Preface

Chapter 6 **Cadherin-Fc Chimeric Protein-Based Biomaterials: Advancing**

Chapter 8 **Human Pluripotent Stem Cell Applications in Drug Discovery**

Shiva Prasad Potta, Tomo Šarić, Michael Heke, Harinath

Hideyuki Kobayashi, Toshihiro Tai, Koichi Nagao and Koichi

**and Toxicology – An overview 181**

Bahudhanapati and Jürgen Hescheler

Chapter 10 **Ethical Implications of Embryo Adoption 213**

Chapter 9 **The Minipig — A New Tool in Stem Cell Research 197**

**Application 137**

**VI** Contents

**Section 4 New Stem Cell Models 179**

Nakajima

Peter A. Clark

**Section 5 Ethics 211**

Chapter 7 **Hepatocyte Selection Medium 165**

**Stem Cell Technology and Regenerative Medicine Towards**

Minoru Tomizawa, Fuminobu Shinozaki, Yasufumi Motoyoshi, Takao Sugiyama, Shigenori Yamamoto and Makoto Sueishi

Kakon Nag, Nihad Adnan, Koichi Kutsuzawa and Toshihiro Akaike

Great progress continues to be made in our understanding of stem cell self-renewal and plu‐ ripotency, and the utilization of stem cells for basic and applied applications. This book em‐ bodies recent advances in the biological mechanisms, methods and models of stem cell selfrenewal, reprograming and regeneration, as well as touching on the ethical and moral dilemmas of embryo donation and adoption. In the first section of this book, 'Stem Cell Self-Renewal and Pluripotency', Agarwal and Zambidis examine the role of the NFκB-STAT3 signaling axis in regulating the induction and maintenance of the pluripotent state. The au‐ thors discuss a novel link between inflammatory pathways and efficient cell reprogram‐ ming, whereby bone marrow stromal-primed human myeloid cell progenitors are significantly more receptive to reprogramming stimuli than other cell types. Myeloid cells appear to harbor a unique epigenetic plasticity and are innately equipped to transcriptional‐ ly and epigenetically activate key inflammatory pathways via an interconnected NFκB and STAT3 signaling machinery. Both pathways act as epigenetic modifiers which promote ESC pluripotency by inducing an open chromatin state that allows transcription factors to regu‐ late cell fates. The importance of the NuRD complex in ensuring that differentiated cells do not reactivate pluripotency genes, which might enable tumorigenesis – is also discussed in this first chapter. The following chapter by Koide examines further the idea that many genes involved in ESC self-renewal also are involved in cancer cell growth. Considerable evidence supports the author's contentions, where self-renewal genes such as Oct 3/4, Sox2, Nanog, STAT3, Klf4 and Zfp57 are not only required for self-renewal in ESCs, but also are highly expressed in neoplastic cells. Conversely, there is evidence that oncogenes also are ex‐ pressed in SCs. That several common transcription factors are involved in the regulation of ESC self-renewal and cancer cell growth raises the intriguing possibility that these common transcription factors are specifically expressed in cancer stem cells involved in tumor growth. Moreover, the authors suggest that since ESCs and induced pluripotent stem cells (iPSCs) have similar gene expression profiles, tumor risk also might be elevated for iPSCs used for therapeutic purposes. As suggested by Agarwal and Zambidis, preventing cancer‐ ous epigenetic patterns in iPSC via more accurate high-fidelity reprogramming methods will be the foundation for future clinical applications.

In the next section on 'Haematopoiesis', Stefanska and colleagues review how ESCs have been used to study the development of the haematopoietic system, that is otherwise very difficult to study in vivo. As the authors write, 'ESCs have been instrumental in identifying and characterizing the elusive haemangioblast …. and more recently, this model system has allowed the merging of two conflicting theories of the origin of blood cells (haemangioblast and haemogenic endothelium) into a single linear model of development'. In addition, the precise roles and requirements of many critical regulators of this process have been elucidat‐ ed using this model system. This system offers many opportunities to further study cell sig‐ naling pathways that support the development of normal haematopoiesis and leukaemogenesis. Compared with the murine haematopoietic system, however, little is known regarding the molecular and cellular regulation of early hematopoiesis in the human as pointed out in the next review by Chen and colleagues. These researchers have establish‐ ed an efficient method to induce large-scale production of multipotential hematopoietic pro‐ genitor cells by co-culturing hESC/hiPSCs with murine hematopoietic niche-derived stromal cells. Their review discusses the origin, evolution and the development of both primitive and definitive hematopoietic waves, especially those derived from hESCs in vitro systems. They also summarize the cellular and molecular characteristics of cells in primitive and/or definitive hematopoiesis as well as the critical problems and challenges facing scientists working in this important area of research. With the advent of these novel hESC/hiPSCs, our understanding of human haematopoietic development should proceed swiftly.

In the final section on 'Ethics', Clark and colleagues remind us of the medical, legal and ethi‐ cal dilemmas associated with embryo donation/adoption. They contend that allowing for embryo donation/adoption is the only viable option that protects and preserves their human life. The other viable options: being discarded, destroyed for research, abandoned or kept in "suspended animation" indefinitely, are unacceptable because they have the potential of harming or intentionally killing these embryos that deserve special respect. To avoid these later situations, Clark puts forth a number of recommendations and safeguards involving: the matching of eggs to be fertilized with those placed in the uterus of the mother, the enact‐ ment of laws at the federal level that regulates Assisted Reproductive Technologies and that regulates the creation, destruction and exploitation of human embryos, that infertile couples and individuals willing to take full responsibility for the upbringing of these children should be encouraged to consider adoption of the presently existing frozen embryos, and that children who are adopted from frozen embryos have the right to know their genetic

The reviews in this book are a reminder of the rapid progress being made in our under‐ standing of stem cell self-renewal and pluripotency, the methodological advances in the cul‐ ture, purification and use of stem cells, and how this basic knowledge and methodological advances can be utilized for future regenerative medicine, drug screening and other applica‐

> **Craig S. Atwood and Sivan Vadakkadath Meethal** Geriatric Research, Education and Clinical Center,

> > Veterans Administration Hospital,

Department of Medicine, University of Wisconsin,

Madison, USA

Preface IX

make-up.

tions of medical benefit.

Major discoveries in the regulation of self-renewal, pluripotency and differentiation are de‐ pendent upon technical developments in the field. In this next section on the 'Technical Ad‐ vances in the Culture and Use of Induced Pluripotent and Embryonic Stem Cells', three research groups present advances and applications of hESCs/iPSCs. Nishishita and collea‐ gues demonstrate a robust, low-cost, stable method for generating and maintaining iPSC clones from cord blood (CB) cells. This feeder-free and serum-free method that utilizes a temperature sensitive Sendai virus vector also solves some of the safety concerns related to tumorigenicity arising from chromosomal integration of exogenous genes and/or infectious hazards associated with the use of by xenogeneic biological products in the culture system. The use of CB cells, the youngest somatic cells, is suggested to alleviate concerns regarding post-natal DNA damage and the ability to cryopreserve CB HSCs long-term in bank confers a unique advantage to CB cells as a suitable material for generating induced pluripotent stem (iPSC) cells for future clinical use. In the next chapter, Nag and colleagues review their development of cell recognizable Fc-chimeric proteins aimed at providing simultaneous support for cell adhesion, proliferation and differentiation as a highly effective novel class of defined biomaterials for stem cell applications. These biomaterials have application in re‐ generative medicine and tissue engineering, including feeder-cell free ESC culture, a simpli‐ fied and cost-effective culture system for stem cells, directed differentiation of stem cells, and on-site stress-free purification of target cells. In the final chapter of this section, Tomiza‐ wa and colleagues describe the development of a hepatocyte selection media that can be used to select hepatoblast-like cells from ESC and other cells. This clever approach utilizes differences in arginine and glucose metabolism between SCs and differentiated hepatocytes to eliminate (induce death of) hiPSCs, for example, and the isolation of differentiated hepa‐ tocytes. This methodology avoids the damage introduced by other selection protocols and is an important contribution to the development of pure hepatocytes for culture or applied ap‐ plications.

The next two chapters examine 'New Stem Cell Models' in two divergent systems. The first, by Potta and colleagues reviews the potential of using stem cells for evaluating the safety of drugs. Drug discovery programs often utilize animals to test the efficacy and safety of new drugs, but the results from such experiments cannot always be extrapolated to humans. This chapter reviews the development of iPSCs as a novel and cost-effective source of organotyp‐ ic cells for assessing drug toxicity. Kobayashi and colleagues review the use and challenges of using domestic pigs and the minipig to both derive and utilize SCs.

In the final section on 'Ethics', Clark and colleagues remind us of the medical, legal and ethi‐ cal dilemmas associated with embryo donation/adoption. They contend that allowing for embryo donation/adoption is the only viable option that protects and preserves their human life. The other viable options: being discarded, destroyed for research, abandoned or kept in "suspended animation" indefinitely, are unacceptable because they have the potential of harming or intentionally killing these embryos that deserve special respect. To avoid these later situations, Clark puts forth a number of recommendations and safeguards involving: the matching of eggs to be fertilized with those placed in the uterus of the mother, the enact‐ ment of laws at the federal level that regulates Assisted Reproductive Technologies and that regulates the creation, destruction and exploitation of human embryos, that infertile couples and individuals willing to take full responsibility for the upbringing of these children should be encouraged to consider adoption of the presently existing frozen embryos, and that children who are adopted from frozen embryos have the right to know their genetic make-up.

ed using this model system. This system offers many opportunities to further study cell sig‐ naling pathways that support the development of normal haematopoiesis and leukaemogenesis. Compared with the murine haematopoietic system, however, little is known regarding the molecular and cellular regulation of early hematopoiesis in the human as pointed out in the next review by Chen and colleagues. These researchers have establish‐ ed an efficient method to induce large-scale production of multipotential hematopoietic pro‐ genitor cells by co-culturing hESC/hiPSCs with murine hematopoietic niche-derived stromal cells. Their review discusses the origin, evolution and the development of both primitive and definitive hematopoietic waves, especially those derived from hESCs in vitro systems. They also summarize the cellular and molecular characteristics of cells in primitive and/or definitive hematopoiesis as well as the critical problems and challenges facing scientists working in this important area of research. With the advent of these novel hESC/hiPSCs, our

understanding of human haematopoietic development should proceed swiftly.

plications.

VIII Preface

Major discoveries in the regulation of self-renewal, pluripotency and differentiation are de‐ pendent upon technical developments in the field. In this next section on the 'Technical Ad‐ vances in the Culture and Use of Induced Pluripotent and Embryonic Stem Cells', three research groups present advances and applications of hESCs/iPSCs. Nishishita and collea‐ gues demonstrate a robust, low-cost, stable method for generating and maintaining iPSC clones from cord blood (CB) cells. This feeder-free and serum-free method that utilizes a temperature sensitive Sendai virus vector also solves some of the safety concerns related to tumorigenicity arising from chromosomal integration of exogenous genes and/or infectious hazards associated with the use of by xenogeneic biological products in the culture system. The use of CB cells, the youngest somatic cells, is suggested to alleviate concerns regarding post-natal DNA damage and the ability to cryopreserve CB HSCs long-term in bank confers a unique advantage to CB cells as a suitable material for generating induced pluripotent stem (iPSC) cells for future clinical use. In the next chapter, Nag and colleagues review their development of cell recognizable Fc-chimeric proteins aimed at providing simultaneous support for cell adhesion, proliferation and differentiation as a highly effective novel class of defined biomaterials for stem cell applications. These biomaterials have application in re‐ generative medicine and tissue engineering, including feeder-cell free ESC culture, a simpli‐ fied and cost-effective culture system for stem cells, directed differentiation of stem cells, and on-site stress-free purification of target cells. In the final chapter of this section, Tomiza‐ wa and colleagues describe the development of a hepatocyte selection media that can be used to select hepatoblast-like cells from ESC and other cells. This clever approach utilizes differences in arginine and glucose metabolism between SCs and differentiated hepatocytes to eliminate (induce death of) hiPSCs, for example, and the isolation of differentiated hepa‐ tocytes. This methodology avoids the damage introduced by other selection protocols and is an important contribution to the development of pure hepatocytes for culture or applied ap‐

The next two chapters examine 'New Stem Cell Models' in two divergent systems. The first, by Potta and colleagues reviews the potential of using stem cells for evaluating the safety of drugs. Drug discovery programs often utilize animals to test the efficacy and safety of new drugs, but the results from such experiments cannot always be extrapolated to humans. This chapter reviews the development of iPSCs as a novel and cost-effective source of organotyp‐ ic cells for assessing drug toxicity. Kobayashi and colleagues review the use and challenges

of using domestic pigs and the minipig to both derive and utilize SCs.

The reviews in this book are a reminder of the rapid progress being made in our under‐ standing of stem cell self-renewal and pluripotency, the methodological advances in the cul‐ ture, purification and use of stem cells, and how this basic knowledge and methodological advances can be utilized for future regenerative medicine, drug screening and other applica‐ tions of medical benefit.

#### **Craig S. Atwood and Sivan Vadakkadath Meethal**

Geriatric Research, Education and Clinical Center, Veterans Administration Hospital, Department of Medicine, University of Wisconsin, Madison, USA

**Section 1**

**Stem Cell Self-Renewal and Pluripotency**

**Stem Cell Self-Renewal and Pluripotency**

**Chapter 1**

**The Role of an NFκB-STAT3 Signaling Axis in Regulating**

**the Induction and Maintenance of the Pluripotent State**

Induced pluripotent stem cells (iPSC) are generated by reprogramming differentiated somatic cells to a pluripotent cell state that highly resembles embryonic stem cells (ESC) [1]. Fully reprogrammed iPSC can differentiate into any adult cell type [2-6]. Takahashi and Yamanaka generated the first iPSC in 2006 by transfecting fibroblasts with four defined factors: SOX2, OCT4, KLF4, c-MYC (SOKM; also referred to as Yamanaka factors) [7]. The clinical use of iPSC offers great potential for regenerative medicine as any cell type can be generated from true pluripotent cells [8-10]. However, human clinical iPSC applications are currently limited by inefficient methods of reprogramming that often generate incompletely reprogrammed pluripotent states that harbor potentially cancerous epigenetic signatures, and possess limited or skewed differentiation capacities [11-13]. Many standard iPSC lines do not fully resemble pluripotent ESC, and often retain an epigenetic memory of their cell of origin [14, 15]. Such incompletely reprogrammed iPSC also display limited differentiation potential to all three

To avoid integrating retroviral constructs that may carry mutagenic risks, many non-viral methods have been described for hiPSC derivation [18, 19]. For example, one successful approach is to transiently express reprogramming factors with EBNA1-based episomal vectors [20-22]. It was initially intuitive to reprogram skin fibroblasts due to their easy accessibility. However, standard episomal reprogramming in fibroblasts occurs at even lower efficiencies (< 0.001-0.1%) than reprogramming with retroviral vectors (0.1%–1%) [23-25]. Subsequent studies revealed that various cell types possess differential receptive‐ ness for being reprogrammed to pluripotency [26-30]. One highly accessible human donor source is blood, which has been demonstrated to reprogram with significantly greater

> © 2014 The Author(s). Licensee InTech. This chapter is 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.

Jasmin Roya Agarwal and Elias T. Zambidis

Additional information is available at the end of the chapter

germ layers (e.g., endoderm, ectoderm, mesoderm) [16, 17].

efficiency than fibroblasts [4, 20, 31-33].

http://dx.doi.org/10.5772/57602

**1. Introduction**

**Chapter 1**
