PSCs has been tested in animal studies with encouraging results [16, 54]. However, no human studies using PSCs for cardiac repair have been attempted so far. A major concern regarding iPSC transplantation as a treatment modality is related to the potential tumor formation. The differentiating cells from PSCs contain derivatives from three germ layers (ectoderm, mesoderm and endoderm), possessing the capacity to differentiate along any or all of these three lineages. This increases the risk of teratoma formation at the transplantation site. Although such teratomas are believed to be largely benign, some teratoma cells have been reported to express markers similar to those seen in malignant tumors [59]. Recently, protocols for generating human iPSCs without genomic integration by utilizing episomal vectors [60] or human artificial chromosome vectors

[61] have been reported. These may reduce tumorigenesis due to mutations, which could

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Considering the results of these basic studies, PSCs (especially iPSCs) are currently recognized to be one of the most promising cell sources for cardiac regeneration. However, further careful exploration for the feasibility of this new modality will be needed to realize the clinical

In addition to the transplanted cell type, the transplantation method is also important to overcome poor efficiency of engraftment associated with needle injection. The low level of grafted cell survival and engraftment diminishes their potential for paracrine effects, besides regeneration of *de novo* myocardium, and is a major technical limitation for stem cell therapy [62]. It is reported that >70% of injected cells die during the first 48 hours after needle injection, progressively diminishing during the following days possibly due to the hypoxic, inflamma‐ tory, and/or fibrotic environment [63]. Another report shows that only 5.4 to 8.8% of micro‐ spheres remain just after direct injection into the beating myocardium due to massive mechanical loss [64]. To overcome this problem, a combination of bioengineering techniques have been developed and investigated for their efficacy, suggesting that these new strategies

Initial experiments were performed by combining the cells with injectable biomaterials such as collagen, fibrin, gelatin or matrigel as a sccafold. In general, early results showed an increased survival of the transplanted cells, and a greater improvement in cardiac function of the treated hearts [66]. However, these approaches did not assure complete cell retention or

The creation of cell sheets without scaffold support would be a more promising approach. The advantages of this method are as follows: 1) Potent increase of the efficiency of transplantation compared to that of needle injection. 2) Potential for construction of three-dimentional tissuelike structure as a graft. 3) Avoidance of inflammatory reactions against the biomaterials constituting the scaffolds. 4) Larger scalability and accessibility due to two-dimensional cell

Several methods have been reported for cell sheet formation [67-69]. Among them, we have utilized temperature-responsive culture surface-based method [16]. This technique was made possible by using a culture dish covalently grafted with temperature-responsive polymer poly (N-isopropylacrylamide) (PIPAAm) which enables the generation of cell sheets without enzymatic digestion, retaining intact extracellular matrices or adhesion molecules [67]. The benefits of this technique have been demonstrated by many experiments of stem cell therapy such as the transplantation of monolayer adipose tissue-derived MSCs to the infarcted rat heart

**4. Cell sheet technology as a novel method for PSC-derived cell**

otherwise limit the clinical application of iPSCs.

may improve the efficiency of stem cell therapies [65].

an adequate distribution of the transplanted cells within the host heart.

application.

culture.

**transplantation**

**Figure 1.** The advantages of PSCs for cardiac regeneration. (A): The capacity for the differentiation towards a desired cardiac cell type. The scheme of directed mouse PSC differentiation system from Flk1+ mesoderm cells as a common progenitor is shown. (B): Effectiveness for further elucidation of regenerative mechanisms. The usage of somatic stem cells (upper) may lead to the transplantation of heterogeneous derivatives in lineage and differentiation stage. On the other hand, the usage of directed PSC differentiation system with purifying processes (lower) clarify which cell popula‐ tions are actually transplanted. ES cell, embryonic stem cell; iPS cell, induced pluripotent stem cell.

[61] have been reported. These may reduce tumorigenesis due to mutations, which could otherwise limit the clinical application of iPSCs.

Considering the results of these basic studies, PSCs (especially iPSCs) are currently recognized to be one of the most promising cell sources for cardiac regeneration. However, further careful exploration for the feasibility of this new modality will be needed to realize the clinical application.

## **4. Cell sheet technology as a novel method for PSC-derived cell transplantation**

In addition to the transplanted cell type, the transplantation method is also important to overcome poor efficiency of engraftment associated with needle injection. The low level of grafted cell survival and engraftment diminishes their potential for paracrine effects, besides regeneration of *de novo* myocardium, and is a major technical limitation for stem cell therapy [62]. It is reported that >70% of injected cells die during the first 48 hours after needle injection, progressively diminishing during the following days possibly due to the hypoxic, inflamma‐ tory, and/or fibrotic environment [63]. Another report shows that only 5.4 to 8.8% of micro‐ spheres remain just after direct injection into the beating myocardium due to massive mechanical loss [64]. To overcome this problem, a combination of bioengineering techniques have been developed and investigated for their efficacy, suggesting that these new strategies may improve the efficiency of stem cell therapies [65].

Initial experiments were performed by combining the cells with injectable biomaterials such as collagen, fibrin, gelatin or matrigel as a sccafold. In general, early results showed an increased survival of the transplanted cells, and a greater improvement in cardiac function of the treated hearts [66]. However, these approaches did not assure complete cell retention or an adequate distribution of the transplanted cells within the host heart.

The creation of cell sheets without scaffold support would be a more promising approach. The advantages of this method are as follows: 1) Potent increase of the efficiency of transplantation compared to that of needle injection. 2) Potential for construction of three-dimentional tissuelike structure as a graft. 3) Avoidance of inflammatory reactions against the biomaterials constituting the scaffolds. 4) Larger scalability and accessibility due to two-dimensional cell culture.

Several methods have been reported for cell sheet formation [67-69]. Among them, we have utilized temperature-responsive culture surface-based method [16]. This technique was made possible by using a culture dish covalently grafted with temperature-responsive polymer poly (N-isopropylacrylamide) (PIPAAm) which enables the generation of cell sheets without enzymatic digestion, retaining intact extracellular matrices or adhesion molecules [67]. The benefits of this technique have been demonstrated by many experiments of stem cell therapy such as the transplantation of monolayer adipose tissue-derived MSCs to the infarcted rat heart

**Figure 1.** The advantages of PSCs for cardiac regeneration. (A): The capacity for the differentiation towards a desired cardiac cell type. The scheme of directed mouse PSC differentiation system from Flk1+ mesoderm cells as a common progenitor is shown. (B): Effectiveness for further elucidation of regenerative mechanisms. The usage of somatic stem cells (upper) may lead to the transplantation of heterogeneous derivatives in lineage and differentiation stage. On the other hand, the usage of directed PSC differentiation system with purifying processes (lower) clarify which cell popula‐

tions are actually transplanted. ES cell, embryonic stem cell; iPS cell, induced pluripotent stem cell.

464 Pluripotent Stem Cells

**Figure 2.** The improvement of infarcted heart function after transplantation of cardiac tissue sheets bioengineered with mouse ES cell-derived defined cardiac cell populations. (A): Cross-sections of the sheet. Upper panel: H&E stain‐ ing showing cell appearance of the sheet. Lower panel: Sirius red staining showing intact extracellular matrix. (B): Im‐ munostaining of sheets for cTnT (red), VE-cadherin (green), and DAPI. (C,D): Echocardiogram (n=9). (C): Representative M-mode image. Note that infarct anterior wall started to move 2–4 weeks after transplantation (Tx). (D): Fractional shortening (FS). (E): LV pressure-volume loop study 4 weeks after Tx (n=8). Ees: End-systolic elastance. (F, G): Capillary formation at Tx-d28. (F): Double staining for vWF (ECs, green) and cTnT (cardiomyocytes [CMs], red) at peri-MI and central-MI areas. Note that newly formed capillaries are clearly observed in transplantation group (dotted circles). (G): Quantification of capillary density (capillary number per square millimeter). Peri-MI area (left panel) and central-MI area (right panel) (15 views each). (H): Triple staining for vWF, cTnT, and species-specific fluorescent in situ hybridiza‐ tion (mouse nuclei, yellow) (Tx-d3). Most of the accumulated vWF-positive cells are negative for mouse nuclear stain‐ ing (arrows). Inset: higher magnification view. \*\*, p <.01; and \*\*\*, p <.001 (unpaired t test), †, p <.05 and ‡, p <.01 (vs. PreTx, paired t test). PreTx; Pretransplantation, Tx2w, Tx4w; 2 and 4 weeks after transplantation, respectively. Scale bars: 200 μm in (B), 100 μm in (F) and (H) (main panel), 50 μm in (H) (inset). HE, Hematoxylin and Eosin; cTnT, cardiac troponin-T; DAPI, 4,6-diamidino-2-phenylindole; vWF, von Willebrandfactor.; MI, myocardial infarction. (quote from ref. 16 with revision)

**Figure 3.** Cell type-controlled sheet analyses. (A): The Scheme of cell sheets with CMs (C+E+M) or without CMs (E+M). (B): ELISA for VEGF secretion (picogram per104 cells) in culture supernatants of C+E+M and E+M sheets. (C,D): Transplantation of sham operation (n=9) versus C+E+M sheets (n=9) versus E+M sheets (n=3) (Tx-d28). (C): Capillary density in peri-MI area (capillary number per square millimeter). (15 views each). (D): Fractional shorten‐ ing (FS) on echocardiogram (fold increase vs. PreTx). \*\*, p <.01, and \*\*\*, p <.001 (unpaired t test). C: cardiomyo‐ cytes, E: endothelial cells, M: vascular mural cells. N.S., not significant; VEGF, vascular endothelial cell growth

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467

factor. (quote from ref. 16 with revision).

Pluripotent Stem Cells for Cardiac Cell Therapy: The Application of Cell Sheet Technology http://dx.doi.org/10.5772/56326 467

**Figure 3.** Cell type-controlled sheet analyses. (A): The Scheme of cell sheets with CMs (C+E+M) or without CMs (E+M). (B): ELISA for VEGF secretion (picogram per104 cells) in culture supernatants of C+E+M and E+M sheets. (C,D): Transplantation of sham operation (n=9) versus C+E+M sheets (n=9) versus E+M sheets (n=3) (Tx-d28). (C): Capillary density in peri-MI area (capillary number per square millimeter). (15 views each). (D): Fractional shorten‐ ing (FS) on echocardiogram (fold increase vs. PreTx). \*\*, p <.01, and \*\*\*, p <.001 (unpaired t test). C: cardiomyo‐ cytes, E: endothelial cells, M: vascular mural cells. N.S., not significant; VEGF, vascular endothelial cell growth factor. (quote from ref. 16 with revision).

**Figure 2.** The improvement of infarcted heart function after transplantation of cardiac tissue sheets bioengineered with mouse ES cell-derived defined cardiac cell populations. (A): Cross-sections of the sheet. Upper panel: H&E stain‐ ing showing cell appearance of the sheet. Lower panel: Sirius red staining showing intact extracellular matrix. (B): Im‐ munostaining of sheets for cTnT (red), VE-cadherin (green), and DAPI. (C,D): Echocardiogram (n=9). (C): Representative M-mode image. Note that infarct anterior wall started to move 2–4 weeks after transplantation (Tx). (D): Fractional shortening (FS). (E): LV pressure-volume loop study 4 weeks after Tx (n=8). Ees: End-systolic elastance. (F, G): Capillary formation at Tx-d28. (F): Double staining for vWF (ECs, green) and cTnT (cardiomyocytes [CMs], red) at peri-MI and central-MI areas. Note that newly formed capillaries are clearly observed in transplantation group (dotted circles). (G): Quantification of capillary density (capillary number per square millimeter). Peri-MI area (left panel) and central-MI area (right panel) (15 views each). (H): Triple staining for vWF, cTnT, and species-specific fluorescent in situ hybridiza‐ tion (mouse nuclei, yellow) (Tx-d3). Most of the accumulated vWF-positive cells are negative for mouse nuclear stain‐ ing (arrows). Inset: higher magnification view. \*\*, p <.01; and \*\*\*, p <.001 (unpaired t test), †, p <.05 and ‡, p <.01 (vs. PreTx, paired t test). PreTx; Pretransplantation, Tx2w, Tx4w; 2 and 4 weeks after transplantation, respectively. Scale bars: 200 μm in (B), 100 μm in (F) and (H) (main panel), 50 μm in (H) (inset). HE, Hematoxylin and Eosin; cTnT, cardiac troponin-T; DAPI, 4,6-diamidino-2-phenylindole; vWF, von Willebrandfactor.; MI, myocardial infarction. (quote from

ref. 16 with revision)

466 Pluripotent Stem Cells

[23]. Recently, we have reported transplantation of a three-layered cardiac tissue sheet bioengineered with mouse ESC-derived defined cardiac cell populations in the infarcted heart (Figure 2) [16]. In both cases, increased tissue neovascularization together with a prominent attenuation of cardiac remodeling responsible for the improvement in cardiac function were demonstrated. Furthermore, our research indicated the potential for cell sheet-based prospec‐ tive elucidation of the cellular mechanisms of cardiac restoration. The combinations of cell populations composing the transplanted cell sheets enabled us to elucidate the contributions of each cell type (for example, the comparison of cell sheets with or without cardiomyocytes is useful for the elucidation of the cellular function of cardiomyocytes). This cell-type control‐ led analysis led us to identify one of the important cellular mechanisms of cardiac restoration following cell therapy, that is, cardiomyocytes are essential for the functional improvement of ischemic heart through neovascularization (Figure 3). These results show that the tissue-like cell sheet system is advantageous for the elucidation of cardiac regenerative mechanism, as well as for therapeutic purposes.

**Author details**

Kyoto, Japan

**References**

Hidetoshi Masumoto1,2\* and Jun K. Yamashita1

index.html accessed 31 August (2012).

Annu Rev Public Health , 32, 5-22.

Circulation 106,II197., 193.

Circulation 100,IIII256., 247.

Nature , 453, 302-305.

myocardial infarction. Lancet , 358, 605-613.

fits and barriers. Expert Rev Mol Med 11,e20.

medicine. Gen Thorac Cardiovasc Surg. In press.

\*Address all correspondence to: masumoto@kuhp.kyoto-u.ac.jp

1 Laboratory of Stem Cell Differentiation, Department of Cell Growth and Differentiation,

Pluripotent Stem Cells for Cardiac Cell Therapy: The Application of Cell Sheet Technology

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469

2 Department of Cardiovascular Surgery, Kyoto University Graduate School of Medicine,

[1] World Health Organization (WHO)The global burden of disease: 2004 update. 2008. http://www.who.int/healthinfo/global\_burden\_disease/2004\_report\_update/en/

[2] Ford, E. S, & Capewell, S. (2011). Proportion of the Decline in Cardiovascular Mortal‐ ity Disease due to Prevention Versus Treatment: Public Health Versus Clinical Care.

[3] Assessment of the Safety and Efficacy of a New Thrombolytic Regimen (ASSENT)-3 Investigators(2001). Efficacy and safety of tenecteplase in combination with enoxa‐ parin, abciximab, or unfractionated heparin: the ASSENT-3 randomised trial in acute

[4] Joggerst, S. J, & Hatzopoulos, A. K. (2009). Stem cell therapy for cardiac repair: bene‐

[5] Masumoto, H, & Sakata, R. Cardiovascular surgery for realization of regenerative

[6] Sakakibara, Y, Tambara, K, Lu, F, Nishina, T, Sakaguchi, G, Nagaya, N, Nishimura, K, Li, R. K, Weisel, R. D, & Komeda, M. (2002). Combined procedure of surgical re‐ pair and cell transplantation for left ventricular aneurysm: an experimental study.

[7] Chien, K. R. (2008). Regenerative medicine and human models of human disease.

[8] Tomita, S, Li, R. K, Weisel, R. D, Mickle, D. A, Kim, E. J, Sakai, T, & Jia, Z. Q. (1999). Autologous transplantation of bone marrow cells improves damaged heart function.

Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan

### **5. Future directions**

One future direction of this PSC-derived cell sheet technology is its utilization as a novel experimental tool for elucidation of regenerative mechanisms. Although the present results of clinical trials using stem cell therapy are marginal, further elucidation of the actual mechanisms of cardiac repair following cell therapy would enhance the potential of stem cell therapy to be a full-scale therapy. It would be a breakthrough for further improvement of cardiac cell therapy to understand the role of each cell population as well as the various cellular interactions in the chaos of heterogeneity.

Another direction is a more efficient survival of transplanted sheets to realize regeneration of functioning *de novo* myocardium. Considering that more cells that survived were observed in peri-infarction than central infarction region in our study [16], it would be possible that the severe ischemic condition may not be suitable for sheet survival. Novel techniques increasing blood supply in the graft should be applied, such as prevascularization in 3-dimensional tissue formation [70,71] or vascularized flap grafts.

#### **6. Conclusion**

In this chapter, we have reviewed the status quo of current cardiac stem cell therapy, and shown the promising potential of PSC-derived cardiac tissue-like sheets. The knowledge yielded from this cell sheet-based study would provide a hallmark for cell therapy with PSCs and a strategic principle for future cardiac restoration therapy.

## **Author details**

[23]. Recently, we have reported transplantation of a three-layered cardiac tissue sheet bioengineered with mouse ESC-derived defined cardiac cell populations in the infarcted heart (Figure 2) [16]. In both cases, increased tissue neovascularization together with a prominent attenuation of cardiac remodeling responsible for the improvement in cardiac function were demonstrated. Furthermore, our research indicated the potential for cell sheet-based prospec‐ tive elucidation of the cellular mechanisms of cardiac restoration. The combinations of cell populations composing the transplanted cell sheets enabled us to elucidate the contributions of each cell type (for example, the comparison of cell sheets with or without cardiomyocytes is useful for the elucidation of the cellular function of cardiomyocytes). This cell-type control‐ led analysis led us to identify one of the important cellular mechanisms of cardiac restoration following cell therapy, that is, cardiomyocytes are essential for the functional improvement of ischemic heart through neovascularization (Figure 3). These results show that the tissue-like cell sheet system is advantageous for the elucidation of cardiac regenerative mechanism, as

One future direction of this PSC-derived cell sheet technology is its utilization as a novel experimental tool for elucidation of regenerative mechanisms. Although the present results of clinical trials using stem cell therapy are marginal, further elucidation of the actual mechanisms of cardiac repair following cell therapy would enhance the potential of stem cell therapy to be a full-scale therapy. It would be a breakthrough for further improvement of cardiac cell therapy to understand the role of each cell population as well as the various cellular interactions in the

Another direction is a more efficient survival of transplanted sheets to realize regeneration of functioning *de novo* myocardium. Considering that more cells that survived were observed in peri-infarction than central infarction region in our study [16], it would be possible that the severe ischemic condition may not be suitable for sheet survival. Novel techniques increasing blood supply in the graft should be applied, such as prevascularization in 3-dimensional tissue

In this chapter, we have reviewed the status quo of current cardiac stem cell therapy, and shown the promising potential of PSC-derived cardiac tissue-like sheets. The knowledge yielded from this cell sheet-based study would provide a hallmark for cell therapy with PSCs

well as for therapeutic purposes.

**5. Future directions**

468 Pluripotent Stem Cells

chaos of heterogeneity.

**6. Conclusion**

formation [70,71] or vascularized flap grafts.

and a strategic principle for future cardiac restoration therapy.

Hidetoshi Masumoto1,2\* and Jun K. Yamashita1

\*Address all correspondence to: masumoto@kuhp.kyoto-u.ac.jp

1 Laboratory of Stem Cell Differentiation, Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan

2 Department of Cardiovascular Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan

## **References**


[9] Asahara, T, Murohara, T, Sullivan, A, Silver, M, Van Der Zee, R, Li, T, Witzenbichler, B, Schatteman, G, & Isner, J. M. (1997). Isolation of putative progenitor endothelial cells for angiogenesis. Science , 275, 964-967.

[20] Jiang, Y, Jahagirdar, B. N, Reinhardt, R. L, Schwartz, R. E, Keene, C. D, Ortiz-gonza‐ lez, X. R, Reyes, M, Lenvik, T, Lund, T, Blackstad, M, et al. (2002). Pluripotency of

Pluripotent Stem Cells for Cardiac Cell Therapy: The Application of Cell Sheet Technology

http://dx.doi.org/10.5772/56326

471

[21] Ishikane, S, Yamahara, K, Sada, M, Harada, K, Kodama, M, Ishibashi-ueda, H, Haya‐ kawa, K, Mishima, K, Iwasaki, K, Fujiwara, M, et al. (2010). Allogeneic administra‐ tion of fetal membrane-derived mesenchymal stem cells attenuates acute myocarditis

[22] Dai, W, Hale, S. L, Martin, B. J, Kuang, J. Q, Dow, J. S, Wold, L. E, & Kloner, R. A. (2005). Allogeneic mesenchymal stem cell transplantation in postinfarcted rat myo‐

[23] Miyahara, Y, Nagaya, N, Kataoka, M, Yanagawa, B, Tanaka, K, Hao, H, Ishino, K, Ishida, H, Shimizu, T, Kangawa, K, et al. (2006). Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med , 12, 459-465.

[24] Yoon, Y. S, Park, J. S, Tkebuchava, T, Luedeman, C, & Losordo, D. W. (2004). Unex‐ pected severe calcification after transplantation of bone marrow cells in acute myo‐

[25] Hristov, M, & Weber, C. (2008). Endothelial progenitor cells in vascular repair and

[26] Llevadot, J, Murasawa, S, Kureishi, Y, Uchida, S, Masuda, H, Kawamoto, A, Walsh, K, Isner, J. M, & Asahara, T. (2001). HMG-CoA reductase inhibitor mobilizes bone

[27] Jujo, K, Ii, M, & Losordo, D. W. (2008). Endothelial progenitor cells in neovasculariza‐

[28] Vasa, M, Fichtlscherer, S, Aicher, A, Adler, K, Urbich, C, Martin, H, Zeiher, A. M, & Dimmeler, S. (2001). Number and migratory activity of circulating endothelial pro‐ genitor cells inversely correlate with risk factors for coronary artery disease. Circ Res

[29] Buckingham, M, & Montarras, D. Skeletal muscle stem cells. ((2008). Curr Opin Gen‐

[30] Taylor, D. A, Atkins, B. Z, Hungspreugs, P, Jones, T. R, Reedy, M. C, Hutcheson, K. A, Glower, D. D, & Kraus, W. E. (1998). Regenerating functional myocardium: im‐ proved performance after skeletal myoblast transplantation. Nat Med , 4, 929-933. [31] Farahmand, P, Lai, T. Y, Weisel, R. D, Fazel, S, Yau, T, Menasche, P, & Li, R. K. (2008). Skeletal myoblasts preserve remote matrix architecture and global function when implanted early or late after coronary ligation into infarcted or remote myocar‐

[32] Menasche, P, Alfieri, O, Janssens, S, Mckenna, W, Reichenspurner, H, Trinquart, L, Vilquin, J. T, Marolleau, J. P, Seymour, B, Larghero, J, et al. (2008). The Myoblast Au‐

marrow-derived endothelial progenitor cells. J Clin Invest , 108, 399-405.

tion of infarcted myocardium. J Mol Cell Cardiol , 45, 530-544.

cardium: short- and long-term effects. Circulation , 112, 214-223.

mesenchymal stem cells derived from adult marrow. Nature , 418, 41-49.

in rats. J Mol Cell Cardiol. , 49, 753-761.

cardial infarction. Circulation , 109, 3154-3157.

remodeling. Pharmacol Res , 58, 148-151.

89,EE7., 1.

et Dev. , 18, 330-336.

dium. Circulation 118,SS137., 130.


[20] Jiang, Y, Jahagirdar, B. N, Reinhardt, R. L, Schwartz, R. E, Keene, C. D, Ortiz-gonza‐ lez, X. R, Reyes, M, Lenvik, T, Lund, T, Blackstad, M, et al. (2002). Pluripotency of mesenchymal stem cells derived from adult marrow. Nature , 418, 41-49.

[9] Asahara, T, Murohara, T, Sullivan, A, Silver, M, Van Der Zee, R, Li, T, Witzenbichler, B, Schatteman, G, & Isner, J. M. (1997). Isolation of putative progenitor endothelial

[10] Murry, C. E, Wiseman, R. W, Schwartz, S. M, & Hauschka, S. D. (1996). Skeletal myo‐ blast transplantation for repair of myocardial necrosis. J Clin Invest , 98, 2512-2523.

[11] Messina, E, De Angelis, L, Frati, G, Morrone, S, Chimenti, S, Fiordaliso, F, Salio, M, Battaglia, M, Latronico, M. V, Coletta, M, et al. (2004). Isolation and expansion of

[12] Rosenzweig, A. (2006). Cardiac cell therapy--mixed results from mixed cells. N Engl J

[13] Thomson, J. A, Itskovitz-eldor, J, Shapiro, S. S, Waknitz, M. A, Swiergiel, J. J, Mar‐ shall, V. S, & Jones, J. M. (1998). Embryonic stem cell lines derived from human blas‐

[14] Takahashi, K, Tanabe, K, Ohnuki, M, Narita, M, Ichisaka, T, Tomoda, K, & Yamana‐ ka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by de‐

[15] Kocher, A. A, Schuster, M. D, Szabolcs, M. J, Takuma, S, Burkhoff, D, Wang, J, Hom‐ ma, S, Edwards, N. M, & Itescu, S. (2001). Neovascularization of ischemic myocardi‐ um by human bone-marrow derived angioblasts prevents cardiomyocyte apoptosis,

[16] Masumoto, H, Matsuo, T, Yamamizu, K, Uosaki, H, Narazaki, G, Katayama, S, Mar‐ ui, A, Shimizu, T, Ikeda, T, Okano, T, et al. (2012). Pluripotent stem cell-engineered cell sheets reassembled with defined cardiovascular populations ameliorate reduc‐ tion in infarct heart function through cardiomyocyte-mediated neovascularization.

[17] Orlic, D, Kajstura, J, Chimenti, S, Jakoniuk, I, Anderson, S. M, Li, B, Pickel, J, Mckay, R, Nadal-ginard, B, Bodine, D. M, et al. (2001). Bone marrow cells regenerate infarct‐

[18] Orlic, D, Kajstura, J, Chimenti, S, Limana, F, Jakoniuk, I, Quaini, F, Nadal-ginard, B, Bodine, D. M, Leri, A, & Anversa, P. (2001). Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci USA. , 98,

[19] Murry, C. E, Soonpaa, M. H, Reinecke, H, Nakajima, H, Nakajima, H. O, Rubart, M, Pasumarthi, K. B, Virag, J. I, Bartelmez, S. H, Poppa, V, et al. (2004). Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Na‐

reduces remodeling and improves cardiac function. Nat Med , 7, 430-436.

adult cardiac stem cells from human and murine heart. Circ Res , 95, 911-921.

cells for angiogenesis. Science , 275, 964-967.

Med , 355, 1274-1277.

470 Pluripotent Stem Cells

tocysts. Science , 282, 1145-1147.

fined factors. Cell , 131, 861-872.

Stem Cells , 30, 1196-1205.

10344-10349.

ture , 428, 664-668.

ed myocardium. Nature , 410, 701-705.


tologous Grafting in Ischemic Cardiomyopathy (MAGIC) trial: first randomized pla‐ cebo-controlled study of myoblast transplantation. Circulation , 117, 1189-1200.

CARE-AMI): final 5-year results suggest longterm safety and efficacy. Clin Res Car‐

Pluripotent Stem Cells for Cardiac Cell Therapy: The Application of Cell Sheet Technology

http://dx.doi.org/10.5772/56326

473

[43] Meyer, G. P, Wollert, K. C, Lotz, J, Steffens, J, Lippolt, P, Fichtner, S, Hecker, H, Schaefer, A, Arseniev, L, Hertenstein, B, et al. (2006). Intracoronary bone marrow cell transfer after myocardial infarction: eighteen months' follow-up data from the randomized, controlled BOOST (BOne marrOw transfer to enhance ST-elevation in‐

[44] Assmus, B, Rolf, A, & Erbs, S. Elsa°sser A, Haberbosch W, Hambrecht R, Tillmanns H, Yu J, Corti R, Mathey DG et al. ((2010). Clinical outcome 2 years after intracorona‐ ry administration of bone marrow-derived progenitor cells in acute myocardial in‐

[45] Traverse, J. H, Henry, T. D, Ellis, S. G, Pepine, C. J, Willerson, J. T, Zhao, D. X, Forder, J. R, Byrne, B. J, Hatzopoulos, A. K, Penn, M. S, et al. (2011). Effect of intracoronary delivery of autologous bone marrow mononuclear cells 2 to 3 weeks following acute myocardial infarction on left ventricular function: the LateTIME randomized trial.

[46] Tendera, M, Wojakowski, W, Ruzyllo, W, Chojnowska, L, Kepka, C, Tracz, W, Musi‐ alek, P, Piwowarska, W, Nessler, J, Buszman, P, et al. (2009). Intracoronary infusion of bone marrowderived selected CD34+CXCR4+ cells and non-selected mononuclear cells in patients with acute STEMI and reduced left ventricular ejection fraction: re‐ sults of randomized, multicenter Myocardial Regeneration by Intracoronary Infusion of Selected Population of Stem Cells in Acute Myocardial Infarction (REGENT) Trial.

[47] Dib, N, Dinsmore, J, Lababidi, Z, White, B, Moravec, S, Campbell, A, Rosenbaum, A, Seyedmadani, K, Jaber, W. A, Rizenhour, C. S, et al. (2009). One-year follow-up of feasibility and safety of the first US, randomized, controlled study using 3-dimen‐ sional guided catheter-based delivery of autologous skeletal myoblasts for ischemic

[48] Makkar, R. R, Smith, R. R, Cheng, K, Malliaras, K, Thomson, L. E, Berman, D, Czer, L. S, Marbán, L, Mendizabal, A, Johnston, P. V, et al. (2012). Intracoronary cardio‐ sphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS):

[49] Bolli, R, Chugh, A. R, Amario, D, Loughran, D, Stoddard, J. H, Ikram, M. F, Beache, S, Wagner, G. M, Leri, S. G, & Hosoda, A. T et al. ((2011). Cardiac stem cells in pa‐ tients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase

[50] Takahashi, K, & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell , 126, 663-676.

cardiomyopathy (CAuSMIC study). JACC Cardiovasc Interv , 2, 9-16.

a prospective, randomised phase 1 trial. Lancet , 379, 895-904.

farct regeneration) trial. Circulation , 113, 1287-1294.

farction. Circ Heart Fail , 3, 89-96.

JAMA , 306, 2110-2119.

Eur Heart J , 30, 1313-1321.

1 trial. Lancet , 378, 1847-1857.

diol , 100, 925-934.


CARE-AMI): final 5-year results suggest longterm safety and efficacy. Clin Res Car‐ diol , 100, 925-934.

[43] Meyer, G. P, Wollert, K. C, Lotz, J, Steffens, J, Lippolt, P, Fichtner, S, Hecker, H, Schaefer, A, Arseniev, L, Hertenstein, B, et al. (2006). Intracoronary bone marrow cell transfer after myocardial infarction: eighteen months' follow-up data from the randomized, controlled BOOST (BOne marrOw transfer to enhance ST-elevation in‐ farct regeneration) trial. Circulation , 113, 1287-1294.

tologous Grafting in Ischemic Cardiomyopathy (MAGIC) trial: first randomized pla‐ cebo-controlled study of myoblast transplantation. Circulation , 117, 1189-1200.

[33] Suzuki, K, Murtuza, B, Beauchamp, J. R, Smolenski, R. T, Varela-carver, A, Fukushi‐ ma, S, Coppen, S. R, Partridge, T. A, & Yacoub, M. H. (2004). Dynamics and media‐ tors of acute graft attrition after myoblast transplantation to the heart. FASEB J , 18,

[34] Anversa, P, Palackal, T, Sonnenblick, E. H, Olivetti, G, & Capasso, J. M. (1990). Hy‐ pertensive cardiomyopathy. Myocyte nuclei hyperplasia in the mammalian rat heart.

[35] Beltrami, A. P, Urbanek, K, Kajstura, J, Yan, S. M, Finato, N, Bussani, R, Nadal-gi‐ nard, B, Silvestri, F, Leri, A, Beltrami, C. A, et al. (2001). Evidence that human cardiac

[36] Bergmann, O, Bhardwaj, R. D, Bernard, S, & Zdunek, S. Barnabe´-Heider F, Walsh S, Zupicich J, Alkass K, Buchholz BA, Druid H et al. ((2009). Evidence for cardiomyo‐

[37] Oyama, T, Nagai, T, Wada, H, Naito, A. T, Matsuura, K, Iwanaga, K, Takahashi, T, Goto, M, Mikami, Y, Yasuda, N, et al. (2007). Cardiac side population cells have a po‐ tential to migrate and differentiate into cardiomyocytes in vitro and in vivo. J Cell Bi‐

[38] Beltrami, A. P, Barlucchi, L, Torella, D, Baker, M, Limana, F, Chimenti, S, Kasahara, H, Rota, M, Musso, E, Urbanek, K, et al. (2003). Adult cardiac stem cells are multipo‐

[39] Oh, H, Bradfute, S. B, Gallardo, T. D, Nakamura, T, Gaussin, V, Mishina, Y, Pocius, J, Michael, L. H, Behringer, R. R, Garry, D. J, et al. (2003). Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl

[40] Takehara, N, Tsutsumi, Y, Tateishi, K, Ogata, T, Tanaka, H, Ueyama, T, Takahashi, T, Takamatsu, T, Fukushima, M, Komeda, M, et al. (2008). Controlled delivery of basic fibroblast growth factor promotes human cardiosphere-derived cell engraftment to enhance cardiac repair for chronic myocardial infarction. J Am Coll Cardiol , 52,

[41] Torella, D, & Ellison, G. M. Me´ndez-Ferrer S, Ibanez B and Nadal-Ginard B. ((2006). Resident human cardiac stem cells: role in cardiac cellular homeostasis and potential

[42] Leistner, D. M, Fischer-rasokat, U, Honold, J, & Seeger, F. H. Scha°chinger V, Leh‐ mann R, Martin H, Burck I, Urbich C, Dimmeler S et al. ((2011). Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOP‐

for myocardial regeneration. Nat Clin Pract Cardiovasc Med 3,SS13., 8.

tent and support myocardial regeneration. Cell , 114, 763-776.

myocytes divide after myocardial infarction. N Engl J Med , 344, 1750-1757.

1153-1155.

472 Pluripotent Stem Cells

J Clin Invest , 85, 994-997.

ol , 176, 329-341.

1858-65.

Acad Sci USA , 100, 12313-12318.

cyte renewal in humans. Science , 324, 98-102.


[51] Murry, C. E, & Keller, G. (2008). Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell , 132, 661-680.

[62] Pelacho, B, Mazo, M, Gavira, J. J, & Prósper, F. (2011). Adult stem cells: from new cell

Pluripotent Stem Cells for Cardiac Cell Therapy: The Application of Cell Sheet Technology

http://dx.doi.org/10.5772/56326

475

[63] Müller-ehmsen, J, Whittaker, P, Kloner, R. A, Dow, J. S, Sakoda, T, Long, T. I, Laird, P. W, & Kedes, L. (2002). Survival and development of neonatal rat cardiomyocytes

[64] Teng, C. J, Luo, J, Chiu, R. C, & Shum-tim, D. (2006). Massive mechanical loss of mi‐ crospheres with direct intramyocardial injection in the beating heart: implications for

[65] Masumoto, H, & Yamashita, J. K. Strategies in cell therapy for cardiac regeneration.

[66] Cortes-morichetti, M, Frati, G, & Schussler, O. Duong Van Huyen JP, Lauret E and Genovese JA. ((2007). Association between a cell-seeded collagen matrix and cellular cardiomyoplasty for myocardial support and regeneration. Tissue Eng , 13,

[67] Okano, T, Yamada, N, Sakai, H, & Sakurai, Y. (1993). A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly (N-isopro‐

[68] Furuta, A, Miyoshi, S, Itabashi, Y, Shimizu, T, Kira, S, Hayakawa, K, Nishiyama, N, Tanimoto, K, Hagiwara, Y, Satoh, T, et al. (2006). Pulsatile cardiac tissue grafts using a novel three-dimensional cell sheet manipulation technique functionally integrates

[69] Ishii, M, Shibata, R, Numaguchi, Y, Kito, T, Suzuki, H, Shimizu, K, Ito, A, Honda, H, & Murohara, T. (2011). Enhanced angiogenesis by transplantation of mesenchymal stem cell sheet created by a novel magnetic tissue engineering method. Arterioscler

[70] Sekine, H, Shimizu, T, Hobo, K, Sekiya, S, Yang, J, Yamato, M, Kurosawa, H, Kobaya‐ shi, E, & Okano, T. (2008). Endothelial cell coculture within tissue-engineered cardio‐ myocyte sheets enhances neovascularization and improves cardiac function of

[71] Stevens, K. R, Kreutziger, K. L, Dupras, S. K, Korte, F. S, Regnier, M, Muskheli, V, Nourse, M. B, Bendixen, K, Reinecke, H, & Murry, C. E. (2009). Physiological func‐ tion and transplantation of scaffold-free and vascularized human cardiac muscle tis‐

sources to changes in methodology. J Cardiovasc Transl Res , 4, 154-160.

transplanted into adult myocardium. J Mol Cell Cardiol , 34, 107-116.

cellular cardiomyoplasty. J Thorac Cardiovasc Surg , 132, 628-632.

Inflammation and Regeneration. In press.

pylacrylamide). J Biomed Mater Res , 27, 1243-1251.

with the host heart, in vivo. Circ Res , 98, 705-712.

Thromb Vasc Biol , 31, 2210-2215.

ischemic hearts. Circulation 118,SS152., 145.

sue. Proc Natl Acad Sci USA , 106, 16568-16573.

2681-2687.


[62] Pelacho, B, Mazo, M, Gavira, J. J, & Prósper, F. (2011). Adult stem cells: from new cell sources to changes in methodology. J Cardiovasc Transl Res , 4, 154-160.

[51] Murry, C. E, & Keller, G. (2008). Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell , 132, 661-680.

[52] Yamashita, J, Itoh, H, Hirashima, M, Ogawa, M, Nishikawa, S, Yurugi, T, Naito, M, Nakao, K, & Nishikawa, S. (2000). Flk1-positive cells derived from embryonic stem

[53] Yamashita, J. K, Takano, M, Hiraoka-kanie, M, Shimazu, C, Yan, P, Yanagi, K, Naka‐ no, A, Inoue, E, Kita, F, & Nishikawa, S. (2005). Prospective identification of cardiac progenitors by a novel single cell-based cardiomyocyte induction. FASEB J , 19,

[54] Laflamme, M. A, Chen, K. Y, Naumova, A. V, Muskheli, V, Fugate, J. A, Dupras, S. K, Reinecke, H, Xu, C, Hassanipour, M, Police, S, et al. (2007). Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarct‐

[55] Sone, M, Itoh, H, Yamahara, K, Yamashita, J. K, Yurugi-kobayashi, T, Nonoguchi, A, Suzuki, Y, Chao, T. H, Sawada, N, Fukunaga, Y, et al. (2007). Pathway for differentia‐ tion of human embryonic stem cells to vascular cell components and their potential

[56] Van Laake, L. W, Passier, R, Monshouwer-kloots, J, Verkleij, A. J, Lips, D. J, & Freund, C. den Ouden K, Ward-van Oostwaard D, Korving J, Tertoolen LG et al. ((2007). Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction. Stem

[57] Narazaki, G, Uosaki, H, Teranishi, M, Okita, K, Kim, B, Matsuoka, S, Yamanaka, S, & Yamashita, J. K. (2008). Directed and systematic differentiation of cardiovascular cells

[58] Uosaki, H, Fukushima, H, Takeuchi, A, Matsuoka, S, Nakatsuji, N, Yamanaka, S, & Yamashita, J. K. (2011). Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression.

[59] Blum, B, & Benvenisty, N. (2008). The tumorigenicity of human embryonic stem cells.

[60] Okita, K, Matsumura, Y, Sato, Y, Okada, A, Morizane, A, Okamoto, S, Hong, H, Na‐ kagawa, M, Tanabe, K, Tezuka, K, et al. (2011). A more efficient method to generate

[61] Hiratsuka, M, Uno, N, Ueda, K, Kurosaki, H, Imaoka, N, Kazuki, K, Ueno, E, Aka‐ kura, Y, Katoh, M, Osaki, M, et al. (2011). Integration-free iPS cells engineered using

integration-free human iPS cells. Nat Method , 8, 409-412.

human artificial chromosome vectors. PLoS One 6,e25961.

from mouse induced pluripotent stem cells. Circulation , 118, 498-506.

for vascular regeneration. Arterioscler Thromb Vasc Biol , 27, 2127-2134.

cells serve as vascular progenitors. Nature , 408, 92-96.

ed rat hearts. Nat Biotechnol , 25, 1015-1024.

1534-1536.

474 Pluripotent Stem Cells

Cell Res. , 1, 9-24.

PLoS One 6,e23657.

Adv Cancer Res , 100, 133-158.


**Chapter 22**

**Human Pluripotent Stem Cells**

**Modeling Neurodegenerative Diseases**

Roxana Nat, Andreas Eigentler and Georg Dechant

Modeling of human neurodegenerative diseases in animals has led to important advances in the understanding of pathogenic mechanisms and has opened avenues for curative ap‐ proaches. However, inherent genetic, developmental and anatomical species differences between humans and animals frequently resulted in imperfect phenotypic correlations between animal models and human diseases. This might account for the observed hampered translation of promising preclinical treatment studies in animal models towards clinics.

Pluripotent stem (PS) cells hold considerable promise as a novel tool for modeling human diseases. Human PS cells include human embryonic stem (hES) cells and induced PS (IPS) cells. IPS cells are generated *via* reprogramming of somatic cells through the forced expression of key transcription factors and share salient characteristics of ES cells, which are derived from

Both types of PS cells show the capacity to self-renew and to differentiate *in vitro* and *in vivo* into the cell types that make up the human body. This includes the various types of mature neurons affected by neurodegenerative diseases. The combination of the key advantages of PS cells allows for the first time to generate large numbers of postmitotic human neurons for preclinical research in cell culture. In particular, the IPS cell technology opens doors for intensified research on human PS-derived neurons because, in comparison to hES cells, ethical concerns can be dispelled. Furthermore, the isolation of patient-derived IPS cell lines from skin biopsies enables the study of pathogenic mechanisms in human cells carrying relevant

During recent years the generation of IPS cell lines from human material has become routine. However, for neurological research a remaining major challenge is to guide *in vitro* differen‐

> © 2013 Nat et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Nat et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55772

the preimplantation blastocyst.

pathogenic allelic constellations.

**1. Introduction**

## **Chapter 22**

## **Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases**

Roxana Nat, Andreas Eigentler and Georg Dechant

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55772

## **1. Introduction**

Modeling of human neurodegenerative diseases in animals has led to important advances in the understanding of pathogenic mechanisms and has opened avenues for curative ap‐ proaches. However, inherent genetic, developmental and anatomical species differences between humans and animals frequently resulted in imperfect phenotypic correlations between animal models and human diseases. This might account for the observed hampered translation of promising preclinical treatment studies in animal models towards clinics.

Pluripotent stem (PS) cells hold considerable promise as a novel tool for modeling human diseases. Human PS cells include human embryonic stem (hES) cells and induced PS (IPS) cells. IPS cells are generated *via* reprogramming of somatic cells through the forced expression of key transcription factors and share salient characteristics of ES cells, which are derived from the preimplantation blastocyst.

Both types of PS cells show the capacity to self-renew and to differentiate *in vitro* and *in vivo* into the cell types that make up the human body. This includes the various types of mature neurons affected by neurodegenerative diseases. The combination of the key advantages of PS cells allows for the first time to generate large numbers of postmitotic human neurons for preclinical research in cell culture. In particular, the IPS cell technology opens doors for intensified research on human PS-derived neurons because, in comparison to hES cells, ethical concerns can be dispelled. Furthermore, the isolation of patient-derived IPS cell lines from skin biopsies enables the study of pathogenic mechanisms in human cells carrying relevant pathogenic allelic constellations.

During recent years the generation of IPS cell lines from human material has become routine. However, for neurological research a remaining major challenge is to guide *in vitro* differen‐

© 2013 Nat et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Nat et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

tiation of IPS cells into defined and homogeneous neuronal populations that are required for modeling neurodegenerative diseases. A hallmark of human neurodegenerative diseases is the chronic and progressive loss of specific types of neurons: cerebral cortex glutamatergic and basal forebrain cholinergic neurons in Alzheimer's disease, midbrain dopaminergic neurons in Parkinson's disease, striatal GABAergic neurons in Huntington's disease, motor neurons in amyotrophic lateral sclerosis and spinal muscular atrophy, cerebellar and peripheral sensory neurons in ataxias and others. To fully tap into the potential of the IPS technology and to progress towards a fundamental understanding of the causes of disease selectivity in the loss of neuron subtypes it will be necessary to establish reproducible and tailored protocols for differentiation of IPS cells specifically into these neuronal subtypes *in vitro.*

factors have been developed, ranging in number between two to six [3;4;9]. Each of these

Skin biopsy Fibroblasts IPS cells

Reprogramming factors

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases

http://dx.doi.org/10.5772/55772

479

Genetic material coding for these reprogramming factors has been introduced into cells via a variety of methods, comprising genome integrating as well as non-integrating techniques [10]. The most commonly used method for factor delivery is the transduction using retroviruses, originally with Moloney murine leukemia virus (MMLV), vectors, later on with modified lentiviral vectors. The efficiency of IPS cell generation using sets of four MMLV-derived retroviruses expressing single genes from the OSKM set separately is ~0.01% in human

Silencing of the permanently integrated transgenes is important because only an IPS cell that has up regulated the endogenous pluripotency gene network but down regulated the expres‐ sion of the transgenes can be considered fully reprogrammed [11]. Although the use of retroviruses is efficient and yields reproducible results, random insertional mutagenesis, permanent alteration of gene expression as well as reactivation of silenced transgenes during differentiation cannot be excluded. The use of Cre-deletable or dox-inducible lentiviruses has overcome some of these problems and allows factor expression in a more controlled manner [12;13]. Other attempts to generate integration-free IPS cells focused on replication-defective adenoviral vectors, or Sendai viral vectors [14;15] which efficiently deliver foreign genes into

To avoid the use of viral vectors, direct delivery of episomal vectors (plasmids) as well as standard DNA transfections using liposomes or electroporation have also been used, but with low transfection efficiency [16-18]. A polycistronic expression cassette flanked by loxP sites enabled the excision of the reprogramming cassette after expressing Cre recombinase also in

Alternatively, Warren et al. [20] developed a novel mRNAs-based system and achieved an efficient conversion of different human somatic donor cells into IPS cells using a direct delivery of high dosages of modified mRNAs encoding OSKM and Lin28 packaged in a cationic vehicle. The efficiency reached with this approach was much higher when compared with other non-

Recently, a potential role of specific microRNAs (miRNAs) for pluripotency has been eluci‐ dated. The miRNAs from the miR-302 cluster contribute to unique ES cells features such as cell cycle and pluripotency maintenance [21;22]. Based on these findings protocols for highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency were reported [23;24]. The resulting miR-IPS cells are subject to a reduced risk of mutations 3

reprogramming factors contributes to the kinetics and efficiency of IPS induction.

Skin biopsie

**Figure 1.** Generation of human IPS cells from a skin biopsy.

fibroblasts.

a multitude of cell types.

the non-viral system [19].

integrative protocols [20].

To date, reprogramming of patient somatic cells into IPS cell-based models has been achieved for several neurodegenerative diseases. The results show that IPS cells or their derivatives can display at least some of the cellular and/or molecular characteristics of the respective diseases. These findings provide first proof for etiological validity of these models. Here, we review the existing reports demonstrating the generation of human PS cell-based models for neurodege‐ nerative diseases, including also the studies showing the differentiation of human PS cells, both ES and IPS cells, toward telencephalic neurons (glutamatergic, GABAergic and choliner‐ gic), midbrain dopaminergic neurons, cerebellar neurons, spinal motor neurons and periph‐ eral neurons. We further discuss the perspectives of these cellular models.

## **2. Generation of human IPS cells**

It was in 2006 when the first IPS cells were generated by Takahashi and Yamanaka *via* reprogramming of mouse somatic fibroblasts through retroviral transduction with a specific set of factors [1]. A screen of pluripotency-associated genes yielded a successful combination of transcription factors, comprising Oct4, Sox2, Klf4 and c-Myc (OSKM), which are commonly referred to as the 'Yamanaka factors'. Shortly afterwards, the same group [2], concurrently with other groups that used different combinations of transcription factors, for example substituting c-Myc and Klf4 by Lin28 or Nanog [3-5], were able to demonstrate that also fibroblasts obtained from adult human beings can be induced to undergo the transformation into PS cells.

Since these first descriptions of IPS cell derivation significant improvements in efficiency of the protocols, in the quality of the resulting IPS lines and in the depth of their analysis have been achieved. So far, fibroblasts remain the most popular donor cell type, and were used in more than 80% of all published reprogramming experiments. Figure 1 illustrates the steps in generating human IPS cell from skin fibroblasts, as well as cell morphology transition in culture.

However, other cell sources for inducing pluripotency have been used, amongst them keratinocytes [6], cord blood cells [7] and mesenchymal stem cells [8] with sometimes higher efficiency compared to fibroblasts. Furthermore, different combinations of reprogramming

**Figure 1.** Generation of human IPS cells from a skin biopsy.

tiation of IPS cells into defined and homogeneous neuronal populations that are required for modeling neurodegenerative diseases. A hallmark of human neurodegenerative diseases is the chronic and progressive loss of specific types of neurons: cerebral cortex glutamatergic and basal forebrain cholinergic neurons in Alzheimer's disease, midbrain dopaminergic neurons in Parkinson's disease, striatal GABAergic neurons in Huntington's disease, motor neurons in amyotrophic lateral sclerosis and spinal muscular atrophy, cerebellar and peripheral sensory neurons in ataxias and others. To fully tap into the potential of the IPS technology and to progress towards a fundamental understanding of the causes of disease selectivity in the loss of neuron subtypes it will be necessary to establish reproducible and tailored protocols for

To date, reprogramming of patient somatic cells into IPS cell-based models has been achieved for several neurodegenerative diseases. The results show that IPS cells or their derivatives can display at least some of the cellular and/or molecular characteristics of the respective diseases. These findings provide first proof for etiological validity of these models. Here, we review the existing reports demonstrating the generation of human PS cell-based models for neurodege‐ nerative diseases, including also the studies showing the differentiation of human PS cells, both ES and IPS cells, toward telencephalic neurons (glutamatergic, GABAergic and choliner‐ gic), midbrain dopaminergic neurons, cerebellar neurons, spinal motor neurons and periph‐

It was in 2006 when the first IPS cells were generated by Takahashi and Yamanaka *via* reprogramming of mouse somatic fibroblasts through retroviral transduction with a specific set of factors [1]. A screen of pluripotency-associated genes yielded a successful combination of transcription factors, comprising Oct4, Sox2, Klf4 and c-Myc (OSKM), which are commonly referred to as the 'Yamanaka factors'. Shortly afterwards, the same group [2], concurrently with other groups that used different combinations of transcription factors, for example substituting c-Myc and Klf4 by Lin28 or Nanog [3-5], were able to demonstrate that also fibroblasts obtained from adult human beings can be induced to undergo the transformation

Since these first descriptions of IPS cell derivation significant improvements in efficiency of the protocols, in the quality of the resulting IPS lines and in the depth of their analysis have been achieved. So far, fibroblasts remain the most popular donor cell type, and were used in more than 80% of all published reprogramming experiments. Figure 1 illustrates the steps in generating human IPS cell from skin fibroblasts, as well as cell morphology transition in

However, other cell sources for inducing pluripotency have been used, amongst them keratinocytes [6], cord blood cells [7] and mesenchymal stem cells [8] with sometimes higher efficiency compared to fibroblasts. Furthermore, different combinations of reprogramming

differentiation of IPS cells specifically into these neuronal subtypes *in vitro.*

eral neurons. We further discuss the perspectives of these cellular models.

**2. Generation of human IPS cells**

into PS cells.

478 Pluripotent Stem Cells

culture.

factors have been developed, ranging in number between two to six [3;4;9]. Each of these reprogramming factors contributes to the kinetics and efficiency of IPS induction.

Genetic material coding for these reprogramming factors has been introduced into cells via a variety of methods, comprising genome integrating as well as non-integrating techniques [10]. The most commonly used method for factor delivery is the transduction using retroviruses, originally with Moloney murine leukemia virus (MMLV), vectors, later on with modified lentiviral vectors. The efficiency of IPS cell generation using sets of four MMLV-derived retroviruses expressing single genes from the OSKM set separately is ~0.01% in human fibroblasts.

Silencing of the permanently integrated transgenes is important because only an IPS cell that has up regulated the endogenous pluripotency gene network but down regulated the expres‐ sion of the transgenes can be considered fully reprogrammed [11]. Although the use of retroviruses is efficient and yields reproducible results, random insertional mutagenesis, permanent alteration of gene expression as well as reactivation of silenced transgenes during differentiation cannot be excluded. The use of Cre-deletable or dox-inducible lentiviruses has overcome some of these problems and allows factor expression in a more controlled manner [12;13]. Other attempts to generate integration-free IPS cells focused on replication-defective adenoviral vectors, or Sendai viral vectors [14;15] which efficiently deliver foreign genes into a multitude of cell types.

To avoid the use of viral vectors, direct delivery of episomal vectors (plasmids) as well as standard DNA transfections using liposomes or electroporation have also been used, but with low transfection efficiency [16-18]. A polycistronic expression cassette flanked by loxP sites enabled the excision of the reprogramming cassette after expressing Cre recombinase also in the non-viral system [19].

Alternatively, Warren et al. [20] developed a novel mRNAs-based system and achieved an efficient conversion of different human somatic donor cells into IPS cells using a direct delivery of high dosages of modified mRNAs encoding OSKM and Lin28 packaged in a cationic vehicle. The efficiency reached with this approach was much higher when compared with other nonintegrative protocols [20].

Recently, a potential role of specific microRNAs (miRNAs) for pluripotency has been eluci‐ dated. The miRNAs from the miR-302 cluster contribute to unique ES cells features such as cell cycle and pluripotency maintenance [21;22]. Based on these findings protocols for highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency were reported [23;24]. The resulting miR-IPS cells are subject to a reduced risk of mutations 3 and tumorigenesis relative to most other protocols because mature miRNAs function without genomic integration [23;24].

Finally, another promising possibility of inducing pluripotency is to deliver the reprogram‐ ming factors directly as proteins. To this end Zhou et al. generated recombinant OSKM proteins fused with a poly-arginine transduction domain [25]. However, this protein-based strategy induced pluripotency with extremely slow kinetics and poor efficiencies [25].

Apart from the delivery methods of reprogramming factors, other parameters, including culture conditions and the application of small pharmacological compounds, exert an influ‐ ence on reprogramming efficiency. For example, it has been demonstrated that culturing IPS cells under hypoxic conditions mimicking the *in vivo* environment, enhances the efficiency rate [26]. The addition of small molecules, that either modifies epigenetic states like DNA methyl‐ ation or histone acetylation, or influences specific receptor mediated signaling pathways, enhances the generation of IPS cells [27-31].

Eventually, the reactivation of endogenous pluripotency genes leads to establishment of cell lines with pluripotent characteristics. However, even though IPS lines share many character‐ istics with hES cells with regard to morphology and pluripotent gene expression, further research is required to establish more precisely communalities and differences between hES and IPS cells. Differences in epigenetic status and *in vitro* and *in vivo* differentiation potential have been reported [32-34].

**Differentiated neural**

General telencephalic

Cortical pyramidal neurons mES cells

Cortical interneurons mES cells

Basal forebrain cholinergic

Striatal medium spiny

Midbrain dopaminergic

neurons

neurons

neurons

hES cells

**Figure 2.** Stem cell fates aligned to nervous system development

hES cells

mES cells

hES cells, hIPS cells

**PS cell type Key patterning**

Neural Induction Neural Patterning and Specification

**differentiation factors**

mES cells DKK, LeftyA, Wnt3a, Shh Watanabe et al. (2005)[43],

hES cells RA, bFGF, FGF8, Shh, BMP9 Wicklund et al (2010)[54],

valproic acid

mES cells, hES cells Shh, BDNF, DKK1, cAMP,

Floor plate cells hES cells Shh, dual SMAD inhibition Fasano et al. (2010)[58]

**References**

Cyclopamine, Fgf2, RA Eiraku et al. (2008)[45],

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases

http://dx.doi.org/10.5772/55772

481

Shh, Fgf2, IGF, Activin Maroof et al. (2010)[49],

Shh, AA, FGF8, bFGF Kawasaki et al. (2000)[59],

Li et al. (2009)[44]

Gaspard et al. (2008)[46], Gaspard et al. (2009)[47], Ideguchi et al. (2010)[48], Nat et al 2012[36]

Danjo et al. (2011)[50], Goulburn et al. (2011, 2012) [51;52], Cambray et al. (2012)[53], Nat et al

Bissonnette et al. (2011)[55]

Lee et al. (2000)[60], Perrier et al. (2004)[61], Yan et al. (2005)[62], Chambers et al. (2009)[63], Sánchez-Danés et

al. (2012)[64]

Aubry et al. (2008)[56], Zhang et al. (2010)[57], Danjo et al. (2011)[50]

2012[36]

**subtype**

neurons

## **3. Neuronal differentiation of human PS cells**

The *in vitro* production of neurons from PS cells, following similar mechanism as in vivo development, involves several sequential steps precisely orchestrated by signaling events (reviewed in [35;36]).

*In vivo*, during embryonic development, the initial step is neural induction, the specification of neuroepithelia from ectoderm cells [37]. When the neuroectodermal fate is determined, the neural plate folds to form the neural tube, from which cells differentiate into various neurons and glia [38;39]. The neural tube is patterned along its anteroposterior (A/P) and dorsoventral (D/V) axes to establish a set of positional cues. The neural plate acquires an anterior character, and is subsequently posteriorized by exposure to Wingless/Int proteins (Wnt), fibroblast growth factors (FGF), bone morphogenic proteins (BMP) and retinoic acid (RA) signals to establish the main subdivisions of the central nervous system (CNS): forebrain, midbrain, hindbrain, and spinal cord, as well as the neural crest from which the peripheral neurons derive [40-42]. Therefore, the precursor cells in each subdivision along the A/P axis are fated to subtypes of neurons and glia depending on its exposure to unique sets of morphogens at specific concentrations (Figure 2).

As reviewed in Petros et al. [35], specific PS cell-bases protocols, following the principles of nervous system development, can generate neuronal types with markers consistent with telencephalic, midbrain, hindbrain spinal cord and peripheral neurons (Table 1).

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases http://dx.doi.org/10.5772/55772 481


and tumorigenesis relative to most other protocols because mature miRNAs function without

Finally, another promising possibility of inducing pluripotency is to deliver the reprogram‐ ming factors directly as proteins. To this end Zhou et al. generated recombinant OSKM proteins fused with a poly-arginine transduction domain [25]. However, this protein-based strategy

Apart from the delivery methods of reprogramming factors, other parameters, including culture conditions and the application of small pharmacological compounds, exert an influ‐ ence on reprogramming efficiency. For example, it has been demonstrated that culturing IPS cells under hypoxic conditions mimicking the *in vivo* environment, enhances the efficiency rate [26]. The addition of small molecules, that either modifies epigenetic states like DNA methyl‐ ation or histone acetylation, or influences specific receptor mediated signaling pathways,

Eventually, the reactivation of endogenous pluripotency genes leads to establishment of cell lines with pluripotent characteristics. However, even though IPS lines share many character‐ istics with hES cells with regard to morphology and pluripotent gene expression, further research is required to establish more precisely communalities and differences between hES and IPS cells. Differences in epigenetic status and *in vitro* and *in vivo* differentiation potential

The *in vitro* production of neurons from PS cells, following similar mechanism as in vivo development, involves several sequential steps precisely orchestrated by signaling events

*In vivo*, during embryonic development, the initial step is neural induction, the specification of neuroepithelia from ectoderm cells [37]. When the neuroectodermal fate is determined, the neural plate folds to form the neural tube, from which cells differentiate into various neurons and glia [38;39]. The neural tube is patterned along its anteroposterior (A/P) and dorsoventral (D/V) axes to establish a set of positional cues. The neural plate acquires an anterior character, and is subsequently posteriorized by exposure to Wingless/Int proteins (Wnt), fibroblast growth factors (FGF), bone morphogenic proteins (BMP) and retinoic acid (RA) signals to establish the main subdivisions of the central nervous system (CNS): forebrain, midbrain, hindbrain, and spinal cord, as well as the neural crest from which the peripheral neurons derive [40-42]. Therefore, the precursor cells in each subdivision along the A/P axis are fated to subtypes of neurons and glia depending on its exposure to unique sets of morphogens at

As reviewed in Petros et al. [35], specific PS cell-bases protocols, following the principles of nervous system development, can generate neuronal types with markers consistent with

telencephalic, midbrain, hindbrain spinal cord and peripheral neurons (Table 1).

induced pluripotency with extremely slow kinetics and poor efficiencies [25].

genomic integration [23;24].

480 Pluripotent Stem Cells

have been reported [32-34].

(reviewed in [35;36]).

specific concentrations (Figure 2).

enhances the generation of IPS cells [27-31].

**3. Neuronal differentiation of human PS cells**



**Neurodegenerative**

**Types of affected neurons**

cholinergic neurons, cortical neurons

nigro-striatal dopaminergic neurons

Alzheimer's disease Basal forebrain

Parkinson's disease Midbrain

Huntington's disease Striatal

Amyotrophic lateral

Spinal muscular atrophy, type I

Friedrich Ataxia Dorsal root

sclerosis

GABAergic medium spiny neurons, cortical neurons

Upper and lower motor neurons

Spinal motor neurons

ganglia (DRG) peripheral

**Histopathology Gene**

Neurofibrillary tangles, Amyloid plaque, Loss of neurons and synapses

Lewy-bodies, loss of dopaminergic neurons

**(Mutation)**

PS1, PS2 mutations; Sporadic and APP duplication

LRRK2 (G2019S)

PINK1 (Q456X; V170G)

SCNA triplication

HTT (CAG repeats)

SMN1 deletion

FXN (GAA expansion)

Neural inclusion bodies, loss of striatal/cortical neurons

Ubiquitinated inclusion bodies, loss of motoneurons

Loss of anterior horn cells

Reduced size of DRG-neurons, iron

**Donor cell**

SF LV: OSKLN RV:OSKM

idiopathic SF LV: Cre-excisable,

**Reprogramming method**

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases

DOX-inducible; OSK or OSKM

SF RV: OSK Increased

SF RV: OSKM Impaired stress-

SF RV: OSKM Increased neural α-

SF RV: OSKM Increase in

SOD1 SF RV: OSKM, OSK NA Dimos et al.

Episomal plasmid OSKMNL combinations

SF RV: OSKM GAA repeat

SF RV: OSNL

**Reported diseaserelated phenotype**

http://dx.doi.org/10.5772/55772

Increased amyloid β42 secretion increased β(1-40) and phospho-τ levels

caspase-3 activation and DA neuron death with various cell stress conditions

induced mitochondrial translocation of Parkin in DA neurons

Synuclein protein

lysosomal activity

Reduced number of motor neurons, decreased soma size, synaptic defects

instability

levels sensitivity to oxidative stress

NA Soldner et al.

**References**

483

Yagi et al. (2011)[83], Israel et al. (2012)[84]

(2009)[12], Hargus et al. (2010)[85]

Nguyen et al. (2011)[86]

Seibler et al. (2011)[87]

Devine et al. (2011)[88], Byers et al. (2011)[89]

Park et al. (2008)[4], Camnasio et al. (2012)[90]

(2008)[73], Boulting et al. (2011)[33]

Ebert et al. (2009)[91] Sareen et al. (2012)[92]

Ku et al. (2010)[93], Liu

**disease**

**Table 1.** Neural cell types derived from PS cells to date (modified from Petros et al. 2011[35])

Recognizing that all resulting cell populations, although enriched in specific neurons, remain heterogeneous, there is a need for additional selection methods to further purify neuronal subtype lineages. Whilst a key aim of positionally specifying human neurons is to work towards the generation of cell-based therapies for diseases that target a sub-population of cells, this system will be particularly powerful in attempting to understand disease specificity when applied to patient-derived IPS cells.

## **4. Neurodegenerative diseases and related models**

Neurodegenerative diseases are characterized by the chronic and progressive loss of neuronal functions in selected neurons. Classical neurodegenerative diseases are Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal muscular atrophy and ataxias. Other rare diseases such as familial dysautonomia and Fragile-X syn‐ drome contain neurodegenerative aspects as well.

Here we aim to present the main characteristics of these diseases, focusing on their pathogen‐ esis and its reflection into the disease models, including the recent cellular models derived via IPS cell technology. The most important publications and aspects regarding the IPS cell-related models for neurological diseases are reviewed in Han et al. 2011[82] and updated for the neurodegenerative diseases in Table 2.


**Differentiated neural**

482 Pluripotent Stem Cells

Spinal cord motor neurons mES cells,

applied to patient-derived IPS cells.

**PS cell type Key patterning**

Cerebellar granule cells mES cells, hES cells Wnt1, Fgf8, RA, BMP 6/7,

hES cells, IPS cells, hMS cells, hADS cells

Neural crest hES cells, hIPS cells SB431542, noggin, BDNF,

**4. Neurodegenerative diseases and related models**

drome contain neurodegenerative aspects as well.

neurodegenerative diseases in Table 2.

**Table 1.** Neural cell types derived from PS cells to date (modified from Petros et al. 2011[35])

**differentiation factors**

Shh, RA, SB431542, Olig2,

GDF7, Shh, JAG1

NGF, AA, dbcAMP

Cerebellar Purkinje cells mES cells BMP4, Fgf8 Su et al. (2006)[67], Tao et al.

HB9

Spinal cord interneurons mES cells Wnt3A, Shh, RA, BMP2 Murashov et al. (2005)[78]

Recognizing that all resulting cell populations, although enriched in specific neurons, remain heterogeneous, there is a need for additional selection methods to further purify neuronal subtype lineages. Whilst a key aim of positionally specifying human neurons is to work towards the generation of cell-based therapies for diseases that target a sub-population of cells, this system will be particularly powerful in attempting to understand disease specificity when

Neurodegenerative diseases are characterized by the chronic and progressive loss of neuronal functions in selected neurons. Classical neurodegenerative diseases are Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal muscular atrophy and ataxias. Other rare diseases such as familial dysautonomia and Fragile-X syn‐

Here we aim to present the main characteristics of these diseases, focusing on their pathogen‐ esis and its reflection into the disease models, including the recent cellular models derived via IPS cell technology. The most important publications and aspects regarding the IPS cell-related models for neurological diseases are reviewed in Han et al. 2011[82] and updated for the

**References**

(2010)[68]

et al. (2005)[70],

Lee et al. (2010)[79], Menendez et al. (2011)[80], Goldstein et al. (2010)[81]

Salero and Hatten (2007) [65], Erceg et al. (2010)[66]

Wichterle et al. (2002)[69], Li

Soundararajan et al. (2006) [71], Lee et al. (2007)[72], Dimos et al. (2008)[73], Peljto et al. (2010)[74], Patani et al. (2011)[75], Park et al. (2012) [76], Liqing et al. (2011)[77]

**subtype**


The majority of AD cases are sporadic; in these cases the major genetic risk factor disease is the *APOE* gene. ApoE is synthesized in astrocytes and acts as a ligand for the receptormediated endocytosis of cholesterol-containing lipoprotein particles. Whether ApoE affects Aβ clearance or operates through its function in lipid metabolism is not yet fully established

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases

http://dx.doi.org/10.5772/55772

485

Few familial AD (FAD) cases are an early-onset autosomal dominant disorder. Three genes have been identified that account for FAD: the first mutations causing Mendelian AD were identified in the *APP* gene [106], although mutations in two other genes, *presenilin 1* and *2* (*PSEN1* and *PSEN2*), that form the γ-secretase complex components, are more commonly found. The mutations cause different clinical phenotypes, but for all the aberrant processing

By classical transgene and knockout approaches, there were established mouse models that reflect different aspects of AD [108]. Representative models are APP mutant strains (such as PDAPP, J20, APP23 or Tg2576) with a robust APP/Aβ pathology and tau mutant strains with NFT formation such as (JNPL3 or pR5). The histopathology in these strains is associated with

The modeling of AD via IPS cell technology was recently reported [83;84]. The first study used AD patient fibroblasts carrying mutations in PS1 and PS2. The IPS cells kept the mutations and differentiated into neural cells, showing increased amyloid β42 secretion as compared to the

In the second study, IPS cells were generated from both patients with sporadic AD or caring APP duplication. Interestingly, increased level of both Aβ (1-40) and pTau were detected in neural cells cultures after neural progenitor expansion of about five weeks, followed by

Parkinson's Disease (PD) is the second most common neurodegenerative disorder, afflicting over 6 million people worldwide. Clinically, there are progressive motor dysfunctions

Pathologically, PD is identified by intracellular inclusions known as Lewy bodies and dopa‐

PD is largely a late onset sporadic neurodegenerative condition. However, 5–10% cases are familial, transmitted in either an autosomal-dominant or autosomal recessive fashion [110]. A number of genes have been linked to our understanding of pathogenesis. The gene αsynuclein (*SNCA)* product is the major component of the Lewy body in sporadic and in some cases of autosomal dominant types and therefore appears to be central to PD pathophysiology [111;112]. The most common mutation related to autosomal-dominant PD occurs in the gene encoding leucine-rich repeat kinase-2 (LRRK2) [113]. One missense mutation, the G2019S mutation, occurs in 5% of familial cases and 1–2% of sporadic cases of PD. Mutations in *PARK2*, *PINK1* and *PARK7* (also known as *DJ1*) cause autosomal-

comprising bradykinesia, rigidity and tremor, as well as non-motor features.

minergic neuronal loss that initiates in the substantia nigra.

[105].

of Aβ led to its aggregation [107].

behavioral impairment [109].

differentiation of about four weeks [84].

healthy controls [83].

**4.2. Parkinson's disease**

SF-skin fibroblasts, RV-retroviruses, LV-lentiviruses and NA- not assessed

**Table 2.** Overview of the iPS the cell-related models for neurodegenerative diseases (modified from Han et al. 2011 [82])

#### **4.1. Alzheimer's disease**

Alzheimer's disease (AD) is the most common neurodegenerative disease, affecting 35 million patients worldwide. Clinically, it is characterized by progressive loss of short-term memory and other cognitive functions toward a state of profound dementia.

AD is histopathologically characterized by neuronal and synapse loss and the appearance of extracellular amyloid plaques (AP) and intracellular neurofibrillary tangles (NFTs) in affected brain regions, especially cerebral cortex, hippocampus and basal forebrain [98;99]. The AP and NFTs form by aggregation of two proteins, beta amyloid (Aβ) and hyperphosphorylated tau (pTau), respectively [100]. Aβ is formed from the cleavage of the amyloid precursor protein (APP) into soluble monomers that then aggregate into fibrils and are eventually deposited in the extracellular space [101]. Tau is a microtubule-associated protein that undergoes hyper‐ phosphorylation and accumulates as intraneuronal inclusions or tangles in the brains of individuals with AD [100;102].

Degeneration of basal forebrain cholinergic neurons is a principal feature of AD and the reduction in the level of acetylcholine and choline acetyltransferase activity in the hippocam‐ pus and cerebral cortex has been reported in the brains of AD patients [103;104].

The majority of AD cases are sporadic; in these cases the major genetic risk factor disease is the *APOE* gene. ApoE is synthesized in astrocytes and acts as a ligand for the receptormediated endocytosis of cholesterol-containing lipoprotein particles. Whether ApoE affects Aβ clearance or operates through its function in lipid metabolism is not yet fully established [105].

Few familial AD (FAD) cases are an early-onset autosomal dominant disorder. Three genes have been identified that account for FAD: the first mutations causing Mendelian AD were identified in the *APP* gene [106], although mutations in two other genes, *presenilin 1* and *2* (*PSEN1* and *PSEN2*), that form the γ-secretase complex components, are more commonly found. The mutations cause different clinical phenotypes, but for all the aberrant processing of Aβ led to its aggregation [107].

By classical transgene and knockout approaches, there were established mouse models that reflect different aspects of AD [108]. Representative models are APP mutant strains (such as PDAPP, J20, APP23 or Tg2576) with a robust APP/Aβ pathology and tau mutant strains with NFT formation such as (JNPL3 or pR5). The histopathology in these strains is associated with behavioral impairment [109].

The modeling of AD via IPS cell technology was recently reported [83;84]. The first study used AD patient fibroblasts carrying mutations in PS1 and PS2. The IPS cells kept the mutations and differentiated into neural cells, showing increased amyloid β42 secretion as compared to the healthy controls [83].

In the second study, IPS cells were generated from both patients with sporadic AD or caring APP duplication. Interestingly, increased level of both Aβ (1-40) and pTau were detected in neural cells cultures after neural progenitor expansion of about five weeks, followed by differentiation of about four weeks [84].

#### **4.2. Parkinson's disease**

**Neurodegenerative**

484 Pluripotent Stem Cells

Spinocerebellar Ataxia Type 3 (Machado-Joseph Disease)

**Types of affected neurons**

neurons, cerebellar neurons

Cerebellar neurons, striatal and cortical neurons

Sensory and autonomic neurons

cerebellar neurons

SF-skin fibroblasts, RV-retroviruses, LV-lentiviruses and NA- not assessed

Fragile-X syndrome Hippocampal,

**4.1. Alzheimer's disease**

individuals with AD [100;102].

**Histopathology Gene**

misdistribution, decreased myelination

Intranuclear inclusion bodies, neuronal loss

Reduced size of DRG neurons, reduced number of nonmyelinated small fibers and intermediolateral column neurons

Dendritic spine abnormalities, neuronal loss

and other cognitive functions toward a state of profound dementia.

**(Mutation)**

ATAXIN 3(CAG expansion)

FMR1 (CGG repeat)

**Table 2.** Overview of the iPS the cell-related models for neurodegenerative diseases (modified from Han et al. 2011

Alzheimer's disease (AD) is the most common neurodegenerative disease, affecting 35 million patients worldwide. Clinically, it is characterized by progressive loss of short-term memory

AD is histopathologically characterized by neuronal and synapse loss and the appearance of extracellular amyloid plaques (AP) and intracellular neurofibrillary tangles (NFTs) in affected brain regions, especially cerebral cortex, hippocampus and basal forebrain [98;99]. The AP and NFTs form by aggregation of two proteins, beta amyloid (Aβ) and hyperphosphorylated tau (pTau), respectively [100]. Aβ is formed from the cleavage of the amyloid precursor protein (APP) into soluble monomers that then aggregate into fibrils and are eventually deposited in the extracellular space [101]. Tau is a microtubule-associated protein that undergoes hyper‐ phosphorylation and accumulates as intraneuronal inclusions or tangles in the brains of

Degeneration of basal forebrain cholinergic neurons is a principal feature of AD and the reduction in the level of acetylcholine and choline acetyltransferase activity in the hippocam‐

pus and cerebral cortex has been reported in the brains of AD patients [103;104].

**Donor cell**

**Reprogramming method**

IKBKAP SF RV: OSKM Defects in

**Reported diseaserelated phenotype**

neurogenesis and migration

SF RV: OSKM NA Koch et al.

SF RV: OSKM NA Urbach et al.

**References**

et al. (2010) [94]

(2011)[95]

Lee et al. (2009)[96]

(2010)[97]

**disease**

Familial dysautonomia

[82])

Parkinson's Disease (PD) is the second most common neurodegenerative disorder, afflicting over 6 million people worldwide. Clinically, there are progressive motor dysfunctions comprising bradykinesia, rigidity and tremor, as well as non-motor features.

Pathologically, PD is identified by intracellular inclusions known as Lewy bodies and dopa‐ minergic neuronal loss that initiates in the substantia nigra.

PD is largely a late onset sporadic neurodegenerative condition. However, 5–10% cases are familial, transmitted in either an autosomal-dominant or autosomal recessive fashion [110]. A number of genes have been linked to our understanding of pathogenesis. The gene αsynuclein (*SNCA)* product is the major component of the Lewy body in sporadic and in some cases of autosomal dominant types and therefore appears to be central to PD pathophysiology [111;112]. The most common mutation related to autosomal-dominant PD occurs in the gene encoding leucine-rich repeat kinase-2 (LRRK2) [113]. One missense mutation, the G2019S mutation, occurs in 5% of familial cases and 1–2% of sporadic cases of PD. Mutations in *PARK2*, *PINK1* and *PARK7* (also known as *DJ1*) cause autosomalrecessive, early onset PD [114-116]. These genetic discoveries have highlighted the impor‐ tance of the ubiquitin proteasome system, mitochondrial dysfunction and oxidative stress in PD pathogenesis.

Byers *et al*. focused on the differences in sensitivity to oxidative stress in correlation with this

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases

http://dx.doi.org/10.5772/55772

487

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder resulting from an expanded CAG triplet repeat in the Huntingtin gene (*HTT*) on chromosome 4 [118]. This expansion accounts for an attachment of a polyglutamine strand of variable length at the N-terminus of the protein leading to a toxic gain of function [119]. HD together with eight other CAG triplet repeat expansion disorders forms the group of PolyQ diseases which share

Although the protein huntingtin is ubiquitously expressed in mammalian cells, mainly striatal GABAergic medium spiny neurons with a dopamine- and cyclic AMP-regulated phospho‐ protein (DARPP-32)-positive phenotype are the most susceptible to neurodegeneration in HD [121]. As a consequence a prominent cell loss and atrophy in the caudate nucleus and putamen can be observed. Other brain regions and neuronal subtypes involved in HD comprise the substantia nigra, hippocampus, cerebellar Purkinje cells and thalamic nuclei [119;122].

One of the histopathological hallmarks of Huntington's disease, as in other PolyQ disorders too, is the appearance of nuclear and cytoplasmic inclusion bodies containing the mutant huntingtin and polyglutamine [123;124]. Much debate regarding the meaning and function of these inclusions is going on, and although indicative of pathological mutant protein processing they do not correlate with cellular dysfunction and might even confer a protective role

Numerous studies indicated that wild-type huntingtin might be involved in a variety of intracellular functions such as in protein trafficking, vesicle and axonal transport, mitochon‐ drial function, postsynaptic signaling; transcriptional regulation, as wells as in anti-apoptotic pathways [127;128]. Therefore a disruption and detrimental impairment of these various intracellular pathways is supposed to be the consequence of accumulation of mutant hun‐

Over the years, several different HD models had been introduced, ranging from invertebrate models like Drosophila and C. elegans to various rodent models [129;130]. Genetically modified animals (especially mouse) models such as transgenic, knock-in and conditional ones

The intrastriatal injection of excitotoxic glutamic acid analogues like kainic acid, quinolinic acid and 3-nitropropionic acid into animals resulted in neuronal cell death similar to the pathology observed in HD patients [132-134]. They proved to be useful in studying pathoge‐ netic processes involved in the progressive disease course although some limitations regarding the selective neuronal cell loss as well as aggregate formation properties and variable pheno‐

Transplantation studies in animal HD models aimed at providing neuroprotective support or intended to replace damaged and lost neuronal subtypes. Successful application of stem cell-

recapitulated some features of HD like neuronal polyglutamine inclusions [131].

mutation [89].

[125;126].

**4.3. Huntington's disease**

some specific pathophysiological features [120].

tingtin, finally leading to neuronal death.

types have to be kept in mind.

The most common genetic risk factor for PD appears to be heterozygous mutations in the *glucocerebrosidase* gene (*GBA*) [117]. The frequency of heterozygous mutations in *GBA* reaches ~4% in sporadic PD populations.

Because PD results from the loss of dopaminergic neurons, the prospect of utilizing cell replacement therapies has attracted substantial interest. Several methods are able to improve the effectiveness of midbrain dopamine neuron generation and/or retrieval from fetal tissue and stem cells.

The ability of deriving large quantities of correctly differentiated dopamine neurons makes stem cells promising cell sources for transplantation in PD; having the transplantation as a main goal, many studies improved the directed differentiation of PS cells toward dopaminer‐ gic neurons, opening the doors to IPS cell-derived models.

Soldner *et al.* induced pluripotency in fibroblasts derived from idiopathic PD patients and controls and subsequently differentiated both into dopaminergic neurons. As they did not find significant differences between the expression of *SNCA* or *LRKK2* between patients and controls*,* they went on to suggest that it might still be necessary to further accelerate PDpathology related phenotypes *in vitro* with neurotoxins such as MPTP, or the overexpression of PD-related genes such as *SNCA* or *LRKK2* in order to obtain a valid PD model [12]*.*

Hargus et al. [85] used a similar protocol of inducing PS cells for idiopathic PD patients and controls, and further differentiated them into dopaminergic neurons. Additionally, they performed intrastriatal transplantation studies into 6-OHDA lesioned rats, demonstrating improvements in motor symptoms.

Regarding familiar PD, Nguyen *et al.* [86] used a classical protocol for IPS cells generation and differentiation and found that IPS cell-derived dopaminergic neurons from patients carrying a LRRK2 mutation had increased expression of oxidative stress response genes and αsynuclein protein. The mutant neurons were also more sensitive to caspase-3 activation and cell death caused by exposure to hydrogen peroxide, MG-132 (a proteasome inhibitor), and 6 hydroxydopamine than control neurons. The finding of increased susceptibility to stress in patient-derived neurons provides insights into the pathogenesis of PD and a potential basis for a cellular screen.

Seibler *et al.*[87] generated IPS cells form PD patients carrying mutation in PINK1 gene (Q456X; V170G). They compared the mitochondrial translocation of Parkin in DA neurons under mitochondrial stress conditions and found a difference between patients and controls, making a step forward into PD pathogenesis *in vitro*.

Two recent studies focused on the IPS cell-derived models of PD carrying a triplication in SNCA genes. Devine *et al.* showed that the levels of α-Synuclein protein were increased in the dopaminergic population derived from patients, compared to the healthy controls [88], while Byers *et al*. focused on the differences in sensitivity to oxidative stress in correlation with this mutation [89].

#### **4.3. Huntington's disease**

recessive, early onset PD [114-116]. These genetic discoveries have highlighted the impor‐ tance of the ubiquitin proteasome system, mitochondrial dysfunction and oxidative stress

The most common genetic risk factor for PD appears to be heterozygous mutations in the *glucocerebrosidase* gene (*GBA*) [117]. The frequency of heterozygous mutations in *GBA* reaches

Because PD results from the loss of dopaminergic neurons, the prospect of utilizing cell replacement therapies has attracted substantial interest. Several methods are able to improve the effectiveness of midbrain dopamine neuron generation and/or retrieval from fetal tissue

The ability of deriving large quantities of correctly differentiated dopamine neurons makes stem cells promising cell sources for transplantation in PD; having the transplantation as a main goal, many studies improved the directed differentiation of PS cells toward dopaminer‐

Soldner *et al.* induced pluripotency in fibroblasts derived from idiopathic PD patients and controls and subsequently differentiated both into dopaminergic neurons. As they did not find significant differences between the expression of *SNCA* or *LRKK2* between patients and controls*,* they went on to suggest that it might still be necessary to further accelerate PDpathology related phenotypes *in vitro* with neurotoxins such as MPTP, or the overexpression

Hargus et al. [85] used a similar protocol of inducing PS cells for idiopathic PD patients and controls, and further differentiated them into dopaminergic neurons. Additionally, they performed intrastriatal transplantation studies into 6-OHDA lesioned rats, demonstrating

Regarding familiar PD, Nguyen *et al.* [86] used a classical protocol for IPS cells generation and differentiation and found that IPS cell-derived dopaminergic neurons from patients carrying a LRRK2 mutation had increased expression of oxidative stress response genes and αsynuclein protein. The mutant neurons were also more sensitive to caspase-3 activation and cell death caused by exposure to hydrogen peroxide, MG-132 (a proteasome inhibitor), and 6 hydroxydopamine than control neurons. The finding of increased susceptibility to stress in patient-derived neurons provides insights into the pathogenesis of PD and a potential basis

Seibler *et al.*[87] generated IPS cells form PD patients carrying mutation in PINK1 gene (Q456X; V170G). They compared the mitochondrial translocation of Parkin in DA neurons under mitochondrial stress conditions and found a difference between patients and controls, making

Two recent studies focused on the IPS cell-derived models of PD carrying a triplication in SNCA genes. Devine *et al.* showed that the levels of α-Synuclein protein were increased in the dopaminergic population derived from patients, compared to the healthy controls [88], while

of PD-related genes such as *SNCA* or *LRKK2* in order to obtain a valid PD model [12]*.*

in PD pathogenesis.

486 Pluripotent Stem Cells

and stem cells.

~4% in sporadic PD populations.

improvements in motor symptoms.

a step forward into PD pathogenesis *in vitro*.

for a cellular screen.

gic neurons, opening the doors to IPS cell-derived models.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder resulting from an expanded CAG triplet repeat in the Huntingtin gene (*HTT*) on chromosome 4 [118]. This expansion accounts for an attachment of a polyglutamine strand of variable length at the N-terminus of the protein leading to a toxic gain of function [119]. HD together with eight other CAG triplet repeat expansion disorders forms the group of PolyQ diseases which share some specific pathophysiological features [120].

Although the protein huntingtin is ubiquitously expressed in mammalian cells, mainly striatal GABAergic medium spiny neurons with a dopamine- and cyclic AMP-regulated phospho‐ protein (DARPP-32)-positive phenotype are the most susceptible to neurodegeneration in HD [121]. As a consequence a prominent cell loss and atrophy in the caudate nucleus and putamen can be observed. Other brain regions and neuronal subtypes involved in HD comprise the substantia nigra, hippocampus, cerebellar Purkinje cells and thalamic nuclei [119;122].

One of the histopathological hallmarks of Huntington's disease, as in other PolyQ disorders too, is the appearance of nuclear and cytoplasmic inclusion bodies containing the mutant huntingtin and polyglutamine [123;124]. Much debate regarding the meaning and function of these inclusions is going on, and although indicative of pathological mutant protein processing they do not correlate with cellular dysfunction and might even confer a protective role [125;126].

Numerous studies indicated that wild-type huntingtin might be involved in a variety of intracellular functions such as in protein trafficking, vesicle and axonal transport, mitochon‐ drial function, postsynaptic signaling; transcriptional regulation, as wells as in anti-apoptotic pathways [127;128]. Therefore a disruption and detrimental impairment of these various intracellular pathways is supposed to be the consequence of accumulation of mutant hun‐ tingtin, finally leading to neuronal death.

Over the years, several different HD models had been introduced, ranging from invertebrate models like Drosophila and C. elegans to various rodent models [129;130]. Genetically modified animals (especially mouse) models such as transgenic, knock-in and conditional ones recapitulated some features of HD like neuronal polyglutamine inclusions [131].

The intrastriatal injection of excitotoxic glutamic acid analogues like kainic acid, quinolinic acid and 3-nitropropionic acid into animals resulted in neuronal cell death similar to the pathology observed in HD patients [132-134]. They proved to be useful in studying pathoge‐ netic processes involved in the progressive disease course although some limitations regarding the selective neuronal cell loss as well as aggregate formation properties and variable pheno‐ types have to be kept in mind.

Transplantation studies in animal HD models aimed at providing neuroprotective support or intended to replace damaged and lost neuronal subtypes. Successful application of stem cellbased therapy in animal models of HD with functional recovery has been reported [135;136]. Different cell types ranging from neural stem/progenitor cells from mouse and rat or human fetal brain tissue to bone marrow and mesenchymal stem cells have been transplanted into excitotoxic animal HD models[137].

early embryonic lethality [148], which has necessitated generating transgenic mice that harbor human SMN2 [149;150] on a SMN-/- background. Although this model provided invaluable protein and disease information, reflecting a gene dosage–dependent phenotype similar to

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It was in 2009 when patient-derived IPS cells were used for the first time to model SMA [91]. Therefore, skin fibroblasts from a three-year old child with SMA as well as from the unaffected mother were successfully reprogrammed via transduction with lentiviral vectors comprising OCT4, SOX2, NANOG, and LIN28. Characterization of the obtained IPS cells demonstrated lack of SMN1 expression and reduced levels of the full-length protein compensated by SMN2. Patient and control IPS cells were further differentiated into neurons. Within these neural cultures, significant differences regarding the number of motoneurons as well as their soma size and synapse formation ability could be observed between patient-specific and control cells, therefore reflecting disease-specific phenotypes. Furthermore, valproic acid and tobra‐ mycin, two drugs known to increase full-length SMN mRNA levels from the SMN2 locus, were tested on this human cellular SMA model. A 2-3-fold increase in SMN protein expression in SMA-IPS cells and an increased nuclear punctuate localization of SMN protein were found.

In a continuative experiment, Sareen et al [92] generated SMA-specific IPS cells using a virusfree plasmid-based approach with subsequent differentiation of IPS cells into NSCs and further MN differentiation. Besides the already described SMA-specific phenotypes, increased apoptosis was detected in SMA-specific cells, which might be another potential target for

ALS, also known as Lou Gehrig's disease is the most common form of MN disease; it as a rapidly progressing, fatal disorder, usually as a result of respiratory failure. In contrast to SMA, it is characterized by a progressive loss of both upper and lower motor neurons in the cerebral cortex, brainstem and spinal cord. Two forms of ALS can be distinguished, the more frequent sporadic form accounts for about 90% of cases and the less common familial form (FALS) for

Mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene are responsible for about 20% of the familial cases [152]. Recently, several other gene mutations were identified as important causing typical FALS, such as the gene encoding the TAR DNA-binding protein 43 (TDP-43) [153]. The role of TDP-43 was first suspected when it was identified as one of the major constituents of the intra-neuronal inclusions characteristically observed in ALS and in frontotemporal lobar degeneration–ubiquitin (FTLD-U); [154]. Subsequently, mutations in the

On macroscopic and microscopic examination of the nervous system in ALS variable neuronal inclusion bodies in lower motor neurons of the spinal cord and brain stem can be detected [155]. Morphologically these inclusions are reliably demonstrated only by their immunoreac‐ tivity to ubiquitin, and have been reported in both sporadic and familial cases and are present in transgenic models of ALS. It is now well established that ALS is typically characterized by

TARDBP gene encoding TDP-43 were identified in some FALS [153].

severe forms of SMA, these mice normally die shortly after birth.

therapeutic intervention.

the remaining 10% [151].

the presence of these inclusion bodies.

In order to facilitate research in the HD field with human material, Bradley *et al*. [138] derived four hES cell lines containing more than 40 CAG repeats from donated embryos obtained through an informed consent. Those hES cells were able to differentiate in to neuronal cells expressing the mutant huntingtin protein.

The first HD-IPS cell lines were successfully generated by Park *et al.* from patient with a 72 CAG repeat tract using the classical lentiviral vectors [4]. In a subsequent study Zhang et al used these patient-specific IPS cells in order to generate HD specific neural stem cells that were then differentiated into striatal neurons. Besides a stable CAG repeat expansion in all patientderived cells, an enhanced caspase 3/7 activity was found.

A second group successfully generated HD-specific IPS cells via lentiviral transduction of transcription factors and was able to demonstrate a stable CAG triple repeat length in all IPS cell clones as well as in IPS cell-derived neurons. Interestingly, they observed an enhanced lysosomal activity in IPS cells and their derived neuronal populations [139].

#### **4.4. Motor neuron diseases**

Motor neurons (MNs) are essential effector cells for the control of motor function. Degenerative MN diseases, such Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) are devastating disorders due to a selective loss of MNs, which in turn leads to progressive muscle atrophy and weakness.

SMA is the most common form of degenerative motor neuron disease in children and young adults, characterized by the selective degeneration of lower MNs in the brainstem and spinal cord [140]. SMA is a classical autosomal recessive disorder with the vast majority of SMA cases caused by homozygous mutations in the gene named Survival of Motor Neuron-1 (SMN1) [141;142].

Interestingly, in humans the SMN exists in a telomeric copy, SMN1, and several centromeric highly homologous copies, SMN2, with both genes being transcribed [143]. Due to the fact that the vast majority of SMN2 transcripts lack an exon due to a splicing defect, it is only partially and poorly able to compensate for reduced SMN1 levels [144;145].

SMN is a ubiquitously expressed gene involved in the biogenesis of small nuclear ribonucleo‐ proteins important for pre-mRNA splicing, but might also have a specific role in RNA transport in neurons [146]. However, it remains to be elucidated how a deficiency in SMN is responsible for the selective degeneration of lower motor neurons [147].

Several experimental models have been used to study the putative cellular and molecular processes involved in SMA. Mouse models have become the most often used, albeit lacking the duplication of the SMN gene in humans. As a consequence, homologous recombination technology of the *Smn* locus in mice leads to complete depletion of the SMN protein, causing early embryonic lethality [148], which has necessitated generating transgenic mice that harbor human SMN2 [149;150] on a SMN-/- background. Although this model provided invaluable protein and disease information, reflecting a gene dosage–dependent phenotype similar to severe forms of SMA, these mice normally die shortly after birth.

based therapy in animal models of HD with functional recovery has been reported [135;136]. Different cell types ranging from neural stem/progenitor cells from mouse and rat or human fetal brain tissue to bone marrow and mesenchymal stem cells have been transplanted into

In order to facilitate research in the HD field with human material, Bradley *et al*. [138] derived four hES cell lines containing more than 40 CAG repeats from donated embryos obtained through an informed consent. Those hES cells were able to differentiate in to neuronal cells

The first HD-IPS cell lines were successfully generated by Park *et al.* from patient with a 72 CAG repeat tract using the classical lentiviral vectors [4]. In a subsequent study Zhang et al used these patient-specific IPS cells in order to generate HD specific neural stem cells that were then differentiated into striatal neurons. Besides a stable CAG repeat expansion in all patient-

A second group successfully generated HD-specific IPS cells via lentiviral transduction of transcription factors and was able to demonstrate a stable CAG triple repeat length in all IPS cell clones as well as in IPS cell-derived neurons. Interestingly, they observed an enhanced

Motor neurons (MNs) are essential effector cells for the control of motor function. Degenerative MN diseases, such Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) are devastating disorders due to a selective loss of MNs, which in turn leads to progressive

SMA is the most common form of degenerative motor neuron disease in children and young adults, characterized by the selective degeneration of lower MNs in the brainstem and spinal cord [140]. SMA is a classical autosomal recessive disorder with the vast majority of SMA cases caused by homozygous mutations in the gene named Survival of Motor Neuron-1 (SMN1)

Interestingly, in humans the SMN exists in a telomeric copy, SMN1, and several centromeric highly homologous copies, SMN2, with both genes being transcribed [143]. Due to the fact that the vast majority of SMN2 transcripts lack an exon due to a splicing defect, it is only partially

SMN is a ubiquitously expressed gene involved in the biogenesis of small nuclear ribonucleo‐ proteins important for pre-mRNA splicing, but might also have a specific role in RNA transport in neurons [146]. However, it remains to be elucidated how a deficiency in SMN is responsible

Several experimental models have been used to study the putative cellular and molecular processes involved in SMA. Mouse models have become the most often used, albeit lacking the duplication of the SMN gene in humans. As a consequence, homologous recombination technology of the *Smn* locus in mice leads to complete depletion of the SMN protein, causing

and poorly able to compensate for reduced SMN1 levels [144;145].

for the selective degeneration of lower motor neurons [147].

lysosomal activity in IPS cells and their derived neuronal populations [139].

excitotoxic animal HD models[137].

488 Pluripotent Stem Cells

**4.4. Motor neuron diseases**

muscle atrophy and weakness.

[141;142].

expressing the mutant huntingtin protein.

derived cells, an enhanced caspase 3/7 activity was found.

It was in 2009 when patient-derived IPS cells were used for the first time to model SMA [91]. Therefore, skin fibroblasts from a three-year old child with SMA as well as from the unaffected mother were successfully reprogrammed via transduction with lentiviral vectors comprising OCT4, SOX2, NANOG, and LIN28. Characterization of the obtained IPS cells demonstrated lack of SMN1 expression and reduced levels of the full-length protein compensated by SMN2. Patient and control IPS cells were further differentiated into neurons. Within these neural cultures, significant differences regarding the number of motoneurons as well as their soma size and synapse formation ability could be observed between patient-specific and control cells, therefore reflecting disease-specific phenotypes. Furthermore, valproic acid and tobra‐ mycin, two drugs known to increase full-length SMN mRNA levels from the SMN2 locus, were tested on this human cellular SMA model. A 2-3-fold increase in SMN protein expression in SMA-IPS cells and an increased nuclear punctuate localization of SMN protein were found.

In a continuative experiment, Sareen et al [92] generated SMA-specific IPS cells using a virusfree plasmid-based approach with subsequent differentiation of IPS cells into NSCs and further MN differentiation. Besides the already described SMA-specific phenotypes, increased apoptosis was detected in SMA-specific cells, which might be another potential target for therapeutic intervention.

ALS, also known as Lou Gehrig's disease is the most common form of MN disease; it as a rapidly progressing, fatal disorder, usually as a result of respiratory failure. In contrast to SMA, it is characterized by a progressive loss of both upper and lower motor neurons in the cerebral cortex, brainstem and spinal cord. Two forms of ALS can be distinguished, the more frequent sporadic form accounts for about 90% of cases and the less common familial form (FALS) for the remaining 10% [151].

Mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene are responsible for about 20% of the familial cases [152]. Recently, several other gene mutations were identified as important causing typical FALS, such as the gene encoding the TAR DNA-binding protein 43 (TDP-43) [153]. The role of TDP-43 was first suspected when it was identified as one of the major constituents of the intra-neuronal inclusions characteristically observed in ALS and in frontotemporal lobar degeneration–ubiquitin (FTLD-U); [154]. Subsequently, mutations in the TARDBP gene encoding TDP-43 were identified in some FALS [153].

On macroscopic and microscopic examination of the nervous system in ALS variable neuronal inclusion bodies in lower motor neurons of the spinal cord and brain stem can be detected [155]. Morphologically these inclusions are reliably demonstrated only by their immunoreac‐ tivity to ubiquitin, and have been reported in both sporadic and familial cases and are present in transgenic models of ALS. It is now well established that ALS is typically characterized by the presence of these inclusion bodies.

Furthermore, it has been reported that an ALS genotype in glial cells (astrocytes) has an effect on the survival of motor neurons and contributes a crucial role in motor neuron degeneration [156].

loss of position and vibration sense [163;164]. Cardiomyopathy and diabetes mellitus are

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491

FRDA is caused in 96% of individuals by a GAA triplet expansion in the first intron of the Frataxin (*FXN*) gene on chromosome 9q13 [166]. The mutation leads to transcriptional silencing as a result of heterochromatin formation, adoption of an abnormal DNA-RNA hybrid structure, or triplex DNA formation [167] with reduced Frataxin protein expression. About 4% of the individuals affected with FRDA are compound heterozygous. Disease-causing expand‐ ed alleles present with 66 to 1700 GAA repeats with the majority ranging from 600 up to 1200 GAA repeats [166;168]. Major neuropathologic findings comprise a degeneration of dorsal root ganglia (DRG), with loss of large sensory neurons, followed by degeneration of posterior columns, corticospinal tracts and spinocerebellar tracts, and the deep nuclei in the cerebellum

The gene product Frataxin is a ubiquitously expressed and evolutionary conserved mitochon‐ drial protein that has been proposed to exhibit roles in mitochondrial iron metabolism and the

Several FRDA disease models, from yeast, C. elegans and Drosophila to mice have been used to get more insight into the disease [170;171]. Viable transgenic mouse models were generated through conditional gene targeting [172] which have been crucial in the development as models for FRDA, although some with ambiguous results. The complete knock-out of Frataxin resulted in embryonic lethality [173], whereas conditional mouse models under the control of different promoters were capable to recapitulate some of the disease phenotypes [174]. In order to circumvent the non-physiologic complete loss of Frataxin at a specific time point in conditional models, GAA based mouse models were introduced [175;176], shedding more light

Despite a general genotype-phenotype correlation it is not possible to predict the specific clinical outcome in any individual based on GAA repeat length. The inherent variability in FRDA may be caused by genetic background, somatic heterogeneity of the GAA expansion

Therefore, FRDA-IPS cell lines have already been established by Ku et al. [93] and Liu et al. [94]. Data showed that, although a specific disease-related phenotype was not reported, these FRDA IPS cells were able to recapitulate some of the molecular genetic aspects of FRDA, including the phenomenon of repeat-length instability, epigenetic silencing of the FXN locus

With regard to GAA repeat instability, IPS cells showed repeat expansions whereas parental fibroblasts did not [93]. Instability was specific to the abnormally expanded FXN as GAA expansions in normal FXN alleles or at two unrelated loci with short GAA repeats remained unchanged. To understand the mechanism of instability in this IPS cell system, analysis of differences in mRNA expression showed that MSH2, a critical component of the DNA mismatch repair (MMR) machinery and important for mediating repeat-length instability, was highly expressed in FRDA-IPS cells relative to donor fibroblasts. ShRNA-mediated silencing

on tissue-dependent GAA dynamics and putative pathophysiologic pathways.

systemic complications in some patients [165].

production of iron-sulfur (Fe-S) clusters.

[177;178], and yet other unidentified factors.

and low levels of Frataxin expression [93].

[165;169].

ALS research has focused mainly on models of the familial SOD1-mediated form, although all forms of ALS share striking similarities in pathology and clinical symptoms. A toxic gain of function of this enzyme with the exact mechanism still unclear is thought to be responsible which subsequently results in mitochondrial dysfunction, oxidative damage, glutamate excitotoxicity, protein aggregation, proteasome dysfunction, cytoskeletal and axonal transport defects and inflammation [151;157].

Transgenic mice or rats overexpressing mutant SOD1 develop MN degeneration with pro‐ gressive muscle weakness, muscle wasting and reduced life span [158]. Furthermore, mutant SOD1 as well as TDP-43 models have been generated in zebrafish and C. elegans, mimicking at least some of the pathological hallmarks (e.g. selective vulnerability of MN and MN dysfunction) and therefore making them suitable for genetic and small compound screening [157;159].

Transgenic ALS models have also already been utilized for stem cell therapies by transplanting different types of cells comprising human as well as rodent fetal neural stem and progenitor cells, umbilical cord blood stem cells, mesenchymal stem cells and bone marrow. In some of the studies, a moderate improvement of motor function and a delayed disease progression could be observed [160]. However, the translation of stem cell transplantation therapies into clinical trials did not show any therapeutic benefit in ALS patients.

In order to get more insight into human pathophysiology, Dimos *et al.* [161] were the first to generate ALS-patient specific IPS cells using retroviral transduction of the classical Yamanaka factors OSKM. They successfully obtained IPS cells from an 82-year old sibling suffering from a familial form of ALS with a mutation in the SOD1 gene. Subsequently, patient-specific IPS cells were forced to differentiate into MN and glia. Due to the fact that more than 90% of ALS cases are sporadic, patient-specific IPS cell models from sporadic ALS might overcome this draw‐ back through the integration of the genetic as well as environmental individual background.

#### **4.5. Ataxias**

The degenerative ataxias are a group of hereditary or idiopathic diseases that are clinically characterized by progressive ataxia resulting from degeneration of cerebellar-brainstem structures and spinal pathways [162].

Autosomal recessive cerebellar ataxias are heterogeneous, complex, disabling inherited neurodegenerative diseases that become manifest usually during childhood and adolescence.

Friedreich Ataxia (FRDA), an autosomal-recessive ataxia, is the most common inherited ataxic disorder in the white Caucasian population with a prevalence of 2-4/100,000 and with an age of onset in the teenage years. Clinical characteristics include progressive ataxia of gait and limbs, dysarthria, muscle weakness, spasticity in the legs, scoliosis, bladder dysfunction, and loss of position and vibration sense [163;164]. Cardiomyopathy and diabetes mellitus are systemic complications in some patients [165].

Furthermore, it has been reported that an ALS genotype in glial cells (astrocytes) has an effect on the survival of motor neurons and contributes a crucial role in motor neuron degeneration

ALS research has focused mainly on models of the familial SOD1-mediated form, although all forms of ALS share striking similarities in pathology and clinical symptoms. A toxic gain of function of this enzyme with the exact mechanism still unclear is thought to be responsible which subsequently results in mitochondrial dysfunction, oxidative damage, glutamate excitotoxicity, protein aggregation, proteasome dysfunction, cytoskeletal and axonal transport

Transgenic mice or rats overexpressing mutant SOD1 develop MN degeneration with pro‐ gressive muscle weakness, muscle wasting and reduced life span [158]. Furthermore, mutant SOD1 as well as TDP-43 models have been generated in zebrafish and C. elegans, mimicking at least some of the pathological hallmarks (e.g. selective vulnerability of MN and MN dysfunction) and therefore making them suitable for genetic and small compound screening

Transgenic ALS models have also already been utilized for stem cell therapies by transplanting different types of cells comprising human as well as rodent fetal neural stem and progenitor cells, umbilical cord blood stem cells, mesenchymal stem cells and bone marrow. In some of the studies, a moderate improvement of motor function and a delayed disease progression could be observed [160]. However, the translation of stem cell transplantation therapies into

In order to get more insight into human pathophysiology, Dimos *et al.* [161] were the first to generate ALS-patient specific IPS cells using retroviral transduction of the classical Yamanaka factors OSKM. They successfully obtained IPS cells from an 82-year old sibling suffering from a familial form of ALS with a mutation in the SOD1 gene. Subsequently, patient-specific IPS cells were forced to differentiate into MN and glia. Due to the fact that more than 90% of ALS cases are sporadic, patient-specific IPS cell models from sporadic ALS might overcome this draw‐ back through the integration of the genetic as well as environmental individual background.

The degenerative ataxias are a group of hereditary or idiopathic diseases that are clinically characterized by progressive ataxia resulting from degeneration of cerebellar-brainstem

Autosomal recessive cerebellar ataxias are heterogeneous, complex, disabling inherited neurodegenerative diseases that become manifest usually during childhood and adolescence.

Friedreich Ataxia (FRDA), an autosomal-recessive ataxia, is the most common inherited ataxic disorder in the white Caucasian population with a prevalence of 2-4/100,000 and with an age of onset in the teenage years. Clinical characteristics include progressive ataxia of gait and limbs, dysarthria, muscle weakness, spasticity in the legs, scoliosis, bladder dysfunction, and

clinical trials did not show any therapeutic benefit in ALS patients.

[156].

490 Pluripotent Stem Cells

[157;159].

**4.5. Ataxias**

structures and spinal pathways [162].

defects and inflammation [151;157].

FRDA is caused in 96% of individuals by a GAA triplet expansion in the first intron of the Frataxin (*FXN*) gene on chromosome 9q13 [166]. The mutation leads to transcriptional silencing as a result of heterochromatin formation, adoption of an abnormal DNA-RNA hybrid structure, or triplex DNA formation [167] with reduced Frataxin protein expression. About 4% of the individuals affected with FRDA are compound heterozygous. Disease-causing expand‐ ed alleles present with 66 to 1700 GAA repeats with the majority ranging from 600 up to 1200 GAA repeats [166;168]. Major neuropathologic findings comprise a degeneration of dorsal root ganglia (DRG), with loss of large sensory neurons, followed by degeneration of posterior columns, corticospinal tracts and spinocerebellar tracts, and the deep nuclei in the cerebellum [165;169].

The gene product Frataxin is a ubiquitously expressed and evolutionary conserved mitochon‐ drial protein that has been proposed to exhibit roles in mitochondrial iron metabolism and the production of iron-sulfur (Fe-S) clusters.

Several FRDA disease models, from yeast, C. elegans and Drosophila to mice have been used to get more insight into the disease [170;171]. Viable transgenic mouse models were generated through conditional gene targeting [172] which have been crucial in the development as models for FRDA, although some with ambiguous results. The complete knock-out of Frataxin resulted in embryonic lethality [173], whereas conditional mouse models under the control of different promoters were capable to recapitulate some of the disease phenotypes [174]. In order to circumvent the non-physiologic complete loss of Frataxin at a specific time point in conditional models, GAA based mouse models were introduced [175;176], shedding more light on tissue-dependent GAA dynamics and putative pathophysiologic pathways.

Despite a general genotype-phenotype correlation it is not possible to predict the specific clinical outcome in any individual based on GAA repeat length. The inherent variability in FRDA may be caused by genetic background, somatic heterogeneity of the GAA expansion [177;178], and yet other unidentified factors.

Therefore, FRDA-IPS cell lines have already been established by Ku et al. [93] and Liu et al. [94]. Data showed that, although a specific disease-related phenotype was not reported, these FRDA IPS cells were able to recapitulate some of the molecular genetic aspects of FRDA, including the phenomenon of repeat-length instability, epigenetic silencing of the FXN locus and low levels of Frataxin expression [93].

With regard to GAA repeat instability, IPS cells showed repeat expansions whereas parental fibroblasts did not [93]. Instability was specific to the abnormally expanded FXN as GAA expansions in normal FXN alleles or at two unrelated loci with short GAA repeats remained unchanged. To understand the mechanism of instability in this IPS cell system, analysis of differences in mRNA expression showed that MSH2, a critical component of the DNA mismatch repair (MMR) machinery and important for mediating repeat-length instability, was highly expressed in FRDA-IPS cells relative to donor fibroblasts. ShRNA-mediated silencing of MSH2 resulted in shorter repeat lengths suggesting that FRDA IPS cells could be a useful system to evaluate the mechanisms of repeat expansions and contractions in disease.

While some of the transgenic mouse models expressing the full-length ATXN3 under control of various exogenous promoters were able to mimic some aspects of the disease, they all overexpress only a single isoform of ATXN3. Taken this into account, a YAC MJD transgenic model was established which more closely recapitulates the human disease as all elements, including regulatory regions of the gene, are present [186]. Research in animal models of SCA has now begun to focus on therapeutic strategies to prevent protein misfolding and aggrega‐

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493

Koch *et al* [95], investigated the formation of early aggregates and their behavior in time by making use of patient- specific IPS cell-derived neurons. They demonstrated that MJD-IPS cell derived neurons constitute an appropriate cellular model in the study of aberrant human protein processing. Moreover, they concluded that neurons are able to cope, at least in the beginning, with the aggregated mutant material and cytotoxicity evolved over time. Besides, a key role for the protease calpain in ATXN3-aggregation formation was found which could

Familial Dysautonomia (FD), also known as Riley-Day Syndrome or Hereditary Sensory Autonomic Neuropathy (HSAN) Type III, is a rare autosomal recessive disease mostly occurring in persons of Ashkenazi Jewish descent [187]. The disease is characterized by degeneration of sensory and autonomic neurons, leading to severe and often lethal central and peripheral autonomic perturbations, as well as small-fiber sensory dysfunction. The underly‐ ing mutation induces a splicing defect in the IkB kinase complex-associated protein (*IKB‐ KAP*) gene, which results in tissue-specific loss of function or reduced levels of the IKAP protein [188]. Individuals affected with FD suffer from incomplete neuronal development as well as progressive neuronal degeneration with the sensory and autonomic neurons mainly

Although the exact function of the IKAP protein is not clearly understood, researchers have identified IKAP as the scaffold protein required for the assembly of a holo-elongator complex [190]. As a consequence, an impaired transcriptional elongation of genes responsible for cell motility is thought to be the cause for the observed cell migration deficiency in FD neurons [191]. Besides, the IKAP protein is also thought to be involved in other cellular processes,

To better understand the function of IKAP, Dietrich *et al.* [193] created a mouse model with two distinct alleles that result in either loss of Ikbkap expression, or expression of the mutated truncated protein. Besides, a humanized IKBKAP transgenic mouse model for FD had been created that recapitulated the tissue-specific splicing defect, i.e. skipping of exon 20, in nervous

In order to untangle the tissue-specific pattern of IKBKAP mRNA splicing in FD, Boone et al. [195] created a human olfactory ecto-mesenchymal stem cell (hOEMSC) model of FD. It has been shown that these multipotent hOE-MSCs exhibit the potential to differentiate in vitro into neurons, astrocytes, and oligodendrocytes as well as other cell types [196]. Classical features

including tRNA and epigenetic modifications and exocytosis [192].

tion in polyglutamine diseases by overexpressing chaperones.

further display a putative benefit of calpain inhibitors.

**4.6. Familial dysautonomia and fragile X syndrome**

affected [189].

tissues [194].

GAA repeat mutations are unstable and progressive and postnatal instability occurs in various tissues throughout life. For example, large GAA repeat expansions are especially prominent in the dorsal root ganglia of FRDA patients, which harbor cell bodies of sensory neurons, a neuronal subtype especially affected in FRDA [179].

Given FRDA-IPS cells can be directed to differentiate into sensory neurons, as well as cardio‐ myocytes [94], the presence and mechanisms of tissue-specific expansion should be testable. The major focus of FRDA IPS cell differentiation research is currently focused on generating appropriate disease-relevant cell types. For example, sensory neurons of the DRG are crucially affected in individuals with FRDA.

The autosomal dominant Spinocerebellar Ataxias (SCAs) comprise a genetically and clinically heterogeneous group of inherited neurodegenerative progressive disorders affecting various parts of the CNS. The number of known SCAs continues to grow and comprises meantime over 30 entities.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is the most frequent entity among the autosomal dominantly inherited cerebellar ataxias in Europe, Japan, and the United States [180].

Genetically, SCA3 belongs to the group of CAG-triple repeat disorders, also known as PolyQdisorders due to abnormally long polyglutamine tracts within the corresponding protein. The majority of patients suffering from SCA3 carry one allele of the ataxin3 (*ATXN3*) gene with 60–82 CAG repeats and a second allele containing the normal number of repeats, which is usually between 13 and 41 [181].

As in most of these polyglutamine diseases, patients with a repeat expansion above a critical threshold form neuronal intranuclear inclusion bodies, one important hallmark of polyQ diseases [182]. Further neuropathological features include a depigmentation of the substan‐ tia nigra as well as a pronounced atrophy of the cerebellum, pons and medulla oblongata, altogether culminating in an overall reduced brain weight compared to healthy individu‐ als [183].

As most of the PolyQ disease proteins are ubiquitously expressed it still remains unclear why only specific neuronal cell populations are prone to neurodegeneration. Many animal models, like rodents, C.elegans and Drosophila, overexpressing specific forms of ATXN3 are available to study the molecular and phenotypic aspects of MJD involving aggregation, proteolysis and toxicity of expanded ATXN3, as well as the apparent neuroprotective role of wild-type ATXN3 [184].

Kakizuka's group was the first to demonstrate neurodegeneration and a neurological pheno‐ type in mice transgenic for the CAG repeat expansion [185]. Mouse models further provided evidence for the subcellular site of pathogenesis, the processing and trafficking of the mutant protein in order to cause cellular dysfunction and neuronal cell loss.

While some of the transgenic mouse models expressing the full-length ATXN3 under control of various exogenous promoters were able to mimic some aspects of the disease, they all overexpress only a single isoform of ATXN3. Taken this into account, a YAC MJD transgenic model was established which more closely recapitulates the human disease as all elements, including regulatory regions of the gene, are present [186]. Research in animal models of SCA has now begun to focus on therapeutic strategies to prevent protein misfolding and aggrega‐ tion in polyglutamine diseases by overexpressing chaperones.

Koch *et al* [95], investigated the formation of early aggregates and their behavior in time by making use of patient- specific IPS cell-derived neurons. They demonstrated that MJD-IPS cell derived neurons constitute an appropriate cellular model in the study of aberrant human protein processing. Moreover, they concluded that neurons are able to cope, at least in the beginning, with the aggregated mutant material and cytotoxicity evolved over time. Besides, a key role for the protease calpain in ATXN3-aggregation formation was found which could further display a putative benefit of calpain inhibitors.

#### **4.6. Familial dysautonomia and fragile X syndrome**

of MSH2 resulted in shorter repeat lengths suggesting that FRDA IPS cells could be a useful

GAA repeat mutations are unstable and progressive and postnatal instability occurs in various tissues throughout life. For example, large GAA repeat expansions are especially prominent in the dorsal root ganglia of FRDA patients, which harbor cell bodies of sensory neurons, a

Given FRDA-IPS cells can be directed to differentiate into sensory neurons, as well as cardio‐ myocytes [94], the presence and mechanisms of tissue-specific expansion should be testable. The major focus of FRDA IPS cell differentiation research is currently focused on generating appropriate disease-relevant cell types. For example, sensory neurons of the DRG are crucially

The autosomal dominant Spinocerebellar Ataxias (SCAs) comprise a genetically and clinically heterogeneous group of inherited neurodegenerative progressive disorders affecting various parts of the CNS. The number of known SCAs continues to grow and comprises meantime

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is the most frequent entity among the autosomal dominantly inherited cerebellar ataxias in Europe,

Genetically, SCA3 belongs to the group of CAG-triple repeat disorders, also known as PolyQdisorders due to abnormally long polyglutamine tracts within the corresponding protein. The majority of patients suffering from SCA3 carry one allele of the ataxin3 (*ATXN3*) gene with 60–82 CAG repeats and a second allele containing the normal number of repeats, which is

As in most of these polyglutamine diseases, patients with a repeat expansion above a critical threshold form neuronal intranuclear inclusion bodies, one important hallmark of polyQ diseases [182]. Further neuropathological features include a depigmentation of the substan‐ tia nigra as well as a pronounced atrophy of the cerebellum, pons and medulla oblongata, altogether culminating in an overall reduced brain weight compared to healthy individu‐

As most of the PolyQ disease proteins are ubiquitously expressed it still remains unclear why only specific neuronal cell populations are prone to neurodegeneration. Many animal models, like rodents, C.elegans and Drosophila, overexpressing specific forms of ATXN3 are available to study the molecular and phenotypic aspects of MJD involving aggregation, proteolysis and toxicity of expanded ATXN3, as well as the apparent neuroprotective role

Kakizuka's group was the first to demonstrate neurodegeneration and a neurological pheno‐ type in mice transgenic for the CAG repeat expansion [185]. Mouse models further provided evidence for the subcellular site of pathogenesis, the processing and trafficking of the mutant

protein in order to cause cellular dysfunction and neuronal cell loss.

system to evaluate the mechanisms of repeat expansions and contractions in disease.

neuronal subtype especially affected in FRDA [179].

affected in individuals with FRDA.

Japan, and the United States [180].

usually between 13 and 41 [181].

of wild-type ATXN3 [184].

over 30 entities.

492 Pluripotent Stem Cells

als [183].

Familial Dysautonomia (FD), also known as Riley-Day Syndrome or Hereditary Sensory Autonomic Neuropathy (HSAN) Type III, is a rare autosomal recessive disease mostly occurring in persons of Ashkenazi Jewish descent [187]. The disease is characterized by degeneration of sensory and autonomic neurons, leading to severe and often lethal central and peripheral autonomic perturbations, as well as small-fiber sensory dysfunction. The underly‐ ing mutation induces a splicing defect in the IkB kinase complex-associated protein (*IKB‐ KAP*) gene, which results in tissue-specific loss of function or reduced levels of the IKAP protein [188]. Individuals affected with FD suffer from incomplete neuronal development as well as progressive neuronal degeneration with the sensory and autonomic neurons mainly affected [189].

Although the exact function of the IKAP protein is not clearly understood, researchers have identified IKAP as the scaffold protein required for the assembly of a holo-elongator complex [190]. As a consequence, an impaired transcriptional elongation of genes responsible for cell motility is thought to be the cause for the observed cell migration deficiency in FD neurons [191]. Besides, the IKAP protein is also thought to be involved in other cellular processes, including tRNA and epigenetic modifications and exocytosis [192].

To better understand the function of IKAP, Dietrich *et al.* [193] created a mouse model with two distinct alleles that result in either loss of Ikbkap expression, or expression of the mutated truncated protein. Besides, a humanized IKBKAP transgenic mouse model for FD had been created that recapitulated the tissue-specific splicing defect, i.e. skipping of exon 20, in nervous tissues [194].

In order to untangle the tissue-specific pattern of IKBKAP mRNA splicing in FD, Boone et al. [195] created a human olfactory ecto-mesenchymal stem cell (hOEMSC) model of FD. It has been shown that these multipotent hOE-MSCs exhibit the potential to differentiate in vitro into neurons, astrocytes, and oligodendrocytes as well as other cell types [196]. Classical features of the FD phenotype, like the expression of the mutant IKBKAP transcript, notably lower IKBKAP levels as well as an impaired migration, were observed. Besides, drug testing experiments with kinetin, which had been shown effective in previous studies [197], had the potential to correct the splicing in a dose-dependent manner in FD hOE-MSCs.

**5. Conclusions and perspectives**

aspect has been in the SMA models [91].

genetic and cellular profile.

In this chapter we have described the first successful attempts to harness the IPS technology for the generation of models for neurodegenerative diseases of the human nervous system. The key advantage of IPS based models over animal models is that they offer researchers for the first time a realistic chance to work in cell culture with large numbers of primary human

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases

http://dx.doi.org/10.5772/55772

495

The first studies in which patient-derived disease-susceptible cellular phenotypes were compared with those of cells derived from healthy individuals, provide strong indications that such cellular models reflect key pathological molecular and cellular aspects of the neurological diseases. Therefore a future concept for patient-derived cellular models will be to correct neuronal malfunctions diseases by *in vitro* treatment of affected cells*.* A first such attempts

These *in vitro* treatments will include hypothesis driven approaches based on knowledge about pathophysiological mechanisms. Equally important patient derived lines will be used as *in vitro* assays for the screening of compound libraries. Drug safety screens with IPS cell-derived neurons will help to reduce the animal dependency of the current drug development pipeline. Finally, IPS cell technology will be an important driver of personalized medicine. Prior to patient treatment drug types and doses can be tested on patient-derived IPS cells or differen‐ tiated progenies in order to tailor a personalized curative approach according to the individual

There is even hope that the novel approach bypasses the laborious, time-consuming and expensive IPS cell generation by direct reprogramming of mouse and human somatic cells into functional neurons, called induced neurons (INs) [206;207], will come to fruition. Several groups have already generated dopaminergic INs [208;209] and motor INs [210]. Patientspecific INs could be generated to enhance the study of developmental disorders and other neurological diseases [211]. The significant decrease in time and resources to derive neurons

But despite the enormous potential of IPS cell derived neurons for studies involving cell biological, physiological and pharmacological methods important question remain to be solved. One major drawback is that we still know very little about the specific cell biology of IPS cells and even less of their neuronal derivatives. This includes for example changes in chromatin structure and epigenetic signatures that accompany the reprogramming process. And there is exceedingly little information about membrane physiology of the IPS cell- derived neurons. Electrophysiological recordings and parallel studies of synaptic proteins and ion

We have already pointed out the difficulties to design specific differentiation protocols for specific neuronal populations from IPS cells. The underlying hypothesis for all existing protocols is that cells should be guided through a shortcut version of embryonic development. A hindrance for progress in this regard is the lack of specific information of human embryonic development since most of our knowledge about vertebrate brain development derives from

directly from somatic cells justifies further investigation into this strategy.

specific channel composition should be a focus of future research.

cells that closely resemble the postmitotic neurons affected by neurodegeneration.

Furthermore, IPS cells were generated from a patient with FD using the classical Yamanaka factors and subsequently differentiated into neural crest derivatives [96]. This was one of the earliest reports of a phenotype for a neurological disease to be modeled with IPS cells. FD-IPS cell derived neural precursors showed particularly low levels of IKBKAP, mis-splicing of IKBKAP, and defects in neurogenic differentiation and migration behavior. Again, the plant hormone kinetin was tested as a candidate and showed a reduction of mutant IKBKAP splice forms, an improvement in neuronal differentiation, but not in cell migration.

Fragile-X (FX) syndrome belongs to the autism spectrum disorders, and is the most common cause of inherited mental retardation with a prevalence of 1/3600 [198]. In the vast majority of cases, the disease is caused by a silencing of the FMR1 gene due to a CGG repeat expansion (>200 repeats) in the 5-UTR of the *FMR1* gene [199]. The FMR1 gene codes for the cytoplasmic protein FMRP, which has RNA-binding properties and is thought to play a role in synaptic plasticity and dendrite maturation. This could be demonstrated in histopathological studies of FX where dendritic spine abnormalities were found [200].

Several animal models revealed important insights into the role of the FMR protein. A Drosophila model showed a role of FRMP in the regulation of the microtubule network [201].

The first fmr1 KO mouse model was generated shortly after the discovery of the diseasecausing gene and showed classical clinical features of FXS like macroorchidism, learning deficits, and hyperactivity[202].

Although current mouse models for FX syndrome are useful for studying the clinical pheno‐ type, they do not recapitulate the hallmark, i.e. silencing of the FMR1 gene due to the triplet repeat expansion [203]. Loss of function studies using morpholino antisense oligonucleotides in zebrafish revealed a function of FMRP in terms of normal axonal branching.

Primary and transformed cell cultures obtained with an unmethylated full mutation in the FMR1 showed that the CGG expansion per se does not block transcription [204]. In undiffer‐ entiated human FX embryonic stem cells (FX-ES cells) derived from affected blastocyst-stage embryos, FMR1 is expressed and gene silencing occurs only upon differentiation [205] indicating a developmentally dependent process.

Recently, Urbach et al. [97] generated FX-IPS cell lines from three patients. In contrast to FX-ES cells, FX-IPS cells presented with a transcriptionally silent FMR1 gene, both in the pluri‐ potent and differentiated states. This was reflected by corresponding epigenetic heterochromatin modifications in the gene promoter. IPS cells were further differentiated into neural derivatives and different potential epigenetic modifiers were tested. Amongst those, 5 azacytidin showed an upregulation of FMR1 transcripts both in pluripotent as well as neuronal FX-cells.

## **5. Conclusions and perspectives**

of the FD phenotype, like the expression of the mutant IKBKAP transcript, notably lower IKBKAP levels as well as an impaired migration, were observed. Besides, drug testing experiments with kinetin, which had been shown effective in previous studies [197], had the

Furthermore, IPS cells were generated from a patient with FD using the classical Yamanaka factors and subsequently differentiated into neural crest derivatives [96]. This was one of the earliest reports of a phenotype for a neurological disease to be modeled with IPS cells. FD-IPS cell derived neural precursors showed particularly low levels of IKBKAP, mis-splicing of IKBKAP, and defects in neurogenic differentiation and migration behavior. Again, the plant hormone kinetin was tested as a candidate and showed a reduction of mutant IKBKAP splice

Fragile-X (FX) syndrome belongs to the autism spectrum disorders, and is the most common cause of inherited mental retardation with a prevalence of 1/3600 [198]. In the vast majority of cases, the disease is caused by a silencing of the FMR1 gene due to a CGG repeat expansion (>200 repeats) in the 5-UTR of the *FMR1* gene [199]. The FMR1 gene codes for the cytoplasmic protein FMRP, which has RNA-binding properties and is thought to play a role in synaptic plasticity and dendrite maturation. This could be demonstrated in histopathological studies

Several animal models revealed important insights into the role of the FMR protein. A Drosophila model showed a role of FRMP in the regulation of the microtubule network [201].

The first fmr1 KO mouse model was generated shortly after the discovery of the diseasecausing gene and showed classical clinical features of FXS like macroorchidism, learning

Although current mouse models for FX syndrome are useful for studying the clinical pheno‐ type, they do not recapitulate the hallmark, i.e. silencing of the FMR1 gene due to the triplet repeat expansion [203]. Loss of function studies using morpholino antisense oligonucleotides

Primary and transformed cell cultures obtained with an unmethylated full mutation in the FMR1 showed that the CGG expansion per se does not block transcription [204]. In undiffer‐ entiated human FX embryonic stem cells (FX-ES cells) derived from affected blastocyst-stage embryos, FMR1 is expressed and gene silencing occurs only upon differentiation [205]

Recently, Urbach et al. [97] generated FX-IPS cell lines from three patients. In contrast to FX-ES cells, FX-IPS cells presented with a transcriptionally silent FMR1 gene, both in the pluri‐ potent and differentiated states. This was reflected by corresponding epigenetic heterochromatin modifications in the gene promoter. IPS cells were further differentiated into neural derivatives and different potential epigenetic modifiers were tested. Amongst those, 5 azacytidin showed an upregulation of FMR1 transcripts both in pluripotent as well as neuronal

in zebrafish revealed a function of FMRP in terms of normal axonal branching.

potential to correct the splicing in a dose-dependent manner in FD hOE-MSCs.

forms, an improvement in neuronal differentiation, but not in cell migration.

of FX where dendritic spine abnormalities were found [200].

deficits, and hyperactivity[202].

494 Pluripotent Stem Cells

FX-cells.

indicating a developmentally dependent process.

In this chapter we have described the first successful attempts to harness the IPS technology for the generation of models for neurodegenerative diseases of the human nervous system. The key advantage of IPS based models over animal models is that they offer researchers for the first time a realistic chance to work in cell culture with large numbers of primary human cells that closely resemble the postmitotic neurons affected by neurodegeneration.

The first studies in which patient-derived disease-susceptible cellular phenotypes were compared with those of cells derived from healthy individuals, provide strong indications that such cellular models reflect key pathological molecular and cellular aspects of the neurological diseases. Therefore a future concept for patient-derived cellular models will be to correct neuronal malfunctions diseases by *in vitro* treatment of affected cells*.* A first such attempts aspect has been in the SMA models [91].

These *in vitro* treatments will include hypothesis driven approaches based on knowledge about pathophysiological mechanisms. Equally important patient derived lines will be used as *in vitro* assays for the screening of compound libraries. Drug safety screens with IPS cell-derived neurons will help to reduce the animal dependency of the current drug development pipeline. Finally, IPS cell technology will be an important driver of personalized medicine. Prior to patient treatment drug types and doses can be tested on patient-derived IPS cells or differen‐ tiated progenies in order to tailor a personalized curative approach according to the individual genetic and cellular profile.

There is even hope that the novel approach bypasses the laborious, time-consuming and expensive IPS cell generation by direct reprogramming of mouse and human somatic cells into functional neurons, called induced neurons (INs) [206;207], will come to fruition. Several groups have already generated dopaminergic INs [208;209] and motor INs [210]. Patientspecific INs could be generated to enhance the study of developmental disorders and other neurological diseases [211]. The significant decrease in time and resources to derive neurons directly from somatic cells justifies further investigation into this strategy.

But despite the enormous potential of IPS cell derived neurons for studies involving cell biological, physiological and pharmacological methods important question remain to be solved. One major drawback is that we still know very little about the specific cell biology of IPS cells and even less of their neuronal derivatives. This includes for example changes in chromatin structure and epigenetic signatures that accompany the reprogramming process. And there is exceedingly little information about membrane physiology of the IPS cell- derived neurons. Electrophysiological recordings and parallel studies of synaptic proteins and ion specific channel composition should be a focus of future research.

We have already pointed out the difficulties to design specific differentiation protocols for specific neuronal populations from IPS cells. The underlying hypothesis for all existing protocols is that cells should be guided through a shortcut version of embryonic development. A hindrance for progress in this regard is the lack of specific information of human embryonic development since most of our knowledge about vertebrate brain development derives from work with rodents. Recent reports about surprising differences between rodent and human developmental processes emphasize the demand for further comparative studies of human and rodent brain development [36;44].

[2] K. Takahashi, K. Tanabe, M. Ohnuki, M. Narita, T. Ichisaka, K. Tomoda, and S. Ya‐ manaka, "Induction of pluripotent stem cells from adult human fibroblasts by de‐

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases

http://dx.doi.org/10.5772/55772

497

[3] J. Yu, M. A. Vodyanik, K. Smuga-Otto, J. Antosiewicz-Bourget, J. L. Frane, S. Tian, J. Nie, G. A. Jonsdottir, V. Ruotti, R. Stewart, I. I. Slukvin, and J. A. Thomson, "Induced pluripotent stem cell lines derived from human somatic cells," *Science*, vol. 318, no.

[4] I. H. Park, N. Arora, H. Huo, N. Maherali, T. Ahfeldt, A. Shimamura, M. W. Lensch, C. Cowan, K. Hochedlinger, and G. Q. Daley, "Disease-specific induced pluripotent

[5] M. Nakagawa, M. Koyanagi, K. Tanabe, K. Takahashi, T. Ichisaka, T. Aoi, K. Okita, Y. Mochiduki, N. Takizawa, and S. Yamanaka, "Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts," *Nat. Biotechnol.*, vol. 26, no. 1,

[6] T. Aasen, A. Raya, M. J. Barrero, E. Garreta, A. Consiglio, F. Gonzalez, R. Vassena, J. Bilic, V. Pekarik, G. Tiscornia, M. Edel, S. Boue, and J. C. Izpisua Belmonte, "Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes,"

[7] A. Giorgetti, N. Montserrat, T. Aasen, F. Gonzalez, I. Rodriguez-Piza, R. Vassena, A. Raya, S. Boue, M. J. Barrero, B. A. Corbella, M. Torrabadella, A. Veiga, and J. C. Izpi‐ sua Belmonte, "Generation of induced pluripotent stem cells from human cord blood

[8] J. Cai, W. Li, H. Su, D. Qin, J. Yang, F. Zhu, J. Xu, W. He, X. Guo, K. Labuda, A. Peter‐ bauer, S. Wolbank, M. Zhong, Z. Li, W. Wu, K. F. So, H. Redl, L. Zeng, M. A. Esteban, and D. Pei, "Generation of human induced pluripotent stem cells from umbilical cord matrix and amniotic membrane mesenchymal cells," *J. Biol. Chem.*, vol. 285, no. 15,

[9] E. P. Papapetrou, M. J. Tomishima, S. M. Chambers, Y. Mica, E. Reed, J. Menon, V. Tabar, Q. Mo, L. Studer, and M. Sadelain, "Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and dif‐

ferentiation," *Proc Natl Acad Sci U S A*, vol. 106, no. 31, pp. 12759-12764, 2009.

[10] F. González, S. Boué, and J. C. I. Belmonte, "Methods for making induced pluripotent stem cells: reprogramming á la carte," *Nat Rev Genet*, vol. 12, no. 4, pp. 231-242, Apr.

[11] A. Hotta and J. Ellis, "Retroviral vector silencing during iPS cell induction: an epige‐ netic beacon that signals distinct pluripotent states," *J. Cell Biochem.*, vol. 105, no. 4,

[12] F. Soldner, D. Hockemeyer, C. Beard, Q. Gao, G. W. Bell, E. G. Cook, G. Hargus, A. Blak, O. Cooper, M. Mitalipova, O. Isacson, and R. Jaenisch, "Parkinson's disease pa‐

using OCT4 and SOX2," *Cell Stem Cell*, vol. 5, no. 4, pp. 353-357, Oct.2009.

fined factors," *Cell*, vol. 131, no. 5, pp. 861-872, 2007.

stem cells," *Cell*, vol. 134, no. 5, pp. 877-886, Sept.2008.

*Nat. Biotechnol.*, vol. 26, no. 11, pp. 1276-1284, Nov.2008.

5858, pp. 1917-1920, Dec.2007.

pp. 101-106, Jan.2008.

pp. 11227-11234, Apr.2010.

pp. 940-948, Nov.2008.

2011.

The biggest limitation of IPS cell models is that they do not offer straightforward possibilities to study functions of neurons *in vivo*, as parts of the brain circuitries that regulate higher brain functions and organismic behavior. Obviously, cellular models alone will never be able to produce clinically important read-outs, such as memory dysfunction and behavioral changes in AD, tremor, bradykinesia, and rigidity in PD, or reduced forced vital capacity, swallowing dysfunction, dysarthria, or limb motor impairment in ALS. Therefore, in the foreseeable future research on neurodegenerative diseases will combine *in vitro* and *in vivo* approaches. *In vivo* transplantation of stem cell derivatives in relevant animal models could bring additional information regarding the potential of hIPS cells for *in vivo* differentiation and their survival in a pathological brain environment. This is first exemplified in a study of directed differen‐ tiation of IPS cells to midbrain neurons and their transplantation into a rat model of PD, which led to functional recovery [64].

This result and many others that we summarized in this chapter raise hopes that IPS cells derived from affected and healthy human individuals will provide a unique opportunity to gain insights into the human pathophysiology and pharmacologic responses in yet incurable neurodegenerative diseases.

## **Acknowledgements**

This work was supported by SPIN FWF W1206-B05, Austria

## **Author details**

Roxana Nat1 , Andreas Eigentler2 and Georg Dechant1

1 Institute for Neuroscience, Innsbruck Medical University, Innsbruck, Austria

2 Department of Neurology, Innsbruck Medical University, Innsbruck, Austria

## **References**

[1] K. Takahashi and S. Yamanaka, "Induction of pluripotent stem cells from mouse em‐ bryonic and adult fibroblast cultures by defined factors," *Cell*, vol. 126, no. 4, pp. 663-676, 2006.

[2] K. Takahashi, K. Tanabe, M. Ohnuki, M. Narita, T. Ichisaka, K. Tomoda, and S. Ya‐ manaka, "Induction of pluripotent stem cells from adult human fibroblasts by de‐ fined factors," *Cell*, vol. 131, no. 5, pp. 861-872, 2007.

work with rodents. Recent reports about surprising differences between rodent and human developmental processes emphasize the demand for further comparative studies of human

The biggest limitation of IPS cell models is that they do not offer straightforward possibilities to study functions of neurons *in vivo*, as parts of the brain circuitries that regulate higher brain functions and organismic behavior. Obviously, cellular models alone will never be able to produce clinically important read-outs, such as memory dysfunction and behavioral changes in AD, tremor, bradykinesia, and rigidity in PD, or reduced forced vital capacity, swallowing dysfunction, dysarthria, or limb motor impairment in ALS. Therefore, in the foreseeable future research on neurodegenerative diseases will combine *in vitro* and *in vivo* approaches. *In vivo* transplantation of stem cell derivatives in relevant animal models could bring additional information regarding the potential of hIPS cells for *in vivo* differentiation and their survival in a pathological brain environment. This is first exemplified in a study of directed differen‐ tiation of IPS cells to midbrain neurons and their transplantation into a rat model of PD, which

This result and many others that we summarized in this chapter raise hopes that IPS cells derived from affected and healthy human individuals will provide a unique opportunity to gain insights into the human pathophysiology and pharmacologic responses in yet incurable

and Georg Dechant1

[1] K. Takahashi and S. Yamanaka, "Induction of pluripotent stem cells from mouse em‐ bryonic and adult fibroblast cultures by defined factors," *Cell*, vol. 126, no. 4, pp.

1 Institute for Neuroscience, Innsbruck Medical University, Innsbruck, Austria

2 Department of Neurology, Innsbruck Medical University, Innsbruck, Austria

and rodent brain development [36;44].

496 Pluripotent Stem Cells

led to functional recovery [64].

neurodegenerative diseases.

**Acknowledgements**

**Author details**

Roxana Nat1

**References**

663-676, 2006.

This work was supported by SPIN FWF W1206-B05, Austria

, Andreas Eigentler2


tient-derived induced pluripotent stem cells free of viral reprogramming factors," *Cell*, vol. 136, no. 5, pp. 964-977, Mar.2009.

[23] F. Anokye-Danso, C. Trivedi, D. Juhr, M. Gupta, Z. Cui, Y. Tian, Y. Zhang, W. Yang, P. Gruber, J. Epstein, and E. Morrisey, "Highly Efficient miRNA-Mediated Reprog‐ ramming of Mouse and Human Somatic Cells to Pluripotency," *Cell Stem Cell*, vol. 8,

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases

http://dx.doi.org/10.5772/55772

499

[24] N. Miyoshi, H. Ishii, H. Nagano, N. Haraguchi, D. Dewi, Y. Kano, S. Nishikawa, M. Tanemura, K. Mimori, F. Tanaka, T. Saito, J. Nishimura, I. Takemasa, T. Mizushima, M. Ikeda, H. Yamamoto, M. Sekimoto, Y. Doki, and M. Mori, "Reprogramming of Mouse and Human Cells to Pluripotency Using Mature MicroRNAs," *Cell Stem Cell*,

[25] H. Zhou, S. Wu, J. Y. Joo, S. Zhu, D. W. Han, T. Lin, S. Trauger, G. Bien, S. Yao, Y. Zhu, G. Siuzdak, H. R. Scholer, L. Duan, and S. Ding, "Generation of induced pluri‐ potent stem cells using recombinant proteins," *Cell Stem Cell*, vol. 4, no. 5, pp.

[26] H. Shimada, Y. Hashimoto, A. Nakada, K. Shigeno, and T. Nakamura, "Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia," *Biochem. Biophys. Res. Commun.*,

[27] D. Huangfu, R. Maehr, W. Guo, A. Eijkelenboom, M. Snitow, A. E. Chen, and D. A. Melton, "Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds," *Nat Biotech*, vol. 26, no. 7, pp. 795-797, July2008.

[28] Y. Shi, J. T. Do, C. Desponts, H. S. Hahm, H. R. Scholer, and S. Ding, "A combined chemical and genetic approach for the generation of induced pluripotent stem cells,"

[29] W. Li, W. Wei, S. Zhu, J. Zhu, Y. Shi, T. Lin, E. Hao, A. Hayek, H. Deng, and S. Ding, "Generation of Rat and Human Induced Pluripotent Stem Cells by Combining Genet‐ ic Reprogramming and Chemical Inhibitors," *Cell Stem Cell*, vol. 4, no. 1, pp. 16-19,

[30] J. Silva, O. Barrandon, J. Nichols, J. Kawaguchi, T. W. Theunissen, and A. Smith, "Promotion of Reprogramming to Ground State Pluripotency by Signal Inhibition,"

[31] T. Lin, R. Ambasudhan, X. Yuan, W. Li, S. Hilcove, R. Abujarour, X. Lin, H. S. Hahm, E. Hao, A. Hayek, and S. Ding, "A chemical platform for improved induction of hu‐

[32] N. Maherali and K. Hochedlinger, "Guidelines and techniques for the generation of induced pluripotent stem cells," *Cell Stem Cell*, vol. 3, no. 6, pp. 595-605, 2008.

[33] G. L. Boulting, E. Kiskinis, G. F. Croft, M. W. Amoroso, D. H. Oakley, B. J. Wainger, D. J. Williams, D. J. Kahler, M. Yamaki, L. Davidow, C. T. Rodolfa, J. T. Dimos, S. Mikkilineni, A. B. MacDermott, C. J. Woolf, C. E. Henderson, H. Wichterle, and K.

man iPSCs," *Nat. Methods*, vol. 6, no. 11, pp. 805-808, Nov.2009.

no. 4, pp. 376-388, Apr.2011.

vol. 8, no. 6, pp. 633-638, June2011.

vol. 417, no. 2, pp. 659-664, Jan.2012.

*Cell Stem Cell*, vol. 2, no. 6, pp. 525-528, June2008.

*PLoS Biol*, vol. 6, no. 10, p. e253, Oct.2008.

381-384, May2009.

Jan.2009.


[23] F. Anokye-Danso, C. Trivedi, D. Juhr, M. Gupta, Z. Cui, Y. Tian, Y. Zhang, W. Yang, P. Gruber, J. Epstein, and E. Morrisey, "Highly Efficient miRNA-Mediated Reprog‐ ramming of Mouse and Human Somatic Cells to Pluripotency," *Cell Stem Cell*, vol. 8, no. 4, pp. 376-388, Apr.2011.

tient-derived induced pluripotent stem cells free of viral reprogramming factors,"

[13] D. Hockemeyer, F. Soldner, E. G. Cook, Q. Gao, M. Mitalipova, and R. Jaenisch, "A drug-inducible system for direct reprogramming of human somatic cells to pluripo‐

[14] W. Zhou and C. R. Freed, "Adenoviral gene delivery can reprogram human fibro‐ blasts to induced pluripotent stem cells," *Stem Cells*, vol. 27, no. 11, pp. 2667-2674,

[15] T. Seki, S. Yuasa, M. Oda, T. Egashira, K. Yae, D. Kusumoto, H. Nakata, S. Tohyama, H. Hashimoto, M. Kodaira, Y. Okada, H. Seimiya, N. Fusaki, M. Hasegawa, and K. Fukuda, "Generation of Induced Pluripotent Stem Cells from Human Terminally Dif‐

ferentiated Circulating T Cells," *Cell Stem Cell*, vol. 7, no. 1, pp. 11-14, July2010.

[16] J. Yu, K. Hu, K. Smuga-Otto, S. Tian, R. Stewart, I. I. Slukvin, and J. A. Thomson, "Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences,"

[17] K. Okita, M. Nakagawa, H. Hyenjong, T. Ichisaka, and S. Yamanaka, "Generation of mouse induced pluripotent stem cells without viral vectors," *Science*, vol. 322, no.

[18] K. Woltjen, I. P. Michael, P. Mohseni, R. Desai, M. Mileikovsky, R. Hamalainen, R. Cowling, W. Wang, P. Liu, M. Gertsenstein, K. Kaji, H. K. Sung, and A. Nagy, "pig‐ gyBac transposition reprograms fibroblasts to induced pluripotent stem cells," *Na‐*

[19] C. A. Sommer, A. G. Sommer, T. A. Longmire, C. Christodoulou, D. D. Thomas, M. Gostissa, F. W. Alt, G. J. Murphy, D. N. Kotton, and G. Mostoslavsky, "Excision of Reprogramming Transgenes Improves the Differentiation Potential of iPS Cells Gen‐ erated with a Single Excisable Vector," *Stem Cells*, vol. 28, no. 1, pp. 64-74, 2010.

[20] L. Warren, P. D. Manos, T. Ahfeldt, Y. H. Loh, H. Li, F. Lau, W. Ebina, P. K. Mandal, Z. D. Smith, A. Meissner, G. Q. Daley, A. S. Brack, J. J. Collins, C. Cowan, T. M. Schlaeger, and D. J. Rossi, "Highly efficient reprogramming to pluripotency and di‐ rected differentiation of human cells with synthetic modified mRNA," *Cell Stem Cell*,

[21] S. L. Lin, D. C. Chang, C. H. Lin, S. Y. Ying, D. Leu, and D. T. Wu, "Regulation of somatic cell reprogramming through inducible mir-302 expression," *Nucleic Acids*

[22] K. N. Ivey, A. Muth, J. Arnold, F. W. King, R. F. Yeh, J. E. Fish, E. C. Hsiao, R. J. Schwartz, B. R. Conklin, H. S. Bernstein, and D. Srivastava, "MicroRNA regulation of cell lineages in mouse and human embryonic stem cells," *Cell Stem Cell*, vol. 2, no. 3,

*Cell*, vol. 136, no. 5, pp. 964-977, Mar.2009.

Nov.2009.

498 Pluripotent Stem Cells

tency," *Cell Stem Cell*, vol. 3, no. 3, pp. 346-353, 2008.

*Science*, vol. 324, no. 5928, pp. 797-801, May2009.

*ture*, vol. 458, no. 7239, pp. 766-770, Apr.2009.

vol. 7, no. 5, pp. 618-630, Nov.2010.

pp. 219-229, Mar.2008.

*Res.*, vol. 39, no. 3, pp. 1054-1065, Feb.2011.

5903, pp. 949-953, Nov.2008.


Eggan, "A functionally characterized test set of human induced pluripotent stem cells," *Nat Biotech*, vol. 29, no. 3, pp. 279-286, Mar.2011.

tion of polarized cortical tissues from ESCs and its active manipulation by extrinsic

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases

http://dx.doi.org/10.5772/55772

501

[46] N. Gaspard, T. Bouschet, R. Hourez, J. Dimidschstein, G. Naeije, J. van den Ameele, I. Espuny-Camacho, A. Herpoel, L. Passante, S. N. Schiffmann, A. Gaillard, and P. Van‐ derhaeghen, "An intrinsic mechanism of corticogenesis from embryonic stem cells,"

[47] N. Gaspard, T. Bouschet, A. Herpoel, G. Naeije, J. van den Ameele, and P. Vander‐ haeghen, "Generation of cortical neurons from mouse embryonic stem cells," *Nat.*

[48] M. Ideguchi, T. D. Palmer, L. D. Recht, and J. M. Weimann, "Murine embryonic stem cell-derived pyramidal neurons integrate into the cerebral cortex and appropriately project axons to subcortical targets," *J. Neurosci.*, vol. 30, no. 3, pp. 894-904, Jan.2010.

[49] A. M. Maroof, K. Brown, S. H. Shi, L. Studer, and S. A. Anderson, "Prospective isola‐ tion of cortical interneuron precursors from mouse embryonic stem cells," *J. Neuro‐*

[50] T. Danjo, M. Eiraku, K. Muguruma, K. Watanabe, M. Kawada, Y. Yanagawa, J. L. Ru‐ benstein, and Y. Sasai, "Subregional specification of embryonic stem cell-derived ventral telencephalic tissues by timed and combinatory treatment with extrinsic sig‐

[51] A. L. Goulburn, D. Alden, R. P. Davis, S. J. Micallef, E. S. Ng, Q. C. Yu, S. M. Lim, C. L. Soh, D. A. Elliott, T. Hatzistavrou, J. Bourke, B. Watmuff, R. J. Lang, J. M. Haynes, C. W. Pouton, A. Giudice, A. O. Trounson, S. A. Anderson, E. G. Stanley, and A. G. Elefanty, "A targeted NKX2.1 human embryonic stem cell reporter line enables iden‐ tification of human basal forebrain derivatives," *Stem Cells*, vol. 29, no. 3, pp. 462-473,

[52] A. L. Goulburn, E. G. Stanley, A. G. Elefanty, and S. A. Anderson, "Generating GA‐ BAergic cerebral cortical interneurons from mouse and human embryonic stem

[53] S. Cambray, C. Arber, G. Little, A. G. Dougalis, P. de, V, M. A. Ungless, M. Li, and T. A. Rodriguez, "Activin induces cortical interneuron identity and differentiation in embryonic stem cell-derived telencephalic neural precursors," *Nat. Commun.*, vol. 3,

[54] L. Wicklund, R. N. Leao, A. M. Stromberg, M. Mousavi, O. Hovatta, A. Nordberg, and A. Marutle, "Beta-amyloid 1-42 oligomers impair function of human embryonic stem cell-derived forebrain cholinergic neurons," *PLoS. One.*, vol. 5, no. 12, p. e15600,

signals," *Cell Stem Cell*, vol. 3, no. 5, pp. 519-532, Nov.2008.

*Nature*, vol. 455, no. 7211, pp. 351-357, Sept.2008.

*Protoc.*, vol. 4, no. 10, pp. 1454-1463, 2009.

*sci.*, vol. 30, no. 13, pp. 4667-4675, Mar.2010.

Mar.2011.

p. 841, 2012.

2010.

nals," *J. Neurosci.*, vol. 31, no. 5, pp. 1919-1933, Feb.2011.

cells," *Stem Cell Res.*, vol. 8, no. 3, pp. 416-426, May2012.


tion of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals," *Cell Stem Cell*, vol. 3, no. 5, pp. 519-532, Nov.2008.

[46] N. Gaspard, T. Bouschet, R. Hourez, J. Dimidschstein, G. Naeije, J. van den Ameele, I. Espuny-Camacho, A. Herpoel, L. Passante, S. N. Schiffmann, A. Gaillard, and P. Van‐ derhaeghen, "An intrinsic mechanism of corticogenesis from embryonic stem cells," *Nature*, vol. 455, no. 7211, pp. 351-357, Sept.2008.

Eggan, "A functionally characterized test set of human induced pluripotent stem

[34] M. Wernig, A. Meissner, R. Foreman, T. Brambrink, M. Ku, K. Hochedlinger, B. E. Bernstein, and R. Jaenisch, "In vitro reprogramming of fibroblasts into a pluripotent

[35] T. J. Petros, J. A. Tyson, and S. A. Anderson, "Pluripotent stem cells for the study of

[36] R. Nat, A. Salti, L. Suciu, S. Strom, and G. Dechant, "Pharmacological modulation of the Hedgehog pathway differentially affects dorsal/ventral patterning in mouse and human embryonic stem cell models of telencephalic development," *Stem Cells Dev.*,

[37] A. J. Levine and A. H. Brivanlou, "Proposal of a model of mammalian neural induc‐

[38] M. Gotz and W. B. Huttner, "The cell biology of neurogenesis," *Nat. Rev. Mol. Cell Bi‐*

[39] R. Nat, M. Nilbratt, S. Narkilahti, B. Winblad, O. Hovatta, and A. Nordberg, "Neuro‐ genic neuroepithelial and radial glial cells generated from six human embryonic stem cell lines in serum-free suspension and adherent cultures," *Glia*, vol. 55, no. 4,

[40] C. D. Stern, "Initial patterning of the central nervous system: how many organizers?,"

[41] T. Kudoh, S. W. Wilson, and I. B. Dawid, "Distinct roles for Fgf, Wnt and retinoic acid in posteriorizing the neural ectoderm," *Development*, vol. 129, no. 18, pp. 4335-4346,

[42] C. Patthey, L. Gunhaga, and T. Edlund, "Early development of the central and pe‐ ripheral nervous systems is coordinated by Wnt and BMP signals," *PLoS. One.*, vol. 3,

[43] K. Watanabe, D. Kamiya, A. Nishiyama, T. Katayama, S. Nozaki, H. Kawasaki, Y. Watanabe, K. Mizuseki, and Y. Sasai, "Directed differentiation of telencephalic pre‐ cursors from embryonic stem cells," *Nat Neurosci*, vol. 8, no. 3, pp. 288-296, Mar.2005.

[44] X. J. Li, X. Zhang, M. A. Johnson, Z. B. Wang, T. Lavaute, and S. C. Zhang, "Coordi‐ nation of sonic hedgehog and Wnt signaling determines ventral and dorsal telence‐ phalic neuron types from human embryonic stem cells," *Development*, vol. 136, no. 23,

[45] M. Eiraku, K. Watanabe, M. Matsuo-Takasaki, M. Kawada, S. Yonemura, M. Matsu‐ mura, T. Wataya, A. Nishiyama, K. Muguruma, and Y. Sasai, "Self-organized forma‐

cells," *Nat Biotech*, vol. 29, no. 3, pp. 279-286, Mar.2011.

tion," *Dev. Biol.*, vol. 308, no. 2, pp. 247-256, Aug.2007.

*Nat. Rev. Neurosci.*, vol. 2, no. 2, pp. 92-98, Feb.2001.

vol. 21, no. 7, pp. 1016-1046, May2012.

*ol.*, vol. 6, no. 10, pp. 777-788, Oct.2005.

pp. 385-399, 2007.

500 Pluripotent Stem Cells

Sept.2002.

no. 2, p. e1625, 2008.

pp. 4055-4063, Dec.2009.

ES-cell-like state," *Nature*, vol. 448, no. 7151, pp. 318-324, July2007.

CNS development," *Frontiers in Molecular Neuroscience*, vol. 4 2011.


[55] C. J. Bissonnette, L. Lyass, B. J. Bhattacharyya, A. Belmadani, R. J. Miller, and J. A. Kessler, "The controlled generation of functional basal forebrain cholinergic neurons from human embryonic stem cells," *Stem Cells*, vol. 29, no. 5, pp. 802-811, May2011.

and M. Stojkovic, "Efficient differentiation of human embryonic stem cells into func‐ tional cerebellar-like cells," *Stem Cells Dev.*, vol. 19, no. 11, pp. 1745-1756, Nov.2010.

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases

http://dx.doi.org/10.5772/55772

503

[67] H. L. Su, K. Muguruma, M. Matsuo-Takasaki, M. Kengaku, K. Watanabe, and Y. Sa‐ sai, "Generation of cerebellar neuron precursors from embryonic stem cells," *Dev. Bi‐*

[68] O. Tao, T. Shimazaki, Y. Okada, H. Naka, K. Kohda, M. Yuzaki, H. Mizusawa, and H. Okano, "Efficient generation of mature cerebellar Purkinje cells from mouse embry‐

[69] H. Wichterle, I. Lieberam, J. A. Porter, and T. M. Jessell, "Directed differentiation of embryonic stem cells into motor neurons," *Cell*, vol. 110, no. 3, pp. 385-397, Aug.2002.

[70] X. J. Li, Z. W. Du, E. D. Zarnowska, M. Pankratz, L. O. Hansen, R. A. Pearce, and S. C. Zhang, "Specification of motoneurons from human embryonic stem cells," *Nat Bio‐*

[71] P. Soundararajan, G. B. Miles, L. L. Rubin, R. M. Brownstone, and V. F. Rafuse, "Mo‐ toneurons derived from embryonic stem cells express transcription factors and de‐ velop phenotypes characteristic of medial motor column neurons," *J. Neurosci.*, vol.

[72] H. Lee, G. A. Shamy, Y. Elkabetz, C. M. Schofield, N. L. Harrsion, G. Panagiotakos, N. D. Socci, V. Tabar, and L. Studer, "Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons," *Stem Cells*, vol. 25, no. 8, pp.

[73] J. T. Dimos, K. T. Rodolfa, K. K. Niakan, L. M. Weisenthal, H. Mitsumoto, W. Chung, G. F. Croft, G. Saphier, R. Leibel, R. Goland, H. Wichterle, C. E. Henderson, and K. Eggan, "Induced pluripotent stem cells generated from patients with ALS can be dif‐ ferentiated into motor neurons," *Science*, vol. 321, no. 5893, pp. 1218-1221, Aug.2008.

[74] M. Peljto, J. S. Dasen, E. O. Mazzoni, T. M. Jessell, and H. Wichterle, "Functional di‐ versity of ESC-derived motor neuron subtypes revealed through intraspinal trans‐

[75] R. Patani, A. J. Hollins, T. M. Wishart, C. A. Puddifoot, S. Alvarez, A. R. de Lera, D. J. Wyllie, D. A. Compston, R. A. Pedersen, T. H. Gillingwater, G. E. Hardingham, N. D. Allen, and S. Chandran, "Retinoid-independent motor neurogenesis from human embryonic stem cells reveals a medial columnar ground state," *Nat Commun.*, vol. 2,

[76] H. W. Park, J. S. Cho, C. K. Park, S. J. Jung, C. H. Park, S. J. Lee, S. B. Oh, Y. S. Park, and M. S. Chang, "Directed Induction of Functional Motor Neuron-Like Cells from Genetically Engineered Human Mesenchymal Stem Cells," *PLoS One*, vol. 7, no. 4, p.

plantation," *Cell Stem Cell*, vol. 7, no. 3, pp. 355-366, Sept.2010.

onic stem cells," *J. Neurosci. Res.*, vol. 88, no. 2, pp. 234-247, Feb.2010.

*ol.*, vol. 290, no. 2, pp. 287-296, Feb.2006.

*tech*, vol. 23, no. 2, pp. 215-221, Feb.2005.

26, no. 12, pp. 3256-3268, Mar.2006.

1931-1939, Aug.2007.

p. 214, 2011.

e35244, Apr.2012.


and M. Stojkovic, "Efficient differentiation of human embryonic stem cells into func‐ tional cerebellar-like cells," *Stem Cells Dev.*, vol. 19, no. 11, pp. 1745-1756, Nov.2010.

[67] H. L. Su, K. Muguruma, M. Matsuo-Takasaki, M. Kengaku, K. Watanabe, and Y. Sa‐ sai, "Generation of cerebellar neuron precursors from embryonic stem cells," *Dev. Bi‐ ol.*, vol. 290, no. 2, pp. 287-296, Feb.2006.

[55] C. J. Bissonnette, L. Lyass, B. J. Bhattacharyya, A. Belmadani, R. J. Miller, and J. A. Kessler, "The controlled generation of functional basal forebrain cholinergic neurons from human embryonic stem cells," *Stem Cells*, vol. 29, no. 5, pp. 802-811, May2011.

[56] L. Aubry, A. Bugi, N. Lefort, F. Rousseau, M. Peschanski, and A. L. Perrier, "Striatal progenitors derived from human ES cells mature into DARPP32 neurons in vitro and in quinolinic acid-lesioned rats," *Proc. Natl. Acad. Sci. U. S. A*, vol. 105, no. 43, pp.

[57] N. Zhang, M. C. An, D. Montoro, and L. M. Ellerby, "Characterization of Human Huntington's Disease Cell Model from Induced Pluripotent Stem Cells," *PLoS. Curr.*,

[58] C. A. Fasano, S. M. Chambers, G. Lee, M. J. Tomishima, and L. Studer, "Efficient deri‐ vation of functional floor plate tissue from human embryonic stem cells," *Cell Stem*

[59] H. Kawasaki, K. Mizuseki, S. Nishikawa, S. Kaneko, Y. Kuwana, S. Nakanishi, S. I. Nishikawa, and Y. Sasai, "Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity," *Neuron*, vol. 28, no. 1, pp. 31-40, Oct.2000.

[60] S. H. Lee, N. Lumelsky, L. Studer, J. M. Auerbach, and R. D. McKay, "Efficient gener‐ ation of midbrain and hindbrain neurons from mouse embryonic stem cells," *Nat. Bi‐*

[61] A. L. Perrier, V. Tabar, T. Barberi, M. E. Rubio, J. Bruses, N. Topf, N. L. Harrison, and L. Studer, "Derivation of midbrain dopamine neurons from human embryonic stem

cells," *Proc. Natl. Acad. Sci. U. S. A*, vol. 101, no. 34, pp. 12543-12548, Aug.2004.

[62] Y. Yan, D. Yang, E. D. Zarnowska, Z. Du, B. Werbel, C. Valliere, R. A. Pearce, J. A. Thomson, and S. C. Zhang, "Directed differentiation of dopaminergic neuronal sub‐ types from human embryonic stem cells," *Stem Cells*, vol. 23, no. 6, pp. 781-790,

[63] S. M. Chambers, C. A. Fasano, E. P. Papapetrou, M. Tomishima, M. Sadelain, and L. Studer, "Highly efficient neural conversion of human ES and iPS cells by dual inhibi‐

[64] A. Sanchez-Danes, A. Consiglio, Y. Richaud, I. Rodriguez-Piza, B. Dehay, M. Edel, J. Bove, M. Memo, M. Vila, A. Raya, and J. C. Izpisua Belmonte, "Efficient generation of A9 midbrain dopaminergic neurons by lentiviral delivery of LMX1A in human em‐ bryonic stem cells and induced pluripotent stem cells," *Hum. Gene Ther.*, vol. 23, no.

[65] E. Salero and M. E. Hatten, "Differentiation of ES cells into cerebellar neurons," *Proc.*

[66] S. Erceg, M. Ronaghi, I. Zipancic, S. Lainez, M. G. Rosello, C. Xiong, V. Moreno-Man‐ zano, F. J. Rodriguez-Jimenez, R. Planells, M. Alvarez-Dolado, S. S. Bhattacharya,

*Natl. Acad. Sci. U. S. A*, vol. 104, no. 8, pp. 2997-3002, Feb.2007.

tion of SMAD signaling," *Nat. Biotechnol.*, vol. 27, no. 3, pp. 275-280, Mar.2009.

16707-16712, Oct.2008.

502 Pluripotent Stem Cells

vol. 2, p. RRN1193, 2010.

June2005.

1, pp. 56-69, Jan.2012.

*Cell*, vol. 6, no. 4, pp. 336-347, Apr.2010.

*otechnol.*, vol. 18, no. 6, pp. 675-679, June2000.


[77] Y. Liqing, G. Jia, C. Jiqing, G. Ran, C. Fei, K. Jie, W. Yanyun, and Z. Cheng, "Directed differentiation of motor neuron cell-like cells from human adipose-derived stem cells in vitro," *Neuroreport*, vol. 22, no. 8, pp. 370-373, June2011.

duced Pluripotent Stem Cells," *The Journal of Neuroscience*, vol. 31, no. 16, pp.

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases

http://dx.doi.org/10.5772/55772

505

[88] M. J. Devine, M. Ryten, P. Vodicka, A. J. Thomson, T. Burdon, H. Houlden, F. Cava‐ leri, M. Nagano, N. J. Drummond, J. W. Taanman, A. H. Schapira, K. Gwinn, J. Har‐ dy, P. A. Lewis, and T. Kunath, "Parkinson's disease induced pluripotent stem cells with triplication of the α-synuclein locus," *Nat Commun*, vol. 2, p. 440, Aug.2011.

[89] B. Byers, B. Cord, H. N. Nguyen, B. Schüle, L. Fenno, P. C. Lee, K. Deisseroth, J. W. Langston, R. R. Pera, and T. D. Palmer, "SNCA Triplication Parkinson's Patient's iPSC-derived DA Neurons Accumulate α-Synuclein and Are Susceptible to Oxida‐

[90] S. Camnasio, A. D. Carri, A. Lombardo, I. Grad, C. Mariotti, A. Castucci, B. Rozell, P. L. Riso, V. Castiglioni, C. Zuccato, C. Rochon, Y. Takashima, G. Diaferia, I. Biunno, C. Gellera, M. Jaconi, A. Smith, O. Hovatta, L. Naldini, D. S. Di, A. Feki, and E. Catta‐ neo, "The first reported generation of several induced pluripotent stem cell lines from homozygous and heterozygous Huntington's disease patients demonstrates mutation related enhanced lysosomal activity," *Neurobiol. Dis.*, vol. 46, no. 1, pp.

[91] A. D. Ebert, J. Yu, F. F. Rose, V. B. Mattis, C. L. Lorson, J. A. Thomson, and C. N. Svendsen, "Induced pluripotent stem cells from a spinal muscular atrophy patient,"

[92] D. Sareen, A. D. Ebert, B. M. Heins, J. V. McGivern, L. Ornelas, and C. N. Svendsen, "Inhibition of Apoptosis Blocks Human Motor Neuron Cell Death in a Stem Cell Model of Spinal Muscular Atrophy," *PLoS ONE*, vol. 7, no. 6, p. e39113, June2012.

[93] S. Ku, E. Soragni, E. Campau, E. A. Thomas, G. Altun, L. C. Laurent, J. F. Loring, M. Napierala, and J. M. Gottesfeld, "Friedreich's ataxia induced pluripotent stem cells model intergenerational GAATTC triplet repeat instability," *Cell Stem Cell*, vol. 7, no.

[94] J. Liu, P. J. Verma, M. V. Evans-Galea, M. B. Delatycki, A. Michalska, J. Leung, D. Crombie, J. P. Sarsero, R. Williamson, M. Dottori, and A. Pebay, "Generation of in‐ duced pluripotent stem cell lines from Friedreich ataxia patients," *Stem Cell Rev.*, vol.

[95] P. Koch, P. Breuer, M. Peitz, J. Jungverdorben, J. Kesavan, D. Poppe, J. Doerr, J. Lade‐ wig, J. Mertens, T. Tuting, P. Hoffmann, T. Klockgether, B. O. Evert, U. Wullner, and O. Brustle, "Excitation-induced ataxin-3 aggregation in neurons from patients with

[96] G. Lee, E. P. Papapetrou, H. Kim, S. M. Chambers, M. J. Tomishima, C. A. Fasano, Y. M. Ganat, J. Menon, F. Shimizu, A. Viale, V. Tabar, M. Sadelain, and L. Studer, "Mod‐ elling pathogenesis and treatment of familial dysautonomia using patient-specific

Machado-Joseph disease," *Nature*, vol. 480, no. 7378, pp. 543-546, Dec.2011.

iPSCs," *Nature*, vol. 461, no. 7262, pp. 402-406, 2009.

tive Stress," *PLoS ONE*, vol. 6, no. 11, p. e26159, Nov.2011.

*Nature*, vol. 457, no. 7227, pp. 277-280, Jan.2009.

5970-5976, Apr.2011.

41-51, Apr.2012.

5, pp. 631-637, Nov.2010.

7, no. 3, pp. 703-713, Sept.2011.


duced Pluripotent Stem Cells," *The Journal of Neuroscience*, vol. 31, no. 16, pp. 5970-5976, Apr.2011.

[88] M. J. Devine, M. Ryten, P. Vodicka, A. J. Thomson, T. Burdon, H. Houlden, F. Cava‐ leri, M. Nagano, N. J. Drummond, J. W. Taanman, A. H. Schapira, K. Gwinn, J. Har‐ dy, P. A. Lewis, and T. Kunath, "Parkinson's disease induced pluripotent stem cells with triplication of the α-synuclein locus," *Nat Commun*, vol. 2, p. 440, Aug.2011.

[77] Y. Liqing, G. Jia, C. Jiqing, G. Ran, C. Fei, K. Jie, W. Yanyun, and Z. Cheng, "Directed differentiation of motor neuron cell-like cells from human adipose-derived stem cells

[78] A. K. Murashov, E. S. Pak, W. A. Hendricks, J. P. Owensby, P. L. Sierpinski, L. M. Tatko, and P. L. Fletcher, "Directed differentiation of embryonic stem cells into dorsal

[79] G. Lee, S. M. Chambers, M. J. Tomishima, and L. Studer, "Derivation of neural crest cells from human pluripotent stem cells," *Nat Protoc.*, vol. 5, no. 4, pp. 688-701, Apr.

[80] L. Menendez, T. A. Yatskievych, P. B. Antin, and S. Dalton, "Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells," *Proceedings of the National Academy of Sciences*, vol. 108,

[81] R. S. Goldstein, O. Pomp, I. Brokhman, and L. Ziegler, "Generation of neural crest cells and peripheral sensory neurons from human embryonic stem cells," *Methods*

[82] S. Han, L. Williams, and K. Eggan, "Constructing and Deconstructing Stem Cell Models of Neurological Disease," *Neuron*, vol. 70, no. 4, pp. 626-644, May2011.

[83] T. Yagi, D. Ito, Y. Okada, W. Akamatsu, Y. Nihei, T. Yoshizaki, S. Yamanaka, H. Oka‐ no, and N. Suzuki, "Modeling familial Alzheimer's disease with induced pluripotent

[84] M. A. Israel, S. H. Yuan, C. Bardy, S. M. Reyna, Y. Mu, C. Herrera, M. P. Hefferan, S. Van Gorp, K. L. Nazor, F. S. Boscolo, C. T. Carson, L. C. Laurent, M. Marsala, F. H. Gage, A. M. Remes, E. H. Koo, and L. S. B. Goldstein, "Probing sporadic and familial Alzheimer/'s disease using induced pluripotent stem cells," *Nature*, vol. advance on‐

[85] G. Hargus, O. Cooper, M. Deleidi, A. Levy, K. Lee, E. Marlow, A. Yow, F. Soldner, D. Hockemeyer, P. J. Hallett, T. Osborn, R. Jaenisch, and O. Isacson, "Differentiated Par‐ kinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats," *Proc. Natl. Acad. Sci. U. S. A*, vol.

[86] H. Nguyen, B. Byers, B. Cord, A. Shcheglovitov, J. Byrne, P. Gujar, K. Kee, B. Schüle, R. Dolmetsch, W. Langston, T. Palmer, and R. Pera, "LRRK2 Mutant iPSC-Derived DA Neurons Demonstrate Increased Susceptibility to Oxidative Stress," *Cell Stem*

[87] P. Seibler, J. Graziotto, H. Jeong, F. Simunovic, C. Klein, and D. Krainc, "Mitochon‐ drial Parkin Recruitment Is Impaired in Neurons Derived from Mutant PINK1 In‐

stem cells," *Human Molecular Genetics*, vol. 20, no. 23, pp. 4530-4539, Dec.2011.

in vitro," *Neuroreport*, vol. 22, no. 8, pp. 370-373, June2011.

interneurons," *The FASEB Journal*, Nov.2004.

no. 48, pp. 19240-19245, Nov.2011.

*Mol. Biol.*, vol. 584, pp. 283-300, 2010.

line publication Jan.2012.

107, no. 36, pp. 15921-15926, Sept.2010.

*Cell*, vol. 8, no. 3, pp. 267-280, Mar.2011.

2010.

504 Pluripotent Stem Cells


[97] A. Urbach, O. Bar-Nur, G. Q. Daley, and N. Benvenisty, "Differential Modeling of Fragile X Syndrome by Human Embryonic Stem Cells and Induced Pluripotent Stem Cells," *Cell Stem Cell*, vol. 6, no. 5, pp. 407-411, May2010.

[111] M. H. Polymeropoulos, C. Lavedan, E. Leroy, S. E. Ide, A. Dehejia, A. Dutra, B. Pike, H. Root, J. Rubenstein, R. Boyer, E. S. Stenroos, S. Chandrasekharappa, A. Athanas‐ siadou, T. Papapetropoulos, W. G. Johnson, A. M. Lazzarini, R. C. Duvoisin, G. Di Iorio, L. I. Golbe, and R. L. Nussbaum, "Mutation in the +¦-Synuclein Gene Identified in Families with Parkinson's Disease," *Science*, vol. 276, no. 5321, pp. 2045-2047,

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases

http://dx.doi.org/10.5772/55772

507

[112] A. B. Singleton, M. Farrer, J. Johnson, A. Singleton, S. Hague, J. Kachergus, M. Huli‐ han, T. Peuralinna, A. Dutra, R. Nussbaum, S. Lincoln, A. Crawley, M. Hanson, D. Maraganore, C. Adler, M. R. Cookson, M. Muenter, M. Baptista, D. Miller, J. Blanca‐ to, J. Hardy, and K. Gwinn-Hardy, "+¦-Synuclein Locus Triplication Causes Parkin‐

[113] W. P. Gilks, P. M. Abou-Sleiman, S. Gandhi, S. Jain, A. Singleton, A. J. Lees, K. Shaw, K. P. Bhatia, V. Bonifati, N. P. Quinn, J. Lynch, D. G. Healy, J. L. Holton, T. Revesz, and N. W. Wood, "A common LRRK2 mutation in idiopathic Parkinson's disease,"

[114] T. Kitada, S. Asakawa, N. Hattori, H. Matsumine, Y. Yamamura, S. Minoshima, M. Yokochi, Y. Mizuno, and N. Shimizu, "Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism," *Nature*, vol. 392, no. 6676, pp. 605-608, Apr.1998.

[115] E. M. Valente, P. M. Abou-Sleiman, V. Caputo, M. M. K. Muqit, K. Harvey, S. Gis‐ pert, Z. Ali, D. Del Turco, A. R. Bentivoglio, D. G. Healy, A. Albanese, R. Nussbaum, R. González-Maldonado, T. Deller, S. Salvi, P. Cortelli, W. P. Gilks, D. S. Latchman, R. J. Harvey, B. Dallapiccola, G. Auburger, and N. W. Wood, "Hereditary Early-On‐ set Parkinson's Disease Caused by Mutations in PINK1," *Science*, vol. 304, no. 5674,

[116] V. Bonifati, P. Rizzu, M. J. van Baren, O. Schaap, G. J. Breedveld, E. Krieger, M. C. J. Dekker, F. Squitieri, P. Ibanez, M. Joosse, J. W. van Dongen, N. Vanacore, J. C. van Swieten, A. Brice, G. Meco, C. M. van Duijn, B. A. Oostra, and P. Heutink, "Mutations in the DJ-1 Gene Associated with Autosomal Recessive Early-Onset Parkinsonism,"

[117] J. Neumann, J. Bras, E. Deas, S. S. O'Sullivan, L. Parkkinen, R. H. Lachmann, A. Li, J. Holton, R. Guerreiro, R. Paudel, B. Segarane, A. Singleton, A. Lees, J. Hardy, H. Houlden, T. Revesz, and N. W. Wood, "Glucocerebrosidase mutations in clinical and pathologically proven Parkinson's disease," *Brain*, vol. 132, no. 7, pp. 1783-1794, Ju‐

[118] "A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The Huntington's Disease Collaborative Re‐

son's Disease," *Science*, vol. 302, no. 5646, p. 841, Oct.2003.

*The Lancet*, vol. 365, no. 9457, pp. 415-416, Jan.2005.

*Science*, vol. 299, no. 5604, pp. 256-259, Jan.2003.

search Group," *Cell*, vol. 72, no. 6, pp. 971-983, Mar.1993.

pp. 1158-1160, May2004.

ly2009.

June1997.


[111] M. H. Polymeropoulos, C. Lavedan, E. Leroy, S. E. Ide, A. Dehejia, A. Dutra, B. Pike, H. Root, J. Rubenstein, R. Boyer, E. S. Stenroos, S. Chandrasekharappa, A. Athanas‐ siadou, T. Papapetropoulos, W. G. Johnson, A. M. Lazzarini, R. C. Duvoisin, G. Di Iorio, L. I. Golbe, and R. L. Nussbaum, "Mutation in the +¦-Synuclein Gene Identified in Families with Parkinson's Disease," *Science*, vol. 276, no. 5321, pp. 2045-2047, June1997.

[97] A. Urbach, O. Bar-Nur, G. Q. Daley, and N. Benvenisty, "Differential Modeling of Fragile X Syndrome by Human Embryonic Stem Cells and Induced Pluripotent Stem

[98] H. Braak and E. Braak, "Frequency of Stages of Alzheimer-Related Lesions in Differ‐ ent Age Categories," *Neurobiology of Aging*, vol. 18, no. 4, pp. 351-357, July1997.

[99] D. J. Selkoe, "Alzheimer's Disease Is a Synaptic Failure," *Science*, vol. 298, no. 5594,

[100] L. M. Ittner and J. Götz, "Amyloid-β and tau - a toxic pas de deux in Alzheimer's dis‐

[101] T. Iwatsubo, T. C. Saido, D. M. Mann, V. M. Lee, and J. Q. Trojanowski, "Full-length amyloid-beta (1-42(43)) and amino-terminally modified and truncated amyloid-beta 42(43) deposit in diffuse plaques," *Am. J. Pathol.*, vol. 149, no. 6, pp. 1823-1830, Dec.

[102] A. Alonso, I. Grundke-Iqbal, H. Barra, and K. Iqbal, "Abnormal phosphorylation of tau and the mechanism of Alzheimer neurofibrillary degeneration: Sequestration of microtubule-associated proteins 1 and 2 and the disassembly of microtubules by the abnormalGÇëtau," *Proceedings of the National Academy of Sciences*, vol. 94, no. 1, pp.

[103] E. Giacobini, "Cholinergic function and Alzheimer's disease," *Int. J. Geriat. Psychiatry*,

[104] J. T. Coyle, D. L. Price, and M. R. DeLong, "Alzheimer's disease: a disorder of cortical cholinergic innervation," *Science*, vol. 219, no. 4589, pp. 1184-1190, Mar.1983.

[105] W. J. Strittmatter and A. D. Roses, "Apolipoprotein E and Alzheimer disease," *Pro‐ ceedings of the National Academy of Sciences*, vol. 92, no. 11, pp. 4725-4727, May1995.

[106] M. C. Chartier-Harlin, F. Crawford, H. Houlden, A. Warren, D. Hughes, L. Fidani, A. Goate, M. Rossor, P. Roques, J. Hardy, and M. Mullan, "Early-onset Alzheimer's dis‐ ease caused by mutations at codon 717 of the [beta]-amyloid precursor protein gene,"

[107] R. E. Tanzi, D. M. Kovacs, T. W. Kim, R. D. Moir, S. Y. Guenette, and W. Wasco, "The gene defects responsible for familial Alzheimer's disease," *Neurobiol. Dis.*, vol. 3, no.

[108] J. Gotz and L. M. Ittner, "Animal models of Alzheimer's disease and frontotemporal

[109] D. Van Dam and P. P. De Deyn, "Drug discovery in dementia: the role of rodent

[110] S. Lesage and A. Brice, "Parkinson's disease: from monogenic forms to genetic sus‐ ceptibility factors," *Hum. Mol. Genet.*, vol. 18, no. R1, p. R48-R59, Apr.2009.

dementia," *Nat Rev Neurosci*, vol. 9, no. 7, pp. 532-544, July2008.

models," *Nat Rev Drug Discov*, vol. 5, no. 11, pp. 956-970, Nov.2006.

Cells," *Cell Stem Cell*, vol. 6, no. 5, pp. 407-411, May2010.

ease," *Nat Rev Neurosci*, vol. 12, no. 2, pp. 67-72, Feb.2011.

pp. 789-791, Oct.2002.

1996.

506 Pluripotent Stem Cells

298-303, Jan.1997.

3, pp. 159-168, 1996.

vol. 18, no. S1, p. S1-S5, 2003.

*Nature*, vol. 353, no. 6347, pp. 844-846, Oct.1991.


[119] Rubinsztein DC, "Molecular biology of Huntington's disease (HD) and HD-like dis‐ orders.," in *Genetics of Movement Disorders*. Pulst S, Ed. San Diego: Academic Press, 2003, pp. 365-377.

[132] M. F. Beal, R. J. Ferrante, K. J. Swartz, and N. W. Kowall, "Chronic quinolinic acid lesions in rats closely resemble Huntington's disease," *J. Neurosci.*, vol. 11, no. 6, pp.

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases

http://dx.doi.org/10.5772/55772

509

[133] M. F. Beal, E. Brouillet, B. G. Jenkins, R. J. Ferrante, N. W. Kowall, J. M. Miller, E. Storey, R. Srivastava, B. R. Rosen, and B. T. Hyman, "Neurochemical and histologic characterization of striatal excitotoxic lesions produced by the mitochondrial toxin 3-

[134] J. T. Coyle and R. Schwarcz, "Lesion of striatal neurones with kainic acid provides a model for Huntington's chorea," *Nature*, vol. 263, no. 5574, pp. 244-246, Sept.1976.

[135] S. T. Lee, K. Chu, J. E. Park, K. Lee, L. Kang, S. U. Kim, and M. Kim, "Intravenous administration of human neural stem cells induces functional recovery in Hunting‐

[136] J. K. Ryu, J. Kim, S. J. Cho, K. Hatori, A. Nagai, H. B. Choi, M. C. Lee, J. G. McLarnon, and S. U. Kim, "Proactive transplantation of human neural stem cells prevents degen‐ eration of striatal neurons in a rat model of Huntington disease," *Neurobiol. Dis.*, vol.

[137] M. Kim, S. T. Lee, K. Chu, and S. U. Kim, "Stem cell-based cell therapy for Hunting‐

[138] C. K. Bradley, H. A. Scott, O. Chami, T. T. Peura, B. Dumevska, U. Schmidt, and T. Stojanov, "Derivation of Huntington's disease-affected human embryonic stem cell

[139] S. Camnasio, A. D. Carri, A. Lombardo, I. Grad, C. Mariotti, A. Castucci, B. Rozell, P. L. Riso, V. Castiglioni, C. Zuccato, C. Rochon, Y. Takashima, G. Diaferia, I. Biunno, C. Gellera, M. Jaconi, A. Smith, O. Hovatta, L. Naldini, S. Di Donato, A. Feki, and E. Cattaneo, "The first reported generation of several induced pluripotent stem cell lines from homozygous and heterozygous Huntington's disease patients demonstrates mutation related enhanced lysosomal activity," *Neurobiology of Disease*, vol. 46, no. 1,

[140] S. Lefebvre, L. Burglen, S. Reboullet, O. Clermont, P. Burlet, L. Viollet, B. Benichou, C. Cruaud, P. Millasseau, M. Zeviani, and ., "Identification and characterization of a spinal muscular atrophy-determining gene," *Cell*, vol. 80, no. 1, pp. 155-165, Jan.1995.

[141] S. Lefebvre, L. Burglen, J. Frezal, A. Munnich, and J. Melki, "The role of the SMN gene in proximal spinal muscular atrophy," *Hum. Mol. Genet.*, vol. 7, no. 10, pp.

[142] D. D. Coovert, T. T. Le, P. E. McAndrew, J. Strasswimmer, T. O. Crawford, J. R. Men‐ dell, S. E. Coulson, E. J. Androphy, T. W. Prior, and A. H. Burghes, "The survival mo‐ tor neuron protein in spinal muscular atrophy," *Hum. Mol. Genet.*, vol. 6, no. 8, pp.

ton disease: A review," *Neuropathology*, vol. 28, no. 1, pp. 1-9, 2008.

lines," *Stem Cells Dev.*, vol. 20, no. 3, pp. 495-502, Mar.2011.

ton's disease rat model," *Neurosci. Res.*, vol. 52, no. 3, pp. 243-249, July2005.

nitropropionic acid," *J. Neurosci.*, vol. 13, no. 10, pp. 4181-4192, Oct.1993.

1649-1659, June1991.

16, no. 1, pp. 68-77, June2004.

pp. 41-51, Apr.2012.

1531-1536, 1998.

1205-1214, Aug.1997.


[132] M. F. Beal, R. J. Ferrante, K. J. Swartz, and N. W. Kowall, "Chronic quinolinic acid lesions in rats closely resemble Huntington's disease," *J. Neurosci.*, vol. 11, no. 6, pp. 1649-1659, June1991.

[119] Rubinsztein DC, "Molecular biology of Huntington's disease (HD) and HD-like dis‐ orders.," in *Genetics of Movement Disorders*. Pulst S, Ed. San Diego: Academic Press,

[120] C. M. Everett and N. W. Wood, "Trinucleotide repeats and neurodegenerative dis‐

[121] I. J. Mitchell, A. J. Cooper, and M. R. Griffiths, "The selective vulnerability of striato‐

[122] N. F. H. S. Gutekunst C, "The neuropathology of Huntington's disease," in *Hunting‐ ton's disease*. H. P. J. L. e. Bates G, Ed. New York: Oxford University Press, 2002, pp.

[123] M. DiFiglia, E. Sapp, K. O. Chase, S. W. Davies, G. P. Bates, J. P. Vonsattel, and N. Aronin, "Aggregation of huntingtin in neuronal intranuclear inclusions and dystro‐

[124] S. W. Davies, M. Turmaine, B. A. Cozens, M. DiFiglia, A. H. Sharp, C. A. Ross, E. Scherzinger, E. E. Wanker, L. Mangiarini, and G. P. Bates, "Formation of neuronal in‐ tranuclear inclusions underlies the neurological dysfunction in mice transgenic for

[125] M. Arrasate, S. Mitra, E. S. Schweitzer, M. R. Segal, and S. Finkbeiner, "Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death,"

[126] H. Mukai, T. Isagawa, E. Goyama, S. Tanaka, N. F. Bence, A. Tamura, Y. Ono, and R. R. Kopito, "Formation of morphologically similar globular aggregates from diverse aggregation-prone proteins in mammalian cells," *Proc. Natl. Acad. Sci. U. S. A*, vol.

[127] J. M. Gil and A. C. Rego, "Mechanisms of neurodegeneration in Huntington's dis‐

[128] C. A. Ross and S. J. Tabrizi, "Huntington's disease: from molecular pathogenesis to

[129] P. Kumar, H. Kalonia, and A. Kumar, "Huntington's disease: pathogenesis to animal

[130] F. O. Walker, "Huntington's Disease," *Semin. Neurol.*, vol. 27, no. 2, pp. 143-150, Apr.

[131] L. Mangiarini, K. Sathasivam, M. Seller, B. Cozens, A. Harper, C. Hetherington, M. Lawton, Y. Trottier, H. Lehrach, S. W. Davies, and G. P. Bates, "Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological

phenotype in transgenic mice," *Cell*, vol. 87, no. 3, pp. 493-506, Nov.1996.

phic neurites in brain," *Science*, vol. 277, no. 5334, pp. 1990-1993, Sept.1997.

the HD mutation," *Cell*, vol. 90, no. 3, pp. 537-548, Aug.1997.

ease," *Eur. J. Neurosci.*, vol. 27, no. 11, pp. 2803-2820, June2008.

models," *Pharmacol. Rep.*, vol. 62, no. 1, pp. 1-14, Jan.2010.

clinical treatment," *Lancet Neurol.*, vol. 10, no. 1, pp. 83-98, Jan.2011.

*Nature*, vol. 431, no. 7010, pp. 805-810, Oct.2004.

102, no. 31, pp. 10887-10892, Aug.2005.

pallidal neurons," *Prog. Neurobiol.*, vol. 59, no. 6, pp. 691-719, Dec.1999.

ease," *Brain*, vol. 127, no. Pt 11, pp. 2385-2405, Nov.2004.

2003, pp. 365-377.

508 Pluripotent Stem Cells

251-275.


[143] U. R. Monani, C. L. Lorson, D. W. Parsons, T. W. Prior, E. J. Androphy, A. H. Burghes, and J. D. McPherson, "A single nucleotide difference that alters splicing pat‐ terns distinguishes the SMA gene SMN1 from the copy gene SMN2," *Hum. Mol. Gen‐ et.*, vol. 8, no. 7, pp. 1177-1183, July1999.

and sporadic amyotrophic lateral sclerosis," *Science*, vol. 319, no. 5870, pp. 1668-1672,

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases

http://dx.doi.org/10.5772/55772

511

[154] M. Neumann, D. M. Sampathu, L. K. Kwong, A. C. Truax, M. C. Micsenyi, T. T. Chou, J. Bruce, T. Schuck, M. Grossman, C. M. Clark, L. F. McCluskey, B. L. Miller, E. Masliah, I. R. Mackenzie, H. Feldman, W. Feiden, H. A. Kretzschmar, J. Q. Trojanow‐ ski, and V. M. Lee, "Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis," *Science*, vol. 314, no. 5796, pp. 130-133, Oct.2006.

[155] J. Lowe, "New pathological findings in amyotrophic lateral sclerosis," *J. Neurol. Sci.*,

[156] M. Nagai, D. B. Re, T. Nagata, A. Chalazonitis, T. M. Jessell, H. Wichterle, and S. Przedborski, "Astrocytes expressing ALS-linked mutated SOD1 release factors selec‐ tively toxic to motor neurons," *Nat Neurosci*, vol. 10, no. 5, pp. 615-622, May2007.

[157] A. Bento-Abreu, D. P. Van, L. Van Den Bosch, and W. Robberecht, "The neurobiology of amyotrophic lateral sclerosis," *Eur. J. Neurosci*, vol. 31, no. 12, pp. 2247-2265,

[158] L. Van Den Bosch, "Genetic rodent models of amyotrophic lateral sclerosis," *J. Bi‐*

[159] M. A. Gama Sosa, G. R. De, and G. A. Elder, "Modeling human neurodegenerative diseases in transgenic systems," *Hum. Genet.*, vol. 131, no. 4, pp. 535-563, Apr.2012.

[160] J. R. Thonhoff, L. Ojeda, and P. Wu, "Stem cell-derived motor neurons: applications and challenges in amyotrophic lateral sclerosis," *Curr. Stem Cell Res. Ther.*, vol. 4, no.

[161] J. T. Dimos, K. T. Rodolfa, K. K. Niakan, L. M. Weisenthal, H. Mitsumoto, W. Chung, G. F. Croft, G. Saphier, R. Leibel, R. Goland, H. Wichterle, C. E. Henderson, and K. Eggan, "Induced Pluripotent Stem Cells Generated from Patients with ALS Can Be Differentiated into Motor Neurons," *Science*, vol. 321, no. 5893, pp. 1218-1221, Aug.

[162] A. E. Harding, "Classification of the hereditary ataxias and paraplegias," *Lancet*, vol.

[163] A. E. Harding, "Friedreich's ataxia: a clinical and genetic study of 90 families with an analysis of early diagnostic criteria and intrafamilial clustering of clinical features,"

[164] J. B. Schulz, S. Boesch, K. Bük, A. Dürr, P. Giunti, C. Mariotti, F. Pousset, L. Schöls, P. Vankan, and M. Pandolfo, "Diagnosis and treatment of Friedreich ataxia: a European

[165] M. Pandolfo, "Friedreich ataxia: the clinical picture," *J. Neurol.*, vol. 256 Suppl 1, pp.

Mar.2008.

June2010.

2008.

3-8, Mar.2009.

vol. 124 Suppl, pp. 38-51, July1994.

*omed. Biotechnol.*, vol. 2011, p. 348765, 2011.

3, pp. 178-199, Sept.2009.

1, no. 8334, pp. 1151-1155, May1983.

*Brain*, vol. 104, no. 3, pp. 589-620, Sept.1981.

perspective," *Nat Rev Neurol*, vol. 5, no. 4, pp. 222-234, 2009.


and sporadic amyotrophic lateral sclerosis," *Science*, vol. 319, no. 5870, pp. 1668-1672, Mar.2008.

[154] M. Neumann, D. M. Sampathu, L. K. Kwong, A. C. Truax, M. C. Micsenyi, T. T. Chou, J. Bruce, T. Schuck, M. Grossman, C. M. Clark, L. F. McCluskey, B. L. Miller, E. Masliah, I. R. Mackenzie, H. Feldman, W. Feiden, H. A. Kretzschmar, J. Q. Trojanow‐ ski, and V. M. Lee, "Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis," *Science*, vol. 314, no. 5796, pp. 130-133, Oct.2006.

[143] U. R. Monani, C. L. Lorson, D. W. Parsons, T. W. Prior, E. J. Androphy, A. H. Burghes, and J. D. McPherson, "A single nucleotide difference that alters splicing pat‐ terns distinguishes the SMA gene SMN1 from the copy gene SMN2," *Hum. Mol. Gen‐*

[144] C. L. Lorson, E. Hahnen, E. J. Androphy, and B. Wirth, "A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy," *Proc.*

[145] U. R. Monani, M. Sendtner, D. D. Coovert, D. W. Parsons, C. Andreassi, T. T. Le, S. Jablonka, B. Schrank, W. Rossoll, T. W. Prior, G. E. Morris, and A. H. Burghes, "The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(-/-) mice and results in a mouse with spinal muscular atrophy," *Hum. Mol.*

[146] A. H. Burghes and C. E. Beattie, "Spinal muscular atrophy: why do low levels of sur‐ vival motor neuron protein make motor neurons sick?," *Nat. Rev. Neurosci.*, vol. 10,

[147] U. R. Monani, "Spinal muscular atrophy: a deficiency in a ubiquitous protein; a mo‐ tor neuron-specific disease," *Neuron*, vol. 48, no. 6, pp. 885-896, Dec.2005.

[148] B. Schrank, R. Gotz, J. M. Gunnersen, J. M. Ure, K. V. Toyka, A. G. Smith, and M. Sendtner, "Inactivation of the survival motor neuron gene, a candidate gene for hu‐ man spinal muscular atrophy, leads to massive cell death in early mouse embryos,"

[149] H. M. Hsieh-Li, J. G. Chang, Y. J. Jong, M. H. Wu, N. M. Wang, C. H. Tsai, and H. Li, "A mouse model for spinal muscular atrophy," *Nat. Genet.*, vol. 24, no. 1, pp. 66-70,

[150] U. R. Monani, M. Sendtner, D. D. Coovert, D. W. Parsons, C. Andreassi, T. T. Le, S. Jablonka, B. Schrank, W. Rossoll, T. W. Prior, G. E. Morris, and A. H. Burghes, "The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(-/-) mice and results in a mouse with spinal muscular atrophy," *Hum. Mol.*

[151] L. I. Bruijn, T. M. Miller, and D. W. Cleveland, "Unraveling the mechanisms involved in motor neuron degeneration in ALS," *Annu. Rev. Neurosci.*, vol. 27, pp. 723-749,

[152] D. R. Rosen, T. Siddique, D. Patterson, D. A. Figlewicz, P. Sapp, A. Hentati, D. Do‐ naldson, J. Goto, J. P. O'Regan, H. X. Deng, and ., "Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis," *Nature*,

[153] J. Sreedharan, I. P. Blair, V. B. Tripathi, X. Hu, C. Vance, B. Rogelj, S. Ackerley, J. C. Durnall, K. L. Williams, E. Buratti, F. Baralle, B. J. de, J. D. Mitchell, P. N. Leigh, A. Al-Chalabi, C. C. Miller, G. Nicholson, and C. E. Shaw, "TDP-43 mutations in familial

*Proc. Natl. Acad. Sci. U. S. A*, vol. 94, no. 18, pp. 9920-9925, Sept.1997.

*Natl. Acad. Sci. U. S. A*, vol. 96, no. 11, pp. 6307-6311, May1999.

*et.*, vol. 8, no. 7, pp. 1177-1183, July1999.

*Genet.*, vol. 9, no. 3, pp. 333-339, Feb.2000.

*Genet.*, vol. 9, no. 3, pp. 333-339, Feb.2000.

vol. 362, no. 6415, pp. 59-62, Mar.1993.

no. 8, pp. 597-609, Aug.2009.

Jan.2000.

510 Pluripotent Stem Cells


[166] V. Campuzano, L. Montermini, M. D. Molt•, L. Pianese, M. Coss'e, F. Cavalcanti, E. Monros, F. Rodius, F. Duclos, A. Monticelli, F. Zara, J. Ca¤izares, H. Koutnikova, S. I. Bidichandani, C. Gellera, A. Brice, P. Trouillas, G. De Michele, A. Filla, R. De Frutos, F. Palau, P. I. Patel, S. Di Donato, J. L. Mandel, S. Cocozza, M. Koenig, and M. Pan‐ dolfo, "Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion," *Science*, vol. 271, no. 5254, pp. 1423-1427, 1996.

[177] L. Montermini, E. Andermann, M. Labuda, A. Richter, M. Pandolfo, F. Cavalcanti, L. Pianese, L. Iodice, G. Farina, A. Monticelli, M. Turano, A. Filla, M. G. De, and S. Co‐ cozza, "The Friedreich ataxia GAA triplet repeat: premutation and normal alleles,"

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases

http://dx.doi.org/10.5772/55772

513

[178] R. Sharma, S. Bhatti, M. Gomez, R. M. Clark, C. Murray, T. Ashizawa, and S. I. Bidi‐ chandani, "The GAA triplet-repeat sequence in Friedreich ataxia shows a high level of somatic instability in vivo, with a significant predilection for large contractions,"

[179] I. De Biase, A. Rasmussen, D. Endres, S. Al-Mahdawi, A. Monticelli, S. Cocozza, M. Pook, and S. I. Bidichandani, "Progressive GAA expansions in dorsal root ganglia of

[180] L. Schols, P. Bauer, T. Schmidt, T. Schulte, and O. Riess, "Autosomal dominant cere‐ bellar ataxias: clinical features, genetics, and pathogenesis," *Lancet Neurol.*, vol. 3, no.

[181] Y. Kawaguchi, T. Okamoto, M. Taniwaki, M. Aizawa, M. Inoue, S. Katayama, H. Ka‐ wakami, S. Nakamura, M. Nishimura, I. Akiguchi, and ., "CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1," *Nat. Genet.*, vol. 8, no. 3,

[182] H. L. Paulson, M. K. Perez, Y. Trottier, J. Q. Trojanowski, S. H. Subramony, S. S. Das, P. Vig, J. L. Mandel, K. H. Fischbeck, and R. N. Pittman, "Intranuclear inclusions of expanded polyglutamine protein in spinocerebellar ataxia type 3," *Neuron*, vol. 19,

[183] U. Rub, E. R. Brunt, and T. Deller, "New insights into the pathoanatomy of spinocere‐ bellar ataxia type 3 (Machado-Joseph disease)," *Curr. Opin. Neurol.*, vol. 21, no. 2, pp.

[184] O. Riess, U. Rüb, A. Pastore, P. Bauer, and L. Schöls, "SCA3: neurological features, pathogenesis and animal models," *Cerebellum*, vol. 7, no. 2, pp. 125-137, 2008.

[185] H. Ikeda, M. Yamaguchi, S. Sugai, Y. Aze, S. Narumiya, and A. Kakizuka, "Expanded polyglutamine in the Machado-Joseph disease protein induces cell death in vitro and

[186] C. K. Cemal, C. J. Carroll, L. Lawrence, M. B. Lowrie, P. Ruddle, S. Al-Mahdawi, R. H. King, M. A. Pook, C. Huxley, and S. Chamberlain, "YAC transgenic mice carrying pathological alleles of the MJD1 locus exhibit a mild and slowly progressive cerebel‐

[187] C. Maayan, E. Kaplan, S. Shachar, O. Peleg, and S. Godfrey, "Incidence of familial dysautonomia in Israel 1977-1981," *Clin. Genet.*, vol. 32, no. 2, pp. 106-108, Aug.1987.

[188] S. A. Slaugenhaupt, A. Blumenfeld, S. P. Gill, M. Leyne, J. Mull, M. P. Cuajungco, C. B. Liebert, B. Chadwick, M. Idelson, L. Reznik, C. Robbins, I. Makalowska, M.

lar deficit," *Hum. Mol. Genet.*, vol. 11, no. 9, pp. 1075-1094, May2002.

in vivo," *Nat. Genet.*, vol. 13, no. 2, pp. 196-202, June1996.

Friedreich's ataxia patients," *Ann Neurol*, vol. 61, no. 1, pp. 55-60, 2007.

*Hum. Mol. Genet.*, vol. 6, no. 8, pp. 1261-1266, Aug.1997.

*Hum. Mol. Genet.*, vol. 11, no. 18, pp. 2175-2187, Sept.2002.

5, pp. 291-304, May2004.

pp. 221-228, Nov.1994.

111-116, Apr.2008.

no. 2, pp. 333-344, Aug.1997.


[177] L. Montermini, E. Andermann, M. Labuda, A. Richter, M. Pandolfo, F. Cavalcanti, L. Pianese, L. Iodice, G. Farina, A. Monticelli, M. Turano, A. Filla, M. G. De, and S. Co‐ cozza, "The Friedreich ataxia GAA triplet repeat: premutation and normal alleles," *Hum. Mol. Genet.*, vol. 6, no. 8, pp. 1261-1266, Aug.1997.

[166] V. Campuzano, L. Montermini, M. D. Molt•, L. Pianese, M. Coss'e, F. Cavalcanti, E. Monros, F. Rodius, F. Duclos, A. Monticelli, F. Zara, J. Ca¤izares, H. Koutnikova, S. I. Bidichandani, C. Gellera, A. Brice, P. Trouillas, G. De Michele, A. Filla, R. De Frutos, F. Palau, P. I. Patel, S. Di Donato, J. L. Mandel, S. Cocozza, M. Koenig, and M. Pan‐ dolfo, "Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA

[167] S. I. Bidichandani, T. Ashizawa, and P. I. Patel, "The GAA triplet-repeat expansion in Friedreich ataxia interferes with transcription and may be associated with an unusu‐

[168] A. Dürr, M. Cossee, Y. Agid, V. Campuzano, C. Mignard, C. Penet, J. L. Mandel, A. Brice, and M. Koenig, "Clinical and genetic abnormalities in patients with Friedrei‐

[169] A. H. Koeppen, "Friedreich's ataxia: pathology, pathogenesis, and molecular genet‐

[170] H. Puccio, "Multicellular models of Friedreich ataxia," *J. Neurol.*, vol. 256 Suppl 1, pp.

[171] A. Martelli, M. Napierala, and H. Puccio, "Understanding the genetic and molecular pathogenesis of Friedreich's ataxia through animal and cellular models," *Dis. Model.*

[172] H. Puccio, "Conditional mouse models for Friedreich ataxia, a neurodegenerative disorder associating cardiomyopathy," *Handb. Exp. Pharmacol.*, no. 178, pp. 365-375,

[173] M. Cossee, H. Puccio, A. Gansmuller, H. Koutnikova, A. Dierich, M. LeMeur, K. Fischbeck, P. Dolle, and M. Koenig, "Inactivation of the Friedreich ataxia mouse gene leads to early embryonic lethality without iron accumulation," *Hum. Mol. Genet.*, vol.

[174] D. Simon, H. Seznec, A. Gansmuller, N. Carelle, P. Weber, D. Metzger, P. Rustin, M. Koenig, and H. Puccio, "Friedreich ataxia mouse models with progressive cerebellar and sensory ataxia reveal autophagic neurodegeneration in dorsal root ganglia," *J.*

[175] C. J. Miranda, M. M. Santos, K. Ohshima, J. Smith, L. Li, M. Bunting, M. Cossee, M. Koenig, J. Sequeiros, J. Kaplan, and M. Pandolfo, "Frataxin knockin mouse," *FEBS*

[176] S. Al-Mahdawi, R. M. Pinto, D. Varshney, L. Lawrence, M. B. Lowrie, S. Hughes, Z. Webster, J. Blake, J. M. Cooper, R. King, and M. A. Pook, "GAA repeat expansion mutation mouse models of Friedreich ataxia exhibit oxidative stress leading to pro‐ gressive neuronal and cardiac pathology," *Genomics*, vol. 88, no. 5, pp. 580-590, Nov.

triplet repeat expansion," *Science*, vol. 271, no. 5254, pp. 1423-1427, 1996.

al DNA structure," *Am. J. Hum. Genet.*, vol. 62, no. 1, pp. 111-121, Jan.1998.

ch's ataxia," *N Engl J Med*, vol. 335, no. 16, pp. 1169-1175, 1996.

ics," *J. Neurol. Sci.*, vol. 303, no. 1-2, pp. 1-12, Apr.2011.

*Mech.*, vol. 5, no. 2, pp. 165-176, Mar.2012.

9, no. 8, pp. 1219-1226, May2000.

*Neurosci.*, vol. 24, no. 8, pp. 1987-1995, Feb.2004.

*Lett.*, vol. 512, no. 1-3, pp. 291-297, Feb.2002.

18-24, Mar.2009.

2007.

512 Pluripotent Stem Cells


Brownstein, D. Krappmann, C. Scheidereit, C. Maayan, F. B. Axelrod, and J. F. Gusel‐ la, "Tissue-specific expression of a splicing mutation in the IKBKAP gene causes fam‐ ilial dysautonomia," *Am. J. Hum. Genet.*, vol. 68, no. 3, pp. 598-605, Mar.2001.

ing a CGG repeat coincident with a breakpoint cluster region exhibiting length varia‐

Human Pluripotent Stem Cells Modeling Neurodegenerative Diseases

http://dx.doi.org/10.5772/55772

515

[200] S. A. Irwin, R. Galvez, and W. T. Greenough, "Dendritic spine structural anomalies in fragile-X mental retardation syndrome," *Cereb. Cortex*, vol. 10, no. 10, pp. 1038-1044,

[201] A. Yao, S. Jin, X. Li, Z. Liu, X. Ma, J. Tang, and Y. Q. Zhang, "Drosophila FMRP regu‐ lates microtubule network formation and axonal transport of mitochondria," *Hum.*

[202] "Fmr1 knockout mice: a model to study fragile X mental retardation. The Dutch-Bel‐

[203] J. R. Brouwer, E. J. Mientjes, C. E. Bakker, I. M. Nieuwenhuizen, L. A. Severijnen, H. C. Van der Linde, D. L. Nelson, B. A. Oostra, and R. Willemsen, "Elevated Fmr1 mRNA levels and reduced protein expression in a mouse model with an unmethylat‐ ed Fragile X full mutation," *Experimental Cell Research*, vol. 313, no. 2, pp. 244-253, Jan.

[204] R. Pietrobono, E. Tabolacci, F. Zalfa, I. Zito, A. Terracciano, U. Moscato, C. Bagni, B. Oostra, P. Chiurazzi, and G. Neri, "Molecular dissection of the events leading to inac‐ tivation of the FMR1 gene," *Hum. Mol. Genet.*, vol. 14, no. 2, pp. 267-277, Jan.2005.

[205] R. Eiges, A. Urbach, M. Malcov, T. Frumkin, T. Schwartz, A. Amit, Y. Yaron, A. Eden, O. Yanuka, N. Benvenisty, and D. Ben-Yosef, "Developmental Study of Fragile X Syn‐ drome Using Human Embryonic Stem Cells Derived from Preimplantation Geneti‐

cally Diagnosed Embryos," *Cell Stem Cell*, vol. 1, no. 5, pp. 568-577, Nov.2007.

[206] T. Vierbuchen, A. Ostermeier, Z. P. Pang, Y. Kokubu, T. C. Sudhof, and M. Wernig, "Direct conversion of fibroblasts to functional neurons by defined factors," *Nature*,

[207] Z. P. Pang, N. Yang, T. Vierbuchen, A. Ostermeier, D. R. Fuentes, T. Q. Yang, A. Citri, V. Sebastiano, S. Marro, T. C. Sudhof, and M. Wernig, "Induction of human neuronal cells by defined transcription factors," *Nature*, vol. 476, no. 7359, pp. 220-223, Aug.

[208] M. Caiazzo, M. T. 'Anno, E. Dvoretskova, D. Lazarevic, S. Taverna, D. Leo, T. D. Sot‐ nikova, A. Menegon, P. Roncaglia, G. Colciago, G. Russo, P. Carninci, G. Pezzoli, R. R. Gainetdinov, S. Gustincich, A. Dityatev, and V. Broccoli, "Direct generation of functional dopaminergic neurons from mouse and human fibroblasts," *Nature*, vol.

[209] U. Pfisterer, A. Kirkeby, O. Torper, J. Wood, J. Nelander, A. Dufour, A. Björklund, O. Lindvall, J. Jakobsson, and M. Parmar, "Direct conversion of human fibroblasts to dopaminergic neurons," *Proceedings of the National Academy of Sciences*, vol. 108, no.

gian Fragile X Consortium," *Cell*, vol. 78, no. 1, pp. 23-33, July1994.

tion in fragile X syndrome," *Cell*, vol. 65, no. 5, pp. 905-914, May1991.

*Mol. Genet.*, vol. 20, no. 1, pp. 51-63, Jan.2011.

vol. 463, no. 7284, pp. 1035-1041, Feb.2010.

476, no. 7359, pp. 224-227, Aug.2011.

25, pp. 10343-10348, June2011.

Oct.2000.

2007.


ing a CGG repeat coincident with a breakpoint cluster region exhibiting length varia‐ tion in fragile X syndrome," *Cell*, vol. 65, no. 5, pp. 905-914, May1991.

[200] S. A. Irwin, R. Galvez, and W. T. Greenough, "Dendritic spine structural anomalies in fragile-X mental retardation syndrome," *Cereb. Cortex*, vol. 10, no. 10, pp. 1038-1044, Oct.2000.

Brownstein, D. Krappmann, C. Scheidereit, C. Maayan, F. B. Axelrod, and J. F. Gusel‐ la, "Tissue-specific expression of a splicing mutation in the IKBKAP gene causes fam‐

ilial dysautonomia," *Am. J. Hum. Genet.*, vol. 68, no. 3, pp. 598-605, Mar.2001.

39, no. 1, pp. 47-59, Nov.1978.

514 Pluripotent Stem Cells

*Biol. Chem.*, vol. 277, no. 4, pp. 3047-3052, Jan.2002.

366, no. 1575, pp. 2286-2296, Aug.2011.

[189] J. Pearson and B. A. Pytel, "Quantitative studies of sympathetic ganglia and spinal cord intermedio-lateral gray columns in familial dysautonomia," *J. Neurol. Sci.*, vol.

[190] N. A. Hawkes, G. Otero, G. S. Winkler, N. Marshall, M. E. Dahmus, D. Krappmann, C. Scheidereit, C. L. Thomas, G. Schiavo, H. Erdjument-Bromage, P. Tempst, and J. Q. Svejstrup, "Purification and characterization of the human elongator complex," *J.*

[191] P. Close, N. Hawkes, I. Cornez, C. Creppe, C. A. Lambert, B. Rogister, U. Siebenlist, M. P. Merville, S. A. Slaugenhaupt, V. Bours, J. Q. Svejstrup, and A. Chariot, "Tran‐ scription impairment and cell migration defects in elongator-depleted cells: implica‐

tion for familial dysautonomia," *Mol. Cell*, vol. 22, no. 4, pp. 521-531, May2006.

[192] G. Lee and L. Studer, "Modelling familial dysautonomia in human induced pluripo‐ tent stem cells," *Philosophical Transactions of the Royal Society B: Biological Sciences*, vol.

[193] P. Dietrich, J. Yue, S. E., and I. Dragatsis, "Deletion of Exon 20 of the Familial Dysau‐ tonomia Gene Ikbkap in Mice Causes Developmental Delay, Cardiovascular Defects,

and Early Embryonic Lethality," *PLoS ONE*, vol. 6, no. 10, p. e27015, Oct.2011.

[194] M. M. Hims, R. S. Shetty, J. Pickel, J. Mull, M. Leyne, L. Liu, J. F. Gusella, and S. A. Slaugenhaupt, "A humanized IKBKAP transgenic mouse models a tissue-specific hu‐

[195] N. Boone, B. Loriod, A. Bergon, O. Sbai, C. Formisano-Treziny, J. Gabert, M. Khrest‐ chatisky, C. Nguyen, F. Feron, F. B. Axelrod, and E. C. Ibrahim, "Olfactory stem cells, a new cellular model for studying molecular mechanisms underlying familial dysau‐

[196] W. Murrell, F. Feron, A. Wetzig, N. Cameron, K. Splatt, B. Bellette, J. Bianco, C. Per‐ ry, G. Lee, and A. Mackay-Sim, "Multipotent stem cells from adult olfactory mucosa,"

[197] S. A. Slaugenhaupt, J. Mull, M. Leyne, M. P. Cuajungco, S. P. Gill, M. M. Hims, F. Quintero, F. B. Axelrod, and J. F. Gusella, "Rescue of a human mRNA splicing defect by the plant cytokinin kinetin," *Hum. Mol. Genet.*, vol. 13, no. 4, pp. 429-436, Feb.2004.

[198] D. C. Crawford, J. M. Acuna, and S. L. Sherman, "FMR1 and the fragile X syndrome: human genome epidemiology review," *Genet. Med.*, vol. 3, no. 5, pp. 359-371, Sept.

[199] A. J. Verkerk, M. Pieretti, J. S. Sutcliffe, Y. H. Fu, D. P. Kuhl, A. Pizzuti, O. Reiner, S. Richards, M. F. Victoria, F. P. Zhang, and ., "Identification of a gene (FMR-1) contain‐

man splicing defect," *Genomics*, vol. 90, no. 3, pp. 389-396, Sept.2007.

tonomia," *PLoS. One.*, vol. 5, no. 12, p. e15590, 2010.

*Dev. Dyn.*, vol. 233, no. 2, pp. 496-515, June2005.


[210] E. Y. Son, J. K. Ichida, B. J. Wainger, J. S. Toma, V. F. Rafuse, C. J. Woolf, and K. Eg‐ gan, "Conversion of mouse and human fibroblasts into functional spinal motor neu‐ rons," *Cell Stem Cell*, vol. 9, no. 3, pp. 205-218, Sept.2011.

**Chapter 23**

**Induced Pluripotent Stem Cells as**

Minoru Tomizawa, Fuminobu Shinozaki, Takao Sugiyama, Shigenori Yamamoto, Makoto Sueishi and Takanobu Yoshida

Additional information is available at the end of the chapter

time. In this chapter, we will cover the following topics:

**•** Production of hepatocytes from human embryonic stem (hES) cells

**•** Current applications of hepatocytes differentiated from hiPS cells

**•** Protocols for differentiation of hiPS cells into hepatocytes

Human induced pluripotent stem (hiPS) cells are generated with cellular reprogramming factors [1], and they have the potential to differentiate into a variety of cells. Ethical issues and graft-versus-host disease may be avoided with hiPS cells because they can be establish‐ ed in each patient individually. hiPS cells may therefore be an ideal cell source for patients. The liver is a single large organ, the cells of which are 70–80% hepatocytes. These liver-spe‐ cific cells play a major role in protein synthesis, glucose metabolism, and detoxification. Methods of producing hepatocytes from hiPS cells have been under development for some

First we will discuss primary hepatocyte culture. The knowledge on primary hepatocyte cul‐ ture is applicable to maintenance of hepatocytes differentiated from hiPS cells. Next, appli‐

> © 2013 Tomizawa et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Tomizawa et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**a Source of Hepatocytes**

http://dx.doi.org/10.5772/54552

**1. Introduction**

**•** Hepatocyte culture

**•** Future directions

**•** Applications of hepatocyte culture

**•** Limitations of differentiation

[211] L. Qiang, R. Fujita, T. Yamashita, S. Angulo, H. Rhinn, D. Rhee, C. Doege, L. Chau, L. Aubry, W. B. Vanti, H. Moreno, and A. Abeliovich, "Directed conversion of Alzheim‐ er's disease patient skin fibroblasts into functional neurons," *Cell*, vol. 146, no. 3, pp. 359-371, Aug.2011.

**Chapter 23**

## **Induced Pluripotent Stem Cells as a Source of Hepatocytes**

Minoru Tomizawa, Fuminobu Shinozaki, Takao Sugiyama, Shigenori Yamamoto, Makoto Sueishi and Takanobu Yoshida

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54552

## **1. Introduction**

[210] E. Y. Son, J. K. Ichida, B. J. Wainger, J. S. Toma, V. F. Rafuse, C. J. Woolf, and K. Eg‐ gan, "Conversion of mouse and human fibroblasts into functional spinal motor neu‐

[211] L. Qiang, R. Fujita, T. Yamashita, S. Angulo, H. Rhinn, D. Rhee, C. Doege, L. Chau, L. Aubry, W. B. Vanti, H. Moreno, and A. Abeliovich, "Directed conversion of Alzheim‐ er's disease patient skin fibroblasts into functional neurons," *Cell*, vol. 146, no. 3, pp.

rons," *Cell Stem Cell*, vol. 9, no. 3, pp. 205-218, Sept.2011.

359-371, Aug.2011.

516 Pluripotent Stem Cells

Human induced pluripotent stem (hiPS) cells are generated with cellular reprogramming factors [1], and they have the potential to differentiate into a variety of cells. Ethical issues and graft-versus-host disease may be avoided with hiPS cells because they can be establish‐ ed in each patient individually. hiPS cells may therefore be an ideal cell source for patients.

The liver is a single large organ, the cells of which are 70–80% hepatocytes. These liver-spe‐ cific cells play a major role in protein synthesis, glucose metabolism, and detoxification. Methods of producing hepatocytes from hiPS cells have been under development for some time. In this chapter, we will cover the following topics:


First we will discuss primary hepatocyte culture. The knowledge on primary hepatocyte cul‐ ture is applicable to maintenance of hepatocytes differentiated from hiPS cells. Next, appli‐

cation of hepatocyte culture will be discussed because the application would provide potential usage of hiPS cells. Then production of hepatocytes from ES cells will be present‐ ed. Methods presented in this section are prototypes of differentiation protocols of hiPS cell into hepatocytes. Sequentially, current protocols of differentiation of hiPS cells into hepato‐ cytes will be summarized. Applications of hepatotyes from hiPS cells will be presented spe‐ cific to human diseases such as hepatitis C virus. Even with the protocols above mentioned, differentiation of hiPS cells to functioning hepatocytes is difficult. Limitations of differentia‐ tion will be discussed. Finally, potential new approaches will be presented in the last sec‐ tion.

**•** In vitro model of liver diseases

treated in this manner.

One of the most important applications of hepatocytes from hiPS cells would be trans‐ plantation into patients with hepatic insufficiency caused by fulminant hepatitis. The disease could be treated perfectly with transplanted hepatocytes because it is caused by significant loss of functioning hepatocytes. Hepatic progenitor cells have potential to differentiate into mature hepatocytes and bile duct epithelial cells. Hepatic progeni‐ tor cells would be expected to construct normal liver structure such as hepatic lobule and bile ducts. Hepatic progenitor cells derived from mouse embryonic stem (ES) cells engraft in host liver tissue and differentiate into hepatocytes when transplanted into partially hepatectomized mice [7]. Hepatocytes will also engraft in mice with acute liv‐ er failure caused by carbon tetrachloride intoxication [8]. This is a promising finding that suggests that hepatocytes from pluripotent cells are transplantable. Hepatocytes have indeed been differentiated from human ES cells and transplanted [9]. One disad‐ vantage of the use of human ES cells is that they may provoke graft-versus-host dis‐ ease. This could be overcome if hepatocytes are derived from iPS cells established from the individual patient. Patients with acute liver failure could be successfully

Induced Pluripotent Stem Cells as a Source of Hepatocytes

http://dx.doi.org/10.5772/54552

519

Another application of hepatocytes from hiPS cells would be metabolic diseases. The disease could be cured with transplantation of functioning hepatocytes because they play pivotal roles in metabolism. High levels of low-density lipoprotein cholesterol (LDL-Chol) in the plasma is known to cause cardiovascular disease. Successful reduction of LDL-Chol may lead to prevention of cardiovascular disease. Mutations in the LDL receptor gene result in familial hypercholesterolemia (FH); iPS cells derived from patients with FH provide a good

Cultured primary hepatocytes do not proliferate but disappear and lose their function quickly. Pluripotent stem cells have been focused as a cell source of hepatocytes. Before the advent of iPS cells, ES cells had been the center of investigation of differentiation methods into hepatocytes. The topics of the investigation have been growth factors, transcription fac‐

Mouse ES cells start differentiation into the hepatocyte lineage once leukemia inhibitory factor (LIF) is deprived and embryoid bodies are formed [11-13]. Hepatocyte-like cells derived from mouse ES cells take up indocyanine green, express albumin, and form bile canaliculi [14]. The induced cells express specific live genes such as α-1-antitrypsin and phosphoenolpyruvate carboxykinase (PEPCK). Withdrawal of LIF is not an appropriate method for inducing hiPS cell differentiation because these cells are not LIF dependent [15]. Human ES cells differentiate into mesoderm, endoderm, and ectoderm after with‐ drawal of the LIF and basic fibroblast growth factor (bFGF) [16], but they do not neces‐

model for analyzing the mechanism of this condition [10].

**4. Differentiation of ES cells into hepatocytes**

tors, extracellular matrix, and three-dimensional (3D) culture

## **2. Primary hepatocyte culture**

Before the era of ES cells or iPS cells, primary hepatocyte culture had been the only method to investigate differentiation and function of hepatocytes. The accumulated knowledge on hepatocytes would be applicable to maintain hepatocytes differentiated from hiPS cells. Hepatocyte culture is useful for developing drugs, cell therapies, and disease models. Pri‐ mary hepatocyte culture is an ideal in vitro model of drug metabolism and toxicology, and primary hepatocytes can be transplanted into patients with liver failure [2]. Hepatocytes from patients with metabolic diseases can be used to investigate disease mechanisms. How‐ ever, primary hepatocyte culture remains technically difficult. Hepatocytes are isolated from a fragment of resected donor liver with a 2-step collagenase perfusion [2]. Fetal hepatocytes (107 cells) have been transplanted into patients with hepatic encephalopathy [3], and while the disease improved, there was no increase in survival time. The speculated reason is that not enough cells were transplanted [4]. Isolated hepatocytes are prone to apoptosis and damage [5] and have difficulty proliferating once cultured [6]. Primary hepatocyte culture also presents ethical issues when cells are harvested from humans. Now hiPS cells could overcome problems that primary hepatocyte culture encounters.

## **3. Application of hepatocytes differentiated from iPS cells**

If hiPS cells could differentiate into hepatocytes, they would be useful for medical practice and biological study. Potential applications would be as follows:


#### **•** In vitro model of liver diseases

cation of hepatocyte culture will be discussed because the application would provide potential usage of hiPS cells. Then production of hepatocytes from ES cells will be present‐ ed. Methods presented in this section are prototypes of differentiation protocols of hiPS cell into hepatocytes. Sequentially, current protocols of differentiation of hiPS cells into hepato‐ cytes will be summarized. Applications of hepatotyes from hiPS cells will be presented spe‐ cific to human diseases such as hepatitis C virus. Even with the protocols above mentioned, differentiation of hiPS cells to functioning hepatocytes is difficult. Limitations of differentia‐ tion will be discussed. Finally, potential new approaches will be presented in the last sec‐

Before the era of ES cells or iPS cells, primary hepatocyte culture had been the only method to investigate differentiation and function of hepatocytes. The accumulated knowledge on hepatocytes would be applicable to maintain hepatocytes differentiated from hiPS cells. Hepatocyte culture is useful for developing drugs, cell therapies, and disease models. Pri‐ mary hepatocyte culture is an ideal in vitro model of drug metabolism and toxicology, and primary hepatocytes can be transplanted into patients with liver failure [2]. Hepatocytes from patients with metabolic diseases can be used to investigate disease mechanisms. How‐ ever, primary hepatocyte culture remains technically difficult. Hepatocytes are isolated from a fragment of resected donor liver with a 2-step collagenase perfusion [2]. Fetal hepatocytes

 cells) have been transplanted into patients with hepatic encephalopathy [3], and while the disease improved, there was no increase in survival time. The speculated reason is that not enough cells were transplanted [4]. Isolated hepatocytes are prone to apoptosis and damage [5] and have difficulty proliferating once cultured [6]. Primary hepatocyte culture also presents ethical issues when cells are harvested from humans. Now hiPS cells could

If hiPS cells could differentiate into hepatocytes, they would be useful for medical practice

overcome problems that primary hepatocyte culture encounters.

and biological study. Potential applications would be as follows:

**•** Transplantation into patients with hepatic insufficiency

**•** In vitro model of hepatitis C virus infection **•** In vitro model of hepatocyte differentiation

**3. Application of hepatocytes differentiated from iPS cells**

**•** A method to support patients with hepatic insufficiency such as hemodialysis

tion.

518 Pluripotent Stem Cells

(107

**•** Drug screening

**•** Toxicology

**2. Primary hepatocyte culture**

One of the most important applications of hepatocytes from hiPS cells would be trans‐ plantation into patients with hepatic insufficiency caused by fulminant hepatitis. The disease could be treated perfectly with transplanted hepatocytes because it is caused by significant loss of functioning hepatocytes. Hepatic progenitor cells have potential to differentiate into mature hepatocytes and bile duct epithelial cells. Hepatic progeni‐ tor cells would be expected to construct normal liver structure such as hepatic lobule and bile ducts. Hepatic progenitor cells derived from mouse embryonic stem (ES) cells engraft in host liver tissue and differentiate into hepatocytes when transplanted into partially hepatectomized mice [7]. Hepatocytes will also engraft in mice with acute liv‐ er failure caused by carbon tetrachloride intoxication [8]. This is a promising finding that suggests that hepatocytes from pluripotent cells are transplantable. Hepatocytes have indeed been differentiated from human ES cells and transplanted [9]. One disad‐ vantage of the use of human ES cells is that they may provoke graft-versus-host dis‐ ease. This could be overcome if hepatocytes are derived from iPS cells established from the individual patient. Patients with acute liver failure could be successfully treated in this manner.

Another application of hepatocytes from hiPS cells would be metabolic diseases. The disease could be cured with transplantation of functioning hepatocytes because they play pivotal roles in metabolism. High levels of low-density lipoprotein cholesterol (LDL-Chol) in the plasma is known to cause cardiovascular disease. Successful reduction of LDL-Chol may lead to prevention of cardiovascular disease. Mutations in the LDL receptor gene result in familial hypercholesterolemia (FH); iPS cells derived from patients with FH provide a good model for analyzing the mechanism of this condition [10].

#### **4. Differentiation of ES cells into hepatocytes**

Cultured primary hepatocytes do not proliferate but disappear and lose their function quickly. Pluripotent stem cells have been focused as a cell source of hepatocytes. Before the advent of iPS cells, ES cells had been the center of investigation of differentiation methods into hepatocytes. The topics of the investigation have been growth factors, transcription fac‐ tors, extracellular matrix, and three-dimensional (3D) culture

Mouse ES cells start differentiation into the hepatocyte lineage once leukemia inhibitory factor (LIF) is deprived and embryoid bodies are formed [11-13]. Hepatocyte-like cells derived from mouse ES cells take up indocyanine green, express albumin, and form bile canaliculi [14]. The induced cells express specific live genes such as α-1-antitrypsin and phosphoenolpyruvate carboxykinase (PEPCK). Withdrawal of LIF is not an appropriate method for inducing hiPS cell differentiation because these cells are not LIF dependent [15]. Human ES cells differentiate into mesoderm, endoderm, and ectoderm after with‐ drawal of the LIF and basic fibroblast growth factor (bFGF) [16], but they do not neces‐ sarily differentiate into hepatocytes. Therefore, growth factors are expected to be needed for hepatocyte differentiation from human ES cells. Nerve growth factor (NGF) and hep‐ atocyte growth factor (HGF) induce differentiation into endoderm and eventually liver cells [17]. Transcription factors also play an important role in hepatocyte differentiation. Transcription factor forkhead box protein (Fox) A2 promotes differentiation of mouse ES cell into the hepatocyte lineage [18], and these hepatocye-like cells express phosphoenol‐ pyruvate (PEPCK) and albumin.

DeLaForest [25] D0-5 D5-10 D10-15 D15-20

S-Tayeb [26] D0-5 D5-10 D10-15 D15-20

Song [27] D0-3 D4-7 D8-13 D14-18 D19-21

D: day; BMP4: bone morphogenic protein 4; FGF: fibroblast growth factor 2; HGF: hepatocyte growth factor; OncoM:

All differentiation protocols apply activin A (a member of the tumor growth factor β super‐ family) at a high concentration of 100-ng/mL. LY294002 (a specific inhibitor of phosphatidylinositol 3 phosphatase), B27 supplement, or bFGF are added, depending on the purpose of the research. After 3–5 days of culture, iPS cells differentiate into endodermal cells. From days 5–10, a combination of bone morphogens 2 or 4 and fibroblast growth factors 2 or 4 is applied. Takayama et al. [28] introduced sex-determining region Y box 17 to promote differ‐ entiation at this stage after incubation with activin A. Sekine et al. [29] used LY294002 in ad‐ dition to 100-ng/mL activin A. In their study, FoxA2 and Sox17 expressions appeared but AFP and albumin were not analyzed. Phosphatidyl inositol (PI) 3 kinase may control differ‐

Hepatocyte growth factor (HGF) or keratinocyte growth factor (KGF) is applied from days 10–14. Inamura et al. introduced hematopoietically expressed homeobox (HEX) to promote

HGF or oncostatin M is added to promote differentiation of hepatoblasts into mature hepa‐ tocytes. Takayama et al. [28] introduced hepatocyte nuclear factor-4 to provide the terminal differentiation of hepatoblasts into hepatocytes. Mature hepatocytes appeared at approxi‐ mately 20 days after the initiation of the differentiation process. Si-Tayeb et al. [26] cultured

In another study, Nakamura et al. [31] derived hepatocytes from human ES and iPS cells un‐ der feeder- and serum-free conditions. They succeeded in producing cholangiocytes and

oncostatin M; KGF: keratinocyte growth factor; Dex: dexamethasone.

**6. Endodermal differentiation**

differentiation into hepatoblasts [30].

cells under 4% oxygen from days 5 to 15.

**Table 1.** Protocols for hepatocyte differentiation from human induced pluripotent stem cells.

entiation of iPS cells into endodemal cells, but other factors are still needed.

**7. Differentiation into immature hepatocytes**

**8. Differentiation into mature hepatocytes**

Activin A, LY294002 BMP4, FGF2 HGF OncoM

Induced Pluripotent Stem Cells as a Source of Hepatocytes

http://dx.doi.org/10.5772/54552

521

O2: 20% O2: 4% O2: 4% O2: 20% Activin A BMP4, FGF2 HGF OncoM

Activin A FGF4, BMP3 HGF, KGF OncoM OncoM, Dex

To search for more efficient protocols to promote differentiation of ES cells into hepatoctyes, combinations of growth factors and extracellular matrices have been investigated [19]. Shira‐ hashi et al. reported that a mixture of Iscove's modified Dulbecco's medium with 20% fetal bovine serum, human insulin, dexamethasone, and type 1 collagen is optimum for mouse and human ES cell differentiation into the hepatocyte lineage. Bovine serum should not be used because xeno-proteins are not suitable for human application. This study suggests that extracellular matrix is important in hepatocyte differentiation.

Hepatic progenitor cells differentiate into hepatocytes in 3D structure in liver. It is ex‐ pected that 3D culture is more suitable environment for ES cells to differentiate into hep‐ atocytes. Indeed, 3D cultures of mouse ES cells have been shown to differentiate into hepatocytes [20]. Embryoid bodies (EB) were inserted into a collagen scaffold 3D culture system and stimulated with exogenous growth factors and hormones to produce hepatic differentiation.

Hepatocytes should be isolated from the other cells because ES cells could be among hepatocytes. Undifferentiated cells have been shown to form teratoma when transplanted into recipient cells mixed with hepatocytes [21]. A practical method to avoid this is to enrich the hepatocytes and eliminate the undifferentiated cells by Percoll discontinuous gradient centrifugation [22, 23].

Rambhatla et al. [24] reported that the addition of sodium butylate leads to significant cell death and induction of hepatocyte differentiation in human ES cells. Cells cultured with so‐ dium butylate express albumin, α-1-antitrypsin, and cytochrome P450 and also accumulate glycogen. However, the induced cells do not express alpha-fetoprotein (AFP). Sodium buty‐ late is a possible candidate for a small molecule to eliminate undifferentiated cells and in‐ duce hepatic differentiation.

## **5. Protocols for differentiation of hiPS cells into hepatocytes**

Protocols for differentiation of hiPS cells into hepatocytes follow those for mouse ES cells as mentioned above. Stepwise protocols are currently used to promote the differentiation [25-28] (Table 1). These protocols consist of sequential application of growth factors and in‐ troduction of transcription factors to mimic hepatocyte differentiation during liver develop‐ ment. The progression is endodermal cell, immature hepatocyte (often referred as hepatoblast), and finally mature hepatocyte.


D: day; BMP4: bone morphogenic protein 4; FGF: fibroblast growth factor 2; HGF: hepatocyte growth factor; OncoM: oncostatin M; KGF: keratinocyte growth factor; Dex: dexamethasone.

**Table 1.** Protocols for hepatocyte differentiation from human induced pluripotent stem cells.

## **6. Endodermal differentiation**

sarily differentiate into hepatocytes. Therefore, growth factors are expected to be needed for hepatocyte differentiation from human ES cells. Nerve growth factor (NGF) and hep‐ atocyte growth factor (HGF) induce differentiation into endoderm and eventually liver cells [17]. Transcription factors also play an important role in hepatocyte differentiation. Transcription factor forkhead box protein (Fox) A2 promotes differentiation of mouse ES cell into the hepatocyte lineage [18], and these hepatocye-like cells express phosphoenol‐

To search for more efficient protocols to promote differentiation of ES cells into hepatoctyes, combinations of growth factors and extracellular matrices have been investigated [19]. Shira‐ hashi et al. reported that a mixture of Iscove's modified Dulbecco's medium with 20% fetal bovine serum, human insulin, dexamethasone, and type 1 collagen is optimum for mouse and human ES cell differentiation into the hepatocyte lineage. Bovine serum should not be used because xeno-proteins are not suitable for human application. This study suggests that

Hepatic progenitor cells differentiate into hepatocytes in 3D structure in liver. It is ex‐ pected that 3D culture is more suitable environment for ES cells to differentiate into hep‐ atocytes. Indeed, 3D cultures of mouse ES cells have been shown to differentiate into hepatocytes [20]. Embryoid bodies (EB) were inserted into a collagen scaffold 3D culture system and stimulated with exogenous growth factors and hormones to produce hepatic

Hepatocytes should be isolated from the other cells because ES cells could be among hepatocytes. Undifferentiated cells have been shown to form teratoma when transplanted into recipient cells mixed with hepatocytes [21]. A practical method to avoid this is to enrich the hepatocytes and eliminate the undifferentiated cells by Percoll discontinuous

Rambhatla et al. [24] reported that the addition of sodium butylate leads to significant cell death and induction of hepatocyte differentiation in human ES cells. Cells cultured with so‐ dium butylate express albumin, α-1-antitrypsin, and cytochrome P450 and also accumulate glycogen. However, the induced cells do not express alpha-fetoprotein (AFP). Sodium buty‐ late is a possible candidate for a small molecule to eliminate undifferentiated cells and in‐

Protocols for differentiation of hiPS cells into hepatocytes follow those for mouse ES cells as mentioned above. Stepwise protocols are currently used to promote the differentiation [25-28] (Table 1). These protocols consist of sequential application of growth factors and in‐ troduction of transcription factors to mimic hepatocyte differentiation during liver develop‐ ment. The progression is endodermal cell, immature hepatocyte (often referred as

**5. Protocols for differentiation of hiPS cells into hepatocytes**

pyruvate (PEPCK) and albumin.

differentiation.

520 Pluripotent Stem Cells

gradient centrifugation [22, 23].

duce hepatic differentiation.

hepatoblast), and finally mature hepatocyte.

extracellular matrix is important in hepatocyte differentiation.

All differentiation protocols apply activin A (a member of the tumor growth factor β super‐ family) at a high concentration of 100-ng/mL. LY294002 (a specific inhibitor of phosphatidylinositol 3 phosphatase), B27 supplement, or bFGF are added, depending on the purpose of the research. After 3–5 days of culture, iPS cells differentiate into endodermal cells. From days 5–10, a combination of bone morphogens 2 or 4 and fibroblast growth factors 2 or 4 is applied. Takayama et al. [28] introduced sex-determining region Y box 17 to promote differ‐ entiation at this stage after incubation with activin A. Sekine et al. [29] used LY294002 in ad‐ dition to 100-ng/mL activin A. In their study, FoxA2 and Sox17 expressions appeared but AFP and albumin were not analyzed. Phosphatidyl inositol (PI) 3 kinase may control differ‐ entiation of iPS cells into endodemal cells, but other factors are still needed.

## **7. Differentiation into immature hepatocytes**

Hepatocyte growth factor (HGF) or keratinocyte growth factor (KGF) is applied from days 10–14. Inamura et al. introduced hematopoietically expressed homeobox (HEX) to promote differentiation into hepatoblasts [30].

### **8. Differentiation into mature hepatocytes**

HGF or oncostatin M is added to promote differentiation of hepatoblasts into mature hepa‐ tocytes. Takayama et al. [28] introduced hepatocyte nuclear factor-4 to provide the terminal differentiation of hepatoblasts into hepatocytes. Mature hepatocytes appeared at approxi‐ mately 20 days after the initiation of the differentiation process. Si-Tayeb et al. [26] cultured cells under 4% oxygen from days 5 to 15.

In another study, Nakamura et al. [31] derived hepatocytes from human ES and iPS cells un‐ der feeder- and serum-free conditions. They succeeded in producing cholangiocytes and proliferating progenitors. The cells produced with their protocol were confirmed to function as mature hepatocytes. Indocyanine green was taken up by 30% of the hepatocytes, and 80% stored glycogen. They also maintained the metabolic activity of CYP3A4.

Hepatocyte-like cells derived from mouse iPS cells have been shown to improve acute liver failure caused by carbon tetrachloride [38]. These cells were transplanted through peritoneal injection and significantly reduced the extent of necrotic liver. The authors concluded that

Induced Pluripotent Stem Cells as a Source of Hepatocytes

http://dx.doi.org/10.5772/54552

523

Cells cultured under the protocols mentioned above are referred to as hepatocyte-like cells. In these cells, detoxification activity is lower than in primary hepatocyte culture [26, 28]. Hepatocytes differentiated from hiPS cells have lower expression levels of FoxA1, FoxA2, FoxA3, and HNF1α, and Takayama et al. [28] speculated that other factors are still needed. iPS cells retain their donor cell gene expressions. Lee et al. [35] generated mouse iPS cells from hepatoblasts and adult hepatocytes. Hepatocytes differentiated from hiPS cells express mRNA that is normally not found in fetal or adult liver [25]. An interesting finding is that hepatocytes are differentiated more efficiently from hepatoblast-derived iPS cells than from adult hepatocytes. This suggests that the efficiency of hepatocyte differentiation may de‐ pend on the origin of the iPS cells. Protocols need to be further developed given that the mentioned liver-specific genes are important for clinical and pharmacological applications.

To overcome these limitations, novel approaches are under investigation. Current research efforts can be categorized into extracellular matrix, 3D culture, and cell sheet approaches.

An extracellular matrix (ECM) provides conditions suitable for cultured cells to differentiate to hepatocytes. M15, a mesonephric cell line, induces differentiation of mouse ES cells into the hepatocyte lineage [39]. Eighty percent of mouse ES cells cultured with M15 express AFP, and 9% express albumin. It is interesting that even the fixed M15 cells can promote mouse ES cell differentiation. Shiraki et al. reported a synthesized basement membrane com‐ posed of human recombinant laminin 511 [40] that induced differentiation of mouse ES cells

A 3D culture system is composed of gelatin and extracellular matrix from Swiss 3T3 cells [41]. This system preserves the functions of hepatocyte-like cells differentiated from hiPS cells. The most important component of the ECM has been determined to be type 1 collagen. Cells are 3D cultured in hollow fibers similar to embryoid bodies. Hollow fibers are useful because the efficiency of embryoid body formation is low compared with mouse ES cells, which also differentiate into hepatocytes in hollow fibers [42]. The organoid culture system efficiently allows mouse ES cells to form cellular aggregates in their lumen. Liver-specific

Primary rat hepatocytes have been successfully cultured for 200 days on temperature-respon‐ sive sheets [43]. These sheets attach on the bottoms of culture dishes at 37ºC and detach at 25ºC.

functions of mouse ES cells are comparable with those of primary hepatocytes.

the hepatoprotective effects were based on antioxidant activity.

**11. Future directions**

into hepatocyte lineages.

**10. Limitations of hepatocytes differentiated from hiPS cells**

Chen et al. [32] proposed another multistep protocol. They do not apply any transcription factors, but growth factors. They have succeeded in differentiation of hiPS cells into mature hepatocytes within only 12 days. The period is significantly shorter than the other research‐ ers. With their method, activin A (100 ng/mL) and HGF (10 ng/mL) were added from days 1 to 3, and prior to that, HGF had been added at the last step of hepatocyte maturation. They also added HGF at the first step of differentiation and successfully derived hepatocyte-like cells. Sox17 and FoxA2, induced by activin A, are important markers of endodermal differ‐ entiation. HGF and activin A may have synergistic effects on the differentiating cells.

Transcription factors play an important role in liver development and hepatocyte differentia‐ tion [33]. Generally, pluripotent stem cells are hard to transfect plasmids. Adenovirus vectors provide highly efficient transduction to hiPS cells [34]. Inamura et al. [30] transduced HEX into hES and hiPS cells to efficiently produce hepatoblasts (Table 2). After differentiation into hepa‐ toblasts, transduction of HNF4α finally produces mature hepatocytes [28].


BMP4: bone morphogen protein 4; FGF4: fibroblast growth factor 4; HGF: hepatocyte growth factor; OncoM: oncosta‐ tin M; Dex: dexamethasone; Ad-Hex, Sox17, HNF4A: adenovirus vector transducing Hex, Sox17, and HNF4A, respec‐ tively; Hex: hematopoietically expressed homeobox; Sox17: sex determining region Y box 7; HNF4A: hepatocyte nuclear factor 4 α.

**Table 2.** Protocols for hepatocyte differentiation from human induced pluripotent stem cells with adenovirus vectors.

## **9. Current applications of hepatocytes differentiated from hiPS cells**

Hepatocytes from hiPS cells are perfect for in vitro model of human diseases because human primary hepatocytes have both ethical and technical issues. Hepatitis C virus (HCV) causes liver cirrhosis and hepatocellular carcinoma (HCC). Primary human hepatocyte culture is a relevant in vitro model for HCV infection, but it presents some ethical issues. Human iPS cells are not permissive to HCV. Interestingly, hepatocyte-like cells derived from hiPS cells recapitulate permissiveness and are infected with HCV [35, 36]. Hepatocyte-like cells de‐ rived from hiPS cells exert an inflammatory response to infection [37] and may provide a suitable in vitro model to study the mechanism of HCV infection. Such a model may poten‐ tially lead to innovative methods to inhibit HCV and prevent liver cirrhosis and HCC.

Hepatocyte-like cells derived from mouse iPS cells have been shown to improve acute liver failure caused by carbon tetrachloride [38]. These cells were transplanted through peritoneal injection and significantly reduced the extent of necrotic liver. The authors concluded that the hepatoprotective effects were based on antioxidant activity.

## **10. Limitations of hepatocytes differentiated from hiPS cells**

Cells cultured under the protocols mentioned above are referred to as hepatocyte-like cells. In these cells, detoxification activity is lower than in primary hepatocyte culture [26, 28]. Hepatocytes differentiated from hiPS cells have lower expression levels of FoxA1, FoxA2, FoxA3, and HNF1α, and Takayama et al. [28] speculated that other factors are still needed. iPS cells retain their donor cell gene expressions. Lee et al. [35] generated mouse iPS cells from hepatoblasts and adult hepatocytes. Hepatocytes differentiated from hiPS cells express mRNA that is normally not found in fetal or adult liver [25]. An interesting finding is that hepatocytes are differentiated more efficiently from hepatoblast-derived iPS cells than from adult hepatocytes. This suggests that the efficiency of hepatocyte differentiation may de‐ pend on the origin of the iPS cells. Protocols need to be further developed given that the mentioned liver-specific genes are important for clinical and pharmacological applications.

## **11. Future directions**

proliferating progenitors. The cells produced with their protocol were confirmed to function as mature hepatocytes. Indocyanine green was taken up by 30% of the hepatocytes, and 80%

Chen et al. [32] proposed another multistep protocol. They do not apply any transcription factors, but growth factors. They have succeeded in differentiation of hiPS cells into mature hepatocytes within only 12 days. The period is significantly shorter than the other research‐ ers. With their method, activin A (100 ng/mL) and HGF (10 ng/mL) were added from days 1 to 3, and prior to that, HGF had been added at the last step of hepatocyte maturation. They also added HGF at the first step of differentiation and successfully derived hepatocyte-like cells. Sox17 and FoxA2, induced by activin A, are important markers of endodermal differ‐

entiation. HGF and activin A may have synergistic effects on the differentiating cells.

Transcription factors play an important role in liver development and hepatocyte differentia‐ tion [33]. Generally, pluripotent stem cells are hard to transfect plasmids. Adenovirus vectors provide highly efficient transduction to hiPS cells [34]. Inamura et al. [30] transduced HEX into hES and hiPS cells to efficiently produce hepatoblasts (Table 2). After differentiation into hepa‐

Activin A BMP4, FGF4 FGF4, HGF, OncoM, Dex

Activin A Activin A BMP4, FGF4 HGF, OncoM, Dex

D5: passage, D6:Ad-Hex D9: passage

BMP4: bone morphogen protein 4; FGF4: fibroblast growth factor 4; HGF: hepatocyte growth factor; OncoM: oncosta‐ tin M; Dex: dexamethasone; Ad-Hex, Sox17, HNF4A: adenovirus vector transducing Hex, Sox17, and HNF4A, respec‐ tively; Hex: hematopoietically expressed homeobox; Sox17: sex determining region Y box 7; HNF4A: hepatocyte

**Table 2.** Protocols for hepatocyte differentiation from human induced pluripotent stem cells with adenovirus vectors.

Hepatocytes from hiPS cells are perfect for in vitro model of human diseases because human primary hepatocytes have both ethical and technical issues. Hepatitis C virus (HCV) causes liver cirrhosis and hepatocellular carcinoma (HCC). Primary human hepatocyte culture is a relevant in vitro model for HCV infection, but it presents some ethical issues. Human iPS cells are not permissive to HCV. Interestingly, hepatocyte-like cells derived from hiPS cells recapitulate permissiveness and are infected with HCV [35, 36]. Hepatocyte-like cells de‐ rived from hiPS cells exert an inflammatory response to infection [37] and may provide a suitable in vitro model to study the mechanism of HCV infection. Such a model may poten‐ tially lead to innovative methods to inhibit HCV and prevent liver cirrhosis and HCC.

**9. Current applications of hepatocytes differentiated from hiPS cells**

stored glycogen. They also maintained the metabolic activity of CYP3A4.

toblasts, transduction of HNF4α finally produces mature hepatocytes [28].

nuclear factor 4 α.

522 Pluripotent Stem Cells

Inamura [34] D0-6 D6-8 D9-18

Takayama [28] D0-3 D3-6 D7-9 D10-20

D3: Ad-Sox17 D5: passage, D6: Ad-Hex D9: Ad-HNF4A

To overcome these limitations, novel approaches are under investigation. Current research efforts can be categorized into extracellular matrix, 3D culture, and cell sheet approaches.

An extracellular matrix (ECM) provides conditions suitable for cultured cells to differentiate to hepatocytes. M15, a mesonephric cell line, induces differentiation of mouse ES cells into the hepatocyte lineage [39]. Eighty percent of mouse ES cells cultured with M15 express AFP, and 9% express albumin. It is interesting that even the fixed M15 cells can promote mouse ES cell differentiation. Shiraki et al. reported a synthesized basement membrane com‐ posed of human recombinant laminin 511 [40] that induced differentiation of mouse ES cells into hepatocyte lineages.

A 3D culture system is composed of gelatin and extracellular matrix from Swiss 3T3 cells [41]. This system preserves the functions of hepatocyte-like cells differentiated from hiPS cells. The most important component of the ECM has been determined to be type 1 collagen.

Cells are 3D cultured in hollow fibers similar to embryoid bodies. Hollow fibers are useful because the efficiency of embryoid body formation is low compared with mouse ES cells, which also differentiate into hepatocytes in hollow fibers [42]. The organoid culture system efficiently allows mouse ES cells to form cellular aggregates in their lumen. Liver-specific functions of mouse ES cells are comparable with those of primary hepatocytes.

Primary rat hepatocytes have been successfully cultured for 200 days on temperature-respon‐ sive sheets [43]. These sheets attach on the bottoms of culture dishes at 37ºC and detach at 25ºC. They provide easy culturing and handling of cells. Primary rat hepatocytes have preserved liv‐ er-specific functions for 28 days in hybrid sheets with endothelial cells [44]. This system ena‐ bles easy manipulation of iPS cells and may promote differentiation into hepatocytes.

**References**

Cell 2007;131(5):861-872.

man disease. Semin Liver Dis 1999;19(1):39-48.

troenterol Hepatol 2011;26 Suppl 1(203-212.

therapeutic application. Hepatology 2003;37(5):983-993.

tor. Hepatology 1991;13(1):21-30.

Gastroenterology 2009;136(3):990-999.

hypercholesterolemia. Hepatology 2012.

Cell Res 1996;229(1):27-34.

[1] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.

Induced Pluripotent Stem Cells as a Source of Hepatocytes

http://dx.doi.org/10.5772/54552

525

[2] Strom SC, Chowdhury JR, Fox IJ. Hepatocyte transplantation for the treatment of hu‐

[3] Habibullah CM, Syed IH, Qamar A, Taher-Uz Z. Human fetal hepatocyte transplan‐ tation in patients with fulminant hepatic failure. Transplantation 1994;58(8):951-952.

[4] Riehle KJ, Dan YY, Campbell JS, Fausto N. New concepts in liver regeneration. J Gas‐

[5] Fisher RA, Bu D, Thompson M, Wolfe L, Ritter JK. Optimization of conditions for

[6] Mitaka T, Sattler CA, Sattler GL, Sargent LM, Pitot HC. Multiple cell cycles occur in rat hepatocytes cultured in the presence of nicotinamide and epidermal growth fac‐

[7] Yin Y, Lim YK, Salto-Tellez M, Ng SC, Lin CS, Lim SK. AFP(+), ESC-derived cells en‐ graft and differentiate into hepatocytes in vivo. Stem Cells 2002;20(4):338-346.

[8] Yamamoto H, Quinn G, Asari A, Yamanokuchi H, Teratani T, Terada M, Ochiya T. Differentiation of embryonic stem cells into hepatocytes: biological functions and

[9] Basma H, Soto-Gutierrez A, Yannam GR, Liu L, Ito R, Yamamoto T, Ellis E, et al. Dif‐ ferentiation and transplantation of human embryonic stem cell-derived hepatocytes.

[10] Cayo MA, Cai J, Delaforest A, Noto FK, Nagaoka M, Clark BS, Collery RF, et al. 'JD' iPS cell-derived hepatocytes faithfully recapitulate the pathophysiology of familial

[11] Chinzei R, Tanaka Y, Shimizu-Saito K, Hara Y, Kakinuma S, Watanabe M, Teramoto K, et al. Embryoid-body cells derived from a mouse embryonic stem cell line show

[12] Abe K, Niwa H, Iwase K, Takiguchi M, Mori M, Abe SI, Abe K, et al. Endoderm-spe‐ cific gene expression in embryonic stem cells differentiated to embryoid bodies. Exp

[13] Jones EA, Tosh D, Wilson DI, Lindsay S, Forrester LM. Hepatic differentiation of

differentiation into functional hepatocytes. Hepatology 2002;36(1):22-29.

murine embryonic stem cells. Exp Cell Res 2002;272(1):15-22.

clinical human hepatocyte infusion. Cell Transplant 2004;13(6):677-689.

## **12. Conclusion**

Human iPS cells are a promising source for hepatocytes and may be used for drug screen‐ ing, for cell transplantation, and as a model for studying human diseases. Protocols have been presented for the differentiation of human iPS cells into hepatocytes; however, the dif‐ ferentiated cells have limited hepatocyte characteristics. In the future, as more sophisticated methods are expected to be developed, new applications of these cells will be realized.

## **Acknowledgements**

This work was supported in part by a Grant-in-Aid for Scientific Research (C) (grant No. 23591002) from the Japan Society for the Promotion of Science (JSPS).

## **Author details**

Minoru Tomizawa1\*, Fuminobu Shinozaki2 , Takao Sugiyama3 , Shigenori Yamamoto4 , Makoto Sueishi3 and Takanobu Yoshida5

\*Address all correspondence to: nihminor-cib@umin.ac.jp

1 Department of Gastroenterology, National Hospital Organization Shimoshizu Hospital, Yotsukaido City, Japan

2 Department of Radiology, National Hospital Organization Shimoshizu Hospital, Yotsukai‐ do City, Japan

3 Department of Rheumatology, National Hospital Organization Shimoshizu Hospital, Yotsukaido City, Japan

4 Department of Pediatrics, National Hospital Organization Shimoshizu Hospital, Yotsukaido City, Japan

5 Department of Internal Medicine, National Hospital Organization Shimoshizu Hospital, Yotsukaido City, Japan

## **References**

They provide easy culturing and handling of cells. Primary rat hepatocytes have preserved liv‐ er-specific functions for 28 days in hybrid sheets with endothelial cells [44]. This system ena‐

Human iPS cells are a promising source for hepatocytes and may be used for drug screen‐ ing, for cell transplantation, and as a model for studying human diseases. Protocols have been presented for the differentiation of human iPS cells into hepatocytes; however, the dif‐ ferentiated cells have limited hepatocyte characteristics. In the future, as more sophisticated methods are expected to be developed, new applications of these cells will be realized.

This work was supported in part by a Grant-in-Aid for Scientific Research (C) (grant No.

1 Department of Gastroenterology, National Hospital Organization Shimoshizu Hospital,

2 Department of Radiology, National Hospital Organization Shimoshizu Hospital, Yotsukai‐

3 Department of Rheumatology, National Hospital Organization Shimoshizu Hospital,

4 Department of Pediatrics, National Hospital Organization Shimoshizu Hospital, Yotsukaido

5 Department of Internal Medicine, National Hospital Organization Shimoshizu Hospital,

, Takao Sugiyama3

, Shigenori Yamamoto4

,

23591002) from the Japan Society for the Promotion of Science (JSPS).

bles easy manipulation of iPS cells and may promote differentiation into hepatocytes.

**12. Conclusion**

524 Pluripotent Stem Cells

**Acknowledgements**

**Author details**

Makoto Sueishi3

do City, Japan

City, Japan

Yotsukaido City, Japan

Yotsukaido City, Japan

Yotsukaido City, Japan

Minoru Tomizawa1\*, Fuminobu Shinozaki2

and Takanobu Yoshida5

\*Address all correspondence to: nihminor-cib@umin.ac.jp


[14] Yamada T, Yoshikawa M, Kanda S, Kato Y, Nakajima Y, Ishizaka S, Tsunoda Y. In vitro differentiation of embryonic stem cells into hepatocyte-like cells identified by cellular uptake of indocyanine green. Stem Cells 2002;20(2):146-154.

[27] Song Z, Cai J, Liu Y, Zhao D, Yong J, Duo S, Song X, et al. Efficient generation of hep‐ atocyte-like cells from human induced pluripotent stem cells. Cell Res 2009;19(11):

Induced Pluripotent Stem Cells as a Source of Hepatocytes

http://dx.doi.org/10.5772/54552

527

[28] Takayama K, Inamura M, Kawabata K, Katayama K, Higuchi M, Tashiro K, Nonaka A, et al. Efficient Generation of Functional Hepatocytes From Human Embryonic Stem Cells and Induced Pluripotent Stem Cells by HNF4alpha Transduction. Mol

[29] Sekine K, Takebe T, Suzuki Y, Kamiya A, Nakauchi H, Taniguchi H. Highly efficient generation of definitive endoderm lineage from human induced pluripotent stem

[30] Inamura M, Kawabata K, Takayama K, Tashiro K, Sakurai F, Katayama K, Toyoda M, et al. Efficient Generation of Hepatoblasts From Human ES Cells and iPS Cells by

[31] Nakamura N, Saeki K, Mitsumoto M, Matsuyama S, Nishio M, Hasegawa M, Miya‐ gawa Y, et al. Feeder-free and serum-free production of hepatocytes, cholangiocytes, and their proliferating progenitors from human pluripotent stem cells: application to liver-specific functional and cytotoxic assays. Cell Reprogram 2012;14(2):171-185. [32] Chen YF, Tseng CY, Wang HW, Kuo HC, Yang VW, Lee OK. Rapid generation of mature hepatocyte-like cells from human induced pluripotent stem cells by an effi‐

[33] Zaret KS, Watts J, Xu J, Wandzioch E, Smale ST, Sekiya T. Pioneer factors, genetic competence, and inductive signaling: programming liver and pancreas progenitors

[34] Xu ZL, Mizuguchi H, Sakurai F, Koizumi N, Hosono T, Kawabata K, Watanabe Y, et al. Approaches to improving the kinetics of adenovirus-delivered genes and gene

[35] Lee SB, Seo D, Choi D, Park KY, Holczbauer A, Marquardt JU, Conner EA, et al. Con‐ tribution of hepatic lineage stage-specific donor memory to the differential potential

[36] Wu X, Robotham JM, Lee E, Dalton S, Kneteman NM, Gilbert DM, Tang H. Produc‐ tive hepatitis C virus infection of stem cell-derived hepatocytes reveals a critical tran‐ sition to viral permissiveness during differentiation. PLoS Pathog 2012;8(4):e1002617.

[37] Schwartz RE, Trehan K, Andrus L, Sheahan TP, Ploss A, Duncan SA, Rice CM, et al. Modeling hepatitis C virus infection using human induced pluripotent stem cells.

[38] Chang HM, Liao YW, Chiang CH, Chen YJ, Lai YH, Chang YL, Chen HL, et al. Im‐ provement of Carbon Tetrachloride-Induced Acute Hepatic Failure by Transplanta‐ tion of Induced Pluripotent Stem Cells without Reprogramming Factor c-Myc. Int J

from the endoderm. Cold Spring Harb Symp Quant Biol 2008;73(119-126.

of induced mouse pluripotent stem cells. Stem Cells 2012;30(5):997-1007.

Transient Overexpression of Homeobox Gene HEX. Mol Ther 2010.

cient three-step protocol. Hepatology 2012;55(4):1193-1203.

products. Adv Drug Deliv Rev 2005;57(5):781-802.

Proc Natl Acad Sci U S A 2012;109(7):2544-2548.

Mol Sci 2012;13(3):3598-3617.

1233-1242.

Ther 2012;20(1):127-137.

cells. Transplant Proc 2012;44(4):1127-1129.


[27] Song Z, Cai J, Liu Y, Zhao D, Yong J, Duo S, Song X, et al. Efficient generation of hep‐ atocyte-like cells from human induced pluripotent stem cells. Cell Res 2009;19(11): 1233-1242.

[14] Yamada T, Yoshikawa M, Kanda S, Kato Y, Nakajima Y, Ishizaka S, Tsunoda Y. In vitro differentiation of embryonic stem cells into hepatocyte-like cells identified by

[15] Hirai H, Firpo M, Kikyo N. Establishment of LIF-Dependent Human iPS Cells Close‐ ly Related to Basic FGF-Dependent Authentic iPS Cells. PLoS One 2012;7(6):e39022.

[16] Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, Soreq H, et al. Differentiation of human embryonic stem cells into embryoid bodies compromis‐

[17] Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton DA, Benvenisty N. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem

[18] Ishizaka S, Shiroi A, Kanda S, Yoshikawa M, Tsujinoue H, Kuriyama S, Hasuma T, et al. Development of hepatocytes from ES cells after transfection with the HNF-3beta

[19] Shirahashi H, Wu J, Yamamoto N, Catana A, Wege H, Wager B, Okita K, et al. Differ‐ entiation of human and mouse embryonic stem cells along a hepatocyte lineage. Cell

[20] Imamura T, Cui L, Teng R, Johkura K, Okouchi Y, Asanuma K, Ogiwara N, et al. Em‐ bryonic stem cell-derived embryoid bodies in three-dimensional culture system form

hepatocyte-like cells in vitro and in vivo. Tissue Eng 2004;10(11-12):1716-1724.

[21] Teramoto K, Hara Y, Kumashiro Y, Chinzei R, Tanaka Y, Shimizu-Saito K, Asahina K, et al. Teratoma formation and hepatocyte differentiation in mouse liver trans‐ planted with mouse embryonic stem cell-derived embryoid bodies. Transplant Proc

[22] Kumashiro Y, Asahina K, Ozeki R, Shimizu-Saito K, Tanaka Y, Kida Y, Inoue K, et al. Enrichment of hepatocytes differentiated from mouse embryonic stem cells as a

[23] Kumashiro Y, Teramoto K, Shimizu-Saito K, Asahina K, Teraoka H, Arii S. Isolation of hepatocyte-like cells from mouse embryoid body cells. Transplant Proc 2005;37(1):

[24] Rambhatla L, Chiu CP, Kundu P, Peng Y, Carpenter MK. Generation of hepatocytelike cells from human embryonic stem cells. Cell Transplant 2003;12(1):1-11.

[25] DeLaForest A, Nagaoka M, Si-Tayeb K, Noto FK, Konopka G, Battle MA, Duncan SA. HNF4A is essential for specification of hepatic progenitors from human pluripo‐

[26] Si-Tayeb K, Noto FK, Nagaoka M, Li J, Battle MA, Duris C, North PE, et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

transplantable source. Transplantation 2005;79(5):550-557.

tent stem cells. Development 2011;138(19):4143-4153.

cells. Hepatology 2010;51(1):297-305.

cellular uptake of indocyanine green. Stem Cells 2002;20(2):146-154.

ing the three embryonic germ layers. Mol Med 2000;6(2):88-95.

cells. Proc Natl Acad Sci U S A 2000;97(21):11307-11312.

gene. Faseb J 2002;16(11):1444-1446.

Transplant 2004;13(3):197-211.

2005;37(1):285-286.

299-300.

526 Pluripotent Stem Cells


[39] Shiraki N, Umeda K, Sakashita N, Takeya M, Kume K, Kume S. Differentiation of mouse and human embryonic stem cells into hepatic lineages. Genes Cells 2008;13(7): 731-746.

**Chapter 24**

**Induced Pluripotent Stem Cells: Therapeutic**

**Regulatory and Bioethical Considerations**

given their ability to differentiate into other more specialized cells [3].

ones); (iii) Multipotent stem cells (adult stem cells) [4,5].

rejection problems in the event of autologous implantation.

Antonio Liras, Cristina Segovia and Aline S. Gabán

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55816

**1. Introduction**

**Applications in Monogenic and Metabolic Diseases, and**

The potential use of stem cells in advanced therapies such as tissue engineering, regenerative medicine, cell therapy and gene therapy by virtue of their significant therapeutic potential and clinical applications has aroused keen interest among scientists [1,2]. Cell therapy is based on the transplantation of living cells into an organism with a view to repairing tissue or restoring a lost or deficient function. Stem cells are the most frequently used cells for such purposes

The chief defining feature of stem cells is their capacity for self-renewal and their ability to differentiate into cells of various lineages. Stem cells can be classified on the basis of their potency and their source into (i) Totipotent stem cells (zygote and 2-4 cell embryo), since these cells are capable of giving rise to the entire organism (both embryonic and extra-embryonic tissues); (ii) Pluripotent stem cells (embryonic stem and embryonic germ cells), which can give rise to derivatives of all three germ layers (embryonic tissues only, but not the extra-embryonic

Adult stem cells are undifferentiated cells that provide a natural reservoir that is available to replace damaged or ageing cells throughout the lifetime of the individual. They can be found in virtually any kind of tissue including bone marrow, trabecular bone, periosteum, synovium, muscle, adipose tissue, breast gland, gastrointestinal tract, central nervous system, lung, peripheral blood, dermis, hair follicle, corneal limbus, etc. [6]. The clinical application of this type of cell is associated with potentially better prospects than that of embryonic stem cells since use of adult stem cells does not raise any ethical conflicts nor does it involve immune

> © 2013 Liras et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Liras et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.


**Chapter 24**

## **Induced Pluripotent Stem Cells: Therapeutic Applications in Monogenic and Metabolic Diseases, and Regulatory and Bioethical Considerations**

Antonio Liras, Cristina Segovia and Aline S. Gabán

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55816

## **1. Introduction**

[39] Shiraki N, Umeda K, Sakashita N, Takeya M, Kume K, Kume S. Differentiation of mouse and human embryonic stem cells into hepatic lineages. Genes Cells 2008;13(7):

[40] Shiraki N, Yamazoe T, Qin Z, Ohgomori K, Mochitate K, Kume K, Kume S. Efficient differentiation of embryonic stem cells into hepatic cells in vitro using a feeder-free

[41] Nagamoto Y, Tashiro K, Takayama K, Ohashi K, Kawabata K, Sakurai F, Tachibana M, et al. The promotion of hepatic maturation of human pluripotent stem cells in 3D co-culture using type I collagen and Swiss 3T3 cell sheets. Biomaterials 2012;33(18):

[42] Amimoto N, Mizumoto H, Nakazawa K, Ijima H, Funatsu K, Kajiwara T. Hepatic differentiation of mouse embryonic stem cells and induced pluripotent stem cells during organoid formation in hollow fibers. Tissue Eng Part A 2011;17(15-16):

[43] Ohashi K, Yokoyama T, Yamato M, Kuge H, Kanehiro H, Tsutsumi M, Amanuma T, et al. Engineering functional two- and three-dimensional liver systems in vivo using

[44] Kim K, Ohashi K, Utoh R, Kano K, Okano T. Preserved liver-specific functions of hepatocytes in 3D co-culture with endothelial cell sheets. Biomaterials 2012;33(5):

basement membrane substratum. PLoS One 2011;6(8):e24228.

hepatic tissue sheets. Nat Med 2007;13(7):880-885.

731-746.

528 Pluripotent Stem Cells

4526-4534.

2071-2078.

1406-1413.

The potential use of stem cells in advanced therapies such as tissue engineering, regenerative medicine, cell therapy and gene therapy by virtue of their significant therapeutic potential and clinical applications has aroused keen interest among scientists [1,2]. Cell therapy is based on the transplantation of living cells into an organism with a view to repairing tissue or restoring a lost or deficient function. Stem cells are the most frequently used cells for such purposes given their ability to differentiate into other more specialized cells [3].

The chief defining feature of stem cells is their capacity for self-renewal and their ability to differentiate into cells of various lineages. Stem cells can be classified on the basis of their potency and their source into (i) Totipotent stem cells (zygote and 2-4 cell embryo), since these cells are capable of giving rise to the entire organism (both embryonic and extra-embryonic tissues); (ii) Pluripotent stem cells (embryonic stem and embryonic germ cells), which can give rise to derivatives of all three germ layers (embryonic tissues only, but not the extra-embryonic ones); (iii) Multipotent stem cells (adult stem cells) [4,5].

Adult stem cells are undifferentiated cells that provide a natural reservoir that is available to replace damaged or ageing cells throughout the lifetime of the individual. They can be found in virtually any kind of tissue including bone marrow, trabecular bone, periosteum, synovium, muscle, adipose tissue, breast gland, gastrointestinal tract, central nervous system, lung, peripheral blood, dermis, hair follicle, corneal limbus, etc. [6]. The clinical application of this type of cell is associated with potentially better prospects than that of embryonic stem cells since use of adult stem cells does not raise any ethical conflicts nor does it involve immune rejection problems in the event of autologous implantation.

© 2013 Liras et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Liras et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The possibility to generate induced pluripotent stem cells (iPSCs) by reprogramming somatic stem cells through the introduction of certain transcription factors [7-12] is radically trans‐ forming received scientific wisdom. The pluripotency of these cells, which enables them to differentiate into cells of all three germ layers (endoderm, mesoderm, and ectoderm), makes them an extremely valuable tool for the potential design of cell therapy protocols. iPSC technology can indeed allow the development of patient-specific cell therapy protocols [13] as the use of cells like iPSCs, which are genetically identical to the donor, may protect the individual from immune rejection. Furthermore, unlike embryonic stem cells, iPSCs are not associated with bioethical problems and are considered a "consensus" alternative that does not require use of human oocytes or embryos and is therefore not subject to any specific regula‐ tions. Lastly, iPSCs are very similar to embryonic stem cells as far as their molecular and functional characteristics are concerned [14-15].

possibility is transplantation of cells, tissues, or organs taken from cultures of stem cell-derived differentiated cells. The US Food and Drug Administration defines somatic cell therapy as the administration to humans of autologous, allogeneic or xenogeneic living non-germline cells, other than transfusion blood products, which have been manipulated, processed, propagated

Induced Pluripotent Stem Cells: Therapeutic Applications in Monogenic and Metabolic Diseases, and Regulatory…

http://dx.doi.org/10.5772/55816

531

The most significant applications of cell therapy as a whole are expected to be related to the treatment of organ-specific conditions such as diabetes —a typically metabolic disease—, liver and cardiovascular conditions, immunological disorders and hereditary monogenic diseases such as haemophilia. As one of the key advanced therapies —together with gene therapy and tissue engineering— cell therapy will require a new legal framework that affords generalized patient accessibility to these products and that allows governments to discharge their regula‐ tory and control duties. In this respect, the main advantage of iPSCs lies in the fact that their use does not raise bioethical questions, which means that regulatory provisions governing

**2. Induced pluripotent stem cells technology and general clinical**

discovery of new drugs and the establishment of cell therapy programs.

rendered the reprogramming process more efficient [9].

iPSCs are obtained through the reprogramming of an individual's somatic stem cells by the introduction of certain transcription factors. Their chief value is based on their pluripotency to differentiate into cells of all three germ layers, which makes them an useful tool for the

iPSC technology makes it possible to develop patient-specific cell therapy protocols as they are genetically identical to the donor and thus prevent the occurrence of an immune rejection in autologous transplantations. Moreover, unlike embryonic stem cells, they are not associated with any ethical controversies and therefore regulatory conditions governing their use are

Induced pluripotent stem cells were generated for the first time by Shinya Yamanaka's team [8] from murine and human fibroblasts by transfecting certain transcription factors (Oct4, Sox2, c-Myc, and Klf4) by means of retroviral vectors. (Figure 1). Thomson *et al*. replicated Yama‐ naka's experiments with human cells and two additional factors: Nanog and Lin28, which

The same group developed an alternative reprogramming method using non-integrating episomal vectors derived from the Epstein-Barr virus (oriP/EBNA1), which may be maintained in a stable form in transfected cells by pharmacological selection [23]. Nonetheless, it was later reported that only two transcription factors (Oct4 and Klf4) are needed for generating the iPSCs

All of these strategies require transfection through retroviral vectors and integration for *in vitro* and *in vivo* modeling, which precludes their clinical use because of the potential risks involved. This is the reason why several research teams have looked into the reprogramming

from neural stem cells that endogenously express high Sox2 concentrations [24].

or expanded *ex vivo*, or are drug-treated.

their use need not be overly stringent.

**applications**

much less stringent.

Although research into iPSCs is still at an early stage, interesting results have already been obtained in a number of monogenic and polygenic diseases of different etiologies: cardiovas‐ cular and liver diseases, immunologic, infectious, metabolic diseases, rare diseases and cancer [16-19]. Researchers have also looked into the application of iPSCs to toxigological and pharmacological screening for the presence of toxic and teratogenic substances [20].

Stem cell therapy is emerging as a new concept of medical application in pharmacology. For all practical purposes, human embryonic stem cells are used in 13% of treatments, whereas fetal stem cells are used in 2%, umbilical cord stem cells in 10%, and adult stem cells in 75% of cases. The most significant treatment indications for gene and cell therapy have so far been cardiovascular and ischemic diseases, diabetes, hematopoietic diseases, liver diseases and, more recently, orthopaedics [21]. For example, over 25,000 transplants of hematopoietic stem cells are performed every year for treatment of lymphoma, leukemia, immunodeficiency disorders, congenital metabolic defects, hemoglobinopathies, and myelodysplastic and myeloproliferative syndromes [22].

Each type of stem cell has its own advantages and disadvantages, which vary depending on the different treatment protocols and the requirements of each clinical condition. Thus, embryonic stem cells have the advantages of being pluripotent, easy to isolate and highly productive in culture, in addition to showing a high capacity to integrate into fetal tissue during development. By contrast, their disadvantages include immune rejection and the possibility that they may spontaneously and uncontrollably differentiate into inadequate cell types or even induce tumors. Adult stem cells have a high differentiation potential, are less likely to induce an undesirable immune response and may be stimulated by drugs. Their disadvantages include that they are scarce and difficult to harvest, grown slowly, differentiate poorly in culture and are difficult to handle and produce in adequate amounts for transplantation. In addition, they behave differently depending on the source tissue, show telomere shortening, and may carry the genetic abnormalities inherited or acquired by the donor.

At least three different strategies are available for proper use of stem cells. The first one is stimulation of endogenous stem cells by growth factors, cytokines, and second messengers, which must be able to induce tissue self-repair. The second alternative is direct administration of the cells so that they differentiate at the damaged or non-functional tissue sites. The third possibility is transplantation of cells, tissues, or organs taken from cultures of stem cell-derived differentiated cells. The US Food and Drug Administration defines somatic cell therapy as the administration to humans of autologous, allogeneic or xenogeneic living non-germline cells, other than transfusion blood products, which have been manipulated, processed, propagated or expanded *ex vivo*, or are drug-treated.

The possibility to generate induced pluripotent stem cells (iPSCs) by reprogramming somatic stem cells through the introduction of certain transcription factors [7-12] is radically trans‐ forming received scientific wisdom. The pluripotency of these cells, which enables them to differentiate into cells of all three germ layers (endoderm, mesoderm, and ectoderm), makes them an extremely valuable tool for the potential design of cell therapy protocols. iPSC technology can indeed allow the development of patient-specific cell therapy protocols [13] as the use of cells like iPSCs, which are genetically identical to the donor, may protect the individual from immune rejection. Furthermore, unlike embryonic stem cells, iPSCs are not associated with bioethical problems and are considered a "consensus" alternative that does not require use of human oocytes or embryos and is therefore not subject to any specific regula‐ tions. Lastly, iPSCs are very similar to embryonic stem cells as far as their molecular and

Although research into iPSCs is still at an early stage, interesting results have already been obtained in a number of monogenic and polygenic diseases of different etiologies: cardiovas‐ cular and liver diseases, immunologic, infectious, metabolic diseases, rare diseases and cancer [16-19]. Researchers have also looked into the application of iPSCs to toxigological and

Stem cell therapy is emerging as a new concept of medical application in pharmacology. For all practical purposes, human embryonic stem cells are used in 13% of treatments, whereas fetal stem cells are used in 2%, umbilical cord stem cells in 10%, and adult stem cells in 75% of cases. The most significant treatment indications for gene and cell therapy have so far been cardiovascular and ischemic diseases, diabetes, hematopoietic diseases, liver diseases and, more recently, orthopaedics [21]. For example, over 25,000 transplants of hematopoietic stem cells are performed every year for treatment of lymphoma, leukemia, immunodeficiency disorders, congenital metabolic defects, hemoglobinopathies, and myelodysplastic and

Each type of stem cell has its own advantages and disadvantages, which vary depending on the different treatment protocols and the requirements of each clinical condition. Thus, embryonic stem cells have the advantages of being pluripotent, easy to isolate and highly productive in culture, in addition to showing a high capacity to integrate into fetal tissue during development. By contrast, their disadvantages include immune rejection and the possibility that they may spontaneously and uncontrollably differentiate into inadequate cell types or even induce tumors. Adult stem cells have a high differentiation potential, are less likely to induce an undesirable immune response and may be stimulated by drugs. Their disadvantages include that they are scarce and difficult to harvest, grown slowly, differentiate poorly in culture and are difficult to handle and produce in adequate amounts for transplantation. In addition, they behave differently depending on the source tissue, show telomere shortening,

At least three different strategies are available for proper use of stem cells. The first one is stimulation of endogenous stem cells by growth factors, cytokines, and second messengers, which must be able to induce tissue self-repair. The second alternative is direct administration of the cells so that they differentiate at the damaged or non-functional tissue sites. The third

and may carry the genetic abnormalities inherited or acquired by the donor.

pharmacological screening for the presence of toxic and teratogenic substances [20].

functional characteristics are concerned [14-15].

530 Pluripotent Stem Cells

myeloproliferative syndromes [22].

The most significant applications of cell therapy as a whole are expected to be related to the treatment of organ-specific conditions such as diabetes —a typically metabolic disease—, liver and cardiovascular conditions, immunological disorders and hereditary monogenic diseases such as haemophilia. As one of the key advanced therapies —together with gene therapy and tissue engineering— cell therapy will require a new legal framework that affords generalized patient accessibility to these products and that allows governments to discharge their regula‐ tory and control duties. In this respect, the main advantage of iPSCs lies in the fact that their use does not raise bioethical questions, which means that regulatory provisions governing their use need not be overly stringent.

## **2. Induced pluripotent stem cells technology and general clinical applications**

iPSCs are obtained through the reprogramming of an individual's somatic stem cells by the introduction of certain transcription factors. Their chief value is based on their pluripotency to differentiate into cells of all three germ layers, which makes them an useful tool for the discovery of new drugs and the establishment of cell therapy programs.

iPSC technology makes it possible to develop patient-specific cell therapy protocols as they are genetically identical to the donor and thus prevent the occurrence of an immune rejection in autologous transplantations. Moreover, unlike embryonic stem cells, they are not associated with any ethical controversies and therefore regulatory conditions governing their use are much less stringent.

Induced pluripotent stem cells were generated for the first time by Shinya Yamanaka's team [8] from murine and human fibroblasts by transfecting certain transcription factors (Oct4, Sox2, c-Myc, and Klf4) by means of retroviral vectors. (Figure 1). Thomson *et al*. replicated Yama‐ naka's experiments with human cells and two additional factors: Nanog and Lin28, which rendered the reprogramming process more efficient [9].

The same group developed an alternative reprogramming method using non-integrating episomal vectors derived from the Epstein-Barr virus (oriP/EBNA1), which may be maintained in a stable form in transfected cells by pharmacological selection [23]. Nonetheless, it was later reported that only two transcription factors (Oct4 and Klf4) are needed for generating the iPSCs from neural stem cells that endogenously express high Sox2 concentrations [24].

All of these strategies require transfection through retroviral vectors and integration for *in vitro* and *in vivo* modeling, which precludes their clinical use because of the potential risks involved. This is the reason why several research teams have looked into the reprogramming

In order for the clinical application of these cells to become a reality both for diagnostic purposes and for the design of cell therapy protocols, a few methodological hurdles must still be resolved in connection, as is often the case with pharmacological products, with their safety profile [30]. This means basically that efforts must be directed at removing the genome in the integrating viral vectors, eliminating the risk of tumor formation and establishing more efficient reprogramming and differentiation protocols. Clearly our knowledge on the reprog‐ ramming mechanisms leading to pluripotency are still insufficient to understand and more importantly control the adverse events that could potentially occur. Therefore the most important goal for research in this field will be to study genetic modifications in animal models by means of large-scale genome sequencing programs. This task will require sharing cell lines with other researchers, with appropriate confidentiality protections and, eventually, patenting scientific discoveries and developing commercial tests and therapies. It will also be necessary to fully ascertain and confirm that pluripotency confers iPSCs with functions similar to those

Induced Pluripotent Stem Cells: Therapeutic Applications in Monogenic and Metabolic Diseases, and Regulatory…

http://dx.doi.org/10.5772/55816

533

of embryonic stem cells regardless of the initial source of somatic cells used [14,15].

pharmacology for screening toxics for embryo and/or teratogenic substances [20].

hematopoietic cells [33], and cardiomyocytes [34,35].

therefore allow physicians to provide well-grounded genetic guidance.

Undoubtedly,themost attractive applicationofthis typeof strategyis theproductionofpatientspecific or healthy individual-specific iPSCs forreplacement ofdamagednon-functionaltissue. Thus for example skin fibroblast-derived iPSCs have been shown to possess a high potential to differentiate into islet-like clusters and to release insulin, which is highly relevant for diabetes [16].Suchdevelopmentsarealsorelevantforamyotrophiclateralsclerosis(LouGehrig´sdisease) [17]; adenosine deaminase deficiency-related severe combined immunodeficiency, Shwach‐ man-Bodian-Diamond syndrome, Gaucher disease type III, Duchenne and Becker muscular dystrophy, Parkinson disease, Huntington disease, juvenile-onset, type 1 diabetes mellitus and Down syndrome (trisomy 21) [31]; spinal muscular atrophy [19]; and in toxicology and

The great promise of iPSCs (Figure 1) is associated to their role in the investigation of the phyisiological mechanisms related with the biology of stem cells themselves; in the modeling of different pathologies; and, fundamentally, in the development of therapies for human diseases and in drug screening. In fact, since they were discovered in 2008, almost onehundred-and-fifty iPSCs have been established from nearly thirty fibroblast cell lines related to over a dozen conditions, including some complex diseases such as schizophrenia and autism and other genetic or acquired disorders such as cardiovascular or infectious diseases. Numer‐ ous types of functional cells have already been derived from iPSCs including neurons [17,32],

Taking into account the far-from-trivial fact that iPSCs can be obtained from individuals affected by a disease and that they are indefinitely self-renewable and fully of human origin, it could well be that these cells, obtained from several individuals suffering from the same disease and presenting with similar clinical manifestations, may provide highly valuable information about certain predisposing genes ―as in the case of diabetes mellitus― and

Human iPSCs have the potential to be used in regenerative medicine for the design of individualized therapies and also in the field of research and development. However, it is still necessary to optimize iPSC protocols, particularly with respect to the possible modifications

**Figure 1.** Generation of human induced pluripotent stem cells for use in cell therapy, in vitro human pathology mod‐ elling and in drug discovery. Reprogramming of human somatic cells can be induced by: Viral transfection of Oct4, Sox2, c-Myc, Klf4, Nanog and Lin28 genes; non-viral methods using a nonintegrating episomal vector derived from Epstein-Barr virus (oriP/EBNA1), plasmid vectors or piggyBac transposon/transposase systems; direct delivery of the re‐ programming proteins (piPSCs) and signal transduction inhibitors and chemical promoters cell survival.

of cells using plasmid vector rather than viral vector transfection [10-12]. Although reprog‐ ramming efficacy with plasmid vectors is lower ―as is also the case with non viral gene therapy― this method significantly increases the safety of the procedure, which makes it clinically applicable and also constitutes a source of valuable cell material that can be used for research into reprogramming and pluripotency.

Another promising strategy consists in the direct release of reprogramming proteins through modified versions of reprogramming factors in some of their molecular domains. These protein-induced pluripotent stem cells (piPSCs) bind to the membrane of cells reaching their nucleus [25]. Ding *et al.*, have also shown that the addition of two signal transduction inhibitors and certain cell-survival promoting chemicals (e.g. thiazovivin) can induce a 200-fold increase in reprogramming efficacy [26].

As explained above, iPSCs technology makes it possible to establish patient-specific cell therapy protocols [13]. On the one hand, this reduces the risk of immune rejection in autologous transplantations by virtue of gene identity. On the other, it provides treatment that is custom‐ ized to the specific characteristics of each patient and takes into account the etiology and severity of the condition. Moreover, induction of pluripotency has been developed for a great variety of tissue types [9,24,27] as it is a relatively straightforward procedure and —as mentioned above— subject to fewer regulatory constraints [28].

Important as these advantages are, there are still a few uncertainties that need to be resolved. One of the most pressing ones is related to determining the likelihood that these iPSCs may undergo genetic aberrations further to the reprogramming process [29].

In order for the clinical application of these cells to become a reality both for diagnostic purposes and for the design of cell therapy protocols, a few methodological hurdles must still be resolved in connection, as is often the case with pharmacological products, with their safety profile [30]. This means basically that efforts must be directed at removing the genome in the integrating viral vectors, eliminating the risk of tumor formation and establishing more efficient reprogramming and differentiation protocols. Clearly our knowledge on the reprog‐ ramming mechanisms leading to pluripotency are still insufficient to understand and more importantly control the adverse events that could potentially occur. Therefore the most important goal for research in this field will be to study genetic modifications in animal models by means of large-scale genome sequencing programs. This task will require sharing cell lines with other researchers, with appropriate confidentiality protections and, eventually, patenting scientific discoveries and developing commercial tests and therapies. It will also be necessary to fully ascertain and confirm that pluripotency confers iPSCs with functions similar to those of embryonic stem cells regardless of the initial source of somatic cells used [14,15].

Undoubtedly,themost attractive applicationofthis typeof strategyis theproductionofpatientspecific or healthy individual-specific iPSCs forreplacement ofdamagednon-functionaltissue. Thus for example skin fibroblast-derived iPSCs have been shown to possess a high potential to differentiate into islet-like clusters and to release insulin, which is highly relevant for diabetes [16].Suchdevelopmentsarealsorelevantforamyotrophiclateralsclerosis(LouGehrig´sdisease) [17]; adenosine deaminase deficiency-related severe combined immunodeficiency, Shwach‐ man-Bodian-Diamond syndrome, Gaucher disease type III, Duchenne and Becker muscular dystrophy, Parkinson disease, Huntington disease, juvenile-onset, type 1 diabetes mellitus and Down syndrome (trisomy 21) [31]; spinal muscular atrophy [19]; and in toxicology and pharmacology for screening toxics for embryo and/or teratogenic substances [20].

of cells using plasmid vector rather than viral vector transfection [10-12]. Although reprog‐ ramming efficacy with plasmid vectors is lower ―as is also the case with non viral gene therapy― this method significantly increases the safety of the procedure, which makes it clinically applicable and also constitutes a source of valuable cell material that can be used for

programming proteins (piPSCs) and signal transduction inhibitors and chemical promoters cell survival.

**Figure 1.** Generation of human induced pluripotent stem cells for use in cell therapy, in vitro human pathology mod‐ elling and in drug discovery. Reprogramming of human somatic cells can be induced by: Viral transfection of Oct4, Sox2, c-Myc, Klf4, Nanog and Lin28 genes; non-viral methods using a nonintegrating episomal vector derived from Epstein-Barr virus (oriP/EBNA1), plasmid vectors or piggyBac transposon/transposase systems; direct delivery of the re‐

Another promising strategy consists in the direct release of reprogramming proteins through modified versions of reprogramming factors in some of their molecular domains. These protein-induced pluripotent stem cells (piPSCs) bind to the membrane of cells reaching their nucleus [25]. Ding *et al.*, have also shown that the addition of two signal transduction inhibitors and certain cell-survival promoting chemicals (e.g. thiazovivin) can induce a 200-fold increase

As explained above, iPSCs technology makes it possible to establish patient-specific cell therapy protocols [13]. On the one hand, this reduces the risk of immune rejection in autologous transplantations by virtue of gene identity. On the other, it provides treatment that is custom‐ ized to the specific characteristics of each patient and takes into account the etiology and severity of the condition. Moreover, induction of pluripotency has been developed for a great variety of tissue types [9,24,27] as it is a relatively straightforward procedure and —as

Important as these advantages are, there are still a few uncertainties that need to be resolved. One of the most pressing ones is related to determining the likelihood that these iPSCs may

research into reprogramming and pluripotency.

mentioned above— subject to fewer regulatory constraints [28].

undergo genetic aberrations further to the reprogramming process [29].

in reprogramming efficacy [26].

532 Pluripotent Stem Cells

The great promise of iPSCs (Figure 1) is associated to their role in the investigation of the phyisiological mechanisms related with the biology of stem cells themselves; in the modeling of different pathologies; and, fundamentally, in the development of therapies for human diseases and in drug screening. In fact, since they were discovered in 2008, almost onehundred-and-fifty iPSCs have been established from nearly thirty fibroblast cell lines related to over a dozen conditions, including some complex diseases such as schizophrenia and autism and other genetic or acquired disorders such as cardiovascular or infectious diseases. Numer‐ ous types of functional cells have already been derived from iPSCs including neurons [17,32], hematopoietic cells [33], and cardiomyocytes [34,35].

Taking into account the far-from-trivial fact that iPSCs can be obtained from individuals affected by a disease and that they are indefinitely self-renewable and fully of human origin, it could well be that these cells, obtained from several individuals suffering from the same disease and presenting with similar clinical manifestations, may provide highly valuable information about certain predisposing genes ―as in the case of diabetes mellitus― and therefore allow physicians to provide well-grounded genetic guidance.

Human iPSCs have the potential to be used in regenerative medicine for the design of individualized therapies and also in the field of research and development. However, it is still necessary to optimize iPSC protocols, particularly with respect to the possible modifications to their genome, and to increase the efficacy of the transfection process leading to iPSC reprogramming [36,37]. The present state of the art of reprogramming mechanisms ―viral transfection of Oct4, Sox2, c-Myc, Klf4, Nanog and Lin28 genes; non-viral transfection using a non-integrating episomal vector derived from the Epstein-Barr virus (oriP/EBNA1), plasmid vectors or piggyback transposon/transposase systems; direct delivery of the reprogramming proteins (piPSCs); and signal transduction inhibitors and chemical promoter cell survival― will allow safe integration and the removal of ectopic transgenes, improving the efficiency of iPSC production using a minimally invasive strategy.

Advanced therapies are applied following three basic approaches: replacement of a deficient gene by a healthy gene so that it generates a certain functional, structural or transport protein (gene therapy); incorporation of a full array of healthy genes and proteins through perfusion or transplantation of healthy cells (cell therapy); or tissue transplantation and formation of healthy organs (tissue engineering). In this context, induced pluripotent stem cells can play a very significant role and hold an enormous therapeutic potential in the fields of cell therapy

Induced Pluripotent Stem Cells: Therapeutic Applications in Monogenic and Metabolic Diseases, and Regulatory…

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**4. Advanced therapies and induced pluripotent stem cells in the treatment**

Haemophilia is a recessive X-linked hereditary disorder caused by a deficiency of coagulation factor VIII (haemophilia A) or IX (haemophilia B). The disease is considered to be severe when factor levels are below 1% of normal values, moderate when they are between 1 and 5% and mild when levels range between 5% and 40%. Haemophilia A is four times more common than haemophilia B and, in terms of severity for both types, 35% of patients have the severe form, 15% the moderate form and 55% have mild haemophilia. Incidence of the disease is 1:6,000

The etiopathogenesis of the disease is related to different kinds of mutations (large deletions and insertions, inversions and point mutations) that occur in the gene expressing the deficient coagulation factor. The clinical characteristics of both types of haemophilia are very similar: spontaneous or traumatic hemorrhages, muscle hematomas, haemophilic arthropathy resulting from the articular damage caused by repetitive bleeding episodes in the target joints, or hemorrhages in the central nervous system. In the absence of appropriate replacement treatment with exogenous coagulation factors, these manifestations of the disease can have disabling or even fatal consequences thus negatively impacting patients' quality of life and

At present, patients with haemophilia benefit from optimized treatment schedules based on the intravenous systemic delivery of exogenous coagulation factors, either prophylactically or on demand. The current policy in developed countries is in general to administer a prophy‐ lactic treatment (2 or 3 times a week) from early childhood into adulthood [39]. Such prophy‐ lactic protocols result in a clear improvement in patients' quality of life on account of the prevention of haemophilic arthropaty and other fatal manifestations of the disease as well as a reduction in the long-term costs of treatment because of a decrease in the need of surgical

Conventional treatment of haemophilia [41,42] is currently based on the use of plasma-derived or recombinant high-purity coagulation factor concentrates. The former are duly treated with heat and detergent to inactivate lipid-coated viruses [43], and the latter are a recently devel‐ oped product that does not contain proteins of human or animal origin [44,45]. Both kinds of factor boast high efficacy and safety profiles, at least for the inactivation-susceptible pathogens

males born alive for haemophilia A and 1:30,000 for haemophilia B [38].

procedures such as arthrodesis, arthroplasty or synovectomy [40].

and tissue engineering.

**of haemophilia**

reducing their life expectancy [39].

## **3. Advanced therapies for monogenic and metabolic diseases**

The progression of the different areas of biology, biotechnology and medicine leads to the development of highly innovative new treatments and pharmacological products. In this regard, advanced therapies based on the by-products of gene therapy, cell therapy and nanomedicine/tissue engineering are of great importance for their potential to radically improve treatment of a large number of conditions. The different schools of thought that advocate the emerging concept of advanced therapies agree that the latter must be used for the treatment of diseases (both hereditary and non-transmissible) caused by the anomalous behavior, or complete lack of function, of a single gene (also called monogenic hereditary diseases) or by an anomaly in several genes (polygenic diseases).

Metabolic diseases, or congenital metabolic errors, are conditions highly amenable to be treated by the new advanced therapies as such treatments have been shown to restore mutation-induced alterations of gene products. Proteins are the most commonly affected gene products, although messenger RNA is also a usual victim. Alterations affect gene products, i.e. proteins, most of which are enzymes but there is also a group of other proteins fulfilling all kinds of different functions (structural proteins, transport proteins and signal cascade activation proteins). Of particular interest are the proteins that participate in homeostasis and exert their functions outside the cells that synthesize them. This is the case of coagulation factors VIII and IX (FVIII and FIX), whose deficiency results in the development of haemophilia A or B, respectively. Another member of this class of proteins is antitrypsin, also of hepatic origin and secreted into the bloodstream, whose function is to prevent the digestion of pulmonary alveoli by proteolytic enzymes. Lastly, mention should be made of proteins with such diverse functions as transcription factors, oncogenes, tumor-suppressing genes and even some hormones and their receptors, the latter being specifically related with diabetes mellitus, a typically metabolic disease.

The nature of the monogenic or metabolic disease is the main factor that determines whether a treatment that can eradicate or at least mitigate its clinical consequences is possible or not. Before the concept of advanced therapies came to be applied to these (wide ranging) condi‐ tions, many of them were treated using both conventional/classical and more advanced approaches.

Advanced therapies are applied following three basic approaches: replacement of a deficient gene by a healthy gene so that it generates a certain functional, structural or transport protein (gene therapy); incorporation of a full array of healthy genes and proteins through perfusion or transplantation of healthy cells (cell therapy); or tissue transplantation and formation of healthy organs (tissue engineering). In this context, induced pluripotent stem cells can play a very significant role and hold an enormous therapeutic potential in the fields of cell therapy and tissue engineering.

to their genome, and to increase the efficacy of the transfection process leading to iPSC reprogramming [36,37]. The present state of the art of reprogramming mechanisms ―viral transfection of Oct4, Sox2, c-Myc, Klf4, Nanog and Lin28 genes; non-viral transfection using a non-integrating episomal vector derived from the Epstein-Barr virus (oriP/EBNA1), plasmid vectors or piggyback transposon/transposase systems; direct delivery of the reprogramming proteins (piPSCs); and signal transduction inhibitors and chemical promoter cell survival― will allow safe integration and the removal of ectopic transgenes, improving the efficiency of

The progression of the different areas of biology, biotechnology and medicine leads to the development of highly innovative new treatments and pharmacological products. In this regard, advanced therapies based on the by-products of gene therapy, cell therapy and nanomedicine/tissue engineering are of great importance for their potential to radically improve treatment of a large number of conditions. The different schools of thought that advocate the emerging concept of advanced therapies agree that the latter must be used for the treatment of diseases (both hereditary and non-transmissible) caused by the anomalous behavior, or complete lack of function, of a single gene (also called monogenic hereditary

Metabolic diseases, or congenital metabolic errors, are conditions highly amenable to be treated by the new advanced therapies as such treatments have been shown to restore mutation-induced alterations of gene products. Proteins are the most commonly affected gene products, although messenger RNA is also a usual victim. Alterations affect gene products, i.e. proteins, most of which are enzymes but there is also a group of other proteins fulfilling all kinds of different functions (structural proteins, transport proteins and signal cascade activation proteins). Of particular interest are the proteins that participate in homeostasis and exert their functions outside the cells that synthesize them. This is the case of coagulation factors VIII and IX (FVIII and FIX), whose deficiency results in the development of haemophilia A or B, respectively. Another member of this class of proteins is antitrypsin, also of hepatic origin and secreted into the bloodstream, whose function is to prevent the digestion of pulmonary alveoli by proteolytic enzymes. Lastly, mention should be made of proteins with such diverse functions as transcription factors, oncogenes, tumor-suppressing genes and even some hormones and their receptors, the latter being specifically related with diabetes mellitus,

The nature of the monogenic or metabolic disease is the main factor that determines whether a treatment that can eradicate or at least mitigate its clinical consequences is possible or not. Before the concept of advanced therapies came to be applied to these (wide ranging) condi‐ tions, many of them were treated using both conventional/classical and more advanced

iPSC production using a minimally invasive strategy.

534 Pluripotent Stem Cells

**3. Advanced therapies for monogenic and metabolic diseases**

diseases) or by an anomaly in several genes (polygenic diseases).

a typically metabolic disease.

approaches.

## **4. Advanced therapies and induced pluripotent stem cells in the treatment of haemophilia**

Haemophilia is a recessive X-linked hereditary disorder caused by a deficiency of coagulation factor VIII (haemophilia A) or IX (haemophilia B). The disease is considered to be severe when factor levels are below 1% of normal values, moderate when they are between 1 and 5% and mild when levels range between 5% and 40%. Haemophilia A is four times more common than haemophilia B and, in terms of severity for both types, 35% of patients have the severe form, 15% the moderate form and 55% have mild haemophilia. Incidence of the disease is 1:6,000 males born alive for haemophilia A and 1:30,000 for haemophilia B [38].

The etiopathogenesis of the disease is related to different kinds of mutations (large deletions and insertions, inversions and point mutations) that occur in the gene expressing the deficient coagulation factor. The clinical characteristics of both types of haemophilia are very similar: spontaneous or traumatic hemorrhages, muscle hematomas, haemophilic arthropathy resulting from the articular damage caused by repetitive bleeding episodes in the target joints, or hemorrhages in the central nervous system. In the absence of appropriate replacement treatment with exogenous coagulation factors, these manifestations of the disease can have disabling or even fatal consequences thus negatively impacting patients' quality of life and reducing their life expectancy [39].

At present, patients with haemophilia benefit from optimized treatment schedules based on the intravenous systemic delivery of exogenous coagulation factors, either prophylactically or on demand. The current policy in developed countries is in general to administer a prophy‐ lactic treatment (2 or 3 times a week) from early childhood into adulthood [39]. Such prophy‐ lactic protocols result in a clear improvement in patients' quality of life on account of the prevention of haemophilic arthropaty and other fatal manifestations of the disease as well as a reduction in the long-term costs of treatment because of a decrease in the need of surgical procedures such as arthrodesis, arthroplasty or synovectomy [40].

Conventional treatment of haemophilia [41,42] is currently based on the use of plasma-derived or recombinant high-purity coagulation factor concentrates. The former are duly treated with heat and detergent to inactivate lipid-coated viruses [43], and the latter are a recently devel‐ oped product that does not contain proteins of human or animal origin [44,45]. Both kinds of factor boast high efficacy and safety profiles, at least for the inactivation-susceptible pathogens known to date. The choice of one product over the other is usually based on the clinical characteristics of the patient and on cost and availability considerations [46,47].

status (Figure 2). For this reason, significant progress has been possible with respect to these kinds of therapies: cell therapy has broken new ground with the use of several types of target cells and gene therapy has shown particular promise with the use of viral and non-viral vectors. In fact, haemophilia is now recognized as a condition amenable to gene therapy [61-64]. Strategies available include use of lentiviral (LVV) [65] and adeno-associated (AAV) [66] vectors in adult stem cells and autologous fibroblasts, in platelets and in hematopoietic stem cells; transfer by means of non-viral vectors; and repair of mutations with chimeric oligonu‐ cleotides. The studies published so far have, in the most part, not reported any severe adverse

Induced Pluripotent Stem Cells: Therapeutic Applications in Monogenic and Metabolic Diseases, and Regulatory…

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537

effect resulting from the application of such strategies in the clinical trials performed.

Specifically, gene therapy trials in haemophilic patients have shown adeno-associated vectors to represent the most promising treatment option given their excellent safety profile, even if on occasion they may create immune response problems. Efforts are currently centered on minimizing the incidence of immune rejection and increasing efficacy and expression time. In this connection, several studies have been published with a view to optimizing the use of this type of viral vectors. Among them, in a landmark study on patients with severe haemophilia B (<1% FIX), Nathwani *et al.* infused their subjects with a dose of a serotype-8-pseudotyped, self-complementary AAV vector that expresses factor IX and can efficiently transduce hepatocytes [66]. Their results showed that factor IX expression ranged between 3 and 11% of normal values. Significant as they may seem, these results must be considered with caution as the expression levels achieved rather than normalize the patient's phenotype convert it to a mild-to-moderate form. Also, concomitant treatment with glucocorticoids is needed to prevent immune rejection and elevation of liver transaminase levels. Due account must also be taken of the fact that the adeno-associated vector has the potential to induce hepatotoxicity. For all these reasons, these undoubtedly encouraging results can only be considered a first step in the development of safe and effective advanced therapies for the treatment of haemophilia.

Non-viral strategies also have a role to play in the treatment of haemophilia as they could in the long term provide a safer alternative than viral vectors which, as we have seen, are fraught with significant biosafety and efficacy-related problems, which have so far limited their clinical application. Sivalingam *et al*. [67] evaluated the genotoxic potential of phiC31 bacteriophage integrase-mediated transgene integration in cord-lining epithelial cells cultured from the human umbilical cord. This non-viral strategy has made it possible to obtain stable factor VIII secretion *in vitro*. Xenoimplantation of these protein-secreting cell lines into immunocompe‐ tent haemophilic mice corrects the severe form of the disease. Such implantation could prove extremely useful as a bioimplant in the context of monogenic diseases such as haemophilia. Our laboratory has advanced the use of nucleofection as a non-viral transfection method to obtain factor IX expression and secretion in adult adipose tissue-derived mesenchymal stem cells [68]. Although it is certainly true that expression efficacy with these types of protocols is lower than when viral vectors are used, it must be underscored that these protocols do offer much higher safety levels, with the additional advantage that increasing factor activity to

above 5% of normal values already places the patient in the mild phenotype group.

The use of cell therapy in the treatment of haemophilia has to date consisted mainly in the transplantation of healthy cells in an attempt to repair or replace a coagulation factor defi‐

Now that infections by pathogenic viruses (HIV, HCV) that were common a few decades ago have been eradicated, the most distressing adverse effect observed when using either product is the development of antibodies (inhibitors) against the perfused exogenous factors [48,49]. The appearance of inhibitors renders current treatment with factor concentrates inefficient, increasing morbidity and mortality, leading to the early onset of haemophilic arthropathy and disability and to a consequent reduction in patients' quality of life. Lastly, inhibitors result in higher costs as treatment must be provided both for bleeding episodes and inhibitor eradica‐ tion (immune tolerance induction). The incidence of inhibitors is around 30% in haemophilia A and 6% in haemophilia B.

The immunologic mechanism whereby these neutralizing antibodies are generated is highly complex and involves several messenger molecules (tumor necrosis factor, interleukins…), and cells (T-lymphocytes B-lymphocytes, macrophages...). They are directed at certain regions in the factor molecule that interact with other components of the coagulation cascade and, depending on their titre level and on whether they are transient or persistent, will bring about greater or lesser alterations in the said cascade. The causes that influence inhibitor develop‐ ment may be genetic, i.e. inherent in the patients themselves [48], such as ethnicity, familial history, type of mutation or certain changes in some of the genes involved in the immune response; or non-genetic, i.e. environmental [50], such as age at first factor infusion, breast‐ feeding, stimulation of the immune system by other antigens or the treatment regimen used (prophylactic vs. on demand). Whether the factor concentrate used is plasma-derived or recombinant does not have a significant influence on the inhibitor incidence rate [51].

Short and medium-term perspectives for the treatment of haemophilia strongly rely on the current research efforts directed at increasing the safety levels of (especially) plasma-derived factors. Such research focuses on the detection and subsequent inactivation of emerging bloodborne pathogens in donors such as the prions causing variant Creutzfeldt-Jakob disease, or other potential emerging agents [52-54]. It is also important to increase the efficiency of recombinant factors increasing their half-life (by PEGylating the factor molecule or using fusion proteins [55-58] and attenuating their immunogenic capacity to produce inhibitors, by chemically modifying them [59] or by developing recombinant factors of human origin [60].

In the long term, efforts must be directed at the development of advanced therapies, particu‐ larly strategies in the field of gene therapy (using of adeno-associated viral vectors) and cell therapy (using of adult stem cells or induced pluripotent stem cells). The chief goal of these new strategies will be to address some of the shortcomings associated with current treatment options such as the short *in vivo* half-life of administered factors, the impending risk of a pathogen-induced infection and the development of inhibitors. Another goal of the advanced therapies (cell therapy) will be palliative treatment of the articular consequences derived from haemophilic arthropathy [40].

Haemophilia is optimally suited for advanced therapies as it is a monogenic condition and does not require very high expression levels of a coagulation factor to reach moderate disease status (Figure 2). For this reason, significant progress has been possible with respect to these kinds of therapies: cell therapy has broken new ground with the use of several types of target cells and gene therapy has shown particular promise with the use of viral and non-viral vectors. In fact, haemophilia is now recognized as a condition amenable to gene therapy [61-64]. Strategies available include use of lentiviral (LVV) [65] and adeno-associated (AAV) [66] vectors in adult stem cells and autologous fibroblasts, in platelets and in hematopoietic stem cells; transfer by means of non-viral vectors; and repair of mutations with chimeric oligonu‐ cleotides. The studies published so far have, in the most part, not reported any severe adverse effect resulting from the application of such strategies in the clinical trials performed.

known to date. The choice of one product over the other is usually based on the clinical

Now that infections by pathogenic viruses (HIV, HCV) that were common a few decades ago have been eradicated, the most distressing adverse effect observed when using either product is the development of antibodies (inhibitors) against the perfused exogenous factors [48,49]. The appearance of inhibitors renders current treatment with factor concentrates inefficient, increasing morbidity and mortality, leading to the early onset of haemophilic arthropathy and disability and to a consequent reduction in patients' quality of life. Lastly, inhibitors result in higher costs as treatment must be provided both for bleeding episodes and inhibitor eradica‐ tion (immune tolerance induction). The incidence of inhibitors is around 30% in haemophilia

The immunologic mechanism whereby these neutralizing antibodies are generated is highly complex and involves several messenger molecules (tumor necrosis factor, interleukins…), and cells (T-lymphocytes B-lymphocytes, macrophages...). They are directed at certain regions in the factor molecule that interact with other components of the coagulation cascade and, depending on their titre level and on whether they are transient or persistent, will bring about greater or lesser alterations in the said cascade. The causes that influence inhibitor develop‐ ment may be genetic, i.e. inherent in the patients themselves [48], such as ethnicity, familial history, type of mutation or certain changes in some of the genes involved in the immune response; or non-genetic, i.e. environmental [50], such as age at first factor infusion, breast‐ feeding, stimulation of the immune system by other antigens or the treatment regimen used (prophylactic vs. on demand). Whether the factor concentrate used is plasma-derived or

recombinant does not have a significant influence on the inhibitor incidence rate [51].

Short and medium-term perspectives for the treatment of haemophilia strongly rely on the current research efforts directed at increasing the safety levels of (especially) plasma-derived factors. Such research focuses on the detection and subsequent inactivation of emerging bloodborne pathogens in donors such as the prions causing variant Creutzfeldt-Jakob disease, or other potential emerging agents [52-54]. It is also important to increase the efficiency of recombinant factors increasing their half-life (by PEGylating the factor molecule or using fusion proteins [55-58] and attenuating their immunogenic capacity to produce inhibitors, by chemically modifying them [59] or by developing recombinant factors of human origin [60].

In the long term, efforts must be directed at the development of advanced therapies, particu‐ larly strategies in the field of gene therapy (using of adeno-associated viral vectors) and cell therapy (using of adult stem cells or induced pluripotent stem cells). The chief goal of these new strategies will be to address some of the shortcomings associated with current treatment options such as the short *in vivo* half-life of administered factors, the impending risk of a pathogen-induced infection and the development of inhibitors. Another goal of the advanced therapies (cell therapy) will be palliative treatment of the articular consequences derived from

Haemophilia is optimally suited for advanced therapies as it is a monogenic condition and does not require very high expression levels of a coagulation factor to reach moderate disease

characteristics of the patient and on cost and availability considerations [46,47].

A and 6% in haemophilia B.

536 Pluripotent Stem Cells

haemophilic arthropathy [40].

Specifically, gene therapy trials in haemophilic patients have shown adeno-associated vectors to represent the most promising treatment option given their excellent safety profile, even if on occasion they may create immune response problems. Efforts are currently centered on minimizing the incidence of immune rejection and increasing efficacy and expression time. In this connection, several studies have been published with a view to optimizing the use of this type of viral vectors. Among them, in a landmark study on patients with severe haemophilia B (<1% FIX), Nathwani *et al.* infused their subjects with a dose of a serotype-8-pseudotyped, self-complementary AAV vector that expresses factor IX and can efficiently transduce hepatocytes [66]. Their results showed that factor IX expression ranged between 3 and 11% of normal values. Significant as they may seem, these results must be considered with caution as the expression levels achieved rather than normalize the patient's phenotype convert it to a mild-to-moderate form. Also, concomitant treatment with glucocorticoids is needed to prevent immune rejection and elevation of liver transaminase levels. Due account must also be taken of the fact that the adeno-associated vector has the potential to induce hepatotoxicity. For all these reasons, these undoubtedly encouraging results can only be considered a first step in the development of safe and effective advanced therapies for the treatment of haemophilia.

Non-viral strategies also have a role to play in the treatment of haemophilia as they could in the long term provide a safer alternative than viral vectors which, as we have seen, are fraught with significant biosafety and efficacy-related problems, which have so far limited their clinical application. Sivalingam *et al*. [67] evaluated the genotoxic potential of phiC31 bacteriophage integrase-mediated transgene integration in cord-lining epithelial cells cultured from the human umbilical cord. This non-viral strategy has made it possible to obtain stable factor VIII secretion *in vitro*. Xenoimplantation of these protein-secreting cell lines into immunocompe‐ tent haemophilic mice corrects the severe form of the disease. Such implantation could prove extremely useful as a bioimplant in the context of monogenic diseases such as haemophilia.

Our laboratory has advanced the use of nucleofection as a non-viral transfection method to obtain factor IX expression and secretion in adult adipose tissue-derived mesenchymal stem cells [68]. Although it is certainly true that expression efficacy with these types of protocols is lower than when viral vectors are used, it must be underscored that these protocols do offer much higher safety levels, with the additional advantage that increasing factor activity to above 5% of normal values already places the patient in the mild phenotype group.

The use of cell therapy in the treatment of haemophilia has to date consisted mainly in the transplantation of healthy cells in an attempt to repair or replace a coagulation factor defi‐

membrane markers for these cells such as CD31, CD34 and Flk1, as well as factor VIII. Following transplantation of these cells into mice with haemophilia A, the latter survived the tail-clip bleeding assay by over 3 months and their factor VIII plasma levels increased to 8%-12%. Yadav *et al*. [73] studied transdifferentiation of iPSC-derived endothelial progenitor cells into hepatocytes (primary cells of FVIII synthesis). These transplanted cells were injected into the liver parenchyma where they integrated functionally and made correction of the haemophilic phenotype. High levels of FVIII mRNA were detected in the spleen, heart, and kidney tissues of injected animals with no indication of tumor formation or any other adverse events in the long-term. Alipio *et al*. [74] for their part also reported on the generation of factor VIII in a haemophilic murine model

Induced Pluripotent Stem Cells: Therapeutic Applications in Monogenic and Metabolic Diseases, and Regulatory…

http://dx.doi.org/10.5772/55816

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**5. Induced pluripotent stem cells in the treatment of diabetes mellitus**

Diseases caused by the destruction or loss of function of a limited number of cells are good

Diabetes mellitus (DM) is classified into two broad categories: type 1 DM, which is a genetic disease, and type 2 DM, a more generalized variety related with insulin resistance. DM, especially the type 1 form, is associated with microvascular complications, such as retinopathy, neuropathy or nephropathy, as well as cardiovascular problems. Type 1 DM is a T-cell mediated autoimmune disease specifically aimed against pancreatic beta cells, which results

Symptoms of DM include episodes of lethargy and fatigue, polyuria, enuresis, noctu‐ ria, polydipsia, polyphagia, weight loss and abdominal pain. The disorder has a strong genetic component related with the susceptibility to inherit and develop the disease through the HLA complex (HLA-DR and HLA-DQ genotypes) and other loci involved in immunologic recognition and cell-to-cell signaling in the immune system (graft

Abnormal T-cell activation in susceptible individuals results in both an inflammatory response within the Langerhans islets and a humoral immune response involving the production of antibodies against insulin-specific beta cell antigens, decarboxylase glutamic acid or the protein tyrosine phosphatase [79]. The presence of one or more types of antibodies may precede the appearance of type 1 diabetes and its subsequent development [80,81]. In any case, the final result is the destruction of beta cells and progressive impairment of the blood glucose metabolism [82]. Some patients with type 1 diabetes may show a higher susceptibility to other conditions such as thyroiditis, Graves disease, Adisson disease, celiac disease, myasthenia gravis or to degenerative skin

The greatest incidence of type 1 DM occurs during childhood and in the early years of adulthood with significant variations across different geographies. Diagnosis is usually made

one year after transplantation of iPSC-derived endothelial cells.

candidates for cell therapy. Such is the case of diabetes mellitus (Figure 2).

in insulin deficiency [75,76].

compatibility) [77,78].

conditions such as vitíligo [83-85].

**Figure 2.** Induced pluripotent stem cells application to the treatment of haemophilia and diabetes mellitus. Autolo‐ gous transplantation of healthy differentiated cells, obtained from iPSCs, into an animal model with haemophilia or diabetes mellitus type 1, normalizes the corresponding altered function by in vivo production of the deficient protein or hormone.

ciency. These procedures have been conducted mainly with adult stem cells and, more recently, with progenitor cells partially differentiated from iPSCs, albeit in most cases the mechanisms by which transplanted cells (to a greater or lesser extent) engraft and go on to proliferate and function remain unknown.

Aronovich *et al*. [69], have shown that transplantation of embryonic spleen tissue (embryonic day 42 spleen tissue) in immunocompetent mice with haemophilia A attenuates the severity of the disease in the 2-3 months after the procedure. These results would seem to indicate that transplantation of a fetal spleen (obtained from a develop‐ mental stage prior to the appearance of T-cells) may potentially be used to treat some genetic disorders. For their part, Follenzi *et al*. [70] reported that once liver sinusoidal endothelial cells were transplanted and successfully engrafted into mice with haemophil‐ ia A, they were seen to proliferate and partially replace some areas of the hepatic endothelium. This resulted in a restoration of factor VIII plasma levels and in the correction of the bleeding phenotype. More recently, this same team [71] demonstrated that transplantation of bone marrow cells (healthy mouse Kupffer cells ―liver macro‐ phage/mononuclear cells― and healthy bone marrow derived mesenchymal stromal cells) can correct the phenotype of haemophilic mice and restore factor VIII levels.

As far as the use of iPSCs is concerned, the first paper came from Xu *et al*. [72], who reported on the generation of murine iPSCs from tail-tip fibroblasts and their differentia‐ tion into endothelial cells and their precursors. These iPSC-derived cells express specific membrane markers for these cells such as CD31, CD34 and Flk1, as well as factor VIII. Following transplantation of these cells into mice with haemophilia A, the latter survived the tail-clip bleeding assay by over 3 months and their factor VIII plasma levels increased to 8%-12%. Yadav *et al*. [73] studied transdifferentiation of iPSC-derived endothelial progenitor cells into hepatocytes (primary cells of FVIII synthesis). These transplanted cells were injected into the liver parenchyma where they integrated functionally and made correction of the haemophilic phenotype. High levels of FVIII mRNA were detected in the spleen, heart, and kidney tissues of injected animals with no indication of tumor formation or any other adverse events in the long-term. Alipio *et al*. [74] for their part also reported on the generation of factor VIII in a haemophilic murine model one year after transplantation of iPSC-derived endothelial cells.

## **5. Induced pluripotent stem cells in the treatment of diabetes mellitus**

Diseases caused by the destruction or loss of function of a limited number of cells are good candidates for cell therapy. Such is the case of diabetes mellitus (Figure 2).

Diabetes mellitus (DM) is classified into two broad categories: type 1 DM, which is a genetic disease, and type 2 DM, a more generalized variety related with insulin resistance. DM, especially the type 1 form, is associated with microvascular complications, such as retinopathy, neuropathy or nephropathy, as well as cardiovascular problems. Type 1 DM is a T-cell mediated autoimmune disease specifically aimed against pancreatic beta cells, which results in insulin deficiency [75,76].

ciency. These procedures have been conducted mainly with adult stem cells and, more recently, with progenitor cells partially differentiated from iPSCs, albeit in most cases the mechanisms by which transplanted cells (to a greater or lesser extent) engraft and go on to

**Figure 2.** Induced pluripotent stem cells application to the treatment of haemophilia and diabetes mellitus. Autolo‐ gous transplantation of healthy differentiated cells, obtained from iPSCs, into an animal model with haemophilia or diabetes mellitus type 1, normalizes the corresponding altered function by in vivo production of the deficient protein

Aronovich *et al*. [69], have shown that transplantation of embryonic spleen tissue (embryonic day 42 spleen tissue) in immunocompetent mice with haemophilia A attenuates the severity of the disease in the 2-3 months after the procedure. These results would seem to indicate that transplantation of a fetal spleen (obtained from a develop‐ mental stage prior to the appearance of T-cells) may potentially be used to treat some genetic disorders. For their part, Follenzi *et al*. [70] reported that once liver sinusoidal endothelial cells were transplanted and successfully engrafted into mice with haemophil‐ ia A, they were seen to proliferate and partially replace some areas of the hepatic endothelium. This resulted in a restoration of factor VIII plasma levels and in the correction of the bleeding phenotype. More recently, this same team [71] demonstrated that transplantation of bone marrow cells (healthy mouse Kupffer cells ―liver macro‐ phage/mononuclear cells― and healthy bone marrow derived mesenchymal stromal cells) can correct the phenotype of haemophilic mice and restore factor VIII levels.

As far as the use of iPSCs is concerned, the first paper came from Xu *et al*. [72], who reported on the generation of murine iPSCs from tail-tip fibroblasts and their differentia‐ tion into endothelial cells and their precursors. These iPSC-derived cells express specific

proliferate and function remain unknown.

or hormone.

538 Pluripotent Stem Cells

Symptoms of DM include episodes of lethargy and fatigue, polyuria, enuresis, noctu‐ ria, polydipsia, polyphagia, weight loss and abdominal pain. The disorder has a strong genetic component related with the susceptibility to inherit and develop the disease through the HLA complex (HLA-DR and HLA-DQ genotypes) and other loci involved in immunologic recognition and cell-to-cell signaling in the immune system (graft compatibility) [77,78].

Abnormal T-cell activation in susceptible individuals results in both an inflammatory response within the Langerhans islets and a humoral immune response involving the production of antibodies against insulin-specific beta cell antigens, decarboxylase glutamic acid or the protein tyrosine phosphatase [79]. The presence of one or more types of antibodies may precede the appearance of type 1 diabetes and its subsequent development [80,81]. In any case, the final result is the destruction of beta cells and progressive impairment of the blood glucose metabolism [82]. Some patients with type 1 diabetes may show a higher susceptibility to other conditions such as thyroiditis, Graves disease, Adisson disease, celiac disease, myasthenia gravis or to degenerative skin conditions such as vitíligo [83-85].

The greatest incidence of type 1 DM occurs during childhood and in the early years of adulthood with significant variations across different geographies. Diagnosis is usually made before the age of 20 (between 16 and 18 in 50-60% of cases) [75]. The factors involved in the development of type 1 DM include the so-called familial predisposing factors, gestational status, age and other iatrogenic causes.

of all three types of advanced therapies: gene therapy, cell therapy and tissue engineering. In this regard, although we are still at a very incipient stage [99,100], procedures based on transplantation of insulin-secreting cells or islets obtained from stem cell differentiation may

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The need to justify the human and financial investment made in the development of new advanced therapies is as strong in diabetes as it is in haemophilia. However, in the case of the former justification is even more compelling taking into account that an optimal and efficient treatment is already available for the disease. The discovery of insulin as a therapeutic tool for DM constituted an important milestone in the history of medicine even if administration of this hormone does not fully compensate for the function lost. This is also the case with factor replacement in haemophilia. Moreover, both coagulation factor and insulin treatment are only palliative, never curative, which is the basic idea underlying treatment of DM and haemo‐ philia. Moreover, it is also important to take into account the potential adverse effects of these therapies, and particularly the complications associated with DM, which derive from the fact

In addition, advances in terms of the clinical transplantation of Langerhans islets have not met with the expected success as a result of the inadequate number of donors available and the incidence of immune rejection of the newly transplanted beta cells [101]. This has intensified efforts aimed at developing insulin-producing cells from stem cells. iPSC technology could turn the tide in this respect as such cells may be induced to form endodermal structures, pancreatic and endocrine progenitors and, naturally,

Built upon the knowledge gained from studies on embryonic cells about the differentiation process, the first studies on iPSCs, whereby human cells were reprogrammed to become *in vitro* differentiated insulin-producing cells, showed great promise [105,106]. However, as only partial cell differentiation was achieved, those studies failed in their attempt to enrich insulin-

Drawing on current knowledge on the embryonic development of the pancreas, Zhu *et al*. [107] recently reported on the generation of insulin-producing pancreatic cells from iPSCs obtained from a rhesus monkey [108]. These authors established a quantitative cytometric method to evaluate the efficacy of cell differentiation. In addition, they increased the level of precision in the assessment of the competence and function of the iPSCs from a rhesus monkey by means of transplantation into immunodeficient mice. These cells were induced to form endodermal structures, pancreatic and endocrine progenitors and insulin-producing cells. By means of a TGF-β inhibitor, generation of endocrine precursor cells capable of generating insulinproducing cells that respond to glucose stimulation *in vitro* was undertaken. Transplantation of these cells into a type 1 DM murine model decreased blood glucose levels in 50% of the mice. These results show the high efficacy that can be achieved by obtaining iPSCs from a superior animal model as well as the capacity of iPSCs to be transformed into insulin-producing cells, which opens up the possibility for carrying out autologous transplantations in the future.

hold valuable hope for the future.

that it is a long-term disease.

differentiated insulin-producing cells [102-104].

producing cell lines or assess their function.

Type 2 DM is characterized by a functional deficiency of insulin per se or by a resistance to the hormone resulting from an alteration of the function or structure of the insulin receptor at the level of the membrane or of any of the molecules involved in the intracytoplasmic signal transduction cascade [86]. The metaboilic effects of insulin vary depending on the action of the molecules that participate in signaling pathways to regulate gene expression in striated muscle cells, adipocytes, hepatocytes and in pancreatic beta cells [87-90]. Thus, for example, insulin resistance caused by the impairment of glucose transporter GLUT4 initially results in a metabolic syndrome, type 2 diabetes, lipodystrophy, hypertension, polycystic ovary syndrome or atherosclerosis.

In general, the morbidity and mortality of DM is related with the different long-term cardio‐ vascular complications associated with the disease, also taking into account other proactivat‐ ing factors such as smoking, obesity, a sedentary lifestyle, hypertension, early onset and prolonged duration of type 1 DM, genetic predisposition and hyperglycemia.

Nephropathy, retinopathy and diabetic neuropathy are the most common microvascular complications of DM. As regards diabetic neuropathy, this can be a focal complication associated with diabetic amyotrophy or with cranial nerve III oculomotor palsy, or a more generalized occurrence that can take the form of a sensorimotor polyneuropathy affecting the autonomic nervous system, gastric motility and cardiac function. Peripheral neuropathy together with peripheral vascular disease may lead to a diabetic foot syndrome, characterized by ulcerations and poor healing in the lower limbs [91]. As a macrovascular complication, cardiovascular disease accounts for 70% of mortality in individuals with type 2 DM, with the incidence of coronary artery disease being higher in women than in men suffering from type 1 DM [92]. Atherosclerotic processes are in turn more common in patients with type 1 DM [93].

Although treatment and diagnosis of diabetes is well-established, there is a constant quest for new drugs that may be more effective at lowering blood glucose levels, controlling their therapeutical management —especially in younger patients—, and preserving patients' longterm quality of life by reducing the incidence of complications resulting from the disease. Current research is centered on unveiling the structure and function of glucose transporters, which may offer significant therapeutic advantages [86], as well as on the development of new fast-acting insulin analogs and more accurate subcutaneous pumps [94-98]. Commendable as these initiatives are, it is difficult to anticipate and control factors that exert a variable influence upon glucose levels such as nutrition, physical activity or stress. These factors alter the glycemic environment and consequently the amount of insulin required at each point in time, which reinforces the need to establish sophisticated artificial pumping systems that may simulate the natural endocrine pancreas.

The continuous advancement of our understanding of the mechanisms that govern the physiopathology of diabetes and gene susceptibility together with the multiple possibilities currently offered by biotechnology have fuelled the researchers' interest in the development of all three types of advanced therapies: gene therapy, cell therapy and tissue engineering. In this regard, although we are still at a very incipient stage [99,100], procedures based on transplantation of insulin-secreting cells or islets obtained from stem cell differentiation may hold valuable hope for the future.

before the age of 20 (between 16 and 18 in 50-60% of cases) [75]. The factors involved in the development of type 1 DM include the so-called familial predisposing factors, gestational

Type 2 DM is characterized by a functional deficiency of insulin per se or by a resistance to the hormone resulting from an alteration of the function or structure of the insulin receptor at the level of the membrane or of any of the molecules involved in the intracytoplasmic signal transduction cascade [86]. The metaboilic effects of insulin vary depending on the action of the molecules that participate in signaling pathways to regulate gene expression in striated muscle cells, adipocytes, hepatocytes and in pancreatic beta cells [87-90]. Thus, for example, insulin resistance caused by the impairment of glucose transporter GLUT4 initially results in a metabolic syndrome, type 2 diabetes, lipodystrophy, hypertension, polycystic ovary

In general, the morbidity and mortality of DM is related with the different long-term cardio‐ vascular complications associated with the disease, also taking into account other proactivat‐ ing factors such as smoking, obesity, a sedentary lifestyle, hypertension, early onset and

Nephropathy, retinopathy and diabetic neuropathy are the most common microvascular complications of DM. As regards diabetic neuropathy, this can be a focal complication associated with diabetic amyotrophy or with cranial nerve III oculomotor palsy, or a more generalized occurrence that can take the form of a sensorimotor polyneuropathy affecting the autonomic nervous system, gastric motility and cardiac function. Peripheral neuropathy together with peripheral vascular disease may lead to a diabetic foot syndrome, characterized by ulcerations and poor healing in the lower limbs [91]. As a macrovascular complication, cardiovascular disease accounts for 70% of mortality in individuals with type 2 DM, with the incidence of coronary artery disease being higher in women than in men suffering from type 1 DM [92]. Atherosclerotic processes are in turn more common in patients with type 1 DM [93].

Although treatment and diagnosis of diabetes is well-established, there is a constant quest for new drugs that may be more effective at lowering blood glucose levels, controlling their therapeutical management —especially in younger patients—, and preserving patients' longterm quality of life by reducing the incidence of complications resulting from the disease. Current research is centered on unveiling the structure and function of glucose transporters, which may offer significant therapeutic advantages [86], as well as on the development of new fast-acting insulin analogs and more accurate subcutaneous pumps [94-98]. Commendable as these initiatives are, it is difficult to anticipate and control factors that exert a variable influence upon glucose levels such as nutrition, physical activity or stress. These factors alter the glycemic environment and consequently the amount of insulin required at each point in time, which reinforces the need to establish sophisticated artificial pumping systems that may

The continuous advancement of our understanding of the mechanisms that govern the physiopathology of diabetes and gene susceptibility together with the multiple possibilities currently offered by biotechnology have fuelled the researchers' interest in the development

prolonged duration of type 1 DM, genetic predisposition and hyperglycemia.

status, age and other iatrogenic causes.

540 Pluripotent Stem Cells

syndrome or atherosclerosis.

simulate the natural endocrine pancreas.

The need to justify the human and financial investment made in the development of new advanced therapies is as strong in diabetes as it is in haemophilia. However, in the case of the former justification is even more compelling taking into account that an optimal and efficient treatment is already available for the disease. The discovery of insulin as a therapeutic tool for DM constituted an important milestone in the history of medicine even if administration of this hormone does not fully compensate for the function lost. This is also the case with factor replacement in haemophilia. Moreover, both coagulation factor and insulin treatment are only palliative, never curative, which is the basic idea underlying treatment of DM and haemo‐ philia. Moreover, it is also important to take into account the potential adverse effects of these therapies, and particularly the complications associated with DM, which derive from the fact that it is a long-term disease.

In addition, advances in terms of the clinical transplantation of Langerhans islets have not met with the expected success as a result of the inadequate number of donors available and the incidence of immune rejection of the newly transplanted beta cells [101]. This has intensified efforts aimed at developing insulin-producing cells from stem cells. iPSC technology could turn the tide in this respect as such cells may be induced to form endodermal structures, pancreatic and endocrine progenitors and, naturally, differentiated insulin-producing cells [102-104].

Built upon the knowledge gained from studies on embryonic cells about the differentiation process, the first studies on iPSCs, whereby human cells were reprogrammed to become *in vitro* differentiated insulin-producing cells, showed great promise [105,106]. However, as only partial cell differentiation was achieved, those studies failed in their attempt to enrich insulinproducing cell lines or assess their function.

Drawing on current knowledge on the embryonic development of the pancreas, Zhu *et al*. [107] recently reported on the generation of insulin-producing pancreatic cells from iPSCs obtained from a rhesus monkey [108]. These authors established a quantitative cytometric method to evaluate the efficacy of cell differentiation. In addition, they increased the level of precision in the assessment of the competence and function of the iPSCs from a rhesus monkey by means of transplantation into immunodeficient mice. These cells were induced to form endodermal structures, pancreatic and endocrine progenitors and insulin-producing cells. By means of a TGF-β inhibitor, generation of endocrine precursor cells capable of generating insulinproducing cells that respond to glucose stimulation *in vitro* was undertaken. Transplantation of these cells into a type 1 DM murine model decreased blood glucose levels in 50% of the mice. These results show the high efficacy that can be achieved by obtaining iPSCs from a superior animal model as well as the capacity of iPSCs to be transformed into insulin-producing cells, which opens up the possibility for carrying out autologous transplantations in the future.

Along the same lines, Jeon *et al*. [109] studied the functionality of iPSC-derived insulinproducing cells generated from pancreas-derived epithelial cells in non-obese diabetic mice. The insulin-producing cells obtained in this way express different pancreatic β cell markers and secrete insulin in response to glucose stimulation. Transplantation of these cells into nonobese diabetic mice (a model of autoimmune type 1 DM very similar to the human form) results in a kidney graft with a functional response to glucose stimulation and a consequent normal‐ ization of blood glucose levels (Figure 2).

protection, control and regulation mechanism. The existing framework regulating advanced therapies will have to be adapted fast in order to keep pace with the proliferation of new knowledge in this rapidly developing field. However, desirable that this may be, the pace of legislative reform is unfortunately slow and inevitably lags behind the development of new

Induced Pluripotent Stem Cells: Therapeutic Applications in Monogenic and Metabolic Diseases, and Regulatory…

http://dx.doi.org/10.5772/55816

543

The aspects to be regulated include mainly those related with controlling the development, manufacturing and quality of release and stability testing programs; non-clinical aspects such as promoting research on biodistribution, cell viability and proliferation levels and ratios, and the persistence of in vivo function; clinical aspects such as dose-specific characteristics, risk stratification; and aspects specifically connected to pharmacovigilance and traceability.

The guidelines for therapeutic products based on human cells must be drawn up by the drug agencies of the different countries [117,118] both as regards the development of clinical and preclinical trials and with respect to pharmacovigilance, taking in all cases a multidisciplinary

For any product based on cells or on tissue, it should be made compulsory to verify that the desired physiological functions are preserved after the preparation process, both in isolation and in combination with other non-biological components, as many of these products will be used with a metabolic purpose [119,120]. Nevertheless, many things remain to be learned about the procedures that should be followed to guarantee the safety and efficacy of cell therapy products, especially with respect to the biology of stem cells, their self-renewal and differen‐

Most cell therapy products are not controversial from a bioethical point of view. The exception to this is therapy with human embryonic stem cells, which raises moral and bioethical problems [121,122]. Such consideration refer to the donor's informed consent and to problems associated with the harvesting of oocytes and the destruction of human embryos. In this regard, the guidelines used by the different countries range from total prohibition to regulated authorization. In general, there is an international consensus that the results obtained in stem cell research should be applied to humans without prior bioethical scrutiny, with the under‐ standing that scientific research and the use of scientific knowledge must respect human rights and the dignity of the individual in accordance with the Universal Declaration of Human

The main advantage of induced pluripotent stem cells is that their use, unlike that of embryonic stem cells, does not raise moral or bioethical issues as the scientific community, as well as society at large, consider it a valid alternative for the generation of pluripotent stem cells without the need to use human oocytes or embryos. Furthermore, these cells have shown themselves to be functionally and molecularly similar to embryonic cells, but without their bioethical problems, which means that their use in humans will not require an overly stringent regulatory framework. The importance of this cannot be overstated as, in many instances, and in some countries more than in others, legislation can hinder the development of science and,

consequently, the application of new knowledge and new therapeutic strategies.

tiation potential and, above all, the evaluation and prediction of potential risks.

Rights and the Universal Declaration of the Human Genome [123].

science.

perspective.

Until recently, iPSC generation from patients with type 2 DM had not been reported in the literature. However, Ohmine *et al*. [110] described not long ago the generation of iPSCs from keratinocytes of elderly patients with type 2 DM. These cells were reprogrammed by lentiviral transduction with human transcription factors OCT4, SOX2, KLF4 and cMYC, telomere elongation, and down-regulation of senescence and apoptosis-related genes, and were subsequently differentiated into insulin-producing islet-like cells. Reprogramming of kerati‐ nocytes from elderly type 2 DM patients produces efficient iPSCs with a "privileged" senes‐ cence status that allows them to transform into insulin-producing islet-like cells, which may lead to the development of a versatile strategy for modeling the disease as well as an advanced therapy for treating it.

Generally speaking, several problems must yet be resolved before iPSCs can be applied clinically, specifically to the treatment of haemophilia or diabetes. In the first place, it is essential to optimize the reprogramming process so that it provides maximum safety assur‐ ances against the potential risks derived from undesirable genetic changes in iPSCs [111]. Recent studies have revealed significant chromosomal changes that take place during the longterm culture of iPSCs as well as variations in the number of copies of certain genes and point mutations, which could clearly be related with the reprogramming of somatic cells and result in damage to the DNA [112-115].

The second hurdle that must be overcome is the high variability that exists between the different cell lines in the context of differentiation into pancreatic lineages [16]. The epigenetic and functional trials that should be performed in this respect are complicated by the fact that iPSCs have a high epigenetic content [116]. The third obstacle has to do with the purification of iPSC-derived β cells to prevent the transplantation of undifferentiated cells, which could result in the formation of teratomas. Moreover, it is necessary to develop new reagents to make direct differentiation of pancreatic progenitors into functional β cells more efficient and to design highly specific surface markers for these cells so that a more precise fluorescence analysis can be performed in order to isolate homogeneous populations of this kind of cell so that their function can be rigorously controlled.

## **6. General regulatory and bioethical issues**

Cell therapy, as one of the bedrocks of the advanced therapies —together with gene therapy and tissue engineering—, requires a new legislative framework in order to guarantee that patients can avail themselves of the products they need and provide governments with a robust protection, control and regulation mechanism. The existing framework regulating advanced therapies will have to be adapted fast in order to keep pace with the proliferation of new knowledge in this rapidly developing field. However, desirable that this may be, the pace of legislative reform is unfortunately slow and inevitably lags behind the development of new science.

Along the same lines, Jeon *et al*. [109] studied the functionality of iPSC-derived insulinproducing cells generated from pancreas-derived epithelial cells in non-obese diabetic mice. The insulin-producing cells obtained in this way express different pancreatic β cell markers and secrete insulin in response to glucose stimulation. Transplantation of these cells into nonobese diabetic mice (a model of autoimmune type 1 DM very similar to the human form) results in a kidney graft with a functional response to glucose stimulation and a consequent normal‐

Until recently, iPSC generation from patients with type 2 DM had not been reported in the literature. However, Ohmine *et al*. [110] described not long ago the generation of iPSCs from keratinocytes of elderly patients with type 2 DM. These cells were reprogrammed by lentiviral transduction with human transcription factors OCT4, SOX2, KLF4 and cMYC, telomere elongation, and down-regulation of senescence and apoptosis-related genes, and were subsequently differentiated into insulin-producing islet-like cells. Reprogramming of kerati‐ nocytes from elderly type 2 DM patients produces efficient iPSCs with a "privileged" senes‐ cence status that allows them to transform into insulin-producing islet-like cells, which may lead to the development of a versatile strategy for modeling the disease as well as an advanced

Generally speaking, several problems must yet be resolved before iPSCs can be applied clinically, specifically to the treatment of haemophilia or diabetes. In the first place, it is essential to optimize the reprogramming process so that it provides maximum safety assur‐ ances against the potential risks derived from undesirable genetic changes in iPSCs [111]. Recent studies have revealed significant chromosomal changes that take place during the longterm culture of iPSCs as well as variations in the number of copies of certain genes and point mutations, which could clearly be related with the reprogramming of somatic cells and result

The second hurdle that must be overcome is the high variability that exists between the different cell lines in the context of differentiation into pancreatic lineages [16]. The epigenetic and functional trials that should be performed in this respect are complicated by the fact that iPSCs have a high epigenetic content [116]. The third obstacle has to do with the purification of iPSC-derived β cells to prevent the transplantation of undifferentiated cells, which could result in the formation of teratomas. Moreover, it is necessary to develop new reagents to make direct differentiation of pancreatic progenitors into functional β cells more efficient and to design highly specific surface markers for these cells so that a more precise fluorescence analysis can be performed in order to isolate homogeneous populations of this kind of cell so

Cell therapy, as one of the bedrocks of the advanced therapies —together with gene therapy and tissue engineering—, requires a new legislative framework in order to guarantee that patients can avail themselves of the products they need and provide governments with a robust

ization of blood glucose levels (Figure 2).

therapy for treating it.

542 Pluripotent Stem Cells

in damage to the DNA [112-115].

that their function can be rigorously controlled.

**6. General regulatory and bioethical issues**

The aspects to be regulated include mainly those related with controlling the development, manufacturing and quality of release and stability testing programs; non-clinical aspects such as promoting research on biodistribution, cell viability and proliferation levels and ratios, and the persistence of in vivo function; clinical aspects such as dose-specific characteristics, risk stratification; and aspects specifically connected to pharmacovigilance and traceability.

The guidelines for therapeutic products based on human cells must be drawn up by the drug agencies of the different countries [117,118] both as regards the development of clinical and preclinical trials and with respect to pharmacovigilance, taking in all cases a multidisciplinary perspective.

For any product based on cells or on tissue, it should be made compulsory to verify that the desired physiological functions are preserved after the preparation process, both in isolation and in combination with other non-biological components, as many of these products will be used with a metabolic purpose [119,120]. Nevertheless, many things remain to be learned about the procedures that should be followed to guarantee the safety and efficacy of cell therapy products, especially with respect to the biology of stem cells, their self-renewal and differen‐ tiation potential and, above all, the evaluation and prediction of potential risks.

Most cell therapy products are not controversial from a bioethical point of view. The exception to this is therapy with human embryonic stem cells, which raises moral and bioethical problems [121,122]. Such consideration refer to the donor's informed consent and to problems associated with the harvesting of oocytes and the destruction of human embryos. In this regard, the guidelines used by the different countries range from total prohibition to regulated authorization. In general, there is an international consensus that the results obtained in stem cell research should be applied to humans without prior bioethical scrutiny, with the under‐ standing that scientific research and the use of scientific knowledge must respect human rights and the dignity of the individual in accordance with the Universal Declaration of Human Rights and the Universal Declaration of the Human Genome [123].

The main advantage of induced pluripotent stem cells is that their use, unlike that of embryonic stem cells, does not raise moral or bioethical issues as the scientific community, as well as society at large, consider it a valid alternative for the generation of pluripotent stem cells without the need to use human oocytes or embryos. Furthermore, these cells have shown themselves to be functionally and molecularly similar to embryonic cells, but without their bioethical problems, which means that their use in humans will not require an overly stringent regulatory framework. The importance of this cannot be overstated as, in many instances, and in some countries more than in others, legislation can hinder the development of science and, consequently, the application of new knowledge and new therapeutic strategies.

## **7. Concluding remarks**

iPSCs offer an unprecedented alternative for basic, clinical and applied biomedical research. The most significant applications of these cells to the field of cell therapy are related to the treatment of such organ-specific conditions as diabetes ―a typically metabolic disease―, hepatic and cardiovascular diseases, immunological disorders and monogenic hereditary conditions in general such as haemophilia.

candidate for such treatments for several reasons: it is a monogenic disease; the expression of low levels of coagulation factor (1-5%) can result in a moderate phenotype; a large variety target cells can be applied; there is no need to regulate factor expression, and a large amount of animal models are available for experimentation. In this regard, application of strategies that are less demanding in terms of efficacy, i.e. level of protein expression, but that afford much greater safety, may be an alternative for this condition, taking into account that both physicians and patients are highly sensitive to the special immunologic situation of the haemophilic population and that viral infections (HIV/HCV) have had lethal consequences for

Induced Pluripotent Stem Cells: Therapeutic Applications in Monogenic and Metabolic Diseases, and Regulatory…

http://dx.doi.org/10.5772/55816

545

As regards diabetes as a typically metabolic disease, advances in the understanding of its physio- and etiopathology, together with the greater biotechnological possibilities available, have made new alternatives possible as a result of the development of advanced therapies to treat it. Transplantation of insulin-secreting cells or of islets obtained a from differentiation of

As in haemophilia, in diabetes it is also necessary to justify the investment of human and financial resources required for the development of new advanced therapeutical strategies, taking account of the fact that patients with this condition also benefit from an optimal and efficient treatment at present. The justification for the said investment is that diabetes gives rise to vascular and neurological complications in the long term and that transplantation of Langerhans islets has not achieved the success that scientists hoped for because of the dearth of donors and the high rate of immune rejection that

In a nutshell, iPSCs technology has the potential to produce an about-face in the way we conceive cell behavior as iPSCs can be induced to form hormone-producing differen‐ tiated cells. In this regard, several authors have reported on the generation of insulinproducing pancreatic cells from iPSCs from rhesus monkey and murine models which, after transplantation, are capable of producing insulin *in vivo* in response to glucose stimulation. Nonetheless, some general issues affecting iPSCs remain to be resolved before these cells can be used clinically in the treatment of diabetes. Prominent among these are optimizing the reprogramming process as well as their genetic safety, control‐ ling the high differentiation variability of the different pancreatic lines by means of epigenetic trials and enhancing the purification, isolation and characterization of

Antonio Liras is Principal Researcher in a preclinical project ―not clinical trial― with adipose mesenchymal stem cells and gene/cell therapy protocols for treatment of haemophilia. This project is supported by funding from a grant from the Royal Foundation Victoria Eugenia of

homogeneous populations of iPSC-derived insulin-producing β cells.

these individuals in the past [76].

characterizes diabetic patients.

**Acknowledgements**

Haemophilia.

stem cells could hold some hope in the long term.

However, many aspects remain to be unveiled about the safety of iPSCs and about their reprogramming mechanisms, although no-one denies that this technology offers new, untilrecently-unimaginable possibilities for correcting alterations in a large number of conditions, particularly in monogenic and metabolic diseases [124]. Also, some technical problems will also have to be resolved such as finding a way to produce these cells using risk-free viral vector transfection as well as safer alternative methods such as viral vector-mediated reprogramming.

Other more general, though no less important, issues that remain to be addressed include optimal extrapolation to humans of the high levels of safety and expression obtained in animal models and finding out whether it is adult mesenchymal stem cells or iPSCs that constitute the best and most easily applicable alternative for the administration of combined cell therapy/ gene therapy.

For the reasons mentioned it is imperative not to create false expectations in patients suffering from a disease that is amenable to advanced therapies, specifically cell therapy, as these strategies are still in their "infancy". In the longer term, once the challenges mentioned above have been overcome, both cell and gene therapy will become plausible alternatives. Optimism is in order, but fantasy is best avoided.

As far as haemophilia is concerned, the first article discussing the benefits of gene therapy for the treatment of the disease was published a decade ago. At that time, experts in the field anticipated that a cure for haemophilia would be found by the first decade of the 21st century [125], a prediction that did not come true because of multiple problems related to biosafety. Although many steps have been taken in the right direction with respect to gene therapy, cellular reprogramming of iPSCs and the safety of transfer vectors, efforts must continue in order to resolve problems related to immune response, insertional mutagenesis, efficacy and expression time, the collateral (particularly hepatotoxic) damage caused by viral vectors and the risk of teratoma and neoplasia derived from the application of certain cell types. Sight should not be lost of the difficulties inherent in recruiting patients for clinical trials and in the large-scale production of vectors and cell lines, needed to facilitate optimal and efficient implementation in the clinical setting.

One of the first things that must be addressed when doing research into advanced therapies is whether the expected benefits of such therapies will be able to offset the investment needed. In the case of haemophilia, the answer is clearly in the affirmative as it is a chronic disease that requires high-frequency life-long treatment, very costly in patients on prophylaxis, and which poses a potential risk of infection by emerging pathogens. The second question is whether advanced therapies are at all feasible. In this regard, haemophilia is considered an optimal candidate for such treatments for several reasons: it is a monogenic disease; the expression of low levels of coagulation factor (1-5%) can result in a moderate phenotype; a large variety target cells can be applied; there is no need to regulate factor expression, and a large amount of animal models are available for experimentation. In this regard, application of strategies that are less demanding in terms of efficacy, i.e. level of protein expression, but that afford much greater safety, may be an alternative for this condition, taking into account that both physicians and patients are highly sensitive to the special immunologic situation of the haemophilic population and that viral infections (HIV/HCV) have had lethal consequences for these individuals in the past [76].

As regards diabetes as a typically metabolic disease, advances in the understanding of its physio- and etiopathology, together with the greater biotechnological possibilities available, have made new alternatives possible as a result of the development of advanced therapies to treat it. Transplantation of insulin-secreting cells or of islets obtained a from differentiation of stem cells could hold some hope in the long term.

As in haemophilia, in diabetes it is also necessary to justify the investment of human and financial resources required for the development of new advanced therapeutical strategies, taking account of the fact that patients with this condition also benefit from an optimal and efficient treatment at present. The justification for the said investment is that diabetes gives rise to vascular and neurological complications in the long term and that transplantation of Langerhans islets has not achieved the success that scientists hoped for because of the dearth of donors and the high rate of immune rejection that characterizes diabetic patients.

In a nutshell, iPSCs technology has the potential to produce an about-face in the way we conceive cell behavior as iPSCs can be induced to form hormone-producing differen‐ tiated cells. In this regard, several authors have reported on the generation of insulinproducing pancreatic cells from iPSCs from rhesus monkey and murine models which, after transplantation, are capable of producing insulin *in vivo* in response to glucose stimulation. Nonetheless, some general issues affecting iPSCs remain to be resolved before these cells can be used clinically in the treatment of diabetes. Prominent among these are optimizing the reprogramming process as well as their genetic safety, control‐ ling the high differentiation variability of the different pancreatic lines by means of epigenetic trials and enhancing the purification, isolation and characterization of homogeneous populations of iPSC-derived insulin-producing β cells.

### **Acknowledgements**

**7. Concluding remarks**

544 Pluripotent Stem Cells

gene therapy.

conditions in general such as haemophilia.

is in order, but fantasy is best avoided.

implementation in the clinical setting.

iPSCs offer an unprecedented alternative for basic, clinical and applied biomedical research. The most significant applications of these cells to the field of cell therapy are related to the treatment of such organ-specific conditions as diabetes ―a typically metabolic disease―, hepatic and cardiovascular diseases, immunological disorders and monogenic hereditary

However, many aspects remain to be unveiled about the safety of iPSCs and about their reprogramming mechanisms, although no-one denies that this technology offers new, untilrecently-unimaginable possibilities for correcting alterations in a large number of conditions, particularly in monogenic and metabolic diseases [124]. Also, some technical problems will also have to be resolved such as finding a way to produce these cells using risk-free viral vector transfection as well as safer alternative methods such as viral vector-mediated reprogramming. Other more general, though no less important, issues that remain to be addressed include optimal extrapolation to humans of the high levels of safety and expression obtained in animal models and finding out whether it is adult mesenchymal stem cells or iPSCs that constitute the best and most easily applicable alternative for the administration of combined cell therapy/

For the reasons mentioned it is imperative not to create false expectations in patients suffering from a disease that is amenable to advanced therapies, specifically cell therapy, as these strategies are still in their "infancy". In the longer term, once the challenges mentioned above have been overcome, both cell and gene therapy will become plausible alternatives. Optimism

As far as haemophilia is concerned, the first article discussing the benefits of gene therapy for the treatment of the disease was published a decade ago. At that time, experts in the field anticipated that a cure for haemophilia would be found by the first decade of the 21st century [125], a prediction that did not come true because of multiple problems related to biosafety. Although many steps have been taken in the right direction with respect to gene therapy, cellular reprogramming of iPSCs and the safety of transfer vectors, efforts must continue in order to resolve problems related to immune response, insertional mutagenesis, efficacy and expression time, the collateral (particularly hepatotoxic) damage caused by viral vectors and the risk of teratoma and neoplasia derived from the application of certain cell types. Sight should not be lost of the difficulties inherent in recruiting patients for clinical trials and in the large-scale production of vectors and cell lines, needed to facilitate optimal and efficient

One of the first things that must be addressed when doing research into advanced therapies is whether the expected benefits of such therapies will be able to offset the investment needed. In the case of haemophilia, the answer is clearly in the affirmative as it is a chronic disease that requires high-frequency life-long treatment, very costly in patients on prophylaxis, and which poses a potential risk of infection by emerging pathogens. The second question is whether advanced therapies are at all feasible. In this regard, haemophilia is considered an optimal

Antonio Liras is Principal Researcher in a preclinical project ―not clinical trial― with adipose mesenchymal stem cells and gene/cell therapy protocols for treatment of haemophilia. This project is supported by funding from a grant from the Royal Foundation Victoria Eugenia of Haemophilia.

## **Author details**

Antonio Liras1,2\*, Cristina Segovia1 and Aline S. Gabán1,3

\*Address all correspondence to: aliras@hotmail.com

1 Department of Physiology, School of Biology, Complutense University of Madrid, and Cell Therapy and Regenerative Medicine Unit, La Paz University Hospital Health Research Insti‐ tute-IdiPAZ. Madrid, Spain

[12] Woltjen K, Michael IP, Mohseni P, et al. PiggyBac transposition reprograms fibro‐

Induced Pluripotent Stem Cells: Therapeutic Applications in Monogenic and Metabolic Diseases, and Regulatory…

http://dx.doi.org/10.5772/55816

547

[13] Yamanaka S. Strategies and new developments in the generation of patient-specific

[14] Amabile G, Meissner A. Induced pluripotent stem cells: current progress and poten‐

[15] Hochedlinger K, Plath K. Epigenetic reprogramming and induced pluripotency. *De‐*

[16] Tateishi K, He J, Taranova O, et al. Generation of insulin-secreting islet-like clusters

[17] Dimos JT, Rodolfa KT, Niakan KK, et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. *Science*

[18] Park IH, Arora N, Huo H, et al. Disease-specific induced pluripotent stem cells. *Cell*

[19] Ebert AD, Yu J, Rose FF Jr, et al. Induced pluripotent stem cells from a spinal muscu‐

[20] Caspi O, Itzhaki I, Kehat I, et al. In vitro electrophysiological drug testing using hu‐ man embryonic stem cell derived cardiomyocytes. *Stem Cells Dev* 2009;18:161-72.

[21] Razvi ES, Oosta GM. Stem Cells for cellular therapy space. *Drug Discov Today*

[22] Hatzimichael E, Tuthill M. Hematopoietic stem cell transplantation. *Stem Cells Clon‐*

[23] Yu J, Hu K, Smuga-Otto K, et al. Human induced pluripotent stem cells free of vector

[24] Park IH, Zhao R, West JA, et al. Reprogramming of human somatic cells to pluripo‐

[25] Zhou H, Wu S, Joo JY, et al. Generation of induced pluripotent stem cells using re‐

[26] Lin T, Ambasudhan R, Yuan X, et al. A chemical platform for improved induction of

[27] Lowry WE, Richter L, Yachechko R, et al. Generation of human induced pluripotent stem cells from dermal fibroblasts. *Proc Natl Acad Sci USA* 2008;105:2883-8.

[28] Alternative sources of human pluripotent stem cells. Available at White paper: The President's Council on Bioethics. Washington, D.C; 2005. Available: http://bioeth‐

blasts to induced pluripotent stem cells. *Nature* 2009;458:766-70.

tial for regenerative medicine. *Trends Mol Med* 2009;15:59-68.

from human skin fibroblasts. *J Biol Chem* 2008;283:31601-7.

lar atrophy patient. *Nature* 2009;457:277-80.

*ing: Advances and Applications* 2010;3:105-17.

and transgene sequences. *Science* 2009;324:797-801.

tency with defined factors. *Nature* 2008;451:141-6.

combinant proteins. *Cell Stem Cell* 2009;4:381-4.

human iPSCs. *Nat Methods* 2009;6:805-8.

pluripotent stem cells. *Cell Stem Cell* 2007;1:39-49.

*velopment* 2009;136:509-23.

2008;321:1218-21.

2008;134:877-86.

2010;11:37-40.

2 Royal Foundation Victoria Eugenia of Haemophilia. Madrid, Spain

3 University for the Development of State and the Pantanal Region. Campo Grande, Brazil

## **References**


[12] Woltjen K, Michael IP, Mohseni P, et al. PiggyBac transposition reprograms fibro‐ blasts to induced pluripotent stem cells. *Nature* 2009;458:766-70.

**Author details**

546 Pluripotent Stem Cells

**References**

Antonio Liras1,2\*, Cristina Segovia1

tute-IdiPAZ. Madrid, Spain

\*Address all correspondence to: aliras@hotmail.com

stem cells. *Sci Prog* 2010;93:113-27.

from Human Blastocysts. *Science* 1998;282:1145-7.

*World* 2009;10:9-16.

2 Royal Foundation Victoria Eugenia of Haemophilia. Madrid, Spain

and Aline S. Gabán1,3

1 Department of Physiology, School of Biology, Complutense University of Madrid, and Cell Therapy and Regenerative Medicine Unit, La Paz University Hospital Health Research Insti‐

3 University for the Development of State and the Pantanal Region. Campo Grande, Brazil

[1] Chagastelles PC, Nardi NB, Camassola M. Biology and applications of mesenchymal

[2] Thiede MA. Stem Cell: applications and opportunities in drug discovery. *Drug Discov*

[3] Ahrlund-Richter L, De Luca M, Marshak DR, et al. Isolation and production of cells

[4] Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic Stem Cell Lines Derived

[5] Aflatoonian B, Moore H. Human primordial germ cells and embryonic germ cells,

[7] Sommer CA, Mostoslavsky G. Experimental approaches for the generation of in‐

[8] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryon‐

[9] Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived

[10] Okita K, Hong H, Takahashi K, et al. Generation of mouse-induced pluripotent stem

[11] Kaji K, Norrby K, Paca A, et al. Virus-free induction of pluripotency and subsequent

suitable for human therapy: challenges ahead. *Cell Stem Cell* 2009;4:20-6.

and their use in cell therapy. *Curr Opin Biotechnol* 2005;16:530-5.

duced pluripotent stem cells. *Stem Cell Res Ther* 2010;1:26.

from human somatic cells. *Science* 2007;318:1917-20.

cells with plasmid vectors. *Nat Protoc* 2010;5:418-28.

excision of reprogramming factors. *Nature* 2009;458:771-5.

[6] Alison MR, Islam S. Attributes of adult stem cells. *J Pathol* 2009;217:144-60.

ic and adult fibroblast cultures by defined factors. *Cell* 2006;126:663-76.


ics.georgetown.edu/pcbe/reports/white\_paper/alternative\_sources\_white\_paper.pdf. (Accessed 2012 July 30).

[46] Batlle J, Villar A, Liras A, et al. Consensus opinion for the selection and use of thera‐ peutic products for the treatment of haemophilia in Spain. *Blood Coagul Fibrinolysis*

Induced Pluripotent Stem Cells: Therapeutic Applications in Monogenic and Metabolic Diseases, and Regulatory…

http://dx.doi.org/10.5772/55816

549

[47] Keeling D, Tait C, Makris M. Guideline on the selection and use of therapeutic prod‐ ucts to treat haemophilia and other hereditary bleeding disorders. A United King‐ dom Haemophilia Center Doctors' Organisation (UKHCDO) Guideline. *Haemophilia*

[48] Astermark J. Inhibitor development: patient-determined risk factors. *Haemophilia*

[50] Astermark J, Altisent C, Batorova A, et al. Non-genetic risk factors and the develop‐ ment of inhibitors in haemophilia: a comprehensive review and consensus report.

[51] Franchini M, Tagliaferri A, Mengoli C, et al. Cumulative inhibitor incidence in previ‐ ously untreated patients with severe hemophilia A treated with plasma-derived ver‐ sus recombinant factor VIII concentrates: A critical systematic review. *Critical Rev*

[52] Andréoletti O, Litaise C, Simmons H, et al. Highly Efficient Prion Transmission by

[53] Peden A, McCardle L, Head MW, et al. Variant CJD infection in the spleen of a neu‐ rologically asymptomatic UK adult patient with haemophilia. *Haemophilia*

[54] Zaman SM, Hill FG, Palmer B, et al. The risk of variant Creutzfeldt-Jakob disease among UK patients with bleeding disorders, known to have received potentially con‐

[55] Mei B, Pan C, Jiang H, et al. Rational design of a fully active, long-acting PEGylated

[56] Negrier C, Knobe K, Tiede A, et al. Enhanced pharmacokinetic properties of a glyco‐ PEGylated recombinant factor IX: a first human dose trial in patients with hemophil‐

[57] Dumont JA, Liu T, Low SC, et al. Prolonged activity of a recombinant factor VIII-Fc

[58] Shapiro AD, Ragni MV, Valentino LA, et al. Recombinant factor IX-Fc fusion protein (rFIXFc) demonstrates safety and prolonged activity in a phase 1/2a study in hemo‐

fusion protein in hemophilia A mice and dogs. *Blood* 2012;119:3024-30.

[49] Green D. Factor VIII inhibitors: a 50-year perspective. *Haemophilia* 2011;17:831-8.

2008;19:333-40.

2008;14:671-84.

2010;16:66-70.

2010;16:296-304.

ia B. *Blood* 2011;118:2695-701.

philia B patients. *Blood* 2012;119:666-72.

*Haemophilia* 2010;16:747-66.

*Oncology/Hematology* 2012;81:82-93.

Blood Transfusion. *PLoS Pathog* 2012;8:e1002782.

taminated plasma products. *Haemophilia* 2011;17:931-7.

factor VIII for hemophilia A treatment. *Blood* 2010;116:270-9.


[46] Batlle J, Villar A, Liras A, et al. Consensus opinion for the selection and use of thera‐ peutic products for the treatment of haemophilia in Spain. *Blood Coagul Fibrinolysis* 2008;19:333-40.

ics.georgetown.edu/pcbe/reports/white\_paper/alternative\_sources\_white\_paper.pdf.

[29] Martins-Taylor K, Xu RH. Concise Review: Genomic Stability of Human Induced

[30] Rolletschek A, Wobus AM. Induced human pluripotent stem cells: promises and

[31] Jang J, Yoo JE, Lee JA, et al. Disease-specific induced pluripotent stem cells: a plat‐ form for human disease modeling and drug discovery. *Exp Mol Med* 2012;44:202-13.

[32] Wernig M, Zhao JP, Pruszak J, et al. Neurons derived from reprogrammed fibro‐ blasts functionally integrate into the fetal brain and improve symptoms of rats with

[33] Hanna J, Wernig M, Markoulaki S, et al. Treatment of sickle cell anemia mouse mod‐

[34] Mauritz C, Schwanke K, Reppel M, et al. Generation of functional murine cardiac myocytes from induced pluripotent stem cells. *Circulation* 2008;118:507-17.

[35] Zhang J, Wilson GF, Soerens AG, et al. Functional cardiomyocytes derived from hu‐

[36] Mason C, Manzotti E. Induced pluripotent stem cells: an emerging technology plat‐

[37] Nelson TJ, Terzic A. Induced pluripotent stem cells: reprogrammed without a trace.

[40] Liras A, Gaban AS, Rodriguez-Merchan EC. Cartilage restoration in haemophilia: ad‐

[41] Schaub RG. Recent advances in the development of coagulation factors and procoa‐ gulants for the treatment of hemophilia. *Biochemical Pharmacology* 2011;82:91-8.

[42] Key NS, Negrier C. Coagulation factor concentrates: past, present, and future. *Lancet*

[44] Hermans C, Brackmann HH, Schinco P, et al. The case for wider use of recombinant

[45] Liras A. Recombinant proteins in therapeutics: Haemophilia treatment as an exam‐

factor VIII concentrates. *Critical Rev Oncology/Hematology* 2012;83:11-20.

[38] Bolton-Maggs PHB, Pasi KJ. Haemophilias A and B. *Lancet* 2003;361:1801-9.

[39] Berntorp E, Shapiro AD. Modern haemophilia care. *Lancet* 2012;379:1447-56.

el with iPS cells generated from autologous skin. *Science* 2007;318:1920-3.

Parkinson's disease. *Proc Natl Acad Sci USA* 2008;105:5856-61.

man induced pluripotent stem cells. *Circ Res* 2009;104:e30-41.

form and the Gartner hype cycle. *Regen Med* 2009;4:329-31.

[43] Farrugia A. Plasma fractionation issues. *Biologicals* 2009;37:88-93.

(Accessed 2012 July 30).

548 Pluripotent Stem Cells

*Regen Med* 2009;4:333-55.

2007;370:439-48.

ple. *Int Arch Med* 2008;1:4.

vanced therapies. *Haemophilia* 2012;1-8.

Pluripotent Stem Cells. *Stem Cells* 2012;30:22–7.

open questions. *Biol Chem* 2009;390:845-9.


[59] Peng A, Kosloski MP, Nakamura G, et al. PEGylation of a factor VIII-phosphatidyli‐ nositol complex: pharmacokinetics and immunogenicity in hemophilia A mice. *AAPS J* 2012;14:35-42.

[74] Alipio Z, Adcock DM, Waner M, et al. Sustained factor VIII production in hemophili‐ ac mice 1 year after engraftment with induced pluripotent stem cell-derived factor

Induced Pluripotent Stem Cells: Therapeutic Applications in Monogenic and Metabolic Diseases, and Regulatory…

http://dx.doi.org/10.5772/55816

551

[76] Retnakaran R, Zinman B. Type 1 diabetes, hyperglycaemia, and the heart. *Lancet*

[77] Sayad A, Akbari MT, Pajouhi M, et al. The influence of the HLA-DRB, HLA-DQB and polymorphic positions of the HLA-DRβ1 and HLA-DQβ1 molecules on risk of

[78] Steck AK, Wong R, Wagner B, et al. Effects of non-HLA gene polymorphisms on de‐ velopment of islet autoimmunity and type 1 diabetes in a population with high-risk

[79] Krischer JP, Cuthbertson DD, Yu L, et al. Screening strategies for the identification of multiple antibody-positive relatives of individuals with type 1 diabetes. *J Clin Endo‐*

[80] Maclaren N, Lan M, Coutant R, et al. Only multiple autoantibodies to islet cells (ICA), insulin, GAD65, IA-2 and IA-2beta predict immune-mediated (Type 1) diabe‐

[81] Barker JM, Barriga KJ, Yu L, et al. Prediction of autoantibody positivity and progres‐ sion to type 1 diabetes: diabetes autoimmunity study in the young (DAISY). *J Clin*

[82] Barker JM, Yu J, Yu L, et al. Autoantibody "subspecificity" in type 1 diabetes: risk for organ-specific autoimmunity clusters in distinct groups. *Diabetes Care* 2005;28:850-5.

[83] Kordonouri O, Hartmann R, Deiss D, et al. Natural course of autoimmune thyroiditis in type 1 diabetes: association with gender, age, diabetes duration, and puberty. *Arch*

[84] Skovbjerg H, Tarnow L, Locht H, et al. The prevalence of coeliac disease in adult Danish patients with type 1 diabetes with and without nephropathy. *Diabetologia*

[85] Norris JM, Barriga K, Hoffenberg EJ, et al. Risk of celiac disease autoimmunity and timing of gluten introduction in the diet of infants at increased risk of disease. *JAMA*

[86] Taton J, Czech A, Piątkiewicz P. Insulin as the main regulator of cellular glucose uti‐ lization-etiological aspects of insulin resistance. *Pol J Endocrinol* 2010;61:388-94.

[87] Azpiazu I, Manchester J, Skurat AV et al. Control of glycogen synthesis is shared be‐ tween glucose transport and glycogen synthase in skeletal muscle fibers. *Am J Physiol*

Iranian type 1 diabetes mellitus patients. *Int J Immunogenet* 2012 (In press).

VIII producing endothelial cells. *Blood Coagul Fibrinolysis* 2010;21:502-4.

[75] Daneman D. Type 1 diabetes. *Lancet* 2006;367:847-58.

HLA-DR,DQ genotypes. Diabetes 2012;61:753-8.

tes in relatives. *J Autoimmun* 1999;12:279-87.

*Endocrinol Metab* 2004;89:3896-902.

*Endocrinol Metab* 2000;278:E234-E43.

*Dis Child* 2005;90:411-4.

2005;48:1416-7.

2005;293:2343-51.

2008;371:1790-9.

*crinol Metab* 2003;88:103-8.


[59] Peng A, Kosloski MP, Nakamura G, et al. PEGylation of a factor VIII-phosphatidyli‐ nositol complex: pharmacokinetics and immunogenicity in hemophilia A mice.

[60] Casademunt E, Martinelle K, Jernberg M, et al. The first recombinant human coagu‐ lation factor VIII of human origin: human cell line and manufacturing characteristics.

[61] Nichols TC, Dillow AM, Franck HW, et al. Protein replacement therapy and gene transfer in canine models of haemophilia A, haemophilia B, von Willebrand disease,

[62] Nienhuis AW. Development of gene therapy for blood disorders. *Blood*

[63] Liras A. Gene therapy for haemophilia: The end of a "royal pathology" in the third

[64] Liras A, Olmedillas S. Gene therapy for haemophilia…yes, but…with non-viral vec‐

[65] Jeon HJ, Oh TK, Kim OH, et al. Delivery of factor VIII gene into skeletal muscle cells

[66] Nathwani AC, Tuddenham EG, Rangarajan S, et al. Adenovirus-associated virus vec‐ tor mediated gene transfer in hemophilia B. *N Engl J Med* 2011;365:2357-65.

[67] Sivalingam J, Krishnan S, Ng WH, et al. Biosafety assessment of site-directed trans‐ gene integration in human umbilical cord-lining cells. *Mol Ther* 2010;18:1346-56.

[68] Liras A, García-Arranz M, García-Gómez I, et al. Factor IX secretion in human adi‐ pose-derived stem cells by non-viral gene transfer. *Haemophilia* 2012;18(Suppl 3):A65.

[69] Aronovich A, Tchorsh D, Katchman H, et al. Correction of hemophilia as a proof of concept for treatment of monogenic diseases by fetal spleen transplantation. *Proc*

[70] Follenzi A, Benten D, Novikoff P, et al. Transplanted endothelial cells repopulate the liver endothelium and correct the phenotype of hemophilia A mice. *J Clin Invest*

[71] Follenzi A, Raut S, Merlin S, et al. Role of bone marrow transplantation for correcting

[72] Xu D, Alipio Z, Fink LM, et al. Phenotypic correction of murine hemophilia A using

[73] Yadav N, Kanjirakkuzhiyil S, Kumar S, et al. The therapeutic effect of bone marrowderived liver cells in the phenotypic correction of murine hemophilia A. *Blood*

an iPSCs cell-based therapy. *Proc Natl Acad Sci USA* 2009;106:808-13.

*AAPS J* 2012;14:35-42.

550 Pluripotent Stem Cells

2009;111:4431-44.

*Eur J Haematol* 2012;89:165-76.

and factor VII deficiency. *ILAR J* 2009;50:144-67.

using lentiviral vector. *Yonsei Med J* 2010;51:52-7.

millennium? *Haemophilia* 2001;7:441-5.

*Natl Acad Sci USA* 2006;103:19075-80.

hemophilia A in mice. *Blood* 2012;119:5532-42.

2008;118:935-45.

2009;114:4552-61.

tors? *Haemophilia* 2009;15:811-6.


[88] Abel ED, Peroni O, Kim JK, et al. Adipose-selective targeting of the GLUT4 gene im‐ pairs insulin action in muscle and liver. *Nature* 2001;409:729-33.

[104] Schroeder IS. Potential of Pluripotent Stem Cells for Diabetes Therapy. *Curr Diab Rep*

Induced Pluripotent Stem Cells: Therapeutic Applications in Monogenic and Metabolic Diseases, and Regulatory…

http://dx.doi.org/10.5772/55816

553

[105] Maehr R, Chen S, Snitow M, et al. Generation of pluripotent stem cells from patients

[106] Zhang D, Jiang W, Liu M, et al. Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. *Cell Res* 2009;19:429-38. [107] Zhu FF, Zhang PB, Zhang DH, et al. Generation of pancreatic insulin-producing cells from rhesus monkey induced pluripotent stem cells. *Diabetologia* 2011;54:2325-36.

[108] Pan FC, Wright C. Pancreas Organogenesis: From Bud to Plexus to Gland. *Dev Dy‐*

[109] Jeon K, Lim H, Kim JH, al. Differentiation and transplantation of functional pancreat‐ ic beta cells generated from induced pluripotent stem cells derived from a type 1 dia‐

[110] Ohmine S, Squillace KA, Hartjes KA, et al. Reprogrammed keratinocytes from elder‐ ly type 2 diabetes patients suppress senescence genes to acquire induced pluripoten‐

[111] Mummery C. Induced Pluripotent Stem Cells - A Cautionary Note. *N Engl J Med*

[112] Hussein SM, Batada NN, Vuoristo S, et al. Copy number variation and selection dur‐

[113] Gore A, Li Z, Fung HL, et al. Somatic coding mutations in human induced pluripo‐

[114] Lister R, Pelizzola M, Kida YS, et al. Hotspots of aberrant epigenomic reprogram‐

[116] Kim K, Doi A, Wen B, et al. Epigenetic memory in induced pluripotent stem cells.

[117] European Medicines Agency. Available: http://www.ema.europa.eu/ema/index.jsp? curl=/pages/home/Home\_Page.jsp&jsenabled=true. (Accessed 2012 July 30).

[118] Halme DG, Kessler DA. FDA Regulation of Stem-Cell-Based Therapies. *N Engl J Med*

[119] CFR - Code of Federal Regulations Title 21. Part 1271-Human Cells, Tissues, and Cel‐ lular and Tissue-Based Products. Available: http://www.accessdata.fda.gov/scripts/

[120] 120. Proposed approach to regulation of cellular and tissue-based products. The Food and Drug Administration. Available: http://www.fda.gov/downloads/Biologi‐

cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=1271. (Accessed 2012 July 30).

ming in human induced pluripotent stem cells. *Nature* 2011;471:68-73.

[115] Pera MF. Stem cells: the dark side of induced pluripotency. *Nature* 2011;471:46-7.

with type 1 diabetes. *Proc Natl Acad Sci USA* 2009;106:15768-73.

betes mouse model. *Stem Cells Dev* 2012 (In press).

ing reprogramming to pluripotency. *Nature* 2011;471:58-62.

cy. *Aging (Albany NY)* 2012;4:60-73.

tent stem cells. *Nature* 2011;471:63-7.

*Nature* 2010;467:285–90.

2006;355:1730-5.

2012 (In press).

*namics* 2011;240:530–65.

2011;364:2160-2.


[104] Schroeder IS. Potential of Pluripotent Stem Cells for Diabetes Therapy. *Curr Diab Rep* 2012 (In press).

[88] Abel ED, Peroni O, Kim JK, et al. Adipose-selective targeting of the GLUT4 gene im‐

[89] Boden G, Shulman GI. Free fatty acids in obesity and type 2 diabetes: defining their role in the development of insulin resistance and β-cell dysfunction. *Eur J Clin Invest*

[90] Bruning JC, Gautam D, Burks DJ, et al. Role of brain insulin receptor in control of

[91] Callaghan BC, Cheng HT, Stables CL, et al. Diabetic neuropathy: clinical manifesta‐

[92] Laing SP, Swerdlow AJ, Slater SD, et al. Mortality from heart disease in a cohort of

[93] Pajunen P, Taskinen MR, Nieminen MS, et al. Angiographic severity and extent of coronary artery disease in patients with type 1 diabetes mellitus. *Am J Cardiol*

[94] Danne T, Bolinder J. New insulins and insulin therapy. *Int J Clin Pract Suppl*

[95] Nicholson G, Hall GM. Diabetes mellitus: new drugs for a new epidemic. *Br J Anaesth*

[96] Boyle ME, Seifert KM, Beer KA, et al. Guidelines for application of continuous subcu‐ taneous insulin infusion (insulin pump) therapy in the perioperative period. *J* Diabe‐

[97] Hanaire H. External insulin pump treatment in the day-to-day management of diabe‐ tes: benefits and future prospectives. Diabetes *Metab* 2011;37(Suppl 4):S40-7.

[98] Schaepelynck P, Darmon P, Molines L, et al. Advances in pump technology: insulin patch pumps, combined pumps and glucose sensors, and implanted pumps. Diabe‐

[99] Hansson M, Tonning A, Frandsen U, et al. Artifactual insulin release from differenti‐

[100] Dor Y, Brown J, Martinez OI, et al. Adult pancreatic beta cells are formed by self-du‐

[101] Azzi J, Geara AS, El-Sayegh S, et al. Immunological aspects of pancreatic islet cell

[102] Maehr R. iPS Cells in Type 1 Diabetes Research and Treatment. *Clin Pharmacol Ther*

[103] Kao DI, Chen S. Pluripotent stem cell-derived pancreatic β-cells: potential for regen‐

plication rather than stem-cell differentiation. *Nature* 2004;429:41-6.

23,000 patients with insulin-treated diabetes. *Diabetologia* 2003;46:760-5.

pairs insulin action in muscle and liver. *Nature* 2001;409:729-33.

body weight and reproduction. *Science* 2000;289:2122-5.

tions and current treatments. *Lancet Neurol* 2012;11:521-34.

2002;32(Suppl 3):14-23.

552 Pluripotent Stem Cells

2000;86:1080-5.

2011;170:26-30.

2011;107:65-73.

2011;89:750-3.

tes *Sci Technol* 2012;6:184-90.

tes *Metab* 2011;37(Suppl 4):S85-93.

ated embryonic stem cells. *Diabetes* 2004;53:2603-9.

transplantation. *Expert Rev Clin Immunol* 2010;6:111-24.

erative medicine in diabetes. *Regen Med* 2012;7:583-93.


csBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Tissue/ UCM062601.pdf. (Accessed 2012 July 30).

**Chapter 25**

**Safety Assessment of Reprogrammed Cells Prior to**

Human pluripotent stem cells (hPSC) include human embryonic stem cells (hESC) and hu‐ man induced pluripotent stem cells (hIPSC). Due to their inherent ability to self-renew in‐ definitely in vitro and to give rise to essentially all cell lineages, both cell types have enormous potential for applications in regenerative medicine, but differ in their origin. HESC are derived from early pre-implantation stage embryos and have the capacity, known as *pluripotency*, to generate any other cell type of the human body. HESC can be differentiat‐ ed in the laboratory, a procedure aimed at the generation of healthy somatic cells that even‐ tually could be used in a large variety of applications including therapeutic options. However, work with hESC raises ethical concerns regarding the use of human early pre-im‐ plantation embryos, as well as concerns regarding the future use of hESC-derived cells in non-autologous cell transplantation therapies due to immune rejection of hESC-derived tis‐ sues, given that hESC are non-self. These concerns appeared to be overcome when it was demonstrated that pluripotency could be induced in differentiated somatic (adult) cells of the body by introduction of a cocktail of pluripotency-associated transcription factors, usu‐ ally *OCT4*, *SOX2*, *KLF4* and *c-MYC* [1]. This process is known as reprogramming, and gener‐ ates human induced pluripotent stem cells (hIPSC), which show an embryonic-like state similar to hESC (for review see [2]). Human iPSC are considered to have immense potential for regenerative medicine, do not require the use of donated human embryos for their gen‐ eration and may provide an alternative and suitable resource for autologous cell-based therapies, in which cells obtained from the patient could be used to generate self-hIPSC fol‐ lowed by differentiation to relevant lineages required for therapeutic intervention. Howev‐ er, disturbingly, mouse experiments have shown that autologous mouse iPSC can induce

> © 2013 Polanco and Laslett; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Polanco and Laslett; licensee InTech. This is a paper distributed under the terms of the Creative Commons

**Clinical Applications: Potential Approaches to**

**Eliminate Teratoma Formation**

Juan Carlos Polanco and Andrew L. Laslett

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54400

**1. Introduction**


## **Safety Assessment of Reprogrammed Cells Prior to Clinical Applications: Potential Approaches to Eliminate Teratoma Formation**

Juan Carlos Polanco and Andrew L. Laslett

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54400

## **1. Introduction**

csBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Tissue/

[121] Rao M, Condic ML. Alternative sources of pluripotent stem cells: scientific solutions

[123] Science for the Twunty-First Century: a New Commitment. UNESCO. http://

[124] Wong GK, Chiu AT. Gene therapy, gene targeting and induced pluripotent stem cells: Applications in monogenic disease treatment. *Biotechnol Adv* 2011;29:1-10.

[125] Mannucci PM, Tuddenham EG. The hemophilias – from royal genes to gene therapy.

[122] Hyun I. The bioethics of stem cell research and therapy. *J Clin Invest* 2010;120:71-5.

UCM062601.pdf. (Accessed 2012 July 30).

554 Pluripotent Stem Cells

*N Engl J Med* 2001;344:1773-9.

to an ethical dilemma. *Stem Cells Dev* 2008;17:1-10.

www.unesco.org/science/wcs/eng/declaration\_e.htm.

Human pluripotent stem cells (hPSC) include human embryonic stem cells (hESC) and hu‐ man induced pluripotent stem cells (hIPSC). Due to their inherent ability to self-renew in‐ definitely in vitro and to give rise to essentially all cell lineages, both cell types have enormous potential for applications in regenerative medicine, but differ in their origin. HESC are derived from early pre-implantation stage embryos and have the capacity, known as *pluripotency*, to generate any other cell type of the human body. HESC can be differentiat‐ ed in the laboratory, a procedure aimed at the generation of healthy somatic cells that even‐ tually could be used in a large variety of applications including therapeutic options. However, work with hESC raises ethical concerns regarding the use of human early pre-im‐ plantation embryos, as well as concerns regarding the future use of hESC-derived cells in non-autologous cell transplantation therapies due to immune rejection of hESC-derived tis‐ sues, given that hESC are non-self. These concerns appeared to be overcome when it was demonstrated that pluripotency could be induced in differentiated somatic (adult) cells of the body by introduction of a cocktail of pluripotency-associated transcription factors, usu‐ ally *OCT4*, *SOX2*, *KLF4* and *c-MYC* [1]. This process is known as reprogramming, and gener‐ ates human induced pluripotent stem cells (hIPSC), which show an embryonic-like state similar to hESC (for review see [2]). Human iPSC are considered to have immense potential for regenerative medicine, do not require the use of donated human embryos for their gen‐ eration and may provide an alternative and suitable resource for autologous cell-based therapies, in which cells obtained from the patient could be used to generate self-hIPSC fol‐ lowed by differentiation to relevant lineages required for therapeutic intervention. Howev‐ er, disturbingly, mouse experiments have shown that autologous mouse iPSC can induce

© 2013 Polanco and Laslett; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Polanco and Laslett; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

unexpected T-cell-dependent immune response in syngeneic recipients [3], suggesting that hIPSC-derived cell types should also be evaluated for immunogenicity before any clinical application.

the generation of *teratomas* in immuno-deficient mice (ie: NOD-SCID and NOD/SCID IL2Rγ-/- mice), by injection of putative pluripotent hPSC into organs like testis, kidney or muscle. Teratomas are benign solid tumours that contain a mixture of differentiated tissues such as nerve cells, muscle cells or cartilage. If a human cell line generates teratomas, it is considered pluripotent, because teratomas emulate differentiation in the developing em‐ bryo, albeit in a disorganised fashion, by generation of tissues resembling different parts of the embryo known as embryonic germ layers (i.e.: Ectoderm, Mesoderm and Endoderm).

Safety Assessment of Reprogrammed Cells Prior to Clinical Applications: Potential Approaches to…

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557

In the clinical context, pluripotent stem cells will not be transplanted, rather the progenitors and/or specialised somatic cell types that are derived from hPSC will be used. It is the hope of researchers working in the expanding field of regenerative medicine that hPSC-derived cell populations will integrate into tissues and receive appropriate cues to functionally cor‐ rect diseased or injured tissue, (i.e.: Parkinson's disease, Huntington's disease, cardiac fail‐ ure, multiple sclerosis or macular degeneration). Therefore, differentiated somatic cell types are the final product for transplantation and therapeutic applications, and pluripotent stem cells are the stable source to generate those somatic cells or their progenitors (depending upon disease context) in the laboratory. In this context, the presence of even low frequency residual undifferentiated stem cells capable of teratoma formation becomes a highly unde‐ sirable feature when considering hPSC-derived somatic cells for transplantation into pa‐ tients. Differentiated cells will not be deemed safe for use in regenerative medicine if they generate tumours at any time after transplantation. To comply with this requirement, we consider that researchers should aim at the generation of pluripotent stem cell-free samples. Therefore, it will be essential to be able to monitor if any undifferentiated pluripotent cells remain after differentiation protocols, and if so, remove them without damaging the poten‐ tially therapeutic differentiated cells. Evidence supporting this statement is that it is known that the numbers of pluripotent cells injected experimentally have a directly proportional ef‐ fect on how fast the teratomas develop and the size of the tumour [11-13]. It has also been reported that at doses of 1,000 pluripotent cells, teratomas developed with 40% efficiency but with 10,000 cells the efficiency increased to 100% [12]. However, as few as two pluripo‐ tent cells have been reported to induce teratoma formation in immuno-deficient mice, al‐ though with lower efficiency [11]. Taken together, this might mean that one remaining pluripotent stem cell in a patient bound cell preparation could lead to teratoma formation. There is some limited evidence that potentially refutes the tumorgenic potential of low doses of pluripotent cells. This evidence is demonstrated by experiments showing that two pluripotent cells transplanted into syngeneic immunocompetent mice practically abolished tumour formation [11], most likely because those stem cells were cleared by the immune system. This could be taken to imply that in the clinical context of immuno-competent pa‐ tients, low contamination with human pluripotent stem cells may be safe, but nevertheless for hPSC-derived cell populations to be approved for use in clinical trials their stringent elimination will be a requirement. Furthermore, the site of transplantation needs to be taken into account as not all places in the body are equally permissive for teratoma growth and development and contaminating hPSC may also migrate to alternative and possibly more permissive sites for teratoma growth post transplantation. For instance, it has been reported that similar number of pluripotent stem cells injected into immuno-deprived mice induced

Given that: (i) the generation of human iPSC does not require destruction of embryos, (ii) that many iPSC lines can be established from a single patient, (iii) hIPSC are predicted to lead to patient specific therapies and (iv) that hIPSC could be used as a source of somatic cells for toxicology and drug screening studies, many research programs have shifted their focus from solely hESC-based research to also include work on hIPSC. However, despite the phenotypic similarities with hESC, recent reports described the worrying phenomena of ele‐ vated genetic [4-6] and epigenetic abnormalities [7-9] in hIPSC, raising concern about the suitability of hiPSC-derived cell types for future clinical applications. Nevertheless, it ap‐ pears that these abnormalities are not present in all iPSC cell lines and that at least in mouse studies the current reprogramming methods can produce pluripotent mouse IPSC lines that lack identifiable genomic alterations [10], a result that calls for additional experiments to ex‐ plain the discrepancies with respect to hIPSC [4-6]. It is becoming increasingly obvious, based on the studies described, that it is extremely important for hIPSC-derived therapies to become a reality in the clinic, that researchers develop diagnostic tools to definitively recog‐ nise clinically "safe" and "unsafe" hIPSC lines. This is likely to be a complex and cumber‐ some task due to the large number of methodological approaches used. To date hIPSC lines have been generated (for review see [2]); using a large number of different vectors to intro‐ duce the transgenes, with variations in the combinations of genes used to induce pluripoten‐ cy, with significant modifications in culture conditions aimed at improving reprogramming efficiency, and from many of the more than 200 cell types in the human body. It will be a challenging undertaking to develop individual safety profiles for the multitude of hIPSC lines developed to date. Additionally, hIPSC-derived cells/tissues intended for clinical appli‐ cations will need to comply with the following conditions: (i) adequate numbers of cells for transplantation therapy, (ii) hIPSC differentiated progeny need to be tolerated (not immu‐ norejected) by a patient's immune system and (iii) hIPSC-derived cells should not generate teratoma-like tumours at any time after transplantation. In vitro and pre-clinical optimisa‐ tions for these parameters are essential before hIPSC-derived technologies reach the clinic.

In this Chapter, we discuss the prospects for clinical applications using pluripotent cells, fo‐ cusing on an evaluation of hIPSC cell potential and on the development of methods for the identification and removal of unwanted residual tumorigenic pluripotent cells from hIPSCderived cell populations following differentiation.

#### **2. The risk of tumour formation from residual pluripotent cells**

In vivo, pluripotent stem cells reside only during a short time in embryonic development. Conversely, in vitro, hESC and hIPSC lines can be propagated indefinitely in the embryoniclike state and remain pluripotent, or with the appropriate cues they can give rise to a range of body cell types. For human cells, the most accepted *in vivo* assay to prove pluripotency is the generation of *teratomas* in immuno-deficient mice (ie: NOD-SCID and NOD/SCID IL2Rγ-/- mice), by injection of putative pluripotent hPSC into organs like testis, kidney or muscle. Teratomas are benign solid tumours that contain a mixture of differentiated tissues such as nerve cells, muscle cells or cartilage. If a human cell line generates teratomas, it is considered pluripotent, because teratomas emulate differentiation in the developing em‐ bryo, albeit in a disorganised fashion, by generation of tissues resembling different parts of the embryo known as embryonic germ layers (i.e.: Ectoderm, Mesoderm and Endoderm).

unexpected T-cell-dependent immune response in syngeneic recipients [3], suggesting that hIPSC-derived cell types should also be evaluated for immunogenicity before any clinical

Given that: (i) the generation of human iPSC does not require destruction of embryos, (ii) that many iPSC lines can be established from a single patient, (iii) hIPSC are predicted to lead to patient specific therapies and (iv) that hIPSC could be used as a source of somatic cells for toxicology and drug screening studies, many research programs have shifted their focus from solely hESC-based research to also include work on hIPSC. However, despite the phenotypic similarities with hESC, recent reports described the worrying phenomena of ele‐ vated genetic [4-6] and epigenetic abnormalities [7-9] in hIPSC, raising concern about the suitability of hiPSC-derived cell types for future clinical applications. Nevertheless, it ap‐ pears that these abnormalities are not present in all iPSC cell lines and that at least in mouse studies the current reprogramming methods can produce pluripotent mouse IPSC lines that lack identifiable genomic alterations [10], a result that calls for additional experiments to ex‐ plain the discrepancies with respect to hIPSC [4-6]. It is becoming increasingly obvious, based on the studies described, that it is extremely important for hIPSC-derived therapies to become a reality in the clinic, that researchers develop diagnostic tools to definitively recog‐ nise clinically "safe" and "unsafe" hIPSC lines. This is likely to be a complex and cumber‐ some task due to the large number of methodological approaches used. To date hIPSC lines have been generated (for review see [2]); using a large number of different vectors to intro‐ duce the transgenes, with variations in the combinations of genes used to induce pluripoten‐ cy, with significant modifications in culture conditions aimed at improving reprogramming efficiency, and from many of the more than 200 cell types in the human body. It will be a challenging undertaking to develop individual safety profiles for the multitude of hIPSC lines developed to date. Additionally, hIPSC-derived cells/tissues intended for clinical appli‐ cations will need to comply with the following conditions: (i) adequate numbers of cells for transplantation therapy, (ii) hIPSC differentiated progeny need to be tolerated (not immu‐ norejected) by a patient's immune system and (iii) hIPSC-derived cells should not generate teratoma-like tumours at any time after transplantation. In vitro and pre-clinical optimisa‐ tions for these parameters are essential before hIPSC-derived technologies reach the clinic.

In this Chapter, we discuss the prospects for clinical applications using pluripotent cells, fo‐ cusing on an evaluation of hIPSC cell potential and on the development of methods for the identification and removal of unwanted residual tumorigenic pluripotent cells from hIPSC-

In vivo, pluripotent stem cells reside only during a short time in embryonic development. Conversely, in vitro, hESC and hIPSC lines can be propagated indefinitely in the embryoniclike state and remain pluripotent, or with the appropriate cues they can give rise to a range of body cell types. For human cells, the most accepted *in vivo* assay to prove pluripotency is

**2. The risk of tumour formation from residual pluripotent cells**

derived cell populations following differentiation.

application.

556 Pluripotent Stem Cells

In the clinical context, pluripotent stem cells will not be transplanted, rather the progenitors and/or specialised somatic cell types that are derived from hPSC will be used. It is the hope of researchers working in the expanding field of regenerative medicine that hPSC-derived cell populations will integrate into tissues and receive appropriate cues to functionally cor‐ rect diseased or injured tissue, (i.e.: Parkinson's disease, Huntington's disease, cardiac fail‐ ure, multiple sclerosis or macular degeneration). Therefore, differentiated somatic cell types are the final product for transplantation and therapeutic applications, and pluripotent stem cells are the stable source to generate those somatic cells or their progenitors (depending upon disease context) in the laboratory. In this context, the presence of even low frequency residual undifferentiated stem cells capable of teratoma formation becomes a highly unde‐ sirable feature when considering hPSC-derived somatic cells for transplantation into pa‐ tients. Differentiated cells will not be deemed safe for use in regenerative medicine if they generate tumours at any time after transplantation. To comply with this requirement, we consider that researchers should aim at the generation of pluripotent stem cell-free samples. Therefore, it will be essential to be able to monitor if any undifferentiated pluripotent cells remain after differentiation protocols, and if so, remove them without damaging the poten‐ tially therapeutic differentiated cells. Evidence supporting this statement is that it is known that the numbers of pluripotent cells injected experimentally have a directly proportional ef‐ fect on how fast the teratomas develop and the size of the tumour [11-13]. It has also been reported that at doses of 1,000 pluripotent cells, teratomas developed with 40% efficiency but with 10,000 cells the efficiency increased to 100% [12]. However, as few as two pluripo‐ tent cells have been reported to induce teratoma formation in immuno-deficient mice, al‐ though with lower efficiency [11]. Taken together, this might mean that one remaining pluripotent stem cell in a patient bound cell preparation could lead to teratoma formation. There is some limited evidence that potentially refutes the tumorgenic potential of low doses of pluripotent cells. This evidence is demonstrated by experiments showing that two pluripotent cells transplanted into syngeneic immunocompetent mice practically abolished tumour formation [11], most likely because those stem cells were cleared by the immune system. This could be taken to imply that in the clinical context of immuno-competent pa‐ tients, low contamination with human pluripotent stem cells may be safe, but nevertheless for hPSC-derived cell populations to be approved for use in clinical trials their stringent elimination will be a requirement. Furthermore, the site of transplantation needs to be taken into account as not all places in the body are equally permissive for teratoma growth and development and contaminating hPSC may also migrate to alternative and possibly more permissive sites for teratoma growth post transplantation. For instance, it has been reported that similar number of pluripotent stem cells injected into immuno-deprived mice induced teratomas with 12.5% efficiency in intramuscular injections, 33% in subcutaneous injections, 60% in intratesticular, and approximately 100% under the kidney capsule [14]. Although many variables can potentially affect teratoma formation, we consider that the most ethical and safest cell population for transplantation into patients should be classified as pluripo‐ tent stem cell-free.

**Antibody Isotype Cell-surface antigen Source/Supplier Literature reference**

Kindly donated by Prof. Mar‐ tin Pera

Safety Assessment of Reprogrammed Cells Prior to Clinical Applications: Potential Approaches to…

Millipore MAB4414 http:// www.millipore.com

Millipore MAB430 http:// www.millipore.com

Millipore MAB4427 http:// www.millipore.com

Millipore MAB4346 http://

www.millipore.com

www.millipore.com

Laslett *et al*., 2003 [27]; Pera *et al*.,2003 [28].

http://dx.doi.org/10.5772/54400

559

Choo *et al*., 2008 [17].

Kerjaschki *et al*., 1986 [29].

Laslett *et al*., 2003 [27]; Pera *et al*., 2003 [28].

Andrews *et al*., 1984 [33].

Andrews *et al*., 1984 [33].

www.millipore.com Kannagi *et al*., 1983 [30].

www.millipore.com Kannagi *et al*., 1983 [31].

www.millipore.com Cooper *et al*., 2002 [32].

**GCTM-2** IgM Keratan sulphate proteogly‐

**mAB 84** IgM Podocalyxin (PODXL); CD34

**PHM-5** IgG1 Podocalyxin (PODXL); CD34

**TG30 (CD9)** IgG2a 25kDa tetraspannin protein

**TG343** IgM

can (KSPG)-protein core

family member.

family member.

**SSEA-3** IgM Globoseries glycolipid Millipore MAB4303 http://

**SSEA-4** IgG3 Globoseries glycolipid Millipore MAB4304 http://

CD9

KSPG-protein core (detects the same antigen as the GCTM-2 antibody).

**TRA-1-60** IgM KSPG-carbohydrate side chain Millipore MAB4360 http://

**TRA-1-81** IgM KSPG-carbohydrate side chain Millipore MAB4381 http://

**Table 1.** Antibodies that are reactive with cell surface markers expressed on human pluripotent stem cells

The studies described above point to FACS technology coupled to antibody detection of sur‐ face markers as a good strategy to eliminate residual undifferentiated pluripotent cells and recover differentiated live cells for further applications such as re-culture or transplantation.

## **3. How to purge residual tumorigenic pluripotent stem cells from differentiated cell types?**

To guarantee that no undifferentiated pluripotent stem cells are present in a hESC or hiPSC-differentiated progeny intended for transplantation into patients, researchers need assays to detect those residual pluripotent cells and efficient methods to purge stem cells from the differentiated cell populations. A good strategy to detect pluripotent cells is us‐ ing antibodies that detect surface markers on live hPSC that are not present on differen‐ tiated cell types. After antibody-mediated detection of stem cells, other technologies could be coupled to the antibodies in order to eliminate residual pluripotent stem cells from the transplantation sample. For instance, Fluorescent or Magnetic Activated Cell Sorting (FACS and MACS) could be used with antibody detection for elimination of the targeted cells.

There are only a few available antibodies that detect cell surface markers on live human pluripotent stem cells (See table 1). Researchers, utilising the available antibodies, have described methods to eliminate residual pluripotent cells from samples of differentiated cell types. For instance the SSEA-4 antibody first demonstrated its utility in purging plu‐ ripotent stem cells from simian ESC-derived hematopoietic precursors used for transplan‐ tations into monkeys [15]. In this study, researchers used SSEA-4 antibody to detect residual pluripotent cells that persisted despite rigorous and extended differentiation protocols for hematopoietic precursors. SSEA-4 negative cells obtained by fluorescence activated cell sorting (FACS) did not develop teratomas, whereas teratomas were consis‐ tently observed in hematopoietic precursors showing presence of SSEA-4 positive cells [15]. The SSEA-4 and Tra-1-60 antibodies have also been compared for their efficiency in detecting and removing residual hPSC, by FACS or magnetic-activated cell sorting MACS [16]. This comparison revealed that MACS technology was not efficient for com‐ plete depletion of hESCs, with an average of 82% retention of hESCs, and highlighted that negative selection via FACS may be a preferred approach to eliminate undesirable hESCs from differentiated populations [16]. However, a note of caution against the use of single antibodies to detect hESCs emerged from data showing that 47% of SSEA-4 low-expressing hESCs exhibited a high level of expression for TRA-1-60. Therefore, de‐ tection of a single cell-surface marker may not be sufficient to eliminate all pluripotent stem cells, and methods that use multiple antibodies detecting different epitopes ex‐ pressed by hESCs are more likely to be successful [16].


teratomas with 12.5% efficiency in intramuscular injections, 33% in subcutaneous injections, 60% in intratesticular, and approximately 100% under the kidney capsule [14]. Although many variables can potentially affect teratoma formation, we consider that the most ethical and safest cell population for transplantation into patients should be classified as pluripo‐

To guarantee that no undifferentiated pluripotent stem cells are present in a hESC or hiPSC-differentiated progeny intended for transplantation into patients, researchers need assays to detect those residual pluripotent cells and efficient methods to purge stem cells from the differentiated cell populations. A good strategy to detect pluripotent cells is us‐ ing antibodies that detect surface markers on live hPSC that are not present on differen‐ tiated cell types. After antibody-mediated detection of stem cells, other technologies could be coupled to the antibodies in order to eliminate residual pluripotent stem cells from the transplantation sample. For instance, Fluorescent or Magnetic Activated Cell Sorting (FACS and MACS) could be used with antibody detection for elimination of the

There are only a few available antibodies that detect cell surface markers on live human pluripotent stem cells (See table 1). Researchers, utilising the available antibodies, have described methods to eliminate residual pluripotent cells from samples of differentiated cell types. For instance the SSEA-4 antibody first demonstrated its utility in purging plu‐ ripotent stem cells from simian ESC-derived hematopoietic precursors used for transplan‐ tations into monkeys [15]. In this study, researchers used SSEA-4 antibody to detect residual pluripotent cells that persisted despite rigorous and extended differentiation protocols for hematopoietic precursors. SSEA-4 negative cells obtained by fluorescence activated cell sorting (FACS) did not develop teratomas, whereas teratomas were consis‐ tently observed in hematopoietic precursors showing presence of SSEA-4 positive cells [15]. The SSEA-4 and Tra-1-60 antibodies have also been compared for their efficiency in detecting and removing residual hPSC, by FACS or magnetic-activated cell sorting MACS [16]. This comparison revealed that MACS technology was not efficient for com‐ plete depletion of hESCs, with an average of 82% retention of hESCs, and highlighted that negative selection via FACS may be a preferred approach to eliminate undesirable hESCs from differentiated populations [16]. However, a note of caution against the use of single antibodies to detect hESCs emerged from data showing that 47% of SSEA-4 low-expressing hESCs exhibited a high level of expression for TRA-1-60. Therefore, de‐ tection of a single cell-surface marker may not be sufficient to eliminate all pluripotent stem cells, and methods that use multiple antibodies detecting different epitopes ex‐

pressed by hESCs are more likely to be successful [16].

**3. How to purge residual tumorigenic pluripotent stem cells from**

tent stem cell-free.

558 Pluripotent Stem Cells

targeted cells.

**differentiated cell types?**

**Table 1.** Antibodies that are reactive with cell surface markers expressed on human pluripotent stem cells

The studies described above point to FACS technology coupled to antibody detection of sur‐ face markers as a good strategy to eliminate residual undifferentiated pluripotent cells and recover differentiated live cells for further applications such as re-culture or transplantation. However, as the viability of hPSC-derived lineage progenitors or more mature cell types can be compromised post-FACS, caused by shearing forces, laser damage or osmotic stress, oth‐ er technologies such as MACS may be better suited in these instances. Although MACS does not completely remove all hESCs in a single pass [16], this technology exhibits higher cell viability than FACS and it is possible that subsequent positive selections by MACS using multiple antibodies for different hESC cell surface markers could completely remove all hESCs. An alternative approach to MACS could be to use cytotoxic antibodies directed against hESC surface antigens or chemicals that could selectively eliminate hESCs without affecting their derivatives. An example of a cytotoxic antibody that detects and removes hESCs is the monoclonal antibody mAB-84 [17], which binds to PODXL (Podocalyxin-like protein 1) on hESCs and initiates a sequence of events that leads to hESC-membrane dam‐ age by formation of leaking pores [18]. It has been proposed that using the monoclonal anti‐ body mAB-84 in a two-step cell-cell separation approach can eliminate teratoma-forming hESC from differentiated cell types [19]. In this strategy, an initial depletion of hESCs was achieved via MACS using a panel of commonly used hESC cell-surface markers, which was followed by selective elimination of residual undifferentiated stem cells post-MACS using the cytotoxic antibody mAB-84, an approach that appears to increase the safety of cell trans‐ plantation [19].

antigens that elicit highly sensitive antibodies that recognize human pluripotent stem cells. Furthermore, a caveat is that stem-cell antibodies could also be immunoreactive with some embryonic tissues, or some mature cell types, becoming problematic with some hESC differentiation protocols. Therefore, depending on the phenotype of the tar‐ get somatic cells, selected antibodies used to detect human pluripotent cells should be selected that do not react with the differentiated cells intended for transplantation. For instance, if working with hESC-derived renal tissues for treatment of kidney disorders, PODXL antibodies should not be used alone to detect stem cells because Podocalyxin

Safety Assessment of Reprogrammed Cells Prior to Clinical Applications: Potential Approaches to…

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561

The information in the previous section demonstrates that FACS and MACS technologies are potential methods for the elimination of residual pluripotent cells following *in vitro* differentiation (Figure 1). Both methodological approaches use cell surface antibodies for the labelling and detection of undifferentiated live hPSC. The advantage of live cell de‐ tection using either FACS or MACS is the ability to retrieve live hESC or hIPSC-deriva‐ tives that could be used for *in vitro* re-culture and expansion, or, ultimately, transplantation. However, FACS and MACS studies have also revealed the immunologi‐ cal complexity of *in-vitro* hESC cultures. HESC cultures contain a continuum of different subpopulations, where some hESC subpopulations express low levels of one surface marker and at the same time high levels of another [16, 21-23]. These findings imply strongly that a single cell-surface marker is not sufficient to eliminate all pluripotent stem cells [16, 21-23]. Therefore, any attempt to eliminate all hESC pluripotent subpopu‐ lations should rely on methods that use multiple antibodies detecting different epitopes expressed by hESCs. For instance, SSEA-4-coupled MACS showed an average 82% reten‐ tion of hESCs [16], but when a panel of cell surface antibodies directed to different epito‐ pes was used with MACS, the removal of undifferentiated hESCs raised to 98% on

In our laboratory, we have been working on the development of monoclonal antibody panels against extracellular markers that allow efficient human pluripotent cell separa‐ tion from mixed populations of cultured cells, an essential requirement for safe hESC or hIPSC-based therapeutics [21-24]. Towards this end, we have reported a FACS-based im‐ muno-transcriptional profiling system based on the detection of two pluripotency-associ‐ ated cell surface antigens TG30 (CD9) and GCTM-2, [25-26]. This method is useful to characterise multiple human pluripotent stem cell lines, and to identify the subpopula‐ tions that are found in hESC *in-vitro* continuous culture [21-22]. Ongoing unpublished observations indicate that this double staining of human stem cells using two cell-surface markers is a better way to eliminate residual and persistent undifferentiated pluripotent cells using FACS in both hESC and hIPSC lines. Nevertheless, we are aware that there will be differentiation contexts in which TG30 (CD9) and GCTM-2 might not be appro‐ priate or sufficient to purge pluripotent cells from particular differentiated hPSC-deriva‐ tives. Therefore there is a real need for new monoclonal antibodies that detect cell

protein is also expressed in glomerular podocytes.

average [19].

surface proteins on live hPSC.

Selective elimination of residual human pluripotent stem cells after differentiation can al‐ so be achieved by targeting apoptosis-meditating receptors that are differentially ex‐ pressed in undifferentiated stem cells and absent in hESC derivatives. Therefore, stimulation of these specific hESC apoptotic receptors induce programmed cell death on‐ ly in the residual stem cells without affecting their differentiated progeny. One example of this kind of receptor is the prostate apoptosis response-4 (PAR-4), which mediates ce‐ ramide or ceramide-analogue-induced apoptosis in proliferating stem cells [20]. The apoptotic response appears to be specific for PAR-4(+) stem cells, and given that ESC-dif‐ ferentiated progenies such as neuro-progenitors express very low levels of PAR-4, they are less sensitive to ceramide induced apoptosis [20]. Using this approach, ceramide treatment appears to prevent teratoma formation when transplanting neural progenitors derived from ES cells [20] although it is likely that regulatory assays will require a more stringent method. Although PAR-4 induced apoptosis by ceramides appears an effective way to eliminate residual pluripotent stem cells following differentiation, this approach has not been broadly tested.

## **4. Antibodies against cell surface markers of human stem cells**

The scarcity of antibodies directed against cell surface markers that recognize live hu‐ man pluripotent stem cells (See table 1) is compounded by the fact that most of these an‐ tibodies lack identification of their encoding gene. Indeed, some cell surface antibodies do not recognize proteins, but complex carbohydrate and lipid moieties for which the corresponding gene is not yet identified. Despite this, these complex moieties are strong antigens that elicit highly sensitive antibodies that recognize human pluripotent stem cells. Furthermore, a caveat is that stem-cell antibodies could also be immunoreactive with some embryonic tissues, or some mature cell types, becoming problematic with some hESC differentiation protocols. Therefore, depending on the phenotype of the tar‐ get somatic cells, selected antibodies used to detect human pluripotent cells should be selected that do not react with the differentiated cells intended for transplantation. For instance, if working with hESC-derived renal tissues for treatment of kidney disorders, PODXL antibodies should not be used alone to detect stem cells because Podocalyxin protein is also expressed in glomerular podocytes.

However, as the viability of hPSC-derived lineage progenitors or more mature cell types can be compromised post-FACS, caused by shearing forces, laser damage or osmotic stress, oth‐ er technologies such as MACS may be better suited in these instances. Although MACS does not completely remove all hESCs in a single pass [16], this technology exhibits higher cell viability than FACS and it is possible that subsequent positive selections by MACS using multiple antibodies for different hESC cell surface markers could completely remove all hESCs. An alternative approach to MACS could be to use cytotoxic antibodies directed against hESC surface antigens or chemicals that could selectively eliminate hESCs without affecting their derivatives. An example of a cytotoxic antibody that detects and removes hESCs is the monoclonal antibody mAB-84 [17], which binds to PODXL (Podocalyxin-like protein 1) on hESCs and initiates a sequence of events that leads to hESC-membrane dam‐ age by formation of leaking pores [18]. It has been proposed that using the monoclonal anti‐ body mAB-84 in a two-step cell-cell separation approach can eliminate teratoma-forming hESC from differentiated cell types [19]. In this strategy, an initial depletion of hESCs was achieved via MACS using a panel of commonly used hESC cell-surface markers, which was followed by selective elimination of residual undifferentiated stem cells post-MACS using the cytotoxic antibody mAB-84, an approach that appears to increase the safety of cell trans‐

Selective elimination of residual human pluripotent stem cells after differentiation can al‐ so be achieved by targeting apoptosis-meditating receptors that are differentially ex‐ pressed in undifferentiated stem cells and absent in hESC derivatives. Therefore, stimulation of these specific hESC apoptotic receptors induce programmed cell death on‐ ly in the residual stem cells without affecting their differentiated progeny. One example of this kind of receptor is the prostate apoptosis response-4 (PAR-4), which mediates ce‐ ramide or ceramide-analogue-induced apoptosis in proliferating stem cells [20]. The apoptotic response appears to be specific for PAR-4(+) stem cells, and given that ESC-dif‐ ferentiated progenies such as neuro-progenitors express very low levels of PAR-4, they are less sensitive to ceramide induced apoptosis [20]. Using this approach, ceramide treatment appears to prevent teratoma formation when transplanting neural progenitors derived from ES cells [20] although it is likely that regulatory assays will require a more stringent method. Although PAR-4 induced apoptosis by ceramides appears an effective way to eliminate residual pluripotent stem cells following differentiation, this approach

The scarcity of antibodies directed against cell surface markers that recognize live hu‐ man pluripotent stem cells (See table 1) is compounded by the fact that most of these an‐ tibodies lack identification of their encoding gene. Indeed, some cell surface antibodies do not recognize proteins, but complex carbohydrate and lipid moieties for which the corresponding gene is not yet identified. Despite this, these complex moieties are strong

plantation [19].

560 Pluripotent Stem Cells

has not been broadly tested.

**of human stem cells**

**4. Antibodies against cell surface markers**

The information in the previous section demonstrates that FACS and MACS technologies are potential methods for the elimination of residual pluripotent cells following *in vitro* differentiation (Figure 1). Both methodological approaches use cell surface antibodies for the labelling and detection of undifferentiated live hPSC. The advantage of live cell de‐ tection using either FACS or MACS is the ability to retrieve live hESC or hIPSC-deriva‐ tives that could be used for *in vitro* re-culture and expansion, or, ultimately, transplantation. However, FACS and MACS studies have also revealed the immunologi‐ cal complexity of *in-vitro* hESC cultures. HESC cultures contain a continuum of different subpopulations, where some hESC subpopulations express low levels of one surface marker and at the same time high levels of another [16, 21-23]. These findings imply strongly that a single cell-surface marker is not sufficient to eliminate all pluripotent stem cells [16, 21-23]. Therefore, any attempt to eliminate all hESC pluripotent subpopu‐ lations should rely on methods that use multiple antibodies detecting different epitopes expressed by hESCs. For instance, SSEA-4-coupled MACS showed an average 82% reten‐ tion of hESCs [16], but when a panel of cell surface antibodies directed to different epito‐ pes was used with MACS, the removal of undifferentiated hESCs raised to 98% on average [19].

In our laboratory, we have been working on the development of monoclonal antibody panels against extracellular markers that allow efficient human pluripotent cell separa‐ tion from mixed populations of cultured cells, an essential requirement for safe hESC or hIPSC-based therapeutics [21-24]. Towards this end, we have reported a FACS-based im‐ muno-transcriptional profiling system based on the detection of two pluripotency-associ‐ ated cell surface antigens TG30 (CD9) and GCTM-2, [25-26]. This method is useful to characterise multiple human pluripotent stem cell lines, and to identify the subpopula‐ tions that are found in hESC *in-vitro* continuous culture [21-22]. Ongoing unpublished observations indicate that this double staining of human stem cells using two cell-surface markers is a better way to eliminate residual and persistent undifferentiated pluripotent cells using FACS in both hESC and hIPSC lines. Nevertheless, we are aware that there will be differentiation contexts in which TG30 (CD9) and GCTM-2 might not be appro‐ priate or sufficient to purge pluripotent cells from particular differentiated hPSC-deriva‐ tives. Therefore there is a real need for new monoclonal antibodies that detect cell surface proteins on live hPSC.

**5. Conclusions**

**Acknowledgements**

are gratefully acknowledged.

Juan Carlos Polanco1,2 and Andrew L. Laslett1,2\*

\*Address all correspondence to: Andrew.Laslett@csiro.au

1 CSIRO, Materials Science and Engineering, Clayton, Victoria, Australia

**Author details**

tralia

Human pluripotent stem cells, namely hESC and hIPSC lines, may be the future main‐ stay of medicine, providing a plethora of medical applications and transplantation thera‐ pies aimed at the correction of an important number of pathological disorders. However, reaching clinical applications based on hPSC-therapies has not been as fast as expected. The ability to generate hIPSC lines from a variety of tissue sources has brought hIPSC re‐ search clearly into the spotlight, but reports on their epigenetic instability and genetic variability suggest that these cells are not yet clinic-ready. In addition, the concern of tu‐ morigenesis or teratoma formation is an unsolved problem for both hESC and hIPSC re‐ search. If differentiation protocols are not 100% efficient and yield a mixture of differentiated and undifferentiated cells, this presents a significant risk of teratoma for‐ mation after transplantation. It is clear that adequate safety assays for hESC or hIPSC-de‐ rived technologies are of the utmost importance to aid in the safe translation from the bench to the clinic. This includes the essential monitoring of any residual undifferentiated pluripotent cells after differentiation protocols, an unavoidable methodological step in any sample to be used in the clinic. A variety of approaches have been discussed in this chapter to help to eliminate the undesirable residual pluripotent stem cells from samples intended for transplantation. However, there is an ongoing need to improve these separa‐ tion methods in order to achieve hPSC free samples in a rapid, easy, safe, cost effective, scalable and clinically applicable way. We expect that novel cell-surface antibodies recog‐

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563

nizing live pluripotent stem cells will strongly contribute to this ongoing search.

Work in ALL's laboratory contributes to Stem Cells Australia and was supported by the Australian Stem Cell Centre, the National Health and Medical Research Council (Australia) and the Victoria-California Stem Cell Alliance (CIRM grant TR1-01250). Comments and sug‐ gestions on draft versions of the manuscript by Drs. Carmel O'Brien and Tung-Liang Chung

2 Department of Anatomy and Developmental Biology, Monash University, Victoria, Aus‐

**Figure 1. Potential approaches to eliminate residual pluripotent stem cells after in vitro differentiation**. Shown are two potential methods that could be used to purge residual tumorigenic pluripotent stem cells from differentiat‐ ed cell types. (A): Human pluripotent stem cells (hPSC) are able to self-renew indefinitely *in vitro*. (B): These pluripotent cells can be induced to differentiate *in vitro* to generate healthy progenitors and/or specialised somatic cell types that could potentially be used for transplantation and therapeutic applications. However, it is essential to monitor if any residual undifferentiated pluripotent cells remain after differentiation protocols. If undifferentiated stem cells remain, these cells should be removed without damaging the potentially therapeutic differentiated cells. Two good strategies for elimination of residual pluripotent cells are Magnetic Activated Cell Sorting (C: MACS) and Fluorescence Activated Cell Sorting (D: FACS). Both technologies are coupled to antibody detection of cell surface markers and allow retrieval of live hPSC-derivatives that could be used for further *in vitro* re-culture and expansion, or in due course transplanta‐ tion (E).

## **5. Conclusions**

Human pluripotent stem cells, namely hESC and hIPSC lines, may be the future main‐ stay of medicine, providing a plethora of medical applications and transplantation thera‐ pies aimed at the correction of an important number of pathological disorders. However, reaching clinical applications based on hPSC-therapies has not been as fast as expected. The ability to generate hIPSC lines from a variety of tissue sources has brought hIPSC re‐ search clearly into the spotlight, but reports on their epigenetic instability and genetic variability suggest that these cells are not yet clinic-ready. In addition, the concern of tu‐ morigenesis or teratoma formation is an unsolved problem for both hESC and hIPSC re‐ search. If differentiation protocols are not 100% efficient and yield a mixture of differentiated and undifferentiated cells, this presents a significant risk of teratoma for‐ mation after transplantation. It is clear that adequate safety assays for hESC or hIPSC-de‐ rived technologies are of the utmost importance to aid in the safe translation from the bench to the clinic. This includes the essential monitoring of any residual undifferentiated pluripotent cells after differentiation protocols, an unavoidable methodological step in any sample to be used in the clinic. A variety of approaches have been discussed in this chapter to help to eliminate the undesirable residual pluripotent stem cells from samples intended for transplantation. However, there is an ongoing need to improve these separa‐ tion methods in order to achieve hPSC free samples in a rapid, easy, safe, cost effective, scalable and clinically applicable way. We expect that novel cell-surface antibodies recog‐ nizing live pluripotent stem cells will strongly contribute to this ongoing search.

## **Acknowledgements**

Work in ALL's laboratory contributes to Stem Cells Australia and was supported by the Australian Stem Cell Centre, the National Health and Medical Research Council (Australia) and the Victoria-California Stem Cell Alliance (CIRM grant TR1-01250). Comments and sug‐ gestions on draft versions of the manuscript by Drs. Carmel O'Brien and Tung-Liang Chung are gratefully acknowledged.

## **Author details**

**Figure 1. Potential approaches to eliminate residual pluripotent stem cells after in vitro differentiation**. Shown are two potential methods that could be used to purge residual tumorigenic pluripotent stem cells from differentiat‐ ed cell types. (A): Human pluripotent stem cells (hPSC) are able to self-renew indefinitely *in vitro*. (B): These pluripotent cells can be induced to differentiate *in vitro* to generate healthy progenitors and/or specialised somatic cell types that could potentially be used for transplantation and therapeutic applications. However, it is essential to monitor if any residual undifferentiated pluripotent cells remain after differentiation protocols. If undifferentiated stem cells remain, these cells should be removed without damaging the potentially therapeutic differentiated cells. Two good strategies for elimination of residual pluripotent cells are Magnetic Activated Cell Sorting (C: MACS) and Fluorescence Activated Cell Sorting (D: FACS). Both technologies are coupled to antibody detection of cell surface markers and allow retrieval of live hPSC-derivatives that could be used for further *in vitro* re-culture and expansion, or in due course transplanta‐

tion (E).

562 Pluripotent Stem Cells

Juan Carlos Polanco1,2 and Andrew L. Laslett1,2\*

\*Address all correspondence to: Andrew.Laslett@csiro.au

1 CSIRO, Materials Science and Engineering, Clayton, Victoria, Australia

2 Department of Anatomy and Developmental Biology, Monash University, Victoria, Aus‐ tralia

## **References**

[1] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T*, et al.* Induction of pluripo‐ tent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5) 861-872.

[14] Prokhorova TA, Harkness LM, Frandsen U, Ditzel N, Schroder HD*, et al.* Teratoma Formation by Human Embryonic Stem Cells Is Site Dependent and Enhanced by the

Safety Assessment of Reprogrammed Cells Prior to Clinical Applications: Potential Approaches to…

http://dx.doi.org/10.5772/54400

565

[15] Shibata H, Ageyama N, Tanaka Y, Kishi Y, Sasaki K*, et al.* Improved safety of hema‐ topoietic transplantation with monkey embryonic stem cells in the allogeneic setting.

[16] Fong CY, Peh GS, Gauthaman K, Bongso A. Separation of SSEA-4 and TRA-1-60 la‐ belled undifferentiated human embryonic stem cells from a heterogeneous cell popu‐ lation using magnetic-activated cell sorting (MACS) and fluorescence-activated cell

[17] Choo AB, Tan HL, Ang SN, Fong WJ, Chin A*, et al.* Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like

[18] Tan HL, Fong WJ, Lee EH, Yap M, Choo A. mAb 84, a Cytotoxic Antibody that Kills Undifferentiated Human Embryonic Stem Cells via Oncosis. Stem Cells. 2009;27(8)

[19] Schriebl K, Satianegara G, Hwang A, Tan HL, Fong WJ*, et al.* Selective removal of un‐ differentiated human embryonic stem cells using magnetic activated cell sorting fol‐ lowed by a cytotoxic antibody. Tissue Engineering Part A. 2012;18(9-10) 899-909.

[20] Bieberich E, Silva J, Wang G, Krishnamurthy K, Condie BG. Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents tera‐ toma formation and enriches for neural precursors in ES cell-derived neural trans‐

[21] Kolle G, Ho M, Zhou Q, Chy HS, Krishnan K*, et al.* Identification of human embryon‐ ic stem cell surface markers by combined membrane-polysome translation state array analysis and immunotranscriptional profiling. Stem Cells. 2009;27(10) 2446-2456.

[22] Laslett AL, Grimmond S, Gardiner B, Stamp L, Lin A*, et al.* Transcriptional analysis of early lineage commitment in human embryonic stem cells. BMC Developmental

[23] Hough SR, Laslett AL, Grimmond SB, Kolle G, Pera MF. A continuum of cell states spans pluripotency and lineage commitment in human embryonic stem cells. PLoS

[24] Wada N, Wang B, Lin NH, Laslett AL, Gronthos S, Bartold PM. Induced pluripotent stem cell lines derived from human gingival fibroblasts and periodontal ligament fi‐

[25] Zhou Q, Chy H, Laslett AL. Preparation of defined human embryonic stem cell pop‐ ulations for transcriptional profiling. Current Protocols in Stem Cell Biology.

broblasts. Journal of Periodontal Research. 2011;46(4) 438-447.

Presence of Matrigel. Stem Cells and Development. 2009;18(1) 47-54.

Stem Cells. 2006;24(6) 1450-1457.

1792-1801.

Biology. 2007;7(1) 12.

One. 2009;4(11) e7708.

2010;Chapter 1 Unit 1B7.

sorting (FACS). Stem Cell Reviews. 2009;5(1) 72-80.

plants. Journal of Cell Biology. 2004;167(4) 723-734.

protein-1. Stem Cells. 2008;26(6) 1454-1463.


[14] Prokhorova TA, Harkness LM, Frandsen U, Ditzel N, Schroder HD*, et al.* Teratoma Formation by Human Embryonic Stem Cells Is Site Dependent and Enhanced by the Presence of Matrigel. Stem Cells and Development. 2009;18(1) 47-54.

**References**

564 Pluripotent Stem Cells

861-872.

58-62.

2011;471(7336) 68-73.

gy. 2011;13(5) 541-549.

893-902.

2000;18(4) 399-404.

[1] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T*, et al.* Induction of pluripo‐ tent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5)

[2] Stadtfeld M, Hochedlinger K. Induced pluripotency: history, mechanisms, and appli‐

[3] Zhao T, Zhang ZN, Rong Z, Xu Y. Immunogenicity of induced pluripotent stem cells.

[4] Gore A, Li Z, Fung HL, Young JE, Agarwal S*, et al.* Somatic coding mutations in hu‐

[5] Hussein SM, Batada NN, Vuoristo S, Ching RW, Autio R*, et al.* Copy number varia‐ tion and selection during reprogramming to pluripotency. Nature. 2011;471(7336)

[6] Laurent LC, Ulitsky I, Slavin I, Tran H, Schork A*, et al.* Dynamic Changes in the Copy Number of Pluripotency and Cell Proliferation Genes in Human ESCs and iPSCs during Reprogramming and Time in Culture. Cell Stem Cell. 2011;8(1) 106-118.

[7] Kim K, Zhao R, Doi A, Ng K, Unternaehrer J*, et al.* Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells.

[8] Lister R, Pelizzola M, Kida YS, Hawkins RD, Nery JR*, et al.* Hotspots of aberrant epi‐ genomic reprogramming in human induced pluripotent stem cells. Nature.

[9] Ohi Y, Qin H, Hong C, Blouin L, Polo JM*, et al.* Incomplete DNA methylation under‐ lies a transcriptional memory of somatic cells in human iPS cells. Nature Cell Biolo‐

[10] Quinlan A, Murat D, Vali H, Komeili A. The HtrA/DegP family protease MamE is a bifunctional protein with roles in magnetosome protein localization and magnetite

[11] Lawrenz B, Schiller H, Willbold E, Ruediger M, Muhs A, Esser S. Highly sensitive bi‐ osafety model for stem-cell-derived grafts. Cytotherapy. 2004;6(3) 212-222.

[12] Shih CC, Forman SJ, Chu P, Slovak M. Human embryonic stem cells are prone to generate primitive, undifferentiated tumors in engrafted human fetal tissues in se‐ vere combined immunodeficient mice. Stem Cells and Development. 2007;16(6)

[13] Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nature Biotechnology.

biomineralization. Molecular Microbiology. 2011;80(4) 1075-1087.

cations. Genes and Development. 2010;24(20) 2239-2263.

man induced pluripotent stem cells. Nature. 2011;471(7336) 63-67.

Nature. 2011;474(7350) 212-215.

Nature Biotechnology. 2011;29(12) 1117-1119.


[26] Ho MS, Fryga A, Laslett AL. Flow cytometric analysis of human pluripotent stem cells. Methods in Molecular Biology. 2011;767221-230.

**Chapter 26**

**Stem Cells in Tissue Engineering**

Shohreh Mashayekhan, Maryam Hajiabbas and

Additional information is available at the end of the chapter

With the increasing number of patients suffering from damaged or diseased organs and the shortage of organ donors, the need for methods to construct human tissues outside the body has arisen. Tissue engineering is a newly emerging biomedical technology and methodology which combines the disciplines of both the materials and life sciences to replace a diseased or damaged tissue or organ with a living, functional engineered substitute [1, 2]. The socalled triad in tissue engineering encompasses three basic components called scaffold, cell

Whatever the approach being used in tissue engineering, the critical issues to optimize any tissue engineering strategy toward producing a functional equivalent tissue are the source of the cells and substrate biomaterial to deliver the cells in particular anatomical sites where a regenerative process is required. Due to their unique properties, stem cells and polymeric biomaterials are key design options. Briefly, stem cells have the ability to self-renew and commit to specific cell lineages in response to appropriate stimuli, providing excellent re‐ generative potential that will most likely lead to functionality of the engineered tissue. Poly‐ meric materials are biocompatible, degradable, and flexible in processing and property design. A major focus of tissue engineering, therefore, is to utilize functional polymers with appropriate characteristics, as a means of controlling stem cell function. Based on their dif‐ ferentiation potential, stem cells used for tissue engineering can be divided into two catego‐ ries: pluripotent stem cells and multipotent stem cells. Pluripotent stem cells include embryonic stem cells (ESCs) as well as induced pluripotent stem cells (iPSCs). Because ESCs are isolated from the inner cell mass of the blastocyst during embryological development, their use in tissue engineering is controversial and more limited while more attention has been paid to adult stem cells, which are multipotent and have a larger capacity to differenti‐

> © 2013 Mashayekhan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Mashayekhan et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons

Ali Fallah

http://dx.doi.org/10.5772/54371

**1. Introduction**

and signaling biomolecule.


## **Chapter 26**

## **Stem Cells in Tissue Engineering**

[26] Ho MS, Fryga A, Laslett AL. Flow cytometric analysis of human pluripotent stem

[27] Laslett AL, Filipczyk AA, Pera MF. Characterization and culture of human embryon‐

[28] Pera MF, Filipczyk AA, Hawes SM, Laslett AL. Isolation, characterization, and differ‐ entiation of human embryonic stem cells. Methods in Enzymology. 2003;365429-446.

[29] Kerjaschki D, Poczewski H, Dekan G, Horvat R, Balzar E*, et al.* Identification of a ma‐ jor sialoprotein in the glycocalyx of human visceral glomerular epithelial cells. Jour‐

[30] Kannagi R, Levery SB, Ishigami F, Hakomori S, Shevinsky LH*, et al.* New globoseries glycosphingolipids in human teratocarcinoma reactive with the monoclonal antibody directed to a developmentally regulated antigen, stage-specific embryonic antigen 3.

[31] Kannagi R, Cochran NA, Ishigami F, Hakomori S, Andrews PW*, et al.* Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells. Embo Journal. 1983;2(12) 2355-2361.

[32] Cooper S, Bennett W, Andrade J, Reubinoff BE, Thomson J, Pera MF. Biochemical properties of a keratan sulphate/chondroitin sulphate proteoglycan expressed in pri‐

[33] Andrews PW, Banting G, Damjanov I, Arnaud D, Avner P. Three monoclonal anti‐ bodies defining distinct differentiation antigens associated with different high molec‐ ular weight polypeptides on the surface of human embryonal carcinoma cells.

mate pluripotent stem cells. Journal of Anatomy. 2002;200(Pt 3) 259-265.

ic stem cells. Trends in Cardiovascular Medicine. 2003;13(7) 295-301.

cells. Methods in Molecular Biology. 2011;767221-230.

566 Pluripotent Stem Cells

nal of Clinical Investigation. 1986;78(5) 1142-1149.

Hybridoma. 1984;3(4) 347-361.

Journal of Biological Chemistry. 1983;258(14) 8934-8942.

Shohreh Mashayekhan, Maryam Hajiabbas and Ali Fallah

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54371

## **1. Introduction**

With the increasing number of patients suffering from damaged or diseased organs and the shortage of organ donors, the need for methods to construct human tissues outside the body has arisen. Tissue engineering is a newly emerging biomedical technology and methodology which combines the disciplines of both the materials and life sciences to replace a diseased or damaged tissue or organ with a living, functional engineered substitute [1, 2]. The socalled triad in tissue engineering encompasses three basic components called scaffold, cell and signaling biomolecule.

Whatever the approach being used in tissue engineering, the critical issues to optimize any tissue engineering strategy toward producing a functional equivalent tissue are the source of the cells and substrate biomaterial to deliver the cells in particular anatomical sites where a regenerative process is required. Due to their unique properties, stem cells and polymeric biomaterials are key design options. Briefly, stem cells have the ability to self-renew and commit to specific cell lineages in response to appropriate stimuli, providing excellent re‐ generative potential that will most likely lead to functionality of the engineered tissue. Poly‐ meric materials are biocompatible, degradable, and flexible in processing and property design. A major focus of tissue engineering, therefore, is to utilize functional polymers with appropriate characteristics, as a means of controlling stem cell function. Based on their dif‐ ferentiation potential, stem cells used for tissue engineering can be divided into two catego‐ ries: pluripotent stem cells and multipotent stem cells. Pluripotent stem cells include embryonic stem cells (ESCs) as well as induced pluripotent stem cells (iPSCs). Because ESCs are isolated from the inner cell mass of the blastocyst during embryological development, their use in tissue engineering is controversial and more limited while more attention has been paid to adult stem cells, which are multipotent and have a larger capacity to differenti‐

© 2013 Mashayekhan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Mashayekhan et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ate into a limited number of cell types [3]. Adult stem cells can be found in many adult tis‐ sue types including bone marrow, peripheral blood, adipose tissues, nervous tissues, muscles, dermis, etc. For instance, mesenchymal stem cells (MSCs) which reside in the bone marrow can differentiate into bone (osteoblasts) [4], muscle (myoblasts) [5], fat (adipocytes) [6] and cartilage (chrondocytes) [3] cells, while neural stem cells (NSCs) either give rise to support cells in the nervous system of vertebrates (astrocytes and oligodendrocytes) or neu‐ rons [7]. *In vivo*, differentiation and self-renewal of stem cells are dominated by signals from their surrounding microenvironment [8]. This microenvironment or "niche" is composed of other cell types as well as numerous chemical, mechanical and topographical cues at microand nano-scales, which are believed to serve as signaling mechanisms to determine cell-spe‐ cific recruitment, migration, proliferation, differentiation as well as the production of numerous proteins required for hierarchical tissue organization [9].

chemical, mechanical, and biological cues mimicking the natural stem cell niche in order to direct the desired stem cell behavior to facilitate the regeneration of desired tissues with particular emphasis on using adult stem cells including MSCs and NSCs. The next part will introduce some new trends emerging in the field of tissue engineering in terms of both cellular biology and biomaterial point of view in order to improve the overall ef‐ ficiency of tissue regeneration for effectively controlling the cell fate and translating the stem cell research into much needed clinical applications in a not-too-distant future. The topics discussed in the latter part include 2D polysaccharide-based hydrogel scaffolds de‐ signed in the authors' studies for muscle tissue engineering applications. Hydrogel scaf‐ folds made of natural polymers with proper handling for surgery and mechanical properties similar to muscle tissue, which could promote the desired muscle-derived

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 569

Damaged tissues often lose deeper layers which contain stem cell niches. In such cases, bio‐ materials could be useful tools for reestablishing the niches' functionality [20]. Artificial niches would need to incorporate appropriate 'homing' signals able to either localize endog‐ enous stem cells or direct the desired incorporated exogenous stem cell behavior by means of developing various microenvironment design parameters including the dynamic control of soluble and surface-bound cytokines, ECM, cell-cell interactions, mechanical forces and

The use of biomaterials as scaffolds is a fundamental component of tissue engineering since these materials serve as templates for tissue formation and are engineered depend‐ ing on the tissue of interest. These scaffolds provide structural and mechanical support for the cells as well as present cues inducing tissue repair. The structure, morphology, degradation and presentation of bioactive sites are all important parameters in material design for these applications and may signal the differentiation of stem cells. Beside all the parameters related to the biomaterials scaffold, there are some other factors such as chemical cues (e.g. soluble reagents in terms of both concentration and their gradient, medium pH), mechanical cues (e.g. fluid shear stress) and other types of cues (electric and magnetic field) which are believed to have significant effect on stem cell behavior.

Figure 1 summarizes the biomimetic microenvironment design strategies for controlling stem cell behavior including chemical/biochemical (e.g. growth/differentiation factor presen‐

Engineering these design parameters will effectively yield materials that create an architec‐ ture resembling the native environment for stem cells, and have controlled mechanical prop‐ erties enabling adhesion and thus enhancing contractility in the cellular cytoskeleton, and present ligands directing intracellular signaling and gene expression. This section provides

tation, density and gradient), structural, mechanical and some other types of cues.

stem cell behavior on the surface were developed in this study.

**2. Biomimetic microenvironment design strategies**

These factors are reviewed extensively elsewhere [23, 24].

physicochemical cues [21, 22].

*In vivo*, the cells are surrounded by a biological matrix comprising of tissue-specific combi‐ nations of insoluble proteins (e.g. collagens, laminins, and fibronectins), glycosaminoglycans (e.g. hyaluronan) and inorganic hydroxyapatite crystals (in bone) that are collectively refer‐ red to as the extracellular matrix (ECM). The varied composition of the ECM components not only contains a reservoir of cell-signaling motifs (ligands) and growth factors that guide cellular anchorage and behavior, but also provides physical architecture and mechanical strength to the tissue. The spatial distribution and concentration of ECM ligands, together with the tissue-specific topography and mechanical properties (in addition to signals from adjacent cells—juxtacrine signalling—and the surrounding fluid), provide signaling gradi‐ ents that direct cell migration and cellular production of ECM constituents. In this dynamic environment, the bidirectional flow of information between the ECM and the cells mediates gene expression, ECM remodeling and ultimately tissue/organ function.

Native ECM exhibits macro- to nano-scale patterns of chemistry and topography [10]. Tissue stiffness is also known to vary depending on the organ type, disease state and ag‐ ing process [11-13]. In tissue culture, stem cell differentiation has traditionally been con‐ trolled by the addition of soluble factors to the growth media [14]. However, most stem cell differentiation protocols yield heterogeneous cell types [15, 16]. Moreover, cells en‐ counter very different, unfamiliar surfaces and environments when cultured *in vitro* or when materials are implanted into the body. Therefore, it is desirable to use more biomi‐ metic *in vitro* culture conditions to regulate stem cell fate so as to advance clinical trans‐ lation of stem cells through better expansion techniques and scaffolding for the regeneration of many tissues. Recent advances have facilitated further the creation of substrates with precise micro- and nano-cues, variable stiffness and chemical composi‐ tion to better mimic the *in vivo* microenvironment [2, 17, and 18]. By employing various novel approaches, tissue engineers aim to incorporate topographical, mechanical and chemical cues into biomaterials to control stem cell fate decisions [2, 18, and 19].

This chapter will present various biomaterial designing considerations and strategies for stem cell-based tissue engineering for development as carriers for stem cells facilitating the *in vivo* use of stem cells in tissue engineering. This part first presents some biomimet‐ ic approaches to designing novel polymeric biomaterials with appropriate physical, chemical, mechanical, and biological cues mimicking the natural stem cell niche in order to direct the desired stem cell behavior to facilitate the regeneration of desired tissues with particular emphasis on using adult stem cells including MSCs and NSCs. The next part will introduce some new trends emerging in the field of tissue engineering in terms of both cellular biology and biomaterial point of view in order to improve the overall ef‐ ficiency of tissue regeneration for effectively controlling the cell fate and translating the stem cell research into much needed clinical applications in a not-too-distant future. The topics discussed in the latter part include 2D polysaccharide-based hydrogel scaffolds de‐ signed in the authors' studies for muscle tissue engineering applications. Hydrogel scaf‐ folds made of natural polymers with proper handling for surgery and mechanical properties similar to muscle tissue, which could promote the desired muscle-derived stem cell behavior on the surface were developed in this study.

## **2. Biomimetic microenvironment design strategies**

ate into a limited number of cell types [3]. Adult stem cells can be found in many adult tis‐ sue types including bone marrow, peripheral blood, adipose tissues, nervous tissues, muscles, dermis, etc. For instance, mesenchymal stem cells (MSCs) which reside in the bone marrow can differentiate into bone (osteoblasts) [4], muscle (myoblasts) [5], fat (adipocytes) [6] and cartilage (chrondocytes) [3] cells, while neural stem cells (NSCs) either give rise to support cells in the nervous system of vertebrates (astrocytes and oligodendrocytes) or neu‐ rons [7]. *In vivo*, differentiation and self-renewal of stem cells are dominated by signals from their surrounding microenvironment [8]. This microenvironment or "niche" is composed of other cell types as well as numerous chemical, mechanical and topographical cues at microand nano-scales, which are believed to serve as signaling mechanisms to determine cell-spe‐ cific recruitment, migration, proliferation, differentiation as well as the production of

*In vivo*, the cells are surrounded by a biological matrix comprising of tissue-specific combi‐ nations of insoluble proteins (e.g. collagens, laminins, and fibronectins), glycosaminoglycans (e.g. hyaluronan) and inorganic hydroxyapatite crystals (in bone) that are collectively refer‐ red to as the extracellular matrix (ECM). The varied composition of the ECM components not only contains a reservoir of cell-signaling motifs (ligands) and growth factors that guide cellular anchorage and behavior, but also provides physical architecture and mechanical strength to the tissue. The spatial distribution and concentration of ECM ligands, together with the tissue-specific topography and mechanical properties (in addition to signals from adjacent cells—juxtacrine signalling—and the surrounding fluid), provide signaling gradi‐ ents that direct cell migration and cellular production of ECM constituents. In this dynamic environment, the bidirectional flow of information between the ECM and the cells mediates

Native ECM exhibits macro- to nano-scale patterns of chemistry and topography [10]. Tissue stiffness is also known to vary depending on the organ type, disease state and ag‐ ing process [11-13]. In tissue culture, stem cell differentiation has traditionally been con‐ trolled by the addition of soluble factors to the growth media [14]. However, most stem cell differentiation protocols yield heterogeneous cell types [15, 16]. Moreover, cells en‐ counter very different, unfamiliar surfaces and environments when cultured *in vitro* or when materials are implanted into the body. Therefore, it is desirable to use more biomi‐ metic *in vitro* culture conditions to regulate stem cell fate so as to advance clinical trans‐ lation of stem cells through better expansion techniques and scaffolding for the regeneration of many tissues. Recent advances have facilitated further the creation of substrates with precise micro- and nano-cues, variable stiffness and chemical composi‐ tion to better mimic the *in vivo* microenvironment [2, 17, and 18]. By employing various novel approaches, tissue engineers aim to incorporate topographical, mechanical and

chemical cues into biomaterials to control stem cell fate decisions [2, 18, and 19].

This chapter will present various biomaterial designing considerations and strategies for stem cell-based tissue engineering for development as carriers for stem cells facilitating the *in vivo* use of stem cells in tissue engineering. This part first presents some biomimet‐ ic approaches to designing novel polymeric biomaterials with appropriate physical,

numerous proteins required for hierarchical tissue organization [9].

568 Pluripotent Stem Cells

gene expression, ECM remodeling and ultimately tissue/organ function.

Damaged tissues often lose deeper layers which contain stem cell niches. In such cases, bio‐ materials could be useful tools for reestablishing the niches' functionality [20]. Artificial niches would need to incorporate appropriate 'homing' signals able to either localize endog‐ enous stem cells or direct the desired incorporated exogenous stem cell behavior by means of developing various microenvironment design parameters including the dynamic control of soluble and surface-bound cytokines, ECM, cell-cell interactions, mechanical forces and physicochemical cues [21, 22].

The use of biomaterials as scaffolds is a fundamental component of tissue engineering since these materials serve as templates for tissue formation and are engineered depend‐ ing on the tissue of interest. These scaffolds provide structural and mechanical support for the cells as well as present cues inducing tissue repair. The structure, morphology, degradation and presentation of bioactive sites are all important parameters in material design for these applications and may signal the differentiation of stem cells. Beside all the parameters related to the biomaterials scaffold, there are some other factors such as chemical cues (e.g. soluble reagents in terms of both concentration and their gradient, medium pH), mechanical cues (e.g. fluid shear stress) and other types of cues (electric and magnetic field) which are believed to have significant effect on stem cell behavior. These factors are reviewed extensively elsewhere [23, 24].

Figure 1 summarizes the biomimetic microenvironment design strategies for controlling stem cell behavior including chemical/biochemical (e.g. growth/differentiation factor presen‐ tation, density and gradient), structural, mechanical and some other types of cues.

Engineering these design parameters will effectively yield materials that create an architec‐ ture resembling the native environment for stem cells, and have controlled mechanical prop‐ erties enabling adhesion and thus enhancing contractility in the cellular cytoskeleton, and present ligands directing intracellular signaling and gene expression. This section provides an overview of biomimetic microenvironment design strategies to direct the stem cell behav‐ ior for tissue engineering applications.

this manner can serve to increase local concentration of the protein by hindering diffusion and receptor-mediated endocytosis. For example, the morphogen Sonic hedgehog (Shh) is modified at its termini by lipids that link it to the cell membrane and thereby limit its mobi‐ lity. Removing the lipids dilutes the factor to a lower concentration and thereby shrinks its effectiveness [28, 29]. Accordingly, mimicking the natural immobilization of cytokines is one approach utilized by engineers to concentrate factors in proximity to the cell surface in a manner that activates target signaling pathways effectively, and reduces, as well, the levels

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 571

An early study exploring this design concept focused on epidermal growth factor (EGF) [30] which is beneficial in repairing the damaged tissues, but is often difficult to deliver at suffi‐ ciently high concentrations to mediate downstream signaling events as it does not contain a matrix-binding domain and rapidly undergoes receptor-mediated endocytosis [31]. In a re‐ cent example involving human and porcine MSCs, amine-targeting chemistry was used to tether EGF to the surface of poly (methyl methacrylate)-graft-poly (ethylene oxide) comb polymers [32]. The tethered EGF led to sustained EGF receptor signaling and subsequent cellular responses including cell spreading and protection from apoptosis, whereas saturat‐ ing levels of soluble EGF did not. Sakiyama-Elbert et al. incorporated heparin into biomate‐ rial scaffolds to allow for immobilization of basic fibroblast growth factor (bFGF) [33]. bFGF was released either passively by diffusion, or actively via heparinases secreted by neighbor‐ ing cells, thereby allowing for a controlled release and presentation of signal which was not possible with soluble growth factor delivery. The same delivery system has been used for differentiation of murine ESCs into mature neural cell types, including neurons and oligo‐ dendrocytes, indicating that biomaterials scaffolds functionalized with immobilized growth factors may be a potential strategy for generation of engineered tissue for treatment of spi‐ nal cord injury [34]. Finally, in a recent study, polymer substrates functionalized with the signaling domain of Shh supported enhanced osteogeneic differentiation of bone marrowderived MSCs, as compared to cells cultured on the same surfaces with soluble Shh at the same concentration [35]. This example further demonstrates how growth factor or morpho‐ gen immobilization serves as an effective means to achieve sustained activation of down‐ stream signaling pathways due in part to the finding that the local concentration in the

of growth factor necessary to elicit a potent cellular response.

scaffold was greater for immobilized growth factor than for soluble form.

There is a significant scope in the application of surface modifications, despite the use of protein biomolecules to provide more cues for cell adhesion, proliferation and differentia‐ tion. Arg-Gly-Asp (RGD) sequence and several natural proteins like collagen, laminin and fibronectin were shown to be essential for cell attachment to polymeric material surfaces de‐ void of any cell recognition sites [36, 37]. The immobilization of these proteins to polymers not only promotes cell adhesion and proliferation but also increases hydrophilicity of the polymers such as aliphatic polyesters. One such surface functionalization for biopolymer substrate surfaces is attachment of RGD peptides that is the most effective and often em‐ ployed peptide sequence for stimulating cell adhesion on synthetic polymer surfaces. This peptide sequence can interact with integrin receptors at the focal adhesion points. Once the RGD sequence is recognized by the integrins, it will initiate an integrin-mediated cell attach‐

**Figure 1.** The biomimetic microenvironment design strategies for controlling stem cell fate

#### **2.1. Chemical and biochemical cues**

Biochemical cues are generally provided by soluble ligands, which may be either secreted by paracrinal cells or supplied by a capillary network in the human body. Insoluble ligands, which are adhesion proteins or molecules such as collagen, laminin and carbohydrates, are also present. Biochemical factors typically influence the cell microenvironment in a concen‐ tration or gradient-dependent manner.

Chemical and biochemical means are the first choice for stem cell differentiation. Small ions, growth factors, and cytokines can exert potent, long-range effects over stem cell mi‐ croenvironments. Owing to their relative ease of study, *soluble biochemical cues* and their downstream signal transduction pathways are the best characterized determinants of stem cell fate and have been extensively used in *ex vivo* stem cell culture systems, as ex‐ tensively discussed elsewhere [24-26]. Therefore, the following section will mainly focus upon the application of other types of *soluble* signals such as dissolved oxygen as well as insoluble chemical and biochemical cues (e.g., immobilized growth factor, extracellular matrix material, etc) to engineered niches.

*In vivo*, numerous growth factors and morphogens are immobilized by binding to the ECM through specific heparin-binding domains or by direct binding to ECM molecules such as collagen, or direct anchoring to cell membranes [27]. Immobilization of growth factors in this manner can serve to increase local concentration of the protein by hindering diffusion and receptor-mediated endocytosis. For example, the morphogen Sonic hedgehog (Shh) is modified at its termini by lipids that link it to the cell membrane and thereby limit its mobi‐ lity. Removing the lipids dilutes the factor to a lower concentration and thereby shrinks its effectiveness [28, 29]. Accordingly, mimicking the natural immobilization of cytokines is one approach utilized by engineers to concentrate factors in proximity to the cell surface in a manner that activates target signaling pathways effectively, and reduces, as well, the levels of growth factor necessary to elicit a potent cellular response.

an overview of biomimetic microenvironment design strategies to direct the stem cell behav‐

**Figure 1.** The biomimetic microenvironment design strategies for controlling stem cell fate

Biochemical cues are generally provided by soluble ligands, which may be either secreted by paracrinal cells or supplied by a capillary network in the human body. Insoluble ligands, which are adhesion proteins or molecules such as collagen, laminin and carbohydrates, are also present. Biochemical factors typically influence the cell microenvironment in a concen‐

Chemical and biochemical means are the first choice for stem cell differentiation. Small ions, growth factors, and cytokines can exert potent, long-range effects over stem cell mi‐ croenvironments. Owing to their relative ease of study, *soluble biochemical cues* and their downstream signal transduction pathways are the best characterized determinants of stem cell fate and have been extensively used in *ex vivo* stem cell culture systems, as ex‐ tensively discussed elsewhere [24-26]. Therefore, the following section will mainly focus upon the application of other types of *soluble* signals such as dissolved oxygen as well as insoluble chemical and biochemical cues (e.g., immobilized growth factor, extracellular

*In vivo*, numerous growth factors and morphogens are immobilized by binding to the ECM through specific heparin-binding domains or by direct binding to ECM molecules such as collagen, or direct anchoring to cell membranes [27]. Immobilization of growth factors in

ior for tissue engineering applications.

570 Pluripotent Stem Cells

**2.1. Chemical and biochemical cues**

tration or gradient-dependent manner.

matrix material, etc) to engineered niches.

An early study exploring this design concept focused on epidermal growth factor (EGF) [30] which is beneficial in repairing the damaged tissues, but is often difficult to deliver at suffi‐ ciently high concentrations to mediate downstream signaling events as it does not contain a matrix-binding domain and rapidly undergoes receptor-mediated endocytosis [31]. In a re‐ cent example involving human and porcine MSCs, amine-targeting chemistry was used to tether EGF to the surface of poly (methyl methacrylate)-graft-poly (ethylene oxide) comb polymers [32]. The tethered EGF led to sustained EGF receptor signaling and subsequent cellular responses including cell spreading and protection from apoptosis, whereas saturat‐ ing levels of soluble EGF did not. Sakiyama-Elbert et al. incorporated heparin into biomate‐ rial scaffolds to allow for immobilization of basic fibroblast growth factor (bFGF) [33]. bFGF was released either passively by diffusion, or actively via heparinases secreted by neighbor‐ ing cells, thereby allowing for a controlled release and presentation of signal which was not possible with soluble growth factor delivery. The same delivery system has been used for differentiation of murine ESCs into mature neural cell types, including neurons and oligo‐ dendrocytes, indicating that biomaterials scaffolds functionalized with immobilized growth factors may be a potential strategy for generation of engineered tissue for treatment of spi‐ nal cord injury [34]. Finally, in a recent study, polymer substrates functionalized with the signaling domain of Shh supported enhanced osteogeneic differentiation of bone marrowderived MSCs, as compared to cells cultured on the same surfaces with soluble Shh at the same concentration [35]. This example further demonstrates how growth factor or morpho‐ gen immobilization serves as an effective means to achieve sustained activation of down‐ stream signaling pathways due in part to the finding that the local concentration in the scaffold was greater for immobilized growth factor than for soluble form.

There is a significant scope in the application of surface modifications, despite the use of protein biomolecules to provide more cues for cell adhesion, proliferation and differentia‐ tion. Arg-Gly-Asp (RGD) sequence and several natural proteins like collagen, laminin and fibronectin were shown to be essential for cell attachment to polymeric material surfaces de‐ void of any cell recognition sites [36, 37]. The immobilization of these proteins to polymers not only promotes cell adhesion and proliferation but also increases hydrophilicity of the polymers such as aliphatic polyesters. One such surface functionalization for biopolymer substrate surfaces is attachment of RGD peptides that is the most effective and often em‐ ployed peptide sequence for stimulating cell adhesion on synthetic polymer surfaces. This peptide sequence can interact with integrin receptors at the focal adhesion points. Once the RGD sequence is recognized by the integrins, it will initiate an integrin-mediated cell attach‐ ment pathway and activate signal transduction between the cell and ECM, thus influencing various cell behaviors on the substrate including proliferation, differentiation, survival and migration [38]. Roeker et al. showed that the composite materials modified by immobilizing poly-L-lysine and BMP-2 as bioactive ligands on the ceramic surface had promising poten‐ tial to enhance the adhesion of hMSCs and directing cell differentiation into osteoblasts [39]. In another study, it was demonstrated that hMSCs encapsulated in poly (ethylene glycol) (PEG)/ RGD hydrogels undergo chondrogenic differentiation in the presence of TGF-β3. More importantly, this effect has been found to be RGD-dose dependent and there is an op‐ timal concentration of RGD present in PEG hydrogels, which improves cell viability and promotes chondrogenesis [40].

strate. Surface modification techniques such as plasma treatment, ion sputtering, oxidation and corona discharge affect the chemical and physical properties of the poly‐ mer surface without significantly changing the bulk material properties. For example, plasma processes makes it possible to change the chemical composition and properties of the polymer system such as hydrophobicity, surface energy, refractive index, hardness, chemical inertness and biocompatibility [48]. Plasma techniques can easily be used to in‐ duce the desired groups or chains onto the surface of a polymer [49, 50]. Appropriate se‐ lection of the plasma source facilitates the introduction of diverse functional groups on the polymer surface to improve biocompatibility or to allow subsequent covalent immo‐ bilization of various bioactive cues. For instance, plasma treatments with oxygen, ammo‐ nia, or air can generate carboxyl groups or amine groups on the polymer surface [51, 52]. A variety of ECM protein components such as gelatin, collagen, laminin, and fibro‐ nectin could be immobilized onto the plasma-treated surface to enhance cellular func‐ tions [53]. Curran et al. show that stem cell differentiation is guided by surface chemistry and energy, independent of inductive media [54]. Although all the surfaces tested main‐ tained cell viability, silanized hydrophobic surfaces with CH3 end groups (with low sur‐ face energy) maintain MSC phenotype, while increasing the surface energy by adding NH2- or SH- terminal groups promotes osteogenesis. Further increase of surface energy by addition of OH or COOH moieties promotes chondrogenesis. However, there are re‐ ports indicating that both hydrophobicity and surface energy play a role in cell adhe‐ sion, but only in the short term until cells themselves modulate their extracellular

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 573

Probably one of the best known soluble reagents is dissolved oxygen. Typical oxygen con‐ centrations *in vivo* vary from 12.5 to 5%, whilst the oxygen concentration in cell culture incu‐ bators is the same as that in the air, which is 20%. Several reports show that lowered oxygen concentrations (5%) increase stem cell proliferation [57-59]. Grayson et al. [60] have shown that even lower oxygen concentrations of about 2% increase MSC proliferation whilst main‐ taining an undifferentiated state, thus suggesting that hypoxic conditions are the character‐ istic of the niche environment. Some authors have observed an induction of adipose-like phenotype in MSCs in severe hypoxia (1%) [61], whilst others showed that adipogenesis is suppressed at 6% oxygen compared to 20% oxygen [62]. Lennon et al. reported that rat MSCs exposed to 5% oxygen during amplification show enhanced osteogenesis after im‐ plantation, compared with cells amplified in 20% which may probably be due to increased proliferation as suggested above [63]. Buckley et al. showed the beneficial response of chon‐ drocyte cells to a low oxygen environment in the absence of TGF-β, suggesting that hypoxia can be used as an alternative to growth factor stimulation to engineer cartilage from culture-

Biomaterial scaffolds take on a variety of structures based on their material composition and processing for forming 3D environments. These materials consist of natural polymers such as collagen, hyaluronic acid, fibrin, alginate, or synthetic polymers such as polyethylene gly‐

environment [55, 56].

expanded chondrocyte [64].

**2.2. Structural cues**

In spite of the addition of differentiation factors in the culture media, the matrix materials which support the cells affect the differentiation of stem cells as well. Mauney et al. found that the matrix-denatured collagen type I is more capable in retaining the osteogenic differ‐ entiation potential *in vitro* and even bone-forming capacity *in vivo* of hMSCs than the con‐ ventional tissue culture plastic [41]. Mwale et al. discerned that bi-axially oriented polypropylene plasma treated in ammonia reduced upregulation of the expression of osteo‐ genic marker genes, such as alkaline phosphatase (ALP), bone sialoprotein and osteocalcin significantly [42]. According to a report presented by Ager et al. [43], collagen I/III and PLLA porous scaffolds showed certain osteoinductive properties without Dex, ascorbic acid, and βGP (DAG) stimulation, verified by immunocytochemical staining against osteoblasttypical markers and completed by calcified matrix detection. Wang et al. demonstrated that ascorbic acid-functionalized poly (methyl methacrylate) can modulate the proliferation and osteogenic differentiation of early and late-passage bone marrow-derived hMSCs [44].

More recently, Xu et al. showed that hMSCs attached, and subsequently proliferated and differentiated toward the osteogenic lineage on the biomimetic bioglass-collagen-hyaluronic acid-phosphatidylserine (BG-COL-HYA-PS) composites to a significantly higher degree compared to those cells on the BG-COL, BG-COL-HYA composites, suggesting the BG-COL-HYA-PS composite porous scaffolds have high potential for bone tissue engineering [45]. In another study, it was shown that the incorporation of gelatin in the poly [(L-lactide)-co-(ecaprolactone)] (PLCL) nano-fibers stimulated the adhesion and osteogenic differentiation of hMSCs, suggesting that the chemical composition of the underlying scaffolds play a key role in regulating the osteogenic differentiation of hMSCs [46].

Regarding chondrogenic differentiation, investigating the effect of cartilage-tissue chon‐ droitin-sulfate (CS) in a fibrin scaffold on the differentiation of adipose-derived adult stem cells into chondrocytes revealed the significant effect of CS on the differentiation ef‐ ficiency. It can be concluded that the fibrin–CS matrices mimicking native cartilage ex‐ tracellular matrix could act as a three-dimensional scaffold for cartilage tissue engineering and have the potential for promoting the differentiation of adipose-derived adult stem cells into chondrocytes [47].

Since the chemical properties of substrates (e.g., hydrophobicity) play an important role in the kinetics of protein adsorption and folding, which in turn influence cellular activi‐ ties, direct the stem cells' fate can be controlled by chemical modification of the sub‐ strate. Surface modification techniques such as plasma treatment, ion sputtering, oxidation and corona discharge affect the chemical and physical properties of the poly‐ mer surface without significantly changing the bulk material properties. For example, plasma processes makes it possible to change the chemical composition and properties of the polymer system such as hydrophobicity, surface energy, refractive index, hardness, chemical inertness and biocompatibility [48]. Plasma techniques can easily be used to in‐ duce the desired groups or chains onto the surface of a polymer [49, 50]. Appropriate se‐ lection of the plasma source facilitates the introduction of diverse functional groups on the polymer surface to improve biocompatibility or to allow subsequent covalent immo‐ bilization of various bioactive cues. For instance, plasma treatments with oxygen, ammo‐ nia, or air can generate carboxyl groups or amine groups on the polymer surface [51, 52]. A variety of ECM protein components such as gelatin, collagen, laminin, and fibro‐ nectin could be immobilized onto the plasma-treated surface to enhance cellular func‐ tions [53]. Curran et al. show that stem cell differentiation is guided by surface chemistry and energy, independent of inductive media [54]. Although all the surfaces tested main‐ tained cell viability, silanized hydrophobic surfaces with CH3 end groups (with low sur‐ face energy) maintain MSC phenotype, while increasing the surface energy by adding NH2- or SH- terminal groups promotes osteogenesis. Further increase of surface energy by addition of OH or COOH moieties promotes chondrogenesis. However, there are re‐ ports indicating that both hydrophobicity and surface energy play a role in cell adhe‐ sion, but only in the short term until cells themselves modulate their extracellular environment [55, 56].

Probably one of the best known soluble reagents is dissolved oxygen. Typical oxygen con‐ centrations *in vivo* vary from 12.5 to 5%, whilst the oxygen concentration in cell culture incu‐ bators is the same as that in the air, which is 20%. Several reports show that lowered oxygen concentrations (5%) increase stem cell proliferation [57-59]. Grayson et al. [60] have shown that even lower oxygen concentrations of about 2% increase MSC proliferation whilst main‐ taining an undifferentiated state, thus suggesting that hypoxic conditions are the character‐ istic of the niche environment. Some authors have observed an induction of adipose-like phenotype in MSCs in severe hypoxia (1%) [61], whilst others showed that adipogenesis is suppressed at 6% oxygen compared to 20% oxygen [62]. Lennon et al. reported that rat MSCs exposed to 5% oxygen during amplification show enhanced osteogenesis after im‐ plantation, compared with cells amplified in 20% which may probably be due to increased proliferation as suggested above [63]. Buckley et al. showed the beneficial response of chon‐ drocyte cells to a low oxygen environment in the absence of TGF-β, suggesting that hypoxia can be used as an alternative to growth factor stimulation to engineer cartilage from cultureexpanded chondrocyte [64].

#### **2.2. Structural cues**

ment pathway and activate signal transduction between the cell and ECM, thus influencing various cell behaviors on the substrate including proliferation, differentiation, survival and migration [38]. Roeker et al. showed that the composite materials modified by immobilizing poly-L-lysine and BMP-2 as bioactive ligands on the ceramic surface had promising poten‐ tial to enhance the adhesion of hMSCs and directing cell differentiation into osteoblasts [39]. In another study, it was demonstrated that hMSCs encapsulated in poly (ethylene glycol) (PEG)/ RGD hydrogels undergo chondrogenic differentiation in the presence of TGF-β3. More importantly, this effect has been found to be RGD-dose dependent and there is an op‐ timal concentration of RGD present in PEG hydrogels, which improves cell viability and

In spite of the addition of differentiation factors in the culture media, the matrix materials which support the cells affect the differentiation of stem cells as well. Mauney et al. found that the matrix-denatured collagen type I is more capable in retaining the osteogenic differ‐ entiation potential *in vitro* and even bone-forming capacity *in vivo* of hMSCs than the con‐ ventional tissue culture plastic [41]. Mwale et al. discerned that bi-axially oriented polypropylene plasma treated in ammonia reduced upregulation of the expression of osteo‐ genic marker genes, such as alkaline phosphatase (ALP), bone sialoprotein and osteocalcin significantly [42]. According to a report presented by Ager et al. [43], collagen I/III and PLLA porous scaffolds showed certain osteoinductive properties without Dex, ascorbic acid, and βGP (DAG) stimulation, verified by immunocytochemical staining against osteoblasttypical markers and completed by calcified matrix detection. Wang et al. demonstrated that ascorbic acid-functionalized poly (methyl methacrylate) can modulate the proliferation and osteogenic differentiation of early and late-passage bone marrow-derived hMSCs [44].

More recently, Xu et al. showed that hMSCs attached, and subsequently proliferated and differentiated toward the osteogenic lineage on the biomimetic bioglass-collagen-hyaluronic acid-phosphatidylserine (BG-COL-HYA-PS) composites to a significantly higher degree compared to those cells on the BG-COL, BG-COL-HYA composites, suggesting the BG-COL-HYA-PS composite porous scaffolds have high potential for bone tissue engineering [45]. In another study, it was shown that the incorporation of gelatin in the poly [(L-lactide)-co-(ecaprolactone)] (PLCL) nano-fibers stimulated the adhesion and osteogenic differentiation of hMSCs, suggesting that the chemical composition of the underlying scaffolds play a key role

Regarding chondrogenic differentiation, investigating the effect of cartilage-tissue chon‐ droitin-sulfate (CS) in a fibrin scaffold on the differentiation of adipose-derived adult stem cells into chondrocytes revealed the significant effect of CS on the differentiation ef‐ ficiency. It can be concluded that the fibrin–CS matrices mimicking native cartilage ex‐ tracellular matrix could act as a three-dimensional scaffold for cartilage tissue engineering and have the potential for promoting the differentiation of adipose-derived

Since the chemical properties of substrates (e.g., hydrophobicity) play an important role in the kinetics of protein adsorption and folding, which in turn influence cellular activi‐ ties, direct the stem cells' fate can be controlled by chemical modification of the sub‐

in regulating the osteogenic differentiation of hMSCs [46].

adult stem cells into chondrocytes [47].

promotes chondrogenesis [40].

572 Pluripotent Stem Cells

Biomaterial scaffolds take on a variety of structures based on their material composition and processing for forming 3D environments. These materials consist of natural polymers such as collagen, hyaluronic acid, fibrin, alginate, or synthetic polymers such as polyethylene gly‐ col (PEG), dextran, or polyvinyl alcohol and can be formed into hydrogels, fibrous struc‐ tures, and microporous scaffolds [65,66]. Figure 1 illustrates examples of the structure of each of these scaffold types. The biomaterial structure controls how a cell interacts with the material and is important in stem cell fate decisions as the presentation of cues and cellular morphology are dependent on this structure.

One of the most widely used biomaterial structures for tissue engineering involves micropo‐ rous scaffolds, which can form interconnected porous networks that allow for cellular infil‐ tration and tissue formation. These scaffolds are often formed with leachable components around which the desired polymer forms a scaffold [84]. Upon removal of the leachable components, a 3D structure can be obtained with varying parameters such as pore size, po‐ rosity, and interconnectivity. Aronin et al. created poly-(*e*-caprolactone) scaffolds with var‐ ied pore sizes and interconnectivity to monitor osteogenesis of dura mater stem cells [85]. High porosity and adequate pore-size are key requisites to increase the surface area availa‐ ble for cell attachment and tissue in-growth in order to facilitate the uniform distribution of cells and the adequate transport of nutrients. Murphy et al. has investigated the effect of mean pore size on cell behavior in collagen–glycosaminoglycan scaffolds for bone tissue en‐ gineering application [86]. The results show that cell number was highest in scaffolds with the largest pore size of 325 μm. While the increased surface area provided by scaffolds with small pores may have a beneficial effect on initial cell adhesion but ultimately the improved cellular infiltration provided by scaffolds with larger pores outweighs this effect and sug‐ gests these scaffolds might be optimal for bone tissue repair. Kasten et al. also showed that porosity, distribution and size of the pores of beta-tricalcium phosphate ceramic scaffold can influence protein production and osteogenic differentiation of hMSCs [87]. Tayton et al. have compared the porous and non-porous versions of poly (DL-lactide) for potential clini‐ cal use as alternatives to allografts in impaction bone grafting [88]. The results showed that the skeletal stem cells differentiated along the osteoblastic lineage in porous samples com‐ pared to the non-porous versions. This feature may result from the fact that the 3D microarchitecture could distribute cellular binding sites in a variety of specific spatial locations rather than on only the single plane of rigid substrate, as in traditional two-dimensional 2D architecture of cell culture plastic or the surface of the non-porous polymers. Cells, there‐ fore, may have cytoskeletal adaptor proteins on a 3D matrix in addition to proteins present in 2D focal adhesions [89, 90]. Such differences in cell adhesion on the porous and non-po‐ rous polymers may therefore lead to different signal transduction and subsequent alteration

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 575

Natural ECM consists of various protein fibrils and fibers interwoven within a hydrated network of glycosaminoglycan chains [91]. The nano-scale structure of the ECM offers a natural network of intricate nano-fibers to support cells and present an instructive back‐ ground to guide their behavior [92-94]. Each nano-fiber provides the way for cells to form tissues as complex as bone, liver, heart, and kidney. Researchers try to fabricate fi‐ bers to mimic the natural ECM as a support for cell growth. The proliferation and osteo‐ genic differentiation of MSCs was investigated in 3D non-woven fabrics prepared from polyethylene terephthalate (PET) microfiber by Takahashi et al. They showed that the at‐ tachment, proliferation and bone differentiation of MSCs were influenced by the fiber di‐ ameter and porosity of non-woven fabrics in the scaffolds [95]. Several reports have demonstrated that nano-fibers are more favorable than micro-fibers, suggesting that cell activities can further be regulated by the size of the fiber [96-98] in terms of the biologi‐ cal response of chondrocytes, NSCs and endothelial cells cultured on nanofibrous and microfibrous scaffolds. Although the mechanisms by which a nano-fibrous scaffold acts

in cellular rearrangement.

Hydrogels are comprised of insoluble networks of cross-linked polymers with high wa‐ ter contents [67]. Hydrogels with the ability to encapsulate stem cells have been used for applications such as cartilage [68, 69] and cardiac [70, 71] tissue regeneration. In order to achieve tissue formation, stem cells must either be encapsulated within or recruited to the hydrogel. Some recently reported applications of hydrogel in tissue engineering are presented the following part.

Hydrogels such as those derived from alginate, collagen and hyaluronic acid have been found to be quite promising – they provide a homogeneous, structureless soft 3D environ‐ ment which is probably ideal for stem cell proliferation and maintenance, as well as for dif‐ ferentiation into softer tissues such as neural or hepatic [72, 73]. Pranga et al. showed the promotion of oriented axonal regrowth in the injured spinal cord by alginate-based aniso‐ tropic capillary hydrogels [74]. In a recent study, Nguyen et al. demonstrated that a threelayer polyethylene glycol-based hydrogel creates native-like articular cartilage with spatially-varying mechanical and biochemical properties that can direct a single MSC popu‐ lation to differentiate into the superficial, transitional, or deep zones of articular cartilage. They concluded that spatially-varying biomaterial compositions within single 3D scaffolds can stimulate efficient regeneration of multi-layered complex tissues from a single stem cell population. The ability to generate such zone-specific tissue could eventually allow tissueengineering of more native-like articular cartilage substitutes with spatially varying ECM composition and mechanical properties [75, 76]. Moreover, injectable hydrogels have been extensively explored as cell delivery systems with the advantage that cells and biomolecules can be readily integrated into the gelling matrix [77, 78]. The injectable nature of the hydro‐ gels provides the attractive feature of facile and homogenous cell distribution within any de‐ fect size or shape prior to gelation. In addition, injectable hydrogels allow good physical integration into the defect and facilitating the use of minimally invasive approaches for ma‐ terial delivery [79, 80]. Tan et al. demonstrated the usefulness of the aminated hyaluronic acid-g-poly (N isopropylacrylamide) copolymer as an injectable hydrogel for adipose tissue engineering [81]. Recently, Tan et al. demonstrated that the thermo-sensitive alginate-based injectable hydrogel has attractive properties that make it suitable as cell or pharmaceutical delivery vehicles for a variety of tissue engineering applications [82].

Although hydrogels provide a highly controlled 3D microenvironment for cells, the nature of this scaffold does not entirely mimic the structure of native ECM. Generally the cells en‐ counter and respond to basement membrane topography in the *in vivo* environment mainly composed of networks of pores, ridges, and fibers made by ECM molecules such as colla‐ gen, fibronectin and laminin at length scales ranging from nano- to micro-scale [83]. It is therefore important to incorporate features at such length scales into the development of bi‐ omaterials suitable for stem cell therapies.

One of the most widely used biomaterial structures for tissue engineering involves micropo‐ rous scaffolds, which can form interconnected porous networks that allow for cellular infil‐ tration and tissue formation. These scaffolds are often formed with leachable components around which the desired polymer forms a scaffold [84]. Upon removal of the leachable components, a 3D structure can be obtained with varying parameters such as pore size, po‐ rosity, and interconnectivity. Aronin et al. created poly-(*e*-caprolactone) scaffolds with var‐ ied pore sizes and interconnectivity to monitor osteogenesis of dura mater stem cells [85]. High porosity and adequate pore-size are key requisites to increase the surface area availa‐ ble for cell attachment and tissue in-growth in order to facilitate the uniform distribution of cells and the adequate transport of nutrients. Murphy et al. has investigated the effect of mean pore size on cell behavior in collagen–glycosaminoglycan scaffolds for bone tissue en‐ gineering application [86]. The results show that cell number was highest in scaffolds with the largest pore size of 325 μm. While the increased surface area provided by scaffolds with small pores may have a beneficial effect on initial cell adhesion but ultimately the improved cellular infiltration provided by scaffolds with larger pores outweighs this effect and sug‐ gests these scaffolds might be optimal for bone tissue repair. Kasten et al. also showed that porosity, distribution and size of the pores of beta-tricalcium phosphate ceramic scaffold can influence protein production and osteogenic differentiation of hMSCs [87]. Tayton et al. have compared the porous and non-porous versions of poly (DL-lactide) for potential clini‐ cal use as alternatives to allografts in impaction bone grafting [88]. The results showed that the skeletal stem cells differentiated along the osteoblastic lineage in porous samples com‐ pared to the non-porous versions. This feature may result from the fact that the 3D microarchitecture could distribute cellular binding sites in a variety of specific spatial locations rather than on only the single plane of rigid substrate, as in traditional two-dimensional 2D architecture of cell culture plastic or the surface of the non-porous polymers. Cells, there‐ fore, may have cytoskeletal adaptor proteins on a 3D matrix in addition to proteins present in 2D focal adhesions [89, 90]. Such differences in cell adhesion on the porous and non-po‐ rous polymers may therefore lead to different signal transduction and subsequent alteration in cellular rearrangement.

col (PEG), dextran, or polyvinyl alcohol and can be formed into hydrogels, fibrous struc‐ tures, and microporous scaffolds [65,66]. Figure 1 illustrates examples of the structure of each of these scaffold types. The biomaterial structure controls how a cell interacts with the material and is important in stem cell fate decisions as the presentation of cues and cellular

Hydrogels are comprised of insoluble networks of cross-linked polymers with high wa‐ ter contents [67]. Hydrogels with the ability to encapsulate stem cells have been used for applications such as cartilage [68, 69] and cardiac [70, 71] tissue regeneration. In order to achieve tissue formation, stem cells must either be encapsulated within or recruited to the hydrogel. Some recently reported applications of hydrogel in tissue engineering are

Hydrogels such as those derived from alginate, collagen and hyaluronic acid have been found to be quite promising – they provide a homogeneous, structureless soft 3D environ‐ ment which is probably ideal for stem cell proliferation and maintenance, as well as for dif‐ ferentiation into softer tissues such as neural or hepatic [72, 73]. Pranga et al. showed the promotion of oriented axonal regrowth in the injured spinal cord by alginate-based aniso‐ tropic capillary hydrogels [74]. In a recent study, Nguyen et al. demonstrated that a threelayer polyethylene glycol-based hydrogel creates native-like articular cartilage with spatially-varying mechanical and biochemical properties that can direct a single MSC popu‐ lation to differentiate into the superficial, transitional, or deep zones of articular cartilage. They concluded that spatially-varying biomaterial compositions within single 3D scaffolds can stimulate efficient regeneration of multi-layered complex tissues from a single stem cell population. The ability to generate such zone-specific tissue could eventually allow tissueengineering of more native-like articular cartilage substitutes with spatially varying ECM composition and mechanical properties [75, 76]. Moreover, injectable hydrogels have been extensively explored as cell delivery systems with the advantage that cells and biomolecules can be readily integrated into the gelling matrix [77, 78]. The injectable nature of the hydro‐ gels provides the attractive feature of facile and homogenous cell distribution within any de‐ fect size or shape prior to gelation. In addition, injectable hydrogels allow good physical integration into the defect and facilitating the use of minimally invasive approaches for ma‐ terial delivery [79, 80]. Tan et al. demonstrated the usefulness of the aminated hyaluronic acid-g-poly (N isopropylacrylamide) copolymer as an injectable hydrogel for adipose tissue engineering [81]. Recently, Tan et al. demonstrated that the thermo-sensitive alginate-based injectable hydrogel has attractive properties that make it suitable as cell or pharmaceutical

delivery vehicles for a variety of tissue engineering applications [82].

omaterials suitable for stem cell therapies.

Although hydrogels provide a highly controlled 3D microenvironment for cells, the nature of this scaffold does not entirely mimic the structure of native ECM. Generally the cells en‐ counter and respond to basement membrane topography in the *in vivo* environment mainly composed of networks of pores, ridges, and fibers made by ECM molecules such as colla‐ gen, fibronectin and laminin at length scales ranging from nano- to micro-scale [83]. It is therefore important to incorporate features at such length scales into the development of bi‐

morphology are dependent on this structure.

presented the following part.

574 Pluripotent Stem Cells

Natural ECM consists of various protein fibrils and fibers interwoven within a hydrated network of glycosaminoglycan chains [91]. The nano-scale structure of the ECM offers a natural network of intricate nano-fibers to support cells and present an instructive back‐ ground to guide their behavior [92-94]. Each nano-fiber provides the way for cells to form tissues as complex as bone, liver, heart, and kidney. Researchers try to fabricate fi‐ bers to mimic the natural ECM as a support for cell growth. The proliferation and osteo‐ genic differentiation of MSCs was investigated in 3D non-woven fabrics prepared from polyethylene terephthalate (PET) microfiber by Takahashi et al. They showed that the at‐ tachment, proliferation and bone differentiation of MSCs were influenced by the fiber di‐ ameter and porosity of non-woven fabrics in the scaffolds [95]. Several reports have demonstrated that nano-fibers are more favorable than micro-fibers, suggesting that cell activities can further be regulated by the size of the fiber [96-98] in terms of the biologi‐ cal response of chondrocytes, NSCs and endothelial cells cultured on nanofibrous and microfibrous scaffolds. Although the mechanisms by which a nano-fibrous scaffold acts as a selective substrate are not known yet, it is clear that the enhanced adsorption of cell adhesion matrix molecules enhances cell adhesion. Xin et al. also confirmed that PLGA nano-fibers accommodate the survival and proliferation of human MSCs. hMSCs, as well as hMSC-derived chondrogenic and osteogenic cells, apparently attach to PLGA nano-fi‐ bers, and yet assume different morphological features [99]. These results demonstrate the full support of multi-lineage differentiation of MSCs within nano-fibrous scaffolds and the feasibility of multi-phasic tissue engineering constructs using a single cell source, which is of particular relevance to the development of multi-phasic tissue constructs. However, there are very few in-depth studies on nano-fiber topographical effects on stem cell differentiation. Other nano-scaled topographical features such as steps, grooves, pillars and pits also modulate cell behavior, as reviewed elsewhere [100].

bone, and potentially brain, it can be hypothesized that the mechanical cues provided by the ECM are particularly instructive in lineage specification. The study carried out by Engler et al. revealed that matrix elasticity influences differentiation of hMSCs into osteo‐ genic, myogenic, and neurogenic cells [113]. Softer gels (0.1–1 kPa) were neurogenic, the hardest (24–40 kPa) were osteogenic, and the gels with intermediate elastic moduli (8–17 kPa) were myogenic. In all three cases, the elastic modulus matches that of the corre‐ sponding native tissue. It has recently been found out that substrate stiffness collaborates with soluble medium conditions to regulate the proliferation and differentiation of adult NSCs [114]. Cells exhibit optimum proliferation (in FGF-2) and optimum neuronal differ‐ entiation (in retinoic acid) at an intermediate stiffness that is characteristic of brain tis‐ sue. Furthermore, under conditions that induce nonspecific cell differentiation, stiff substrates support the differentiation of GFAP-expressing astrocytes, whereas soft sub‐ strates preferentially support the differentiation of β-tubulin III expressing neurons. This research demonstrates how the mechanical and biochemical properties of an adult NSCs microenvironment can be tuned to regulate the self-renewal and differentiation of adult NSCs. In another study, Leipzig et al. demonstrated that an optimal stiffness exists for both proliferation (3.5 kPa) as well as differentiation of neural stem/progenitor cell to

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 577

The study conducted by Banerjee et al. [116] provided insights into the influence of the me‐ chanical properties of 3D alginate hydrogel scaffolds on the proliferation and differentiation of NSCs, where varying the concentrations of alginate and calcium chloride provided facile control over the elastic modulus of the hydrogels. They demonstrated that the properties of the 3D scaffolds significantly impacted both the proliferation and the neuronal differentia‐ tion of encapsulated NSCs. In addition, they observed the greatest enhancement in expres‐ sion of the neuronal marker β-tubulin III within hydrogels having an elastic modulus comparable to that of brain tissues. They noted that the optimal value of the elastic modulus might depend on the stem cell type and the lineage to which differentiation is being direct‐ ed. Wang et al. reported an injectable hydrogel scaffold composed of gelatin-hydroxyphe‐ nylpropionic acid conjugate system with tunable stiffness for controlling the proliferation rate and differentiation of hMSCs in a 3D context in normal growth media. The rate of hMSC proliferation increased with the decrease in the stiffness of the hydrogel. Also, the neurogenesis of hMSCs was controlled by the hydrogel stiffness in a 3D context without the use of any additional biochemical signal. These cells which were cultured for 3 weeks in hy‐ drogels with lower stiffness expressed much more neuronal protein markers compared to those cultured in stiffer hydrogels for the same period of time [117]. In another study, lower cross-linked matrix of hydrogel system comprising hyaluronic acid-tyramine conjugates en‐ hanced chondrogenesis with increases in the percentage of cells with chondrocytic morphol‐ ogy, biosynthetic rates of glycosaminoglycan and type II collagen, and hyaline cartilage tissue formation. By increasing cross-linking degree and matrix stiffness, a shift in MSC dif‐ ferentiation toward fibrous phenotypes with the formation of fibrocartilage and fibrous tis‐ sues was observed [118]. In general, the ability to control stem cell fate – possibly without the use of chemical inducers – would be broadly useful for applications in regenerative

neurons (<1 kPa) [115].

medicine and tissue engineering [116].

Currently, there are three techniques available for the synthesis of nano-fibers: electrospin‐ ning, self-assembly, and phase separation. In particular, electrospinning technique is the most widely studied technique which has attracted wide attention due to its applicability for a variety of synthetic and natural polymers, exhibiting the most promising results for tissue engineering applications. Electrospinning is a spinning method to generate submicron to nanometer scale fibers from polymer melts or solutions. It is a physical process to obtain fi‐ bers from a bulk polymer of interest under the applied electric field. The most commonly used polymers for nano-fiber fabrication using electrospinning are the aliphatic polyesters [101]. There are several reports describing the potential of nanofibers fabricated by electro‐ spinning method for neural [102-104], bone [105-108] and cartilage [109, 110] tissue engi‐ neering which mimic the native tissue environment and support the cell adhesion, proliferation and differentiation.

Nano-fibers hold great promise as potential scaffolds owing to their high porosity and high surface area-to-volume ratio, which are favorable parameters for cell attachment, growth, and proliferation in addition to possessing favorable mechanical properties [111]. Further‐ more the effect of nano-fibers for stem cells' differentiation is promising further applications of nano-fibers for tissue engineering. Stem cells can be induced to differentiate into different cell types by growth/differentiation factors in the media, and we can incorporate such bio‐ molecules into the nano-fibers to direct differentiation to a desired cell type. The biomimetic morphology of nano-fibers with different patterns may also help to direct the stem cells' dif‐ ferentiation, which is particularly attractive given differentiation induction by some of me‐ dium supplements, although successful, is not physiologically relevant and offers the possibility for development of improved clinical prostheses with topographies that can di‐ rectly modulate stem cell fate.

#### **2.3. Mechanical cues**

Importantly, the various tissues of the body exhibit a range of matrix stiffness, and such differences in substrate stiffness have long been known to influence cell fate decisions in differentiated cell types [112]. An emerging area of study in stem cell biology and engi‐ neering is investigation of the role of these mechanical cues in stem cell fate decisions. Because MSCs can differentiate *in vitro* into cell types from tissues ranging from muscle, bone, and potentially brain, it can be hypothesized that the mechanical cues provided by the ECM are particularly instructive in lineage specification. The study carried out by Engler et al. revealed that matrix elasticity influences differentiation of hMSCs into osteo‐ genic, myogenic, and neurogenic cells [113]. Softer gels (0.1–1 kPa) were neurogenic, the hardest (24–40 kPa) were osteogenic, and the gels with intermediate elastic moduli (8–17 kPa) were myogenic. In all three cases, the elastic modulus matches that of the corre‐ sponding native tissue. It has recently been found out that substrate stiffness collaborates with soluble medium conditions to regulate the proliferation and differentiation of adult NSCs [114]. Cells exhibit optimum proliferation (in FGF-2) and optimum neuronal differ‐ entiation (in retinoic acid) at an intermediate stiffness that is characteristic of brain tis‐ sue. Furthermore, under conditions that induce nonspecific cell differentiation, stiff substrates support the differentiation of GFAP-expressing astrocytes, whereas soft sub‐ strates preferentially support the differentiation of β-tubulin III expressing neurons. This research demonstrates how the mechanical and biochemical properties of an adult NSCs microenvironment can be tuned to regulate the self-renewal and differentiation of adult NSCs. In another study, Leipzig et al. demonstrated that an optimal stiffness exists for both proliferation (3.5 kPa) as well as differentiation of neural stem/progenitor cell to neurons (<1 kPa) [115].

as a selective substrate are not known yet, it is clear that the enhanced adsorption of cell adhesion matrix molecules enhances cell adhesion. Xin et al. also confirmed that PLGA nano-fibers accommodate the survival and proliferation of human MSCs. hMSCs, as well as hMSC-derived chondrogenic and osteogenic cells, apparently attach to PLGA nano-fi‐ bers, and yet assume different morphological features [99]. These results demonstrate the full support of multi-lineage differentiation of MSCs within nano-fibrous scaffolds and the feasibility of multi-phasic tissue engineering constructs using a single cell source, which is of particular relevance to the development of multi-phasic tissue constructs. However, there are very few in-depth studies on nano-fiber topographical effects on stem cell differentiation. Other nano-scaled topographical features such as steps, grooves,

Currently, there are three techniques available for the synthesis of nano-fibers: electrospin‐ ning, self-assembly, and phase separation. In particular, electrospinning technique is the most widely studied technique which has attracted wide attention due to its applicability for a variety of synthetic and natural polymers, exhibiting the most promising results for tissue engineering applications. Electrospinning is a spinning method to generate submicron to nanometer scale fibers from polymer melts or solutions. It is a physical process to obtain fi‐ bers from a bulk polymer of interest under the applied electric field. The most commonly used polymers for nano-fiber fabrication using electrospinning are the aliphatic polyesters [101]. There are several reports describing the potential of nanofibers fabricated by electro‐ spinning method for neural [102-104], bone [105-108] and cartilage [109, 110] tissue engi‐ neering which mimic the native tissue environment and support the cell adhesion,

Nano-fibers hold great promise as potential scaffolds owing to their high porosity and high surface area-to-volume ratio, which are favorable parameters for cell attachment, growth, and proliferation in addition to possessing favorable mechanical properties [111]. Further‐ more the effect of nano-fibers for stem cells' differentiation is promising further applications of nano-fibers for tissue engineering. Stem cells can be induced to differentiate into different cell types by growth/differentiation factors in the media, and we can incorporate such bio‐ molecules into the nano-fibers to direct differentiation to a desired cell type. The biomimetic morphology of nano-fibers with different patterns may also help to direct the stem cells' dif‐ ferentiation, which is particularly attractive given differentiation induction by some of me‐ dium supplements, although successful, is not physiologically relevant and offers the possibility for development of improved clinical prostheses with topographies that can di‐

Importantly, the various tissues of the body exhibit a range of matrix stiffness, and such differences in substrate stiffness have long been known to influence cell fate decisions in differentiated cell types [112]. An emerging area of study in stem cell biology and engi‐ neering is investigation of the role of these mechanical cues in stem cell fate decisions. Because MSCs can differentiate *in vitro* into cell types from tissues ranging from muscle,

pillars and pits also modulate cell behavior, as reviewed elsewhere [100].

proliferation and differentiation.

576 Pluripotent Stem Cells

rectly modulate stem cell fate.

**2.3. Mechanical cues**

The study conducted by Banerjee et al. [116] provided insights into the influence of the me‐ chanical properties of 3D alginate hydrogel scaffolds on the proliferation and differentiation of NSCs, where varying the concentrations of alginate and calcium chloride provided facile control over the elastic modulus of the hydrogels. They demonstrated that the properties of the 3D scaffolds significantly impacted both the proliferation and the neuronal differentia‐ tion of encapsulated NSCs. In addition, they observed the greatest enhancement in expres‐ sion of the neuronal marker β-tubulin III within hydrogels having an elastic modulus comparable to that of brain tissues. They noted that the optimal value of the elastic modulus might depend on the stem cell type and the lineage to which differentiation is being direct‐ ed. Wang et al. reported an injectable hydrogel scaffold composed of gelatin-hydroxyphe‐ nylpropionic acid conjugate system with tunable stiffness for controlling the proliferation rate and differentiation of hMSCs in a 3D context in normal growth media. The rate of hMSC proliferation increased with the decrease in the stiffness of the hydrogel. Also, the neurogenesis of hMSCs was controlled by the hydrogel stiffness in a 3D context without the use of any additional biochemical signal. These cells which were cultured for 3 weeks in hy‐ drogels with lower stiffness expressed much more neuronal protein markers compared to those cultured in stiffer hydrogels for the same period of time [117]. In another study, lower cross-linked matrix of hydrogel system comprising hyaluronic acid-tyramine conjugates en‐ hanced chondrogenesis with increases in the percentage of cells with chondrocytic morphol‐ ogy, biosynthetic rates of glycosaminoglycan and type II collagen, and hyaline cartilage tissue formation. By increasing cross-linking degree and matrix stiffness, a shift in MSC dif‐ ferentiation toward fibrous phenotypes with the formation of fibrocartilage and fibrous tis‐ sues was observed [118]. In general, the ability to control stem cell fate – possibly without the use of chemical inducers – would be broadly useful for applications in regenerative medicine and tissue engineering [116].

Except mechanical properties of the matrix, the external mechanical stimulus can also in‐ duce stem cell differentiation. Bioreactors provide various active environments for stem cell growth under specific mechanical conditions. Flow perfusion culture of scaffold/cell con‐ structs has been witnessed to enhance the osteoblastic differentiation of rat MSCs over static culture in the presence of osteogenic supplements such as Dex. Although Dex is known to be a powerful induction agent of osteogenic differentiation in MSCs, Holtorf et al. showed that the mechanical shear force caused by fluid flow in a flow perfusion bioreactor would be sufficient to induce osteoblast differentiation in the absence of Dex [119]. Flow perfusion al‐ so accelerates the proliferation and differentiation of rat MSCs seeded on non-woven PLLA microfibrous scaffolds toward the osteoblastic phenotype, and improves the distribution of the calcified extracellular matrix generated *in vitro* [120]. Li et al. reported that MSCs are also mechano-sensitive and that Ca2+ may play a role in the signaling pathway since MSCs sub‐ jected to oscillatory fluid flow exhibited increased intracellular Ca2+ mobilization [121]. More recently, studies have shown that *shear stress* can induce *differentiation* of *stem cells* toward both endothelial and bone-producing cell phenotypes. The current data supporting the role of shear stress in stem cell fate and potential mechanisms and signaling cascades for trans‐ ducing shear stress into a biological signal are reviewed elsewhere [122].

astrocyte differentiation over neuronal differentiation in the 1 Hz cultures. In another study, Park et al. discovered the enhanced neuronal differentiation of hNSCs on graphene, which had a good electrical coupling with the differentiated neurons for electrical stimulation [126]. The application of an electrical stimulus causes fibroblasts to change cell shape and reorient in the 3D collagen scaffold perpendicularly to the direction of electrical stimulus, while the same electrical stimulus applied to MSCs induces much less significant reorienta‐ tion. A stimulus as strong as 10 V/cm is needed to induce a δV of 50 mV or greater, which would be sufficient to activate voltage-gated Ca2+ channels and regulate Ca2+-dependent sub-cellular processes, including cytoskeletal reorganization that is likely to cause changes in the cell morphology and reorientation signaling pathways [127]. It needs to be identified as to whether the differentiation of stem cell following adhesion will change under electrical stimulus. Endothelial progenitor cells and muscle precursor cells can also be stimulated by electromagnetic fields to promote myocyte differentiation [128,129]. Interestingly, electrical stimulation (10–40 V, 5 ms, 0.5 Hz pulses) of human embryonic fibroblasts was found to cause loss of cell proliferation and cell number but also led to differentiation of fibroblasts

Ultrasound has also been shown to induce differentiation. In low-intensity ultrasound field studies, MSCs differentiate towards a chondrocytic phenotype [131]. In one study, Abramo‐ vitch-Gottlib L et al. have illustrated that the use of low level laser irradiation (~0.5 mW/cm2

applied to a MSC/coralline construct stimulates the proliferation and differentiation of MSC into an osteoblastic phenotype during the initial culture period and significantly induced *in vitro* osteogenesis over time [132]. Thus, low level laser irradiation quickens the differentia‐ tion of MSC into an osteoblastic phenotype during bone formation processes in early culture

Numerous recent papers have sprouted showing how even minor experimental modifica‐ tions can change cell phenotype. Indeed, stem cells are so sensitive and unstable that even cell seeding density and seeding protocol have been observed to influence cell shape and

In the following part we will introduce some novel trends emerging in the field of tissue en‐ gineering in terms of both cellular biology (cell reprogramming) and biomaterial (multifac‐ torial design strategies) point of view in order to improve the overall efficiency of tissue

Though all somatic cells of the human body have the same genome structure, differences in chromatin organization and expression pattern of genes lead to the formation of various types of cells with different physiology, function and morphology [134,135]. Therefore, one could speculate that by changing chromatin structure and pattern of gene expression, all

**3. Some novel trends emerging in the field of tissue engineering**

)

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 579

into multinucleated myotube-like structures [130].

periods.

gene expression [133].

regeneration.

**3.1. Cell reprogramming**

In another study, it was shown that the cyclic compressive loading alone will induce chon‐ drogenic differentiation as effectively as the TGF-β alone or TGF-β plus loading in short term culture. Regarding MSCs angiogenesis, DNA microarray experiments [123] showed that uniaxial strain increased smooth muscle cell (SMC) markers. But cyclic equiaxial strain downregulated SM α-actin and SM-22α in MSCs on collagen- or elastin-coated membranes after 1 day, and decreased α-actin in stress fibers. This result suggests that uniaxial strain, which better mimics the type of mechanical strain experienced by SMCs, may promote MSCs differentiation into SMCs if cell orientation can be controlled. Solvig Diederichs et al. applied singular and repetitive cyclic strain of short- and long-time strains [124]. Additional‐ ly, a gradually increasing strain scheme commencing with short-time strain and continuing elongated strain periods was applied. Adipose tissue–derived MSCs on planar silicone and a three-dimensionally structured collagen I mesh were exposed to these strain regimes. The results revealed that even short-time strain can enhance osteogenic differentiation. Elonga‐ tion and repetition of strain, however, resulted in a decline of the observed short-time strain effects, which was interpreted as positively induced cellular adaptation to the mechanically active surroundings. With regard to cellular adaptation, the gradually increasing strain scheme was especially advantageous.

Taken together, these results suggest that the design of *ex vivo* stem cell culture systems should consider all types of mechanical cues in the microenvironment including matrix stiff‐ ness, compressive loading and shear stress as factors in guiding proper lineage specification.

#### **2.4. Electrical stimulus and other cues**

Several studies have recently shown the response of NSCs to electric fields. The studies re‐ ported by Matos et al. showed the response of murine NSCs encapsulated in alginate hydro‐ gel beads to alternating current electric fields [125]. They found an enhanced propensity for astrocyte differentiation over neuronal differentiation in the 1 Hz cultures. In another study, Park et al. discovered the enhanced neuronal differentiation of hNSCs on graphene, which had a good electrical coupling with the differentiated neurons for electrical stimulation [126]. The application of an electrical stimulus causes fibroblasts to change cell shape and reorient in the 3D collagen scaffold perpendicularly to the direction of electrical stimulus, while the same electrical stimulus applied to MSCs induces much less significant reorienta‐ tion. A stimulus as strong as 10 V/cm is needed to induce a δV of 50 mV or greater, which would be sufficient to activate voltage-gated Ca2+ channels and regulate Ca2+-dependent sub-cellular processes, including cytoskeletal reorganization that is likely to cause changes in the cell morphology and reorientation signaling pathways [127]. It needs to be identified as to whether the differentiation of stem cell following adhesion will change under electrical stimulus. Endothelial progenitor cells and muscle precursor cells can also be stimulated by electromagnetic fields to promote myocyte differentiation [128,129]. Interestingly, electrical stimulation (10–40 V, 5 ms, 0.5 Hz pulses) of human embryonic fibroblasts was found to cause loss of cell proliferation and cell number but also led to differentiation of fibroblasts into multinucleated myotube-like structures [130].

Ultrasound has also been shown to induce differentiation. In low-intensity ultrasound field studies, MSCs differentiate towards a chondrocytic phenotype [131]. In one study, Abramo‐ vitch-Gottlib L et al. have illustrated that the use of low level laser irradiation (~0.5 mW/cm2 ) applied to a MSC/coralline construct stimulates the proliferation and differentiation of MSC into an osteoblastic phenotype during the initial culture period and significantly induced *in vitro* osteogenesis over time [132]. Thus, low level laser irradiation quickens the differentia‐ tion of MSC into an osteoblastic phenotype during bone formation processes in early culture periods.

Numerous recent papers have sprouted showing how even minor experimental modifica‐ tions can change cell phenotype. Indeed, stem cells are so sensitive and unstable that even cell seeding density and seeding protocol have been observed to influence cell shape and gene expression [133].

## **3. Some novel trends emerging in the field of tissue engineering**

In the following part we will introduce some novel trends emerging in the field of tissue en‐ gineering in terms of both cellular biology (cell reprogramming) and biomaterial (multifac‐ torial design strategies) point of view in order to improve the overall efficiency of tissue regeneration.

#### **3.1. Cell reprogramming**

Except mechanical properties of the matrix, the external mechanical stimulus can also in‐ duce stem cell differentiation. Bioreactors provide various active environments for stem cell growth under specific mechanical conditions. Flow perfusion culture of scaffold/cell con‐ structs has been witnessed to enhance the osteoblastic differentiation of rat MSCs over static culture in the presence of osteogenic supplements such as Dex. Although Dex is known to be a powerful induction agent of osteogenic differentiation in MSCs, Holtorf et al. showed that the mechanical shear force caused by fluid flow in a flow perfusion bioreactor would be sufficient to induce osteoblast differentiation in the absence of Dex [119]. Flow perfusion al‐ so accelerates the proliferation and differentiation of rat MSCs seeded on non-woven PLLA microfibrous scaffolds toward the osteoblastic phenotype, and improves the distribution of the calcified extracellular matrix generated *in vitro* [120]. Li et al. reported that MSCs are also mechano-sensitive and that Ca2+ may play a role in the signaling pathway since MSCs sub‐ jected to oscillatory fluid flow exhibited increased intracellular Ca2+ mobilization [121]. More recently, studies have shown that *shear stress* can induce *differentiation* of *stem cells* toward both endothelial and bone-producing cell phenotypes. The current data supporting the role of shear stress in stem cell fate and potential mechanisms and signaling cascades for trans‐

In another study, it was shown that the cyclic compressive loading alone will induce chon‐ drogenic differentiation as effectively as the TGF-β alone or TGF-β plus loading in short term culture. Regarding MSCs angiogenesis, DNA microarray experiments [123] showed that uniaxial strain increased smooth muscle cell (SMC) markers. But cyclic equiaxial strain downregulated SM α-actin and SM-22α in MSCs on collagen- or elastin-coated membranes after 1 day, and decreased α-actin in stress fibers. This result suggests that uniaxial strain, which better mimics the type of mechanical strain experienced by SMCs, may promote MSCs differentiation into SMCs if cell orientation can be controlled. Solvig Diederichs et al. applied singular and repetitive cyclic strain of short- and long-time strains [124]. Additional‐ ly, a gradually increasing strain scheme commencing with short-time strain and continuing elongated strain periods was applied. Adipose tissue–derived MSCs on planar silicone and a three-dimensionally structured collagen I mesh were exposed to these strain regimes. The results revealed that even short-time strain can enhance osteogenic differentiation. Elonga‐ tion and repetition of strain, however, resulted in a decline of the observed short-time strain effects, which was interpreted as positively induced cellular adaptation to the mechanically active surroundings. With regard to cellular adaptation, the gradually increasing strain

Taken together, these results suggest that the design of *ex vivo* stem cell culture systems should consider all types of mechanical cues in the microenvironment including matrix stiff‐ ness, compressive loading and shear stress as factors in guiding proper lineage specification.

Several studies have recently shown the response of NSCs to electric fields. The studies re‐ ported by Matos et al. showed the response of murine NSCs encapsulated in alginate hydro‐ gel beads to alternating current electric fields [125]. They found an enhanced propensity for

ducing shear stress into a biological signal are reviewed elsewhere [122].

scheme was especially advantageous.

578 Pluripotent Stem Cells

**2.4. Electrical stimulus and other cues**

Though all somatic cells of the human body have the same genome structure, differences in chromatin organization and expression pattern of genes lead to the formation of various types of cells with different physiology, function and morphology [134,135]. Therefore, one could speculate that by changing chromatin structure and pattern of gene expression, all cells can be converted to other cell types [136]. The first cell reprogramming report has been presented in an earlier report [137] in which fibroblast cells converted into myocyte through the overexpression of MyoD gene. In a later study, the nucleus of the fibroblast cell has been transferred to the enucleated oocytes which finally led to the birth of Dolly sheep [135]. Ya‐ manaka (2006) shed some light on the biology underlying cell differentiation and cell fate by converting the mouse fibroblast to iPS cells in his study; one year later, Yamanaka and Thompson [138-140] reported the generation of human iPS cells from fibroblast cells.

cells is that all types of cells which are necessary for the regenerating of that specific tissue will be provided in the former approach. For instance, it has recently been well demonstrat‐ ed that convection of fibroblast cells to NSC is more promising than the conversion of the same cells to the neuron [149]. Moreover, adult stem cell generation through direct reprog‐ ramming has more capacity for self-renewal, which can be expanded and stored for differ‐ ent clinical applications. Tissue specific adult stem cells are natural stem cells of any tissue and match the normal homing tissue [149] and can respond to niche messages under both

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 581

Human body is a complex system that works with many regulatory and check points in co‐ ordination with many flexible programs. Using direct reprogramming, progression in regen‐ erative therapy will be possible if all demanding material such as adult stem cells, ES, iPS

In contrast to elements of living systems' ECM, the designed scaffolds are very poor in infor‐ mation, which make them suboptimal for many tissue engineering applications. These pas‐ sive biomaterials are unlikely to guide cell migration and differentiation or controlled matrix deposition, a problem that becomes even more evident in complex tissues with more than one cell type. Furthermore, they also cannot induce tissue neo-formation while pre‐ venting other undesirable tissue repair processes such as scarring; they are also unable to promote functional tissue integrations, such as vascular and/or nervous connectivity, in the host. Finally, these passive scaffolds largely lack the capacity to induce cell differentiation, thus resulting in a major limitation for their use together with current stem cell-based thera‐ pies [150]. A promising strategy to overcome these limitations is to consider the *multi-factori‐ al design strategies* by combining various external cues with one another for efficient and

While combining the structural and biological cues, a bioactive scaffold can be constructed in which biological functionality has been integrated to provide an information-rich support material for tissue engineering. Bioactive scaffolds are designed to control cell and tissue re‐ sponses, and to provide a more efficient integration with the host. Indeed, bioactive scaf‐ folds can also be prepared from synthetic materials by physical adsorption or chemical immobilization of biomolecules or oligopeptides on the scaffold surface, or by physical en‐ trapment of bioactive molecules alone or incorporated in a drug delivery system into the scaffold. These strategies can also be applied to enhance the bioactivity of scaffolds made

Engineered tissues need not only to remedy a defect and to integrate into a host tissue, but they also need to meet the demands of a constantly changing tissue. It was hypothesized that those tissues capable of growing with time could be engineered by supplying growth stimulus signals to cells from the biomaterials used for cell transplantation [151]. Smart drug

are well prepared in a suitable place and appropriate manner.

*3.2.1. Combining structural and biological cues for scaffold bioactivation*

stress and damage condition.

**3.2. Multifactorial design strategies**

controlled formation of complex tissues.

from ECM-native materials.

The possibility of directing lineage specific reprogramming of cells opens a window to a vast range of new possibilities in tissue engineering and regenerative medicines [141]. Here‐ in, generation of iPS cell lines is an important issue in the way to derive pluripotent cells from somatic cells. Instability of the genome, high cost of culture, lack of an efficient proto‐ col for differentiation as well as the presence of tumorigenic potential upon transplantation are among the main reasons for the slow progress of its clinical application [142].

Differentiation of stem cells into different types of tissue or organ is still a major limiting fac‐ tor in the area of tissue engineering mainly due to the complexity and multicellular struc‐ ture of the tissues and organs. To overcome such a limitation, it is highly demanded to have different types of cells for tissue engineering which is considered to be as important as mim‐ icking the physiological condition *in vivo*. Self-renewing and pluripotency are unique prop‐ erties of pluripotent stem cells that make the embryonic developmental process possible for the complex and integrated tissue-engineered systems. Accordingly, to make complex and integrated tissues, intrinsic developmental programs of inner cell mass of blastocysts such as those of post gastrulation events can be followed. Eiraku et al. [143] in a recent study man‐ aged to recreate the 3D structure of an organ for the first time in the world. They succeeded in growing a structure like the optic cup with the six cell types present in normal retina tis‐ sue. They mimicked aggregation and self-induction of mESCs as embryoid body and neuro‐ sphere formation to make optic cup that can be the source of retinal neurons like embryonic process of eye formation. For this, they used genetic engineered mES with tissue specific re‐ porter RX-venues DNA construct for capturing the early stages of optic cup-cell mass forma‐ tion and their separation for more maturation. Scientists hope to begin applying the same technologies used for retinal tissue to make 3D structure of other organs such as the brain, lung and kidney. However, despite advances like these, it is quick to note that we can deter‐ mine as to whether pluripotent stem cells can be used for regenerative therapy. The best idea is not always to uprise the cells to the tip of potency pyramid and then downrise it to a low level with differentiation, whereas one can directly convert one cell type to another [143]. It has been shown that the fibroblast cells can be converted to myocyte, neuron, hepa‐ tocyte, cardiomyocyte simply with direct reprogramming [137, 144-146]. This provides us good tools for having wide ranges of cells for regenerative medicines [147]. New approaches to cell reprogramming such as direct reprogramming of somatic cells to tissue-specific stem cells and conversion of fibroblast to neural stem cells have been proposed [148]. Providing three types of cells, namely astrocyte, oligodendrocyte and neuron, which are required in neural systems, is the advantage of cell reprogramming [148]. Another advantage of using direct reprogramming to tissue specific stem cells instead of reprogramming to full matured cells is that all types of cells which are necessary for the regenerating of that specific tissue will be provided in the former approach. For instance, it has recently been well demonstrat‐ ed that convection of fibroblast cells to NSC is more promising than the conversion of the same cells to the neuron [149]. Moreover, adult stem cell generation through direct reprog‐ ramming has more capacity for self-renewal, which can be expanded and stored for differ‐ ent clinical applications. Tissue specific adult stem cells are natural stem cells of any tissue and match the normal homing tissue [149] and can respond to niche messages under both stress and damage condition.

Human body is a complex system that works with many regulatory and check points in co‐ ordination with many flexible programs. Using direct reprogramming, progression in regen‐ erative therapy will be possible if all demanding material such as adult stem cells, ES, iPS are well prepared in a suitable place and appropriate manner.

#### **3.2. Multifactorial design strategies**

cells can be converted to other cell types [136]. The first cell reprogramming report has been presented in an earlier report [137] in which fibroblast cells converted into myocyte through the overexpression of MyoD gene. In a later study, the nucleus of the fibroblast cell has been transferred to the enucleated oocytes which finally led to the birth of Dolly sheep [135]. Ya‐ manaka (2006) shed some light on the biology underlying cell differentiation and cell fate by converting the mouse fibroblast to iPS cells in his study; one year later, Yamanaka and

The possibility of directing lineage specific reprogramming of cells opens a window to a vast range of new possibilities in tissue engineering and regenerative medicines [141]. Here‐ in, generation of iPS cell lines is an important issue in the way to derive pluripotent cells from somatic cells. Instability of the genome, high cost of culture, lack of an efficient proto‐ col for differentiation as well as the presence of tumorigenic potential upon transplantation

Differentiation of stem cells into different types of tissue or organ is still a major limiting fac‐ tor in the area of tissue engineering mainly due to the complexity and multicellular struc‐ ture of the tissues and organs. To overcome such a limitation, it is highly demanded to have different types of cells for tissue engineering which is considered to be as important as mim‐ icking the physiological condition *in vivo*. Self-renewing and pluripotency are unique prop‐ erties of pluripotent stem cells that make the embryonic developmental process possible for the complex and integrated tissue-engineered systems. Accordingly, to make complex and integrated tissues, intrinsic developmental programs of inner cell mass of blastocysts such as those of post gastrulation events can be followed. Eiraku et al. [143] in a recent study man‐ aged to recreate the 3D structure of an organ for the first time in the world. They succeeded in growing a structure like the optic cup with the six cell types present in normal retina tis‐ sue. They mimicked aggregation and self-induction of mESCs as embryoid body and neuro‐ sphere formation to make optic cup that can be the source of retinal neurons like embryonic process of eye formation. For this, they used genetic engineered mES with tissue specific re‐ porter RX-venues DNA construct for capturing the early stages of optic cup-cell mass forma‐ tion and their separation for more maturation. Scientists hope to begin applying the same technologies used for retinal tissue to make 3D structure of other organs such as the brain, lung and kidney. However, despite advances like these, it is quick to note that we can deter‐ mine as to whether pluripotent stem cells can be used for regenerative therapy. The best idea is not always to uprise the cells to the tip of potency pyramid and then downrise it to a low level with differentiation, whereas one can directly convert one cell type to another [143]. It has been shown that the fibroblast cells can be converted to myocyte, neuron, hepa‐ tocyte, cardiomyocyte simply with direct reprogramming [137, 144-146]. This provides us good tools for having wide ranges of cells for regenerative medicines [147]. New approaches to cell reprogramming such as direct reprogramming of somatic cells to tissue-specific stem cells and conversion of fibroblast to neural stem cells have been proposed [148]. Providing three types of cells, namely astrocyte, oligodendrocyte and neuron, which are required in neural systems, is the advantage of cell reprogramming [148]. Another advantage of using direct reprogramming to tissue specific stem cells instead of reprogramming to full matured

Thompson [138-140] reported the generation of human iPS cells from fibroblast cells.

580 Pluripotent Stem Cells

are among the main reasons for the slow progress of its clinical application [142].

In contrast to elements of living systems' ECM, the designed scaffolds are very poor in infor‐ mation, which make them suboptimal for many tissue engineering applications. These pas‐ sive biomaterials are unlikely to guide cell migration and differentiation or controlled matrix deposition, a problem that becomes even more evident in complex tissues with more than one cell type. Furthermore, they also cannot induce tissue neo-formation while pre‐ venting other undesirable tissue repair processes such as scarring; they are also unable to promote functional tissue integrations, such as vascular and/or nervous connectivity, in the host. Finally, these passive scaffolds largely lack the capacity to induce cell differentiation, thus resulting in a major limitation for their use together with current stem cell-based thera‐ pies [150]. A promising strategy to overcome these limitations is to consider the *multi-factori‐ al design strategies* by combining various external cues with one another for efficient and controlled formation of complex tissues.

#### *3.2.1. Combining structural and biological cues for scaffold bioactivation*

While combining the structural and biological cues, a bioactive scaffold can be constructed in which biological functionality has been integrated to provide an information-rich support material for tissue engineering. Bioactive scaffolds are designed to control cell and tissue re‐ sponses, and to provide a more efficient integration with the host. Indeed, bioactive scaf‐ folds can also be prepared from synthetic materials by physical adsorption or chemical immobilization of biomolecules or oligopeptides on the scaffold surface, or by physical en‐ trapment of bioactive molecules alone or incorporated in a drug delivery system into the scaffold. These strategies can also be applied to enhance the bioactivity of scaffolds made from ECM-native materials.

Engineered tissues need not only to remedy a defect and to integrate into a host tissue, but they also need to meet the demands of a constantly changing tissue. It was hypothesized that those tissues capable of growing with time could be engineered by supplying growth stimulus signals to cells from the biomaterials used for cell transplantation [151]. Smart drug delivery system is able to transmit multiple signals to the cells in a timely controlled release pattern. This release may be controlled through properties of the drug delivery system itself such as biodegradation-controlled release devices or stimulisensitive systems. Polymeric materials can be used as tissue-engineering *scaffolds and drug release* carriers, a strategy that has been mainly used for soluble signaling molecules such as growth factors. Cell recruit‐ ment and migration to the site of injury may be promoted through various signaling mole‐ cules. Many of these factors, e.g. TGF-βs, BMPs and IGF-1, are not only involved in cell attraction but also affect stem cell proliferation and differentiation [152-155].

which will then have improved cell substrate interactions. The orientation of neurites from chick embryonic dorsal root ganglia is enhanced on aligned blended polycaprolactone/colla‐ gen (PCL/collagen) (72:25) nano-fibers compared with that on aligned, pure PCL [163]. The migration and proliferation of Schwann cells is also significantly improved on aligned PCL/ collagen nano-fibers, indicating more specific biomolecular interactions between cells and

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 583

Instead of direct electrospinning the naturally derived polymers such as collagen together with synthetic polymers to provide biomemitic nano-fibrous scaffolds, one can immobilize some specific peptide motifs derived from ECM protein, which have been discerned to play an important role in tissue regeneration to the synthetic nano-fiber surface, which provides an alternative method to render the fibers bioactive. For instance, immobilization of mole‐ cules, such as specific peptide motifs derived from fibronectin and collagen VI, to the syn‐ thetic nano-fiber surface provides an alternative method to render the fibers bioactive. Therefore, surface immobilization of these small molecules that are neuroactive can provide a great advantage for neural tissue engineering. In addition, immobilized growth factors such as brain-derived neurotrophic factor [165] and basic fibroblast growth factor [166] can

*3.2.2. Combining structural and mechanical cues for engineering large-scale and/or complex tissues*

The successful replacement of large-scale defects using tissue-engineering approaches will likely require composite biomaterial scaffolds that have biomimetic structural and mechani‐ cal properties and can provide cell-instructive cues to control the growth and differentiation

The depth-dependent composition and structure of articular cartilage gives rise to its complex, non-homogeneous mechanical properties. Articular cartilage is generally composed of chon‐ drocytes and a dense ECM, which mainly includes type II collagen and proteoglycans [167]. Articular cartilage is structurally comprised of four different layers that can be distinguished from one another by collagen fiber alignment and proteoglycan composition. The depth-de‐ pendent alignment of collagen leads to important tensile and shear properties, whereas the depth-dependent proteoglycan content contributes more to the compressive properties of each zone [168, 169]. Nguyen et al. demonstrated in a recent study that layer-by-layer organization of specific biomaterial compositions creates 3D niches that allow a single MSC population to differentiate into zone-specific chondrocytes and organize into a complex tissue structure [75]. The results indicated that a three-layer polyethylene glycol (PEG)-based hydrogel with chon‐ droitin sulfate (CS) and matrix metalloproteinase-sensitive peptides (MMP-pep) incorporated into the top layer (superficial zone, PEG:CS:MMP-pep), CS incorporated into the middle layer (transitional zone, PEG:CS) and hyaluronic acid incorporated into the bottom layer (deep zone, PEG:HA) which ultimately created native-like articular cartilage with spatially-varying me‐ chanical and biochemical properties. They concluded that spatially-varying biomaterial com‐ positions within single 3D scaffolds can stimulate efficient regeneration of multi-layered

the collagen polymers on the nano-fiber surface [164].

also promote cell survival and neurite outgrowth.

complex tissues from a single stem cell population.

of embedded stem or progenitor cells.

Drug delivery strategies are designed to provide a platform for the localized delivery of the growth factors at the site of implantation. This is to protect the bioactivity of the molecule, to provide a controlled release pattern of the drug over a desired time frame, and *deliver angio‐ genic factors so as to promote angiogenesis*.

Two approaches have been mainly used for scaffold bioactivation: growth factors can be en‐ capsulated in a selected drug delivery system such as a microsphere or nanoparticle formu‐ lation, and these can be incorporated into the scaffolds. Otherwise, growth factors can be incorporated directly into the scaffold itself [156-158]. For example, IGF-1 has been directly incorporated into porous 3D silk fibroin scaffolds [159]. Silk scaffolds incorporating IGF-1 were able to preserve growth factor bioactivity, and prompted chondrogenic stimuli to seed‐ ed MSCs *in vitro*. By definition, implantation of growth factor-loaded scaffolds results in the localized delivery of the signaling molecule. Still, a certain fraction of the incorporated drug can reach the lymphatics or the circulation, and then distribute to non-target tissues. There‐ fore, even for these localized therapies, potential adverse effects of growth factor need to be carefully monitored.

Silk fibroin nano-fibrous scaffolds containing BMP-2 and*/*or nanoparticles of hydroxyapatite which were prepared via electrospinning were selected as matrix for *in vitro* bone formation from human bone marrow derived hMSCs. Li et al. [160] reported that silk fibroin nano-fibrous scaffolds with BMP-2 supported higher calcium deposition and enhanced transcript levels of bone-specific markers in comparison with controls without BMP-2, suggesting that nano-fi‐ brous electrospun silk scaffolds can be an efficient delivery system for BMP-2. The mild aque‐ ous process required for electrospinning, offers an important option for delivery of labile cytokines and other biomolecules. Lee et al. reported that calcium phosphate cement (CPC( combined with alginate solution to form a porous scaffold showed the capability to safe‐ ly load biological proteins (BSA and lysozyme) during preparation and to release them *in vitro* for over a month [161]. CPC–alginate scaffolds can further be developed into tissue engineered constructs which deliver biological molecules for bone regeneration stimulation.

In case of building biofunctionality into electrospun nano-fibers for neural tissue engineer‐ ing, the challenge to produce nano-fibers with more bioactive surfaces, significantly improv‐ ing specific targeting of cell substrate interactions and consequently creating a more biomimetic microenvironment for implanted cells remains. There are several methods, such as polymer blending and surface biofunctionalization, for improvement of nano-fibrous scaffolds bioactivity for nerve tissue engineering which are reviewed elsewhere [162]. It is possible to fabricate electrospun scaffolds from blends of synthetic and natural polymers, which will then have improved cell substrate interactions. The orientation of neurites from chick embryonic dorsal root ganglia is enhanced on aligned blended polycaprolactone/colla‐ gen (PCL/collagen) (72:25) nano-fibers compared with that on aligned, pure PCL [163]. The migration and proliferation of Schwann cells is also significantly improved on aligned PCL/ collagen nano-fibers, indicating more specific biomolecular interactions between cells and the collagen polymers on the nano-fiber surface [164].

delivery system is able to transmit multiple signals to the cells in a timely controlled release pattern. This release may be controlled through properties of the drug delivery system itself such as biodegradation-controlled release devices or stimulisensitive systems. Polymeric materials can be used as tissue-engineering *scaffolds and drug release* carriers, a strategy that has been mainly used for soluble signaling molecules such as growth factors. Cell recruit‐ ment and migration to the site of injury may be promoted through various signaling mole‐ cules. Many of these factors, e.g. TGF-βs, BMPs and IGF-1, are not only involved in cell

Drug delivery strategies are designed to provide a platform for the localized delivery of the growth factors at the site of implantation. This is to protect the bioactivity of the molecule, to provide a controlled release pattern of the drug over a desired time frame, and *deliver angio‐*

Two approaches have been mainly used for scaffold bioactivation: growth factors can be en‐ capsulated in a selected drug delivery system such as a microsphere or nanoparticle formu‐ lation, and these can be incorporated into the scaffolds. Otherwise, growth factors can be incorporated directly into the scaffold itself [156-158]. For example, IGF-1 has been directly incorporated into porous 3D silk fibroin scaffolds [159]. Silk scaffolds incorporating IGF-1 were able to preserve growth factor bioactivity, and prompted chondrogenic stimuli to seed‐ ed MSCs *in vitro*. By definition, implantation of growth factor-loaded scaffolds results in the localized delivery of the signaling molecule. Still, a certain fraction of the incorporated drug can reach the lymphatics or the circulation, and then distribute to non-target tissues. There‐ fore, even for these localized therapies, potential adverse effects of growth factor need to be

Silk fibroin nano-fibrous scaffolds containing BMP-2 and*/*or nanoparticles of hydroxyapatite which were prepared via electrospinning were selected as matrix for *in vitro* bone formation from human bone marrow derived hMSCs. Li et al. [160] reported that silk fibroin nano-fibrous scaffolds with BMP-2 supported higher calcium deposition and enhanced transcript levels of bone-specific markers in comparison with controls without BMP-2, suggesting that nano-fi‐ brous electrospun silk scaffolds can be an efficient delivery system for BMP-2. The mild aque‐ ous process required for electrospinning, offers an important option for delivery of labile cytokines and other biomolecules. Lee et al. reported that calcium phosphate cement (CPC( combined with alginate solution to form a porous scaffold showed the capability to safe‐ ly load biological proteins (BSA and lysozyme) during preparation and to release them *in vitro* for over a month [161]. CPC–alginate scaffolds can further be developed into tissue engineered

constructs which deliver biological molecules for bone regeneration stimulation.

In case of building biofunctionality into electrospun nano-fibers for neural tissue engineer‐ ing, the challenge to produce nano-fibers with more bioactive surfaces, significantly improv‐ ing specific targeting of cell substrate interactions and consequently creating a more biomimetic microenvironment for implanted cells remains. There are several methods, such as polymer blending and surface biofunctionalization, for improvement of nano-fibrous scaffolds bioactivity for nerve tissue engineering which are reviewed elsewhere [162]. It is possible to fabricate electrospun scaffolds from blends of synthetic and natural polymers,

attraction but also affect stem cell proliferation and differentiation [152-155].

*genic factors so as to promote angiogenesis*.

carefully monitored.

582 Pluripotent Stem Cells

Instead of direct electrospinning the naturally derived polymers such as collagen together with synthetic polymers to provide biomemitic nano-fibrous scaffolds, one can immobilize some specific peptide motifs derived from ECM protein, which have been discerned to play an important role in tissue regeneration to the synthetic nano-fiber surface, which provides an alternative method to render the fibers bioactive. For instance, immobilization of mole‐ cules, such as specific peptide motifs derived from fibronectin and collagen VI, to the syn‐ thetic nano-fiber surface provides an alternative method to render the fibers bioactive. Therefore, surface immobilization of these small molecules that are neuroactive can provide a great advantage for neural tissue engineering. In addition, immobilized growth factors such as brain-derived neurotrophic factor [165] and basic fibroblast growth factor [166] can also promote cell survival and neurite outgrowth.

#### *3.2.2. Combining structural and mechanical cues for engineering large-scale and/or complex tissues*

The successful replacement of large-scale defects using tissue-engineering approaches will likely require composite biomaterial scaffolds that have biomimetic structural and mechani‐ cal properties and can provide cell-instructive cues to control the growth and differentiation of embedded stem or progenitor cells.

The depth-dependent composition and structure of articular cartilage gives rise to its complex, non-homogeneous mechanical properties. Articular cartilage is generally composed of chon‐ drocytes and a dense ECM, which mainly includes type II collagen and proteoglycans [167]. Articular cartilage is structurally comprised of four different layers that can be distinguished from one another by collagen fiber alignment and proteoglycan composition. The depth-de‐ pendent alignment of collagen leads to important tensile and shear properties, whereas the depth-dependent proteoglycan content contributes more to the compressive properties of each zone [168, 169]. Nguyen et al. demonstrated in a recent study that layer-by-layer organization of specific biomaterial compositions creates 3D niches that allow a single MSC population to differentiate into zone-specific chondrocytes and organize into a complex tissue structure [75]. The results indicated that a three-layer polyethylene glycol (PEG)-based hydrogel with chon‐ droitin sulfate (CS) and matrix metalloproteinase-sensitive peptides (MMP-pep) incorporated into the top layer (superficial zone, PEG:CS:MMP-pep), CS incorporated into the middle layer (transitional zone, PEG:CS) and hyaluronic acid incorporated into the bottom layer (deep zone, PEG:HA) which ultimately created native-like articular cartilage with spatially-varying me‐ chanical and biochemical properties. They concluded that spatially-varying biomaterial com‐ positions within single 3D scaffolds can stimulate efficient regeneration of multi-layered complex tissues from a single stem cell population.

In another study, the potency of scaffold stiffness and topology in driving cardiac stem cell differentiation in a 3D culture context was confirmed by Forte et al. [170]. Cardiac stem cells adopted the cardiomyocytic phenotype only when cultured in strictly controlled conditions characterized by a critical combination of chemical, biochemical, structural and mechanical factors, and emulation of the inner myocardial environment. In these studies, the mimicry of myocardial environment was achieved by fine-tuning the array of growth factors dissolved in the culture medium and the chemistry, topology and stiffness of three-dimensional sup‐ ports on which stem cells were seeded. Scaffold stiffness was modulated in this study by changing the topology of the structure using a rapid prototyping technique. The optimal stiffness to induce cardiomyocyte differentiation was around 300 kPa on the scaffolds with square pores of about 150 μm.

elasticity of hydrogel sheets, most of the cells on the hydrogel surfaces with high elastici‐ ty (E=100 kPa, CS=4.5% w/v) didn't fully expand on the hydrogel surface, while the cells on the hydrogel surfaces with low and intermediate elasticity (E=15 kPa, CS=1.5 % w/v; E=25 kPa, CS=3% w/v) had more spindle shape (data not presented). Gelatin concentra‐ tion was fixed (18% w/v) for all the samples. The greatest proliferation of the cells was found on the hydrogels with intermediate elasticity (25 kPa) and the number of cells in‐ creased over time during the 7-day culture (Figure2). Hydrogel blends with lower or higher gelatin concentration showed significantly lower attached cell numbers (data not presented). Recent studies have illustrated the profound *dependence of cellular behavior* on *the stiffness* of 2D hydrogel sheets. Boontheekul et al. demonstrated that alginate gel with higher mechanical strength (increasing from 13 kPa to 45 kPa) increased myoblast adhe‐ sion, proliferation, and differentiation in a 2D cell culture model [182]. They also showed that primary mouse myoblasts were more highly responsive to this cue than the C2C12

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 585

An innovative approach has recently been described by Gilbert et al. as well. Using a bi‐ oengineered substrate in conjunction with a highly automated single-cell tracking algo‐ rithm, the authors showed that substrate elasticity is a potent regulator of muscle stem cells' fate in culture. In fact, muscle stem cells cultured on soft hydrogel substrates that mimic the elasticity of muscle self-renew *in vitro*, and contribute extensively to muscle regeneration when subsequently transplanted into mice. This study has provided novel evidence showing that recapitulating physiological tissue rigidity allows the propagation

In the current study, the authors investigated the behavior of MDSCs and SMCs cul‐ tured on the prepared hydrogel surfaces. The results indicate that increasing the hydro‐ gel mechanical strength from E=15 kPa to E=25 kPa, increases MDSCs adhesion and proliferation. The authors further found that MDSCs were more responsive to mechani‐ cal properties of the hydrogel sheets compared to SMCs, due to their higher ability and relatively smaller size (Data not presented). In contrast, for engineering central nervous system tissue, Leipzig et al. demonstrated that gels with lower mechanical properties of methacrylamide chitosan hydrogel sheet (E ≤3.5 kPa) were more appropriate for neural stem progenitor cell differentiation and proliferation [115]. As mentioned above, mechan‐ ical properties of hydrogel can regulate the cell adhesion, proliferation, and differentia‐ tion. However, the response and sensitivity to this variable is highly dependent on the cell source. In the current work, MDSCs exhibited maximal proliferation on hydrogel surface with 25 kPa elasticity. The same hydrogel sheet showed also the best handling qualities for surgery, with elasticity in the range of elastic modulus for muscle tissues [184], showing its potential for being used in muscle tissue engineering applications.

The strategy applied in the current study provides an opportunity to independently control mechanical and bioadhesive properties of the hydrogels so as to probe stem cell behavior. By changing both material mechanical and biochemical properties of the hydrogel blend, we could find the optimum condition for MDSCs attachment and proliferation in contact with

myoblast cell line.

of adult muscle stem cells [183].

CS-G hydrogel sheets.

## **4. 2D Polysaccharide-based hydrogel scaffolds for muscle tissue engineering**

Hydrogels have been used for a variety of biomedical applications [171-175], and because of their viscoelastic characteristics [176], similarities with ECM, excellent biological perform‐ ance, inherent cellular interaction capability [177], ability to allow transfer of gases and nu‐ trients [177], and their amiability of fabrication into specific shapes, they have recently been explored as scaffolding materials for tissue engineering applications [178-180]. On the other hand, in the recent decade, researchers realized that the mechanical properties of the used hydrogel material had to be adapted to the elastic properties of the damaged tissue [181]. Hydrogels such as alginate, chitosan, collagen and hyaluronic acid, which are derived from natural polymers, have been proved to be quite promising for stem cell proliferation, main‐ tenance and differentiation for tissue engineering applications.

The authors of this paper tried to prepare hydrogels made of natural polymers (chitosan (CS) and gelatin (G)) with proper handling for surgery, and with mechanical properties similar to those of muscle tissues as well as good cell adhesion properties. In the current study, we investigated the effect of CS and G concentration in blend scaffolds on me‐ chanical properties of the CS-G hydrogel sheets as well as the seeded muscle-derived stem cells (MDSCs) and smooth muscle cells' (SMCs) behavior on the CS-G hydrogel sheets. MDSCs and SMCs were isolated, expanded in culture and characterized with re‐ spect to the expression of surface markers with flow cytometry analysis. After crosslink‐ ing of CS and G, the CS-G blend hydrogel sheets were prepared by a casting method and used for 2D cell culture.

While the elasticity of the CS-G hydrogel sheets increased by increasing the CS concen‐ tration, the gelatin concentration did not have any notable effect on the hydrogel me‐ chanical properties.

The MDSCs attachment on the surface with elastic modulus of 25 kPa stiffness and pro‐ liferation on different CS-G hydrogel sheet surfaces having varying modulus of elasticity is shown in Figure 2. The cell observation result on day 1 showed that by increasing the elasticity of hydrogel sheets, most of the cells on the hydrogel surfaces with high elastici‐ ty (E=100 kPa, CS=4.5% w/v) didn't fully expand on the hydrogel surface, while the cells on the hydrogel surfaces with low and intermediate elasticity (E=15 kPa, CS=1.5 % w/v; E=25 kPa, CS=3% w/v) had more spindle shape (data not presented). Gelatin concentra‐ tion was fixed (18% w/v) for all the samples. The greatest proliferation of the cells was found on the hydrogels with intermediate elasticity (25 kPa) and the number of cells in‐ creased over time during the 7-day culture (Figure2). Hydrogel blends with lower or higher gelatin concentration showed significantly lower attached cell numbers (data not presented). Recent studies have illustrated the profound *dependence of cellular behavior* on *the stiffness* of 2D hydrogel sheets. Boontheekul et al. demonstrated that alginate gel with higher mechanical strength (increasing from 13 kPa to 45 kPa) increased myoblast adhe‐ sion, proliferation, and differentiation in a 2D cell culture model [182]. They also showed that primary mouse myoblasts were more highly responsive to this cue than the C2C12 myoblast cell line.

In another study, the potency of scaffold stiffness and topology in driving cardiac stem cell differentiation in a 3D culture context was confirmed by Forte et al. [170]. Cardiac stem cells adopted the cardiomyocytic phenotype only when cultured in strictly controlled conditions characterized by a critical combination of chemical, biochemical, structural and mechanical factors, and emulation of the inner myocardial environment. In these studies, the mimicry of myocardial environment was achieved by fine-tuning the array of growth factors dissolved in the culture medium and the chemistry, topology and stiffness of three-dimensional sup‐ ports on which stem cells were seeded. Scaffold stiffness was modulated in this study by changing the topology of the structure using a rapid prototyping technique. The optimal stiffness to induce cardiomyocyte differentiation was around 300 kPa on the scaffolds with

**4. 2D Polysaccharide-based hydrogel scaffolds for muscle tissue**

tenance and differentiation for tissue engineering applications.

Hydrogels have been used for a variety of biomedical applications [171-175], and because of their viscoelastic characteristics [176], similarities with ECM, excellent biological perform‐ ance, inherent cellular interaction capability [177], ability to allow transfer of gases and nu‐ trients [177], and their amiability of fabrication into specific shapes, they have recently been explored as scaffolding materials for tissue engineering applications [178-180]. On the other hand, in the recent decade, researchers realized that the mechanical properties of the used hydrogel material had to be adapted to the elastic properties of the damaged tissue [181]. Hydrogels such as alginate, chitosan, collagen and hyaluronic acid, which are derived from natural polymers, have been proved to be quite promising for stem cell proliferation, main‐

The authors of this paper tried to prepare hydrogels made of natural polymers (chitosan (CS) and gelatin (G)) with proper handling for surgery, and with mechanical properties similar to those of muscle tissues as well as good cell adhesion properties. In the current study, we investigated the effect of CS and G concentration in blend scaffolds on me‐ chanical properties of the CS-G hydrogel sheets as well as the seeded muscle-derived stem cells (MDSCs) and smooth muscle cells' (SMCs) behavior on the CS-G hydrogel sheets. MDSCs and SMCs were isolated, expanded in culture and characterized with re‐ spect to the expression of surface markers with flow cytometry analysis. After crosslink‐ ing of CS and G, the CS-G blend hydrogel sheets were prepared by a casting method

While the elasticity of the CS-G hydrogel sheets increased by increasing the CS concen‐ tration, the gelatin concentration did not have any notable effect on the hydrogel me‐

The MDSCs attachment on the surface with elastic modulus of 25 kPa stiffness and pro‐ liferation on different CS-G hydrogel sheet surfaces having varying modulus of elasticity is shown in Figure 2. The cell observation result on day 1 showed that by increasing the

square pores of about 150 μm.

and used for 2D cell culture.

chanical properties.

**engineering**

584 Pluripotent Stem Cells

An innovative approach has recently been described by Gilbert et al. as well. Using a bi‐ oengineered substrate in conjunction with a highly automated single-cell tracking algo‐ rithm, the authors showed that substrate elasticity is a potent regulator of muscle stem cells' fate in culture. In fact, muscle stem cells cultured on soft hydrogel substrates that mimic the elasticity of muscle self-renew *in vitro*, and contribute extensively to muscle regeneration when subsequently transplanted into mice. This study has provided novel evidence showing that recapitulating physiological tissue rigidity allows the propagation of adult muscle stem cells [183].

In the current study, the authors investigated the behavior of MDSCs and SMCs cul‐ tured on the prepared hydrogel surfaces. The results indicate that increasing the hydro‐ gel mechanical strength from E=15 kPa to E=25 kPa, increases MDSCs adhesion and proliferation. The authors further found that MDSCs were more responsive to mechani‐ cal properties of the hydrogel sheets compared to SMCs, due to their higher ability and relatively smaller size (Data not presented). In contrast, for engineering central nervous system tissue, Leipzig et al. demonstrated that gels with lower mechanical properties of methacrylamide chitosan hydrogel sheet (E ≤3.5 kPa) were more appropriate for neural stem progenitor cell differentiation and proliferation [115]. As mentioned above, mechan‐ ical properties of hydrogel can regulate the cell adhesion, proliferation, and differentia‐ tion. However, the response and sensitivity to this variable is highly dependent on the cell source. In the current work, MDSCs exhibited maximal proliferation on hydrogel surface with 25 kPa elasticity. The same hydrogel sheet showed also the best handling qualities for surgery, with elasticity in the range of elastic modulus for muscle tissues [184], showing its potential for being used in muscle tissue engineering applications.

The strategy applied in the current study provides an opportunity to independently control mechanical and bioadhesive properties of the hydrogels so as to probe stem cell behavior. By changing both material mechanical and biochemical properties of the hydrogel blend, we could find the optimum condition for MDSCs attachment and proliferation in contact with CS-G hydrogel sheets.

Overall, this chapter provides an overview of recent progresses made by application of nov‐ el engineering strategies that have been developed to emulate the stem cell niche for effec‐ tively controlling the cell fate and translating the stem cell research into much needed

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 587

Future directions in tissue engineering will involve elucidation of molecular mechanisms by which all types of external cues influence stem cells' behavior, followed by translation of these scientific data to clinical applications. Further advances in controlling stem cell fate can be achieved by combining the above mentioned parameters in a more scalable and com‐ binatorial manner to address the complexity of the natural stem cell niche. To this end, col‐ laborative efforts between cell biologists and materials scientists are critical for answering the key biological questions and promoting interdisciplinary stem-cell researches in the di‐

and Ali Fallah2,3

1 Department of Chemical & Petroleum Engineering, Sharif University of Technology,

2 Molecular Medicine Group, Faculty of Medicine, Shahid Beheshti University of Medical

[1] Griffith LG, Naughton G. Tissue engineering-current challenges and expanding op‐

[2] Khademhosseini A, Langer R, Borenstein J, Vacanti JP. Microscale technologies for tissue engineering and biology. Proc. Natl. Acad. Sci. USA 2006;103:2480-2487.

[3] Zandstra PW, Nagy A. Stem cell bioengineering. Annu. Rev. Biomed. Eng.

[4] Oh S, Brammer KS, Li JYS, Teng D, Engler AJ, Chien S, Jin S. Stem cell fate dictated solely by altered nanotube dimension. Proc. Natl. Acad. Sci. USA 2009;106:2130-2135.

[5] Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage

clinical applications in the not-too-distant future.

Shohreh Mashayekhan1\*, Maryam Hajiabbas1

3 Maad Systems Biomedicine, Tehran, Iran

\*Address all correspondence to: mashayekhan@sharif.edu

portunities. Science 2002;295(5557):1009-1014.

specification. Cell 2006;126:677-689.

rection of clinical relevance.

**Author details**

Tehran, Iran

**References**

Sciences, Tehran, Iran

2001;3;275-305.

**Figure 2.** MDSCs adhesion and proliferation on CS-G hydrogel surfaces. Photomicrographs of MDSCs attachment on the surface with intermediate elasticity (25kPa, CS=3 % w/v) at: (A) day 1, (B) day 7 and (C) cell proliferation on CS-G hydrogel surfaces with different mechanical strength. CS-G hydrogel sheets prepared at different chitosan concentra‐ tion (4.5, 3 & 1.5 % w/v) with constant gelatin (18 %w/v). MDSCs were seeded onto all hydrogel surfaces at the densi‐ ty of 7500cells/cm2.

#### **5. Conclusion and outlook for the future**

In tissue engineering, directing the cells to differentiate at the right time, in the right place, and into the right phenotype, requires an environment providing the same factors that govern cellular processes *in vivo*. The current chapter described various biomaterials and external cues designing considerations mimicking the natural stem cell microenvir‐ onment in order to direct the desired stem cell fate, facilitating the regeneration of de‐ sired tissues. In addition we introduced our approach to designing a 2D polysaccharidebased hydrogel scaffolds as a potential and suitable biomaterial for muscle tissue engineering applications.

Overall, this chapter provides an overview of recent progresses made by application of nov‐ el engineering strategies that have been developed to emulate the stem cell niche for effec‐ tively controlling the cell fate and translating the stem cell research into much needed clinical applications in the not-too-distant future.

Future directions in tissue engineering will involve elucidation of molecular mechanisms by which all types of external cues influence stem cells' behavior, followed by translation of these scientific data to clinical applications. Further advances in controlling stem cell fate can be achieved by combining the above mentioned parameters in a more scalable and com‐ binatorial manner to address the complexity of the natural stem cell niche. To this end, col‐ laborative efforts between cell biologists and materials scientists are critical for answering the key biological questions and promoting interdisciplinary stem-cell researches in the di‐ rection of clinical relevance.

## **Author details**

Shohreh Mashayekhan1\*, Maryam Hajiabbas1 and Ali Fallah2,3

\*Address all correspondence to: mashayekhan@sharif.edu

1 Department of Chemical & Petroleum Engineering, Sharif University of Technology, Tehran, Iran

2 Molecular Medicine Group, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

3 Maad Systems Biomedicine, Tehran, Iran

## **References**

**Figure 2.** MDSCs adhesion and proliferation on CS-G hydrogel surfaces. Photomicrographs of MDSCs attachment on the surface with intermediate elasticity (25kPa, CS=3 % w/v) at: (A) day 1, (B) day 7 and (C) cell proliferation on CS-G hydrogel surfaces with different mechanical strength. CS-G hydrogel sheets prepared at different chitosan concentra‐ tion (4.5, 3 & 1.5 % w/v) with constant gelatin (18 %w/v). MDSCs were seeded onto all hydrogel surfaces at the densi‐

In tissue engineering, directing the cells to differentiate at the right time, in the right place, and into the right phenotype, requires an environment providing the same factors that govern cellular processes *in vivo*. The current chapter described various biomaterials and external cues designing considerations mimicking the natural stem cell microenvir‐ onment in order to direct the desired stem cell fate, facilitating the regeneration of de‐ sired tissues. In addition we introduced our approach to designing a 2D polysaccharidebased hydrogel scaffolds as a potential and suitable biomaterial for muscle tissue

ty of 7500cells/cm2.

586 Pluripotent Stem Cells

engineering applications.

**5. Conclusion and outlook for the future**


[6] Kilian KA, Bugarija B, Lahn BT, Mrksich M. Geometric cues for directing the differ‐ entiation of mesenchymal stem cells. Proc. Natl. Acad. Sci.USA 2010;107:4872-7487.

[22] Peerani R, Zandstra PW. Enabling stem cell therapies through synthetic stem cell-ni‐

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 589

[23] Nardo PD, Minieri M, Ahluwalia A. Engineering the Stem Cell Niche and the Differ‐ entiative Micro- and Macroenvironment: Technologies and Tools for Applying Bio‐ chemical, Physical and Structural Stimuli and Their Effects on Stem Cells. Stem Cell

[24] Liao S, Chan CK, Ramakrishna S. Stem cells and biomimetic materials strategies for tissue engineering, Materials Science and Engineering C 2008;28(8):1189–1202.

[25] Lowry WE, Richter L. Signaling in adult stem cells. Front Biosci. 2007;12:3911–3927.

[26] Molofsky AV, Pardal R, Morrison SJ. Diverse mechanisms regulate stem cell self-re‐

[27] Rider CC. Heparin/heparan sulphate binding in the TGF-beta cytokine superfamily.

[28] Li Y, Zhang H, Litingtung Y, Chiang C. Cholesterol modification restricts the spread of Shh gradient in the limb bud. Proc Natl Acad Sci U S A 2006;103:6548–6553.

[29] Saha K, Schaffer DV. Signal dynamics in Sonic hedgehog tissue patterning. Develop‐

[30] Kuhl PR, Griffith-Cima LG. Tethered epidermal growth factor as a paradigm for growth factor-induced stimulation from the solid phase. Nat Med 1996;2:1022–1027.

[31] Reddy CC, Niyogi SK, Wells A, Wiley HS, Lauffenburger DA. Engineering epider‐ mal growth factor for enhanced mitogenic potency. Nat Biotechnol 1996;14:1696–

[32] Fan VH, Tamama K, Au A, Littrell R, Richardson LB, Wright JW, Wells A, Griffith LG. Tethered epidermal growth factor provides a survival advantage to mesenchy‐

[33] Sakiyama-Elbert SE, Hubbell JA. Development of fibrin derivatives for controlled re‐ lease of heparin binding growth factors. J Control Release 2000;65:389–402.

[34] Willerth SM, Rader AR, Sakiyama-Elbert SE. The Effect of Controlled Growth Factor Delivery on Embryonic Stem Cell Differentiation Inside of Fibrin Scaffolds. Stem Cell

[35] Ho JE, Chung EH, Wall S, Schaffer DV, Healy KE. Immobilized sonic hedgehog Nterminal signaling domain enhances differentiation of bone marrow-derived mesen‐

[37] Mrksich M. What can surface chemistry do for cell biology? Curr. Opin. Chem. Biol.

[36] Anselme K. Osteoblast adhesion on biomaterials. Biomaterials 2000;21(7):667-81.

chymal stem cells. J Biomed Mater Res A 2007;83:1200–1208.

cheengineering. J Clin Invest 2010;120:60–70.

newal. Curr Op in Cell Biol 2004;16:700–707.

mal stem cells. Stem Cells 2007;25:1241–1251.

Biochem Soc Trans 2006;34:458–460.

Engineering 2011;41-59.

ment 2006;133:889–900.

Res. 2008;1(3):205-218.

2002;6: 794.


[22] Peerani R, Zandstra PW. Enabling stem cell therapies through synthetic stem cell-ni‐ cheengineering. J Clin Invest 2010;120:60–70.

[6] Kilian KA, Bugarija B, Lahn BT, Mrksich M. Geometric cues for directing the differ‐ entiation of mesenchymal stem cells. Proc. Natl. Acad. Sci.USA 2010;107:4872-7487.

[7] Solanki A, Shah S, Memoli KA, Park SY, Hong S, Lee K-B. Controlling differentiation of neural stem cells using extracellular matrix protein patterns. Small

[8] Moore KA, Lemischka IR. Stem cells and their niches. Science 2006;311:1880-1885.

[9] Place ES, Evans ND, Stevens MM. Complexity in biomaterials for tissue engineering.

[10] Stevens MM, George JH. Exploring and engineering the cell surface interface. Science

[11] Gefen A, Gefen N, Zhu Q, Raghupathi R, Margulies SS. Age-dependent changes in material properties of the brain and braincase of the rat. J. Neurotrauma

[12] Engler AJ, Griffin MA, Sen S, Bönnemann CG, Sweeney HL, Discher DE. Myotubes differentiate optimally on substrates with tissue-like stiffness: Pathological implica‐

[13] Mitchell GF, Parise H, Benjamin EJ, Larson MG, Keyes MJ, Vita JA, Vasan RS, Levy D. Changes in arterial stiffness and wave reflection with advancing age in healthy men and women: The Framingham Heart Study. Hypertension 2004;43:1239-1245.

[14] Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human

[15] Ding S, Schultz PG. A role for chemistry in stem cell biology. Nat. Biotechnol.

[16] Hwang NS, Varghese S, Elisseeff J. Controlled differentiation of stem cells. Adv.

[17] Park TH, Shuler ML. Integration of cell culture and microfabrication technology. Bio‐

[18] Bettinger CJ, Langer R, Borenstein JT. Engineering substrate topography at the mi‐ cro- and nanoscale to control cell function. Angew. Chem. Int. Ed. 2009;48:5406-5415.

[19] Pollard TD, Earnshaw WC, Lippincott-Schwartz J. Cell Biology. 2nd ed. Philadel‐

[20] Lutolf MP, Doyonnas R, Havenstrite K, Koleckar K, Blau HM. Perturbation of single‐ hematopoietic stem cell fates in artificial niches. Integr Biol (Camb) 2009;1:59–69.

[21] Dickinson LE, Kusuma S, Gerecht S. Reconstructing the differentiation niche ofem‐

bryonic stem cells using biomaterials. Macromol Biosci 2011;1:36–49.

tions for soft or stiff microenvironments. J. Cell Biol. 2004;166:877-887.

mesenchymal stem cells. Science 1999;284:143-147.

Drug Delivery Rev. 2008;60:199-214.

technol. Prog. 2003;19:243-253.

phia,PA,USA: Elsevier Inc.; 2008.

2010;6:2509-2513.

588 Pluripotent Stem Cells

2005;310: 1135-8.

2003;20:1163-1177.

2004;22:833-840.

Nat. Mater. 2009;8:457-470.


[38] Hersel U, Dahmen C, Kessler H. RGD modified polymers: biomaterials for stimulat‐ ed cell adhesion and beyond. Biomaterials 2003;24(24):4385-415.

[50] Hsu SH, Chen WC. Improved cell adhesion by plasma-induced grafting of l-lactide

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 591

[51] Hegemann D, Brunner H, Oehr C, Instrum N. Plasma treatment of polymers for sur‐ face and adhesion improvement.Methods Phys. Res. B Beam Interact. Mater. Atoms

[52] Park H, Lee KY, Lee SJ, Park KE, Park WH. Plasma-treated poly (lacticco-glycolic

[53] Baek HS, Park YH, Ki CS, Park JC, Rah DK. Enhanced chondrogenic responses of ar‐ ticular chondrocytes onto porous silk fibroin scaffolds treated with microwave-in‐

[54] Curran JM, Chen R, Hunt JA. The guidance of human mesenchymal stem cell differ‐ entiation in vitro by controlled modifications to the cell substrate. Biomaterials

[55] Bianchi F, Vozzi G, Pescia C, Domenici C, Ahluwalia A. A comparative study of chemical derivatisation methods for spatially differentiated cell adhesion on 2-di‐ mensional microfabricated polymeric matrices. J Biomater Sci Polymer Edn.

[56] Liu X, Lim JY, Donahue HJ, Dhurjati R, Mastro AM, Vogler EM. Influence of substra‐ tum surface chemistry/energy and topography on the human fetal osteoblastic cell line hFOB 1.19: phenotypic and genotypic responses observed in vitro. Biomaterials

[57] Csete M. Oxygen in the cultivation of stem cells. Ann NY Acad Sci. 2005;1049:1–8.

[58] Studet L, Csete M, Lee SH. Enhanced proliferation, survival, and dopaminergic dif‐ ferentiation of CNS precursors in lowered oxygen. J Neurosci. 2000;20:7377–7383.

[59] Moussavi-Harami F, Duwayri Y, Martin JA, Moussavi-Harami F, Buckwalter JA. Oxygen effects on senescence in chondrocytes and mesenchymal stem cells: conse‐

[60] Grayson WL, Zhao F, Izadpanah R, Bunnell B, Ma T. Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs. J Cell Physiol. 2006

[61] Fink T, Abildtrup L, Fogd K, Abdallah BM, Kassem M, Ebbesen P, Zachara V. Induc‐ tion of adipocyte-like phenotype in human mesenchymal stem cells by hypoxia.

[62] Csete M, Walkikonis J, Slawany N, Wei Y, Korsnes S, Doyle JC, Wold B. Oxygenmediated regulation of skeletal muscle satellite cell proliferation and adipogenesis in

quences for tissue engineering. Iowa Orthop J. 2004;24:15–20.

acid) nanofibers for tissue engineering.Macromol. Res. 2007;15:238-243.

duced argon plasma.Surf. Coat. Technol. 2008;202:5794-5797.

onto polyurethane surface.Biomaterials 2000;21:359-367.

2003;208:281-286.

2006;27(27):4783–4793.

2003;14:1077–1096.

2007;28:4535–4550.

May;207(2):331–339.

Stem Cells. 2004; 22:1346–1355.

culture. J Cell Physiol. 2001;189:189–196.


[50] Hsu SH, Chen WC. Improved cell adhesion by plasma-induced grafting of l-lactide onto polyurethane surface.Biomaterials 2000;21:359-367.

[38] Hersel U, Dahmen C, Kessler H. RGD modified polymers: biomaterials for stimulat‐

[39] Roeker S, Böhm S, Diederichs S, Bode F, Quade A, Korzhikov V, van Griensven M, Tennikova TB, Kasper C. A study on the influence of biocompatible composites with bioactive ligands toward their effect on cell adhesion and growth for the application in bone tissue engineering. J Biomed Mater Res B Appl Biomater. 2009;91(1):153-62.

[40] Liu SQ, Tian Q, Wang L, Hedrick JL, Hui JHP, Yang YY, Ee PLR. Injectable Biode‐ gradable Poly(ethylene glycol)/ RGD Peptide Hybrid Hydrogels for in vitro Chon‐ drogenesis of Human Mesenchymal Stem Cells. Macromolecular Rapid

[41] Mauney JR, Kirker-Head C, Abrahamson L, Gronowicz G, Volloch V, Kaplan DL. Matrix-mediated retention of in vitro osteogenic differentiation potential and in vivo bone-forming capacity by human adult bone marrow-derived mesenchymal stem

[42] Mwale F, Wang HT, Nelea V, Li L, John A, Wertheimer MR. The effect of glow dis‐ charge plasma surface modification of polymers on osteogenic differentiation of committed human mesenchymal stem cell. Biomaterials 2006;27(10):2258-2264.

[43] Ager MJ, Feser T, Denck H, Krauspe R. Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells Cultured onto Three Different Polymers In Vitro. Annals

[44] Wang YZ, Singh A, Xu P, Pindrus MA, Blasioli DJ, Kaplan DL. Expansion and osteo‐ genic differentiation of bone marrow-derived mesenchymal stem cells on a vitamin C

[45] Xu C, Wang Y, Yu X, Chen X, Li X, Yang X, Li S, Zhan X, Xiang AP. Evaluation of human mesenchymal stem cells response to biomimetic bioglass-collagen-hyaluronic acid-phosphatidylserine composite scaffolds for bone tissue engineering. Materials

[46] Rim NG, Lee JH, Jeong SI, Lee BK, Kim CH, Shin H. Modulation of Osteogenic Dif‐ ferentiation of Human Mesenchymal Stem Cells by Poly[(Llactide)-co-(e caprolac‐

[47] Wei Y, Hu Y, Hao W, Han Y, Meng G, Zhang D, Wu Z, Wang H. A Novel Injectable Scaffold for Cartilage Tissue Engineering Using Adipose-Derived Adult Stem Cells.

[48] Wan Y, Qu X, Lu J, Zhu C, Wan L, Yang J. Characterization of surface property of poly(lactide-co-glycolide) after oxygen plasma treatment. Biomaterials

[49] Favia P, d'Agostino R. Plasma treatments and plasma deposition of polymers for bio‐

medical applications. Surf. Coat. Technol. 1998;98:1102-1106.

tone)]/Gelatin Nanofibers. Macromolecular Bioscience 2009;9(8):795–804.

cells during ex vivo expansion. J Biomed Mater Res A. 2006;79(3):464-75.

ed cell adhesion and beyond. Biomaterials 2003;24(24):4385-415.

Communications 2010;31(13): 1148-54

590 Pluripotent Stem Cells

of Biomedical Engineering 2005;33(10):1319-1332.

Research Part A 2009;88(1):264-73.

Orthopaedic Research 2008;26(1):27–33.

2004;25:4777-4783.

functionalized polymer. Biomaterials 2006; 27(17) 3265-3273.


[63] Lennon DP, Edminson JM, Caplan AI. Cultivation of rat marrow-derived mesenchy‐ mal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochon‐ drogenesis. J Cell Physiol. 2001;187:345–355.

[75] Nguyen LH, Kudva AK, Saxena NS, Roy K. Engineering articular cartilage with spa‐ tially-varying matrix composition and mechanical properties from a single stem cell

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 593

[76] Nguyen LH, Kudva AK, Guckert NL, Linse KD, Roy K. Unique biomaterial composi‐ tions direct bone marrow stem cells into specific chondrocytic phenotypes corre‐ sponding to the various zones of articular cartilage. Biomaterials 2011;32:1327-1338.

[77] Nuttelman CR, Rice MA, Rydholm AE, Salinas CN, Shah DN, Anseth KS. Macromo‐ lecular monomers for the synthesis of hydrogel niches and their application in cell

[78] Hou QP, De Bank PA, Shakesheff KM. Injectable scaffolds for tissue regeneration. J

[79] Pratt AB, Weber FE, Schmoekel HG, Müller R, Hubbell JA. Synthetic extracellular

[80] Hennink WE, van Nostrum CF. Novel crosslinking methods to design hydrogels.

[81] Tan H, Ramirez CM, Miljkovic N, Li H, Rubin JP, Marra KG. Thermosensitive inject‐ able hyaluronic acid hydrogel for adipose tissue engineering. Biomaterials

[82] Tana R, She Zh, Wang M, Fang Zh, Liu Y, Feng Q. Thermo-sensitive alginate-based injectable hydrogel for tissue engineering. Carbohydrate Polymers 2012;87:1515–

[83] Stevens MM, George JH. Exploring and engineering the cell surface interface. Sci‐

[84] Liu X, Ma P, Ann. Polymeric scaffolds for bone tissue engineering. Biomed. Eng.

[85] Petrie Aronin CE, Cooper JA, Sefcik LS, Tholpady SS, Ogle RC, Botchwey EA. Osteo‐ genic differentiation of dura mater stem cells cultured in vitro on three-dimensional porous scaffolds of poly(ε-caprolactone) fabricated via co-extrusion and gas foaming.

[86] Murphy CM, Haugh MG, O'Brien FJ. The effect of mean pore size on cell attachment, proliferation and migration in collagen–glycosaminoglycan scaffolds for bone tissue

[87] Kasten P, Beyen I, Niemeyer P, Luginbühl R, Bohner M, Richter W. Porosity and pore size of b-tricalcium phosphate scaffold can influence protein production and os‐ teogenic differentiation of human mesenchymal stem cells: An in vitro and in vivo

[88] Tayton E, Purcell M, Aarvold A, Smith JO, Kalra S, Briscoe A, Shakesheff K, Howdle SM, Dunlop DG, Oreffo ROC. Supercritical CO2 fluid-foaming of polymers to in‐

encapsulation and tissue engineering. Prog Polym Sci 2008;33:167–79.

matrices for in situ tissue engineering. Biotechnol Bioeng 2004;86:27–36.

Mater Chem 2004;14:1915–23.

2009;30:6844–6853.

ence2005;310: 1135-1138.

Acta Biomater. 2008;4:1187-1197.

engineering. Biomaterials 2010;31(3):461–6.

study. Acta Biomater. 2008;4(6):1904-15.

2004;32:477-86.

1521.

Adv Drug Delivery Rev 2002;54:13–36.

population using a multi-layered hydrogel. Biomaterials 2011;32:6946-6952.


[75] Nguyen LH, Kudva AK, Saxena NS, Roy K. Engineering articular cartilage with spa‐ tially-varying matrix composition and mechanical properties from a single stem cell population using a multi-layered hydrogel. Biomaterials 2011;32:6946-6952.

[63] Lennon DP, Edminson JM, Caplan AI. Cultivation of rat marrow-derived mesenchy‐ mal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochon‐

[64] Buckley CT, Vinardell T, Kelly DJ. Oxygen tension differentially regulates the func‐ tional properties of cartilaginous tissues engineered from infrapatellar fat pad de‐ rived MSCs and articular chondrocytes. Osteoarthritis and Cartilage 2010;18(10):

[65] Burdick JA, Vunjak-Novakovic G. Engineered Microenvironments for Controlled

[66] Ifkovits JL, Burdick JA. Photopolymerizable and Degradable Biomaterials for Tissue

[67] Lutolf M, Hubbell JA. Synthetic biomaterials as instructive extracellular microenvir‐ onments for morphogenesis in tissue engineering. Nature Biotechnology 2005;23(1):

[68] Chung C, Burdick JA. Influence of three-dimensional hyaluronic acid microenviron‐ ments on mesenchymal stem cell chondrogenesis. Tissue Engineering. Part A

[69] Xu JA, Wang W, Ludeman M, Cheng K, Hayami T, Lotz JA, Kapila S. Chondrogenic differentiation of human mesenchymal stem cells in three-dimensional alginate gels.

[70] Kraehenbuehl TP, Zammaretti P, Van der Vlies AJA, Schoenmakers RG, Lutolf MP, Jaconi ME, Hubbell JA. Three-dimensional extracellular matrix-directed cardioproge‐ nitor differentiation: systematic modulation of a synthetic cell-responsive PEG-hy‐

[71] Yeo Y, Geng W, Ito T, Kohane D, Burdick JA, Radisic M. A photocrosslinkable hydro‐ gel for myocyte cell culture and injection. J. Biomed. Mater. Res. 2007;81B:312-322.

[72] Baharvand H, Hashemi SM, Ashtiani SM, Farrokhi A. Differentiation of human em‐ bryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro. Int J Dev

[73] Brännvall K, Bergman K, Wallenquist U, Svahn S, Bowden T, Hilborn J, Forsberg-Nilsson K. Enhanced neuronal differentiation in a three-dimensional collagen–hya‐

[74] Pranga P, Müller R, Eljaouharib A, Heckmannb K, Kunzb W,Weberc T, Faberc C, Vroemena M, Bogdahna U, Weidner N. Thepromotion of oriented axonal regrowth in the injured spinal cord by alginate-based anisotropic capillary hydrogels. Biomate‐

luronan matrix. J Neurosci Res. 2007 Aug 1;85(10):2138–2146.

Stem Cell Differentiation. Tissue Eng. Part A 2008;15(2):205-219.

Engineering Applications. Tissue Eng. 2007;13:2369-2385.

drogenesis. J Cell Physiol. 2001;187:345–355.

1345-54.

592 Pluripotent Stem Cells

47-55.

2009;15:243-254.

Biol. 2006;50:645–652.

rials 2006;27:3560–3569.

Tissue Eng. Part A 2008;14:667-680.

drogel. Biomaterials 2008;29(18):2757-2766.


crease porosity: A method to improve the mechanical and biocompatibility charac‐ teristics for use as a potential alternative to allografts in impaction bone grafting? Acta Biomaterialia 2012;8:1918–1927.

[102] Eberli D. Tissue Engineering.Bio-nanotechnology Approaches to Neural Tissue Engi‐

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 595

[103] Yang F, Xu CY, Kotaki M, Wang S, Ramakrishna S. Characterization of neural stem cells on electrospun poly(L-lactic acid) nanofibrous scaffold. J. Biomater. Sci. Polym.

[104] Christopherson GT, Song H, Mao H-Q. The influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation. Biomaterials

[105] Zilberman M. Active implants and scaffolds for tissue regeneration(Studies in Me‐

[106] Shin M, Yoshimoto H, Vacanti JP. In vivo bone tissue engineering using mesenchy‐ mal stem cells on a novel electrospun nanofibrous scaffold. Tissue Eng. 2004;10(1–2):

[107] Yoshimoto H, Shin YM, Terai H, Vacanti JP. A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering. Biomaterials

[108] Li WJ, Tuli R, Huang X, Laquerriere P, Tuan RS. Multilineage differentiation of hu‐ man mesenchymal stem cells in a three-dimensional nanofibrous scaffold. Biomateri‐

[109] Li W-J, Tuli R, Okafor C, Derfoul A, Danielson KG, Hall DJ, Tuan RS. A three-dimen‐ sional nanofibrous scaffold for cartilage tissue engineering using human mesenchy‐

[110] Xin X, Hussain M, Mao JJ. Continuing differentiation of human mesenchymal stem cells and induced chondrogenic and osteogenic lineages in electrsopun PLGA nano‐

[111] Li W-J, Laurencin CT, Caterson EJ, Tuan RS, Ko FK, Electrospun nanofibrous struc‐ ture: A novel scaffold for tissue engineering. J. Biomed. Mater. Res. 2002;60(4):613–

[112] Discher DE, JanmeyP, WangYL. Tissue cells feel and respond to the stiffness of their

[113] Engler AJ, Sen S, Sweeney HL, DischerDE. Matrix elasticity directs stem cell lineage

[114] Saha K, Keung AJ, Irwin EF, Li Y, Little L, Schaffer DV, Healy KE.Substrate modulus

[115] Leipzig ND, Shoichet MS. The effect of substrate stiffness on adult neural stem cell

directs neural stem cell behaviour. Biophys J. 2008;95(9):4426-4438.

chanobiology, Tissue Engineering and Biomaterials). Springer; 2011.

neering. Published by In-The; 2010,Chap-23.

Ed. 2004; 15(12):1483–1497.

2009;30(4):556–564.

2003;24(12):2077–2082.

als 2005;26(25):5158–5166.

mal stem cells. Biomaterials 2005;26:599–609.

fiber scaffold. Biomaterials 2007;28:316–325.

substrate. Science 2005;310:1139–1143.

specification. Cell 2006;126:677–689.

behaviour. Biomaterials 2009;30:6867–6878.

33–41.


[102] Eberli D. Tissue Engineering.Bio-nanotechnology Approaches to Neural Tissue Engi‐ neering. Published by In-The; 2010,Chap-23.

crease porosity: A method to improve the mechanical and biocompatibility charac‐ teristics for use as a potential alternative to allografts in impaction bone grafting?

[89] Chang Z, Meyer K, Rapraeger AC, Friedl A. Differential ability of heparan sulfate proteoglycans to assemble the fibroblast growth factor receptor complex in situ. FA‐

[90] Cukierman E, Pankov R, Stevens DR, Yamada KM. Taking cell-matrix adhesions to

[91] Lutolf MP, Hubbell JA. Synthetic biomaterials as instructive extracellular microenvir‐ onments for morphogenesis in tissue engineering. Nature biotechnology 2005;23(1):

[92] Stevens MM, George JH. Exploring and Engineering the Cell Surface Interface. Sci‐

[93] Griffith LG, Naughton G. Tissue Engineering--Current Challenges and Expanding

[94] Langer R, Tirrell DA. Designing materials for biology and medicine. Nature

[95] Takahashi Y, Tabata Y. Effect of the fiber diameter and porosity of non-woven PET fabrics on the osteogenic differentiation of mesenchymal stem cells. Journal of Bio‐

[96] Li WJ, Jiang YJ, Tuan RS. Chondrocyte phenotype in engineered fibrous matrix is

[97] Yang F, Murugan R, Wang S, Ramakrishna S. Electrospinning of nano/micro scale poly (L-lactic acid) aligned fibers and their potential in neural tissue engineering. Bi‐

[98] Xu CY, Yang F, Wang S, Ramakrishna S. in vitro study of human vascular endothe‐ lial cell function on materials with various surface roughness. Journal of Biomedical

[99] Xin XJ, Hussain M, Mao JJ. Continuing differentiation of human mesenchymal stem cells and induced chondrogenic and osteogenic lineages in electrospun PLGA nano‐

[100] Liao S, Chan CK, Ramakrishna S. Stem cells and biomimetic materials strategies for tissue engineering. Materials Science and Engineering C 2008;28:1189–1202.

[101] Boland ED, Wnek GE, Simpson DG, Pawlowski KJ, Bowlin GL, Tailoring tissue engi‐ neering scaffolds using electrostatic processing techniques: A study of poly(glycolic acid) electrospinning. J. Macromol. Sci. A, Pure Appl. Chem., 2001;38(12):1231–1243.

regulated by fiber size. Tissue Engineering 2006;12(7):1775-1785.

Acta Biomaterialia 2012;8:1918–1927.

the third dimension. Science 2001;294:1708–12.

Opportunities. Science 2002;295:1009-1014.

materials Science Polymer Ed 2004;15(1):41-57.

Materials Research Part A 2004;71A: 154-161.

fiber scaffold. Biomaterials 2007;28(2):316-325.

SEB J 2000;14:137–44.

ence 2005;310:1135-1138.

omaterials 2005;26:2603-2610.

2004;428:487-92.

47-55.

594 Pluripotent Stem Cells


[116] Banerjee A, Arha M, Choudhary S, Ashton RS, Bhatia SR, Schaffer DV, Kane RS. The influence of hydrogel modulus on the proliferation and differentiation of encapsulat‐ ed neural stem cells. Biomaterials 2009;30:4695–4699.

[129] Serena E, Flaibani M, Carnio S, Boldrin L, Vitiello L, De Coppi P, Elvassore N. Elec‐ trophysiologic stimulation improves myogenic potential of muscle precursor cells

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 597

[130] Genovese JA, Spadaccio C, Langer J, Habe J, Jackson J, Patel AN. Electrostimulation induces cardiomyocyte predifferentiation of fibroblasts. Biochem Biophys Res Com‐

[131] Ebisawa K, Hata K, Okada K, Kimata K, Ueda M, Torii S, Watanabe H. Ultrasound enhances transforming growth factor beta-mediated chondrocyte differentiation of

[132] Abramovitch-Gottlib L, Naveh TGD, Geresh S, Rosenwaks S, Bar I, Vago R. Low lev‐ el laser irradiation stimulates osteogenic phenotype of mesenchymal stem cells seed‐

[133] McBride SH, Knothe Tate ML. Modulation of stem cell shape and fate A: the role of density and seeding protocol on nucleus shape and gene expression. Tissue Eng Part

[134] Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH. Viable offspring derived

[135] MacArthur BD, Ma'ayan A, Lemischka IR. Systems biology of stem cell fate and cel‐ lular reprogramming. Nature Reviews Molecular Cell Biology 2009;10: 672-681.

[136] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.

[137] Choi J, Costa ML, Mermelstein CS, Chagas C, Holtzer S, Holtzer H. MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes.

[138] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryon‐ ic and adult fibroblast cultures bydefined factors. Cell 2006;126(4):663-76.

[139] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.

[140] Yu J, Vodyanik MA, Smuqa-Otta K, Antosiewica-Bourqet J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. Science 2007;318(5858):1917–

[141] Blanpain C, Daley GQ, Hochedlinger K, Passegué E, Rossant J, Yamanaka S. Stem

cells assessed. Nature Reviews Molecular Cell Biology 2012;13:471-476

from fetal and adult mammalian cells. Nature 1997;385(6619): 810–3.

grown in a 3D collagen scaffold. Neurol Res. 2008;30(2):207–214.

human mesenchymal stem cells. Tissue Eng. 2004; 10(5–6):921–929.

ed on a three-dimensional biomatrix. Lasers in Medical Science

mun. 2008;370(3):450–455.

A. 2008;14(9):1561–1572.

Cell 2007;131(5):861-872.

Cell 2007;131(5):861-72.

1920.

Proc Natl Acad Sci USA 1990;87(20):7988–7992.


[129] Serena E, Flaibani M, Carnio S, Boldrin L, Vitiello L, De Coppi P, Elvassore N. Elec‐ trophysiologic stimulation improves myogenic potential of muscle precursor cells grown in a 3D collagen scaffold. Neurol Res. 2008;30(2):207–214.

[116] Banerjee A, Arha M, Choudhary S, Ashton RS, Bhatia SR, Schaffer DV, Kane RS. The influence of hydrogel modulus on the proliferation and differentiation of encapsulat‐

[117] Wang LS, Chung JE, Chan PP, Kurisawa M. Injectable biodegradable hydrogels with tunable mechanical properties for the stimulation of neurogenesic differentiation of

human mesenchymal stem cells in 3D culture. Biomaterials 2010;31(6):1148-57. [118] Toh WS, Lim TCh, Kurisawa M, Spector M. Modulation of mesenchymal stem cell chondrogenesis in a tunable hyaluronic acid hydrogel microenvironment. Biomateri‐

[119] Holtorf HL, Jansen JA, Mikos AG. Flow perfusion culture induces the osteoblastic differentiation of marrow stromal cell-scaffold constructs in the absence of dexame‐

[120] Sikavitsas VI, Bancroft GN, Lemoine JJ, Liebschner MAK, Dauner M, Mikos AG. Flow perfusion enhances the calcified matrix deposition of marrow stromal cells in biodegradable nonwoven fiber mesh scaffolds. Annals of Biomedical Engineering

[121] Li YJ, Batra NN, You LD, Meier SC, Coe IA, Yellowley CE, Jacobs CR. Oscillatory flu‐ id flow affects human marrow stromal cell proliferation and differentiation. Journal

[122] Stolberg S, McCloskey KE. Can shear stress direct stem cell fate? Biotechnol Prog.

[123] Park JS, Chu JSF, Cheng C, Chen F, Chen D, Li S. Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells. Biotechnology and Bioengineering

[124] Diederichs S, Bo°hm S, Peterbauer A, Kasper C, Scheper T, van Griensven M.Appli‐ cation of different strain regimes in two-dimensional and three-dimensional adipose tissue–derived stem cell cultures induces osteogenesis: Implications for bone tissue engineering. Journal of Biomedical Materials Research Part A 2010;94(A):927-936.

[125] Matos MA, Cicerone MT. Alternating current electric field effects on neural stem cell viability and differentiation. Biotechnology Progress 2010; 26(3):664–670.

[126] Park SY, Park J, Sim SH, Sung MG, Kim KS, Hong BH, Hong S. Enhanced Differen‐ tiation of Human Neural Stem Cells into Neurons on Graphene. Adv. Mater. 2011;23:

[127] Schienbein M, Gruler H. Physical Review. E- Statistical Physics, Plasmas, Fluids, and Related Interdisciplinary Topics. The American Physical Society 1995;52: 4183-4197.

[128] Abilez O, Benharash P, Miyamoto E, Gale A, Xu C, Zarins CK. P19 progenitor cells progress to organized contracting myocytes after chemical and electrical stimulation: implications for vascular tissue engineering. Endovasc Ther 2006;13(3):377–388.

thasone. Journal of Biomedical Materials Research 2005;72(A): 326-334.

ed neural stem cells. Biomaterials 2009;30:4695–4699.

of Orthopaedic Research 2004;22:1283-1289.

als 2012;33: 3835-3845.

596 Pluripotent Stem Cells

2005;33:63-70.

2009; 25(1):10-19.

2004;88:359-368.

263–267.


[142] Okita K, Yamanaka S. Induced pluripotent stem cells: opportunities and challenges. Philos Trans R Soc Lond B Biol Sci. 2011;366(1575):2198-207.

[155] Sundelacruz S, Kaplan DL. Stem Cell-And Scaffold-Based Tissue Engineering Ap‐ proaches to Osteochondral Regenerative Medicine. Seminars in Cell &Developmen‐

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 599

[156] Holland TA, Mikos AG. Biodegradable Polymeric Scaffolds. Improvements in bone tissue engineering through controlled drug delivery. Advances inBiochemical Engi‐

[157] Holland TA, Bodde EWH, Cuijpers V, Baggett LS, Tabata Y, Mikos AG, Jansen JA. Degradable Hydrogel Scaffolds for in Vivo Delivery of Single and Dual Growth Fac‐

[158] Liu H, Zhang L, Shi P, Zou Q, Zuo Y, Li Y. Hydroxyapatite/Polyurethane Scaffold Incorporated with Drug-Loaded Ethyl Cellulose Microspheres for Bone Regenera‐ tion. Journal of Biomedical Materials Research Part B: Applied Biomaterials

[159] Uebersax L, Merkle HP, Meinel L. Insulin-Like Growth Factor I Releasing Silk Fi‐ broin Scaffolds Induce Chondrogenic Differentiation of Human Mesenchymal Stem

[160] Li C, Vepari C, Jina HJ, Kim HJ, Kaplan DL. Electrospun silk-BMP-2 scaffolds for

[161] Lee GS, Park JH, Shin US, Kim HW. Direct deposited porous scaffolds of calcium phosphate cement with alginatefor drug delivery and bone tissue engineering. Acta

[162] Zhou K, Nisbet DR, Thouas G, Bernard C, Forsythe JS., editor. Eberli. Bio-nanotech‐ nological approaches to neural tissue engineering in Tissue Engineering. In-Tech;

[163] Schnell E, Klinkhammer K, Balzer S, Brook G, Klee D, Datton P, Mey J. Guidance of glial cell migration and axonal growth on electrospun nanofibers of poly-e-caprolac‐ tone and a collagen/poly-ecaprolactone blend. Biomaterials 2007;28(19): 3012-3025.

[164] Geiger B, Bershadsky A, Pankov R, Yamada KM. Transmembrane crosstalk between the extracellular matrix and the cytoskeleton. Nat Rev Mol Cell Biol 2001;2:793-805.

[165] Horne MK, NisbetDR,Forsythe JS, Parish CL. Three dimensional nanofibrous scaf‐ folds incorporating immobilized BDNF promote proliferation and differentiation of

[166] Patel S, Kurpinski K, Quigley R, Gao H, Hsiao BS, Poo MM, Li S. Bioactive Nanofib‐ ers: Synergistic Effects of Nanotopography and Chemical Signaling on Cell Guid‐

[167] Bobick BE, Chen FH, Le AM, Tuan RS. Regulation of the chondrogenic phenotype in

culture. Birth Defects Res C Embryo Today 2009;87:351-71.

cortical neural stem cells. Stem Cells and Development 2010;19(6):843-852.

tors in Cartilage Repair. Osteoarthritis and Cartilage 2007;15(2):187-197.

tal Biology 2009;20(6):646-655.

2010;95(1):36-46.

2010. p459-483.

neering/Biotechnology 2006;102:161-185.

Cells. Journal of Controlled Release 2008;127(1):12-21.

Biomaterialia 2011;7(8):3178–3186.

ance. Nano Letters 2007;7(7): 2122-2128.

bone tissue engineering. Biomaterials 2006;27(16): 3115–3124.


[155] Sundelacruz S, Kaplan DL. Stem Cell-And Scaffold-Based Tissue Engineering Ap‐ proaches to Osteochondral Regenerative Medicine. Seminars in Cell &Developmen‐ tal Biology 2009;20(6):646-655.

[142] Okita K, Yamanaka S. Induced pluripotent stem cells: opportunities and challenges.

[143] Eiraku M, Takata N, Ishibashi H, Kawada M, Sakakura E, Okuda S, Sekiguchi K, Adachi T, Sasai Y. Self-organizing optic-cup morphogenesis in three-dimensional

[144] Selvaraj V, Plane JM, Williams AJ, Deng W. Switching cell fate: the remarkable rise of induced pluripotent stem cells and lineage reprogramming technologies. Trends in

[145] Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Sudhof TC, Wernig M. Direct con‐ version of fibroblasts to functional neurons by defined factors. Nature 2010 Feb

[146] Huang P, He Z, Ji S, Sun H, Xiang D, Liu C, Hu Y, Wang X, Hui L. Induction of func‐ tional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature 2011

[147] Ieda M, Fu JD, Delgado-Olguin P, Vedantham V, Hayashi Y, Bruneau BG, Srivastava D. Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined

[148] Nicholas CR, Kriegstein AR. Regenerative medicine: Cell reprogramming gets direct.

[149] Ring KL, Tong LM, Balestra ME, Javier R, Andrews-Zwilling Y, Li G, Walker D, Zhang WR, Kreitzer AC, Huang Y. Direct reprogramming of mouse and human fi‐ broblasts into multipotent neural stem cells with a single factor. Cell Stem Cell

[150] Huebsch N, Mooney DJ. Inspiration and Application in the Evolution of Biomateri‐

[151] Alsberg E, Anderson KW, Albeiruti A, Rowley JA, Mooney DJ. Engineering Growing Tissues. Proceedings of the National Academy of Sciences of the United States of

[152] Lieberman JR, Daluiski A, Einhorn TA. The Role of Growth Factors in the Repair of Bone: Biology and Clinical Applications. The Journal of Bone and Joint Surgery

[153] Reddi AH. Bone Morphogenetic Proteins: From Basic Science to Clinical Applica‐

[154] Reddi AH. Interplay Between Bone Morphogenetic Proteins and Cognate Binding Proteins in Bone and Cartilage Development: Noggin, Chordin and DAN. Arthritis‐

tions. The Journal of Bone and Joint Surgery 2001;83(S1):S1-S6.

Philos Trans R Soc Lond B Biol Sci. 2011;366(1575):2198-207.

culture. Nature 2011;472:51–56.

25;463(7284):1035-41.

598 Pluripotent Stem Cells

May 11;475(7356):386-9.

Factors. Cell 2010;142(3):375-386.

als. Nature 2009;462(7272):426-432.

America 2002;99(19):12025.

2002;84(6):1032-1044.

Research 2001;3(1):1-5.

Nature 2010;463:1031-1032.

2012;11(1):100-9.

Biotechnology 2010 Feb 9;28(4):214-223.


[168] Schinagl RM, Ting MK, Price JH, Sah RL. Video microscopy to quantitate the inho‐ mogeneous equilibrium strain within articular cartilage during confined compres‐ sion. Ann Biomed Eng 1996;24:500-512.

[180] Atala A, Kim W, Paige KT, Vacanti CA, Retik AB. Endoscopic treatment of vesicoure‐ teral reflux with a chondrocyte-alginate suspension. Journal of Urology 1994; 152(2

Stem Cells in Tissue Engineering http://dx.doi.org/10.5772/54371 601

[181] Rosellini E, Cristallini C, Barbani N, Vozzi G, Giusti P. Preparation and characteriza‐ tion of alginate/gelatin blend films for cardiac tissue engineering. Journal of Biomedi‐

[182] Boontheekul T, Hill EE, Kong HU, Mooney DJ. Regulating myoblast phenotype through controlled gel stiffness and degradation. Tissue engineering 2007; 13 (7):

[183] Gilbert PM, Havenstrite KL, Magnusson KEG, Sacco A, Leonardi NA, Kraft P, Nguy‐ en NK, Thrun S, Lutolf MP, Blau HM. Substrate elasticity regulates skeletal muscle

[184] Fisher OZ, Khademhosseini A, Langer R, Peppas NA. Bioinspired Materials for Con‐ trolling Stem Cell Fate. Accounts of chemical research 2010; 43 (3):419-428.

Pt 2) 641-643; discussion 644.

1431-1442.

cal Material Research A 2009 Nov;91(2):447-53.

stem cell self-renewal in culture. Science 2010;329:1078–81.


[180] Atala A, Kim W, Paige KT, Vacanti CA, Retik AB. Endoscopic treatment of vesicoure‐ teral reflux with a chondrocyte-alginate suspension. Journal of Urology 1994; 152(2 Pt 2) 641-643; discussion 644.

[168] Schinagl RM, Ting MK, Price JH, Sah RL. Video microscopy to quantitate the inho‐ mogeneous equilibrium strain within articular cartilage during confined compres‐

[169] Wang CC, Hung CT, Mow VC. An analysis of the effects of depth-dependent aggre‐ gate modulus on articular cartilage stress-relaxation behavior in compression. J Bio‐

[170] Forte G, Carotenuto F, Pagliari F, Pagliari S, Cossa P, Fiaccavento R, Ahluwalia A, Vozzi G, Vinci B, Serafino A, Rinaldi A, Traversa E, Carosella L, Minieri M, Di Nardo P. Criticality of the biological and physical stimuli array inducing resident cardiac

[171] Lowman AM, Morishita M, Kajita M, Nagai T, Peppas NA. Oral delivery of insulin using pH-responsive complexation gels. Journal Pharmacy Science 1999;88(9):

[172] Elisseeff J, Anseth K, Sims D, McIntosh W, Randolph M, Langer R. Transdermal pho‐ topolymerization for minimally invasive implantation. Proc Natl Academic Science

[173] Mongia NK, Anseth KS, Peppas NA. Mucoadhesive poly (vinyl alcohol) hydrogels produced by freezing/thawing processes: applications in the development of wound healing systems. Journal of Biomaterial Science Polymer Edition 1996; 7(12):

[174] Lu S, Anseth KS. Photopolymerization of multilaminated poly (HEMA) hydrogels

[175] Babensee JE, Cornelius RM, Brash JL, Sefton MV. Immunoblot analysis of proteins associated with HEMAMMA microcapsules: human serum proteins in vitro and rat

[176] Ahearne M, Yang Y, El Haj AJ, Then KY, Liu KK.Characterizing the viscoelastic properties of thin hydrogel-based constructs for tissue engineering applications. J. R.

[177] Annabi N, Nichol JW, Zhong X, Ji Ch, Koshy S, Khademhosseini A, Dehghani F. Controlling the Porosity and Microarchitecture of Hydrogels for Tissue Engineering.

[178] Suggs LJ, Payne RG, Yaszemski MJ, Alemany LB, Mikos AG. Preparation and charac‐ terization of poly (propylene fumarate-co-ethylene glycol) hydrogels. Journal of Bio‐

[179] Hadlock TA, Elisseeff J, Langer R, Vacanti J, Cheney MA. Tissue engineered conduit for peripheral nerve repair. Arch Otolaryngol Head Neck Surg 1998; 124(10):1081-6.

for controlled release. Journal of Control Release 1999; 57(3):291-300.

proteins following implantation. Biomaterials 1998; 19(7-9):839-849.

Tissue engineering Part B 2010 August;16(4):371–383.

material Science Polymer Edition 1998; 9(7): 653-666.

stem cell determination. Stem Cells 2008;26(8):2093–2103.

sion. Ann Biomed Eng 1996;24:500-512.

mech 2001;34:75-84.

USA 1999; 96(6):3104-3107.

Soc. Interface 2005; 2:455-463.

933-937.

600 Pluripotent Stem Cells

1055-1064.


**Chapter 27**

**Ethical Considerations on Stem Cell Research**

Definitions: First we have to clearly define what we are talking about in the field of stem cells. The zygote (fertilized egg cell) and the cells of the very young embryo up until the eigth-cell stage are totipotent. This expression means that in the appropriate environment

In contrast to this notion, the embryonal stem cells of mammals are derived from the in‐ ner cell mass of the blastocyst, a slightly later stage of embryonal development. These cells are no longer totipotent, but pluripotent. This means that those cells, if artificially inserted into a heterologous young embryo, survive and give rise to all tissues and cell types in this embryo including cells of the germ line, thus creating a chimeric embryo, which consists of two types of cells that are genetically different form each other. Embry‐ onal stem cells (ES cells) display a few properties that make them highly interesting for regenerative medicine: they can be grown and multiplied indefinitely in the presence of the appropriate "factors" (proteins, growth factors, small molecules) without major genet‐ ic changes and without loss of pluripotency, and they can be modified by genetic engi‐ neering without major chromosomal changes and without using viral vehicles [1]. The latter property is essential for the future application of those cells for gene therapy. Mam‐ malian ES cell technology was first developed in the mouse model system beginning with the landmark paper of Martin [2]. Human ES cells (hESC) were first isolated by Thomson [3]. The patenting of the isolation of hESC (the so-called WARF patents) led to a huge public discussion regarding the moral and legal implications of those patents [4]. Ultimately the US supreme court acknowledged those patents as being legal, while the Court of Justice of the European Union ruled that no procedures can be patented, which use embryo research, i.e. the destruction of human embryos [81]. However, human in‐

> © 2013 Weiss et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Weiss et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

Andreas M. Weiss, Michael Breitenbach,

Additional information is available at the end of the chapter

(the uterus) these cells can form a complete and normal individual.

Mark Rinnerthaler and Günter Virt

http://dx.doi.org/10.5772/54375

**1. Introduction**

## **Ethical Considerations on Stem Cell Research**

Andreas M. Weiss, Michael Breitenbach, Mark Rinnerthaler and Günter Virt

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54375

## **1. Introduction**

Definitions: First we have to clearly define what we are talking about in the field of stem cells. The zygote (fertilized egg cell) and the cells of the very young embryo up until the eigth-cell stage are totipotent. This expression means that in the appropriate environment (the uterus) these cells can form a complete and normal individual.

In contrast to this notion, the embryonal stem cells of mammals are derived from the in‐ ner cell mass of the blastocyst, a slightly later stage of embryonal development. These cells are no longer totipotent, but pluripotent. This means that those cells, if artificially inserted into a heterologous young embryo, survive and give rise to all tissues and cell types in this embryo including cells of the germ line, thus creating a chimeric embryo, which consists of two types of cells that are genetically different form each other. Embry‐ onal stem cells (ES cells) display a few properties that make them highly interesting for regenerative medicine: they can be grown and multiplied indefinitely in the presence of the appropriate "factors" (proteins, growth factors, small molecules) without major genet‐ ic changes and without loss of pluripotency, and they can be modified by genetic engi‐ neering without major chromosomal changes and without using viral vehicles [1]. The latter property is essential for the future application of those cells for gene therapy. Mam‐ malian ES cell technology was first developed in the mouse model system beginning with the landmark paper of Martin [2]. Human ES cells (hESC) were first isolated by Thomson [3]. The patenting of the isolation of hESC (the so-called WARF patents) led to a huge public discussion regarding the moral and legal implications of those patents [4]. Ultimately the US supreme court acknowledged those patents as being legal, while the Court of Justice of the European Union ruled that no procedures can be patented, which use embryo research, i.e. the destruction of human embryos [81]. However, human in‐

© 2013 Weiss et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Weiss et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

duced pluripotent stem cells (hiPSC) can now be created from differentiated adult cells, like dermal fibroblasts (see below), which according to biochemical criteria (transcrip‐ tome, proteome), are very near identical to hESC [5]. It has been shown, in the mouse, that not only by biochemical criteria, but also in terms of the developmental potential, mouse iPSC are identical with mouse ESC [6].

and/or a combination of stem cell and gene therapy. This promise is, as we now know, a realistic one, but the enormous technical difficulties and the requirements imposed by clinical safety (for instance concerning the cancer risk) are not easily overcome and we estimate that many years will pass before these methods become clinical routine for many diseases. Presently, very few clinical examples exist that successfully show the effi‐

Ethical Considerations on Stem Cell Research

http://dx.doi.org/10.5772/54375

605

The theoretical and biological basis for the techniques to be discussed here are, among oth‐ ers, the fact that somatic cells of animals (and of the human animal, of course) contain the same genetic complement as the fertilized egg cell (the zygote). This means that every gene needed for the complete development of an individual is present in every somatic cell of a mature individual. The direct and undisputable proof for this is shown by the cloning of an‐ imals [15]. However a similar result was obtained decades before "Dolly the sheep" by John Gurdon [16], working with frogs. Therefore, the phenotypic differences between different somatic cells of an adult individual must depend on differences in gene expression, or to use a modern term on the "epigenome" of those cells. At present Bio-medicine is, at an increas‐ ing speed, discovering methods to change this differentiated state from one well defined cell type (say fibroblasts) to another (say, for example, a specific subtype of neurons needed for an individual patient) [7]. Previously, the differentiated state of somatic cells was believed to be immutable, at least *in vivo*, but this paradigm clearly is no longer true. Why are such pro‐ cedures needed in regenerative medicine? This question leads us to the genetic differences between human individuals and the immunological incompatibility between humans who are not monozygotic twins. For reasons that are not entirely clear to scientists who study the evolutionary history of mankind, it appears that differences in the antigens of the HLA type (human lymphocyte antigen; displayed on cell surfaces) occur between any two humans and are large enough to lead to immunological attack (host versus graft disease) after the transplantation of cells and organs. Therefore, it is desirable to use autologous (HLA-com‐ patible) cells for therapy, which raise no immune response and make immune suppression of the patient superfluous. In organ transplantation, this problem is generally overcome (al‐ though, perhaps, insufficiently) by the pharmacological immune suppression of the patient who receives a transplant. For the combination of gene and cell therapy, the idea is to use autologous cells which, however, must conform to strict safety standards before a clinical trial is granted by the authorities and can be started. There are also a number of unresolved problems if the autologous cells to be transplanted need a genetic "repair" because the pa‐ tient to be treated suffers from a genetic disease whose underlying mutation is known and

will be corrected by sophisticated genetic engineering as is applicable to human cells.

Genome editing: For several reasons which have to do with differences that exist between mouse and human iPSCs, as well as with the low success rate of current methods for ge‐ nome editing [17], the originally developed ingenious method of selection and counter-se‐ lection in mouse ESCs [1] seems not to be suitable for a safe repair of known mutations in genes of a patient suffering from a particular and genetically well-known inherited disease. Ideally, the presence of the mutation in question should be known by DNA sequencing of the relevant part (or the whole genome) of the patient. Instead, the scientific community is

cacy of stem cell and gene therapy [14].

In contrast to the pluripotent ES cells, somatic stem cells are multipotent, meaning that their developmental potential is rather limited to a number of related cell types. For instance, the well-known hematopoietic stem cells of the red bone marrow can generate *in vivo* all cells that are found in the blood of humans. Until recently it was believed that this commitment to a number of related developmental fates is absolute, however it is now known that even in normal individuals *in vivo,* a low percentage of bone marrow stem cells can become quite different cells [7], and, to give just one example, fibroblasts can be induced, by expression of two to three transcription factors, to become *bona fide* heart muscle cells [8].

Currently, an ever increasing number of papers on hiPSC (human induced pluripotent stem cells) are being published as documented by indexing services such as PubMed. In vitro methods of creating hiPSC from the easily available dermal fibroblasts were first described in 2006 and 2007 [9, 10]. Due to longer experience with the stem cells of the mouse and due to ethical and legal considerations, there is still a technical gap between procedures applicable to mouse iPSC and hiPSC. Since 2008, a nearly exponential in‐ crease in papers dealing with hiPSC is appearing and well over 1000 papers are now be‐ ing published every year. Many of those papers mention that hiPSCs in contrast to hESCs (human embryonal stem cells) are considered to be ethically acceptable while an intensive debate was and is going on concerning the ethical implications of hESCs [4, 11]; (see below in the next part of this chapter).

Another unsolved probem in stem cell therapy is "homing" of the repaired cells to the "ni‐ che" in the body where they are needed and can function. Only in exceptional cases does homing occur automatically (bone marrow stem cells in the mouse), but in other cases (brain) the cells must be directly injected into the relevant area. Modern nanotechnological methods may be helpful for this immense task in the future [12].

What we would like to do in the current paper is (paragraph 2) to give a very short over‐ view of the present and the anticipated future status of hiPSCs and their use in biomedicine including the new topic of differentiated cell plasticity [7]; (paragraph 3) to explain the ethi‐ cal arguments that were brought forward concerning hESCs; and (paragraph 4) to discuss some remaining ethical arguments concerning hiPSCs with special emphasis on the argu‐ ment of complicity [13].

## **2. Short overview of the present and the anticipated future status of hiPSCs**

Stem cell and related techniques, such as direct reprogramming of differentiated cells, of‐ fer an immense promise for the future of regenerative medicine using stem cell therapy and/or a combination of stem cell and gene therapy. This promise is, as we now know, a realistic one, but the enormous technical difficulties and the requirements imposed by clinical safety (for instance concerning the cancer risk) are not easily overcome and we estimate that many years will pass before these methods become clinical routine for many diseases. Presently, very few clinical examples exist that successfully show the effi‐ cacy of stem cell and gene therapy [14].

duced pluripotent stem cells (hiPSC) can now be created from differentiated adult cells, like dermal fibroblasts (see below), which according to biochemical criteria (transcrip‐ tome, proteome), are very near identical to hESC [5]. It has been shown, in the mouse, that not only by biochemical criteria, but also in terms of the developmental potential,

In contrast to the pluripotent ES cells, somatic stem cells are multipotent, meaning that their developmental potential is rather limited to a number of related cell types. For instance, the well-known hematopoietic stem cells of the red bone marrow can generate *in vivo* all cells that are found in the blood of humans. Until recently it was believed that this commitment to a number of related developmental fates is absolute, however it is now known that even in normal individuals *in vivo,* a low percentage of bone marrow stem cells can become quite different cells [7], and, to give just one example, fibroblasts can be induced, by expression of

Currently, an ever increasing number of papers on hiPSC (human induced pluripotent stem cells) are being published as documented by indexing services such as PubMed. In vitro methods of creating hiPSC from the easily available dermal fibroblasts were first described in 2006 and 2007 [9, 10]. Due to longer experience with the stem cells of the mouse and due to ethical and legal considerations, there is still a technical gap between procedures applicable to mouse iPSC and hiPSC. Since 2008, a nearly exponential in‐ crease in papers dealing with hiPSC is appearing and well over 1000 papers are now be‐ ing published every year. Many of those papers mention that hiPSCs in contrast to hESCs (human embryonal stem cells) are considered to be ethically acceptable while an intensive debate was and is going on concerning the ethical implications of hESCs [4, 11];

Another unsolved probem in stem cell therapy is "homing" of the repaired cells to the "ni‐ che" in the body where they are needed and can function. Only in exceptional cases does homing occur automatically (bone marrow stem cells in the mouse), but in other cases (brain) the cells must be directly injected into the relevant area. Modern nanotechnological

What we would like to do in the current paper is (paragraph 2) to give a very short over‐ view of the present and the anticipated future status of hiPSCs and their use in biomedicine including the new topic of differentiated cell plasticity [7]; (paragraph 3) to explain the ethi‐ cal arguments that were brought forward concerning hESCs; and (paragraph 4) to discuss some remaining ethical arguments concerning hiPSCs with special emphasis on the argu‐

**2. Short overview of the present and the anticipated future status of**

Stem cell and related techniques, such as direct reprogramming of differentiated cells, of‐ fer an immense promise for the future of regenerative medicine using stem cell therapy

two to three transcription factors, to become *bona fide* heart muscle cells [8].

mouse iPSC are identical with mouse ESC [6].

604 Pluripotent Stem Cells

(see below in the next part of this chapter).

ment of complicity [13].

**hiPSCs**

methods may be helpful for this immense task in the future [12].

The theoretical and biological basis for the techniques to be discussed here are, among oth‐ ers, the fact that somatic cells of animals (and of the human animal, of course) contain the same genetic complement as the fertilized egg cell (the zygote). This means that every gene needed for the complete development of an individual is present in every somatic cell of a mature individual. The direct and undisputable proof for this is shown by the cloning of an‐ imals [15]. However a similar result was obtained decades before "Dolly the sheep" by John Gurdon [16], working with frogs. Therefore, the phenotypic differences between different somatic cells of an adult individual must depend on differences in gene expression, or to use a modern term on the "epigenome" of those cells. At present Bio-medicine is, at an increas‐ ing speed, discovering methods to change this differentiated state from one well defined cell type (say fibroblasts) to another (say, for example, a specific subtype of neurons needed for an individual patient) [7]. Previously, the differentiated state of somatic cells was believed to be immutable, at least *in vivo*, but this paradigm clearly is no longer true. Why are such pro‐ cedures needed in regenerative medicine? This question leads us to the genetic differences between human individuals and the immunological incompatibility between humans who are not monozygotic twins. For reasons that are not entirely clear to scientists who study the evolutionary history of mankind, it appears that differences in the antigens of the HLA type (human lymphocyte antigen; displayed on cell surfaces) occur between any two humans and are large enough to lead to immunological attack (host versus graft disease) after the transplantation of cells and organs. Therefore, it is desirable to use autologous (HLA-com‐ patible) cells for therapy, which raise no immune response and make immune suppression of the patient superfluous. In organ transplantation, this problem is generally overcome (al‐ though, perhaps, insufficiently) by the pharmacological immune suppression of the patient who receives a transplant. For the combination of gene and cell therapy, the idea is to use autologous cells which, however, must conform to strict safety standards before a clinical trial is granted by the authorities and can be started. There are also a number of unresolved problems if the autologous cells to be transplanted need a genetic "repair" because the pa‐ tient to be treated suffers from a genetic disease whose underlying mutation is known and will be corrected by sophisticated genetic engineering as is applicable to human cells.

Genome editing: For several reasons which have to do with differences that exist between mouse and human iPSCs, as well as with the low success rate of current methods for ge‐ nome editing [17], the originally developed ingenious method of selection and counter-se‐ lection in mouse ESCs [1] seems not to be suitable for a safe repair of known mutations in genes of a patient suffering from a particular and genetically well-known inherited disease. Ideally, the presence of the mutation in question should be known by DNA sequencing of the relevant part (or the whole genome) of the patient. Instead, the scientific community is now seeking to improve the efficiency of point-directed genome editing to clinically accepta‐ ble levels [17]. The cells to be used for these procedures should be as close as possible to the original patient-derived cells, avoiding prolonged proliferation of hiPSCs. The tools that must be developed to achieve this are the so-called ZNF-nucleases (zinc finger nucleases) based on a concept by Kim [18] which can produce a double strand break at a precisely de‐ fined point in the whole human genome [17]. This double strand break is then recombino‐ genic enough to lead to homologous recombination with a co-transformed plasmid that carries the corrected DNA sequence [19]. Alternatively, the TALEN strategy can be used [20]. One problem that must be overcome here in the future, is the limited capacity for pro‐ liferation of differentiated cells and their general reluctance to be transformed by plasmids, which is true for instance for dermal fibroblasts.

have to ask, if a human embryo is recognized as a being endowed with human dignity and a right to life comparable to that of born human beings. Destruction for research purposes raises the serious ethical issues of exploitation, instrumentalisation and killing of human be‐ ings. Concerning both ethical issues, human dignity and the prohibition of killing, in re‐ gards to human embryos in spite of the long discussions an ethical consensus is nowhere in sight. In the following passage some explanations will be given regarding the fundamental

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Further intensively discussed issues in hESC research are research cloning (the procurement of embryos for research purposes by nuclear transfer in enucleated egg cells) and the dona‐ tion of egg-cells. For a long time, the development of therapeutic applications seemed to in‐ volve research cloning (also called "therapeutic cloning"). Research cloning of humans would represent a clear instance of exploiting humans solely for the benefit and interests of others. Establishing this technique in humans requires further destructive embryo research and is feared to prepare a slippery slope for reproductive cloning of humans, which is gen‐

If this way to therapeutic applications had succeeded, the demand for a high number of do‐ nated egg-cells would have been a consequence. For women, egg donation causes health risks and the danger of commercial exploitation. The alternative to produce hybrids of hu‐ mans and animals is also seen as offending human dignity [38]. These ethical problems have lost some urgency, since this strategy doesn't seem to be succeeding. The fundamental ques‐ tion of the moral status of human embryos is still a matter of open discussion in ethics.

hESCs needed for research are obtained from different sources that entail a different ethi‐ cal evaluation. While extraction of stem cells from adults, from umbilical cord blood or from aborted foetuses, is considered to be ethically acceptable under certain conditions, the procurement of hESCs is confronted with ethical objections, since it is necessarily connected with the destruction of human embryos. It is a kind of consuming embryo re‐ search. The possible sources are already established embryonic stem cell lines, supernum‐ erary embryos from IVF-treatment, embryos produced specifically for research purposes or even embryos cloned by nuclear transfer as a logical consequence in case of successful

Different regulations worldwide and in the EU, as well as an on-going discussion about the funding of research projects are taking place [12, 39]. As a minimal consensus, creation of embryos solely for research purposes is forbidden in the European Council's Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Appli‐

By obtaining hESCs from the inner cell mass of a blastocyst for research purposes begin‐ ning human life is destroyed. The embryo is obviously a human being, a member of the human species, has an individual genome, neither identical with that of the mother nor that of the father, in contrast to other human tissue, can develop into the full shape of a

question of the moral status of embryos [12, 24-31].

erally considered as ethically unacceptable [32-37].

therapeutic applications.

cation of Biology and Medicine [32].

**3.1. The discussion about the moral status of early human embryos**

Cancer risk: One of the greatest obstacles that must be overcome before stem cell therapy can become clinical routine is the inherent cancer risk conferred by both ESCs and iPSCs. In one of the very few and frequently-quoted clinical trials for gene therapy of X-SCID, some of the affected and essentially cured children came down with leukemia. The rea‐ son for the cancer incidence in this case was the lack of control of the point of integra‐ tion of the viral vector used to introduce the genetically corrected gene sequence, which was inserted at locations in the genome where it caused leukemia [21, 22]. However, even ESCs or iPSCs which are not genetically manipulated, by their "stemness" alone can cause cancer. It must not be forgotten that embryonal stem cells were first discovered during the study of teratocarcinomas and one of the most important decisive traits was the ability to form teratocarcinomas in nude mice [2]. Therefore, for some time, the idea was to re-differentiate the hiPSCs to the needed cells after genetic manipulation and then purify these cells until they were essentially free of remaining stem cells [23]. This proved to be a difficult job. The other solution to this problem is to directly produce the desired cell type using the action of transcription factors and small molecule signalling substances without ever going through a stage of stem cells [7]. This way is very promis‐ ing but also not yet matured enough for clinical practice.

In summary, we may say that it is still too early to decide in which direction future cell and gene therapy will go. For some time, hESCs, and even more importantly, hiPSCs will be needed for biomedical research. This is not restricted to gene therapy and cell therapy with‐ out genetic corrections (as in the case of acquired diseases), but equally is needed for the es‐ tablishment of disease models and for drug testing, which is, however, not the topic of this chapter. For all of those reasons, we think it is timely to discuss the ethical implications of stem cell research.

#### **3. Ethical arguments brought forward concerning hESCs**

The central ethical concern that is raised by production and use of hESC is the question con‐ cerning the moral status of human embryos. The derivation of hESCs from early embryos (blastocysts) is, in practice, necessarily connected with their destruction. Because of that, we have to ask, if a human embryo is recognized as a being endowed with human dignity and a right to life comparable to that of born human beings. Destruction for research purposes raises the serious ethical issues of exploitation, instrumentalisation and killing of human be‐ ings. Concerning both ethical issues, human dignity and the prohibition of killing, in re‐ gards to human embryos in spite of the long discussions an ethical consensus is nowhere in sight. In the following passage some explanations will be given regarding the fundamental question of the moral status of embryos [12, 24-31].

now seeking to improve the efficiency of point-directed genome editing to clinically accepta‐ ble levels [17]. The cells to be used for these procedures should be as close as possible to the original patient-derived cells, avoiding prolonged proliferation of hiPSCs. The tools that must be developed to achieve this are the so-called ZNF-nucleases (zinc finger nucleases) based on a concept by Kim [18] which can produce a double strand break at a precisely de‐ fined point in the whole human genome [17]. This double strand break is then recombino‐ genic enough to lead to homologous recombination with a co-transformed plasmid that carries the corrected DNA sequence [19]. Alternatively, the TALEN strategy can be used [20]. One problem that must be overcome here in the future, is the limited capacity for pro‐ liferation of differentiated cells and their general reluctance to be transformed by plasmids,

Cancer risk: One of the greatest obstacles that must be overcome before stem cell therapy can become clinical routine is the inherent cancer risk conferred by both ESCs and iPSCs. In one of the very few and frequently-quoted clinical trials for gene therapy of X-SCID, some of the affected and essentially cured children came down with leukemia. The rea‐ son for the cancer incidence in this case was the lack of control of the point of integra‐ tion of the viral vector used to introduce the genetically corrected gene sequence, which was inserted at locations in the genome where it caused leukemia [21, 22]. However, even ESCs or iPSCs which are not genetically manipulated, by their "stemness" alone can cause cancer. It must not be forgotten that embryonal stem cells were first discovered during the study of teratocarcinomas and one of the most important decisive traits was the ability to form teratocarcinomas in nude mice [2]. Therefore, for some time, the idea was to re-differentiate the hiPSCs to the needed cells after genetic manipulation and then purify these cells until they were essentially free of remaining stem cells [23]. This proved to be a difficult job. The other solution to this problem is to directly produce the desired cell type using the action of transcription factors and small molecule signalling substances without ever going through a stage of stem cells [7]. This way is very promis‐

In summary, we may say that it is still too early to decide in which direction future cell and gene therapy will go. For some time, hESCs, and even more importantly, hiPSCs will be needed for biomedical research. This is not restricted to gene therapy and cell therapy with‐ out genetic corrections (as in the case of acquired diseases), but equally is needed for the es‐ tablishment of disease models and for drug testing, which is, however, not the topic of this chapter. For all of those reasons, we think it is timely to discuss the ethical implications of

The central ethical concern that is raised by production and use of hESC is the question con‐ cerning the moral status of human embryos. The derivation of hESCs from early embryos (blastocysts) is, in practice, necessarily connected with their destruction. Because of that, we

which is true for instance for dermal fibroblasts.

606 Pluripotent Stem Cells

ing but also not yet matured enough for clinical practice.

**3. Ethical arguments brought forward concerning hESCs**

stem cell research.

Further intensively discussed issues in hESC research are research cloning (the procurement of embryos for research purposes by nuclear transfer in enucleated egg cells) and the dona‐ tion of egg-cells. For a long time, the development of therapeutic applications seemed to in‐ volve research cloning (also called "therapeutic cloning"). Research cloning of humans would represent a clear instance of exploiting humans solely for the benefit and interests of others. Establishing this technique in humans requires further destructive embryo research and is feared to prepare a slippery slope for reproductive cloning of humans, which is gen‐ erally considered as ethically unacceptable [32-37].

If this way to therapeutic applications had succeeded, the demand for a high number of do‐ nated egg-cells would have been a consequence. For women, egg donation causes health risks and the danger of commercial exploitation. The alternative to produce hybrids of hu‐ mans and animals is also seen as offending human dignity [38]. These ethical problems have lost some urgency, since this strategy doesn't seem to be succeeding. The fundamental ques‐ tion of the moral status of human embryos is still a matter of open discussion in ethics.

#### **3.1. The discussion about the moral status of early human embryos**

hESCs needed for research are obtained from different sources that entail a different ethi‐ cal evaluation. While extraction of stem cells from adults, from umbilical cord blood or from aborted foetuses, is considered to be ethically acceptable under certain conditions, the procurement of hESCs is confronted with ethical objections, since it is necessarily connected with the destruction of human embryos. It is a kind of consuming embryo re‐ search. The possible sources are already established embryonic stem cell lines, supernum‐ erary embryos from IVF-treatment, embryos produced specifically for research purposes or even embryos cloned by nuclear transfer as a logical consequence in case of successful therapeutic applications.

Different regulations worldwide and in the EU, as well as an on-going discussion about the funding of research projects are taking place [12, 39]. As a minimal consensus, creation of embryos solely for research purposes is forbidden in the European Council's Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Appli‐ cation of Biology and Medicine [32].

By obtaining hESCs from the inner cell mass of a blastocyst for research purposes begin‐ ning human life is destroyed. The embryo is obviously a human being, a member of the human species, has an individual genome, neither identical with that of the mother nor that of the father, in contrast to other human tissue, can develop into the full shape of a human being (totipotent) and has a small, but realistic chance to be born and live its own life.

**a. Personalistic positions** claim that already the embryo must be respected as a person and, therefore, has a right to life in the earliest stage and also outside the mother [42,

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The reason for personal positions is a certain view of the embryonic development. The de‐ velopment from fertilization up to birth is understood as a continuous development (*argu‐ ment of continuity*) of something that is, basically, already present, and under natural conditions, has the inner capability of further development into a fully evolved person (*ar‐ gument of potentiality*) and remains the same being (*argument of identity*). The embryo is not a preliminary stage of a human but a human in the earliest stage. Although it doesn't have the actual abilities of a person (self-consciousness, reason, freedom), the embryo must be treated as a person because of its inner potential to develop these qualities and, under normal cir‐

This reasoning can be combined with two additional arguments. The *species argument* points out that the embryo's membership in the human species is a biological fact. Biological facts alone are not sufficient reasonings for moral judgement. However, the argument may serve as a determination of the scope, the application area of dignity: All members of the human species are included. Being a member of the human species and being endowed with digni‐ ty and certain rights is actually coinciding with each other. Therefore, the species-member‐ ship suffices to claim the corresponding rights. If this argument also applies to embryos, then this is controversial and presupposes the first three arguments. The four previously mentioned arguments are often described as a "SKIP-quadrology": species, continuity, iden‐

Sometimes another argument is added in respect to the remaining uncertainties of empirical knowledge, as well as philosophical interpretation of early embryonic development. The *precautionary principle* generally calls for a careful proceeding in small steps and imposes the burden of proof on those, who want to change existing attitudes and moral norms. They have to offer evidence, not those who defend them. According to this position, doubts about being a person may not lead to an arbitrary restriction of human dignity. No man is subject to the constraint of having to justify his existence. This corresponds to the basic structure of the human dignity argument, which should, primarily, serve as protection of the weak against any kind of discrimination. Everyone is basically interested in safe conditions, in which she/he need not fear being excluded from the common protection area due to some

The consequences of the personal position are unambiguous: Destroying embryos for re‐ search purposes and research cloning is forbidden. Freedom of research is subjected to mo‐ ral limits. Therapies, which cost the lives of other humans, are not acceptable. Even the hope for therapy for serious diseases is no adequate reason for the specific production and de‐ struction of human embryos. Nevertheless, each mentioned argument is subjected to criti‐

cism and the personal position hasn't turned out to provide a consensus [42, 43].

**b. Non-personalistic positions** deny what personalistic positions proclaim.

48-52].

cumstances, become such a person.

actual lack of abilities or characteristics [45, 53].

tity, potentiality [44].

Since hESC research, on the one hand, gives hope in terms of therapeutic applications for severe diseases and, on the other hand, is connected with the destruction of embryos as nec‐ essary means to this end, two ethically high standing aims are opposed. Basic research (free‐ dom of research) and the hopes connected with therapeutic application (principle of beneficence, value of health and life of patients) are confronted with the respect for human dignity and the right to life of human embryos. The question is: May human embryos be produced and destroyed as biological material for research and therapy or even for industri‐ al applications?

In relation to already born humans we would never accept such destruction or killing no matter how great the benefit for research or therapy could be. For born humans there is a strong agreement: They have moral status and equal human dignity independent of their ac‐ tual abilities or disabilities. The statement about the moral status is a value judgement. At first it means that humans have intrinsic value. If the moral status of humans is determined in the tradition of the German philosopher, Immanuel Kant, with the term "dignity", an un‐ conditional value is proclaimed, which goes beyond the intrinsic value of non-human beings and can't be balanced with the benefit of others. Kant makes this clear in a well-known quote regarding his categorical imperative: "Act in such a way that you treat humanity, whether in your own person or in the person of any other, never merely as a means to an end, but always at the same time as an end." [40, 41].

The central consequences of the recognition of equal human dignity are the fundamental equality of all humans with regard to this dignity, the same right to welfare and the prohibi‐ tion of arbitrary instrumentalisation and exploitation for the purposes of others. Killing for research purposes definitely falls under this prohibition. Whether and to which degree these moral demands are already valid in the early stages of development, is a matter of the con‐ troversy concerning the ontological, moral, and legal status of human embryos [36, 42-47].

It is therefore clear why this discussion is unavoidable. Before discussing freedom of re‐ search, hopes for therapeutic applications, and different possibilities of regulations, the question, of whether or not embryos, in an ethical respect, belong to the community of beings deserving equal and impartial consideration, must be answered. Is impartiality (the "golden rule"), to be applied even to embryos, or not at all, or merely in a gradually weaker sense?

These issues were discussed extensively in the last decades and, regrettably, have not ach‐ ieved a consensus. Here we will shortly explain the general lines of reasoning. Summarized in a simplified overview there are three types of answers: (a) Personalistic positions main‐ tain human dignity and a right to life of human embryos. (b) Non-personalistic positions de‐ ny that and impute to embryos a status similar to human tissues or cadavers. A third group proposes to find a kind of middle position by giving several types of (c) relative or gradual‐ istic answers.

**a. Personalistic positions** claim that already the embryo must be respected as a person and, therefore, has a right to life in the earliest stage and also outside the mother [42, 48-52].

human being (totipotent) and has a small, but realistic chance to be born and live its own

Since hESC research, on the one hand, gives hope in terms of therapeutic applications for severe diseases and, on the other hand, is connected with the destruction of embryos as nec‐ essary means to this end, two ethically high standing aims are opposed. Basic research (free‐ dom of research) and the hopes connected with therapeutic application (principle of beneficence, value of health and life of patients) are confronted with the respect for human dignity and the right to life of human embryos. The question is: May human embryos be produced and destroyed as biological material for research and therapy or even for industri‐

In relation to already born humans we would never accept such destruction or killing no matter how great the benefit for research or therapy could be. For born humans there is a strong agreement: They have moral status and equal human dignity independent of their ac‐ tual abilities or disabilities. The statement about the moral status is a value judgement. At first it means that humans have intrinsic value. If the moral status of humans is determined in the tradition of the German philosopher, Immanuel Kant, with the term "dignity", an un‐ conditional value is proclaimed, which goes beyond the intrinsic value of non-human beings and can't be balanced with the benefit of others. Kant makes this clear in a well-known quote regarding his categorical imperative: "Act in such a way that you treat humanity, whether in your own person or in the person of any other, never merely as a means to an

The central consequences of the recognition of equal human dignity are the fundamental equality of all humans with regard to this dignity, the same right to welfare and the prohibi‐ tion of arbitrary instrumentalisation and exploitation for the purposes of others. Killing for research purposes definitely falls under this prohibition. Whether and to which degree these moral demands are already valid in the early stages of development, is a matter of the con‐ troversy concerning the ontological, moral, and legal status of human embryos [36, 42-47].

It is therefore clear why this discussion is unavoidable. Before discussing freedom of re‐ search, hopes for therapeutic applications, and different possibilities of regulations, the question, of whether or not embryos, in an ethical respect, belong to the community of beings deserving equal and impartial consideration, must be answered. Is impartiality (the "golden rule"), to be applied even to embryos, or not at all, or merely in a gradually

These issues were discussed extensively in the last decades and, regrettably, have not ach‐ ieved a consensus. Here we will shortly explain the general lines of reasoning. Summarized in a simplified overview there are three types of answers: (a) Personalistic positions main‐ tain human dignity and a right to life of human embryos. (b) Non-personalistic positions de‐ ny that and impute to embryos a status similar to human tissues or cadavers. A third group proposes to find a kind of middle position by giving several types of (c) relative or gradual‐

life.

608 Pluripotent Stem Cells

al applications?

weaker sense?

istic answers.

end, but always at the same time as an end." [40, 41].

The reason for personal positions is a certain view of the embryonic development. The de‐ velopment from fertilization up to birth is understood as a continuous development (*argu‐ ment of continuity*) of something that is, basically, already present, and under natural conditions, has the inner capability of further development into a fully evolved person (*ar‐ gument of potentiality*) and remains the same being (*argument of identity*). The embryo is not a preliminary stage of a human but a human in the earliest stage. Although it doesn't have the actual abilities of a person (self-consciousness, reason, freedom), the embryo must be treated as a person because of its inner potential to develop these qualities and, under normal cir‐ cumstances, become such a person.

This reasoning can be combined with two additional arguments. The *species argument* points out that the embryo's membership in the human species is a biological fact. Biological facts alone are not sufficient reasonings for moral judgement. However, the argument may serve as a determination of the scope, the application area of dignity: All members of the human species are included. Being a member of the human species and being endowed with digni‐ ty and certain rights is actually coinciding with each other. Therefore, the species-member‐ ship suffices to claim the corresponding rights. If this argument also applies to embryos, then this is controversial and presupposes the first three arguments. The four previously mentioned arguments are often described as a "SKIP-quadrology": species, continuity, iden‐ tity, potentiality [44].

Sometimes another argument is added in respect to the remaining uncertainties of empirical knowledge, as well as philosophical interpretation of early embryonic development. The *precautionary principle* generally calls for a careful proceeding in small steps and imposes the burden of proof on those, who want to change existing attitudes and moral norms. They have to offer evidence, not those who defend them. According to this position, doubts about being a person may not lead to an arbitrary restriction of human dignity. No man is subject to the constraint of having to justify his existence. This corresponds to the basic structure of the human dignity argument, which should, primarily, serve as protection of the weak against any kind of discrimination. Everyone is basically interested in safe conditions, in which she/he need not fear being excluded from the common protection area due to some actual lack of abilities or characteristics [45, 53].

The consequences of the personal position are unambiguous: Destroying embryos for re‐ search purposes and research cloning is forbidden. Freedom of research is subjected to mo‐ ral limits. Therapies, which cost the lives of other humans, are not acceptable. Even the hope for therapy for serious diseases is no adequate reason for the specific production and de‐ struction of human embryos. Nevertheless, each mentioned argument is subjected to criti‐ cism and the personal position hasn't turned out to provide a consensus [42, 43].

**b. Non-personalistic positions** deny what personalistic positions proclaim.

A far-reaching objection to the personal position is, for example, represented by the Australian moral philosopher, Peter Singer. He denies the human dignity of embryos, foetuses and even newborn children due to a very narrow concept of personality based solely on actual abilities: "My suggestion, then, is that we accord the life of a fetus no greater value than the life of a nonhuman animal at a similar level of rationality, selfconsciousness, awareness, capacity to feel, etc. Since no fetus is a person, no fetus has the same claim to life as a person." [54]. For these positions there is, in principle, no ob‐ jection to hESC research as long as the rights of the donors of gametes or embryos are respected.

Austrian Bioethics Commission these attempts are summarized as follows: "The first argu‐ ment chooses the comparison with the removal of organs from brain dead patients. This does not violate the prohibition of killing nor the prohibition of the complete instrumentali‐ sation of a human life that is derived from the concept of human dignity. Even less should the use of fertilised egg cells at a stage in which one cannot speak of either an organ or brain development be rejected as such on ethical reasons. The second argument compares the ob‐ taining of embryonic stem cells from surplus embryos with the medical use of tissue from aborted foetuses, which can be ethically justified in so far as the abortion was not performed for the purpose of obtaining foetal tissue. Both lines of argument imply that at the moment it is no longer used for reproduction, the embryo created in vitro undergoes a change of status that is equivalent to that of a person's transition from life into death. Even if one wishes to accord the fertilised egg cell personhood, this does not mean that there is an irresolvable conflict of values between the protection of life for the embryo and the freedom of research in the service of present and future patients"[31]. These arguments cannot be discussed here [63, 64]. The intention to escape the endless discussion about the moral status of embryos is clever, the hope to prevent the weakening of the human dignity argument may be honoura‐ ble, but as a matter of fact, the relevant embryos are not dead prior to the destruction for research. One might wonder, what results this kind of reasoning could have, when applied to disabled persons or patients at the end of life, which could also be said to have no chance

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for further development (a logical version of the slippery slope-argument).

Each modification of ethical reasoning and central moral attitudes must be paid attention to in terms of consistency, rationality and possible side effects for other areas of life. This ex‐ amination of the arguments is sometimes more important than the solution itself. Bad argu‐ ments are counterproductive, promote distrust against ethical reasoning and science in the long run, and weaken their aptitude to give orientation. The first task of ethics is the effort to obtain good reasons, not fast answers [65]. The personalistic positions are consistent in the protection of the right to life, but have trouble convincing society and researchers. The nonpersonalistic positions will not find approval because of the openly declared consequences for new-born children, disabled, or dying persons. The middle positions try to release re‐ search from some ethical boarders, without damaging the conviction of equal human digni‐ ty. But their methods of reasoning don't really convince and, in the long run, leave open too

Nevertheless two fundamental considerations seem to support maintaining a rejection of the

**a.** If someone wants to justify hESC research, either within a limited extent or up to re‐ search cloning, she/he must be able to give convincing arguments, why embryos might be treated in a different way than born humans. This seems to be impossible without weakening or denying the moral status of early human embryos. This method of rea‐ soning possesses danger of weakening the protection of the human dignity in general. If the coincidence of human species and human dignity is given up and exchanged for a

destruction of human embryos for research purposes:

**3.2. Results of the status debate**

many options.

**c. Gradualistic positions** try to find a way of maintaining special respect for human em‐ bryos and restrictions of research purposes and, at the same time, allowing research for high standing objectives. They are quite frequently supported [29, 31, 55-58].

According to this kind of reasoning full protection of human embryos starts at a later stage of development. The time before the moral status is gradually weakened, but not reduced to that of some other human tissue. Most frequently nidation, or the end of the possibility of twin formation, is seen as the relevant moment. When nidation is com‐ plete, the embryos' chance of survival increases significantly. Sometimes other stages of development are argued as being relevant e.g. the beginning of the first nerve cells in the fifth or sixth week. This is seen as relevant, if the ability to feel pain is seen as a deci‐ sive ethical quality.

Finally, there are suggestions in which the moral status of embryos isn't differentiated de‐ pending on the stage of development, but according to the context and target of its creation. In such an "extrinsic" determination of the moral status surplus embryos from IVF-treat‐ ment and research embryos don't have any dignity, because they lack the necessary condi‐ tions for further development, or according to their creators' intentions, never should be born at all, while embryos produced for IVF-treatment already have this dignity in a very early stage, since the intention and hope is that they be born [24, 59, 60]. In this way of rea‐ soning, dignity and the right to life are conferred or awarded by society. Dignity depends on the allocation to the research department or the IVF department. Some authors turn this rea‐ soning into the field of metaethics and proclaim, that human dignity is always invented and awarded by society and not based on an objective moral reality [58].

If the protection of some early stage or research embryos or surplus embryos is weakened, the interests and well-being of embryos and patients can be balanced against each other and destruction of embryos can be justified for high standing objectives. Strict embryo protection is argued to be valid for later stages and a clear limit seems possible for the time being. Nev‐ ertheless this reasoning is not free of some arbitrariness and, if the restrictions are sustained, one can fear for the time, when interests for research with later stages of embryonic develop‐ ment will emerge. In principle, everything seems justifiable, if dignity depends on society or the intentions of the embryo's creators.

Some authors try to justify hESC research without weakening the moral status of embryos through a special reasoning within the prohibition of killing [61, 62]. In an opinion of the Austrian Bioethics Commission these attempts are summarized as follows: "The first argu‐ ment chooses the comparison with the removal of organs from brain dead patients. This does not violate the prohibition of killing nor the prohibition of the complete instrumentali‐ sation of a human life that is derived from the concept of human dignity. Even less should the use of fertilised egg cells at a stage in which one cannot speak of either an organ or brain development be rejected as such on ethical reasons. The second argument compares the ob‐ taining of embryonic stem cells from surplus embryos with the medical use of tissue from aborted foetuses, which can be ethically justified in so far as the abortion was not performed for the purpose of obtaining foetal tissue. Both lines of argument imply that at the moment it is no longer used for reproduction, the embryo created in vitro undergoes a change of status that is equivalent to that of a person's transition from life into death. Even if one wishes to accord the fertilised egg cell personhood, this does not mean that there is an irresolvable conflict of values between the protection of life for the embryo and the freedom of research in the service of present and future patients"[31]. These arguments cannot be discussed here [63, 64]. The intention to escape the endless discussion about the moral status of embryos is clever, the hope to prevent the weakening of the human dignity argument may be honoura‐ ble, but as a matter of fact, the relevant embryos are not dead prior to the destruction for research. One might wonder, what results this kind of reasoning could have, when applied to disabled persons or patients at the end of life, which could also be said to have no chance for further development (a logical version of the slippery slope-argument).

#### **3.2. Results of the status debate**

A far-reaching objection to the personal position is, for example, represented by the Australian moral philosopher, Peter Singer. He denies the human dignity of embryos, foetuses and even newborn children due to a very narrow concept of personality based solely on actual abilities: "My suggestion, then, is that we accord the life of a fetus no greater value than the life of a nonhuman animal at a similar level of rationality, selfconsciousness, awareness, capacity to feel, etc. Since no fetus is a person, no fetus has the same claim to life as a person." [54]. For these positions there is, in principle, no ob‐ jection to hESC research as long as the rights of the donors of gametes or embryos are

**c. Gradualistic positions** try to find a way of maintaining special respect for human em‐ bryos and restrictions of research purposes and, at the same time, allowing research for

According to this kind of reasoning full protection of human embryos starts at a later stage of development. The time before the moral status is gradually weakened, but not reduced to that of some other human tissue. Most frequently nidation, or the end of the possibility of twin formation, is seen as the relevant moment. When nidation is com‐ plete, the embryos' chance of survival increases significantly. Sometimes other stages of development are argued as being relevant e.g. the beginning of the first nerve cells in the fifth or sixth week. This is seen as relevant, if the ability to feel pain is seen as a deci‐

Finally, there are suggestions in which the moral status of embryos isn't differentiated de‐ pending on the stage of development, but according to the context and target of its creation. In such an "extrinsic" determination of the moral status surplus embryos from IVF-treat‐ ment and research embryos don't have any dignity, because they lack the necessary condi‐ tions for further development, or according to their creators' intentions, never should be born at all, while embryos produced for IVF-treatment already have this dignity in a very early stage, since the intention and hope is that they be born [24, 59, 60]. In this way of rea‐ soning, dignity and the right to life are conferred or awarded by society. Dignity depends on the allocation to the research department or the IVF department. Some authors turn this rea‐ soning into the field of metaethics and proclaim, that human dignity is always invented and

If the protection of some early stage or research embryos or surplus embryos is weakened, the interests and well-being of embryos and patients can be balanced against each other and destruction of embryos can be justified for high standing objectives. Strict embryo protection is argued to be valid for later stages and a clear limit seems possible for the time being. Nev‐ ertheless this reasoning is not free of some arbitrariness and, if the restrictions are sustained, one can fear for the time, when interests for research with later stages of embryonic develop‐ ment will emerge. In principle, everything seems justifiable, if dignity depends on society or

Some authors try to justify hESC research without weakening the moral status of embryos through a special reasoning within the prohibition of killing [61, 62]. In an opinion of the

awarded by society and not based on an objective moral reality [58].

high standing objectives. They are quite frequently supported [29, 31, 55-58].

respected.

610 Pluripotent Stem Cells

sive ethical quality.

the intentions of the embryo's creators.

Each modification of ethical reasoning and central moral attitudes must be paid attention to in terms of consistency, rationality and possible side effects for other areas of life. This ex‐ amination of the arguments is sometimes more important than the solution itself. Bad argu‐ ments are counterproductive, promote distrust against ethical reasoning and science in the long run, and weaken their aptitude to give orientation. The first task of ethics is the effort to obtain good reasons, not fast answers [65]. The personalistic positions are consistent in the protection of the right to life, but have trouble convincing society and researchers. The nonpersonalistic positions will not find approval because of the openly declared consequences for new-born children, disabled, or dying persons. The middle positions try to release re‐ search from some ethical boarders, without damaging the conviction of equal human digni‐ ty. But their methods of reasoning don't really convince and, in the long run, leave open too many options.

Nevertheless two fundamental considerations seem to support maintaining a rejection of the destruction of human embryos for research purposes:

**a.** If someone wants to justify hESC research, either within a limited extent or up to re‐ search cloning, she/he must be able to give convincing arguments, why embryos might be treated in a different way than born humans. This seems to be impossible without weakening or denying the moral status of early human embryos. This method of rea‐ soning possesses danger of weakening the protection of the human dignity in general. If the coincidence of human species and human dignity is given up and exchanged for a dignity awarded by society, corresponding to actual research interests, serious doubts may arise, whether the desired protection standard can be maintained in other areas of life, e.g. for coma patients, disabled people or new born children.

period, some ethicists argue for the use of surplus embryos from IVF-treatment [31]. The ethical objections were indicated above. This way is surely not acceptable, if, according to our appraisal, existing cell lines are sufficient. If not, the use of surplus embryos needs to be justified in a consistent way without denying the human dignity of embryos and without opening the way to the creation of research embryos on demand and even for non-therapeu‐

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How can the cell donors' right to voluntary and informed consent, as well as the protection of personal data, especially in the case of application of hiPSCs as disease models, be guar‐ anteed? How can the relevant questions of property rights and patent law be solved [69]?

Even hiPSC-research is, in several ways, confronted with the ethical problem of "complicity in others' wrongdoing": How can someone consistently reject the destruction of human em‐ bryos and, at the same time, use the result of former destructive research [13, 47, 70, 71]? Katrien Devolder draws attention to this problem of complicity. She contradicts the opinion, that hiPSC research is ethically correct, while hESC research is wrong because it involves de‐ struction of human embryos: "Many who object to human embryonic stem cell (hESC) re‐ search because they believe it involves complicity in embryo destruction have welcomed induced pluripotent stem cell (iPSC) research as an ethical alternative. This opinion article aims to show that complicity arguments against hESC research are *prima facie* inconsistent

In this passage we would like to scrutinize her theses and her suggestions for a solution. We are convinced that the problem of complicity is no obstacle for hiPSC research, if certain re‐

In theological and philosophical ethics, problems like this (cooperation with another's sin, "cooperatio in malo") can be discussed in relation to the so-called "principle of an action with double effect", in brief "principle of double effect", or "double-effect reasoning" [72-74]. In this principle, a distinction is drawn between direct consequences of an action and side effects, which are only indirectly wanted or accepted as unavoidable. The principle wasn't interpreted and used uniformly and has undergone some changes. In philosophical and theological ethics, it is relevant in two different contexts. The first and original context is the question of cooperating with the sin of another person. In these cases, the wrongness of the action is presupposed and the question concerns only the legitimacy, or culpability of the cooperation. Furthermore the principle of double effect is relevant in the context of some specific moral norms, such as the prohibition of killing to determine moral rightness or wrongness. In these cases it is a principle of restrictive interpretation of deontological moral norms [75]. This is an issue of high complexity and not necessary for the question of com‐ plicity. In the first context, the principle draws one's attention to several relevant aspects

that may be helpful for our question of complicity in hESC and hiPSC research.

The basis of the argument of complicity with another's wrongdoing is the estimation that somebody, who cooperates in, or profits from the morally reprehensable actions of other

with accepting iPSC research as it is currently done." [13].

tic applications.

quirements are met.

**4.1. Double effect reasoning**

**b.** hESC research including destruction of human embryos is not without alternatives. The promised therapeutic applications of hESC research are still lacking, while research in adult stem cells and hiPSC research seem very promising and are reducing the ethical objections. When opposition to hESC research is still accused of impeding research and preventing necessary new therapies, this could also be seen as a clever policy of small steps to deceive moral convictions. Also other objectives are highly relevant, for exam‐ ple industrial applications in toxicity testing with human embryos as a substitute for animal experiments: "These cell lines may provide more clinically relevant biological systems than animal models for drug testing and are therefore expected to contribute to the development of safer and more effective drugs for human diseases and ultimately to reduce the use of animals. They also offer the possibility to develop better in vitro models to enhance the hazard identification of chemicals. It is possible that these appli‐ cations will turn out to be the major medical impact of human ES cell research..." [66].

## **4. Remaining ethical arguments concerning hiPSCs with special emphasis on the argument of complicity in another's wrongdoing and double effect reasoning**

If it is true that successful therapeutic applications are more likely to result from hiPSC research than from hESC research, ethical problems would be reduced significantly [4, 26, 67, 68]. Research cloning could be avoided. It would never be necessary for therapeutic application. hESC-research would, at least, be reduced to the domain of basic research and control experiments. For this remaining need it seems realistic that already existing cell-lines will be sufficient [12]. In this case, the destruction of human embryos for re‐ search is completely avoidable in the future and even the destruction of surplus human embryos may be unnecessary.

Nevertheless, even in hiPSC-research, some ethical issues remain and are in need of inten‐ sive consideration:

Can the distinction between hESC and hiPSC be explained in a consistent and convincing way? Is it possible to find a reliable delimitation between pluripotent and totipotent stem cells? Is it possible to prevent the production of germ cells out of hiPSCs, as well as their use to create new research-embryos [26]?

Is the assumption that hESC-research is completely dispensable, or will be after a period of time, justified, or is it only a means of sedating the conscience? Some scientist say, that is too early to decide [11]. Even a temporal limited "exception", or a limited number cannot be seen as an exception of ethical principles but must be justified. If further destruction of a limited number of human embryos for research purposes would be necessary during a transition period, some ethicists argue for the use of surplus embryos from IVF-treatment [31]. The ethical objections were indicated above. This way is surely not acceptable, if, according to our appraisal, existing cell lines are sufficient. If not, the use of surplus embryos needs to be justified in a consistent way without denying the human dignity of embryos and without opening the way to the creation of research embryos on demand and even for non-therapeu‐ tic applications.

How can the cell donors' right to voluntary and informed consent, as well as the protection of personal data, especially in the case of application of hiPSCs as disease models, be guar‐ anteed? How can the relevant questions of property rights and patent law be solved [69]?

Even hiPSC-research is, in several ways, confronted with the ethical problem of "complicity in others' wrongdoing": How can someone consistently reject the destruction of human em‐ bryos and, at the same time, use the result of former destructive research [13, 47, 70, 71]? Katrien Devolder draws attention to this problem of complicity. She contradicts the opinion, that hiPSC research is ethically correct, while hESC research is wrong because it involves de‐ struction of human embryos: "Many who object to human embryonic stem cell (hESC) re‐ search because they believe it involves complicity in embryo destruction have welcomed induced pluripotent stem cell (iPSC) research as an ethical alternative. This opinion article aims to show that complicity arguments against hESC research are *prima facie* inconsistent with accepting iPSC research as it is currently done." [13].

In this passage we would like to scrutinize her theses and her suggestions for a solution. We are convinced that the problem of complicity is no obstacle for hiPSC research, if certain re‐ quirements are met.

#### **4.1. Double effect reasoning**

dignity awarded by society, corresponding to actual research interests, serious doubts may arise, whether the desired protection standard can be maintained in other areas of

**b.** hESC research including destruction of human embryos is not without alternatives. The promised therapeutic applications of hESC research are still lacking, while research in adult stem cells and hiPSC research seem very promising and are reducing the ethical objections. When opposition to hESC research is still accused of impeding research and preventing necessary new therapies, this could also be seen as a clever policy of small steps to deceive moral convictions. Also other objectives are highly relevant, for exam‐ ple industrial applications in toxicity testing with human embryos as a substitute for animal experiments: "These cell lines may provide more clinically relevant biological systems than animal models for drug testing and are therefore expected to contribute to the development of safer and more effective drugs for human diseases and ultimately to reduce the use of animals. They also offer the possibility to develop better in vitro models to enhance the hazard identification of chemicals. It is possible that these appli‐ cations will turn out to be the major medical impact of human ES cell research..." [66].

life, e.g. for coma patients, disabled people or new born children.

**4. Remaining ethical arguments concerning hiPSCs with special**

**double effect reasoning**

612 Pluripotent Stem Cells

embryos may be unnecessary.

to create new research-embryos [26]?

sive consideration:

**emphasis on the argument of complicity in another's wrongdoing and**

If it is true that successful therapeutic applications are more likely to result from hiPSC research than from hESC research, ethical problems would be reduced significantly [4, 26, 67, 68]. Research cloning could be avoided. It would never be necessary for therapeutic application. hESC-research would, at least, be reduced to the domain of basic research and control experiments. For this remaining need it seems realistic that already existing cell-lines will be sufficient [12]. In this case, the destruction of human embryos for re‐ search is completely avoidable in the future and even the destruction of surplus human

Nevertheless, even in hiPSC-research, some ethical issues remain and are in need of inten‐

Can the distinction between hESC and hiPSC be explained in a consistent and convincing way? Is it possible to find a reliable delimitation between pluripotent and totipotent stem cells? Is it possible to prevent the production of germ cells out of hiPSCs, as well as their use

Is the assumption that hESC-research is completely dispensable, or will be after a period of time, justified, or is it only a means of sedating the conscience? Some scientist say, that is too early to decide [11]. Even a temporal limited "exception", or a limited number cannot be seen as an exception of ethical principles but must be justified. If further destruction of a limited number of human embryos for research purposes would be necessary during a transition

In theological and philosophical ethics, problems like this (cooperation with another's sin, "cooperatio in malo") can be discussed in relation to the so-called "principle of an action with double effect", in brief "principle of double effect", or "double-effect reasoning" [72-74]. In this principle, a distinction is drawn between direct consequences of an action and side effects, which are only indirectly wanted or accepted as unavoidable. The principle wasn't interpreted and used uniformly and has undergone some changes. In philosophical and theological ethics, it is relevant in two different contexts. The first and original context is the question of cooperating with the sin of another person. In these cases, the wrongness of the action is presupposed and the question concerns only the legitimacy, or culpability of the cooperation. Furthermore the principle of double effect is relevant in the context of some specific moral norms, such as the prohibition of killing to determine moral rightness or wrongness. In these cases it is a principle of restrictive interpretation of deontological moral norms [75]. This is an issue of high complexity and not necessary for the question of com‐ plicity. In the first context, the principle draws one's attention to several relevant aspects that may be helpful for our question of complicity in hESC and hiPSC research.

The basis of the argument of complicity with another's wrongdoing is the estimation that somebody, who cooperates in, or profits from the morally reprehensable actions of other persons, makes himself responsible in a certain way as an accomplice. "Complicity" means a culpable cooperation in the ethically wrong action of another person. The conviction that we are responsible not only for the immediate results of our behaviour, but also for the influ‐ ence we exert by our behaviour on convictions and behaviour of others in the long run, as far as this is foreseeable, is fundamental.

rect causation or a direct intention, in which the wrong action is intended itself (per se), or as a means to an end [80]. In these cases the sin must be seen as directly intended. As a mini‐ mum for speaking of an indirect causation of an evil, it was demanded that good and bad

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In casuistry, further types of a "material" cooperation were distinguished: A *positive* cooper‐ ation by an active action is more serious than a *negative* cooperation by omission of an ac‐ tion. An *immediate* cooperation is more serious than *a mediate.* A *near* cooperation is more serious than a *remote* one. Necessary cooperation, without which the wrong action of anoth‐ er one wouldn't have happened at all, is worse than cooperation, when it would have been performed anyway. A direct intention could be suspected, the more immediate and more near one's own action is connected with another one's sin and the more probably the other one wouldn't sin without this cooperation. Here the principle includes a difficult question: Does the indirectness and justifiability of complicity primarily depend on the causal proxim‐ ity, or on the probability of another person's wrong action? Is it really less problematic to promote a wrong action with high probability, if the number of mediating instances is in‐ creased? In the theological tradition there was no agreement on this matter. According to a teleological method, responsibility refers to all foreseeable consequences that can be influ‐ enced by one's actions. In this point of view, probability is more important than proximity.

consequences must result from the action "at least equal immediately" [73, 74, 78].

For the credibility of the inner consent, proximity may be the greater problem.

the more a cooperation is near, immediately and necessarily.

be necessary to maintain one's credibility.

**4.2. Complicity according to Devolder**

the following variants [13]:

These distinctions show the difficulties in dissociating oneself consistently from another's wrongdoing while cooperating or profiting from it. While the distinction between formal and material cooperation is a clear alternative, the distinctions of types of material coopera‐ tion seems in real life often to be a matter of degree. Al least one could say, that the effort to make one's own inner rejection of anothers' wrongdoing credible to other people is greater,

The principle of double effect includes at least three relevant aspects that may help to evaluate the problem of complicity in hESC and iPSC research: (a) In any case, the rejec‐ tion of another one's action, which one determines as ethically wrong, is required as mat‐ ter of inner consistency. (b) A material cooperation can, nevertheless, be ethically justified, if intention and causal relation can be seen as indirect, which is sometimes clearly identifiable, but is often a matter of degree. (c) In any case, a proportionate reason for accepting the others' sin must be given. Additionally sometimes symbolic actions will

Devolder's statements to complicity partly correspond with these arguments. She introduces

**1.** "Causally contributing": "When I induce or encourage you, or provide you with the means to commit a murder, and as a result you commit it, I am complicit in that mur‐ der." In these cases, the other's wrong action is also the result of one's own action.

Just as the demands of morality are aimed at the inner attitude as well as the outer ac‐ tions of man, accusations of complicity are not only aimed at a voluntary and deliberate cooperation in the wrong actions of others, but also at inadequate attitudes towards the wrong actions of others. Our inner disposition, our fundamental attitude, our character is the central content of our moral obligation. Morality primarily consists in the fundamen‐ tal attitude of impartial benevolence, in the respect for the equal dignity of all humans. Motives cannot be recognized directly but only inferred from our behaviour. Sometimes adequate symbolic actions can help to express the inner attitudes and prevent misunder‐ standings. Symbolic actions partly get credibility by the costs they cause and by the dis‐ advantages somebody is ready to accept [76].

This effort especially is necessary if somebody profits from the wrong actions of others and thus, gives the impression of approval or inner consent of these actions. This can even be the case, if one wasn't involved in the wrong actions at all. The use of research results from mo‐ rally reprehensible experiments in the past [77] without an explicit dissociation can give the impression of lacking sensibility and missing respect for the victims or even the impression of an inner consent, of condoning or justifying these actions. If there are scientific reasons to use the results, the rejection of these crimes must be articulated by explicitly remembering the victims and condemning the crimes.

Complicity with another's wrongdoing can happen in different constellations. In the tradi‐ tion of moral theology, different types of cooperation with the sin of another one were dis‐ tinguished and relevant distinctions were made for the degree of guilt [74, 78, 79].

In any case, the rejection of a sin, a willingly performed wrong action of another person, is required. Complicity, as an inner consent when another one's sin "is wanted as such", is called "*formal*" cooperation and is always wrong. Even an implicit inner consent is seen as a formal cooperation, especially in the case of serious offenses. If the inner consent is missing because the cooperation happens involuntarily or without knowledge, this is called a "*mate‐ rial"* cooperation. However, this kind of cooperation requires a justification, but, in contrast to a formal cooperation, this is possible. According to traditional arguments a material coop‐ eration is permitted, if the other's sin is "wanted only indirectly" and the action corresponds to the rules of the "principle of double effect".

Within the principle of double effect, a distinction is drawn between direct consequences of an action and side effects, which are only indirectly wanted, or accepted as unavoidable. While direct cooperation is regarded as forbidden, the indirect one can be justified by ade‐ quately important, so-called *proportionate reasons* for accepting the others' sin. In this way, teleological reasoning, on the basis of balancing good and bad consequences, is made possi‐ ble for the indirect causation of the others' sin. Nevertheless, this remains excluded for a di‐ rect causation or a direct intention, in which the wrong action is intended itself (per se), or as a means to an end [80]. In these cases the sin must be seen as directly intended. As a mini‐ mum for speaking of an indirect causation of an evil, it was demanded that good and bad consequences must result from the action "at least equal immediately" [73, 74, 78].

In casuistry, further types of a "material" cooperation were distinguished: A *positive* cooper‐ ation by an active action is more serious than a *negative* cooperation by omission of an ac‐ tion. An *immediate* cooperation is more serious than *a mediate.* A *near* cooperation is more serious than a *remote* one. Necessary cooperation, without which the wrong action of anoth‐ er one wouldn't have happened at all, is worse than cooperation, when it would have been performed anyway. A direct intention could be suspected, the more immediate and more near one's own action is connected with another one's sin and the more probably the other one wouldn't sin without this cooperation. Here the principle includes a difficult question: Does the indirectness and justifiability of complicity primarily depend on the causal proxim‐ ity, or on the probability of another person's wrong action? Is it really less problematic to promote a wrong action with high probability, if the number of mediating instances is in‐ creased? In the theological tradition there was no agreement on this matter. According to a teleological method, responsibility refers to all foreseeable consequences that can be influ‐ enced by one's actions. In this point of view, probability is more important than proximity. For the credibility of the inner consent, proximity may be the greater problem.

These distinctions show the difficulties in dissociating oneself consistently from another's wrongdoing while cooperating or profiting from it. While the distinction between formal and material cooperation is a clear alternative, the distinctions of types of material coopera‐ tion seems in real life often to be a matter of degree. Al least one could say, that the effort to make one's own inner rejection of anothers' wrongdoing credible to other people is greater, the more a cooperation is near, immediately and necessarily.

The principle of double effect includes at least three relevant aspects that may help to evaluate the problem of complicity in hESC and iPSC research: (a) In any case, the rejec‐ tion of another one's action, which one determines as ethically wrong, is required as mat‐ ter of inner consistency. (b) A material cooperation can, nevertheless, be ethically justified, if intention and causal relation can be seen as indirect, which is sometimes clearly identifiable, but is often a matter of degree. (c) In any case, a proportionate reason for accepting the others' sin must be given. Additionally sometimes symbolic actions will be necessary to maintain one's credibility.

#### **4.2. Complicity according to Devolder**

persons, makes himself responsible in a certain way as an accomplice. "Complicity" means a culpable cooperation in the ethically wrong action of another person. The conviction that we are responsible not only for the immediate results of our behaviour, but also for the influ‐ ence we exert by our behaviour on convictions and behaviour of others in the long run, as

Just as the demands of morality are aimed at the inner attitude as well as the outer ac‐ tions of man, accusations of complicity are not only aimed at a voluntary and deliberate cooperation in the wrong actions of others, but also at inadequate attitudes towards the wrong actions of others. Our inner disposition, our fundamental attitude, our character is the central content of our moral obligation. Morality primarily consists in the fundamen‐ tal attitude of impartial benevolence, in the respect for the equal dignity of all humans. Motives cannot be recognized directly but only inferred from our behaviour. Sometimes adequate symbolic actions can help to express the inner attitudes and prevent misunder‐ standings. Symbolic actions partly get credibility by the costs they cause and by the dis‐

This effort especially is necessary if somebody profits from the wrong actions of others and thus, gives the impression of approval or inner consent of these actions. This can even be the case, if one wasn't involved in the wrong actions at all. The use of research results from mo‐ rally reprehensible experiments in the past [77] without an explicit dissociation can give the impression of lacking sensibility and missing respect for the victims or even the impression of an inner consent, of condoning or justifying these actions. If there are scientific reasons to use the results, the rejection of these crimes must be articulated by explicitly remembering

Complicity with another's wrongdoing can happen in different constellations. In the tradi‐ tion of moral theology, different types of cooperation with the sin of another one were dis‐

In any case, the rejection of a sin, a willingly performed wrong action of another person, is required. Complicity, as an inner consent when another one's sin "is wanted as such", is called "*formal*" cooperation and is always wrong. Even an implicit inner consent is seen as a formal cooperation, especially in the case of serious offenses. If the inner consent is missing because the cooperation happens involuntarily or without knowledge, this is called a "*mate‐ rial"* cooperation. However, this kind of cooperation requires a justification, but, in contrast to a formal cooperation, this is possible. According to traditional arguments a material coop‐ eration is permitted, if the other's sin is "wanted only indirectly" and the action corresponds

Within the principle of double effect, a distinction is drawn between direct consequences of an action and side effects, which are only indirectly wanted, or accepted as unavoidable. While direct cooperation is regarded as forbidden, the indirect one can be justified by ade‐ quately important, so-called *proportionate reasons* for accepting the others' sin. In this way, teleological reasoning, on the basis of balancing good and bad consequences, is made possi‐ ble for the indirect causation of the others' sin. Nevertheless, this remains excluded for a di‐

tinguished and relevant distinctions were made for the degree of guilt [74, 78, 79].

far as this is foreseeable, is fundamental.

614 Pluripotent Stem Cells

advantages somebody is ready to accept [76].

the victims and condemning the crimes.

to the rules of the "principle of double effect".

Devolder's statements to complicity partly correspond with these arguments. She introduces the following variants [13]:

**1.** "Causally contributing": "When I induce or encourage you, or provide you with the means to commit a murder, and as a result you commit it, I am complicit in that mur‐ der." In these cases, the other's wrong action is also the result of one's own action.

**2.** "Promoting wrongdoing through increasing demand for embryonic stem cell lines": "One can be complicit in wrongdoing by increasing the likelihood of that wrongdoing (or future instances of it) in certain ways, even if one does not in fact cause it."

discarded IVF embryos. Since they are available in a large number, hESC research will not increase the likelihood of embryo-destruction in any way. Of course this objection presup‐ poses the acceptability of the destruction of surplus IVF embryos, which is an open discus‐ sion. In addition to this, the question arises, of whether or not research interests truly have

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Furthermore, Devolder indicates complicity by contributing to altering attitudes in society, changing moral beliefs, legislation or incentives. In this way, the potential benefits of hESC research for many people and the good reputation of biomedical research in general may

Finally, hESC research is accused of "implicitly condoning wrongdoing and disrespecting its victims". If the destruction of embryos is evaluated as a kind of wrongdoing, it is inconsis‐ tent and not credible, when researchers, who benefit from it, would regret or try to distance themselves from the practice of destruction of embryos. By using the stem cell lines, they

hiPSC research enables the development of illness specific or patient specific pluripotent stem cells without supply of oocytes and without the creation and destruction of embryos. Thus, the central ethical objections seem to be removed. Contrary to widespread opinion, Devolders thesis is that, regarding complicity with the destruction of human embryos, hiPSC research is in a similar situation as hESC research. hiPSC research wouldn't be a solu‐ tion for the ethical problems connected to hESC research. She "aims to show that complicity arguments against hESC research are *prima facie* inconsistent with accepting iPSC research as it is currently done." [13]. She suggests that, in a consistent way, both should be accepted or

Devolder accuses hiPSC research of "promoting and condoning embryonic stem cell re‐ search". The connections between hiPSC and hESC research seem to be similar to the con‐ nections between hESC research and embryo destruction: "Research on hESCs arguably promotes embryo destruction through increasing demand; similarly iPSC research arguably promotes hESC research in the same way. Engaging in hESC research arguably also implic‐ itly condones embryo destruction, in part because it involves significant interaction with those who destroy embryos. Engaging in iPSC research involves even more significant inter‐ action with hESC researchers and thus, even more plausibly, implicitly condones hESC re‐ search.... Consistency requires that considerations of complicity are invoked in both cases." [13]. To a great extent, hiPSC research uses results of hESC research and therefore cannot dissociate itself in a credible way from it. It seems to be contributing at least implicitly to weakening the rejection of the destruction of embryos. If hESC research is opposed because of complicity, according to Devolder, even hiPSC research must be seen as highly problem‐

no effect on the production of surplus IVF embryos [71].

seem to condone the way, they were obtained.

atic, unless several modifications are implemented [13].

*4.2.2. Devolder's Criticism of hiPSC research*

rejected.

weaken efforts to reduce the number of embryos discarded in IVF.


In the terminology of theological ethics, paradigms 1-3 refer to different forms of material cooperation. The first includes examples of direct and indirect cooperation specified as near forms of cooperation. Category 2 and 3 are examples of mediate cooperation of a more re‐ mote type, the acceptance of a wrong action as a side effect. One's own action is not suffi‐ cient for the realization of this side effect, but increases its probability in connection with others. In contrast to Devolder, this can also be seen as a kind of causation, but an indirect one. In Example 3, the side effect is a problematic change of social attitudes. This effect is even more remote. The connection is a very complex one. It is unquestionable that research often changes social attitudes. Researchers should think about such consequences, which oc‐ cur as a result of their work. But they aren't alone responsible for it and their actions are sel‐ dom a sufficient condition for a change of social attitudes. Category 4 refers to the appearance of an inner consent, which is called an implicit formal cooperation. Either the actual inner attitude or the publicly noticeable expression is not adequate.

#### *4.2.1. Devolder's criticism of hESC research*

According to Devolder hESC research is confronted with the problem of complicity even if researchers use already existing cell lines and don't themselves destroy human embryos. Even if there is no direct causal contribution, they contribute to an "increasing demand for embryonic cell lines" [13, p 2176] and, in this way, promote the likelihood of "further em‐ bryo destruction" [13, p 2176]. At least at a collective level, this mediate and remote effect is a reality. Presupposition for this criticism is that destroying human embryos is determined as ethically not justified.

A strategy to prevent this contribution is "separating the use of hESCs from their derivation by instituting a cut-off date" [13, p 2176]. This method was used by the jurisdiction in Ger‐ many when trying to deal with the problem in 2002. When the cut-off date was moved in 2007, the credibility of the proclaimed objection to the destruction of embryos was damaged. If the shift of a cut-off date can be anticipated, contribution to an increasing demand is not prevented any more. Devolder emphasizes, that even when using hESCs produced before a cut-off date successful research may promote the destruction of embryos in less restrictive countries. As a counter-argument, she points out that hESC lines are mostly derived from discarded IVF embryos. Since they are available in a large number, hESC research will not increase the likelihood of embryo-destruction in any way. Of course this objection presup‐ poses the acceptability of the destruction of surplus IVF embryos, which is an open discus‐ sion. In addition to this, the question arises, of whether or not research interests truly have no effect on the production of surplus IVF embryos [71].

Furthermore, Devolder indicates complicity by contributing to altering attitudes in society, changing moral beliefs, legislation or incentives. In this way, the potential benefits of hESC research for many people and the good reputation of biomedical research in general may weaken efforts to reduce the number of embryos discarded in IVF.

Finally, hESC research is accused of "implicitly condoning wrongdoing and disrespecting its victims". If the destruction of embryos is evaluated as a kind of wrongdoing, it is inconsis‐ tent and not credible, when researchers, who benefit from it, would regret or try to distance themselves from the practice of destruction of embryos. By using the stem cell lines, they seem to condone the way, they were obtained.

#### *4.2.2. Devolder's Criticism of hiPSC research*

**2.** "Promoting wrongdoing through increasing demand for embryonic stem cell lines": "One can be complicit in wrongdoing by increasing the likelihood of that wrongdoing

**3.** "Promoting wrongdoing through altering attitudes to embryo destruction": Further ways of promoting wrongdoing "include condoning a wrong or fostering more permis‐ sive social attitudes towards it." Profiting from the use of the results of a wrong action can awake the assumption that one excuses this action. This can in the long run weaken

**4.** "Implicitly condoning wrongdoing and disrespecting its victims": Complicity can also be supposed, independent of the consequences, if an implicit excuse of a wrong action,

In the terminology of theological ethics, paradigms 1-3 refer to different forms of material cooperation. The first includes examples of direct and indirect cooperation specified as near forms of cooperation. Category 2 and 3 are examples of mediate cooperation of a more re‐ mote type, the acceptance of a wrong action as a side effect. One's own action is not suffi‐ cient for the realization of this side effect, but increases its probability in connection with others. In contrast to Devolder, this can also be seen as a kind of causation, but an indirect one. In Example 3, the side effect is a problematic change of social attitudes. This effect is even more remote. The connection is a very complex one. It is unquestionable that research often changes social attitudes. Researchers should think about such consequences, which oc‐ cur as a result of their work. But they aren't alone responsible for it and their actions are sel‐ dom a sufficient condition for a change of social attitudes. Category 4 refers to the appearance of an inner consent, which is called an implicit formal cooperation. Either the

According to Devolder hESC research is confronted with the problem of complicity even if researchers use already existing cell lines and don't themselves destroy human embryos. Even if there is no direct causal contribution, they contribute to an "increasing demand for embryonic cell lines" [13, p 2176] and, in this way, promote the likelihood of "further em‐ bryo destruction" [13, p 2176]. At least at a collective level, this mediate and remote effect is a reality. Presupposition for this criticism is that destroying human embryos is determined

A strategy to prevent this contribution is "separating the use of hESCs from their derivation by instituting a cut-off date" [13, p 2176]. This method was used by the jurisdiction in Ger‐ many when trying to deal with the problem in 2002. When the cut-off date was moved in 2007, the credibility of the proclaimed objection to the destruction of embryos was damaged. If the shift of a cut-off date can be anticipated, contribution to an increasing demand is not prevented any more. Devolder emphasizes, that even when using hESCs produced before a cut-off date successful research may promote the destruction of embryos in less restrictive countries. As a counter-argument, she points out that hESC lines are mostly derived from

(or future instances of it) in certain ways, even if one does not in fact cause it."

social attitudes and promote wrong behaviour.

616 Pluripotent Stem Cells

or disrespect towards the victims seems to be expressed.

actual inner attitude or the publicly noticeable expression is not adequate.

*4.2.1. Devolder's criticism of hESC research*

as ethically not justified.

hiPSC research enables the development of illness specific or patient specific pluripotent stem cells without supply of oocytes and without the creation and destruction of embryos. Thus, the central ethical objections seem to be removed. Contrary to widespread opinion, Devolders thesis is that, regarding complicity with the destruction of human embryos, hiPSC research is in a similar situation as hESC research. hiPSC research wouldn't be a solu‐ tion for the ethical problems connected to hESC research. She "aims to show that complicity arguments against hESC research are *prima facie* inconsistent with accepting iPSC research as it is currently done." [13]. She suggests that, in a consistent way, both should be accepted or rejected.

Devolder accuses hiPSC research of "promoting and condoning embryonic stem cell re‐ search". The connections between hiPSC and hESC research seem to be similar to the con‐ nections between hESC research and embryo destruction: "Research on hESCs arguably promotes embryo destruction through increasing demand; similarly iPSC research arguably promotes hESC research in the same way. Engaging in hESC research arguably also implic‐ itly condones embryo destruction, in part because it involves significant interaction with those who destroy embryos. Engaging in iPSC research involves even more significant inter‐ action with hESC researchers and thus, even more plausibly, implicitly condones hESC re‐ search.... Consistency requires that considerations of complicity are invoked in both cases." [13]. To a great extent, hiPSC research uses results of hESC research and therefore cannot dissociate itself in a credible way from it. It seems to be contributing at least implicitly to weakening the rejection of the destruction of embryos. If hESC research is opposed because of complicity, according to Devolder, even hiPSC research must be seen as highly problem‐ atic, unless several modifications are implemented [13].

#### **4.3. Application of double effect reasoning**

The argument of complicity legitimately asks for justification of the involvement of hESC re‐ search and in a more remote way hiPSC research in the destruction of human embryos, even if researchers don't perform it themselves. Double effect reasoning can give some general guidance for performing research with including benefits from objected research in the past and unintended side-effects in the future. Researchers must look back and consider, how they think about the way cell lines, were obtained via the destruction of human embryos in the past. Their research should be in consistency with this judgement. They should also think about their contribution to further destruction of human embryos in the future. They should pay attention to the way their research changes the attitudes of society. Both kinds of consequences are part of the responsibility of researches to the extent they can be foreseen as being in some direct or indirect, close or remote way connected to their scientific work.

Rejection or radical separation of the two research areas are regarded as unappealing by De‐ volder, because this would be connected with considerable disadvantages for research. A complete renunciation would retard important research projects and be a disadvantage for potential patients hoping for new therapies. The renunciation would be a credible sign, but

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A possible solution might be seen in a combination of Devolder's suggestions 2 and 3. The change in the ways hiPSC research is performed could be a radical constraint on the already existing stem cell lines and a credible renunciation of obtaining new stem cell lines, or using new ones from other countries, such as e.g. the European Group on Ethics proposes in its opinion 22: "The derivation of new toti-potent cells or pluri-potent stem cell lines from do‐ nated pre-implantation human embryos or embryonic cells, or via nuclear reprogramming, is not funded by the EU Research Programme." [12]. If existing cell lines are sufficient for the necessary comparison studies, research for therapeutic applications will not be ham‐ pered or retarded any way and no direct or near contribution to further destruction of em‐ bryos is remaining. If applicable regulations were found on a broad basis, protected in a credible way and maintained in the long run, complicity arguments pertaining to embryo destruction in the future wouldn't be applicable anymore to hiPSC research. If, according to the latest reports, the stage of pluripotency were dispensable for therapeutic applications and adult stem cells could be developed into desired cell types without this step [7], even

An important step in the direction of a limitation of research to existing hESC lines is the European registry of existing hESC lines: "The European Commission has therefore decided to establish and fund a European registry for human embryonic stem cell lines in order to help researchers to optimise the hESC resources available, avoid duplication of work and/or the creation of new cell lines where possible." [12]. This kind of policy helps to avoid the new destruction of embryos and enables transparency and credibility. Regulated in such a way hiPSC has a good chance, not to contribute to a weakening of the social sensibility for the victims of research and to changing attitudes to the dignity of human embryos. More

likely it is a step towards the opposite direction of more respect for human dignity.

usage of results out of this kind of rejected research is not secretly hoped for.

Devolder's suggestion 3 and 4 refer to the distinction of causally direct and indirect action. The argument, "that the complicity arguments for rejecting hESC research are stronger than the complicity arguments for rejecting iPSC research" [13] seems appropriate to us. Con‐ forming to the principle of double effect, the distinction between immediate consequences and side effects, which are only wanted or accepted indirectly, opens a way to justify these kind of consequences by proportionate reasons like the high benefit of research for fighting diseases in the future. The remaining indirect and remote contribution to the destruction of embryos can be estimated as balanced as long as it is not actively supported and possible

Of course clarification is needed, which research objectives are regarded as adequately high for the use of hESC lines. Therapeutical applications for humans can be regarded as ade‐ quate, also necessary control experiments for research with adult stem cells or hiPSCs. But serious doubts appear in relation to non-therapeutic industrial applications like toxicity test‐

a burden for others is a problematic proof of one's own integrity.

the control studies with hESCs would become less important.

The possible indirect and more remote consequences of hiPSC research on the destruction of embryos cannot be denied. Who opposes the destruction of embryos for ethical reasons and nevertheless participates in hiPSC research, can be justified in the line of double effect rea‐ soning only, if the rejection of the destruction of embryos and of possible problematic re‐ search in other countries is honest and proven by the attempt to minimize the effect of one's own research on promoting further embryo destruction. This objection should also be made public in some clear and unambiguous way and should be accompanied by institutional or legal precautions to avoid further embryo destruction and weakening of social attitudes. The remaining indirect or remote contributing can be justified, if the benefit of the research is adequately high.

#### **4.4. Consistent solutions?**

Devolder suggests 5 possible solutions [13]:


Rejection or radical separation of the two research areas are regarded as unappealing by De‐ volder, because this would be connected with considerable disadvantages for research. A complete renunciation would retard important research projects and be a disadvantage for potential patients hoping for new therapies. The renunciation would be a credible sign, but a burden for others is a problematic proof of one's own integrity.

**4.3. Application of double effect reasoning**

618 Pluripotent Stem Cells

is adequately high.

ly.

**4.4. Consistent solutions?**

Devolder suggests 5 possible solutions [13]:

**1.** Rejection of hESC research, as well as hiPSC research.

search with stem cells obtained by others.

nent to both ways of research.

The argument of complicity legitimately asks for justification of the involvement of hESC re‐ search and in a more remote way hiPSC research in the destruction of human embryos, even if researchers don't perform it themselves. Double effect reasoning can give some general guidance for performing research with including benefits from objected research in the past and unintended side-effects in the future. Researchers must look back and consider, how they think about the way cell lines, were obtained via the destruction of human embryos in the past. Their research should be in consistency with this judgement. They should also think about their contribution to further destruction of human embryos in the future. They should pay attention to the way their research changes the attitudes of society. Both kinds of consequences are part of the responsibility of researches to the extent they can be foreseen as being in some direct or indirect, close or remote way connected to their scientific work.

The possible indirect and more remote consequences of hiPSC research on the destruction of embryos cannot be denied. Who opposes the destruction of embryos for ethical reasons and nevertheless participates in hiPSC research, can be justified in the line of double effect rea‐ soning only, if the rejection of the destruction of embryos and of possible problematic re‐ search in other countries is honest and proven by the attempt to minimize the effect of one's own research on promoting further embryo destruction. This objection should also be made public in some clear and unambiguous way and should be accompanied by institutional or legal precautions to avoid further embryo destruction and weakening of social attitudes. The remaining indirect or remote contributing can be justified, if the benefit of the research

**2.** Radical separation of the two research areas and "a change in the ways iPSC research is

**3.** One could argue that hiPSC research is considerably more remote from the destruction of human embryos and is, in this respect, less contributing to a weakening of the social sensibility for the victims. In this respect, the "moral costs" could be justified more easi‐

**4.** Complicity arguments could be rejected or limited to cases "when one actually and sig‐ nificantly causally contributes to more embryo deaths", which is not the case for re‐

**5.** The wrongness of the destruction of human embryos for important research areas could be denied. In this case, the discussed complicity arguments would no longer be perti‐

done so that it would no longer involve complicity in hESC research."

A possible solution might be seen in a combination of Devolder's suggestions 2 and 3. The change in the ways hiPSC research is performed could be a radical constraint on the already existing stem cell lines and a credible renunciation of obtaining new stem cell lines, or using new ones from other countries, such as e.g. the European Group on Ethics proposes in its opinion 22: "The derivation of new toti-potent cells or pluri-potent stem cell lines from do‐ nated pre-implantation human embryos or embryonic cells, or via nuclear reprogramming, is not funded by the EU Research Programme." [12]. If existing cell lines are sufficient for the necessary comparison studies, research for therapeutic applications will not be ham‐ pered or retarded any way and no direct or near contribution to further destruction of em‐ bryos is remaining. If applicable regulations were found on a broad basis, protected in a credible way and maintained in the long run, complicity arguments pertaining to embryo destruction in the future wouldn't be applicable anymore to hiPSC research. If, according to the latest reports, the stage of pluripotency were dispensable for therapeutic applications and adult stem cells could be developed into desired cell types without this step [7], even the control studies with hESCs would become less important.

An important step in the direction of a limitation of research to existing hESC lines is the European registry of existing hESC lines: "The European Commission has therefore decided to establish and fund a European registry for human embryonic stem cell lines in order to help researchers to optimise the hESC resources available, avoid duplication of work and/or the creation of new cell lines where possible." [12]. This kind of policy helps to avoid the new destruction of embryos and enables transparency and credibility. Regulated in such a way hiPSC has a good chance, not to contribute to a weakening of the social sensibility for the victims of research and to changing attitudes to the dignity of human embryos. More likely it is a step towards the opposite direction of more respect for human dignity.

Devolder's suggestion 3 and 4 refer to the distinction of causally direct and indirect action. The argument, "that the complicity arguments for rejecting hESC research are stronger than the complicity arguments for rejecting iPSC research" [13] seems appropriate to us. Con‐ forming to the principle of double effect, the distinction between immediate consequences and side effects, which are only wanted or accepted indirectly, opens a way to justify these kind of consequences by proportionate reasons like the high benefit of research for fighting diseases in the future. The remaining indirect and remote contribution to the destruction of embryos can be estimated as balanced as long as it is not actively supported and possible usage of results out of this kind of rejected research is not secretly hoped for.

Of course clarification is needed, which research objectives are regarded as adequately high for the use of hESC lines. Therapeutical applications for humans can be regarded as ade‐ quate, also necessary control experiments for research with adult stem cells or hiPSCs. But serious doubts appear in relation to non-therapeutic industrial applications like toxicity test‐ ing to replace or reduce animal experimentation. Here the opinions are divided and depend‐ ing on the ethical background, using hESCs for applications like these are seen as a welcome improvement by the one side [12, 66], or as a disproportionate means and a way of damag‐ ing human dignity that is not acceptable by the other side. The European Group on Ethics stated clearly: "Although the Group is aware of the importance of respecting animal wel‐ fare, it is concerned that respect for human dignity may not be maintained when hESCs are used in toxicity testing of industrial or other commercially produced chemicals not related to drugs, such as cosmetics, or for replacement of animal testing. Therefore, particular atten‐ tion is to be drawn to this issue." [12, 38, 69, 81]. The demand for further destruction of em‐ bryos would be increasing enormously and one can suppose that social attitudes would really change in the long run, if cell lines derived from human embryos are used as com‐ modity, as raw material in industrial dimensions.

**5. Conclusion**

within a shared basic consensus.

world of future generations" [82].

**Acknowledgements**

A consensus conferring the moral status of human embryos and the ethical evaluation of creating and destructing human embryos hasn't been achieved in the past and doesn't seem probable in the near future. Attempts to justify the destruction of human embryos for re‐ search have not succeeded in answering the ethical objections in a sufficient and convincing way. Since fundamental moral attitudes and convictions are concerned, it is adequate to im‐ pose the burden of proof on those, who advocate these ways of research. Liberty of research

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621

In areas of close scientific cooperation the search for agreement in fundamental ethical questions remains an urgent challenge. In a pluralistic society, despite all efforts for an ethical basic consensus, it is possible that over a longer period of time, a consensus on a certain moral question cannot be found. In such cases, the principle of tolerance is appli‐ cable only if both positions, at least, share a common basis that allows to include the contradicting positions as rational and consistent lines of reasoning. The problem is that the positions regarding the moral status of human embryos don't seem to be reconcilable

In this situation, the only rational way seems to be the renunciation of any further destruc‐ tion of human embryos, a concentration on research with adult stem cells, iPSCs, and, where necessary, with existing hESC-lines. According to the newest developments in stem cell re‐ search, this position doesn't retard research for therapeutic objectives. It has a chance to serve as a minimal consensus and, in the long run, possibly will prove to be the better way, scientifically, ethically, in relation to social acceptability and maybe even economically.

The concern for common and strong ethical standards is part of the external responsibility of science. Science itself is dependent on social agreement and legal certainty and would suffer from a distrust and hostility towards science. In the end, there should be no difference be‐ tween ethical requirements and a science that is striving for an improvement of human liv‐ ing conditions in a sustainable and comprehensive way: "An ethics turned towards the future and a politics of comprehensive ecological, social and humane sustainability are guided by the insight, that there cannot be a double truth. Both, ethics and politics, should be guided by the conviction that in a humane society the moral right in the long run will also be the really beneficial for humans. Though one must realistically anticipate that single groups and perhaps even societies will try to provide themselves with short-term advantag‐ es by overriding ethical boundaries, this won't be to the advantage of most people and the

This work was supported by Austrian Science Fund (FWF) Grant S9302-B05 (to M.B.).

finds its limits where the basic moral convictions of a society are violated.

Devolder's fourth solution, narrowing "complicity" to cases "when one actually and signifi‐ cantly causally contributes to more embryo deaths" [13], is no convenient way. It tends to reduce researchers responsibility too much. Mediate and remote consequences of research are part of the researchers' moral responsibility. Abuse of discoveries and inventions, the promotion of personally rejected methods and applications and even a problematic modifi‐ cation of social attitudes are relevant objects of responsibility, as far as they can be foreseen and are enabled or promoted by one's own activity. Taking responsibility of course doesn't mean being accused for every effect, but being willing to give a justification for accepting unwanted side effects or long term consequences. If appropriate reasons are given, research is justifiable despite these problems. Thus, the principle of double effect opens a way of dealing with negative and unwanted side effects in a responsible way. Research does not justify everything. But complicity is reduced to cases of voluntary and deliberate coopera‐ tion in the actions of others, which one claims to evaluate as morally wrong, (1) when there is formal inner consent, even an implicit one, which is inconsistent, (2) when the cooperation is so near and direct, that an inner rejection is not credible any more, or (3) when the dam‐ age and harm caused by the wrong action is not balanced by a proportionate high benefit.

Devolder's fifth solution shows the necessary precondition for this discussion about com‐ plicity of hiPSC research, the determination of the destruction of human embryos for re‐ search purposes as morally wrong. This judgement mostly corresponds to a personalistic position regarding the moral status of human embryos. Non-personalistic and gradualistic positions don't determine destruction of embryos as morally wrong generally or under spe‐ cific conditions. Of course they don't have a problem with the discussed type of complicity. As indicated in section 2 of this chapter, the ways of justifying the destruction of human em‐ bryos haven't been able to obtain an agreement until now: Denying or weakening of the mo‐ ral status and dignity of early human embryos, of research embryos or at least of surplus IVF-embryos, always contains the risk of weakening this basic ethical argument of equal hu‐ man dignity in general and causing bad effects for humans in other stages of life. The sec‐ ond way, a justification of their destruction, as a legitimate way of killing without denying dignity of human embryos, is not convincing and may cause similar side-effects.

## **5. Conclusion**

ing to replace or reduce animal experimentation. Here the opinions are divided and depend‐ ing on the ethical background, using hESCs for applications like these are seen as a welcome improvement by the one side [12, 66], or as a disproportionate means and a way of damag‐ ing human dignity that is not acceptable by the other side. The European Group on Ethics stated clearly: "Although the Group is aware of the importance of respecting animal wel‐ fare, it is concerned that respect for human dignity may not be maintained when hESCs are used in toxicity testing of industrial or other commercially produced chemicals not related to drugs, such as cosmetics, or for replacement of animal testing. Therefore, particular atten‐ tion is to be drawn to this issue." [12, 38, 69, 81]. The demand for further destruction of em‐ bryos would be increasing enormously and one can suppose that social attitudes would really change in the long run, if cell lines derived from human embryos are used as com‐

Devolder's fourth solution, narrowing "complicity" to cases "when one actually and signifi‐ cantly causally contributes to more embryo deaths" [13], is no convenient way. It tends to reduce researchers responsibility too much. Mediate and remote consequences of research are part of the researchers' moral responsibility. Abuse of discoveries and inventions, the promotion of personally rejected methods and applications and even a problematic modifi‐ cation of social attitudes are relevant objects of responsibility, as far as they can be foreseen and are enabled or promoted by one's own activity. Taking responsibility of course doesn't mean being accused for every effect, but being willing to give a justification for accepting unwanted side effects or long term consequences. If appropriate reasons are given, research is justifiable despite these problems. Thus, the principle of double effect opens a way of dealing with negative and unwanted side effects in a responsible way. Research does not justify everything. But complicity is reduced to cases of voluntary and deliberate coopera‐ tion in the actions of others, which one claims to evaluate as morally wrong, (1) when there is formal inner consent, even an implicit one, which is inconsistent, (2) when the cooperation is so near and direct, that an inner rejection is not credible any more, or (3) when the dam‐ age and harm caused by the wrong action is not balanced by a proportionate high benefit.

Devolder's fifth solution shows the necessary precondition for this discussion about com‐ plicity of hiPSC research, the determination of the destruction of human embryos for re‐ search purposes as morally wrong. This judgement mostly corresponds to a personalistic position regarding the moral status of human embryos. Non-personalistic and gradualistic positions don't determine destruction of embryos as morally wrong generally or under spe‐ cific conditions. Of course they don't have a problem with the discussed type of complicity. As indicated in section 2 of this chapter, the ways of justifying the destruction of human em‐ bryos haven't been able to obtain an agreement until now: Denying or weakening of the mo‐ ral status and dignity of early human embryos, of research embryos or at least of surplus IVF-embryos, always contains the risk of weakening this basic ethical argument of equal hu‐ man dignity in general and causing bad effects for humans in other stages of life. The sec‐ ond way, a justification of their destruction, as a legitimate way of killing without denying

dignity of human embryos, is not convincing and may cause similar side-effects.

modity, as raw material in industrial dimensions.

620 Pluripotent Stem Cells

A consensus conferring the moral status of human embryos and the ethical evaluation of creating and destructing human embryos hasn't been achieved in the past and doesn't seem probable in the near future. Attempts to justify the destruction of human embryos for re‐ search have not succeeded in answering the ethical objections in a sufficient and convincing way. Since fundamental moral attitudes and convictions are concerned, it is adequate to im‐ pose the burden of proof on those, who advocate these ways of research. Liberty of research finds its limits where the basic moral convictions of a society are violated.

In areas of close scientific cooperation the search for agreement in fundamental ethical questions remains an urgent challenge. In a pluralistic society, despite all efforts for an ethical basic consensus, it is possible that over a longer period of time, a consensus on a certain moral question cannot be found. In such cases, the principle of tolerance is appli‐ cable only if both positions, at least, share a common basis that allows to include the contradicting positions as rational and consistent lines of reasoning. The problem is that the positions regarding the moral status of human embryos don't seem to be reconcilable within a shared basic consensus.

In this situation, the only rational way seems to be the renunciation of any further destruc‐ tion of human embryos, a concentration on research with adult stem cells, iPSCs, and, where necessary, with existing hESC-lines. According to the newest developments in stem cell re‐ search, this position doesn't retard research for therapeutic objectives. It has a chance to serve as a minimal consensus and, in the long run, possibly will prove to be the better way, scientifically, ethically, in relation to social acceptability and maybe even economically.

The concern for common and strong ethical standards is part of the external responsibility of science. Science itself is dependent on social agreement and legal certainty and would suffer from a distrust and hostility towards science. In the end, there should be no difference be‐ tween ethical requirements and a science that is striving for an improvement of human liv‐ ing conditions in a sustainable and comprehensive way: "An ethics turned towards the future and a politics of comprehensive ecological, social and humane sustainability are guided by the insight, that there cannot be a double truth. Both, ethics and politics, should be guided by the conviction that in a humane society the moral right in the long run will also be the really beneficial for humans. Though one must realistically anticipate that single groups and perhaps even societies will try to provide themselves with short-term advantag‐ es by overriding ethical boundaries, this won't be to the advantage of most people and the world of future generations" [82].

## **Acknowledgements**

This work was supported by Austrian Science Fund (FWF) Grant S9302-B05 (to M.B.).

## **Author details**

Andreas M. Weiss1 , Michael Breitenbach2 , Mark Rinnerthaler2 and Günter Virt3

1 Department of Practical Theology, University of Salzburg, Austria

2 Department of Cell Biology, University of Salzburg, Austria

3 Department of Moral Theology, University of Vienna, Austria

## **References**

[1] Capecchi MR. Altering the genome by homologous recombination. Science 1989; 244:1288-92.

[11] Hug K, Hermeren G. Do we still need human embryonic stem cells for stem cellbased therapies? Epistemic and ethical aspects. Stem Cell Rev 2011; 7:761-74.

Ethical Considerations on Stem Cell Research

http://dx.doi.org/10.5772/54375

623

[12] Recommendations on the ethical review of hESC FP7 research projects. Opinion No 22.: EGE (The European Group on Ethics in Science and New Technologies to the Eu‐

[13] Devolder K. Complicity in stem cell research: the case of induced pluripotent stem

[14] Yoshida Y, Yamanaka S. Recent stem cell advances: induced pluripotent stem cells for disease modeling and stem cell-based regeneration. Circulation 2010; 122:80-7.

[15] Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH. Viable offspring derived

[16] Gurdon JB, Elsdale TR, Fischberg M. Sexually mature individuals of Xenopus laevis

[17] Cheng LT, Sun LT, Tada T. Genome editing in induced pluripotent stem cells. Genes

[18] Kim YG, Cha J, Chandrasegaran S. Hybrid restriction enzymes: zinc finger fusions to

[19] Sebastiano V, Maeder ML, Angstman JF, Haddad B, Khayter C, Yeo DT, Goodwin MJ, Hawkins JS, Ramirez CL, Batista LF, Artandi SE, Wernig M, Joung JK. In situ ge‐ netic correction of the sickle cell anemia mutation in human induced pluripotent

stem cells using engineered zinc finger nucleases. Stem Cells 2011; 29:1717-26.

[20] Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ. A TALE nuclease architecture for effi‐

[21] Hacein-Bey-Abina S, Hauer J, Lim A, Picard C, Wang GP, Berry CC, Martinache C, Rieux-Laucat F, Latour S, Belohradsky BH, Leiva L, Sorensen R, Debre M, Casanova JL, Blanche S, Durandy A, Bushman FD, Fischer A, Cavazzana-Calvo M. Efficacy of gene therapy for X-linked severe combined immunodeficiency. N Engl J Med

[22] Kaiser J. Gene therapy. Seeking the cause of induced leukemias in X-SCID trial. Sci‐

[23] Chen HF, Chuang CY, Lee WC, Huang HP, Wu HC, Ho HN, Chen YJ, Kuo HC. Sur‐ face marker epithelial cell adhesion molecule and E-cadherin facilitate the identifica‐ tion and selection of induced pluripotent stem cells. Stem Cell Rev 2011; 7:722-35.

[24] Maio G, Hilt A. Der Status des extrakorporalen Embryos im interdisziplinären Zu‐ gang – Grundlagen, Herausforderungen, Ergebnisse. In: Maio G (ed.) Der Status des

from the transplantation of single somatic nuclei. Nature 1958; 182:64-5.

Fok I cleavage domain. Proc Natl Acad Sci U S A 1996; 93:1156-60.

cient genome editing. Nat Biotechnol 2011; 29:143-8.

from fetal and adult mammalian cells. Nature 1997; 385:810-3.

ropean Commission); 2007.

Cells 2012; 17:431-8.

363:355-64.

ence 2003; 299:495.

cells. Hum Reprod 2010; 25:2175-80.


[11] Hug K, Hermeren G. Do we still need human embryonic stem cells for stem cellbased therapies? Epistemic and ethical aspects. Stem Cell Rev 2011; 7:761-74.

**Author details**

622 Pluripotent Stem Cells

Andreas M. Weiss1

**References**

244:1288-92.

78:7634-8.

282:1145-7.

2009; 461:91-4.

Cell Biol 2011; 13:215-22.

Cell 2007; 131:861-72.

, Michael Breitenbach2

2 Department of Cell Biology, University of Salzburg, Austria

3 Department of Moral Theology, University of Vienna, Austria

ic issues revisited. Stem Cells Dev 2010; 19:1121-9.

and induced pluripotent stem cells. Cell Stem Cell 7:249-57.

ty' in stem cell biology. EMBO Mol Med 2012; 4:353-61.

1 Department of Practical Theology, University of Salzburg, Austria

, Mark Rinnerthaler2

[1] Capecchi MR. Altering the genome by homologous recombination. Science 1989;

[2] Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 1981;

[3] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science 1998;

[4] Condic ML, Rao M. Alternative sources of pluripotent stem cells: ethical and scientif‐

[5] Guenther MG, Frampton GM, Soldner F, Hockemeyer D, Mitalipova M, Jaenisch R, Young RA. Chromatin structure and gene expression programs of human embryonic

[6] Boland MJ, Hazen JL, Nazor KL, Rodriguez AR, Gifford W, Martin G, Kupriyanov S, Baldwin KK. Adult mice generated from induced pluripotent stem cells. Nature

[7] Bonfanti P, Barrandon Y, Cossu G. 'Hearts and bones': the ups and downs of 'plastici‐

[8] Efe JA, Hilcove S, Kim J, Zhou H, Ouyang K, Wang G, Chen J, Ding S. Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat

[9] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.

[10] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryon‐

ic and adult fibroblast cultures by defined factors. Cell 2006; 126:663-76.

and Günter Virt3


extrakorporalen Embryos. Perspektiven eines interdisziplinären Zugangs (Medizin und Philosophie 9). Stuttgart/Bad Cannstadt: frommann-holzboog; 2007. p 11-44.

[38] Directive 98/44/EC OF THE on the legal protection of biotechnological inventions.

Ethical Considerations on Stem Cell Research

http://dx.doi.org/10.5772/54375

625

[39] Ethical Aspects of Human Stem Cell Research and Use. Opinion No 15. EGE (The Eu‐ ropean Group on Ethics in Science and New Technologies to the European Commis‐

[40] Kant I. Groundwork of the Metaphysic of Morals, translated by James W. Ellington.

[41] Wolbert W. Der Mensch als Mittel und Zweck. Die Idee der Menschenwürde in nor‐

[42] Wolbert W. Du sollst nicht töten. Systematische Überlegungen zum Tötungsverbot.

[43] Knoepffler N. Der Beginn der menschlichen Person und bioethische Konfliktfälle.

[44] Damschen G, Schönecker D (eds.) Der moralische Status menschlicher Embryonen. Pro und contra Spezies-, Kontinuums-, Identitäts- und Potentialitätsargument. In:

[45] Düwell M. Der moralische Status von Embryonen und Feten. In: Düwell M, Steigled‐

[46] Oduncu FS. Moralischer Status von Embryonen. In: Düwell M, Steigleder K (eds.) Bi‐

[47] Maio G. Mittelpunkt Mensch. Ethik in der Medizin. Ein Lehrbuch. Stuttgart: Schatta‐

[48] Congregation for the Doctrine of the Faith, Instruction on respect for human life in its origin and on the dignity of procreation – Donum vitae (February 22, 1987). Donum

[49] Congregation for the Doctrine of the Faith, Instruction Dignitas Personae on Certain Bioethical Questions (September 8, 2008). Dignitas personae AAS 100; 2008. p

[50] Bormann FJ. Embryonen, Menschen und die Stammzellforschung. Plädoyer für eine differenzierte Identitätsthese in der Statusfrage. In: Theologie und Philosophie. 2002.

[51] Virt G. Verantwortung für das Menschenleben an seinem Beginn. In: Marschütz G, Prüller-Jagenteufel G (eds.) Damit Menschsein Zukunft hat. Theologische Ethik im Einsatz für eine humane Gesellschaft. Würzburg: Echter Verlag; 2007. p 170-186.

[52] Schockenhoff E. Ethik des Lebens. Grundlagen und neue Herausforderungen. Frei‐

Freiburg i.Ue./Freiburg i. Br.: Herder/Academic Press Fribourg; 2009.

er K (eds.) Bioethik. Eine Einführung. Frankfurt 2003. p 221-229.

oethik. Eine Einführung. Frankfurt: Suhrkamp; 2003. p 213-220.

mativer Ethik und Metaethik. Münster: Aschendorff; 1987.

Anfragen an das Lehramt. Freiburg i. Br.: Herder; 2012.

Berlin/New York: Walter de Gruyter; 2003.

European Parliament and the Council; 1998.

sion); 2000.

Hackett; 1785/1993.

uer; 2012. p 201-220.

858-887.

p 216-232.

vitae AAS 80; 1987. p 70-102.

burg/Basel/Wien: Herder; 2009.


[38] Directive 98/44/EC OF THE on the legal protection of biotechnological inventions. European Parliament and the Council; 1998.

extrakorporalen Embryos. Perspektiven eines interdisziplinären Zugangs (Medizin und Philosophie 9). Stuttgart/Bad Cannstadt: frommann-holzboog; 2007. p 11-44.

[25] Maio G. Der Status des extrakorporalen Embryos. Perspektiven eines interdisziplinä‐ ren Zugangs (Medizin und Philosophie 9). Stuttgart/Bad Cannstadt: frommann-holz‐

[26] Kummer C. Induzierte pluripotente Stammzellen und Totipotenz. Die Bedeutung der Reprogrammierbarkeit von Körperzellen für die Potentialitätsproblematik in der Stammzellforschung. In: Hilpert K (ed.) Forschung contra Lebensschutz. Der Streit um die Stammzellforschung (Quaestiones disputatae 233). Freiburg i. Br./Basel/Wien:

[27] Holland S, Lebacqz K, Zoloth L editors. The Human Embryonic Stem Cell Debate. Science Ethics and Public Policy. Cambridge Mass: Massachusetts Institute of Tech‐

[28] Lenzen W (ed.) Wie bestimmt man den "moralischen Status" von Embryonen? Pader‐

[29] Wagner-Westerhausen K. Die Statusfrage in der Bioethik (Ethik in der Praxis, Kon‐

[30] Ostnor L. Stem Cells, Human Embryos and Ethics. Interdisciplinary Perspectives.

[31] Research on Human Embryonic Stem Cells. Opinion of the Austrian Bioethics Com‐

[32] Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine (Convention on Human

[33] Additional Protocol to the Convention for the Protection of Human Rights and Dig‐ nity of the Human Being with regard to the Application of Biology and Medicine, on

[34] Cloning in Biomedical Research and Reproduction. Scientific Aspects – Ethical, Legal

[35] Virt G. Zur ethischen Debatte um das Klonen. In: Marschütz G, Prüller-Jagenteufel G (eds.) Damit Menschsein Zukunft hat. Theologische Ethik im Einsatz für eine hu‐

[36] Stammzellenforschung und therapeutisches Klonen. Göttingen: Vandenhoeck und

[37] Human Cloning and Human Dignity. With a foreword by Leon R. Kass and M.D. Chairman. New York: Public Affairs: The Report of the President's Council on Bio‐

the Prohibition of Cloning Human Beings. Paris: Council of Europe 1998.

boog; 2007.

624 Pluripotent Stem Cells

nology; 2001.

born: 2004.

Oslo: Springer; 2008.

Ruprecht; 2002.

ethics 2002.

Herder; 2009. p 322-338.

troversen 26). Münster: LIT-Verlag; 2008.

mission. Wien: Austrian Bioethics Commission 2009

Rights and Biomedicine). Oviedo: Council of Europe; 1997.

and Social Limits. Bonn: Bonn University Press; 2003.

mane Gesellschaft. Würzburg: Echter Verlag; 2007. p 196-203.


[53] Virt G. Die Spaltung des menschlichen im Horizont der modernen Lebenswissen‐ schaften. In: Oduncu FS, Schroth U, Vossenkuhl W (eds.) Stammzellenforschung und therapeutisches Klonen. Göttingen: Vandenhoeck und Ruprecht; 2002. p 201-210.

[65] Morscher E. Why is it morally wrong to clone a human being? How to evaluate argu‐ ments of biopolitics, biomorality, and bioethics. In: Thiele F, Ashcroft RE (eds.) Bio‐

Ethical Considerations on Stem Cell Research

http://dx.doi.org/10.5772/54375

627

[66] Commission Staff Working Paper: Report on Human Embryonic Stem Cell Research.

[67] Müller AM, Obier N, Choi SW, Li X, Dinger TC, Brousos N. Möglichkeiten und Chancen der Stammzellenforschung: Stammzellen für Alle? In: Hilpert K (ed.) For‐ schung contra Lebensschutz. Der Streit um die Stammzellforschung (Quaestiones

[68] Breitenbach M, Laun P. Einige biologische Grundlagen der modernen Reproduktion‐ smedizin und der Stammzell- bzw. Gentherapie. In: Fischer M, Zänker KS (eds.) Medizin- und Bioethik (Ethik transdisziplinär 1). Frankfurt: Peter Lang; 2006. p

[69] Ethical Aspects of Patenting Inventions involving Human Stem Cells. Opinion No 16. EGE (The European Group on Ethics in Science and New Technologies to the Euro‐

[70] Green RM. The human embryo research debates. Oxford: Oxford University Press;

[71] Wolbert W. Zum Vorwurf der Doppelmoral in der Diskussion um die embryonale Stammzellforschung. Deutsche Medizinische Wochenschrift 2003; 128:453-456.

[72] Cavanaugh TA. Double Effect reasoning. A Critique and Defense. Dissertation. Uni‐

[73] Kaczor C. Proportionalism and the Natural Law Tradition. Washington: The Catholic

[74] Weiss AM. Sittlicher Wert und nichtsittliche Werte. Zur Relevanz der Unterschei‐ dung in der moraltheologischen Diskussion um deontologische Normen (Studien zur theologischen Ethik 73). Freiburg i. Ue./Freiburg i. Br.: Universitätsverlag Frei‐

[75] Schüller B. Die Begründung sittlicher Urteile. Typen ethischer Argumentation in der

[76] Ginters R. Die Ausdruckshandlung. Eine Untersuchung ihrer sittlichen Bedeutsam‐ keit (Moraltheologische Studien. Systematische Abteilung 4). Düsseldorf: Patmos

[77] Fragwürdige Medizin. Unmoralische Forschung in Deutschland, Japan und den USA

[78] Mausbach J. Katholische Moraltheologie, überarbeitet von G. Ermecke. Münster:

ethics in a Small World. Berlin-Heidelberg Springer; 2005. p 121-128.

("Matthiessen report"). Commission of the European Union; 2003.

disputatae 233). Freiburg i. Br. / Basel / Wien: Herder; 2009. p 30-44.

29-50.

2001.

pean Commission); 2002

versity of Notre Dame: 1995.

burg/Herder; 1996.

Verlag; 1976.

Aschendorff; 1959.

University of America Press; 2002.

Moraltheologie. Düsseldorf: Patmos Verlag 1987.

im 20. Jahrhundert. Frankfurt a. M.: Campus Verlag; 2008.


[65] Morscher E. Why is it morally wrong to clone a human being? How to evaluate argu‐ ments of biopolitics, biomorality, and bioethics. In: Thiele F, Ashcroft RE (eds.) Bio‐ ethics in a Small World. Berlin-Heidelberg Springer; 2005. p 121-128.

[53] Virt G. Die Spaltung des menschlichen im Horizont der modernen Lebenswissen‐ schaften. In: Oduncu FS, Schroth U, Vossenkuhl W (eds.) Stammzellenforschung und therapeutisches Klonen. Göttingen: Vandenhoeck und Ruprecht; 2002. p 201-210.

[55] Körtner HJ, Bünker M. Verantwortung für das Leben. Eine evangelische Denkschrift zu Fragen der Biomedizin. Im Auftrag des Evangelischen Oberkirchenrats A. und H.B. der Evangelischen Kirche A. und H.B. in Österreich erarbeitet von Ulrich H. J.

[57] Vieth A. Einführung in die Angewandte Ethik. Darmstadt Wissenschaftliche Buchge‐

[58] Wiesemann C. Von der Verantwortung, ein Kind zu bekommen. Eine Ethik der El‐

[59] Beckmann JP. Ontologische Status- oder pragmatische Umgangsanalyse? Zur Ergän‐ zungsbedürftigkeit des Fragens nach dem Seinsstatus des extrakorporalen frühen menschlichen Embryos in ethischen Analysen. In: Maio G (ed.) Der Status des extra‐ korporalen Embryos. Perspektiven eines interdisziplinären Zugangs (Medizin und

Philosophie 9). Stuttgart/Bad Cannstadt: frommann-holzboog; 2007. p 275-304.

[60] Koch HG. Disziplinspezifische Vorannahmen: Intrinsische und extrinsische Status‐ bestimmung des extrakorporalen Embryos – Vermittlung und Kombinatorik der ver‐ schiedenen Kriterien. In: Maio G (ed.) Der Status des extrakorporalen Embryos. Perspektiven eines interdisziplinären Zugangs (Medizin und Philosophie 9). Stutt‐

[61] Körtner UHJ. Embryonenschutz und Embryonenforschung aus der Sicht evangel‐ ischer Theologie. In: Körtner UHJ, Kopetzki C (eds.) Embryonenschutz – Hemm‐

[62] Dabrock P, Klinnert P. Verbrauchende Embryonenforschung. Kommt allen Embry‐ onen Menschenwürde zu? In: Dabrock P, Klinnert P, Schardien S (eds.) Menschen‐ würde und Lebensschutz. Herausforderungen theologischer Bioethik. Gütersloh:

[63] Weiss AM. Moralischer Status von Embryonen – Hindernis für die Forschung? An‐ merkungen zu Thesen von Ulrich Körtner. In: Fischer M, Zänker KS (eds.) Medizin-

und Bioethik (Ethik transdisziplinär 1). Frankfurt: Peter Lang; 2006. p 117-138.

[64] Weiss AM. Abwägung im Tötungsverbot? Zur Kontroverse um die Forschung an überzähligen Embryonen. In: Haering S, Hirnsberger J, Katzinger G, Rees W (eds.) In mandatis meditari. FS f. Hans Paarhammer zum 65. Geburtstag. Berlin: Duncker &

[54] Singer P. Practical Ethics. Cambridge: Cambridge University Press; 1993.

Körtner in Zusammenarbeit mit Michael Bünker. Wien: 2001.

[56] Fischer J. Vom Etwas zum Jemand. Zeitzeichen 2002; 3(1) 13. 2002.

sellschaft; 2006.

626 Pluripotent Stem Cells

ternschaft. München: C.H. Beck; 2006.

gart/Bad Cannstadt: frommann-holzboog; 2007.

Gütersloher Verlagshaus; 2004. p 173-210.

Humblot; 2012. p 387-402.

schuh für die Biomedizin? Wien: Springer; 2003. p 84-111.


[79] Göpfert FA. Moraltheologie. Paderborn: Schöningh; 1905.

sellschaft. Würzburg: Echter Verlag; 2007. p 106-114.

ington D.C.: Georgetown University Press; 1986.

ropean Union 2011.

628 Pluripotent Stem Cells

[80] Schüller B. Wholly Human. Essays on the Theory and Language of Morality. Wash‐

[81] Judgement in case C-34/10 Oliver Brüstle vs. Greenpeace. Court of Justice of the Eu‐

[82] Virt G. Zukunftswelten – Lebenswelten. In: Marschütz G, Prüller-Jagenteufel G (eds.) Damit Menschsein Zukunft hat. Theologische Ethik im Einsatz für eine humane Ge‐

## *Edited by Deepa Bhartiya and Nibedita Lenka*

Stem cells have generated a lot of excitement among the researchers, clinicians and the public alike. Various types of stem cells are being evaluated for their regenerative potential. Marginal benefit resulting by transplanting autologus stem cells (deemed to be absolutely safe) in various clinical conditions has been proposed to be a growth factor effect rather than true regeneration. In contrast, various pre-clinical studies have been undertaken, using differentiated cells from embryonic stem cells or induced pluripotent stem cells have shown promise, functional improvement and no signs of teratoma formation. The scientists are not in a rush to reach the clinic but a handful of clinical studies have shown promise. This book is a collection of studies/reviews, beginning with an introduction to the pluripotent stem cells and covering various aspects like derivation, differentiation, ethics, etc., and hence would provide insight into the recent standing on the pluripotent stem cells biology. The chapters have been categorized into three sections, covering subjects ranging from the generation of pluripotent stem cells and various means of their derivation from embryonic as well as adult tissues, the mechanistic understanding of pluripotency and narrating the potential therapeutic implications of these in vitro generated cells in various diseases, in addition to the associated pros and cons in the same.

Photo by selvanegra / iStock

Pluripotent Stem Cells

Pluripotent Stem Cells

*Edited by Deepa Bhartiya and Nibedita Lenka*