**Meet the editor**

Kamran Alimoghaddam, M.D. was born in 1964 in Tehran (Iran), and his primary research field is in hematology, oncology and bone marrow transplantation. His past positions include: Internship at Hamadan University of Medical sciences, Hamadan (1990-1992); Residency of Internal Medicine at Tehran University of Medical Sciences (1992-1995); Fellowship of Hematolo-

gy, Oncology and BMT, Tehran University of Medical Sciences (1996-1999); Assistant Professor of Medicine, Tehran University of Medical Sciences (1999-2005); and Associated Professor of Medicine, Tehran University of Medical Sciences (2005-2012). From 2012, Dr. Alimoghaddam is Professor of Medicine, at Tehran University of Medical Sciences. He is a member of EBMT, ESMO, EHA, ASCO, ASH, ECAR, Iranian society of Hematology/Oncology and Iranian society of Bone Marrow Transplantation. Dr. Alimoghaddam is also co-editor of International Journal of Hematology, Oncology and BMT and serves as a member for several editorial boards journals (Hematology, Oncology and Stem Cell Therapy Journal; Khoon (blood) journal; Cell Journal (Yakhteh)).

Contents

**Preface VII**

**Section 1 Stem Cell Biology 1**

Pereira

Chapter 1 **Regulators of the Proliferation of Hematopoietic Stem and**

Chapter 2 **Hematopoietic Stem Cell Niche: Role in Normal and Malignant**

Chapter 3 **Expression Profile of Galectins (Gal-1, Gal-9, Gal-11 and Gal-13)**

Yasushi Kubota and Shinya Kimura

**Different Culture Mediums 33**

Chapter 6 **Stem Cell Predictive Hemotoxicology 79** Holli Harper and Ivan N. Rich

Chapter 4 **Canonical HSC Markers and Recent Achievements 51**

Yukiko Doi, Kenji Oritani and Yuzuru Kanakura

Chapter 5 **Hematopoietic Stem Cells and Response to Interferon 65**

**Hematopoiesis 17**

Faouzi Jenhani

Atsuko Masumi

**Progenitor Cells During Hematopoietic Regeneration 3**

Sergio P. Bydlowski, Debora Levy, Jorge M.L. Ruiz and Juliana

**in Human Bone Marrow Derived Mesenchymal Stem Cells in**

Takao Sudo, Takafumi Yokota, Tomohiko Ishibashi, Michiko Ichii,

## Contents

**Preface XI**


#### **Section 2 Stem Cells in Disease 109**

Chapter 7 **Stem Cell-Based (Auto)Grafting: From Innovative Research Toward Clinical Use in Regenerative Medicine 111** Bela Balint, Slobodan Obradovic, Milena Todorovic, Mirjana Pavlovic and Biljana Mihaljevic

Preface

lost/died cells by new normal cells.

safe manipulation.

cers (chronic myeloid leukemia).

logical property of them is not completely understood.

cells in an organ and start process of carcinogenesis.

marrow, due to environmental or immunologic dysregulation.

potentially applicable in regenerative medicine and also cancer treatment.

Stem cells have a prominent role in biology of normal life and also in pathogenesis of diseases. Life starts with generation of an embryo (the most primitive form of a stem cell) and goes through all of the embryonic and fetal stages of life. It forms all of the tissue and organs of a living organism and then has a role in maintenance of normal cellular composition of body. And finally these cells maintain body in a steady state of cell loss and substitution of normal

Nearly in every organ of body we have these cells, although location characteristic and bio‐

Despite of their role in normal life, these cells also have a prominent role in many diseases. Deficiency/depletion of these cells is very important in pathogenesis diseases. For example aplastic anemia (a fatal form of hematopoietic stem cell disease) is stem cell damage in bone

Cancer is also a stem cell disease. Genetic/metabolic abnormalities of tissue stem cells (or some times more mature progenitor cells) may disrupt normal proliferation of immature

Although clinical application of the best known stem cells (Hematopoietic stem cells) is pos‐ sible today and they are useful for treatment of many malignant and non-malignant disor‐ ders, clinical knowledge and application of these cells is in infancy. These cells are

For safe and effective application of these cells, we need better knowledge of their biology, their interaction with other cells (especially supporting niche cells), growth, maturation and also immigration of stem cells through body in normal and abnormal conditions. Also for clinical application we need to know their characterization, their separation methods and

This book is written to clarify some aspects of stem cell biology, their characteristics, assess‐ ment of damage to cells during ex vivo manipulation and also their role in a model of can‐

Institute for Oncology, Hematology and Stem Cell Transplantation

Tehran University of Medical Sciences, Tehran, Iran

**Kamran Alimoghaddam, MD.**


## Preface

**Section 2 Stem Cells in Disease 109**

**VI** Contents

and Biljana Mihaljevic

Aisha Nasef

Chapter 7 **Stem Cell-Based (Auto)Grafting: From Innovative Research Toward Clinical Use in Regenerative Medicine 111**

Chapter 8 **Hematopoietic Stem Cells in Chronic Myeloid Leukemia 137**

Chapter 9 **Role of Bone Marrow Derived Mesenchymal Stem Cells in Management of Graft Versus Host Disease 165**

Moreno-Lorenzana and Héctor Mayani

Antonieta Chávez-González, Sócrates Avilés-Vázquez, Dafne

Bela Balint, Slobodan Obradovic, Milena Todorovic, Mirjana Pavlovic

Stem cells have a prominent role in biology of normal life and also in pathogenesis of diseases.

Life starts with generation of an embryo (the most primitive form of a stem cell) and goes through all of the embryonic and fetal stages of life. It forms all of the tissue and organs of a living organism and then has a role in maintenance of normal cellular composition of body. And finally these cells maintain body in a steady state of cell loss and substitution of normal lost/died cells by new normal cells.

Nearly in every organ of body we have these cells, although location characteristic and bio‐ logical property of them is not completely understood.

Despite of their role in normal life, these cells also have a prominent role in many diseases. Deficiency/depletion of these cells is very important in pathogenesis diseases. For example aplastic anemia (a fatal form of hematopoietic stem cell disease) is stem cell damage in bone marrow, due to environmental or immunologic dysregulation.

Cancer is also a stem cell disease. Genetic/metabolic abnormalities of tissue stem cells (or some times more mature progenitor cells) may disrupt normal proliferation of immature cells in an organ and start process of carcinogenesis.

Although clinical application of the best known stem cells (Hematopoietic stem cells) is pos‐ sible today and they are useful for treatment of many malignant and non-malignant disor‐ ders, clinical knowledge and application of these cells is in infancy. These cells are potentially applicable in regenerative medicine and also cancer treatment.

For safe and effective application of these cells, we need better knowledge of their biology, their interaction with other cells (especially supporting niche cells), growth, maturation and also immigration of stem cells through body in normal and abnormal conditions. Also for clinical application we need to know their characterization, their separation methods and safe manipulation.

This book is written to clarify some aspects of stem cell biology, their characteristics, assess‐ ment of damage to cells during ex vivo manipulation and also their role in a model of can‐ cers (chronic myeloid leukemia).

#### **Kamran Alimoghaddam, MD.**

Institute for Oncology, Hematology and Stem Cell Transplantation Tehran University of Medical Sciences, Tehran, Iran

**Section 1**

**Stem Cell Biology**

**Section 1**

**Stem Cell Biology**

**Chapter 1**

**Regulators of the Proliferation of**

Additional information is available at the end of the chapter

Yasushi Kubota and Shinya Kimura

http://dx.doi.org/10.5772/55639

**1. Introduction**

(e.g., CD34low/- Kit+

**During Hematopoietic Regeneration**

**Hematopoietic Stem and Progenitor Cells**

Adult hematopoietic stem cells (HSCs) ensure the maintenance of the HSC pool via their selfrenewal capacity, and replenish mature cells throughout life via their proliferation and differentiation into lineage-restricted cells [1, 2]. HSCs are the only stem cells that have been used in the clinic to treat diseases, such as leukemia, germ cell tumors, and congenital immunodeficiencies. Since HSCs were first proposed [3], advances in multicolor flow cytom‐ etry have allowed the purification of mouse HSCs close to homogeneity. Several groups have succeeded in long-term hematopoietic reconstitution by transplanting a single lineage of HSCs

Sca-1+ lineage marker-negative cells and CD150+

that divide only about five times throughout the mouse life span [8, 9].

marker-negative cells), providing direct proof of the existence of HSCs [4-7]. More recently, two groups analyzed the cycling status of HSCs by monitoring their proliferation rate over several months in vivo. The results of these studies suggested the presence of dormant HSCs

In the bone marrow (BM), HSCs are located in a specialized microenvironment, called the niche. Under steady-state conditions, signals from niches maintain some HSCs in a dormant state. Acquisition of dormancy is critical for the preservation of the self-renewal ability of HSCs and for the prevention of premature stem cell exhaustion [10-12]. However, in response to external stresses, such as bleeding, myeloablative chemotherapy and total body irradiation, HSCs proliferate extensively to produce very large numbers of primitive progenitor cells, thereby enabling rapid hematological regeneration [13]. Once recovery from myelosuppres‐ sion or other stresses has been achieved, the activated HSCs return to a quiescent state via a number of negative feedback mechanisms [14]. This ability is a hallmark of HSCs and is

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

© 2013 Kubota and Kimura; 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 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,

CD48-

Kit+

Sca-1+

lineage

**Chapter 1**

## **Regulators of the Proliferation of Hematopoietic Stem and Progenitor Cells During Hematopoietic Regeneration**

Yasushi Kubota and Shinya Kimura

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55639

#### **1. Introduction**

Adult hematopoietic stem cells (HSCs) ensure the maintenance of the HSC pool via their selfrenewal capacity, and replenish mature cells throughout life via their proliferation and differentiation into lineage-restricted cells [1, 2]. HSCs are the only stem cells that have been used in the clinic to treat diseases, such as leukemia, germ cell tumors, and congenital immunodeficiencies. Since HSCs were first proposed [3], advances in multicolor flow cytom‐ etry have allowed the purification of mouse HSCs close to homogeneity. Several groups have succeeded in long-term hematopoietic reconstitution by transplanting a single lineage of HSCs (e.g., CD34low/- Kit+ Sca-1+ lineage marker-negative cells and CD150+ CD48- Kit+ Sca-1+ lineage marker-negative cells), providing direct proof of the existence of HSCs [4-7]. More recently, two groups analyzed the cycling status of HSCs by monitoring their proliferation rate over several months in vivo. The results of these studies suggested the presence of dormant HSCs that divide only about five times throughout the mouse life span [8, 9].

In the bone marrow (BM), HSCs are located in a specialized microenvironment, called the niche. Under steady-state conditions, signals from niches maintain some HSCs in a dormant state. Acquisition of dormancy is critical for the preservation of the self-renewal ability of HSCs and for the prevention of premature stem cell exhaustion [10-12]. However, in response to external stresses, such as bleeding, myeloablative chemotherapy and total body irradiation, HSCs proliferate extensively to produce very large numbers of primitive progenitor cells, thereby enabling rapid hematological regeneration [13]. Once recovery from myelosuppres‐ sion or other stresses has been achieved, the activated HSCs return to a quiescent state via a number of negative feedback mechanisms [14]. This ability is a hallmark of HSCs and is

© 2013 Kubota and Kimura; 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 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.

fundamental for the maintenance of hematopoietic function throughout the life of the organ‐ ism. Although this property has long been recognized and is very important for organism survival, the molecular basis underlying how HSCs react to a hematologic emergency remains enigmatic. However, some key players have been identified. In this chapter, we briefly review the recent advances in our knowledge of HSC cell-intrinsic and cell-extrinsic regulators that are critical for hematopoietic regeneration under stress hematopoiesis.

**2.3. Necdin**

**2.4. Slug**

SLAM, and EPCR+

Necdin belongs to the melanoma antigen family of molecules, whose physiological roles have not been well characterized [25]. Necdin was originally identified as a gene product induced in neurally differentiated embryonal carcinoma cells [26]. Interestingly, recent genetic analyses demonstrate that aberrant genomic imprinting of *NDN* on the human 15q11-q13 chromosomal region is, at least in part, responsible for the pathogenesis of Prader-Willi syndrome [27-29], a disease associated with a mildly increased risk of myeloid leukemia [30]. Necdin interacts with multiple cell cycle-related proteins, such as SV-40 large T antigen, adenovirus E1A, E2F1, and p53 [31-34]. We reported that necdin is one of 32 genes that show higher expression in HSCs than in differentiated hematopoietic cells [35]. Other groups also found that necdin is highly expressed in HSCs [36, 37]. Necdin-deficient mice show accelerated recovery of hematopoietic systems after myelosuppressive stress, such as after 5-FU treatment and BM transplantation, whereas no overt abnormality is seen under conditions of steady-state hematopoiesis. As necdin is a potential negative cell cycle regulator, the enhanced hematologic recovery in necdin-null mice was suggested to result from an increase in the number of proliferating HSCs and progenitor cells. As expected, after 5-FU treatment, necdin-deficient mice had an increased number of HSCs, but this increase was transient and was observed only during the recovery phase [35]. These data suggest that the repression of necdin function in HSCs could form the basis of a novel strategy for the acceleration of hematopoietic recovery, thereby providing therapeutic benefits after clinical myelosuppressive treatments (e.g., cytoablative chemother‐ apy or HSC transplantation). Necdin is a p53 target gene, and in vitro overexpression and knockdown experiments demonstrated that necdin plays a role in the maintenance of HSC quiescence and self-renewal [37]. However, another group reported that necdin overexpres‐

Regulators of the Proliferation of Hematopoietic Stem and Progenitor Cells During Hematopoietic Regeneration

Slug is a member of the highly conserved Slug/Snail family of zinc-finger transcriptional repressors found in diverse species ranging from C. elegans to humans. SLUG is a target gene for the E2A-HLF chimeric oncoprotein in pro-B cell acute leukemia [39]. Slug is highly expressed in immature hematopoietic cells, and a study using *Slug*-/- mice revealed that slug is essential for the radioprotection of HPCs [40]. Slug is induced by p53 and protects immature hematopoietic cells from apoptosis triggered by DNA damage. Slug exerts this function by repressing Puma, a proapoptotic target of p53 [41]. Sun et al. investigated the effects of Slug under steady-state and stress hematopoiesis [42]. The numbers of HSCs (LSK, Flk2—LSK,

) and progenitor cells (multipotent progenitors: CD150—CD48—CD244+

) were comparable regardless of the Slug

http://dx.doi.org/10.5772/55639

5

CD244+

genotype under steady-state conditions. Consistent with previous reports [40, 43], they found that hematopoiesis in the BM was normal in *Slug*-/- mice, suggesting that Slug is not required for steady-state hematopoiesis. On the other hand, an *in vivo* competitive repopulation assay revealed that Slug-deficient BM cells had a higher long-term reconstitution capacity. However, HSC homing and differentiation were not affected by the deficiency of Slug. These results suggest that Slug deficiency increases HSC self-renewal. Next, to assess whether Slug dosage

;

sion does not result in enhanced HSC quiescence [38].

lineage-restricted progenitors: CD150—CD48+

#### **2. Cell-intrinsic factors**

#### **2.1. Hemeoxygenase-1**

Heme plays many important roles, such as in the promotion of proliferation and differentiation of hematopoietic progenitor cells (HPCs) [15], and in the stimulation of hematopoiesis [16, 17]. Heme oxygenase (HO) catalyzes the degradation of heme. HO-1, encoded by the *Hmox1* gene, is a stress-inducible isozyme of HO and is highly expressed in the BM and spleen [17]. Cao et al reported that the hematopoietic lineages of heterozygous HO-1-deficient mice (*HO-1*+/-) display accelerated recovery from myelotoxic injury induced by 5-FU treatment. BM transplantation experiments also revealed that mice transplanted with *HO-1*+/- BM cells reconstituted hematopoietic lineages more rapidly than those transplanted with *HO-1*+/+ BM cells. However, *HO-1*+/- HSCs could not rescue lethally irradiated recipient mice or serially reconstitute irradiated mice [18]. These results suggest that HO-1 restricts the proliferation and differentiation of HSCs/HPCs under stress conditions, and that the dysregulation of HO-1 can lead to precocious HSC exhaustion.

#### **2.2. PSF-1**

PSF-1, a partner of sld five-1, forms a multiprotein complex, termed GINS. The GINS complex contains Psf2, Psf3, and Sld5 in addition to Psf-1 [19-21]. All genes encoding this complex are evolutionally conserved and are essential for cell growth [22]. Ueno et al isolated the mouse orthlog of *PSF1* from a BM Linc-Kit+ Sca-1+ hematopoietic stem cell cDNA library and found that PSF1 was specifically expressed in immature cells including blastocysts and spermato‐ gonia. They also generated *PSF1*-/- mice lacking functional *PSF1*, which died in utero around the implantation stage [23]. In hematopoietic cells, PSF1 was highly expressed in CD34+ KSL progenitor cells but not in CD34- KSL cells. In addition, proliferating CD34+ KSL cells sorted from BM 4 days after 5-FU exposure were PSF1 positive. Next, Ueno et al investigated the function of PSF1 in hematopoiesis using *PSF1*+/- mice. The pool size of HSCs and progenitor cells was decreased in aged *PSF1*+/- mice compared to wild-type mice. Whereas young *PSF1*+/ mice showed normal hematopoiesis under steady-state conditions, 5-FU treatment was lethal in *PSF1*+/- mice as a result of a delay in the induction of HSC proliferation after BM ablation [24]. These results suggest that PSF1 is essential for the acute proliferation of HSCs during the regeneration phase after BM suppression.

#### **2.3. Necdin**

fundamental for the maintenance of hematopoietic function throughout the life of the organ‐ ism. Although this property has long been recognized and is very important for organism survival, the molecular basis underlying how HSCs react to a hematologic emergency remains enigmatic. However, some key players have been identified. In this chapter, we briefly review the recent advances in our knowledge of HSC cell-intrinsic and cell-extrinsic regulators that

Heme plays many important roles, such as in the promotion of proliferation and differentiation of hematopoietic progenitor cells (HPCs) [15], and in the stimulation of hematopoiesis [16, 17]. Heme oxygenase (HO) catalyzes the degradation of heme. HO-1, encoded by the *Hmox1* gene, is a stress-inducible isozyme of HO and is highly expressed in the BM and spleen [17]. Cao et al reported that the hematopoietic lineages of heterozygous HO-1-deficient mice (*HO-1*+/-) display accelerated recovery from myelotoxic injury induced by 5-FU treatment. BM transplantation experiments also revealed that mice transplanted with *HO-1*+/- BM cells reconstituted hematopoietic lineages more rapidly than those transplanted with *HO-1*+/+ BM cells. However, *HO-1*+/- HSCs could not rescue lethally irradiated recipient mice or serially reconstitute irradiated mice [18]. These results suggest that HO-1 restricts the proliferation and differentiation of HSCs/HPCs under stress conditions, and that the dysregulation of HO-1 can

PSF-1, a partner of sld five-1, forms a multiprotein complex, termed GINS. The GINS complex contains Psf2, Psf3, and Sld5 in addition to Psf-1 [19-21]. All genes encoding this complex are evolutionally conserved and are essential for cell growth [22]. Ueno et al isolated the mouse

that PSF1 was specifically expressed in immature cells including blastocysts and spermato‐ gonia. They also generated *PSF1*-/- mice lacking functional *PSF1*, which died in utero around the implantation stage [23]. In hematopoietic cells, PSF1 was highly expressed in CD34+

progenitor cells but not in CD34- KSL cells. In addition, proliferating CD34+ KSL cells sorted from BM 4 days after 5-FU exposure were PSF1 positive. Next, Ueno et al investigated the function of PSF1 in hematopoiesis using *PSF1*+/- mice. The pool size of HSCs and progenitor cells was decreased in aged *PSF1*+/- mice compared to wild-type mice. Whereas young *PSF1*+/ mice showed normal hematopoiesis under steady-state conditions, 5-FU treatment was lethal in *PSF1*+/- mice as a result of a delay in the induction of HSC proliferation after BM ablation [24]. These results suggest that PSF1 is essential for the acute proliferation of HSCs during the

Sca-1+ hematopoietic stem cell cDNA library and found

KSL

c-Kit+

are critical for hematopoietic regeneration under stress hematopoiesis.

**2. Cell-intrinsic factors**

4 Stem Cell Biology in Normal Life and Diseases

lead to precocious HSC exhaustion.

orthlog of *PSF1* from a BM Lin-

regeneration phase after BM suppression.

**2.2. PSF-1**

**2.1. Hemeoxygenase-1**

Necdin belongs to the melanoma antigen family of molecules, whose physiological roles have not been well characterized [25]. Necdin was originally identified as a gene product induced in neurally differentiated embryonal carcinoma cells [26]. Interestingly, recent genetic analyses demonstrate that aberrant genomic imprinting of *NDN* on the human 15q11-q13 chromosomal region is, at least in part, responsible for the pathogenesis of Prader-Willi syndrome [27-29], a disease associated with a mildly increased risk of myeloid leukemia [30]. Necdin interacts with multiple cell cycle-related proteins, such as SV-40 large T antigen, adenovirus E1A, E2F1, and p53 [31-34]. We reported that necdin is one of 32 genes that show higher expression in HSCs than in differentiated hematopoietic cells [35]. Other groups also found that necdin is highly expressed in HSCs [36, 37]. Necdin-deficient mice show accelerated recovery of hematopoietic systems after myelosuppressive stress, such as after 5-FU treatment and BM transplantation, whereas no overt abnormality is seen under conditions of steady-state hematopoiesis. As necdin is a potential negative cell cycle regulator, the enhanced hematologic recovery in necdin-null mice was suggested to result from an increase in the number of proliferating HSCs and progenitor cells. As expected, after 5-FU treatment, necdin-deficient mice had an increased number of HSCs, but this increase was transient and was observed only during the recovery phase [35]. These data suggest that the repression of necdin function in HSCs could form the basis of a novel strategy for the acceleration of hematopoietic recovery, thereby providing therapeutic benefits after clinical myelosuppressive treatments (e.g., cytoablative chemother‐ apy or HSC transplantation). Necdin is a p53 target gene, and in vitro overexpression and knockdown experiments demonstrated that necdin plays a role in the maintenance of HSC quiescence and self-renewal [37]. However, another group reported that necdin overexpres‐ sion does not result in enhanced HSC quiescence [38].

#### **2.4. Slug**

Slug is a member of the highly conserved Slug/Snail family of zinc-finger transcriptional repressors found in diverse species ranging from C. elegans to humans. SLUG is a target gene for the E2A-HLF chimeric oncoprotein in pro-B cell acute leukemia [39]. Slug is highly expressed in immature hematopoietic cells, and a study using *Slug*-/- mice revealed that slug is essential for the radioprotection of HPCs [40]. Slug is induced by p53 and protects immature hematopoietic cells from apoptosis triggered by DNA damage. Slug exerts this function by repressing Puma, a proapoptotic target of p53 [41]. Sun et al. investigated the effects of Slug under steady-state and stress hematopoiesis [42]. The numbers of HSCs (LSK, Flk2—LSK, SLAM, and EPCR+ ) and progenitor cells (multipotent progenitors: CD150—CD48—CD244+ ; lineage-restricted progenitors: CD150—CD48+ CD244+ ) were comparable regardless of the Slug genotype under steady-state conditions. Consistent with previous reports [40, 43], they found that hematopoiesis in the BM was normal in *Slug*-/- mice, suggesting that Slug is not required for steady-state hematopoiesis. On the other hand, an *in vivo* competitive repopulation assay revealed that Slug-deficient BM cells had a higher long-term reconstitution capacity. However, HSC homing and differentiation were not affected by the deficiency of Slug. These results suggest that Slug deficiency increases HSC self-renewal. Next, to assess whether Slug dosage affects HSC self-renewal capability, they performed a serial transplantation assay. *Slug*-/- BM cells showed an enhanced repopulation capacity during serial transplantation. Furthermore, the repopulating and proliferation potential of *Slug*-/- HSCs treated with 5-FU were also examined in vivo and in vitro. Slug deficiency increased the reconstituting potential of 5-FUactivated HSCs in vivo and accelerated HSC expansion in vitro. Taken together, these results suggest that Slug negatively regulates HSC self-renewal under stress hematopoiesis.

[36]. Cx43-deficiency in the BM (*Mx1*-Cre/*Cx43*flox/flox) led to impaired hematopoietic recovery after 5-FU treatment [62]. To clarify the mechanism of impaired hematopoietic regeneration after myeloablation by 5-FU, Taniguchi-Ishikawa et al generated hematopoietic-specific Cx43 (H-Cx43)-deficient mice (*Vav1*-Cre/*Cx43*flox/flox) and analyzed their hematopoietic phenotype [63]. The lack of Cx43 in hematopoiesis did not impair long-term competitive repopulation capacity but impaired hematopoietic recovery after 5-FU administration. 5-FU-treated H-Cx43-deficient HSCs failed to enter the cell cycle and showed decreased cell survival. More detailed analyses revealed that enhanced quiescence in H-Cx43-deficient HSCs treated with 5-FU is associated with up-regulation of the expression of quiescence markers, p16INK4a [64] and p38 [65], and with an increased level of intracellular reactive oxygen species (ROS).

Regulators of the Proliferation of Hematopoietic Stem and Progenitor Cells During Hematopoietic Regeneration

http://dx.doi.org/10.5772/55639

7

The same group also investigated the role of Cx43 in the BM osteoblastic niche. For this, Gonzalez-Nieto et al used conditionally osteoblast lineage-specific Cx43-deficient mice: *Col1 α1*-Cre; *Cx43*flox/flox mice (OB/P Cx43-deficient mice) [66]. The OB/P Cx43-deficient mice showed normal hematopoiesis under steady-state conditions. However, engraftment and migration of normal HSCs was impaired by the loss of Cx43 in the osteoblast lineage. Interestingly, in nonmyeloablated mice, OB/P Cx43 deficiency did not cause a homing defect but increased the endosteal lodgment of HSCs, which was associated with the expansion of Cxcl-12-expressing mesenchymal/osteolineage cells in the BM niche [67]. Another group reported that Cx43 and Cx45 gap junctions mediate the secretion of CXCL12 from BM cells, resulting in HSCs adhesion

Metalloproteinases (MMPs) modulate the extracellular matrix (ECM) environment [69-71]. Several studies have indicated that MMP-9 and MT1-MMP are important for the cleavage and inactivation of the KIT ligand (Stem cell factor: SCF) and CXCL12 during G-CSF-induced mobilization and hematopoietic recovery after cytotoxic stress in the BM [72-75]. Tissue inhibitors of the metalloproteinase (TIMP) family consist of four members (TIMP-1 to -4), all of which are endogenous regulators of metalloproteinases (MMPs) [76, 77]. Although TIMPs were initially identified as inhibitors of MMPs, recent findings suggest that they might have more diverse functions [78, 79]. Previous work suggested that TIMP-1 deficiency or enforced expression of TIMP-1 or TIMP-2 does not alter steady-state hematopoiesis and stress hemato‐ poiesis, such as those induced by G-CSF stimulation and myelotoxic insult, respectively [80]. However, Rossi et al recently found that increased expression of p53 in *TIMP-1*-/- HSCs resulted in dysregulation of the transition from G1 to S phase of the cell cycle, indicating that TIMP-1

Among TIMP family members, TIMP-3 has unique properties. TIMP-3 binds firmly to ECM, a disintegrin [82-84], and inhibits metalloproteinase domain-containing proteins, such as ADAMs and ADAMTSs (ADAM proteins with Thrombospondin Motifs) [85]. Nakajima et al found decreased expression of TIMP-3 in immune suppressor factor (ISF)/short form of ISF (ShIF)-transfected cell lines, and partial reduction of HSC-supporting activity following the restoration of TIMP-3 expression in stromal cells expressing ISF [86]. These authors also investigated the role of TIMP-3 in HSC regulation. TIMP-3 expression in BM was increased

has a role in controlling the cell cycle dynamics of LT-HSCs [81].

to stromal cells [68].

**3.2. TIMP-3**

#### **2.5. Erg**

The E-twenty-six (ETS)-related gene (ERG) belongs to the ETS family of transcription factors [44]. ERG rearrangement has been reported in acute myeloid leukemia (AML) [45] and Ewing sarcoma [46]. ERG overexpression has also been observed in prostate cancer [47]. A recent study revealed that overexpression of ERG is an adverse prognostic factor in AML with a normal karyotype [48]. More recently, the role of Erg in hematopoietic development and normal hematopoiesis was investigated. *Mld2*, an allele of the murine *Erg* gene with a missense mutation in the ETS domain-encoding region, disrupts Erg transactivation of gene expression. Mice homozygous for the *ErgMld2* allele die at midgestation because of a failure in definitive hematopoiesis [49, 50]. Ng et al studied hematopoiesis in mice heterozygous for the *ErgMld2* mutation [51]. While *Erg+/Mld2* mice showed normal steady-state hematopoiesis, *Erg+/Mld2* BM cells exhibited defective HSC self-renewal in BM transplantation or during recovery from exposure to sublethal γ-irradiation. The TPO/c-Mpl pathway is critical for the self-renewal and proliferation of HSCs [52-54]. Next, the phenotype of *Erg+/Mld2Mpl-/-* mice was examined because the Mld2 mutation was originally uncovered during a sensitized ENU mutagenesis screen of Mpl-deficient mice [49]. The double mutant mice died of BM failure following an exacerbation of a defect in HSC proliferation. Thus, Erg is required for HSC self-renewal during stress hematopoiesis. ERG is also expressed in endothelial cells [55, 56]. A recent study showed that ERG plays a role in endothelial tube formation and angiogenesis [57]. More recently, Yuan et al identified Rhoj, a Rho GTPase family member, as a novel downstream target of ERG [58]. Interestingly, Rhoj is also highly expressed in HSCs [35].

#### **3. Cell-extrinsic factors**

The interaction between HSCs and their microenvironment (niche) is essential for HSC maintenance, self-renewal, and survival. However, recent studies have revealed that the cellextrinsic factors provided by the BM niche are also important for HSC responses during hematopoietic regeneration. Some key players have been identified.

#### **3.1. Connexin-43**

The exchange of ions, metabolites, and other small molecules (up to ~1,200 Da) occurs via gap junctions. Gap junctions are configured by a large family of proteins known as connexins (Cxs). In the connexin family, connexin-43 (Cx43) is highly expressed in BM stromal cells [59], endothelial cells [7], osteoblasts [60], and mesenchymal stem cells [61]. HSCs also express Cx43 [36]. Cx43-deficiency in the BM (*Mx1*-Cre/*Cx43*flox/flox) led to impaired hematopoietic recovery after 5-FU treatment [62]. To clarify the mechanism of impaired hematopoietic regeneration after myeloablation by 5-FU, Taniguchi-Ishikawa et al generated hematopoietic-specific Cx43 (H-Cx43)-deficient mice (*Vav1*-Cre/*Cx43*flox/flox) and analyzed their hematopoietic phenotype [63]. The lack of Cx43 in hematopoiesis did not impair long-term competitive repopulation capacity but impaired hematopoietic recovery after 5-FU administration. 5-FU-treated H-Cx43-deficient HSCs failed to enter the cell cycle and showed decreased cell survival. More detailed analyses revealed that enhanced quiescence in H-Cx43-deficient HSCs treated with 5-FU is associated with up-regulation of the expression of quiescence markers, p16INK4a [64] and p38 [65], and with an increased level of intracellular reactive oxygen species (ROS).

The same group also investigated the role of Cx43 in the BM osteoblastic niche. For this, Gonzalez-Nieto et al used conditionally osteoblast lineage-specific Cx43-deficient mice: *Col1 α1*-Cre; *Cx43*flox/flox mice (OB/P Cx43-deficient mice) [66]. The OB/P Cx43-deficient mice showed normal hematopoiesis under steady-state conditions. However, engraftment and migration of normal HSCs was impaired by the loss of Cx43 in the osteoblast lineage. Interestingly, in nonmyeloablated mice, OB/P Cx43 deficiency did not cause a homing defect but increased the endosteal lodgment of HSCs, which was associated with the expansion of Cxcl-12-expressing mesenchymal/osteolineage cells in the BM niche [67]. Another group reported that Cx43 and Cx45 gap junctions mediate the secretion of CXCL12 from BM cells, resulting in HSCs adhesion to stromal cells [68].

#### **3.2. TIMP-3**

affects HSC self-renewal capability, they performed a serial transplantation assay. *Slug*-/- BM cells showed an enhanced repopulation capacity during serial transplantation. Furthermore, the repopulating and proliferation potential of *Slug*-/- HSCs treated with 5-FU were also examined in vivo and in vitro. Slug deficiency increased the reconstituting potential of 5-FUactivated HSCs in vivo and accelerated HSC expansion in vitro. Taken together, these results

The E-twenty-six (ETS)-related gene (ERG) belongs to the ETS family of transcription factors [44]. ERG rearrangement has been reported in acute myeloid leukemia (AML) [45] and Ewing sarcoma [46]. ERG overexpression has also been observed in prostate cancer [47]. A recent study revealed that overexpression of ERG is an adverse prognostic factor in AML with a normal karyotype [48]. More recently, the role of Erg in hematopoietic development and normal hematopoiesis was investigated. *Mld2*, an allele of the murine *Erg* gene with a missense mutation in the ETS domain-encoding region, disrupts Erg transactivation of gene expression. Mice homozygous for the *ErgMld2* allele die at midgestation because of a failure in definitive hematopoiesis [49, 50]. Ng et al studied hematopoiesis in mice heterozygous for the *ErgMld2* mutation [51]. While *Erg+/Mld2* mice showed normal steady-state hematopoiesis, *Erg+/Mld2* BM cells exhibited defective HSC self-renewal in BM transplantation or during recovery from exposure to sublethal γ-irradiation. The TPO/c-Mpl pathway is critical for the self-renewal and proliferation of HSCs [52-54]. Next, the phenotype of *Erg+/Mld2Mpl-/-* mice was examined because the Mld2 mutation was originally uncovered during a sensitized ENU mutagenesis screen of Mpl-deficient mice [49]. The double mutant mice died of BM failure following an exacerbation of a defect in HSC proliferation. Thus, Erg is required for HSC self-renewal during stress hematopoiesis. ERG is also expressed in endothelial cells [55, 56]. A recent study showed that ERG plays a role in endothelial tube formation and angiogenesis [57]. More recently, Yuan et al identified Rhoj, a Rho GTPase family member, as a novel downstream target of ERG [58].

The interaction between HSCs and their microenvironment (niche) is essential for HSC maintenance, self-renewal, and survival. However, recent studies have revealed that the cellextrinsic factors provided by the BM niche are also important for HSC responses during

The exchange of ions, metabolites, and other small molecules (up to ~1,200 Da) occurs via gap junctions. Gap junctions are configured by a large family of proteins known as connexins (Cxs). In the connexin family, connexin-43 (Cx43) is highly expressed in BM stromal cells [59], endothelial cells [7], osteoblasts [60], and mesenchymal stem cells [61]. HSCs also express Cx43

suggest that Slug negatively regulates HSC self-renewal under stress hematopoiesis.

Interestingly, Rhoj is also highly expressed in HSCs [35].

hematopoietic regeneration. Some key players have been identified.

**3. Cell-extrinsic factors**

**3.1. Connexin-43**

**2.5. Erg**

6 Stem Cell Biology in Normal Life and Diseases

Metalloproteinases (MMPs) modulate the extracellular matrix (ECM) environment [69-71]. Several studies have indicated that MMP-9 and MT1-MMP are important for the cleavage and inactivation of the KIT ligand (Stem cell factor: SCF) and CXCL12 during G-CSF-induced mobilization and hematopoietic recovery after cytotoxic stress in the BM [72-75]. Tissue inhibitors of the metalloproteinase (TIMP) family consist of four members (TIMP-1 to -4), all of which are endogenous regulators of metalloproteinases (MMPs) [76, 77]. Although TIMPs were initially identified as inhibitors of MMPs, recent findings suggest that they might have more diverse functions [78, 79]. Previous work suggested that TIMP-1 deficiency or enforced expression of TIMP-1 or TIMP-2 does not alter steady-state hematopoiesis and stress hemato‐ poiesis, such as those induced by G-CSF stimulation and myelotoxic insult, respectively [80]. However, Rossi et al recently found that increased expression of p53 in *TIMP-1*-/- HSCs resulted in dysregulation of the transition from G1 to S phase of the cell cycle, indicating that TIMP-1 has a role in controlling the cell cycle dynamics of LT-HSCs [81].

Among TIMP family members, TIMP-3 has unique properties. TIMP-3 binds firmly to ECM, a disintegrin [82-84], and inhibits metalloproteinase domain-containing proteins, such as ADAMs and ADAMTSs (ADAM proteins with Thrombospondin Motifs) [85]. Nakajima et al found decreased expression of TIMP-3 in immune suppressor factor (ISF)/short form of ISF (ShIF)-transfected cell lines, and partial reduction of HSC-supporting activity following the restoration of TIMP-3 expression in stromal cells expressing ISF [86]. These authors also investigated the role of TIMP-3 in HSC regulation. TIMP-3 expression in BM was increased after 5-FU injection, and addition or overexpression of TIMP-3 resulted in enhanced prolifer‐ ation of HSCs in vitro. BM regeneration after myelotoxic stress was impaired in TIMP-3 deficient mice, but was accelerated by enforced expression of TIMP-3 in vivo [87]. Another group also studied the role of TIMP-3 in hematopoiesis. They found that TIMP-3 was highly expressed in the endosteal region of the BM, the HSC niche, whereas its expression was low in HSCs and progenitor cells. They also examined the effect of human TIMP-3 (huTIMP-3) overexpression in HSCs in vivo. TIMP-3 overexpression resulted in increased myelopoiesis and decreased lymphopoiesis. Consistent with the study of Nakajima et al [87], HSC prolifer‐ ation was increased by huTIMP-3 overexpression in vitro and in vivo [88]. These results suggest that TIMP-3 is important for the cellular response to myelosuppression.

**Acknowledgements**

**Author details**

**References**

Yasushi Kubota1,2\* and Shinya Kimura1

\*Address all correspondence to: kubotay@cc.saga-u.ac.jp

Medicine, Faculty of Medicine, Saga University, Japan

gy. Cell 2008; 132(4): 631-644.

273(5272): 242-245.

2256-2259.

2 Department of Transfusion Medicine, Saga University Hospital, Japan

plication. Annu Rev Immunol 2003; 21: 759-806.

niches for stem cells. Cell 2005; 121(7): 1109-1121.

mal mouse bone marrow cells. Radiat Res 1961; 14(2): 213-222.

This work was supported by a Grant-in-Aid for Young Scientists to Y.K. (no. 23791083) from

Regulators of the Proliferation of Hematopoietic Stem and Progenitor Cells During Hematopoietic Regeneration

http://dx.doi.org/10.5772/55639

9

1 Division of Hematology, Respiratory Medicine and Oncology, Department of Internal

[1] Orkin, S.H., & Zon, L. I. Hematopoiesis: An Evolving Paradigm for Stem Cell Biolo‐

[2] Kondo, M., Wagers, A.J., Manz, M.G., Prohaska, S.S., Scherer, D.C., Beilhack, G.F., et al. Biology of hematopoietic stem cells and progenitors: implications for clinical ap‐

[3] Till, J.E., & McCulloch, C. E. A direct measurement of the radiation sensitivity of nor‐

[4] Osawa, M., Hanada, K., Hamada, H. & Nakauchi, H. Long-term lymphohemato‐ poietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 1996;

[5] Wagers, A. J., Sherwood, R. I., Christensen, J. L. & Weissman, I. L. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 2002; 297(5590):

[6] Matsuzaki, Y., Kinjo, K., Mulligan, R.C. & Okano, H. Unexpectedly efficient homing capacity of purified murine hematopoietic stem cells. Immunity 2004; 20(1): 87-93.

[7] Kiel, M.J., Yilmaz, O.H., Iwashita, T., Terhorst, C. & Morrison, S.J. SLAM family re‐ ceptors distinguish hematopoietic stem and progenitor cells and reveal endosteal

the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

#### **3.3. Tenascin-C**

Tenascin-C (TN-C) is a large extracellular matrix (ECM) glycoprotein that is expressed mainly in the developing embryo [89]. In the adult BM, expression of TN-C is restricted to the endosteal region [90, 91]. Although TN-C-deficient mice exhibit grossly normal development, the colonyforming capacity of *TN-C*-/- BM cells is lower than that of wild-type BM cells [92]. This suggests that TN-C makes a significant contribution under stress hematopoiesis because the mononu‐ clear cell count and BM architecture of TN-C-deficient mice are essentially normal. Nakamura-Ishizu et al studied the function of TN-C during hematopoiesis in vivo using TN-C knockout mice. First, they examined the expression pattern of various ECM proteins in the BM under different conditions (steady-state, immediately after myeloablation, and during the hemato‐ poietic recovery phase). TN-C was predominantly expressed in stromal cells and endothelial cells, which are components of the BM niche, and was markedly up-regulated in the BM during hematopoietic regeneration. *TN-C*-/- mice showed defects in hematopoietic recovery after BM ablation caused by 5-FU treatment and sublethal irradiation. The transplantation of wild-type BM cells into *TN-C*-/- recipient mice demonstrated that a supporting ability of hematopoiesis in BM microenvironment lacking TN-C is inadequate for the proliferation of transplanted wildtype BM cells for the regeneration of hematopoiesis [93]. These findings suggest that TN-C is a critical component of the BM microenvironment during hematopoietic regeneration.

#### **4. Conclusions**

In this chapter, we have briefly summarized regulators that have recently been shown to be involved in the control of HSCs and progenitors during the hematopoietic regeneration phase. Recent studies have revealed that, when myelopoiesis is compromised following infection, HSC proliferation involves not only the factors described in this review, but also inflammatory signaling molecules such as interferons [94-96], tumor necrosis factor-α [97, 98], and Toll-like receptors [99-101]. Because HSC proliferation potential is critical for organism survival during stress conditions, further understanding of the mechanism of stem cell activation will be needed before stem cells can be used in regenerative medicine.

#### **Acknowledgements**

after 5-FU injection, and addition or overexpression of TIMP-3 resulted in enhanced prolifer‐ ation of HSCs in vitro. BM regeneration after myelotoxic stress was impaired in TIMP-3 deficient mice, but was accelerated by enforced expression of TIMP-3 in vivo [87]. Another group also studied the role of TIMP-3 in hematopoiesis. They found that TIMP-3 was highly expressed in the endosteal region of the BM, the HSC niche, whereas its expression was low in HSCs and progenitor cells. They also examined the effect of human TIMP-3 (huTIMP-3) overexpression in HSCs in vivo. TIMP-3 overexpression resulted in increased myelopoiesis and decreased lymphopoiesis. Consistent with the study of Nakajima et al [87], HSC prolifer‐ ation was increased by huTIMP-3 overexpression in vitro and in vivo [88]. These results

Tenascin-C (TN-C) is a large extracellular matrix (ECM) glycoprotein that is expressed mainly in the developing embryo [89]. In the adult BM, expression of TN-C is restricted to the endosteal region [90, 91]. Although TN-C-deficient mice exhibit grossly normal development, the colonyforming capacity of *TN-C*-/- BM cells is lower than that of wild-type BM cells [92]. This suggests that TN-C makes a significant contribution under stress hematopoiesis because the mononu‐ clear cell count and BM architecture of TN-C-deficient mice are essentially normal. Nakamura-Ishizu et al studied the function of TN-C during hematopoiesis in vivo using TN-C knockout mice. First, they examined the expression pattern of various ECM proteins in the BM under different conditions (steady-state, immediately after myeloablation, and during the hemato‐ poietic recovery phase). TN-C was predominantly expressed in stromal cells and endothelial cells, which are components of the BM niche, and was markedly up-regulated in the BM during hematopoietic regeneration. *TN-C*-/- mice showed defects in hematopoietic recovery after BM ablation caused by 5-FU treatment and sublethal irradiation. The transplantation of wild-type BM cells into *TN-C*-/- recipient mice demonstrated that a supporting ability of hematopoiesis in BM microenvironment lacking TN-C is inadequate for the proliferation of transplanted wildtype BM cells for the regeneration of hematopoiesis [93]. These findings suggest that TN-C is a critical component of the BM microenvironment during hematopoietic regeneration.

In this chapter, we have briefly summarized regulators that have recently been shown to be involved in the control of HSCs and progenitors during the hematopoietic regeneration phase. Recent studies have revealed that, when myelopoiesis is compromised following infection, HSC proliferation involves not only the factors described in this review, but also inflammatory signaling molecules such as interferons [94-96], tumor necrosis factor-α [97, 98], and Toll-like receptors [99-101]. Because HSC proliferation potential is critical for organism survival during stress conditions, further understanding of the mechanism of stem cell activation will be

needed before stem cells can be used in regenerative medicine.

suggest that TIMP-3 is important for the cellular response to myelosuppression.

**3.3. Tenascin-C**

8 Stem Cell Biology in Normal Life and Diseases

**4. Conclusions**

This work was supported by a Grant-in-Aid for Young Scientists to Y.K. (no. 23791083) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

### **Author details**

Yasushi Kubota1,2\* and Shinya Kimura1

\*Address all correspondence to: kubotay@cc.saga-u.ac.jp

1 Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Japan

2 Department of Transfusion Medicine, Saga University Hospital, Japan

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[78] Lambert, E., Dassé, E., Haye, B., & Petitfrère, E. TIMPs as multifacial proteins. Crit

[79] Chirco, R., Liu, X.W., Jung, K.K., & Kim, H.R. Novel functions of TIMPs in cell sig‐

[80] Haviernik, P., Diaz, M.T., Haviernikova, E., Tse, W., Stetler-Stevenson, W.G., & Bunt‐ ing, K.D. Hematopoiesis in mice is extremely resilient to wide variation in

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**Chapter 2**

**Hematopoietic Stem Cell Niche:**

Additional information is available at the end of the chapter

testicular *D. melanogaster* niches (Wilson & Trumpp, 2006).

Sergio P. Bydlowski, Debora Levy, Jorge M.L. Ruiz and Juliana Pereira

http://dx.doi.org/10.5772/55508

**1. Introduction**

**Role in Normal and Malignant Hematopoiesis**

A stem-cell niche can be defined as a spatial structure in which stem cells are housed and maintained by allowing self-renewal in the absence of differentiation. The concept of the stemcell niche was initially proposed in invertebrates where they were first characterized; the control of germ line stem cell maintenance and differentiation was observed in ovarian and

Hematological stem cells (HSCs) can be initially identified in the developing embryo in aortagonada-mesonephro regions, form where they migrate into the fetal liver and subsequently move into the bone marrow (BM) after birth. During development, HSCs undergo active selfrenewal to expand the pool size, but then become largely quiescent and stay in a steady state

The link between bone-marrow formation (hematopoiesis) and bone development (osteogen‐ esis) was first recognized in the 1970s. It was noted that whilst HSCs in the bone marrow drive hematopoiesis during the whole life of organisms, when they are removed from the bone marrow, they lose the ability to self-renewal, indicating similar dependence of HSCs on extrinsic signals (Lilly *et al.,* 2011). The term 'niche' used for the specific HSC bone-marrow microenvironment was first coined by Schofield (1978), who observed that HSC growth was not supported in the spleen in the same manner as in the bone marrow; he also proposed that HSCs are in intimate contact with bone, and that cell–cell contact was responsible for the

This concept has since been further developed, and it is now widely accepted that specific anatomical regions within the bone marrow comprise specialized niches for HSC development

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

© 2013 Bydlowski 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 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,

in adult BM, where their maintenance is tightly regulated (Oh & Kwon, 2010).

apparently unlimited proliferative capacity and inhibition of maturation of HSCs.


## **Hematopoietic Stem Cell Niche: Role in Normal and Malignant Hematopoiesis**

Sergio P. Bydlowski, Debora Levy, Jorge M.L. Ruiz and Juliana Pereira

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55508

#### **1. Introduction**

[88] Shen, Y., Winkler, I.G., Barbier, V., Sims, N.A., Hendy, J., & Lévesque, J.P. Tissue in‐ hibitor of metalloproteinase-3 (TIMP-3) regulates hematopoiesis and bone formation

[89] Hsia, H.C., & Schwarzbauer, J.E. Meet the tenascins: multifunctional and mysterious.

[90] Klein, G., Beck, S., & Muller, C.A. Tenescin is a cytoadhesive extracellular matrix component of the human hematopoietic microenvironment. J Cell Biol 1993; 123(4):

[91] Soini, Y., Kamel, D., Apaja-Sarkkinen, M., Virtanen, I., & Lehto, V.P. Tenascin immu‐ noreactivity in normal and pathological bone marrow. J Clin Pathol 1993; 46(3):

[92] Ohta, M., Sakai, T., Saga, Y., Aizawa, S-I., Saito, M. Suppression of hematopoietic ac‐

[93] Nakamura-Ishizu, A., Okuno, Y., Omatsu, Y., et al. Extracellular matrix protein te‐ nascin-C is required in the bone marrow microenvironment primed for hematopoiet‐

[94] Essers, M.A., Offner, S., Blanco-Bose, W.E., et al. IFNalpha activates dormant haema‐

[95] Zhao, X., Ren, G., Liang, L., et al. Brief report: interferon-gamma induces expansion

[96] Baldridge, M.T., King, K.Y., Boles, N.C., Weksberg, D.C., & Goodell, M.A. Quiescent haematopoietic stem cells are activated by IFN-gamma in response to chronic infec‐

[97] Rezzoug, F., Huang, Y., Tanner, M.K., et al. TNF-α is critical to facilitate hemato‐ poietic stem cell engraftment and function. J Immunol 2008; 180(1): 49-57.

[98] Rebel, V.I., Hartnett, S., Hill, G.R., Lazo-Kallanian, S.B., Ferrara, J.L., & Sieff, C.A. Es‐ sential role for the p55 tumor necrosis factor receptor in regulating hematopoiesis at

[99] Nagai, Y., Garrett, K.P., Ohta, S., et al. Toll-like receotors on hematopoietic progeni‐ tor cells stimulate innate immune system replenishment. Immunity 2006; 24(6):

[100] Sioud, M., Floisand, Y., Forfang, L, & Lund-Johansen, F. Signaling through toll-like receptor 7/8 induces the differentiation of human bone marrow CD34+ progenitor

[101] Sioud, M., & Floisand, Y. TLR agonists induce the differentiation of human bone marrow CD34+ progenitor cells into CD11c+ CD80/86+ DC capable of inducing a

cells along the myeloid lineage. J Mol Biol 2006; 364(5): 945-954.

Th1-type response. Eur J Immunol 2007; 37(10): 2834-2846.

tivity in Tenascin-C-deficient mice. Blood 1998; 91(11): 4074-4083.

topoietic stem cells in vivo. Nature 2009; 458(7240): 904-908.

of Lin(-)Sca-1(+)C-Kit(+) Cells. Stem Cells 2010; 28(1): 122-126.

a stem cell level. J Exp Med 1999; 190(10): 1493-1504.

in vivo. PLoS One. 2010; 5(9). doi:pii: e13086.

ic regeneration. Blood 2012; 119(23): 5429-5437.

tion. Nature 2010; 465(7299): 793-797.

J Biol Chem 2005; 280(29): 26641-26644.

1027-1035.

16 Stem Cell Biology in Normal Life and Diseases

218-221.

801-812.

A stem-cell niche can be defined as a spatial structure in which stem cells are housed and maintained by allowing self-renewal in the absence of differentiation. The concept of the stemcell niche was initially proposed in invertebrates where they were first characterized; the control of germ line stem cell maintenance and differentiation was observed in ovarian and testicular *D. melanogaster* niches (Wilson & Trumpp, 2006).

Hematological stem cells (HSCs) can be initially identified in the developing embryo in aortagonada-mesonephro regions, form where they migrate into the fetal liver and subsequently move into the bone marrow (BM) after birth. During development, HSCs undergo active selfrenewal to expand the pool size, but then become largely quiescent and stay in a steady state in adult BM, where their maintenance is tightly regulated (Oh & Kwon, 2010).

The link between bone-marrow formation (hematopoiesis) and bone development (osteogen‐ esis) was first recognized in the 1970s. It was noted that whilst HSCs in the bone marrow drive hematopoiesis during the whole life of organisms, when they are removed from the bone marrow, they lose the ability to self-renewal, indicating similar dependence of HSCs on extrinsic signals (Lilly *et al.,* 2011). The term 'niche' used for the specific HSC bone-marrow microenvironment was first coined by Schofield (1978), who observed that HSC growth was not supported in the spleen in the same manner as in the bone marrow; he also proposed that HSCs are in intimate contact with bone, and that cell–cell contact was responsible for the apparently unlimited proliferative capacity and inhibition of maturation of HSCs.

This concept has since been further developed, and it is now widely accepted that specific anatomical regions within the bone marrow comprise specialized niches for HSC development

© 2013 Bydlowski 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 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.

and normal blood cell production (Noll *et al.,* 2012). The niche is composed of a vast milieu of cellular and humoral factors.

mouse was used to show that an increase in the number of osteoblastic cells was correlat‐

Hematopoietic Stem Cell Niche: Role in Normal and Malignant Hematopoiesis

http://dx.doi.org/10.5772/55508

19

HSCs proliferation and differentiation is regulated by a wide variety of cytokines, growth factors, and other signaling molecules. Osteoblastic cells synthesize a number of cell-signaling molecules that appear to contribute to the maintenance and regulation of HSCs by the

**•** Jagged, a ligand for Notch receptors expressed on HSCs which is markedly upregulated with activation of the osteoblast by the parathyroid hormone. Activation of Notch receptors on HSCs has been shown to inhibit differentiation and enhance their self renewal capacity in vitro (Butler *et al.,* 2011). Notch signalling is important *in vivo* for controlling HSC self renewal and differentiation during hematopoietic stress conditions (Lilly *et al.,* 2011). **•** Thrombopoietin (THPO) and angiopoietin (Ang-1), which bind to cell surface receptors MPL and Tie2, respectively, are expressed on HSCs. These cytokines are thought to be important as THPO and Ang-1 knockout mice have decreased numbers or defects in bone marrow HSCs. The interaction of Ang-1 with its Tie2 receptor activates β1-integrin and Ncadherin, enhances quiescence, and maintains the long term repopulating ability of HSCs;

it also protects against apoptosis by activating the PI3K pathway (Lilly *et al.,* 2011)

the maintenance of a quiescent state (Guerrouahen *et al.,* 2011).

EPCs could form blood vessels (Chotinantakul & Leeanansaksiri, 2012).

differentiation (Coskun & Hirschi, 2010).

**•** Both HSC and osteoblasts express N-cadherin, and bone marrow imaging studies suggest that spindle-shaped bone-lining osteoblasts (so-called SNO cells) communicate with HSC through N-cadherin interactions. BrdU incorporation studies demonstrated that quiescent HSCs with moderate N-cadherin reside close to osteoblasts which have high N-cadherin expression. Furthermore, upregulation of N-cadherin on osteoblasts increases adherence of HSC on the endosteal surface, which is associated with HSC quiescence and diminished

**•** Osteopontin, a matrix glycoprotein expressed by the osteoblasts supports the adhesion of HSC to the osteoblastic niche and negatively regulates HSC proliferation, contributing to

**•** The chemokines and their receptors can control HSC behaviour The best understood chemokine, in this regards, is the stromal-derived factor-1 (SDF-1), also called chemo‐ kine C-X-C motif ligand 12 (CXCL12). The SDF-1 receptor is the C-X-C chemokine receptor type 4 (CXCR4) and is expressed on HSCs and progenitors (Lilly *et al.,* 2011). SDF-1 belongs to α-chemokines that functions as chemoattractant for both committed and primitive hematopoietic progenitors and regulates embryonic development includ‐ ing organ homeostasis (Ratajczak *et al.,* 2006). SDF-1 counteracts with its cognate receptor CXCR4, that is widely expressed in several tissues including hematopoietic and endothelial cells. SDF-1/CXCR4 signaling plays a critical role during embryonic develop‐ ment by regulating B-cell lymphopoiesis, myelopoiesis in bone marrow and heart ventricular septum formation. In addition, SDF-1 has been shown to mediate the recruitment of endothelial progenitor cells (EPCs) from the bone marrow through a CXCR4 dependent mechanism suggesting the functional role in vasculogenesis in which

ed with an increase in the number of HSCs.

endosteal niche such as (table1):

The advancement of our understanding of the interactions between bone, the microenviron‐ ment and hematopoiesis is rapidly accelerating. Research continues on the identification and characterization of individual cell populations constituting the hematopoietic stem cell (HSC) niche. Whether there are one or multiple HSC niches and how cells within the HSC niche interact with each other are also subjects of interest (Shen *et al.,* 2012).

Recently it has been suggested that two main microenvironments form the bone marrow niches: 1. the endosteal niche, where osteoblasts derived from mesenchymal precursors are localized in the endosteal regions, and 2. The vascular niche, which might be involved in HSC maintenance within the bone marrow (Chotinantakul *et al.,* 2012).

#### **2. The endosteal niche**

The first direct evidence for cells having stem cell-supporting activity in bone formation was provided by studies in which both mouse and human osteoblast cell lines were shown to secrete a large number of cytokines that promote the proliferation of hematopoietic cells in culture (Wilson & Trumpp, 2006). Studies in the 1970s indicated that undifferentiated hema‐ topoietic cells are localized close to the endosteal bone surface, and that differentiated cells move toward the central axis of the marrow. The endosteum is the interface between bone and bone marrow and constitutes a reservoir of HSCs that can be mobilized, restoring hematopoi‐ esis in response to tissue injury. Although bone is a hard and stiff organ, the endosteal niche is not so; it rather presents plasticity and is under self and systemic regulation.

After the identification of niches in the endosteal region, cells in the endothelial lining were proposed as a cellular component of the niche. However, heterogeneous cell populations were found in endosteal regions that included mature bone lining cells, osteoblasts, and preosteo‐ blasts (Oh *et al.,* 2010).

#### **2.1. Osteoblastic cells**

Osteoblasts are responsible for the production of matrix, which they secrete at the site of the bone, as well as for bone mineralization. Eventually, osteoblasts either get surround‐ ed by matrix and end up as osteocytes or they become bone-lining cells, which is a reversible process (ter Ruurne *et al.,* 2010). The bone-forming osteoblastic cells are crucial players for the homeostasis of the hematopoietic tissue with its high turnover. In 1994, primary human osteoblasts were shown to stimulate the proliferation of primitive CD34+ hematopoietic progenitors in vitro, which raised the possibility that osteoblasts are involved in hematopoiesis (Nagasawa *et al.,* 2011).

Involvement of osteoblastic cells in HSC regulation and maintenance in vivo was first reported by two groups using engineered mouse models (Lymperi *et al.,* 2010). The first study, a bone morphogenic protein (BMP) receptor IA (BMPRIA) conditional knockout mouse was used to show that an increase in the number of osteoblastic cells was correlat‐ ed with an increase in the number of HSCs.

and normal blood cell production (Noll *et al.,* 2012). The niche is composed of a vast milieu of

The advancement of our understanding of the interactions between bone, the microenviron‐ ment and hematopoiesis is rapidly accelerating. Research continues on the identification and characterization of individual cell populations constituting the hematopoietic stem cell (HSC) niche. Whether there are one or multiple HSC niches and how cells within the HSC niche

Recently it has been suggested that two main microenvironments form the bone marrow niches: 1. the endosteal niche, where osteoblasts derived from mesenchymal precursors are localized in the endosteal regions, and 2. The vascular niche, which might be involved in HSC

The first direct evidence for cells having stem cell-supporting activity in bone formation was provided by studies in which both mouse and human osteoblast cell lines were shown to secrete a large number of cytokines that promote the proliferation of hematopoietic cells in culture (Wilson & Trumpp, 2006). Studies in the 1970s indicated that undifferentiated hema‐ topoietic cells are localized close to the endosteal bone surface, and that differentiated cells move toward the central axis of the marrow. The endosteum is the interface between bone and bone marrow and constitutes a reservoir of HSCs that can be mobilized, restoring hematopoi‐ esis in response to tissue injury. Although bone is a hard and stiff organ, the endosteal niche

After the identification of niches in the endosteal region, cells in the endothelial lining were proposed as a cellular component of the niche. However, heterogeneous cell populations were found in endosteal regions that included mature bone lining cells, osteoblasts, and preosteo‐

Osteoblasts are responsible for the production of matrix, which they secrete at the site of the bone, as well as for bone mineralization. Eventually, osteoblasts either get surround‐ ed by matrix and end up as osteocytes or they become bone-lining cells, which is a reversible process (ter Ruurne *et al.,* 2010). The bone-forming osteoblastic cells are crucial players for the homeostasis of the hematopoietic tissue with its high turnover. In 1994, primary human osteoblasts were shown to stimulate the proliferation of primitive CD34+ hematopoietic progenitors in vitro, which raised the possibility that osteoblasts are

Involvement of osteoblastic cells in HSC regulation and maintenance in vivo was first reported by two groups using engineered mouse models (Lymperi *et al.,* 2010). The first study, a bone morphogenic protein (BMP) receptor IA (BMPRIA) conditional knockout

is not so; it rather presents plasticity and is under self and systemic regulation.

interact with each other are also subjects of interest (Shen *et al.,* 2012).

maintenance within the bone marrow (Chotinantakul *et al.,* 2012).

cellular and humoral factors.

18 Stem Cell Biology in Normal Life and Diseases

**2. The endosteal niche**

blasts (Oh *et al.,* 2010).

**2.1. Osteoblastic cells**

involved in hematopoiesis (Nagasawa *et al.,* 2011).

HSCs proliferation and differentiation is regulated by a wide variety of cytokines, growth factors, and other signaling molecules. Osteoblastic cells synthesize a number of cell-signaling molecules that appear to contribute to the maintenance and regulation of HSCs by the endosteal niche such as (table1):


**•** Early HSC characterization studies led to the discovery of a growth factor secreted by osteoblasts, called stem cell factor (SCF) that regulates HSC activity *in vivo* and self-renewal *in vitro*. HSCs express a transmembrane receptor tyrosine kinase called stem cell factor receptor (C-Kit) that can bind to SCF, activating intracellular signaling important for HSC regulation (Audet *et al.,* 2002).

**2.3. Osteoclasts**

**3. Vascular niche**

(Coskun & Hirschi, 2010).

production (Avecilla *et al*., 2004).

cell behavior (Sugiyama & Nagasawa *et al*., 2012).

These cells are formed by fusion of multiple granulocyte–macrophage progenitor cells, a process mediated by osteoblasts. It reabsorb the mineralized bone matrix formed by chondro‐ cytes or osteoblasts and located in endosteal niches. The role of osteoclasts in hematopoiesis remains a controversial issue. It has been reported that osteoclasts degrade endosteal niche components and enhance mobilization of hematopoietic progenitor cells (Kollet *et al.,* 2006). Enzymes secreted by osteoclasts are responsible for the release of HSCs from the endosteal niche. These enzymes are able to cleave factors that promote the interaction between HSCs and their niche. On the other hand, results have been published that suggest that osteoclast activity can promote lodgment of HSCs at the endosteal niche (ter Huurner *et al.,* 2010). Further studies will be needed to clarify how osteoclasts regulate hematopoietic stem and progenitor

Hematopoietic Stem Cell Niche: Role in Normal and Malignant Hematopoiesis

http://dx.doi.org/10.5772/55508

21

Hematopoiesis and vascularization occur concurrently during development. In fact, HSCs and endothelial cells are derived from the same progenitor cells (termed hemangioblasts) at the embryonic stage and are closely related to the ontogeny of hematopoiesis (Yin & Li, 2006). The presence of a second specialized HSC microenvironment in the bone marrow has recently been postulated, as a large proportion of CD150+ HSCs was observed to be attached to the fenes‐

The vascular niche promotes proliferation and differentiation, active cycling, and shortterm HSCs. Most purified HSCs, containing CD150+ CD48− CD41− Lin− cells, were found to be mainly associated with the sinusoidal endothelium lining blood vessels, suggesting that endothelial cells create a cellular niche for HSCs. Endothelial cells in the vascular niche environment contacting HSCs also provide maintenance signals on the HSC behavior (Can, 2008). However, it is essential to keep in mind that vasculature is not compartmentalized to the central region of bone marrow and, in fact, the endosteal region of bone is also vascularized. Therefore, the proposed osteoblast and vascular niches within marrow are not completely separable, and may function interdependently to generate and sustain HSCs

The vascular niche has been shown to produce factors important for mobilization, homing, and engraftment of HSC. Endothelial cells expressing vascular cell-adhesion molecule-1 (VCAM-1) associate closely with megakaryocytes and their progenitors through VLA-4 in response to chemotactic factors, stromal cell-derived factor-1 (SDF1) and fibroblast growth factor-4 (FGF4); thus, they provide a niche for megakaryocyte maturation and platelet

Two perivascular cell groups that possess mesenchymal cell properties function as niche cells: 1. CXC chemokine ligand 12 (CXCL-12)-abundant reticular cells (CAR cells), and 2. Nestin

234+ mesenchymal stem cells (Nakamura-Ishizu & Suda, 2012).

trated endothelium of bone marrow sinusoids (Wilson & Trumpp, 2006).

**•** Members of the Wingless (Wnt) family of lipid-modified proteins have been also investi‐ gated in hematopoiesis. The Frizzled (Fzd) receptors act as Wnt receptors which activate downstream signaling in the Ctnnb1-dependent canonical and non-canonical pathways. Several components of the Wnt signaling machinery have been shown to play a role in HSC self-renewal. Wnt signaling is important in bone formation and in enlargement of endosteal surfaces. Several lines of evidence suggest that Wnt signaling in endosteal stromal cells may affect HSC maintenance, not by intrinsic signals, but by signals originated from the stromal cells. Wnt signaling may not be intrinsically involved in the maintenance of normal HSC during hemostasis or self-renewal. However, there are data suggesting that changes of Wnt signaling in endosteal stromal cells affect HSC maintenance through extrinsical mechanisms (Renstrom *et al.,* 2010).


**Table 1.** Molecules that regulate HSC activity

#### **2.2. Endothelial cells**

Endothelial cells were proposed to be important in the HSC microenvironment. *In vivo* and tissue section imaging studies localize HSCs next to endothelial cells. Also, endothelial cells secrete soluble factors that can expand human primitive hematopoietic cells *ex vivo*. However, endothelial cells have not yet been shown to be a necessary regulatory component of the HSC microenvironment *in vivo* (Frisch *et al.,* 2008).

#### **2.3. Osteoclasts**

**•** Early HSC characterization studies led to the discovery of a growth factor secreted by osteoblasts, called stem cell factor (SCF) that regulates HSC activity *in vivo* and self-renewal *in vitro*. HSCs express a transmembrane receptor tyrosine kinase called stem cell factor receptor (C-Kit) that can bind to SCF, activating intracellular signaling important for HSC

**•** Members of the Wingless (Wnt) family of lipid-modified proteins have been also investi‐ gated in hematopoiesis. The Frizzled (Fzd) receptors act as Wnt receptors which activate downstream signaling in the Ctnnb1-dependent canonical and non-canonical pathways. Several components of the Wnt signaling machinery have been shown to play a role in HSC self-renewal. Wnt signaling is important in bone formation and in enlargement of endosteal surfaces. Several lines of evidence suggest that Wnt signaling in endosteal stromal cells may affect HSC maintenance, not by intrinsic signals, but by signals originated from the stromal cells. Wnt signaling may not be intrinsically involved in the maintenance of normal HSC during hemostasis or self-renewal. However, there are data suggesting that changes of Wnt signaling in endosteal stromal cells affect HSC maintenance through extrinsical mechanisms

Jagged (Notch receptor) Control HSC self-renewal and differentiation during

Thrombopoietin and angiopoietin Maintenance of HSC in the niche in quiescence state

Osteopontin Maintenance of a quiescent state

CXCR-4 Expressed in HSCs; receptor of SDF-1

Wnt HSC self-renewal

**Table 1.** Molecules that regulate HSC activity

microenvironment *in vivo* (Frisch *et al.,* 2008).

**2.2. Endothelial cells**

SDF-1 (or CXCL12) Expressed in the stromal cells; attracts HSC

N-cadherin HSC quiescence and decrease in differentiation

SCF Activation of intracellular signaling important for HSC

Endothelial cells were proposed to be important in the HSC microenvironment. *In vivo* and tissue section imaging studies localize HSCs next to endothelial cells. Also, endothelial cells secrete soluble factors that can expand human primitive hematopoietic cells *ex vivo*. However, endothelial cells have not yet been shown to be a necessary regulatory component of the HSC

regulation

hematopoietic stress conditions

with a link to cell-cycle control

regulation (Audet *et al.,* 2002).

20 Stem Cell Biology in Normal Life and Diseases

(Renstrom *et al.,* 2010).

**Molecule Function**

These cells are formed by fusion of multiple granulocyte–macrophage progenitor cells, a process mediated by osteoblasts. It reabsorb the mineralized bone matrix formed by chondro‐ cytes or osteoblasts and located in endosteal niches. The role of osteoclasts in hematopoiesis remains a controversial issue. It has been reported that osteoclasts degrade endosteal niche components and enhance mobilization of hematopoietic progenitor cells (Kollet *et al.,* 2006). Enzymes secreted by osteoclasts are responsible for the release of HSCs from the endosteal niche. These enzymes are able to cleave factors that promote the interaction between HSCs and their niche. On the other hand, results have been published that suggest that osteoclast activity can promote lodgment of HSCs at the endosteal niche (ter Huurner *et al.,* 2010). Further studies will be needed to clarify how osteoclasts regulate hematopoietic stem and progenitor cell behavior (Sugiyama & Nagasawa *et al*., 2012).

#### **3. Vascular niche**

Hematopoiesis and vascularization occur concurrently during development. In fact, HSCs and endothelial cells are derived from the same progenitor cells (termed hemangioblasts) at the embryonic stage and are closely related to the ontogeny of hematopoiesis (Yin & Li, 2006). The presence of a second specialized HSC microenvironment in the bone marrow has recently been postulated, as a large proportion of CD150+ HSCs was observed to be attached to the fenes‐ trated endothelium of bone marrow sinusoids (Wilson & Trumpp, 2006).

The vascular niche promotes proliferation and differentiation, active cycling, and shortterm HSCs. Most purified HSCs, containing CD150+ CD48− CD41− Lin− cells, were found to be mainly associated with the sinusoidal endothelium lining blood vessels, suggesting that endothelial cells create a cellular niche for HSCs. Endothelial cells in the vascular niche environment contacting HSCs also provide maintenance signals on the HSC behavior (Can, 2008). However, it is essential to keep in mind that vasculature is not compartmentalized to the central region of bone marrow and, in fact, the endosteal region of bone is also vascularized. Therefore, the proposed osteoblast and vascular niches within marrow are not completely separable, and may function interdependently to generate and sustain HSCs (Coskun & Hirschi, 2010).

The vascular niche has been shown to produce factors important for mobilization, homing, and engraftment of HSC. Endothelial cells expressing vascular cell-adhesion molecule-1 (VCAM-1) associate closely with megakaryocytes and their progenitors through VLA-4 in response to chemotactic factors, stromal cell-derived factor-1 (SDF1) and fibroblast growth factor-4 (FGF4); thus, they provide a niche for megakaryocyte maturation and platelet production (Avecilla *et al*., 2004).

Two perivascular cell groups that possess mesenchymal cell properties function as niche cells: 1. CXC chemokine ligand 12 (CXCL-12)-abundant reticular cells (CAR cells), and 2. Nestin 234+ mesenchymal stem cells (Nakamura-Ishizu & Suda, 2012).

#### **3.1. CAR cells**

In human bone marrow, such reticular cells constitute the subendothelial (adventitial) layer of sinusoidal walls projecting a reticular process that is in close contact with HSCs. Interestingly, these reticular cells were derived from a specific subset of mesenchymal cells (CD146þ) that had been shown to produce either reticular or endosteum of the ectopic hematopoietic microenvironment (HME), referred to as skeletal stem cells (Sugiyama & Nagasawa, 2012).

**4. The relationship between the endosteal and vascular niches**

& Li, 2006).

G0 state (Parmar *et al.,* 2007).

**5. HSCs outside their niches**

It is well known that HSC circulation involves HSCs leaving the bone marrow, entering in the vascular system (mobilization), and returning to the bone marrow (homing). However, the underlying physiological function of these events is not well understood (Yin

Hematopoietic Stem Cell Niche: Role in Normal and Malignant Hematopoiesis

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23

The endosteal niche, localized at the inner surface of the bone cavity and with abundant osteoblasts, might serve as a reservoir for long-term HSC storage in a quiescent state, whereas the vascular niche, which consists of sinusoidal endothelial cell lining blood vessel, provides an environment for short-term HSC proliferation and differentiation. Both niches act together to maintain hematopoietic homeostasis or to restore it after damage (Guerrouahen *et al.,* 2011).

Based on *in vivo* immunofluorescence with signaling lymphocytic activation molecule (SLAM), a family of cell surface receptors, Kiel & Morrison (2006) identified the vascular niche of HSCs in several tissues, also known as the sinusoidal endothelial niche. Though the two kinds of HSCs niches were anatomically and functionally defined, accumulated data suggests that endosteal and vascular niches overlap in both location and function (Figure 1). Three dimension imaging determined that there are abundant vascular structures on the surfaces of trabecular bones, and that those vessels and endosteal surfaces are intimately coupled with each other within a trabecular region (Wang & Wargers*,* 2011).

The major difference between both microenvironments is the oxygen level. Higher in the vascular niche than in the endosteal niche under hypoxia, HSCs would move to the vascular niche and resume then the cell cycle in order to restore hematopoiesis. HSCs would then return to the endosteal niche where they would again be maintained in the

Numerous examples of HSC-endothelial cross-talk exist, although most studies have focused on the function of the endothelial cell in HSC homing. Recent investigations have suggested roles for the vascular CAMs E- and P-selectin and VCAM-1 in HSC homing to bone marrow,

To turn the situation even more complex, HSCs are not static. Although the vast majority of HSCs in adult humans is located in the bone marrow, HSCs show remarkable mobility. In response to specific signals they can exit and re-enter the endosteal bone-marrow HSC niche,

The use of mobilizing regimens for the collection of HSCs from the peripheral blood of donors rather than from the BM soon became common clinical practice in transplanta‐ tion settings far before understanding the mechanisms underlying this phenomenon. The most efficient cytokine currently used in the clinical practice to mobilize HSCs is the

processes known as mobilization and homing, respectively (Wilson *et al.,* 2006).

as well as for the chemokine SDF-1 (Calmone & Sipkins, 2008).

These cells have recently been shown to be high secretors of SDF-1 (CXCL12), and as a result they have been named CXCL12 abundant reticular (CAR) cells. Phenotypically they express VCAM-1, CD44, 238 platelet-derived growth factor receptor (PDGFRα and PDGFRβ) and possess adipogenic and osteogenic differentiation capacity (Nakamura-Ishizu & Suda, 2012). Histochemical analysis revealed that all bone marrow sinusoidal endothelial cells are surrounded by a proportion of CAR cells, however, CAR cells do not express the pan-endothelial marker platelet/endothelial cell-adhesion molecule 1 (PECAM-1)/CD31 or the smooth muscle cell maker and smooth muscle α-actin (SMαA), suggesting that they are different from endothelial cells and smooth muscle cells (Sugiyama & Nagasawa, 2012).

The depletion of CAR cells using a diphtheria toxin mouse model reduces the cycling of lymphoid and erythroid progenitors, as well as the total HSC cell number and cell cycling, reflecting that CAR cells regulate the proliferation of HSC rather than its quiescence. Ablation of CAR cells did not influence other niche cell compartments such as endothelial cells or osteoblasts (Lilly *et al*., 2011). However, results based on the short term duration of the CAR cell deletion underestimate the influence that CAR cells may convey on other niches.

#### **3.2. NES+ cells**

Nestin is an intermediate filament protein that was originally identified as a marker of neural progenitor. Its expression has subsequently been detected in a wide range of progenitor cells and endothelial cells (Sugiyama & Nagasawa, 2012). NES+ MSCs can be differentiated into adipocytes, osteoblasts and chondrocytes, and their HSC regulatory function is modified by sympathectomy or by treatment with G‑CSF (which downregulates HSC ability to express CXCL12, SCF, angiopoietin, IL7, and vascular cell adhesion molecule 1 (VCAM1)) (Wang & Wagers, 2011). Nestin+ cells exhibited multilineage differentiation into various mesenchymal cell lineages including osteoblasts (Nagasawa *et al.,* 2011).

Nestin+ cells express high levels of genes involved in the regulation of HSCs: Cxcl12, c-kit ligand, angiopoietin-1, interleukin-7, vascular cell adhesion molecule-1 and osteopontin. Recently, it was demonstrated that bone marrow CD169+ macrophages are able to maintain the HSCs through CXCL12 levels and through nestin 265+ niche cells, which emphasizes the dense crosstalk among various niches (Nakamura-Ishizu & Suda, 2012).

### **4. The relationship between the endosteal and vascular niches**

It is well known that HSC circulation involves HSCs leaving the bone marrow, entering in the vascular system (mobilization), and returning to the bone marrow (homing). However, the underlying physiological function of these events is not well understood (Yin & Li, 2006).

The endosteal niche, localized at the inner surface of the bone cavity and with abundant osteoblasts, might serve as a reservoir for long-term HSC storage in a quiescent state, whereas the vascular niche, which consists of sinusoidal endothelial cell lining blood vessel, provides an environment for short-term HSC proliferation and differentiation. Both niches act together to maintain hematopoietic homeostasis or to restore it after damage (Guerrouahen *et al.,* 2011).

Based on *in vivo* immunofluorescence with signaling lymphocytic activation molecule (SLAM), a family of cell surface receptors, Kiel & Morrison (2006) identified the vascular niche of HSCs in several tissues, also known as the sinusoidal endothelial niche. Though the two kinds of HSCs niches were anatomically and functionally defined, accumulated data suggests that endosteal and vascular niches overlap in both location and function (Figure 1). Three dimension imaging determined that there are abundant vascular structures on the surfaces of trabecular bones, and that those vessels and endosteal surfaces are intimately coupled with each other within a trabecular region (Wang & Wargers*,* 2011).

The major difference between both microenvironments is the oxygen level. Higher in the vascular niche than in the endosteal niche under hypoxia, HSCs would move to the vascular niche and resume then the cell cycle in order to restore hematopoiesis. HSCs would then return to the endosteal niche where they would again be maintained in the G0 state (Parmar *et al.,* 2007).

Numerous examples of HSC-endothelial cross-talk exist, although most studies have focused on the function of the endothelial cell in HSC homing. Recent investigations have suggested roles for the vascular CAMs E- and P-selectin and VCAM-1 in HSC homing to bone marrow, as well as for the chemokine SDF-1 (Calmone & Sipkins, 2008).

#### **5. HSCs outside their niches**

**3.1. CAR cells**

(Sugiyama & Nagasawa, 2012).

22 Stem Cell Biology in Normal Life and Diseases

(Sugiyama & Nagasawa, 2012).

convey on other niches.

**3.2. NES+ cells**

In human bone marrow, such reticular cells constitute the subendothelial (adventitial) layer of sinusoidal walls projecting a reticular process that is in close contact with HSCs. Interestingly, these reticular cells were derived from a specific subset of mesenchymal cells (CD146þ) that had been shown to produce either reticular or endosteum of the ectopic hematopoietic microenvironment (HME), referred to as skeletal stem cells

These cells have recently been shown to be high secretors of SDF-1 (CXCL12), and as a result they have been named CXCL12 abundant reticular (CAR) cells. Phenotypically they express VCAM-1, CD44, 238 platelet-derived growth factor receptor (PDGFRα and PDGFRβ) and possess adipogenic and osteogenic differentiation capacity (Nakamura-Ishizu & Suda, 2012). Histochemical analysis revealed that all bone marrow sinusoidal endothelial cells are surrounded by a proportion of CAR cells, however, CAR cells do not express the pan-endothelial marker platelet/endothelial cell-adhesion molecule 1 (PECAM-1)/CD31 or the smooth muscle cell maker and smooth muscle α-actin (SMαA), suggesting that they are different from endothelial cells and smooth muscle cells

The depletion of CAR cells using a diphtheria toxin mouse model reduces the cycling of lymphoid and erythroid progenitors, as well as the total HSC cell number and cell cycling, reflecting that CAR cells regulate the proliferation of HSC rather than its quiescence. Ablation of CAR cells did not influence other niche cell compartments such as endothelial cells or osteoblasts (Lilly *et al*., 2011). However, results based on the short term duration of the CAR cell deletion underestimate the influence that CAR cells may

Nestin is an intermediate filament protein that was originally identified as a marker of neural progenitor. Its expression has subsequently been detected in a wide range of progenitor cells and endothelial cells (Sugiyama & Nagasawa, 2012). NES+ MSCs can be differentiated into adipocytes, osteoblasts and chondrocytes, and their HSC regulatory function is modified by sympathectomy or by treatment with G‑CSF (which downregulates HSC ability to express CXCL12, SCF, angiopoietin, IL7, and vascular cell adhesion molecule 1 (VCAM1)) (Wang & Wagers, 2011). Nestin+ cells exhibited multilineage differentiation into various mesenchymal

Nestin+ cells express high levels of genes involved in the regulation of HSCs: Cxcl12, c-kit ligand, angiopoietin-1, interleukin-7, vascular cell adhesion molecule-1 and osteopontin. Recently, it was demonstrated that bone marrow CD169+ macrophages are able to maintain the HSCs through CXCL12 levels and through nestin 265+ niche cells, which emphasizes the

cell lineages including osteoblasts (Nagasawa *et al.,* 2011).

dense crosstalk among various niches (Nakamura-Ishizu & Suda, 2012).

To turn the situation even more complex, HSCs are not static. Although the vast majority of HSCs in adult humans is located in the bone marrow, HSCs show remarkable mobility. In response to specific signals they can exit and re-enter the endosteal bone-marrow HSC niche, processes known as mobilization and homing, respectively (Wilson *et al.,* 2006).

The use of mobilizing regimens for the collection of HSCs from the peripheral blood of donors rather than from the BM soon became common clinical practice in transplanta‐ tion settings far before understanding the mechanisms underlying this phenomenon. The most efficient cytokine currently used in the clinical practice to mobilize HSCs is the granulocyte colony-stimulating factor (G-CSF) or its pegylated form (Peg G-CSF) used in a single administration. It was then shown that G-CSF-induced mobilization involved the modulation of the SDF-1/CXCR4 axis, whereby the reduction of the SDF-1 levels and the upregulation of its receptor CXCR4 were correlated with stem cell mobilization. Howev‐ er, although evidence suggested that the mobilization effect of G-CSF lies in its capacity to modify the SDF1 gradient (CXCL12) between the bone marrow and the peripheral blood, favoring the release of HSCs, the exact mechanism by which this occurs has not been completely clarified (Lymperi *et al.,* 2010).

**6.1. Leukemic Stem Cells**

ing mutations (Blair *et al.,* 1998).

wa *et al.,* 2011).

Leukemic stem cells (LSCs) were first described in 1994. The paradigm of cancer stem cells considers leukemia a hierarchical disease process whose growth is sustained by a rare population of LSCs. LSCs maintain the capacity to self-renewal and give rise to malignant

Hematopoietic Stem Cell Niche: Role in Normal and Malignant Hematopoiesis

http://dx.doi.org/10.5772/55508

25

It has been speculated that the transformation involves at least a 2-step process, one mutation blocking differentiation and another event conferring a proliferative advantage to its progeny (figure 2). Other LSCs may result from dedifferentiation of more commit‐ ted progenitors that reacquire the ability of self-renewal prior to accumulating transform‐

**Figure 1. Reticular niches created by mesenchymal progenitors might maintain and regulate HSCs.** A model for the localization of HSCs and their association with candidate cell niches in the bone marrow (Modified from Nagasa‐

There is much greater heterogeneity in the phenotype of LSCs than has been previously thought, indicating the inadequacy of the currently used surface antigens or biochemical markers as criteria for LSCs isolation. Evidences from the literature suggest that LSCs are a moving target and its identification depends on many factors including the receptiveness of the murine model used in the experimental design. In addition, it is debatable whether results derived from highly artificial animal models could be extrapolated for the situation in human

progeny with extensive proliferative potential (Flynn & Kaufman*,* 2007).

The release of HSCs not only occurs during mobilization but it is also observed during homeostasis, when a small number of HSCs are constantly released into the circulation. Although their precise physiological role remains unclear, they might provide a rapidly accessible source of HSCs to repopulate areas of injured bone marrow (Wilson *et al.,* 2006).

#### **6. HSC niches and disease**

The elucidation of the cellular components and molecular effectors of the HSC niche has raised obvious interest on whether analogous regulatory processes are involved in the biology of bone marrow tumors. Increasing evidences point toward critical roles of nonautonomous, microenvironmental factors in the development, progression, and drug resistance of different malignancies evolving in the bone marrow (Carlesso & Cardoso*,* 2010). Although most hematopoietic malignancies are likely to arise from mutations that inappropriately activate hematopoietic cell proliferation and survival pathways, recent data demonstrate that hematopoiesis can also be dysregulated by alterations in the niche, with defects in HSCs themselves arising secondarily (Wang *et al.,* 2012). The extent of the reliance of these tumors on the microenvironment appears to be dependent upon the type and stage of malignancy. At one extreme is a neoplastic growth that is dependent on dysregulated cell-cell interactions and signalling pathways within the microenvironment. At the other end of the spectrum there are malignancies that exhibit an absolute depend‐ ence on normal microenvironmental cues for disease progression, such as the expression of specific cytokines and growth factors (Guerrouahen *et al.,* 2011).

Several studies have provided insights into the role of altered microenvironment signaling leading to myelofibrosis, myeloma, and myelodysplastic syndromes (Noll *et al.,* 2012). Lataillade *et al.* (2008) suggested that an imbalance between endosteal and vascular niches may be important in idiopathic disorder characterized by bone marrow fibrosis (primary myelo‐ fibrosis), leading to the development of clonal stem cell proliferation. The most compelling recent example comes from work in which miRNA processing was disrupted in osteoblast precursors and mice developed myelodysplasia, which in rare cases progressed to myeloid leukaemia by 3 weeks of age; notably, HSCs transplanted from these mice into wild-type recipients did not transfer myelodysplasia, indicating that the HSCs were not intrinsically competent to produce pathologic changes (Hosokawa *et al.,* 2010).

#### **6.1. Leukemic Stem Cells**

granulocyte colony-stimulating factor (G-CSF) or its pegylated form (Peg G-CSF) used in a single administration. It was then shown that G-CSF-induced mobilization involved the modulation of the SDF-1/CXCR4 axis, whereby the reduction of the SDF-1 levels and the upregulation of its receptor CXCR4 were correlated with stem cell mobilization. Howev‐ er, although evidence suggested that the mobilization effect of G-CSF lies in its capacity to modify the SDF1 gradient (CXCL12) between the bone marrow and the peripheral blood, favoring the release of HSCs, the exact mechanism by which this occurs has not been

The release of HSCs not only occurs during mobilization but it is also observed during homeostasis, when a small number of HSCs are constantly released into the circulation. Although their precise physiological role remains unclear, they might provide a rapidly accessible source of HSCs to repopulate areas of injured bone marrow (Wilson *et al.,* 2006).

The elucidation of the cellular components and molecular effectors of the HSC niche has raised obvious interest on whether analogous regulatory processes are involved in the biology of bone marrow tumors. Increasing evidences point toward critical roles of nonautonomous, microenvironmental factors in the development, progression, and drug resistance of different malignancies evolving in the bone marrow (Carlesso & Cardoso*,* 2010). Although most hematopoietic malignancies are likely to arise from mutations that inappropriately activate hematopoietic cell proliferation and survival pathways, recent data demonstrate that hematopoiesis can also be dysregulated by alterations in the niche, with defects in HSCs themselves arising secondarily (Wang *et al.,* 2012). The extent of the reliance of these tumors on the microenvironment appears to be dependent upon the type and stage of malignancy. At one extreme is a neoplastic growth that is dependent on dysregulated cell-cell interactions and signalling pathways within the microenvironment. At the other end of the spectrum there are malignancies that exhibit an absolute depend‐ ence on normal microenvironmental cues for disease progression, such as the expression

Several studies have provided insights into the role of altered microenvironment signaling leading to myelofibrosis, myeloma, and myelodysplastic syndromes (Noll *et al.,* 2012). Lataillade *et al.* (2008) suggested that an imbalance between endosteal and vascular niches may be important in idiopathic disorder characterized by bone marrow fibrosis (primary myelo‐ fibrosis), leading to the development of clonal stem cell proliferation. The most compelling recent example comes from work in which miRNA processing was disrupted in osteoblast precursors and mice developed myelodysplasia, which in rare cases progressed to myeloid leukaemia by 3 weeks of age; notably, HSCs transplanted from these mice into wild-type recipients did not transfer myelodysplasia, indicating that the HSCs were not intrinsically

of specific cytokines and growth factors (Guerrouahen *et al.,* 2011).

competent to produce pathologic changes (Hosokawa *et al.,* 2010).

completely clarified (Lymperi *et al.,* 2010).

**6. HSC niches and disease**

24 Stem Cell Biology in Normal Life and Diseases

Leukemic stem cells (LSCs) were first described in 1994. The paradigm of cancer stem cells considers leukemia a hierarchical disease process whose growth is sustained by a rare population of LSCs. LSCs maintain the capacity to self-renewal and give rise to malignant progeny with extensive proliferative potential (Flynn & Kaufman*,* 2007).

It has been speculated that the transformation involves at least a 2-step process, one mutation blocking differentiation and another event conferring a proliferative advantage to its progeny (figure 2). Other LSCs may result from dedifferentiation of more commit‐ ted progenitors that reacquire the ability of self-renewal prior to accumulating transform‐ ing mutations (Blair *et al.,* 1998).

**Figure 1. Reticular niches created by mesenchymal progenitors might maintain and regulate HSCs.** A model for the localization of HSCs and their association with candidate cell niches in the bone marrow (Modified from Nagasa‐ wa *et al.,* 2011).

There is much greater heterogeneity in the phenotype of LSCs than has been previously thought, indicating the inadequacy of the currently used surface antigens or biochemical markers as criteria for LSCs isolation. Evidences from the literature suggest that LSCs are a moving target and its identification depends on many factors including the receptiveness of the murine model used in the experimental design. In addition, it is debatable whether results derived from highly artificial animal models could be extrapolated for the situation in human

In analogy to normal HSC, LSC also need the marrow niche for their malignant self-renewal and dormant state. Perturbing the binding of LSC to the marrow niche through disruption of the adhesive forces might therefore "mobilize" LSC from their protective environment. Molecules such as CD44, CXCR4, N-cadherin, among others, appear to play significant regulatory roles for HSC and LSC trafficking, signaling and homing to their marrow niche (Sugiyama *et al.,* 2006; Spoo *et al.,* 2007; Teicher & Fricker, 2010; Lutz *et al.,* 2012). LSC may also hijack these pathways in a number of ways, for example, up-regulation of the α4β1 integrin, VLA-4, which mediates adhesion to fibronectin and VCAM-1. Patients with undetectable VLA-4 levels on leukemic blasts had an excellent response to chemotherapy, perhaps indicat‐ ing that this pathway may mediate a stromal influence on sensitivity to chemotherapy (Matsunaga *et al.,* 2003). These data suggest that interactions between VLA-4 on leukemic cells and fibronectin expressed on BM stromal cells may modulate chemotherapy sensitivity (Doan & Chute*,* 2012). Another example is the CD44, the receptor for hyaluronic acid, which is also important for hematopoietic stem and progenitor cell homing to the vascular niche. In both, a mouse model of chronic myelogenous leukemia and a xenograft model of human AML, treatment with an anti-CD44 antibody resulted in LSC mobilization from the niche, LSC

Hematopoietic Stem Cell Niche: Role in Normal and Malignant Hematopoiesis

http://dx.doi.org/10.5772/55508

27

A number of pathways and cell types have been shown to affect the behaviour of both HSCs in normal hematopoiesis and the malignant myeloma plasma cells. It is through these regulated interactions with cell populations and signalling pathways within the bone marrow microenvironment that myeloma cells are believed to 'hijack' the normal hematopoietic niche to aid the extensive growth and proliferation of tumour cells (Noll *et al.,* 2012). Multiple myeloma (MM) is characterized by the proliferation of a malignant plasma cell clone, initially located in the bone marrow microenvironment. This illness is unique among hematological malignancies in its capacity to cause great bone destruction, leading to pathologic bone fractures and intractable bone pain. This result is the consequence of an imbalance between

Normal plasma cells are dependent on specific signals from bone marrow stem cells to regulate their differentiation, growth and localization. These same signals are required for myeloma cell growth and survival, supporting the notion that the bone marrow provides a permissive environment for disease development (Degrassi *et al.,* 1993). It is evident that the presence of myeloma cells in the bone marrow modulates the expression of cytokines from stromal cells, which enhances their ability to modify the microenvironment to support

During the past few years, the theoretical concept of a specific stem cell microenvironment that was proposed in the 1960s and 1970s, has finally received greater attention. As mentioned,

osteoblastic and osteoclastic activity induced by MM cells (Corre *et al.,* 2007).

differentiation and LSC eradication (Krause *et al.,* 2006).

**6.2. Multiple myeloma**

malignant growth (Noll *et al.,* 2012).

**7. Conclusion**

**Figure 2. Putative mechanisms for AML stem cell and niche interactions** *in vivo.* The niche provides support for self-renewal, quiescence, homing, engraftment, and proliferative potential for HSCs. LSCs may impair the function of the normal HSC niche by direct invasion or secretion of substances such as stem cell factor. LSCs can infiltrate these niches leading to enhanced self-renewal and proliferation, enforced quiescence, and resistance to chemotherapeutic agents. (Modified from Lane *et al.,* 2009).

(Lutz *et al.,* 2012). So far there are only few data analyzing the diversity of LSC in individual patients with acute myeloid leukemia (AML). It has been shown that CD34+CD38− subpopu‐ lations are heterogeneous in the distribution of mutations compared to the whole blast population at diagnosis. The situation might be similar to the genetic heterogeneity detected in childhood acute lymphoblastic leukemia (Anderson *et al.,* 2011).

In BCR-ABL–associated leukemias, the transformation occurs at the stem cell and progen‐ itor cell level depending on the phenotype and fusion transcript isoform (Castor *et al.,* 2005). It is a population of highly quiescent HSCs expressing BCR-ABL1 that can be isolated from untreated chronic myeloid leukemia (CML) patients and from imatinib-treated CML patients. These quiescent, non-proliferating CD34+ CML cells have been shown to be resistant to a range of pro-apoptotic stimuli including chemotherapy and tyrosine kinase inhibition with imatinib. The quiescent BCR-ABL1-expressing HSCs can be regarded as LSCs. By way of comparison, proliferating CD34+ progenitors in CML are sensitive to imatinib-induced apoptosis that is significantly mediated by the pro-apoptotic BCL-2 family proteins Bim and Bad (Kuroda *et al.,* 2006; Ng, 2012).

In analogy to normal HSC, LSC also need the marrow niche for their malignant self-renewal and dormant state. Perturbing the binding of LSC to the marrow niche through disruption of the adhesive forces might therefore "mobilize" LSC from their protective environment. Molecules such as CD44, CXCR4, N-cadherin, among others, appear to play significant regulatory roles for HSC and LSC trafficking, signaling and homing to their marrow niche (Sugiyama *et al.,* 2006; Spoo *et al.,* 2007; Teicher & Fricker, 2010; Lutz *et al.,* 2012). LSC may also hijack these pathways in a number of ways, for example, up-regulation of the α4β1 integrin, VLA-4, which mediates adhesion to fibronectin and VCAM-1. Patients with undetectable VLA-4 levels on leukemic blasts had an excellent response to chemotherapy, perhaps indicat‐ ing that this pathway may mediate a stromal influence on sensitivity to chemotherapy (Matsunaga *et al.,* 2003). These data suggest that interactions between VLA-4 on leukemic cells and fibronectin expressed on BM stromal cells may modulate chemotherapy sensitivity (Doan & Chute*,* 2012). Another example is the CD44, the receptor for hyaluronic acid, which is also important for hematopoietic stem and progenitor cell homing to the vascular niche. In both, a mouse model of chronic myelogenous leukemia and a xenograft model of human AML, treatment with an anti-CD44 antibody resulted in LSC mobilization from the niche, LSC differentiation and LSC eradication (Krause *et al.,* 2006).

#### **6.2. Multiple myeloma**

A number of pathways and cell types have been shown to affect the behaviour of both HSCs in normal hematopoiesis and the malignant myeloma plasma cells. It is through these regulated interactions with cell populations and signalling pathways within the bone marrow microenvironment that myeloma cells are believed to 'hijack' the normal hematopoietic niche to aid the extensive growth and proliferation of tumour cells (Noll *et al.,* 2012). Multiple myeloma (MM) is characterized by the proliferation of a malignant plasma cell clone, initially located in the bone marrow microenvironment. This illness is unique among hematological malignancies in its capacity to cause great bone destruction, leading to pathologic bone fractures and intractable bone pain. This result is the consequence of an imbalance between osteoblastic and osteoclastic activity induced by MM cells (Corre *et al.,* 2007).

Normal plasma cells are dependent on specific signals from bone marrow stem cells to regulate their differentiation, growth and localization. These same signals are required for myeloma cell growth and survival, supporting the notion that the bone marrow provides a permissive environment for disease development (Degrassi *et al.,* 1993). It is evident that the presence of myeloma cells in the bone marrow modulates the expression of cytokines from stromal cells, which enhances their ability to modify the microenvironment to support malignant growth (Noll *et al.,* 2012).

#### **7. Conclusion**

(Lutz *et al.,* 2012). So far there are only few data analyzing the diversity of LSC in individual patients with acute myeloid leukemia (AML). It has been shown that CD34+CD38− subpopu‐ lations are heterogeneous in the distribution of mutations compared to the whole blast population at diagnosis. The situation might be similar to the genetic heterogeneity detected

**Figure 2. Putative mechanisms for AML stem cell and niche interactions** *in vivo.* The niche provides support for self-renewal, quiescence, homing, engraftment, and proliferative potential for HSCs. LSCs may impair the function of the normal HSC niche by direct invasion or secretion of substances such as stem cell factor. LSCs can infiltrate these niches leading to enhanced self-renewal and proliferation, enforced quiescence, and resistance to chemotherapeutic

In BCR-ABL–associated leukemias, the transformation occurs at the stem cell and progen‐ itor cell level depending on the phenotype and fusion transcript isoform (Castor *et al.,* 2005). It is a population of highly quiescent HSCs expressing BCR-ABL1 that can be isolated from untreated chronic myeloid leukemia (CML) patients and from imatinib-treated CML patients. These quiescent, non-proliferating CD34+ CML cells have been shown to be resistant to a range of pro-apoptotic stimuli including chemotherapy and tyrosine kinase inhibition with imatinib. The quiescent BCR-ABL1-expressing HSCs can be regarded as LSCs. By way of comparison, proliferating CD34+ progenitors in CML are sensitive to imatinib-induced apoptosis that is significantly mediated by the pro-apoptotic BCL-2 family

in childhood acute lymphoblastic leukemia (Anderson *et al.,* 2011).

agents. (Modified from Lane *et al.,* 2009).

26 Stem Cell Biology in Normal Life and Diseases

proteins Bim and Bad (Kuroda *et al.,* 2006; Ng, 2012).

During the past few years, the theoretical concept of a specific stem cell microenvironment that was proposed in the 1960s and 1970s, has finally received greater attention. As mentioned, pinpointing the exact location of the hematopoietic stem cell *in vivo* within the bone marrow is difficult, despite advancements in immunohistochemistry, genetic marking of cells, and *in vivo* imaging. Early studies showed that hematopoietic progenitor and stem cells were highly present near the endosteal bone surface, whereas more mature cells were selectively localized centrally within the bone marrow cavity.

mura, Y, Kitajewski, J, Rosenwaks, Z, Bernstein, I. D, & Rafii, S. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic

Hematopoietic Stem Cell Niche: Role in Normal and Malignant Hematopoiesis

http://dx.doi.org/10.5772/55508

29

[6] Can, A. Haematopoietic stem cells niches: interrelations between structure and func‐

[7] Carlesso, N, & Cardoso, A. A. Stem cell regulatory niches and their role in normal

[8] Castor, A, Nilsson, L, Astrand-grundström, I, Buitenhuis, M, Ramirez, C, Anderson, K, Strömbeck, B, Garwicz, S, Békássy, A. N, Schmiegelow, K, Lausen, B, Hokland, P, Lehmann, S, Juliusson, G, Johansson, B, & Jacobsen, S. E. Distinct patterns of hemato‐ poietic stem cell involvement in acute lymphoblastic leukemia. Nat Med. (2005). , 11,

[9] Chotinantakul, K, & Leeanansaksiri, W. Hematopoietic stem cell development, nich‐

[10] Corre, J, Mahtouk, K, Attal, M, Gadelorge, M, Huynh, A, Fleury-cappellesso, S, Dan‐ ho, C, Laharrague, P, Klein, B, Rème, T, & Bourin, P. Bone marrow mesenchymal stem cells are abnormal in multiple myeloma. Leukemia. (2007). , 21, 1079-1088.

[11] Coskun, S, & Hirschi, K. K. Establishment and regulation of the HSC niche: Roles of osteoblastic and vascular compartments. Birth Defects Res C Embryo Today. (2010). ,

[12] Degrassi, A, Hilbert, D. M, Rudikoff, S, Anderson, A. O, Potter, M, & Coon, H. G. In vitro culture of primary plasmacytomas requires stromal cell feeder layers. Proc Natl

[13] Doan, P. L, & Chute, J. P. The vascular niche: home for normal and malignant hema‐

[14] Flynn, C. M, & Kaufman, D. S. Donor cell leukemia: insight into cancer stem cells and

[15] Frisch, B. J, Porter, R. L, & Calvi, L. M. Hematopoietic niche and bone meet. Curr

[16] Guerrouahen, B. S, Al-hijji, I, & Tabrizi, A. R. Osteoblastic and vascular endothelial niches, their control on normal hematopoietic stem cells, and their consequences on

[17] Hosokawa, K, Arai, F, Yoshihara, H, Iwasaki, H, Hembree, M, Yin, T, Nakamura, Y, Gomei, Y, Takubo, K, Shiama, H, Matsuoka, S, Li, L, & Suda, T. Cadherin-based ad‐ hesion is a potential target for niche manipulation to protect hematopoietic stem cells

and malignant hematopoiesis. Curr Opin Hematol. (2010). , 17, 281-286.

stem cells. Cell Stem Cell. (2011). , 6, 251-264.

tion. Transfus Apher Sci. (2008). , 38, 261-268.

es, and signaling pathways. Bone Marrow Res. (2012).

Acad Sci USA. (1993). , 90, 2060-2064.

topoietic stem cells. Leukemia. (2012). , 26, 54-62.

the stem cell niche. Blood. (2007). , 109, 2688-2692.

the development of leukemia. Stem Cells Int. (2011).

in adult bone marrow. Cell Stem Cell. (2010). , 6, 194-198.

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630-637.

90, 229-242.

The development of hematologic malignancies may be a multi-step process involving muta‐ tions both in the hematopoietic cells and/or in cells present in the supportive microenviron‐ ment. Targeting the niche-HSCs, niche-leukemic cells, or niche itself in hematopoietic malignancies is an attractive potential addition to the therapeutic possibilities. The challenge of stem cell biology is to translate the expansion of biological insights into clinically meaningful improvements for patients with hematological malignancies and related disorders.

#### **Author details**

Sergio P. Bydlowski, Debora Levy, Jorge M.L. Ruiz and Juliana Pereira

Laboratory of Genetics and Molecular Hematology, Department of Medicine, University of São Paulo School of Medicine, São Paulo/SP,, Brazil

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[6] Can, A. Haematopoietic stem cells niches: interrelations between structure and func‐ tion. Transfus Apher Sci. (2008). , 38, 261-268.

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The development of hematologic malignancies may be a multi-step process involving muta‐ tions both in the hematopoietic cells and/or in cells present in the supportive microenviron‐ ment. Targeting the niche-HSCs, niche-leukemic cells, or niche itself in hematopoietic malignancies is an attractive potential addition to the therapeutic possibilities. The challenge of stem cell biology is to translate the expansion of biological insights into clinically meaningful

Laboratory of Genetics and Molecular Hematology, Department of Medicine, University of

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[18] Kiel, M. J, & Morrison, S. J. Maintaining hematopoietic stem cells in the vascular ni‐ che. Immunity. (2006). , 25, 862-864.

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[31] Oh, I. H, & Kwon, K. R. Concise review: multiple niches for hematopoietic stem cell

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[34] Renström, J, Kröger, M, Peschel, C, & Oostendorp, R. A. How the niche regulates

[35] Schofield, R. The relationship between the spleen colony-forming cell and the hae‐

[36] Shen, Y, & Nilsson, S. K. Bone, microenvironment and hematopoiesis. Curr Opin

[37] Spoo, A. C, Lübbert, M, Wierda, W. G, & Burger, J. A. CXCR4 is a prognostic marker

[38] Sugiyama, T, Kohara, H, Noda, M, & Nagasawa, T. Maintenance of the hematopoiet‐ ic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal

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[40] Teicher, B. A, & Fricker, S. P. CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin Can‐

[41] ter Huurne MFigdor CG, Torensma R. Hematopoietic stem cells are coordinated by the molecular cues of the endosteal niche. Stem Cells Dev. (2010). , 19, 1131-1141. [42] Wang, H, Zhang, P, Liu, L, & Zou, L. Hierarchical organization and regulation of the hematopoietic stem cell osteoblastic niche. Crit Rev Oncol Hematol. (2012). Epub

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[27] Matsunaga, T, Takemoto, N, Sato, T, Takimoto, R, Tanaka, I, Fujimi, A, Akiyama, T, Kuroda, H, Kawano, Y, Kobune, M, Kato, J, Hirayama, Y, Sakamaki, S, Kohda, K, Miyake, K, & Niitsu, Y. Interaction between leukemic-cell VLA-4 and stromal fibro‐ nectin is a decisive factor for minimal residual disease of acute myelogenous leuke‐

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**Chapter 3**

**Expression Profile of Galectins (Gal-1, Gal-9, Gal-11 and**

**Gal-13) in Human Bone Marrow Derived Mesenchymal**

Mesenchymal stem cells (MCSs) are refined as undifferencitaed cells that are capable of self renewal and differentiation into several cell types such chondrocyte, adipocyte osteocyte, myocyte and neuron-like cells. MSC can be isolated from bone marrow umbilical cord blood adipose tissue placenta. Although bone marrow(BM) has been regarded as a major source of MSC umbilical cord blood has been regarded as an alternative source for isolation of MSC. Human umbilical cord blood derived mesenchymal stem cell (hUCB-MSCs) have a capacity

Researchers are interested in these cells because of their ability of differentiating into multiple mesenchymal and non mesenchymal lineages. Furthermore, MSCs evoke only minimal immunoreactivity and they display trophic, anti-inflammatory, and immunomodulatory capacities, through secretion of bioactive soluble factors with anti-inflammatory and immu‐ nomodulatory effects in vivo. These properties were confirmed by both experimental and clinical studies which demonstrated that the MSCs support hematopoiesis and enhance the engraftment of hematopoietic stem cells (HSC) after cotransplantation, which may contribute

The galectins are a family of soluble lectins characterized by their affinity for b-galactoside residues. These proteins have recently attracted increasing attention because of theirinvolve‐ ment in various physiological and pathological processes. In addition, these proteins have recently attracted increasing attention of cancer biologists because of their essential functions including development, differentiation, cell–cell adhesion, cell–matrix interaction, growth

> © 2013 Jenhani; 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 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,

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

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

**Stem Cells in Different Culture Mediums**

Additional information is available at the end of the chapter

similar to that of BM-MCSs for multi-lineage differentiation

to a reduced incidence of graft versus host disease (GVHD). [1,2,3,4,5,6,7]

Faouzi Jenhani

**1. Introduction**

http://dx.doi.org/10.5772/55383

**Chapter 3**

## **Expression Profile of Galectins (Gal-1, Gal-9, Gal-11 and Gal-13) in Human Bone Marrow Derived Mesenchymal Stem Cells in Different Culture Mediums**

Faouzi Jenhani

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55383

#### **1. Introduction**

Mesenchymal stem cells (MCSs) are refined as undifferencitaed cells that are capable of self renewal and differentiation into several cell types such chondrocyte, adipocyte osteocyte, myocyte and neuron-like cells. MSC can be isolated from bone marrow umbilical cord blood adipose tissue placenta. Although bone marrow(BM) has been regarded as a major source of MSC umbilical cord blood has been regarded as an alternative source for isolation of MSC. Human umbilical cord blood derived mesenchymal stem cell (hUCB-MSCs) have a capacity similar to that of BM-MCSs for multi-lineage differentiation

Researchers are interested in these cells because of their ability of differentiating into multiple mesenchymal and non mesenchymal lineages. Furthermore, MSCs evoke only minimal immunoreactivity and they display trophic, anti-inflammatory, and immunomodulatory capacities, through secretion of bioactive soluble factors with anti-inflammatory and immu‐ nomodulatory effects in vivo. These properties were confirmed by both experimental and clinical studies which demonstrated that the MSCs support hematopoiesis and enhance the engraftment of hematopoietic stem cells (HSC) after cotransplantation, which may contribute to a reduced incidence of graft versus host disease (GVHD). [1,2,3,4,5,6,7]

The galectins are a family of soluble lectins characterized by their affinity for b-galactoside residues. These proteins have recently attracted increasing attention because of theirinvolve‐ ment in various physiological and pathological processes. In addition, these proteins have recently attracted increasing attention of cancer biologists because of their essential functions including development, differentiation, cell–cell adhesion, cell–matrix interaction, growth

© 2013 Jenhani; 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 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.

regulation, apoptosis, RNA splicing, and tumor metastasis. Also, it has been shown that galectins' levels are altered in various cancers. [8,9,10]

*2.2.1. Osteogenic induction (O)for analysis galectins expression*

*2.2.2. Adipogenic induction (A) for analysis galectins expression*

with Nile Red (Sigma) and observed by fluorescent microscopy.

*2.2.3. Vascular smooth muscle induction (V) for analysis galectins expression*

days. The VSM differentiation was evaluated after 3 weeks of induction.

**2.3. Immunophenotyping of mesenchymal stem cells by flow cytometry**

**2.4. Detection of galectins (Gal-1, Gal-9 Gal-11 and Gal-13), by flow cytometry**

recombinant galectin (Gal-1, Gal-9, Gal-11 and Gal-13). Thus we incubated 1,5 106

software (Becton–Dickinson, BD Biosciences San Jose, CA).

the presence of calcium deposition into osteocytes.

Jose CA).

primary antibody.

At 50% confluence, the cells were cultured for 14 days in DMEM-HG (High Glucose 4. 5 g/L) containing 2% FBS, 0. 1 µM dexamethasone (Sigma), 2 mM β-glycerolphosphate (Sigma), and 100 µM ascorbate-2-phosphate (Sigma) with medium changes every 3 days. After 2 weeks of induction, the cells were stained according to Von Kossa's and Alizarin Red methods to detect

Human Platelet Lysate for MSC Expression Galectins

http://dx.doi.org/10.5772/55383

35

The adipogenic induction medium consisted of DMEM-LG (Low Glucose 1g/L) supplemented with 20% FBS, 1 µM/L dexamethasone, 0. 5 mmol/L isobutylmethylxanthine (IBMX; Invitro‐ gen), and 60 µmol/L indomethacin (Sigma). Adipogenic differentiation was evaluated after 2 weeks of induction, by the cellular accumulation of neutral lipid vacuoles that were stained

Vascular smooth muscle (VSM) differentiation was obtained in a Mc Coy'5A medium supplemented with 12. 5% FBS, 12. 5% HS (Horse Serum, Invitrogen), 20 µM L-glutamine, 0,8 mM L-serine, 0,15 mM L-asparagine, 1 Mm sodium pyruvate, 5 mM sodium bicarbonate, 1 µM hydrocortisone and amphotericin B and antibodies. The medium was changed every 4

MSCs were immunophenotypically characterized by flow cytometry using the following fluorochrome (FITC PE, PerCP) marked monoclonal antibodies anti : CD45, CD105, CD106, CD90,CD49, CD34, et CD73 and CD14 (Becton Dickinson and Company BD Biosciences San

To reveal the expression of the galactin we used biotynaled antibodies against human

with the antibody which is specific to the focused galectin, for 30 minutes, at 4° C. We incubated also the MSCs with a control antibody which doesn't recognize any protein, but has the same isotype to evaluate a background noise which correspond to non-specific fixation of the

The MSCs were washed 2 times with PBS 1X solution, then incubated for 30 minutes with Streptavidin coupled with a phycoerythrin (PE) fluorochrome phycoerythrin (PE). Flow cytometry analysis was performed on a FACS calibur, and data were analyzed using CellQuest

MSCs cells

Galectins-1,3,9 and 13 were among the best characterized members of this family. These galactins possesses several functions and were expressed in many tissues.

Given the importance of galectins, we investigated in this work, their expression by BM MSCs in different culture mediums

### **2. Materiel and methods**

#### **2.1. Preparation of hBM-MSC**

MSCs were extracted and isolated from two sources adult the bone marrow. H- *BM cells* was harvested from the sternum or the iliac crest of healthy donors [20] after obtaining an informed consent was obtained in the Bone Marrow Graft National Center of Tunisia. The mean age of donors was 30 ± 2 years (range, 15-40 years).

MSCs were isolated using the classical plastic adhesion method. Mononuclear cells (MNCs) were isolated from hBM by density gradient (Ficoll Hypaque solution d=1. 077) centrifugation. After centrifugation at 800 g for 20 min at room temperature. MNC layer was removed from the interphase and washed twice with Hank's buffered salt solution and seeded into uncoated T25 or T75 flasks (Becton Dickinson, Bedford, MA, USA) at a cell concentration of 1×105 cells/ cm2 for BM cells and cultured in three condition mediums:


#### **2.2. Induction of the differentiation directions**

We induced the differentiation of the MSCs when they reached the confluence. We used three differentiation mediums according to the focused differentiation line:

#### *2.2.1. Osteogenic induction (O)for analysis galectins expression*

regulation, apoptosis, RNA splicing, and tumor metastasis. Also, it has been shown that

Galectins-1,3,9 and 13 were among the best characterized members of this family. These

Given the importance of galectins, we investigated in this work, their expression by BM MSCs

MSCs were extracted and isolated from two sources adult the bone marrow. H- *BM cells* was harvested from the sternum or the iliac crest of healthy donors [20] after obtaining an informed consent was obtained in the Bone Marrow Graft National Center of Tunisia. The mean age of

MSCs were isolated using the classical plastic adhesion method. Mononuclear cells (MNCs) were isolated from hBM by density gradient (Ficoll Hypaque solution d=1. 077) centrifugation. After centrifugation at 800 g for 20 min at room temperature. MNC layer was removed from the interphase and washed twice with Hank's buffered salt solution and seeded into uncoated T25 or T75 flasks (Becton Dickinson, Bedford, MA, USA) at a cell concentration of 1×105

**i.** *(M1) : basic growth* medium consisting of alpha-Minimum Essential Mediums (alpha

**ii.** (M2): basic growth medium with fetal bovine serum and FGF2 replaced by 5 % (v/v)

**iii.** (M3): basic growth medium with fetal bovine serum and FGF2 replaced by 10 %

We induced the differentiation of the MSCs when they reached the confluence. We used three

differentiation mediums according to the focused differentiation line:

(v/v) h (HPL). Cell cultures were incubated at 37°C in a 5% CO2 humidified atmos‐ phere. The medium was changed twice weekly thereafter. When reaching 60% -80% confluence, the adherent cells were detached after treatment with 0. 05% (v/v) trypsin/ 1 mM EDTA solution (Gibco BRL) and expanded by replating at a lower density at

. All the studies were performed after 2 passages (P2).


cells/

galactins possesses several functions and were expressed in many tissues.

galectins' levels are altered in various cancers. [8,9,10]

in different culture mediums

34 Stem Cell Biology in Normal Life and Diseases

**2. Materiel and methods**

**2.1. Preparation of hBM-MSC**

France)

103

cells per cm2

cm2

donors was 30 ± 2 years (range, 15-40 years).

human patelet lysed (HPL)

**2.2. Induction of the differentiation directions**

for BM cells and cultured in three condition mediums:

At 50% confluence, the cells were cultured for 14 days in DMEM-HG (High Glucose 4. 5 g/L) containing 2% FBS, 0. 1 µM dexamethasone (Sigma), 2 mM β-glycerolphosphate (Sigma), and 100 µM ascorbate-2-phosphate (Sigma) with medium changes every 3 days. After 2 weeks of induction, the cells were stained according to Von Kossa's and Alizarin Red methods to detect the presence of calcium deposition into osteocytes.

#### *2.2.2. Adipogenic induction (A) for analysis galectins expression*

The adipogenic induction medium consisted of DMEM-LG (Low Glucose 1g/L) supplemented with 20% FBS, 1 µM/L dexamethasone, 0. 5 mmol/L isobutylmethylxanthine (IBMX; Invitro‐ gen), and 60 µmol/L indomethacin (Sigma). Adipogenic differentiation was evaluated after 2 weeks of induction, by the cellular accumulation of neutral lipid vacuoles that were stained with Nile Red (Sigma) and observed by fluorescent microscopy.

#### *2.2.3. Vascular smooth muscle induction (V) for analysis galectins expression*

Vascular smooth muscle (VSM) differentiation was obtained in a Mc Coy'5A medium supplemented with 12. 5% FBS, 12. 5% HS (Horse Serum, Invitrogen), 20 µM L-glutamine, 0,8 mM L-serine, 0,15 mM L-asparagine, 1 Mm sodium pyruvate, 5 mM sodium bicarbonate, 1 µM hydrocortisone and amphotericin B and antibodies. The medium was changed every 4 days. The VSM differentiation was evaluated after 3 weeks of induction.

#### **2.3. Immunophenotyping of mesenchymal stem cells by flow cytometry**

MSCs were immunophenotypically characterized by flow cytometry using the following fluorochrome (FITC PE, PerCP) marked monoclonal antibodies anti : CD45, CD105, CD106, CD90,CD49, CD34, et CD73 and CD14 (Becton Dickinson and Company BD Biosciences San Jose CA).

#### **2.4. Detection of galectins (Gal-1, Gal-9 Gal-11 and Gal-13), by flow cytometry**

To reveal the expression of the galactin we used biotynaled antibodies against human recombinant galectin (Gal-1, Gal-9, Gal-11 and Gal-13). Thus we incubated 1,5 106 MSCs cells with the antibody which is specific to the focused galectin, for 30 minutes, at 4° C. We incubated also the MSCs with a control antibody which doesn't recognize any protein, but has the same isotype to evaluate a background noise which correspond to non-specific fixation of the primary antibody.

The MSCs were washed 2 times with PBS 1X solution, then incubated for 30 minutes with Streptavidin coupled with a phycoerythrin (PE) fluorochrome phycoerythrin (PE). Flow cytometry analysis was performed on a FACS calibur, and data were analyzed using CellQuest software (Becton–Dickinson, BD Biosciences San Jose, CA).

#### **2.5. Galectins immunofluorescence detection**

MSCs were fixed using 4% Methanol for 30 minutes at +4°C and washed with PBS. The primary antibodies against many anti-antibodies: Gal-1-Streptavidine, Gal-3-Streptavidine, Gal-9- Streptavidin and Gal-13 Streptavidin were diluted in 10% BSA and 0. 2% Tween 20 (1:100) and were incubated at 4°C for 12h followed by washings 3 times with PBS. For immunofluorescence non-specific binding sites were blocked with 10% BSA-PBS. For secondary immunofluorecence Biotine –fluoroscein were diluted in 10% BSA-PBS and incubated for 60 min at +4°C in the dark. Images were taken with camera and with inverse fluorescence microscopy

*2.6.3. Reverse transcription – Polymerase chain reaction before and after induction for*

min, 42°C for 60 min and 95°C for 5 min. Primers used are shown in Table 2.

**Construct Sequence forward Sequence reverse**

After denaturation at 65°C for 5 min, amplification was carried out by 30 cycles at 30°C for 10

Human Platelet Lysate for MSC Expression Galectins

http://dx.doi.org/10.5772/55383

37

GAPDH 5'AATCCCATCACCATCTTCCAGG3' 5'AGAGGCAGGGATGATGTTCTGG3' Gal 1 5'ATGGCTTGTGGTCTGGTC3' 5'TCAGTCAAAGGCCACACA3' Gal 9 5'ATGGCCTTCAGCGGTTCC3' 5'CTATGTCTGCACATGGGTCAG3' Gal 11 5'ATGAGTCAGCCCAGTGGG3' 5'TCAGGAGTGGACACAGTAGAG3' Gal 13 5'ATGTCCCTGACCCACAG3' 5'TCAATCGCTGATAAGCACT3'

Thermocycling was performed with a gradient thermocycler (Takara, Japan and Applied Biosystems). The analyse of the PCR products was carried out in an electrophoresis gel 1%

Data are expressed as mean ± standard error of the mean. Statistical comparisons were performed using the t-test student or Mann Whitney test with the program Graphpad, version. 5. A one-way analysis of variance (ANOVA) was done for paired samples; p < 0. 05 was

**3.1. Morphologic analysis of MSCs derived from bone marrow in terms of culture mediums**

To prepare MSCs cultures, we isolated mononuclear cells (MNCs) from bone marrow (hBM).

Adherent cell populations from the MNC fraction of hBM samples were generated by expansion culture using 3 different media: (M1) Alpha MEM with 10% FBS and 1 ng/mL FGF2, (M2) Alpha MEM with 5% HPL, (M3) Alpha with 10% HPL. After two weeks of culture, an adherent and stable cell layer was obtained from BM derived MNC with all medium Mean time for the primary culture to reach subconfluence was 15 days. After one passage(P1), adherent cells displayed a fibroblast-like morphology in culture plate (n=20). The figure 1

The MNCs derived from bone marrow were put in a density of 1. 10⁶ cells / cm².

*Galectin1,Gal-9,Gal-11 and Gal-13 expression*

**Table 2.** Sequences of the primers used in RT-PCR study

considered to be statistically significant.

showed a particular morphology of cultured MSC.

(Sigma).

**3. Results**

**2.7. Statistical analysis**

#### **2.6. RNA isolation and reverse transcription – Polymerase chain reaction before and after induction**

#### *2.6.1. RNA isolation*

For RT-PCR, Total RNA was extracted from cell culture in total confluence [90% ) using TRIZOL reagent (Gibco BRL) according to the manufacturer's instructions. Reverse transcrip‐ tion was carried out using PrimeScript™RTase (Takara: Japan and Applied Biosystems) and the cDNA fragments were amplified using RNase Inhibitor (Takara: Japan and Applied Biosystems): For each reaction, we mixed 5 µl of ARN or 5µl of distilled water (for the control), 6 µl desoxyribonucleotides (dNTP)(TaKaRa Japan and Applied Biosystems), 1µl primers specific of the gene sequence (table 1) et 0. 5 µl of enzyme la Taq polymerase enzyme (Takara: Japan and Applied Biosystems).

#### *2.6.2. RT-PCR before and after induction for the confirmation of the three directions of differentiation*

After denaturation at 65°C for 5 min, amplification was carried out by 30 cycles at 30°C for 10 min, 42°C for 60 min and 95°C for 5 min. Primers used are shown in Table 1. The expression of the following genes were analyzed before (D0) and after differentiation (D14) into three directions of differentiation: osteogenesis (O), adipogenesis (A) and vascular smooth muscle (V). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression was used as control. The sequences of the oligonucleotides were reported in Table 1. Thermocycling was performed with a gradient thermocycler (Takara, Japan and Applied Biosystems). The analyse of the PCR products was carried out in an electrophoresis gel 1% (Sigma).


**Table 1.** Sequences of the primers used in RT-PCR study

*2.6.3. Reverse transcription – Polymerase chain reaction before and after induction for Galectin1,Gal-9,Gal-11 and Gal-13 expression*

After denaturation at 65°C for 5 min, amplification was carried out by 30 cycles at 30°C for 10 min, 42°C for 60 min and 95°C for 5 min. Primers used are shown in Table 2.


**Table 2.** Sequences of the primers used in RT-PCR study

Thermocycling was performed with a gradient thermocycler (Takara, Japan and Applied Biosystems). The analyse of the PCR products was carried out in an electrophoresis gel 1% (Sigma).

#### **2.7. Statistical analysis**

Data are expressed as mean ± standard error of the mean. Statistical comparisons were performed using the t-test student or Mann Whitney test with the program Graphpad, version. 5. A one-way analysis of variance (ANOVA) was done for paired samples; p < 0. 05 was considered to be statistically significant.

#### **3. Results**

**2.5. Galectins immunofluorescence detection**

36 Stem Cell Biology in Normal Life and Diseases

**induction**

*2.6.1. RNA isolation*

Japan and Applied Biosystems).

**Table 1.** Sequences of the primers used in RT-PCR study

MSCs were fixed using 4% Methanol for 30 minutes at +4°C and washed with PBS. The primary antibodies against many anti-antibodies: Gal-1-Streptavidine, Gal-3-Streptavidine, Gal-9- Streptavidin and Gal-13 Streptavidin were diluted in 10% BSA and 0. 2% Tween 20 (1:100) and were incubated at 4°C for 12h followed by washings 3 times with PBS. For immunofluorescence non-specific binding sites were blocked with 10% BSA-PBS. For secondary immunofluorecence Biotine –fluoroscein were diluted in 10% BSA-PBS and incubated for 60 min at +4°C in the

**2.6. RNA isolation and reverse transcription – Polymerase chain reaction before and after**

For RT-PCR, Total RNA was extracted from cell culture in total confluence [90% ) using TRIZOL reagent (Gibco BRL) according to the manufacturer's instructions. Reverse transcrip‐ tion was carried out using PrimeScript™RTase (Takara: Japan and Applied Biosystems) and the cDNA fragments were amplified using RNase Inhibitor (Takara: Japan and Applied Biosystems): For each reaction, we mixed 5 µl of ARN or 5µl of distilled water (for the control), 6 µl desoxyribonucleotides (dNTP)(TaKaRa Japan and Applied Biosystems), 1µl primers specific of the gene sequence (table 1) et 0. 5 µl of enzyme la Taq polymerase enzyme (Takara:

*2.6.2. RT-PCR before and after induction for the confirmation of the three directions of differentiation*

After denaturation at 65°C for 5 min, amplification was carried out by 30 cycles at 30°C for 10 min, 42°C for 60 min and 95°C for 5 min. Primers used are shown in Table 1. The expression of the following genes were analyzed before (D0) and after differentiation (D14) into three directions of differentiation: osteogenesis (O), adipogenesis (A) and vascular smooth muscle (V). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression was used as control. The sequences of the oligonucleotides were reported in Table 1. Thermocycling was performed with a gradient thermocycler (Takara, Japan and Applied Biosystems). The analyse

of the PCR products was carried out in an electrophoresis gel 1% (Sigma).

**Construct Sequence forward Sequence reverse**

GAPDH 5'- AATCCCATCACCATCTTCCAGG-3' 5'-AGAGGCAGGGATGATGTTCTGG-3' PAL 5'-CTGGACCTCGTTGACACCTG-3' 5'-GACATTCTCTCGTTCACCGC-3' Runx2 5'-AACTTCCTGTGCTCGGTGCTG-3' 5'-GGGGAGGATTTGTGAAGACGG-3' LPL 5'-AAAGCCCTGCTCGTGCTGAC-3' 5'-TAAACCGGGCCACATCCTGT-3' PPRγ 5'-GGAGAAGCTGTTGGCGGAGA-3' 5'-TCAAGGAGGCCAGCATTGTG-3' ASMA 5'-TCATGATGCTGTTGTAGGTGGT-3' 5'-CTGTTCCAGCCATCCTTCAT-3'

dark. Images were taken with camera and with inverse fluorescence microscopy

#### **3.1. Morphologic analysis of MSCs derived from bone marrow in terms of culture mediums**

To prepare MSCs cultures, we isolated mononuclear cells (MNCs) from bone marrow (hBM). The MNCs derived from bone marrow were put in a density of 1. 10⁶ cells / cm².

Adherent cell populations from the MNC fraction of hBM samples were generated by expansion culture using 3 different media: (M1) Alpha MEM with 10% FBS and 1 ng/mL FGF2, (M2) Alpha MEM with 5% HPL, (M3) Alpha with 10% HPL. After two weeks of culture, an adherent and stable cell layer was obtained from BM derived MNC with all medium Mean time for the primary culture to reach subconfluence was 15 days. After one passage(P1), adherent cells displayed a fibroblast-like morphology in culture plate (n=20). The figure 1 showed a particular morphology of cultured MSC.

Isotype

CD90

CD105

CD106

CD73

CD49

CD34

CD14

CD45

Control

αMEM+FGF2 + 10 % FBS

**Figure 2.** Representative flow cytometry analysis of BM-MSCs.Comparison of membrane antigen expression of BM-

MSCs cultured at P2 in different media: M1 :(10% FBS+FGF2) M2: (5% HPL), M3: (10% HPL)

αMEM+ 5%HPL

αMEM+ 10%HPL

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**Figure 1.** Morphological characterization of hMSC-BM cultured in different media in the P2 (passage): M1: (10% FBS +FGF2), M2:(5% HPL), M3:(10% HPL).

**hM**

**MSC-BM**

**M1** 

38 Stem Cell Biology in Normal Life and Diseases

**M2** 

**M3** 

+FGF2), M2:(5% HPL), M3:(10% HPL).

**Figure 1.** Morphological characterization of hMSC-BM cultured in different media in the P2 (passage): M1: (10% FBS

**Figure 2.** Representative flow cytometry analysis of BM-MSCs.Comparison of membrane antigen expression of BM-MSCs cultured at P2 in different media: M1 :(10% FBS+FGF2) M2: (5% HPL), M3: (10% HPL)

     

Also MSCs were immunophenotypically characterized by flow cytometry. This analysis revealed that MSCs were uniformly positive for CD73 CD90 CD105 CD106 but negative for CD14 CD34 and CD45 (figure 2).

The influence of HPL or FBS on the osteogenic and adipogenic differentiation potential of MSCs was investigated after appropriate induction at the second passage (P2). Cells grown in HPL or FBS deposited an extensive mineralized matrix when cultured for 2 weeks in an osteogenic medium [2% FBS), as demonstrated by strong alizarin red staining. These cells also efficiently differentiated into the adipogenic lineage, as indicated by Oil Red O staining of lipid droplets in the cytoplasm following culture in an adipogenic medium.

All populations of BM-MSCs cultured in different conditions with the HPL showed osteogenic, Vascular smooth muscle and chondrogenic differentiation capacity. The difference appears in adipogenic differentiation with 10% HPL (Fig 3 ).

ogenic, adipog

Osteo

 differentia ation capacity assessed by s taining of BM M-derived MS Cs

scular smooth

muscular

To confirm their differentiation potential, after P1 MSCs were plated in specific induction

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To select the differentiated MSCs, we used the RT-PCR to search markers specific for the three differentiation directions. These markers are the alkalin phosphatase (PAL) as osteoblast specific marker the lipoprotein lipase (LPL)as specific of the adipocyte line and the actin-ASMA specific of vascular smooth muscles line. The positive control is the GAPDH. These markers were not expressed before the induction of the differentiation and presented an overexpression after the 14th day from the induction of specific differentiation in the three expansion mediums (M1, M2,

mediums to generate for adipocytes, osteoblasts, or chondrocytes.

et M3) demonstrating the multipotent nature of the MSCs (figure 4).

 **A B**

 **BM (D0) BM (D14)**  M1 M2 M3 M1 M2 M3

**ASMA** 

**PPRγ** 

**Runx 2** 

**PAL** 

**GAPDH** 

**Figure 4.** RT-PCR analyses of vascular smooth muscle (ASMA), adipogenic (PPRγ), and osteogenic (Runx2, PAL) mark‐ ers prior to (D0) (**A**) and after 14 days of differentiation induction (D14) (**B**) of BM-derived MSCs previously cultured in

**3.2. Detection of galectins in MSCs in terms of the culture mediums by flow cytometry**

MSCs from different BM samples were characterized by flow cytometry with a panel of biotynaled antibodies against human recombinant galectin (Gal-1, Gal-9, Gal-11 and Gal-13) at P2 after culture in the three mediums (M1, M2, et M3). Flow cytometry analysis revealed that MSC derived-hBM constitutively expressed galectins (Gal-1, Gal-9, Gal-11, Gal-13) at

expansion media (M1, M2, M3)

genic, and vas

**Figure 3.** Morphological characterization of hBM- MSCs derived cultured in different media M1, M2 and M3 at P2 and Osteogenic, chondrogenic, adipogenic, and vascular smooth muscular differentiation capacity assessed by staining

To confirm their differentiation potential, after P1 MSCs were plated in specific induction mediums to generate for adipocytes, osteoblasts, or chondrocytes.

Also MSCs were immunophenotypically characterized by flow cytometry. This analysis revealed that MSCs were uniformly positive for CD73 CD90 CD105 CD106 but negative for

The influence of HPL or FBS on the osteogenic and adipogenic differentiation potential of MSCs was investigated after appropriate induction at the second passage (P2). Cells grown in HPL or FBS deposited an extensive mineralized matrix when cultured for 2 weeks in an osteogenic medium [2% FBS), as demonstrated by strong alizarin red staining. These cells also efficiently differentiated into the adipogenic lineage, as indicated by Oil Red O staining of lipid

All populations of BM-MSCs cultured in different conditions with the HPL showed osteogenic, Vascular smooth muscle and chondrogenic differentiation capacity. The difference appears in

> ogenic, adipog ation capacity **\_\_\_\_\_\_\_\_\_\_ arin Red**

genic, and vas assessed by s **\_\_\_\_\_\_\_\_\_\_ Nil R (A)**

scular smooth taining of BM **\_\_\_\_\_\_\_\_\_\_**

muscular M-derived MS **\_\_\_\_\_\_\_\_\_\_ ASMA (VSM)** 

Cs **\_\_**

**Red )** 

**Figure 3.** Morphological characterization of hBM- MSCs derived cultured in different media M1, M2 and M3 at P2 and Osteogenic, chondrogenic, adipogenic, and vascular smooth muscular differentiation capacity assessed by staining

droplets in the cytoplasm following culture in an adipogenic medium.

Osteo differentia  **\_\_\_\_\_\_ Aliza (0)** 

adipogenic differentiation with 10% HPL (Fig 3 ).

 **gical Cs** 

CD14 CD34 and CD45 (figure 2).

40 Stem Cell Biology in Normal Life and Diseases

 **M h**

**Morpholog hBM MSC**

**M1**

 **M2**

**M2**

To select the differentiated MSCs, we used the RT-PCR to search markers specific for the three differentiation directions. These markers are the alkalin phosphatase (PAL) as osteoblast specific marker the lipoprotein lipase (LPL)as specific of the adipocyte line and the actin-ASMA specific of vascular smooth muscles line. The positive control is the GAPDH. These markers were not expressed before the induction of the differentiation and presented an overexpression after the 14th day from the induction of specific differentiation in the three expansion mediums (M1, M2, et M3) demonstrating the multipotent nature of the MSCs (figure 4).

**Figure 4.** RT-PCR analyses of vascular smooth muscle (ASMA), adipogenic (PPRγ), and osteogenic (Runx2, PAL) mark‐ ers prior to (D0) (**A**) and after 14 days of differentiation induction (D14) (**B**) of BM-derived MSCs previously cultured in expansion media (M1, M2, M3)

#### **3.2. Detection of galectins in MSCs in terms of the culture mediums by flow cytometry**

MSCs from different BM samples were characterized by flow cytometry with a panel of biotynaled antibodies against human recombinant galectin (Gal-1, Gal-9, Gal-11 and Gal-13) at P2 after culture in the three mediums (M1, M2, et M3). Flow cytometry analysis revealed that MSC derived-hBM constitutively expressed galectins (Gal-1, Gal-9, Gal-11, Gal-13) at different mediums but with unequal percentages. We noticed that galectins 9 and 11 are more expressed by cells derived from hBM with an average of [88. 1 ± 1. 8 % ) in M1 Meduim in comparison to M2 (16,8 ± 3. 9 % ) and M3 (11,6 ± 3. 9% ) with p < 0. 01.

We noticed also that galectin 1 is expressed with in average of (91. 2 ± 1. 4 % ) at P2 after culture in the three mediums (M1,M2,M3). In the contrary no expression of galactin 13 have been noted in any medium. (figure 5)

Gal 1

 st (a

tandard med alpha MEM+

dium (M1) +FGF)

Medium HPL 5% (M2) Medium HPL 10% (M3)

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**Figure 6.** Immunofluorescence Galectin-1, Galectin-9, Galectin -11 and Galactin-13 expression by cultured at P2 in dif‐

ferent media: M1 (10% FBS+FGF2) M2 (5% HPL), M3 (10% HPL)

Gal 9

Gal 11

Gal 13

**Figure 5.** Galectins Gal-1, Gal-9,Gal-11 and Gal-13 antigen expression of BM-MSCs cultured at P2 in different media: M1 (10% FBS+FGF2) M2(5% HPL), M3 (10% HPL) by flow cytometry

#### *3.2.1. Expression of galectins of MSC-BM by microscopy immunofluorecence*

Immunofluorecsence of galectin-1 in MSC of normal adult bone marrow was showed expres‐ sion in three mediums M1,M2,M3. Following this technique, we are also identified galectins gal-9 and gal-11 expression but only in standard medium M1. No expression of gal- 13 have been reveled by hBM-CSM cultured in all media M1,M2 and M3. we are determined the localization of galectin mostly in nucleus cells. (Fig6).

different mediums but with unequal percentages. We noticed that galectins 9 and 11 are more expressed by cells derived from hBM with an average of [88. 1 ± 1. 8 % ) in M1 Meduim in

We noticed also that galectin 1 is expressed with in average of (91. 2 ± 1. 4 % ) at P2 after culture in the three mediums (M1,M2,M3). In the contrary no expression of galactin 13 have been noted

**Figure 5.** Galectins Gal-1, Gal-9,Gal-11 and Gal-13 antigen expression of BM-MSCs cultured at P2 in different media:

Immunofluorecsence of galectin-1 in MSC of normal adult bone marrow was showed expres‐ sion in three mediums M1,M2,M3. Following this technique, we are also identified galectins gal-9 and gal-11 expression but only in standard medium M1. No expression of gal- 13 have been reveled by hBM-CSM cultured in all media M1,M2 and M3. we are determined the

M1 (10% FBS+FGF2) M2(5% HPL), M3 (10% HPL) by flow cytometry

localization of galectin mostly in nucleus cells. (Fig6).

*3.2.1. Expression of galectins of MSC-BM by microscopy immunofluorecence*

comparison to M2 (16,8 ± 3. 9 % ) and M3 (11,6 ± 3. 9% ) with p < 0. 01.

in any medium. (figure 5)

42 Stem Cell Biology in Normal Life and Diseases

**Figure 6.** Immunofluorescence Galectin-1, Galectin-9, Galectin -11 and Galactin-13 expression by cultured at P2 in dif‐ ferent media: M1 (10% FBS+FGF2) M2 (5% HPL), M3 (10% HPL)

MSCs cultivated in M2, M3, and M4. And After the MSCs differentiation Gal-11 was no more expressed in the standard culture's medium (M1 : MEM+ FGF2), while MSCs culti‐ vated in mediums containing the HPL (M2, M3) expressed the Gal- 11 in adipocytes (A) and vascular smooth muscle cells (VML). As to Gal-13, we reported that this galectin wasn't expressed by the undiferenciated MSCs neither in the standard medium nor in the mediums containing the HPL (M1, M2, and M3). Whereas, after the MSCs differentiation, Gal-13 was

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Multipotent mesenchymal stromal cells (MSCs) have generated a great debate as a potential source of cells for cell-based therapeutic strategies primarily owing to their intrinsic ability to selfrenewanddifferentiate intofunctional celltypes that constitutethe tissueinwhichtheyexist and also because MSCs express a large number of molecules including the galectins. [11,12]

The galectins are a family of soluble lectins characterized by their affinity for β-galactoside residues. In recent years, galectins have become a major focus of investigation because of their

Given that galectins have been shown to have important effects [13,14] we decided to evaluate

In this study we analyzed MSCs cultured and expanded in three conditions mediums: *basic growth* medium consisting of alpha-Minimum Essential Medium in which HPL replaced FBS in order to investigate their morphology, plasticity, proliferation differentiation to osteoblasts, adipocytes, and vascular smooth muscles and their capacity to express galectins in terms of

In our laboratory MSCs were characterized and defined according to the International Society for Cellular Therapy minimal criteria. [17,18]. So first, we demonstrated that BM-MSC adhered to plastic. Second as measured by flow cytometry all of MSC derived from BM-MSC were nearly 100% positive for the makers CD90 CD105, and CD73 (≥95% ), while they lack the expression of CD45 CD34, and CD14 (≤ 2% ). Third, BM-MSC differentiated to osteoblasts

First, we investigated the effect of culture mediums on the adherence and the proliferation of MSCs. Then we demonstrated that the three mediums allow the adherence of MSCs to plastic and their proliferation, but the percentage of confluence and the times it takes to get it, change in terms of the composition of the culture medium. So, the culture mediums containing 5% HPL induced the best MSCs confluence. Thus the most important confluence of the bone marrow's MSCs was reached at the first passage (P1) in the culture's medium 5% HPL after 30 days from their implantation. So, we confirmed that HPL-expanded MSCs show a significantly higher proliferation rate, in comparison with MSCs expanded in the standard medium. So, we can use the HPL as a substitute of FBS in mediums specific to the culture of MSCs destined for clinical

applications, to avoid a possible xenogenic contamination caused by the FBS. [19]

involvement in various physiological and pathological processes. [13,14]

the expression of galectins (Gal-1, Gal-3, Gal-9 and Gal-13) by MSCs.

only expressed by adipocytes. (figure 5)

the mediums' compositions. [15,16]

adipocytes and vascular smooth cells.

**4. Discussion**

**Figure 7.** RT –PCR Galectin-1, Galectin-9, Galectin -11 and Galactin-13 expression by cultured at P2 in different media: M1 (10% FBS+FGF2) M2 (5% HPL), M3 (10% HPL) and after differentiation process ( O: osteogenic induction, A: Adipo‐ genic induction,V: Vascular Smooth Muscle induction, GAPDH : Glyceraldheyde 3-phospate dehydrogenase, PM: Weight molecular )

#### **3.3. RT –PCR galectin-1,Gal-9,Gal-11 and Gal-13 expression by hBM-MSCs**

By RT-PCR, we evaluated the messenger RNA expression of Galectin-1,Galectin-9, Galectin-11 and Galectin -13 in hBM-MSCs before and after differentiation to the three direction : osteo‐ blastic [0), adipocyte (A) and to vascular smooth muscle (VSM).

We had a positive results for hBM-MSCs both before their differentiation and after the induction of the three differentiation directions:


MSCs cultivated in M2, M3, and M4. And After the MSCs differentiation Gal-11 was no more expressed in the standard culture's medium (M1 : MEM+ FGF2), while MSCs culti‐ vated in mediums containing the HPL (M2, M3) expressed the Gal- 11 in adipocytes (A) and vascular smooth muscle cells (VML). As to Gal-13, we reported that this galectin wasn't expressed by the undiferenciated MSCs neither in the standard medium nor in the mediums containing the HPL (M1, M2, and M3). Whereas, after the MSCs differentiation, Gal-13 was only expressed by adipocytes. (figure 5)

### **4. Discussion**

Gal 1

standar (alpha

44 Stem Cell Biology in Normal Life and Diseases

rd medium ( MEM+FGF)

M1) Medium HPL 5% (M2) Medium HPL 10% (M3)

**Figure 7.** RT –PCR Galectin-1, Galectin-9, Galectin -11 and Galactin-13 expression by cultured at P2 in different media: M1 (10% FBS+FGF2) M2 (5% HPL), M3 (10% HPL) and after differentiation process ( O: osteogenic induction, A: Adipo‐ genic induction,V: Vascular Smooth Muscle induction, GAPDH : Glyceraldheyde 3-phospate dehydrogenase, PM:

By RT-PCR, we evaluated the messenger RNA expression of Galectin-1,Galectin-9, Galectin-11 and Galectin -13 in hBM-MSCs before and after differentiation to the three direction : osteo‐

We had a positive results for hBM-MSCs both before their differentiation and after the

**•** before the induction of the differentiation process :Gal-1 was expressed by hBM- MSCs in the standard medium M1 (MEM+ FGF2) and also in the three mediums (M2, M3, et M4) which contain the human platelet lysat (HPL). While, after the induction of the MSCs differentiation, Gal-1 was only expressed in MSCs cultivated in mediums containing the

**•** Concerning the expression of Gal-9 by BM-MSCs: we observed that Gal-9 was expressed by undifferentiated BM-MSCs cultivated in the standard medium (M1: MEM+ FGF2), but absent in MSCs cultivated in mediums containing the HPL (M2, M3). However, after inducing the differentiation process, this galectin was absent in MSCs of the standard

**•** Regarding the expression of Gal-11 by BM-MSCs, we found that Gal- 11 was expressed by undifferenciated BM-MSCs, in the standard medium M1 (MEM+ FGF2) but absent in BM-

medium and had a very low expression in mediums containing the HPL (M2, M3).

**3.3. RT –PCR galectin-1,Gal-9,Gal-11 and Gal-13 expression by hBM-MSCs**

blastic [0), adipocyte (A) and to vascular smooth muscle (VSM).

HPL (M2, M3), in the three differentiation directions (O, A, VML)

induction of the three differentiation directions:

Gal 9

Gal 11

Gal 13

Weight molecular )

Multipotent mesenchymal stromal cells (MSCs) have generated a great debate as a potential source of cells for cell-based therapeutic strategies primarily owing to their intrinsic ability to selfrenewanddifferentiate intofunctional celltypes that constitutethe tissueinwhichtheyexist and also because MSCs express a large number of molecules including the galectins. [11,12]

The galectins are a family of soluble lectins characterized by their affinity for β-galactoside residues. In recent years, galectins have become a major focus of investigation because of their involvement in various physiological and pathological processes. [13,14]

Given that galectins have been shown to have important effects [13,14] we decided to evaluate the expression of galectins (Gal-1, Gal-3, Gal-9 and Gal-13) by MSCs.

In this study we analyzed MSCs cultured and expanded in three conditions mediums: *basic growth* medium consisting of alpha-Minimum Essential Medium in which HPL replaced FBS in order to investigate their morphology, plasticity, proliferation differentiation to osteoblasts, adipocytes, and vascular smooth muscles and their capacity to express galectins in terms of the mediums' compositions. [15,16]

In our laboratory MSCs were characterized and defined according to the International Society for Cellular Therapy minimal criteria. [17,18]. So first, we demonstrated that BM-MSC adhered to plastic. Second as measured by flow cytometry all of MSC derived from BM-MSC were nearly 100% positive for the makers CD90 CD105, and CD73 (≥95% ), while they lack the expression of CD45 CD34, and CD14 (≤ 2% ). Third, BM-MSC differentiated to osteoblasts adipocytes and vascular smooth cells.

First, we investigated the effect of culture mediums on the adherence and the proliferation of MSCs. Then we demonstrated that the three mediums allow the adherence of MSCs to plastic and their proliferation, but the percentage of confluence and the times it takes to get it, change in terms of the composition of the culture medium. So, the culture mediums containing 5% HPL induced the best MSCs confluence. Thus the most important confluence of the bone marrow's MSCs was reached at the first passage (P1) in the culture's medium 5% HPL after 30 days from their implantation. So, we confirmed that HPL-expanded MSCs show a significantly higher proliferation rate, in comparison with MSCs expanded in the standard medium. So, we can use the HPL as a substitute of FBS in mediums specific to the culture of MSCs destined for clinical applications, to avoid a possible xenogenic contamination caused by the FBS. [19]

The expansion-promoting effect of the HPL is likely to result from the high concentration of natural growth factors that HPLcontains.In factplatelet granules containmany growth factors, including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), transforming growth factor (TGFb), platelet factor 4 (PF-4), and plateletderivedepidermalgrowthfactor(PDEGF)[20,21].Thesegrowthfactors,whicharereleasedfrom platelet lysate, have been shown to enhance MSC expansion in vitro. [20, 21,22,23,24]

We noted that MSCs expressed the galectins constitutively, but this expression was modulated differently depending on the type of the galectin the culture's stage and the composition of

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47

These findings are in agreement with previous reports. Thus, Friederike Gieseke and al. [27] claim that m RNAs of Gal-1, Gal-3, -8 and -9 were detected in MSCs by the RT-PCR and when performing a quantitative real time –PCR the m RNAs of Gal-3, -8 and -9 were detected to a lesser extent than Gal-1 which is similar to the results we obtained when performing the flow

Najar M and al. [14] confirmed the constitutive expression of galectin-1 m RNA by BM-MSCs. Regarding Gal-3 Ju-Yeon Kim and al. [28] demonstrated that Gal-3 is secreted by UCB-MSC under pathological conditions whereas low level of GAL-3 was detected by Western blot analysis in conditioned medium of naïve UCB-MSC. This report is in contrast with our results since we confirmed the absence of galectins' in both membranes and RNAs of UC-MSCs.

The present study provides new insights concerning the expression of the galectins by the MSCs. According to our observations and the reports mentioned above the galectins are located both in nuclear and membrane levels. This suggests that the galectins are released by MSCs. Because of their their multilineage differentiation potential, immunomodulatory and anti-inflammatory properties, MSCs have become increasingly attractive as a therapeutic approach. Careful characterization of MSC physiology and the effects of culture mediums context on their proliferation differentiation and molecules' secretion will increase the safety

Address all correspondence to: faouzi.jenhani@yahoo.tn, tunisiacelltherapy@ymail.com

2 Unit Immunology Research, Faculty of pharmacy, Monastir, Tunisia

transplantation. Transplantation,2003, 75, 389–397.

1 Cell Immunology and Cytometry and Cell Therapy Laboratories, Blood National Center,

[1] Tse, W. T. , Pendleton, J. D. , Beyer, W. M. , Egalka, M. C. , Guinan, E. C. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in

the culture medium.

and efficiency of MSCs in clinical settings.

**Author details**

Faouzi Jenhani1,2\*

Tunisia

**References**

cytometry.

Second, we analyzed the morphology of MSC, and observed fibroblast-like cells in BM. Thus the MSCs formed a monolayer of homogenous spindle-shaped cells with a whirlpool-like array. This result is in accordance with the literature. [25]. However, two cell phenotypes were observed among fibroblast-like BM-MSC colonies: thin spindle shaped MSC and star-shaped MSC. These BM-MSC two phenotypes were described by Wolgang et Wagnera and al [25]

To detect the expression of galectins Gal-1, Gal-9, Gal-11 and Gal-13 by MSCs, we used two techniques which are the flow cytometry (FC) and the RT-PCR. We searched the expression of galectins in MSCs cultivated in the three culture mediums: the standard medium: alpha-MEM+ FGF2, the medium (with 5% HPL), the medium (with 10% HPL).

The flow cytometry revealed that all the galectins of interest (Gal-1, Gal-3, Gal-9, and Gal-13) are expressed in BM-MSCs, in above mentioned culture mediums,. It thus becomes clear that expression percents of galacetins revealed by the flow cytometry in hBM-MCSs depend on two parameters which are the composition of the medium and the type of the galectin.

Thus, we observed mainly that the best Galectins' expression was visualized in the standard medium (alpha-MEM /FGF2). Previous studies have shown that the supplementation of fibroblast growth factor 2 (FGF2) in vitro selects for the survival of a large number of of MSCs enriched in pluripotent mesenchymal precursors enhances the growth of MSCs maintains their multilineagedifferentiationpotentialduringinvitroexpansionandprolongs theirlifespan[26]. So, this data and our observation suggest that the increase of galectins in the presence of FGF2 isdue to the improvement ofthe growth theproliferation anddifferentiationpotential ofMSCs.

Then, when comparing the means of the galectins' expression by the MSCs we concluded that the best means of protein expression were those of Gal-1; this galectin had the highest means of expression in all culture mediums. Friederike Gieseke and al. confirm our result; thus, they report that Gal-1 is highly expressed in MSCs. [27]

The results of the RT-PCR are in accordance with those of the flow cytometry. Thus, the RT-PCR applied to RNA of the MSCs revealed that galectins (Gal-1, Gal-9, Gal-11, and Gal-13) are expressed by both undifferentiated and differentiated BM-MSCs. So, before the induction of the differentiation process, the MSCs cultivated in the standard medium expressed Gal-1, Gal-9, Gal-11, but not Gal-13, while the MSCs cultivated in the mediums enriched by the HPL expressed only the Gal-1. Nevertheless, after the induction of the MSCs differentiation, first we observed that Gal-1, Gal-9, Gal-11 were no more expressed in the standard culture's medium (alpha MEM+ FGF2) while we detected the expression of Gal-13 in the adipocytes. Second, when examining the expression of galectins by MSCs cultivated in mediums contain‐ ing the HPL we noticed the presence of Gal-1, Gal-9 with a low expression, Gal-11 in both adipocytes and VMLs and finally Gal-13 in adipocytes.

We noted that MSCs expressed the galectins constitutively, but this expression was modulated differently depending on the type of the galectin the culture's stage and the composition of the culture medium.

These findings are in agreement with previous reports. Thus, Friederike Gieseke and al. [27] claim that m RNAs of Gal-1, Gal-3, -8 and -9 were detected in MSCs by the RT-PCR and when performing a quantitative real time –PCR the m RNAs of Gal-3, -8 and -9 were detected to a lesser extent than Gal-1 which is similar to the results we obtained when performing the flow cytometry.

Najar M and al. [14] confirmed the constitutive expression of galectin-1 m RNA by BM-MSCs. Regarding Gal-3 Ju-Yeon Kim and al. [28] demonstrated that Gal-3 is secreted by UCB-MSC under pathological conditions whereas low level of GAL-3 was detected by Western blot analysis in conditioned medium of naïve UCB-MSC. This report is in contrast with our results since we confirmed the absence of galectins' in both membranes and RNAs of UC-MSCs.

The present study provides new insights concerning the expression of the galectins by the MSCs. According to our observations and the reports mentioned above the galectins are located both in nuclear and membrane levels. This suggests that the galectins are released by MSCs. Because of their their multilineage differentiation potential, immunomodulatory and anti-inflammatory properties, MSCs have become increasingly attractive as a therapeutic approach. Careful characterization of MSC physiology and the effects of culture mediums context on their proliferation differentiation and molecules' secretion will increase the safety and efficiency of MSCs in clinical settings.

#### **Author details**

The expansion-promoting effect of the HPL is likely to result from the high concentration of natural growth factors that HPLcontains.In factplatelet granules containmany growth factors, including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), transforming growth factor (TGFb), platelet factor 4 (PF-4), and plateletderivedepidermalgrowthfactor(PDEGF)[20,21].Thesegrowthfactors,whicharereleasedfrom

Second, we analyzed the morphology of MSC, and observed fibroblast-like cells in BM. Thus the MSCs formed a monolayer of homogenous spindle-shaped cells with a whirlpool-like array. This result is in accordance with the literature. [25]. However, two cell phenotypes were observed among fibroblast-like BM-MSC colonies: thin spindle shaped MSC and star-shaped MSC. These BM-MSC two phenotypes were described by Wolgang et Wagnera and al [25] To detect the expression of galectins Gal-1, Gal-9, Gal-11 and Gal-13 by MSCs, we used two techniques which are the flow cytometry (FC) and the RT-PCR. We searched the expression of galectins in MSCs cultivated in the three culture mediums: the standard medium: alpha-

The flow cytometry revealed that all the galectins of interest (Gal-1, Gal-3, Gal-9, and Gal-13) are expressed in BM-MSCs, in above mentioned culture mediums,. It thus becomes clear that expression percents of galacetins revealed by the flow cytometry in hBM-MCSs depend on two parameters which are the composition of the medium and the type of the galectin.

Thus, we observed mainly that the best Galectins' expression was visualized in the standard medium (alpha-MEM /FGF2). Previous studies have shown that the supplementation of fibroblast growth factor 2 (FGF2) in vitro selects for the survival of a large number of of MSCs enriched in pluripotent mesenchymal precursors enhances the growth of MSCs maintains their multilineagedifferentiationpotentialduringinvitroexpansionandprolongs theirlifespan[26]. So, this data and our observation suggest that the increase of galectins in the presence of FGF2 isdue to the improvement ofthe growth theproliferation anddifferentiationpotential ofMSCs. Then, when comparing the means of the galectins' expression by the MSCs we concluded that the best means of protein expression were those of Gal-1; this galectin had the highest means of expression in all culture mediums. Friederike Gieseke and al. confirm our result; thus, they

The results of the RT-PCR are in accordance with those of the flow cytometry. Thus, the RT-PCR applied to RNA of the MSCs revealed that galectins (Gal-1, Gal-9, Gal-11, and Gal-13) are expressed by both undifferentiated and differentiated BM-MSCs. So, before the induction of the differentiation process, the MSCs cultivated in the standard medium expressed Gal-1, Gal-9, Gal-11, but not Gal-13, while the MSCs cultivated in the mediums enriched by the HPL expressed only the Gal-1. Nevertheless, after the induction of the MSCs differentiation, first we observed that Gal-1, Gal-9, Gal-11 were no more expressed in the standard culture's medium (alpha MEM+ FGF2) while we detected the expression of Gal-13 in the adipocytes. Second, when examining the expression of galectins by MSCs cultivated in mediums contain‐ ing the HPL we noticed the presence of Gal-1, Gal-9 with a low expression, Gal-11 in both

platelet lysate, have been shown to enhance MSC expansion in vitro. [20, 21,22,23,24]

MEM+ FGF2, the medium (with 5% HPL), the medium (with 10% HPL).

report that Gal-1 is highly expressed in MSCs. [27]

46 Stem Cell Biology in Normal Life and Diseases

adipocytes and VMLs and finally Gal-13 in adipocytes.

Faouzi Jenhani1,2\*

Address all correspondence to: faouzi.jenhani@yahoo.tn, tunisiacelltherapy@ymail.com

1 Cell Immunology and Cytometry and Cell Therapy Laboratories, Blood National Center, Tunisia

2 Unit Immunology Research, Faculty of pharmacy, Monastir, Tunisia

#### **References**

[1] Tse, W. T. , Pendleton, J. D. , Beyer, W. M. , Egalka, M. C. , Guinan, E. C. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation,2003, 75, 389–397.

[2] Selmani Z, Naji A, Zidi I, Favier B, Gaiffe E, Obert L, et al. Human leukocyte antigen-G5 secretion by human mesenchymal stem cells is required to suppress T lympho‐ cyte and natural killer function and to induce CD4 1 CD25 high FOXP31 regulatory T cells. Stem Cells 2008;26:212-222.

mal cells from small samples of bone marrow aspirates or marrow filter washouts.

Human Platelet Lysate for MSC Expression Galectins

http://dx.doi.org/10.5772/55383

49

[16] Doucet C, Ernou I, Zhang Y, Llense JR, Begot L, Holy X, et al. Platelet lysates pro‐ mote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-

[17] M Dominici K Le Blanc I Mueller I Slaper-Cortenbach FC Marini DJ Prockop and EM Horwitz. Minimal Criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement. Cytotherapy (2006) Vol

[18] Horwitz EM, Le Blanc K, Dominici M, et al. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytothera‐

[19] Berger,M. G. ,Veyrat-Masson,R. ,Rapatel,C. ,Descamps, S. , Chassagne,J. ,Boiret-Dupre,N. ,2006. Cellculture medium compositionandtranslationaladultbone marrow-

[21] Kilian O, Flesch I, Wenisch S, Taborski B, Jork A, Schnettler R, et al. Effects of platelet growth factors on human mesenchymal stem cells and human endothelial cells in vi‐

[22] Van den Dolder J, Mooren R, Vloon AP, Stoelinga PJ, Jansen JA. Platelet-rich plasma: quantification of growth factor levels and the effect on growth and differentiation of

[23] Capelli C, Domenghini M, Borleri G, Bellavita P, Poma R, Carobbio A, et al. Human platelet lysate allows expansion and clinical grade production of mesenchymal stro‐ mal cells from small samples of bone marrow aspirates or marrow filter washouts.

[24] Doucet C, Ernou I, Zhang Y, Llense JR, Begot L, Holy X, et al. Platelet lysates pro‐ mote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-

[25] Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Wolfgang Wagnera, Frederik Weina, Anja Seckingera, Maria Frankhauserb, Ute Wirknerc, Ulf Krausea, Jonathon Blakec, Chris‐ tian Schwagerc, Volker Ecksteina, Wilhelm Ansorgec, and Anthony D. Hoa. Experi‐

[26] Bianchi et al : Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2. Giordano Bianchi,a,b,c,2 Andrea Banfi,a,b,c,1,2 Maddalena Mastro‐ giacomo,a,b,c Rosario Notaro,a Lucio Luzzatto,a Ranieri Cancedda,a,b,c and Rodolfo

[20] Harrison P, Cramer EM. Platelet alpha-granules. Blood Rev 1993;7(1):52–62.

based therapy applications. J Cell Physiol 2005;205(2):228–36.

derived stemcellresearch. StemCells24 (12), 2888–2890.

rat bone marrow cells. Tissue Eng 2006;12(11):3067–73.

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Quartoa,b,c,\*. Experimental Cell Research 287 (2003) 98–105.

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[16] Doucet C, Ernou I, Zhang Y, Llense JR, Begot L, Holy X, et al. Platelet lysates pro‐ mote mesenchymal stem cell expansion: a safety substitute for animal serum in cellbased therapy applications. J Cell Physiol 2005;205(2):228–36.

[2] Selmani Z, Naji A, Zidi I, Favier B, Gaiffe E, Obert L, et al. Human leukocyte antigen-G5 secretion by human mesenchymal stem cells is required to suppress T lympho‐ cyte and natural killer function and to induce CD4 1 CD25 high FOXP31 regulatory T

[3] Le Blanc K, Rasmusson I, Sundberg B, Gotherstrom C, Hassan M, Uzunel M, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical

[4] Frank MH, Sayegh MH. Immunomodulatory functions of mesenchymal stem cells.

[5] Danguy, A. , Camby, I. , Kiss, R. , 2002. Galectins and cancer. Biochim. Biophys. Acta

[6] Yagi H, Soto-Gutierrez A, Parekkadan B, Kitagawa Y, Tompkins RG, Kobayashi N, Yarmush ML: Mesenchymal stem cells: mechanisms of immunomodulation and

[7] Pittenger MF, Mackay AM, Beck SC, Jaiswal RK: Multilineage potential of adult hu‐

[8] Maria Virginia Tribulatti, Galectin-8 provides costimulatory and proliferative signals

[9] Rabinovich GA. Galectins: an evolutionarily conserved family of animal lectins with multifunctional properties; a trip from the gene to clinical therapy. Cell Death Differ

[10] Camby I, Le Mercier M, Lefranc F, Kiss R. Galectin-1: a small protein with major

[11] D. Baksh, L. Song R. S. Tuan. Adult mesenchymal stem cells : Characterization differ‐ entiation and application in cell and gene therapy. J. Cell. Mol. Med. Vol 8, No 3,

[12] Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses.

[13] Camby I, Le Mercier M, Lefranc F, Kiss R. Galectin-1: a small protein with major

[14] Najar et al : Mehdi Najar, Gordana Raicevic, Hicham Id Boufker, Basile Stamatopou‐ los, Ce´cile De Bruyn, Nathalie Meuleman, Dominique Bron, Michel Toungouz, and Laurence Lagneaux. Modulated expression of adhesion molecules and galectin-1: Role during mesenchymal stromal cell immunoregulatory functions. Experimental

[15] Capelli C, Domenghini M, Borleri G, Bellavita P, Poma R, Carobbio A, et al. Human platelet lysate allows expansion and clinical grade production of mesenchymal stro‐

to T lymphocytes. Journal of Leukocyte Biology 2009,Volume 86, August.

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functions. Glycobiology. 2006;16:137R–157R.

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mesenchymal stem cells. Lancet. 2004;363:1439–1441.

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[27] Friederike Gieseke and al : Friederike Gieseke, Judith Böhringer Rita Bussolari Massi‐ mo Dominci Rupert Handgretinger and Ingo Müller. Human multipotent mesenchy‐ mal stromal cells employ galectin-1 to inhibit immune effector cells. Jul 19, 2010; doi: 10. 1182/Blood-2010-02-270777.

**Chapter 4**

**Canonical HSC Markers and Recent Achievements**

A specific feature of hematopoietic stem cells (HSC) is the potency to supply all types of blood cells throughout life by self-renewal and differentiation. Bone marrow (BM) is actively producing differentiated blood cells with enormous cellular turnover. Under homeostatic state, primitive HSC in adult BM divide only rarely and are located in specialized regulatory environment to avoid exhaustion and DNA damages that are supposed to cumulatively de‐ velop hematopoietic disorders such as myelodysplastic syndrome or leukemia. However, those quiescent HSC can be proliferative on demand, particularly on systemic infection or myelo-suppressive treatment. Therefore, elaborate mechanisms regulating the self-renewal and differentiation of BM HSC is indispensable to maintain normal hematopoiesis through‐ out life. The fluctuating feature of HSC is thought to be associated with their regulatory en‐

Technical improvement for purifying authentic HSC from heterogeneous cellular popula‐ tions is necessary to understand the features of those extremely rare and precious cells and promote their therapeutic application. Many studies have attempted to identify their specif‐ ic markers, and now flow cytometry- based strategies have made it possible to sort HSC with high purity in mice. However, the source and the stage of HSC change along ontogeny, which consequently influence not only their functional abilities but also their surface immu‐ nophenotypes. In the light of fluctuating nature of HSC, it should be very important to un‐ derstand their phenotype specific to reconstitution activity of the immune system after

Hematopoietic cells and endothelial cells are both generated from mesodermal precursor cells in ontogeny [1]. Thereafter, HSC pool is formed in several anatomical sites such as aor‐

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

© 2013 Sudo 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 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,

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

Takao Sudo, Takafumi Yokota, Tomohiko Ishibashi,

Michiko Ichii, Yukiko Doi, Kenji Oritani and

Additional information is available at the end of the chapter

Yuzuru Kanakura

**1. Introduction**

http://dx.doi.org/10.5772/54474

vironment, generally called "HSC niche".

myelo-suppressive events.

[28] Ju-Yeon Kim and al: Galectin-3 secreted by human umbilical cord blood-derived mesenchymal stem cells reduces amyloid-b42 neurotoxicity in vitro Ju-Yeon Kim a,b, Dong Hyun Kim a, Dal-Soo Kim a, Ji Hyun Kim a, Sang Young Jeong a, Hong Bae Jeon a, Eun Hui Lee b, Yoon Sun Yang a, Wonil Oh a, Jong Wook Chang a,\* FEBS Letters 584 (2010) 3601–3608.

## **Canonical HSC Markers and Recent Achievements**

Takao Sudo, Takafumi Yokota, Tomohiko Ishibashi, Michiko Ichii, Yukiko Doi, Kenji Oritani and Yuzuru Kanakura

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54474

#### **1. Introduction**

[27] Friederike Gieseke and al : Friederike Gieseke, Judith Böhringer Rita Bussolari Massi‐ mo Dominci Rupert Handgretinger and Ingo Müller. Human multipotent mesenchy‐ mal stromal cells employ galectin-1 to inhibit immune effector cells. Jul 19, 2010; doi:

[28] Ju-Yeon Kim and al: Galectin-3 secreted by human umbilical cord blood-derived mesenchymal stem cells reduces amyloid-b42 neurotoxicity in vitro Ju-Yeon Kim a,b, Dong Hyun Kim a, Dal-Soo Kim a, Ji Hyun Kim a, Sang Young Jeong a, Hong Bae Jeon a, Eun Hui Lee b, Yoon Sun Yang a, Wonil Oh a, Jong Wook Chang a,\* FEBS

10. 1182/Blood-2010-02-270777.

50 Stem Cell Biology in Normal Life and Diseases

Letters 584 (2010) 3601–3608.

A specific feature of hematopoietic stem cells (HSC) is the potency to supply all types of blood cells throughout life by self-renewal and differentiation. Bone marrow (BM) is actively producing differentiated blood cells with enormous cellular turnover. Under homeostatic state, primitive HSC in adult BM divide only rarely and are located in specialized regulatory environment to avoid exhaustion and DNA damages that are supposed to cumulatively de‐ velop hematopoietic disorders such as myelodysplastic syndrome or leukemia. However, those quiescent HSC can be proliferative on demand, particularly on systemic infection or myelo-suppressive treatment. Therefore, elaborate mechanisms regulating the self-renewal and differentiation of BM HSC is indispensable to maintain normal hematopoiesis through‐ out life. The fluctuating feature of HSC is thought to be associated with their regulatory en‐ vironment, generally called "HSC niche".

Technical improvement for purifying authentic HSC from heterogeneous cellular popula‐ tions is necessary to understand the features of those extremely rare and precious cells and promote their therapeutic application. Many studies have attempted to identify their specif‐ ic markers, and now flow cytometry- based strategies have made it possible to sort HSC with high purity in mice. However, the source and the stage of HSC change along ontogeny, which consequently influence not only their functional abilities but also their surface immu‐ nophenotypes. In the light of fluctuating nature of HSC, it should be very important to un‐ derstand their phenotype specific to reconstitution activity of the immune system after myelo-suppressive events.

Hematopoietic cells and endothelial cells are both generated from mesodermal precursor cells in ontogeny [1]. Thereafter, HSC pool is formed in several anatomical sites such as aor‐

© 2013 Sudo 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 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.

ta-gonad-mesonephros (AGM) region, placenta, fetal liver, and BM. At the early stage of on‐ togeny, HSC frequently undergo symmetrical and/or asymmetrical division to form entire hematopoietic system compared to adult HSC. Those early HSC and endothelial cells bear some common surface antigens, of which expression levels on HSC decline along aging. In‐ terestingly, some of the endothelial-related surface molecules revive on HSC after BM in‐ jury, when the cells actively divide to regenerate BM cells.

CD34-

(21%) of CD150+ CD48-

of CD150+ CD48-

in Table 1.

 KSL cells, which are extremely rare, representing only 0.001-0.01% of BM mononu‐ clear cells, are almost pure primitive hematopoietic cells that have long-term multilineage repopulating potency [10]. More recently, the endothelial protein C receptor CD201 was found as a new endothelial-related HSC marker which marks approximately 70% of the SP cells. The marker seems to be useful to purify LT-HSC among the SP cells because only the

In recent years, Kiel *et al.* demonstrated that a simple combination of SLAM family markers (CD150, CD244 and CD48) could enrich primitive murine HSC. That is, one out of every 4.8

constitution [12]. Furthermore, they observed that one out of every 2.1 (47%) of CD150+ CD48- LSK cells had long-term multilineage reconstituting potential. Approximately 15~20%

the mouse life span, have more long-term repopulating potential than other cells [13,14]. We can now purify dormant LT-HSC from murine BM using the SLAM family markers in com‐ bination with LSK gating. The information regarding murine HSC markers is summarized

**Markers References**

 Thy-1Lo Sca-1+ Spangrude *et al. Science* (1988) [4] CD34-/Lo LSK Osawa *et al*. *Science* (1996) [7] Side population (SP) Goodell *et al. J Exp Med* (1996) [9]

Tip-SP LSK Matsuzaki *et al. Immunity* (2004) [10]

SP Uchida *et al. Exp Hematol* (2003) [15]

LSK Kiel *et al. Cell* (2005) [12]

CD150+ LSK Foudi *et al. Nat Biotechnol* (2009) [13]

LSK Wilson *et al. Cell* (2008) [14]

cells from young adult murine BM gave long-term multilineage re‐

LSK cells, which divide only 5-6 times during

Canonical HSC Markers and Recent Achievements

http://dx.doi.org/10.5772/54474

53

CD201 + subpopulation exhibited repopulating ability [11].

LSK or CD34- CD150+ CD48-

Lin-

CD48-

BrdU and histone 2B-retaining CD48-

Lin, lineage; Rho, Rhodamine-123; LSK, Lin-

Histone 2B-retaining CD48-

**Table 1.** Markers for adult murine hematopoietic stem cells.

Lin- Rho-

CD150+ CD41-

**3. HSC markers during developmental stages**

CD150+ CD34-

HSC markers during developmental stages are not identical to those of adult HSC. In the embryo, functional HSC that can reconstitute hematopoiesis in adult recipients are firstly found in the aorta-gonad-mesonephros (AGM) region at approximately embryonic day 10 (E10) [16-18]. Many reports have demonstrated that those earliest authentic HSC bud from endothelial-related cells, which involve the concept of "hemangioblast" or "hemogenic en‐ dothelium" [19-23]. In fact, emerging HSC and endothelial cells share various surface mark‐

Sca-1+ c-kit+

We recently reported endothelial cell-selective adhesion molecule (ESAM) as a new marker for HSC [2]. Interestingly, ESAM levels on HSC clearly mirror the shift of HSC between qui‐ escence and activation, and the up-regulation amplitude is prominent in comparison to oth‐ er HSC-related antigens [3]. Furthermore, we found that ESAM is functionally indispensable for HSC to re-establish homeostatic hematopoiesis [3]. In this chapter, we review a wealth of information about traditional HSC markers, and introduce our recent findings.

#### **2. Development of the strategy for purifying HSC from murine BM**

HSC are defined by their capacity for both self-renewal and differentiation into all the blood cell types. In 1988, Spangrude *et al.* reported lineage (Lin; Mac-1, Gr-1, B220, CD4, and CD8)- Thy-1Lo Sca-1+ cells in mouse BM as a multipotent HSC population. When these cells were transplanted into lethally irradiated mice, only thirty cells were sufficient to save 50% of the recipient mice and reconstitute B, T, and myeloid cells [4]. In 1991, Ogawa *et al.* reported that half of the c-kit+ BM cells do not express Lin (Mac-1, Gr-1, Ter119, and B220) markers, and ckit- population do not include hematopoietic progenitor cells [5]. From then on, Lin- Sca-1+ ckit+ (LSK ) cells has been used as the population in which HSC are highly concentrated [6,7]. HSC can be functionally classified as either long-term (LT-HSC) or short-term (ST-HSC) ac‐ cording to their capacity to give rise to life-long or transient hematopoiesis. Osawa *et al.* showed that CD34+ LSK cells are capable of only short-term multilineage differentiation. In contrast, CD34-/Lo LSK cells have long-term multilineage reconstitution capacity. They also showed that CD34-/Lo LSK cells can differentiate into CD34+ LSK cells [7]. LSK fraction also can be divided into two populations by expression level of Flk-2. While LT-HSC are en‐ riched in the Flk-2- LSK fraction, the Flk-2+ LSK cells are mainly ST-HSC [8].

While the techniques of purifying HSC by use of surface markers had been promoted, Goodell *et al.* reported the method for purifying HSC without use of surface markers. Hoechst33342 is a fluorescent dye which binds to DNA of live cells. When Hoechst fluores‐ cence on whole BM was examined simultaneously at two emission wavelength (red and blue), one population of cells with increased ability to efflux Hoechst dye was observed. Goodell *et al.* named it "side population (SP)", and showed that a majority of HSC were en‐ riched in the SP by competitive repopulating experiments [9]. Subsequently, Matsuzaki *et al.* described a method of further purifying HSC by combining staining with antibodies to sur‐ face molecules with the Hoechst dye efflux. They showed the fraction of cells with the stron‐ gest dye efflux activity (termed as "Tip"-SP) has the highest marrow-repopulating activity. While 20% of "Tip"-SP cells are primitive hematopoietic cells, more than 90% of "Tip"-SP CD34- KSL cells, which are extremely rare, representing only 0.001-0.01% of BM mononu‐ clear cells, are almost pure primitive hematopoietic cells that have long-term multilineage repopulating potency [10]. More recently, the endothelial protein C receptor CD201 was found as a new endothelial-related HSC marker which marks approximately 70% of the SP cells. The marker seems to be useful to purify LT-HSC among the SP cells because only the CD201 + subpopulation exhibited repopulating ability [11].

In recent years, Kiel *et al.* demonstrated that a simple combination of SLAM family markers (CD150, CD244 and CD48) could enrich primitive murine HSC. That is, one out of every 4.8 (21%) of CD150+ CD48 cells from young adult murine BM gave long-term multilineage re‐ constitution [12]. Furthermore, they observed that one out of every 2.1 (47%) of CD150+ CD48- LSK cells had long-term multilineage reconstituting potential. Approximately 15~20% of CD150+ CD48- LSK or CD34- CD150+ CD48- LSK cells, which divide only 5-6 times during the mouse life span, have more long-term repopulating potential than other cells [13,14]. We can now purify dormant LT-HSC from murine BM using the SLAM family markers in com‐ bination with LSK gating. The information regarding murine HSC markers is summarized in Table 1.


**Table 1.** Markers for adult murine hematopoietic stem cells.

ta-gonad-mesonephros (AGM) region, placenta, fetal liver, and BM. At the early stage of on‐ togeny, HSC frequently undergo symmetrical and/or asymmetrical division to form entire hematopoietic system compared to adult HSC. Those early HSC and endothelial cells bear some common surface antigens, of which expression levels on HSC decline along aging. In‐ terestingly, some of the endothelial-related surface molecules revive on HSC after BM in‐

We recently reported endothelial cell-selective adhesion molecule (ESAM) as a new marker for HSC [2]. Interestingly, ESAM levels on HSC clearly mirror the shift of HSC between qui‐ escence and activation, and the up-regulation amplitude is prominent in comparison to oth‐ er HSC-related antigens [3]. Furthermore, we found that ESAM is functionally indispensable for HSC to re-establish homeostatic hematopoiesis [3]. In this chapter, we review a wealth of

information about traditional HSC markers, and introduce our recent findings.

**2. Development of the strategy for purifying HSC from murine BM**

kit- population do not include hematopoietic progenitor cells [5]. From then on, Lin-

showed that CD34-/Lo LSK cells can differentiate into CD34+

LSK fraction, the Flk-2+

HSC are defined by their capacity for both self-renewal and differentiation into all the blood cell types. In 1988, Spangrude *et al.* reported lineage (Lin; Mac-1, Gr-1, B220, CD4, and CD8)-

transplanted into lethally irradiated mice, only thirty cells were sufficient to save 50% of the recipient mice and reconstitute B, T, and myeloid cells [4]. In 1991, Ogawa *et al.* reported that half of the c-kit+ BM cells do not express Lin (Mac-1, Gr-1, Ter119, and B220) markers, and c-

kit+ (LSK ) cells has been used as the population in which HSC are highly concentrated [6,7]. HSC can be functionally classified as either long-term (LT-HSC) or short-term (ST-HSC) ac‐ cording to their capacity to give rise to life-long or transient hematopoiesis. Osawa *et al.*

contrast, CD34-/Lo LSK cells have long-term multilineage reconstitution capacity. They also

can be divided into two populations by expression level of Flk-2. While LT-HSC are en‐

While the techniques of purifying HSC by use of surface markers had been promoted, Goodell *et al.* reported the method for purifying HSC without use of surface markers. Hoechst33342 is a fluorescent dye which binds to DNA of live cells. When Hoechst fluores‐ cence on whole BM was examined simultaneously at two emission wavelength (red and blue), one population of cells with increased ability to efflux Hoechst dye was observed. Goodell *et al.* named it "side population (SP)", and showed that a majority of HSC were en‐ riched in the SP by competitive repopulating experiments [9]. Subsequently, Matsuzaki *et al.* described a method of further purifying HSC by combining staining with antibodies to sur‐ face molecules with the Hoechst dye efflux. They showed the fraction of cells with the stron‐ gest dye efflux activity (termed as "Tip"-SP) has the highest marrow-repopulating activity. While 20% of "Tip"-SP cells are primitive hematopoietic cells, more than 90% of "Tip"-SP

cells in mouse BM as a multipotent HSC population. When these cells were

LSK cells are capable of only short-term multilineage differentiation. In

LSK cells are mainly ST-HSC [8].

 Sca-1+ c-

LSK cells [7]. LSK fraction also

jury, when the cells actively divide to regenerate BM cells.

52 Stem Cell Biology in Normal Life and Diseases

Thy-1Lo Sca-1+

showed that CD34+

riched in the Flk-2-

#### **3. HSC markers during developmental stages**

HSC markers during developmental stages are not identical to those of adult HSC. In the embryo, functional HSC that can reconstitute hematopoiesis in adult recipients are firstly found in the aorta-gonad-mesonephros (AGM) region at approximately embryonic day 10 (E10) [16-18]. Many reports have demonstrated that those earliest authentic HSC bud from endothelial-related cells, which involve the concept of "hemangioblast" or "hemogenic en‐ dothelium" [19-23]. In fact, emerging HSC and endothelial cells share various surface mark‐ ers such as CD34 and VE-cadherin that do not mark adult murine LT-HSC [24-26]. On the contrary, the emerging HSC do not express either Sca-1 or CD45, a pan-hematopoietic mark‐ er [19,27]. Interestingly, those developing HSC express CD41/Integrin-αv, a marker for meg‐ akaryocytes [28].

**4. Niche signals regulating HSC pool**

controlled the number of HSC by regulating SNO cells [37].

On the other hand, Kiel *et al.* reported that many CD150+ CD48- CD41- Lin-

**5. Differences between murine and human HSC markers**

long-term HSC of human BM are enriched in CD34+ CD38-

[26,47,48].

cadherin+

CD150+

CD48-

tain HSC hibernation [46].

HSC are CD34-

CD38+

We think it seems meaningful to deal with the "HSC niche" briefly here, although another chapter in this book provides more detailed information about its function. Molecular cross‐ talk between HSC and their niches has been considered to be important to provide signals for self-renewing division that maintain HSC pool. Although precise mechanisms regulating HSC status still remain unknown, there are accumulating evidences to involve several spe‐

In 1994, human osteoblasts were shown to maintained hematopoiesis by constitutively pro‐ ducing G-CSF in vitro [41]. In the first decade of the 21st century, a notion that connects os‐ teoblasts with the HSC niche rapidly developed. Parathyroid hormone (PTH), which is a main regulator of calcium homeostasis, was reported to increase in the number of both os‐ teoblasts and HSC, suggesting osteoblasts as the candidate for HSC niche [42]. In addition, it was also reported that BrdU label retaining cells (LRC) were attached to spindle-shaped N-

contact with sinusoidal endothelial cells in spleen or BM, suggesting that endothelial cells are also essential components of the HSC niche. With regard to cytokine-chemokine sinal‐ ings, the CXC chemokine ligand 12 (CXCL12) -CXC chemokine receptor 4 (CXCR4) pathway was found to be important. In vitro, HSC expressing CXCR4 migrate in response to CXCL12 which is the ligand for CXCR4 [43]. Nagasawa's laboratory reported that a majority of

that the numbers of HSC in CAR cell-depleted mice were reduced in comparison with con‐ trol mice. These data are supportive of the idea that CXCL12-CXCR4 pathway is essential for HSC pool [39,44]. Recently, Yamazaki *et al.* reported TGF- β as a candidate niche signal in the control of HSC hibernation [45]. The same group advocated that glial cells, regulating activation of TGF-β signal, might be a component of the HSC niche in adult BM and main‐

A critical issue that has been an obstacle in applying the information of murine HSC to hu‐ man is the lack of common HSC markers between the two species. Researchers described above have made great efforts to purify authentic HSC from murine hematopoietic organs. Owing to those achievements, we can now sort LT-HSC with very high purity from the murine BM. However, human HSC cannot be purified with the same markers. Human HSC do not express Sca-1 or CD150 that are the established HSC markers in mice. In addition, the

osteoblasts (SNO) cells, and that bone morphogenetic protein (BMP) signalling

CD41- HSC were in contact with CXCL12-abundant reticular (CAR) cells, and

LT-HSC were in

Canonical HSC Markers and Recent Achievements

http://dx.doi.org/10.5772/54474

55

population, while murine BM

cific cells, or cytokines and chemokines secreted from stromal cells in this process.

Although HSC do not emerge in the fetal liver de novo, the organ is the main site of HSC expansion before birth. Circulating HSC seed in the fetal liver, where they robustly expand and differentiate. Indeed, numbers of HSC increase ~40-fold in the fetal liver between E12 and E16 [29]. Unlike the emerging HSC in the AGM region, HSC in fetal liver express CD45 and Sca-1. Morrison *et al.* showed that HSC are highly enriched in Thy1Lo Sca-1+ Lin- Mac1+ fraction of fetal liver cells [30]. His group later demonstrated that the SLAM family markers (CD150+ CD48- ) are also useful to enrich for HSC in E14.5 fetal liver just as in adult BM by the fact that 37% of CD150+ CD48- Sca-1+ Lin- Mac1+ fetal liver cells had long-term reconsti‐ tuting capacity [31]. Although the expression levels of AA4.1 and VE-cadherin are very high at the early stage of fetal hematopoiesis, they become gradually down-regulated after E12- E13 [32,33]. Interestingly, the phenotype of HSC in fetal liver rapidly changes after E16, when their number is reaching to a plateau level [30]. Recently, our group reported ESAM as a novel HSC marker in fetal liver (see below). HSC markers during mouse ontogeny are summarized in Table 2.


**Table 2.** Markers for hematopoietic stem cells during mouse ontogeny.

Adult and fetal HSC are not the same with regard to not only surface phenotypes but also cell-cycle status. Recent studies have shown that the long-term reconstituting activity of adult BM is sustained mostly in very quiescent HSC [13,14]. However, cycling HSC from the fetal liver give rise to higher levels of reconstitution than HSC obtained from adult BM [30,36]. The microenvironments, known as "HSC niches", are believed to influence cell-cycle status of HSC, and adult HSC niches in BM seem to be different from HSC niches in the fetal liver [12,37-40]. More precise analyses of hematopoietic environment in the embryo should give us valuable information regarding what are the imperative conditions for HSC expan‐ sion and how the alteration of surface molecules on HSC is functionally involved in that process. Furthermore, such cell surface antigens that mirror the HSC state are invaluable for understanding the relationship between HSC and their niches.

### **4. Niche signals regulating HSC pool**

ers such as CD34 and VE-cadherin that do not mark adult murine LT-HSC [24-26]. On the contrary, the emerging HSC do not express either Sca-1 or CD45, a pan-hematopoietic mark‐ er [19,27]. Interestingly, those developing HSC express CD41/Integrin-αv, a marker for meg‐

Although HSC do not emerge in the fetal liver de novo, the organ is the main site of HSC expansion before birth. Circulating HSC seed in the fetal liver, where they robustly expand and differentiate. Indeed, numbers of HSC increase ~40-fold in the fetal liver between E12 and E16 [29]. Unlike the emerging HSC in the AGM region, HSC in fetal liver express CD45 and Sca-1. Morrison *et al.* showed that HSC are highly enriched in Thy1Lo Sca-1+ Lin- Mac1+ fraction of fetal liver cells [30]. His group later demonstrated that the SLAM family markers

tuting capacity [31]. Although the expression levels of AA4.1 and VE-cadherin are very high at the early stage of fetal hematopoiesis, they become gradually down-regulated after E12- E13 [32,33]. Interestingly, the phenotype of HSC in fetal liver rapidly changes after E16, when their number is reaching to a plateau level [30]. Recently, our group reported ESAM as a novel HSC marker in fetal liver (see below). HSC markers during mouse ontogeny are

VE-

c-kit+

**Fetal age Location Markers References**

cadherin+ Sca-1- AA4.1+ ESAM+

AA4.1+ VE-cadherin+/- CD150+ CD48-

Sca-1+ Mac1+ Tie-2+ Flt3-

CD34+ CD45+ CD31+

ESAM+

Adult and fetal HSC are not the same with regard to not only surface phenotypes but also cell-cycle status. Recent studies have shown that the long-term reconstituting activity of adult BM is sustained mostly in very quiescent HSC [13,14]. However, cycling HSC from the fetal liver give rise to higher levels of reconstitution than HSC obtained from adult BM [30,36]. The microenvironments, known as "HSC niches", are believed to influence cell-cycle status of HSC, and adult HSC niches in BM seem to be different from HSC niches in the fetal liver [12,37-40]. More precise analyses of hematopoietic environment in the embryo should give us valuable information regarding what are the imperative conditions for HSC expan‐ sion and how the alteration of surface molecules on HSC is functionally involved in that process. Furthermore, such cell surface antigens that mirror the HSC state are invaluable for

) are also useful to enrich for HSC in E14.5 fetal liver just as in adult BM by

fetal liver cells had long-term reconsti‐

Petrenko *et al. Immunity* (1999) [33], Hsu *et al. Blood* (2000) [34], Baumann *et al. Blood* (2004) [24], Fraser *et al. Exp Hematol* (2002) [25], Ogawa *Exp Hematol* (2002) [26], de Brujin *et al. Immunity* (2002) [19], Mikkola *et al. Blood* (2003) [28], Matsubara *et al. J Exp Med* (2005) [27], Kim *et al. Blood* (2005) [32], Kim *et al. Blood* (2006) [31], Mansson *et al. Immunity* (2007) [35], Yokota *et al.* Blood (2009) [2]

akaryocytes [28].

54 Stem Cell Biology in Normal Life and Diseases

(CD150+ CD48-

summarized in Table 2.

the fact that 37% of CD150+ CD48- Sca-1+ Lin- Mac1+

E8.5-E10.5 AGM region CD41+ CD34+ CD45-

**Table 2.** Markers for hematopoietic stem cells during mouse ontogeny.

understanding the relationship between HSC and their niches.

E11.5-E16.5 Fetal liver CD41-

We think it seems meaningful to deal with the "HSC niche" briefly here, although another chapter in this book provides more detailed information about its function. Molecular cross‐ talk between HSC and their niches has been considered to be important to provide signals for self-renewing division that maintain HSC pool. Although precise mechanisms regulating HSC status still remain unknown, there are accumulating evidences to involve several spe‐ cific cells, or cytokines and chemokines secreted from stromal cells in this process.

In 1994, human osteoblasts were shown to maintained hematopoiesis by constitutively pro‐ ducing G-CSF in vitro [41]. In the first decade of the 21st century, a notion that connects os‐ teoblasts with the HSC niche rapidly developed. Parathyroid hormone (PTH), which is a main regulator of calcium homeostasis, was reported to increase in the number of both os‐ teoblasts and HSC, suggesting osteoblasts as the candidate for HSC niche [42]. In addition, it was also reported that BrdU label retaining cells (LRC) were attached to spindle-shaped Ncadherin+ osteoblasts (SNO) cells, and that bone morphogenetic protein (BMP) signalling controlled the number of HSC by regulating SNO cells [37].

On the other hand, Kiel *et al.* reported that many CD150+ CD48- CD41- Lin- LT-HSC were in contact with sinusoidal endothelial cells in spleen or BM, suggesting that endothelial cells are also essential components of the HSC niche. With regard to cytokine-chemokine sinal‐ ings, the CXC chemokine ligand 12 (CXCL12) -CXC chemokine receptor 4 (CXCR4) pathway was found to be important. In vitro, HSC expressing CXCR4 migrate in response to CXCL12 which is the ligand for CXCR4 [43]. Nagasawa's laboratory reported that a majority of CD150+ CD48- CD41- HSC were in contact with CXCL12-abundant reticular (CAR) cells, and that the numbers of HSC in CAR cell-depleted mice were reduced in comparison with con‐ trol mice. These data are supportive of the idea that CXCL12-CXCR4 pathway is essential for HSC pool [39,44]. Recently, Yamazaki *et al.* reported TGF- β as a candidate niche signal in the control of HSC hibernation [45]. The same group advocated that glial cells, regulating activation of TGF-β signal, might be a component of the HSC niche in adult BM and main‐ tain HSC hibernation [46].

#### **5. Differences between murine and human HSC markers**

A critical issue that has been an obstacle in applying the information of murine HSC to hu‐ man is the lack of common HSC markers between the two species. Researchers described above have made great efforts to purify authentic HSC from murine hematopoietic organs. Owing to those achievements, we can now sort LT-HSC with very high purity from the murine BM. However, human HSC cannot be purified with the same markers. Human HSC do not express Sca-1 or CD150 that are the established HSC markers in mice. In addition, the long-term HSC of human BM are enriched in CD34+ CD38 population, while murine BM HSC are CD34- CD38+ [26,47,48].

Early studies in the 1980s proved by using monoclonal antibody technique that CD34+ popu‐ lation of human BM includes immature hematopoietic progenitors [49-51]. Berenson *et al.* showed that autologous CD34+ cells enriched from baboon BM were able to reconstitute nor‐ mal hematopoiesis after lethal irradiation. Animals transplanted with CD34 cells, however, did not recover sufficient hematopoiesis [52]. Afterwards, over the past two decades, CD34 positive has been used as a reliable marker for human HSC or hematopoietic progenitor cells (HPC). Indeed, transplantation of CD34+ cells obtained from donor BM, peripheral blood, or cord blood (CB) can provide long-term and multilineage hematopoietic reconstitu‐ tion in recipients.

dition. While activated HSC increase the expression level of Sca-1, CD150, Tie2, Endo‐ glin, Mac-1, and CD34, they clearly decrease that of c-kit and N-cadherin [26,56,57]. Some endothelial-related antigens, which mark actively dividing fetal HSC but do not mark quiescent adult HSC, are up-regulated again on the activated HSC after BM injury. The characteristics of those activated HSC are reminiscent of fetal HSC. Since no obvious phenotypes have been documented regarding CD34 or CD150-deficient mice, how the up-regulation of those molecules contributes to the functions and/or characteristics of ac‐ tivated HSC remains unknown [12,58]. Tie2 and Endoglin, which are the receptors for angiopoietin and TGF, respectively, might transduce important signals to regulate divid‐ ing speed of HSC. If we could accurately monitor the fluctuation of HSC status with a set of surface markers, that should yield significant insight regarding HSC biology and HSC applications for clinical purposes. As a very recent achievement, our group has

demonstrated that ESAM is a useful marker for activated HSC.

[61,62]. We found the ESAMHi population of Rag1- c-kitHi Sca-1+

cantly different between Rag1- c-kitHi Sca-1+

.

creased with age after reaching adulthood 2

pression is clearly detectable on human CB CD34+

E10.5 AGM cells, only ESAM+

transcripts were detected in Lin-

manuscript in preparation).

myeloid cells 2

that ESAM+ Sca-1+ Lin-

Mac1+

**7. An endothelial-related antigen ESAM as a new novel HSC marker**

We previously reported sorting strategy of HSC and early lymphoid progenitors (ELP) from Rag1/GFP knockin mice [59,60]. We searched for genes whose expression levels are signifi‐

micro-array data. Among the HSC related genes ESAM drew our attention because its tran‐ scripts were conspicuous in the HSC fraction whereas the expression was drastically downregulated in the ELP fraction. ESAM molecule is an immunoglobulin superfamily protein that is exposed on cell surface and originally identified as an endothelial cell-specific protein

was highly enriched for LT-HSC compared with ESAM-/Lo subset. Among Rag1/GFP- Tie2Hi

ESAM is also expressed on adult murine HSC-enriched fraction in BM. Ooi *et al.* reported

tional HSC-enriched LSK cells, and that ESAM expression on HSC was conserved among various mouse strains [63]. ESAM levels on HSC are variable according to developing stages or advancing age. Interestingly, the intensity of ESAM expression on HSC gradually in‐

The usefulness of ESAM as a HSC marker has been further enhanced by the findings that its expression in human HSC is also detected. Ooi *et al.* reported that robust levels of ESAM

transcripts in unfractionated CB cells were very low [63]. We have confirmed that ESAM ex‐

flow cytometry [64]. In addition, our group has also observed that the marker is effective as well for adult human HSC in both BM and mobilized peripheral blood. (Ishibashi *et al.*

CD38-

novel murine HSC marker throughout life including developmental stages.

CD34+

cells could effectively produce both CD19+

BM cells could more effectively enrich for LT-HSC than the conven‐

HSC and Rag1Lo c-kitHi Sca-1+ ELP by analyzing

Canonical HSC Markers and Recent Achievements

http://dx.doi.org/10.5772/54474

57

. Based on these observations, ESAM can be a

CD90+ human HSC, while the levels of ESAM

cells by using its specific antibody and

fraction of E14.5 fetal liver

lymphoid cells and

As CD34 marks human HSC as well as more differentiated progenitor cells, researchers have sought additional markers to further enrich CD34+ population for LT-HSC. Baum *et al.* reported that CD90/Thy-1+ population in Lin- CD34+ cells contained pluripotent hemato‐ poietic progenitors [53]. Recently a series of studies of John Dick's laboratory have success‐ fully improved the techniques to more purify human HSC. His group reported that human HSC activity was restricted to CD49f+ fraction, and that single Lin- CD34+ CD38- CD45RA-Thy-1+ Rhodamin123Lo CD49f+ cells in CB cells accomplished multilineage engraftment in immune-deficient mice [54].

While LT-HSC can be enriched mainly in the CD34+ population, the possibility that CD34 cells also contain LT-HSC has been reported. Bhatia *et al.* showed human CD34- population in Lin cells of BM and CB also contained LT-HSC [55]. It should be important to compare the features of primate CD34- HSC with those of murine CD34- LSK cells. In addition, a new positive marker for human HSC could resolve the relationship between the CD34+ and CD34- HSC. Markers for human HSC are summarized in Table 3.


**Table 3.** Markers for human hematopoietic stem cells.

#### **6. Differences between quiescent and activated HSC markers**

After mice are treated with cytotoxic agents or irradiation, most of cell-cycling hemato‐ poietic cells are killed and dormant primitive HSC start to proliferate. The patterns of surface molecules expressed on activated HSC change from those under steady-state con‐ dition. While activated HSC increase the expression level of Sca-1, CD150, Tie2, Endo‐ glin, Mac-1, and CD34, they clearly decrease that of c-kit and N-cadherin [26,56,57]. Some endothelial-related antigens, which mark actively dividing fetal HSC but do not mark quiescent adult HSC, are up-regulated again on the activated HSC after BM injury. The characteristics of those activated HSC are reminiscent of fetal HSC. Since no obvious phenotypes have been documented regarding CD34 or CD150-deficient mice, how the up-regulation of those molecules contributes to the functions and/or characteristics of ac‐ tivated HSC remains unknown [12,58]. Tie2 and Endoglin, which are the receptors for angiopoietin and TGF, respectively, might transduce important signals to regulate divid‐ ing speed of HSC. If we could accurately monitor the fluctuation of HSC status with a set of surface markers, that should yield significant insight regarding HSC biology and HSC applications for clinical purposes. As a very recent achievement, our group has demonstrated that ESAM is a useful marker for activated HSC.

Early studies in the 1980s proved by using monoclonal antibody technique that CD34+

mal hematopoiesis after lethal irradiation. Animals transplanted with CD34-

showed that autologous CD34+

56 Stem Cell Biology in Normal Life and Diseases

tion in recipients.

in Lin-

CD34-

Lin-

CD34+ CD38-

reported that CD90/Thy-1+

Thy-1+ Rhodamin123Lo CD49f+

immune-deficient mice [54].

cells (HPC). Indeed, transplantation of CD34+

have sought additional markers to further enrich CD34+

While LT-HSC can be enriched mainly in the CD34+

CD34+ Lin-

Lin- CD34-

CD34+ CD38-

**Table 3.** Markers for human hematopoietic stem cells.

CD45RA-

Lin-

HSC. Markers for human HSC are summarized in Table 3.

**6. Differences between quiescent and activated HSC markers**

After mice are treated with cytotoxic agents or irradiation, most of cell-cycling hemato‐ poietic cells are killed and dormant primitive HSC start to proliferate. The patterns of surface molecules expressed on activated HSC change from those under steady-state con‐

lation of human BM includes immature hematopoietic progenitors [49-51]. Berenson *et al.*

did not recover sufficient hematopoiesis [52]. Afterwards, over the past two decades, CD34 positive has been used as a reliable marker for human HSC or hematopoietic progenitor

blood, or cord blood (CB) can provide long-term and multilineage hematopoietic reconstitu‐

As CD34 marks human HSC as well as more differentiated progenitor cells, researchers

poietic progenitors [53]. Recently a series of studies of John Dick's laboratory have success‐ fully improved the techniques to more purify human HSC. His group reported that human HSC activity was restricted to CD49f+ fraction, and that single Lin- CD34+ CD38- CD45RA-

cells also contain LT-HSC has been reported. Bhatia *et al.* showed human CD34- population

 cells of BM and CB also contained LT-HSC [55]. It should be important to compare the features of primate CD34- HSC with those of murine CD34- LSK cells. In addition, a new positive marker for human HSC could resolve the relationship between the CD34+ and

**Markers References**

CD34+ Berenson *et al. J Clin Invest* (1988) [52] CD34+ CD38- Terstappen *et al. Blood* (1991) [48]

CD38- Bhatia *et al. Nat Med* (1998) [55]

Thy-1+ RhoLo CD49f+ Notta *et al. Science* (2011) [54]

RhoLo McKenzie *et al. Blood* (2007) [47]

Thy-1+ Baum *et al. Proc Natl Acad Sci USA* (1992) [53]

population in Lin- CD34+

cells enriched from baboon BM were able to reconstitute nor‐

cells in CB cells accomplished multilineage engraftment in

cells obtained from donor BM, peripheral

population for LT-HSC. Baum *et al.*

cells contained pluripotent hemato‐

population, the possibility that CD34-

popu‐

cells, however,

### **7. An endothelial-related antigen ESAM as a new novel HSC marker**

We previously reported sorting strategy of HSC and early lymphoid progenitors (ELP) from Rag1/GFP knockin mice [59,60]. We searched for genes whose expression levels are signifi‐ cantly different between Rag1- c-kitHi Sca-1+ HSC and Rag1Lo c-kitHi Sca-1+ ELP by analyzing micro-array data. Among the HSC related genes ESAM drew our attention because its tran‐ scripts were conspicuous in the HSC fraction whereas the expression was drastically downregulated in the ELP fraction. ESAM molecule is an immunoglobulin superfamily protein that is exposed on cell surface and originally identified as an endothelial cell-specific protein [61,62]. We found the ESAMHi population of Rag1- c-kitHi Sca-1+ fraction of E14.5 fetal liver was highly enriched for LT-HSC compared with ESAM-/Lo subset. Among Rag1/GFP- Tie2Hi E10.5 AGM cells, only ESAM+ cells could effectively produce both CD19+ lymphoid cells and Mac1+ myeloid cells 2 .

ESAM is also expressed on adult murine HSC-enriched fraction in BM. Ooi *et al.* reported that ESAM+ Sca-1+ Lin- BM cells could more effectively enrich for LT-HSC than the conven‐ tional HSC-enriched LSK cells, and that ESAM expression on HSC was conserved among various mouse strains [63]. ESAM levels on HSC are variable according to developing stages or advancing age. Interestingly, the intensity of ESAM expression on HSC gradually in‐ creased with age after reaching adulthood 2 . Based on these observations, ESAM can be a novel murine HSC marker throughout life including developmental stages.

The usefulness of ESAM as a HSC marker has been further enhanced by the findings that its expression in human HSC is also detected. Ooi *et al.* reported that robust levels of ESAM transcripts were detected in Lin- CD34+ CD38- CD90+ human HSC, while the levels of ESAM transcripts in unfractionated CB cells were very low [63]. We have confirmed that ESAM ex‐ pression is clearly detectable on human CB CD34+ cells by using its specific antibody and flow cytometry [64]. In addition, our group has also observed that the marker is effective as well for adult human HSC in both BM and mobilized peripheral blood. (Ishibashi *et al.* manuscript in preparation).

#### **8. ESAM monitors HSC status between quiescence and self-renewal**

As mentioned above, the expression pattern of surface antigens on activated HSC after BM injury substantially differs from that on quiescent HSC. Administration of an anti-cancer drug 5-FU causes apoptosis of dividing hematopoietic progenitors, while the treatment re‐ tains quiescent LT-HSC and induces their proliferation afterward. We have observed that re‐ markable increase of ESAM expression levels transiently occurs on BM HSC after a 5-FU treatment. Furthermore, we have proved that the long-term hematopoietic reconstituting ac‐ tivity is almost exclusive to LSK cells bearing up-regulated ESAM expression [3].

Although expression levels of CD34, Tie2, and Endoglin on LSK show modest increases af‐ ter 5-FU injection, up-regulation of ESAM is remarkable (Figure 1). Why does ESAM need to revive so vividly on HSC after BM injury? One possible reason is that HSC might directly receive necessary signals which regulate self-renewal or differentiation via interaction with ESAM. Another possibility is that high amounts of ESAM might change the polarity or mo‐ bility of HSC, which consequently facilitate them to settle in adequate supporting niches (Figure 2). The latter assumption is likely because Wegmann and colleagues reported that ESAM deficiency causes insufficient Rho signalling in endothelial cells, which regulates the stabilization of endothelial tight junctions [65]. Rho is also expressed in hematopoietic pro‐ genitors and involved in their polarity and mobility [66]. It is noteworthy that more than 80% of ESAMHi HSC were located around perivascular areas in 5-FU-treated BM3 .

In any case, ESAM is likely to play an indispensable role during the recovery from BM in‐ jury. Because, while ESAM deficient mice do not show significant hematopoietic defects in homeostatic stage, the mice fall into severe and prolonged pancytopenia after myelo-sup‐ pressive treatment. In particular, they suffer from severe anemia and frequently die before hematopoietic recovery. Our findings indicate that ESAM not only marks activated HSC but also functionally supports their proliferation and differentiation.

**Figure 2.** Tentative models of ESAM function. (A) In this model, activated HSC directly receive necessary signals which regulate self-renewal or differentiation via interaction with ESAM. (B) In this model, HSC change their polarity or mobi‐ lity, and consequently, they can move to appropriate niches. ESAM may function as an adhesion factor between HSC

Canonical HSC Markers and Recent Achievements

http://dx.doi.org/10.5772/54474

59

In this chapter, we summarized achievements for identification of murine and human HSC, and introduced endothelial-related antigen ESAM as a useful HSC marker. While we can now purify murine LT-HSC with high efficiency, characterization of human HSC is less well understood because of insufficient information about surface antigens. Over two decades CD34-positive has been believed to be a reliable marker for human HSC/HPC. Although there are now accumulating evidences regarding surface markers to further enrich human LT-HSC in the CD34+ fraction, more information about human HSC-related antigens should be useful to improve strategies of HSC application to the clinical medicine. Although ESAM was originally identified with an endothelial specific molecule, we have demonstrated that it is a positive marker for both murine and human HSC. Because ESAM seems to play an essential role for hematopoietic recovery after BM injury, it would be significant to elucidate downstream signals of ESAM, and the possibility of ESAM as niche components. In addi‐ tion, we now know that the up-regulation of ESAM is observed on cultured murine embry‐ onic stem (ES) cells cultured in the OP9 system which recapitulate very primitive stages of

and their niches.

**9. Concluding remarks**

**Figure 1.** Overview of cell surface expression levels on quiescent steady-state HSC and activated HSC.

**Figure 2.** Tentative models of ESAM function. (A) In this model, activated HSC directly receive necessary signals which regulate self-renewal or differentiation via interaction with ESAM. (B) In this model, HSC change their polarity or mobi‐ lity, and consequently, they can move to appropriate niches. ESAM may function as an adhesion factor between HSC and their niches.

#### **9. Concluding remarks**

**8. ESAM monitors HSC status between quiescence and self-renewal**

58 Stem Cell Biology in Normal Life and Diseases

tivity is almost exclusive to LSK cells bearing up-regulated ESAM expression [3].

80% of ESAMHi HSC were located around perivascular areas in 5-FU-treated BM3

**Figure 1.** Overview of cell surface expression levels on quiescent steady-state HSC and activated HSC.

also functionally supports their proliferation and differentiation.

In any case, ESAM is likely to play an indispensable role during the recovery from BM in‐ jury. Because, while ESAM deficient mice do not show significant hematopoietic defects in homeostatic stage, the mice fall into severe and prolonged pancytopenia after myelo-sup‐ pressive treatment. In particular, they suffer from severe anemia and frequently die before hematopoietic recovery. Our findings indicate that ESAM not only marks activated HSC but

.

As mentioned above, the expression pattern of surface antigens on activated HSC after BM injury substantially differs from that on quiescent HSC. Administration of an anti-cancer drug 5-FU causes apoptosis of dividing hematopoietic progenitors, while the treatment re‐ tains quiescent LT-HSC and induces their proliferation afterward. We have observed that re‐ markable increase of ESAM expression levels transiently occurs on BM HSC after a 5-FU treatment. Furthermore, we have proved that the long-term hematopoietic reconstituting ac‐

Although expression levels of CD34, Tie2, and Endoglin on LSK show modest increases af‐ ter 5-FU injection, up-regulation of ESAM is remarkable (Figure 1). Why does ESAM need to revive so vividly on HSC after BM injury? One possible reason is that HSC might directly receive necessary signals which regulate self-renewal or differentiation via interaction with ESAM. Another possibility is that high amounts of ESAM might change the polarity or mo‐ bility of HSC, which consequently facilitate them to settle in adequate supporting niches (Figure 2). The latter assumption is likely because Wegmann and colleagues reported that ESAM deficiency causes insufficient Rho signalling in endothelial cells, which regulates the stabilization of endothelial tight junctions [65]. Rho is also expressed in hematopoietic pro‐ genitors and involved in their polarity and mobility [66]. It is noteworthy that more than

> In this chapter, we summarized achievements for identification of murine and human HSC, and introduced endothelial-related antigen ESAM as a useful HSC marker. While we can now purify murine LT-HSC with high efficiency, characterization of human HSC is less well understood because of insufficient information about surface antigens. Over two decades CD34-positive has been believed to be a reliable marker for human HSC/HPC. Although there are now accumulating evidences regarding surface markers to further enrich human LT-HSC in the CD34+ fraction, more information about human HSC-related antigens should be useful to improve strategies of HSC application to the clinical medicine. Although ESAM was originally identified with an endothelial specific molecule, we have demonstrated that it is a positive marker for both murine and human HSC. Because ESAM seems to play an essential role for hematopoietic recovery after BM injury, it would be significant to elucidate downstream signals of ESAM, and the possibility of ESAM as niche components. In addi‐ tion, we now know that the up-regulation of ESAM is observed on cultured murine embry‐ onic stem (ES) cells cultured in the OP9 system which recapitulate very primitive stages of

hematopoietic development [67] (Doi et al. manuscript in preparation). ESAM might have some roles in embryonic hematopoiesis at very early stages. As an on-going study, we are now investigating whether ESAM can be a useful biomarker for inducing hematopoietic cells from ES or induced pluripotent stem cells.

[10] Matsuzaki Y, Kinjo K, Mulligan RC, et al. Unexpectedly efficient homing capacity of

Canonical HSC Markers and Recent Achievements

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61

[11] Balazs AB, Fabian AJ, Esmon CT, et al. Endothelial protein C receptor (CD201) ex‐ plicitly identifies hematopoietic stem cells in murine bone marrow. Blood.

[12] Kiel MJ, Yilmaz OH, Iwashita T, et al. SLAM family receptors distinguish hemato‐ poietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell.

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[16] Cumano A, Ferraz JC, Klaine M, et al. Intraembryonic, but not yolk sac hematopoiet‐ ic precursors, isolated before circulation, provide long-term multilineage reconstitu‐

[17] Medvinsky A, Dzierzak E. Definitive hematopoiesis is autonomously initiated by the

[18] Muller AM, Medvinsky A, Strouboulis J, et al. Development of hematopoietic stem

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#### **Author details**

Takao Sudo, Takafumi Yokota\* , Tomohiko Ishibashi, Michiko Ichii, Yukiko Doi, Kenji Oritani and Yuzuru Kanakura

\*Address all correspondence to: yokotat@bldon.med.osaka-u.ac.jp

Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita, Japan

#### **References**


[10] Matsuzaki Y, Kinjo K, Mulligan RC, et al. Unexpectedly efficient homing capacity of purified murine hematopoietic stem cells. Immunity. 2004;20:87-93.

hematopoietic development [67] (Doi et al. manuscript in preparation). ESAM might have some roles in embryonic hematopoiesis at very early stages. As an on-going study, we are now investigating whether ESAM can be a useful biomarker for inducing hematopoietic

Department of Hematology and Oncology, Osaka University Graduate School of Medicine,

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Suita, Japan

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**Chapter 5**

**Hematopoietic Stem Cells and Response to Interferon**

Homeostasis in the bone marrow is dependent on the ability of hematopoietic stem cells (HSCs) to self-renew faithfully, differentiate into various lineages of the hematopoietic system, and form blood cells of several types (Figure 1) [1,2]. Under homeostatic conditions, HSCs are thought to be quiescent, and they are referred to as long-term reconstituting HSCs (LT-HSC) or dormant HSCs (dHSCs) [3,4]. Blood and immune cells are produced by the more differen‐ tiated short-term reconstituting HSCs (ST-HSCs) or multipotent progenitors (MPPs). Genetic and molecular studies of HSC self-renewal have identified candidate regulatory factors, in‐ cluding cell-intrinsic regulators, such as transcription factors and cell surface receptors, and cell-extrinsic regulators, such as the bone marrow niche and cytokines. Under certain condi‐ tions, such as inflammatory stress, HSCs differentiate into progenitor cells with less ability to self-renew, and they can be stimulated to divide and/or differentiate into all cell types in the peripheral blood [5,6]. Under inflammatory conditions, such as during bacterial infection or sepsis, an apparent expansion of lineage-negative Sca-1+c-Kit+ bone marrow cells (KSL) has been observed [7–11]. HSCs and progenitor cells are involved in the expansion of KSL; and expansion of the KSL population in the bone marrow has been associated with a loss of dor‐ mant LT-HSCs, reduced engraftment, and a bias towards myeloid lineage differentiation within that population. The process of the transition of HSCs from dormancy to activity is

Interferon is produced by cells of the immune system in response to challenge by agents, such as viruses, bacteria, and tumor cells. Type I IFNs are induced by the genomes of many RNA viruses during viral infection, and they suppress viral replication and have immunomodula‐ tory activity. IFNs are used clinically to treat viral diseases and malignancies, such as chronic myeloid leukemia (CML)[12]. In addition, under steady state conditions in the absence of in‐ fection, a small amount of intrinsic IFN is produced constitutively [13]. Recently, Essers et al. demonstrated that chronic activation of the IFN-α pathway impairs the function of HSCs and

> © 2013 Masumi; 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 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,

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

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

Atsuko Masumi

**1. Introduction**

http://dx.doi.org/10.5772/54689

Additional information is available at the end of the chapter

mediated by type I interferon (IFN) and type II IFN.


### **Hematopoietic Stem Cells and Response to Interferon**

### Atsuko Masumi

[55] Bhatia M, Bonnet D, Murdoch B, et al. A newly discovered class of human hemato‐

[56] Haug JS, He XC, Grindley JC, et al. N-cadherin expression level distinguishes re‐ served versus primed states of hematopoietic stem cells. Cell Stem Cell.

[57] Randall TD, Weissman IL. Phenotypic and functional changes induced at the clonal level in hematopoietic stem cells after 5-fluorouracil treatment. Blood.

[58] Cheng J, Baumhueter S, Cacalano G, et al. Hematopoietic defects in mice lacking the

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[61] Hirata K, Ishida T, Penta K, et al. Cloning of an immunoglobulin family adhesion molecule selectively expressed by endothelial cells. J Biol Chem.

[62] Nasdala I, Wolburg-Buchholz K, Wolburg H, et al. A transmembrane tight junction protein selectively expressed on endothelial cells and platelets. J Biol Chem.

[63] Ooi AG, Karsunky H, Majeti R, et al. The adhesion molecule esam1 is a novel hema‐

[64] Yokota T, Oritani K, Butz S, et al. Markers for Hematopoietic Stem Cells: Histories and Recent Achievements. Advances in Hematopoietic Stem Cell Research: InTech;

[65] Wegmann F, Petri B, Khandoga AG, et al. ESAM supports neutrophil extravasation, activation of Rho, and VEGF-induced vascular permeability. J Exp Med.

[66] Fonseca AV, Freund D, Bornhauser M, et al. Polarization and migration of hemato‐ poietic stem and progenitor cells rely on the RhoA/ROCK I pathway and an active reorganization of the microtubule network. J Biol Chem. 2010;285:31661-31671. [67] Nakano T, Kodama H, Honjo T. Generation of lymphohematopoietic cells from em‐

topoietic stem cell marker. Stem Cells. 2009;27:653-661.

bryonic stem cells in culture. Science. 1994;265:1098-1101.

poietic cells with SCID-repopulating activity. Nat Med. 1998;4:1038-1045.

2008;2:367-379.

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1997;89:3596-3606.

2001;276:16223-16231.

2002;277:16294-16303.

2012:77-88.

2006;203:1671-1677.

sialomucin CD34. Blood. 1996;87:479-490.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54689

#### **1. Introduction**

Homeostasis in the bone marrow is dependent on the ability of hematopoietic stem cells (HSCs) to self-renew faithfully, differentiate into various lineages of the hematopoietic system, and form blood cells of several types (Figure 1) [1,2]. Under homeostatic conditions, HSCs are thought to be quiescent, and they are referred to as long-term reconstituting HSCs (LT-HSC) or dormant HSCs (dHSCs) [3,4]. Blood and immune cells are produced by the more differen‐ tiated short-term reconstituting HSCs (ST-HSCs) or multipotent progenitors (MPPs). Genetic and molecular studies of HSC self-renewal have identified candidate regulatory factors, in‐ cluding cell-intrinsic regulators, such as transcription factors and cell surface receptors, and cell-extrinsic regulators, such as the bone marrow niche and cytokines. Under certain condi‐ tions, such as inflammatory stress, HSCs differentiate into progenitor cells with less ability to self-renew, and they can be stimulated to divide and/or differentiate into all cell types in the peripheral blood [5,6]. Under inflammatory conditions, such as during bacterial infection or sepsis, an apparent expansion of lineage-negative Sca-1+c-Kit+ bone marrow cells (KSL) has been observed [7–11]. HSCs and progenitor cells are involved in the expansion of KSL; and expansion of the KSL population in the bone marrow has been associated with a loss of dor‐ mant LT-HSCs, reduced engraftment, and a bias towards myeloid lineage differentiation within that population. The process of the transition of HSCs from dormancy to activity is mediated by type I interferon (IFN) and type II IFN.

Interferon is produced by cells of the immune system in response to challenge by agents, such as viruses, bacteria, and tumor cells. Type I IFNs are induced by the genomes of many RNA viruses during viral infection, and they suppress viral replication and have immunomodula‐ tory activity. IFNs are used clinically to treat viral diseases and malignancies, such as chronic myeloid leukemia (CML)[12]. In addition, under steady state conditions in the absence of in‐ fection, a small amount of intrinsic IFN is produced constitutively [13]. Recently, Essers et al. demonstrated that chronic activation of the IFN-α pathway impairs the function of HSCs and

© 2013 Masumi; 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 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.



**Chapter 6**

**Stem Cell Predictive Hemotoxicology**

Stem cells, those elusive entities that have the capacity for producing, maintaining and reconstituting the integrity of a biological system, also demonstrate the potential to predict partial or life-threatening damage in response to drugs, environmental compounds and other agents. It is ironic however, that in the animal or human, prior to the manifestation of such potential biological damage most, if not all of the stem cells might have been eradicated. To predict possible damage, surrogate *in vitro* stem cell assays have been developed that utilize specific properties and characteristics that divulge, through a measured response, how the

*In vitro* assays that detect toxicity to stem cells of a biological system allow potentially life-threatening damage to be predicted prior to human clinical trials taking place and environmental agents from causing harm. Discussions about stem cells usually focuses not on primary cells, but rather on embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, their ability to produce virtually any type of functional cell and their use in cellular therapy and regenerative medicine. The ES and iPS types of stem cells are, in fact, the least understood of all stem cell types. For many companies investigating or considering using these stem cells routinely as surrogate *in vitro* models for toxicity test‐ ing many questions remain, including (1) what is the relevance of these cells, (2) how do they compare with primary stem cell populations, and (3) can they be validated? In many cases, it is not the stem cells themselves, but rather the cells derived from ES and iPS cells that are of interest. Several companies already produce ES- or iPS-derived cardi‐ omyocytes, hepatocytes, neural cells and many other cell types not only for toxicity test‐

ing, but also for basic research, cellular therapy and regenerative medicine.

How can stem cells be used to predict toxicity? The answer to this question lies in the characteristics and properties of stem cells and how they respond to different situations.

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

© 2013 Harper and Rich; 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 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,

Additional information is available at the end of the chapter

Holli Harper and Ivan N. Rich

system will react to different agents.

http://dx.doi.org/10.5772/54430

**1. Introduction**

## **Stem Cell Predictive Hemotoxicology**

Holli Harper and Ivan N. Rich

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54430

### **1. Introduction**

Stem cells, those elusive entities that have the capacity for producing, maintaining and reconstituting the integrity of a biological system, also demonstrate the potential to predict partial or life-threatening damage in response to drugs, environmental compounds and other agents. It is ironic however, that in the animal or human, prior to the manifestation of such potential biological damage most, if not all of the stem cells might have been eradicated. To predict possible damage, surrogate *in vitro* stem cell assays have been developed that utilize specific properties and characteristics that divulge, through a measured response, how the system will react to different agents.

*In vitro* assays that detect toxicity to stem cells of a biological system allow potentially life-threatening damage to be predicted prior to human clinical trials taking place and environmental agents from causing harm. Discussions about stem cells usually focuses not on primary cells, but rather on embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, their ability to produce virtually any type of functional cell and their use in cellular therapy and regenerative medicine. The ES and iPS types of stem cells are, in fact, the least understood of all stem cell types. For many companies investigating or considering using these stem cells routinely as surrogate *in vitro* models for toxicity test‐ ing many questions remain, including (1) what is the relevance of these cells, (2) how do they compare with primary stem cell populations, and (3) can they be validated? In many cases, it is not the stem cells themselves, but rather the cells derived from ES and iPS cells that are of interest. Several companies already produce ES- or iPS-derived cardi‐ omyocytes, hepatocytes, neural cells and many other cell types not only for toxicity test‐ ing, but also for basic research, cellular therapy and regenerative medicine.

How can stem cells be used to predict toxicity? The answer to this question lies in the characteristics and properties of stem cells and how they respond to different situations.

© 2013 Harper and Rich; 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 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.

To understand this better, stem cell systems can be divided into "definitive" and "nondefinitive" systems as illustrated in Fig. 1. Definitive stem cell systems are responsible for maintaining a specific biological system. They can be divided into continuously pro‐ liferating systems such as the blood-forming or lympho-hematopoietic system, the gastrointestinal system, hair and skin, reproductive organs and cells of the eye cornea. Although not necessarily a continuously proliferating system, the mesenchymal stem cell (MSC), also called the multipotent mesenchymal stromal cell [1] system can been includ‐ ed, because in culture, the MSCs proliferate and can be passaged over a long period of time. Definitive stem cells systems can also demonstrate partial proliferation. These in‐ clude, but are not limited to, the liver, lung, kidney, heart, pancreas, and the neural/ neuronal system. From a toxicological viewpoint, these are not usually considered stem cell systems. Yet, the different types of lineage cells present in these organs and the abili‐ ty to maintain a specific cell mass has all the intricacies of a stem cell system, especially during development, even though the cell turnover in the adult may be very low. Nondefinitive stem cells systems are represented by the ES and iPS cell systems, which can, theoretically, give rise to any of the definitive stem cell systems. Indeed, it is a prerequi‐ site that the production of functionally, mature cells from ES or iPS cells first pass through a definitive stem cell compartment.

ever happens at the stem cell level will ultimately affect all downstream events. These char‐

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 81

**Figure 1. Definitive and Non-Definitive Stem Cell Systems.** Definitive stem cell systems can be further divided into continuously and partially proliferating systems. Non-definitive stem cell systems such as embryonic stem cells and in‐ duced pluripotent stem cells can produce definitive stem cell systems, which in turn, give rise to mature functional

All definitive stem cell systems have a common organization shown in Fig. 2. There is a continuum of stem cells within the stem cell compartment that exhibit different degrees of primitiveness or "stemness", which in turn, implies changing proliferating potential or potency as a stem cell moves through the compartment to the point of determination. These characteristic properties actually provide the information that allows stem cells to be predictors of potential toxicity. Once a stem cell becomes a progenitor cell, prolifera‐ tion continues and actually increases for a certain time so that the compartment can be amplified, until it ceases completely and the differentiation and maturation processes takes over. These changes have important implications for the types of assays that can

From Fig. 2, it is clear that proliferation occurs prior to differentiation. Although there is considerable overlap between proliferation and differentiation, they are two separate proc‐ esses that cannot be measured using the same assay readout. Since stem cells only proliferate, it follows that a proliferation assay is required to detect the presence and response of stem cells to a compound or agent. Using a differentiation assay to detect the effect of a compound or agent that targets one or more steps in the proliferation process can influence the interpretation

cells.

be used *in vitro* to detect potential toxicity.

acteristics enable stem cells to be the most important predictors of potential toxicity.

#### **2. Stem cell characteristics and properties used for toxicity testing**

Stem cells of primary, definitive systems always represent a very small proportion of the tissue or organ cellularity. This proportion is between 0.1 and 0.01% or less. The basic definition of a stem cell is that it possesses the capacity for self-renewal. In fact, stem cell systems are usually termed self-renewal cell systems, meaning that one stem cell can produce two daughter cells that are exact replicas of the parent. However, self-renewal is a difficult property to measure. The capacity for either serial *in vivo* repopulation or *in vitro* serial re-plating is considered a property of stem cells that implicates not only the presence of stem cells, but also their selfrenewal capability. The fact that serial *in vitro* re-plating or *in vivo* repopulation cannot be performed ad infinitum is not only an indication for a stem cell hierarchy [2-7], but also for an alternative hypothesis to stem cell self-renewal. This hypothesis states that tissues and organs are endowed with a specific number of stem cells. Once used up, the system ceases to function [8]. Regardless of the hypothesis, this important property can be utilized in a toxicological setting by employing secondary re-plating technology. This allows not only the presence of residual stem cells to be detected that have not been affected by a compound, but any change in sensitivity to a compound that might be important during repeated dose administration.

Stem cells have two other important properties that can be applied to toxicity testing. The first is that they are undifferentiated. The second is that stem cells proliferate. Stem cells can be "determined" into one or more lineages of mature functional cells. When a stem cell be‐ comes determined, it ceases to be a stem cell and becomes a progenitor cell that proliferates and differentiates. The fact that stem cells can be induced to differentiate means that what‐ ever happens at the stem cell level will ultimately affect all downstream events. These char‐ acteristics enable stem cells to be the most important predictors of potential toxicity.

To understand this better, stem cell systems can be divided into "definitive" and "nondefinitive" systems as illustrated in Fig. 1. Definitive stem cell systems are responsible for maintaining a specific biological system. They can be divided into continuously pro‐ liferating systems such as the blood-forming or lympho-hematopoietic system, the gastrointestinal system, hair and skin, reproductive organs and cells of the eye cornea. Although not necessarily a continuously proliferating system, the mesenchymal stem cell (MSC), also called the multipotent mesenchymal stromal cell [1] system can been includ‐ ed, because in culture, the MSCs proliferate and can be passaged over a long period of time. Definitive stem cells systems can also demonstrate partial proliferation. These in‐ clude, but are not limited to, the liver, lung, kidney, heart, pancreas, and the neural/ neuronal system. From a toxicological viewpoint, these are not usually considered stem cell systems. Yet, the different types of lineage cells present in these organs and the abili‐ ty to maintain a specific cell mass has all the intricacies of a stem cell system, especially during development, even though the cell turnover in the adult may be very low. Nondefinitive stem cells systems are represented by the ES and iPS cell systems, which can, theoretically, give rise to any of the definitive stem cell systems. Indeed, it is a prerequi‐ site that the production of functionally, mature cells from ES or iPS cells first pass

**2. Stem cell characteristics and properties used for toxicity testing**

Stem cells of primary, definitive systems always represent a very small proportion of the tissue or organ cellularity. This proportion is between 0.1 and 0.01% or less. The basic definition of a stem cell is that it possesses the capacity for self-renewal. In fact, stem cell systems are usually termed self-renewal cell systems, meaning that one stem cell can produce two daughter cells that are exact replicas of the parent. However, self-renewal is a difficult property to measure. The capacity for either serial *in vivo* repopulation or *in vitro* serial re-plating is considered a property of stem cells that implicates not only the presence of stem cells, but also their selfrenewal capability. The fact that serial *in vitro* re-plating or *in vivo* repopulation cannot be performed ad infinitum is not only an indication for a stem cell hierarchy [2-7], but also for an alternative hypothesis to stem cell self-renewal. This hypothesis states that tissues and organs are endowed with a specific number of stem cells. Once used up, the system ceases to function [8]. Regardless of the hypothesis, this important property can be utilized in a toxicological setting by employing secondary re-plating technology. This allows not only the presence of residual stem cells to be detected that have not been affected by a compound, but any change in sensitivity to a compound that might be important during repeated dose administration.

Stem cells have two other important properties that can be applied to toxicity testing. The first is that they are undifferentiated. The second is that stem cells proliferate. Stem cells can be "determined" into one or more lineages of mature functional cells. When a stem cell be‐ comes determined, it ceases to be a stem cell and becomes a progenitor cell that proliferates and differentiates. The fact that stem cells can be induced to differentiate means that what‐

through a definitive stem cell compartment.

80 Stem Cell Biology in Normal Life and Diseases

**Figure 1. Definitive and Non-Definitive Stem Cell Systems.** Definitive stem cell systems can be further divided into continuously and partially proliferating systems. Non-definitive stem cell systems such as embryonic stem cells and in‐ duced pluripotent stem cells can produce definitive stem cell systems, which in turn, give rise to mature functional cells.

All definitive stem cell systems have a common organization shown in Fig. 2. There is a continuum of stem cells within the stem cell compartment that exhibit different degrees of primitiveness or "stemness", which in turn, implies changing proliferating potential or potency as a stem cell moves through the compartment to the point of determination. These characteristic properties actually provide the information that allows stem cells to be predictors of potential toxicity. Once a stem cell becomes a progenitor cell, prolifera‐ tion continues and actually increases for a certain time so that the compartment can be amplified, until it ceases completely and the differentiation and maturation processes takes over. These changes have important implications for the types of assays that can be used *in vitro* to detect potential toxicity.

From Fig. 2, it is clear that proliferation occurs prior to differentiation. Although there is considerable overlap between proliferation and differentiation, they are two separate proc‐ esses that cannot be measured using the same assay readout. Since stem cells only proliferate, it follows that a proliferation assay is required to detect the presence and response of stem cells to a compound or agent. Using a differentiation assay to detect the effect of a compound or agent that targets one or more steps in the proliferation process can influence the interpretation and conclusion of the results. This can have far-reaching consequences on the decision to move forward with the development of a new drug candidate.

of human origin) nor animal testing provide good extrapolation to the human situation. It is not uncommon for unexpected results or toxicity to rear its head during animal studies be‐ cause of the lack of predictive information obtained during previous screening and testing [13]. Many published articles have dealt with this problem, one of the most notable being the monograph on Toxicity Testing in the 21st Century [14]. Despite the goals of the drug development pipeline and the considerable effort being undertaken by regulatory agencies [15-20] to determine the effect of environmental agents on human cells, interpretation and conclusions often fall short due to lack of understanding of the mechanism of action of the molecule, incorrect assay readout and/or incorrect target cell, to name but a few reasons. If the goal is to determine the effect on human cells, then mouse, rat, dog or even non-human primate cells will not provide the required information; human cells must be used. It goes without saying that drug development or testing xenobiotic agents cannot be performed on human subjects. It is for this reason why surrogate *in vitro* assays using primary human cells obtained from donors under the auspices of regulatory controlled internal review boards (IRBs) provide the best alternative. However, even under these circumstances, detailed knowledge of the biological system under study is necessary in order to interpret and make

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 83

Of all the biological systems of the body, the one most studied is also one of the systems that is given the least priority with respect to toxicity. The blood-forming or hematopoietic system and the gastrointestinal system are two continuously proliferating systems that are expected to be dramatically affected by anti-proliferating agents such as anti-cancer drugs. As a result, the only relevant questions are (a) how severe would toxicity be, and (b) would use of the drug

conclusions in the most objective manner.

provide a favorable therapeutic index?

**Figure 3.** The Major Stages of the Drug Development Pipeline

**Figure 2.** The Common Organization of Definitive Stem Cell Systems

Toxicity represents between 30-40% of the drug attrition rate [9,10]. It is therefore not sur‐ prising that biopharmaceutical companies are eager to employ assays that allow early pre‐ diction of toxicity prior to starting human clinical trials. Once the drug discovery phase has been concluded, the drug development phase begins (Fig. 3) by screening thousands of com‐ pounds in a battery of tests to determine absorption, distribution, metabolism and excretion (ADME) as well as preliminary toxicity (ADME/Tox). Many of the ADME/Tox assays as well as those in the lead optimization phase use transformed cell lines as cell targets, such as the NCI60 cell line panel [11,12]. Once these tests have whittled down the number of possi‐ ble drug candidates, pre-clinical animal models are used. Neither cell lines (even if they are of human origin) nor animal testing provide good extrapolation to the human situation. It is not uncommon for unexpected results or toxicity to rear its head during animal studies be‐ cause of the lack of predictive information obtained during previous screening and testing [13]. Many published articles have dealt with this problem, one of the most notable being the monograph on Toxicity Testing in the 21st Century [14]. Despite the goals of the drug development pipeline and the considerable effort being undertaken by regulatory agencies [15-20] to determine the effect of environmental agents on human cells, interpretation and conclusions often fall short due to lack of understanding of the mechanism of action of the molecule, incorrect assay readout and/or incorrect target cell, to name but a few reasons. If the goal is to determine the effect on human cells, then mouse, rat, dog or even non-human primate cells will not provide the required information; human cells must be used. It goes without saying that drug development or testing xenobiotic agents cannot be performed on human subjects. It is for this reason why surrogate *in vitro* assays using primary human cells obtained from donors under the auspices of regulatory controlled internal review boards (IRBs) provide the best alternative. However, even under these circumstances, detailed knowledge of the biological system under study is necessary in order to interpret and make conclusions in the most objective manner.

Of all the biological systems of the body, the one most studied is also one of the systems that is given the least priority with respect to toxicity. The blood-forming or hematopoietic system and the gastrointestinal system are two continuously proliferating systems that are expected to be dramatically affected by anti-proliferating agents such as anti-cancer drugs. As a result, the only relevant questions are (a) how severe would toxicity be, and (b) would use of the drug provide a favorable therapeutic index?

**Figure 3.** The Major Stages of the Drug Development Pipeline

and conclusion of the results. This can have far-reaching consequences on the decision to move

forward with the development of a new drug candidate.

82 Stem Cell Biology in Normal Life and Diseases

**Figure 2.** The Common Organization of Definitive Stem Cell Systems

Toxicity represents between 30-40% of the drug attrition rate [9,10]. It is therefore not sur‐ prising that biopharmaceutical companies are eager to employ assays that allow early pre‐ diction of toxicity prior to starting human clinical trials. Once the drug discovery phase has been concluded, the drug development phase begins (Fig. 3) by screening thousands of com‐ pounds in a battery of tests to determine absorption, distribution, metabolism and excretion (ADME) as well as preliminary toxicity (ADME/Tox). Many of the ADME/Tox assays as well as those in the lead optimization phase use transformed cell lines as cell targets, such as the NCI60 cell line panel [11,12]. Once these tests have whittled down the number of possi‐ ble drug candidates, pre-clinical animal models are used. Neither cell lines (even if they are

Hemotoxicity testing is traditionally performed during the last stage of drug development, namely pre-clinical animal testing. Circulating blood parameters are measured and at necropsy, bone marrow, spleen and even liver hematopathology are performed. Primary stem and progenitor cells cannot be morphologically identified. Morphological identification of cells is only possible once the cells start to differentiate and mature. Consequently, traditional hemotoxicity testing provides little, if any, predictive value since most of the toxic effects have occurred on more primitive cells.

As mentioned above, although proliferation is required to produce colonies, the number of cells produced as a quantitative measure of proliferation cannot be ascertained. Although proliferation is assumed, the CFC assay actually detects differentiation ability or potential.

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 85

Over several years, the European Center for the Validation of Alternative Methods (ECVAM) undertook a series of studies in which a number of drugs and chemicals were tested using the CFC assay. These studies are noteworthy because they represented the first attempt to validate a prediction model for assessing the maximum tolerated dose (MTD, equivalent to the IC90 value) for drugs that induce neutropenia [26,27] using the CFC assay. The studies were performed in different laboratories and were later extended to compounds that caused thrombocytopenia [28]. Potential neutropenia was detected by the effect on the granulocytemacrophage colony-forming cell or GM-CFC (also called CFC or CFC-GM), while thrombo‐ cytopenia was detected by the effect on the megakaryocyte colony-forming cell or Mk-CFC (also called CFC-Mk). A decrease or inhibition in the number of colonies counted derived from GM-CFC or Mk-CFC predicted a reduction in neutrophils or platelets in the circulation. The authors demonstrated that the model could correctly predict the MTD of 20 out of 23 drugs

There are two points worth emphasizing. First, not all compounds will produce an estimated IC90 value and may not even produce an IC50 value, when tested using the CFC assay. Does that mean that these compounds will not produce neutropenia or thrombocytopenia? It is interesting to note that the same CFC assay that is used to predict toxicity causing neutropenia or thrombocytopenia, is also used in an opposite manner to predict time to neutrophil or platelet engraftment after bone marrow, mobilized peripheral blood or umbilical cord blood stem cell transplantation for cellular therapy [39-31]. In either case, the GM-CFC or MK-CFC populations provide no information on the response of the more sensitive and more important stem cells. After all, it is the hematopoietic stem cells that give rise to both of these populations. This leads to the second point, namely that many compounds target one or more steps in the proliferation process, either at a molecular and/or cellular level. Although both GM-CFC and Mk-CFC populations are proliferating progenitor cell populations, they are not always the primary targets. When a compound affects more than one lineage, the primary effect is not on those lineages individually, but on the common cell that gives rise to those lineages, namely the stem cells [32]. From a practical viewpoint, however, the CFC assay posses daunting problems. The ECVAM studies summarized previously were exceptional in that the authors took the trouble to try and verify and standardize the readout of the assay that is inherently subjective and lacks the necessary external standards and controls by which the assay could be properly validated. In studies performed by the National Marrow Donor Program (NMDP), the results showed very high variability in CFU colony counting for cord blood [33]. This high variability, primarily due to the inaccuracy of dispensing methylcellulose and colony counting, together with the lack of high throughput capability does not provide the biopharmaceutical industry, environmental agencies or other areas of toxicology, risk or efficacy assessment with a routine and trustworthy assay platform. To negate all of these problems, the HALO Predic‐

This has important consequences for toxicity testing.

tested (87% predictive rate).

tive Hemotoxicity Platform was developed.

Much of our knowledge about the characterization, properties and responses of hematopoietic stem cells and the system as a whole has been provided through the use of drugs and other agents (e.g. radiation) using both *in vivo* and later, *in vitro* assays. The information obtained has allowed the organization and hierarchy within the different compartments of the hema‐ topoietic and lymphopoietic systems to be elucidated. By utilizing the knowledge that has accrued over more than six decades, analysis of the lympho-hematopoietic stem and progen‐ itor cells provide the highest degree of predictive toxicity of any biological system.

#### **3. The Colony-Forming Cell (CFC) assay and ECVAM studies**

In 1966, Bradley and Metcalf in Melbourne, Australia [21] and Pluznik and Sachs in Rehovot, Israel, [22] independently published what is now known as the colony-forming unit (CFU) or colony-forming cell (CFC) assay. In its original form, mouse bone marrow target cells were suspended in agar containing a conditioned medium that we now know contained granulo‐ cyte-macrophage colony stimulating factor or GM-CSF as well as other soluble factors. In the semi-solid medium, the cells underwent proliferation and later differentiation to produce colonies of cells that were identified either as neutrophils, macrophages or a combination of the two cell types. The number of colonies counted under an inverted microscope was proportional to both the number of cells plated and the dose of the conditioned medium added. In the same year, Cole and Paul [23] in Glasgow, Scotland reported the first *in vitro* suspension culture of murine erythropoietic cells from the yolk sac and fetal liver. Culture of erythropoietic cells under clonal conditions did not occur until 1971, when the Axelrad group [24] in Toronto, Canada, demonstrated that erythroid colonies could be produced using a plasma clot techni‐ que. In 1974, Iscove and colleagues [25] in Basel, Switzerland introduced the methylcellulose CFC assay that is still used today. Since that time, colony assays have been developed to detect multiple cell populations of every blood cell lineage, including several different stem cell populations. In addition, conditioned media has been replaced with recombinant growth factors and cytokines.

In Section 2, emphasis was placed on the importance between proliferation and differentiation. The cell populations detected using the CFC assay must all be proliferating populations, otherwise the production of colonies would not occur. However, to identify the type of colony, the *in vitro* culture must be allowed to proceed long enough so that the cells produced can themselves be identified as being derived from a morphologically unidentifiable stem, progenitor or precursor cell, all of which are capable of proliferation, but to different extents. As mentioned above, although proliferation is required to produce colonies, the number of cells produced as a quantitative measure of proliferation cannot be ascertained. Although proliferation is assumed, the CFC assay actually detects differentiation ability or potential. This has important consequences for toxicity testing.

Hemotoxicity testing is traditionally performed during the last stage of drug development, namely pre-clinical animal testing. Circulating blood parameters are measured and at necropsy, bone marrow, spleen and even liver hematopathology are performed. Primary stem and progenitor cells cannot be morphologically identified. Morphological identification of cells is only possible once the cells start to differentiate and mature. Consequently, traditional hemotoxicity testing provides little, if any, predictive value since most of the toxic effects have

Much of our knowledge about the characterization, properties and responses of hematopoietic stem cells and the system as a whole has been provided through the use of drugs and other agents (e.g. radiation) using both *in vivo* and later, *in vitro* assays. The information obtained has allowed the organization and hierarchy within the different compartments of the hema‐ topoietic and lymphopoietic systems to be elucidated. By utilizing the knowledge that has accrued over more than six decades, analysis of the lympho-hematopoietic stem and progen‐

In 1966, Bradley and Metcalf in Melbourne, Australia [21] and Pluznik and Sachs in Rehovot, Israel, [22] independently published what is now known as the colony-forming unit (CFU) or colony-forming cell (CFC) assay. In its original form, mouse bone marrow target cells were suspended in agar containing a conditioned medium that we now know contained granulo‐ cyte-macrophage colony stimulating factor or GM-CSF as well as other soluble factors. In the semi-solid medium, the cells underwent proliferation and later differentiation to produce colonies of cells that were identified either as neutrophils, macrophages or a combination of the two cell types. The number of colonies counted under an inverted microscope was proportional to both the number of cells plated and the dose of the conditioned medium added. In the same year, Cole and Paul [23] in Glasgow, Scotland reported the first *in vitro* suspension culture of murine erythropoietic cells from the yolk sac and fetal liver. Culture of erythropoietic cells under clonal conditions did not occur until 1971, when the Axelrad group [24] in Toronto, Canada, demonstrated that erythroid colonies could be produced using a plasma clot techni‐ que. In 1974, Iscove and colleagues [25] in Basel, Switzerland introduced the methylcellulose CFC assay that is still used today. Since that time, colony assays have been developed to detect multiple cell populations of every blood cell lineage, including several different stem cell populations. In addition, conditioned media has been replaced with recombinant growth

In Section 2, emphasis was placed on the importance between proliferation and differentiation. The cell populations detected using the CFC assay must all be proliferating populations, otherwise the production of colonies would not occur. However, to identify the type of colony, the *in vitro* culture must be allowed to proceed long enough so that the cells produced can themselves be identified as being derived from a morphologically unidentifiable stem, progenitor or precursor cell, all of which are capable of proliferation, but to different extents.

itor cells provide the highest degree of predictive toxicity of any biological system.

**3. The Colony-Forming Cell (CFC) assay and ECVAM studies**

occurred on more primitive cells.

84 Stem Cell Biology in Normal Life and Diseases

factors and cytokines.

Over several years, the European Center for the Validation of Alternative Methods (ECVAM) undertook a series of studies in which a number of drugs and chemicals were tested using the CFC assay. These studies are noteworthy because they represented the first attempt to validate a prediction model for assessing the maximum tolerated dose (MTD, equivalent to the IC90 value) for drugs that induce neutropenia [26,27] using the CFC assay. The studies were performed in different laboratories and were later extended to compounds that caused thrombocytopenia [28]. Potential neutropenia was detected by the effect on the granulocytemacrophage colony-forming cell or GM-CFC (also called CFC or CFC-GM), while thrombo‐ cytopenia was detected by the effect on the megakaryocyte colony-forming cell or Mk-CFC (also called CFC-Mk). A decrease or inhibition in the number of colonies counted derived from GM-CFC or Mk-CFC predicted a reduction in neutrophils or platelets in the circulation. The authors demonstrated that the model could correctly predict the MTD of 20 out of 23 drugs tested (87% predictive rate).

There are two points worth emphasizing. First, not all compounds will produce an estimated IC90 value and may not even produce an IC50 value, when tested using the CFC assay. Does that mean that these compounds will not produce neutropenia or thrombocytopenia? It is interesting to note that the same CFC assay that is used to predict toxicity causing neutropenia or thrombocytopenia, is also used in an opposite manner to predict time to neutrophil or platelet engraftment after bone marrow, mobilized peripheral blood or umbilical cord blood stem cell transplantation for cellular therapy [39-31]. In either case, the GM-CFC or MK-CFC populations provide no information on the response of the more sensitive and more important stem cells. After all, it is the hematopoietic stem cells that give rise to both of these populations. This leads to the second point, namely that many compounds target one or more steps in the proliferation process, either at a molecular and/or cellular level. Although both GM-CFC and Mk-CFC populations are proliferating progenitor cell populations, they are not always the primary targets. When a compound affects more than one lineage, the primary effect is not on those lineages individually, but on the common cell that gives rise to those lineages, namely the stem cells [32]. From a practical viewpoint, however, the CFC assay posses daunting problems. The ECVAM studies summarized previously were exceptional in that the authors took the trouble to try and verify and standardize the readout of the assay that is inherently subjective and lacks the necessary external standards and controls by which the assay could be properly validated. In studies performed by the National Marrow Donor Program (NMDP), the results showed very high variability in CFU colony counting for cord blood [33]. This high variability, primarily due to the inaccuracy of dispensing methylcellulose and colony counting, together with the lack of high throughput capability does not provide the biopharmaceutical industry, environmental agencies or other areas of toxicology, risk or efficacy assessment with a routine and trustworthy assay platform. To negate all of these problems, the HALO Predic‐ tive Hemotoxicity Platform was developed.

#### **4. Predictive stem cell hemotoxicity testing**

Whereas the CFU assay may be used to predict neutropenia, thrombocytopenia, anemia and the MTD indicated by the IC90 values [27-29], stem cells assays allow potential hemotoxicity to be taken to a different system-wide "global" level. The reason is provided in Fig. 2 and in more detail in Fig. 4, which shows the different lympho-hematopoietic cell populations that can be detected using a hemotoxicity screening and testing platform specifically developed for this purpose. This platform, called HALO, will be described in more detail in the next section. Figures 2 and 4 demonstrate that functionally mature cells from definitive continu‐ ously proliferating and partially proliferating cell systems, are derived from stem cells. As such, any perturbation or damage to the stem cell compartment will ultimately affect all downstream cell populations. In other words, examining the effect on stem cells allows the "global" effect on the system to be predicted. Since more is known about the organization, hierarchy and regulation of the lympho-hematopoietic system than probably any other biological system in the body, this knowledge can be used to predict and explain potentially deleterious effects to the system. Changes in the response to hematopoietic stem cells will affect all three primary hematopoietic lineages, namely the erythropoietic, myelomonocytic and magakaryopoietic lineages. Changes in the response to lympho-hematopoietic stem cells, i.e. those stem cells that can give rise to both the lymphopoietic and hematopoietic cells, will be expected to affect most, if not all cell lineages, including the T- and B-cell lineages and therefore the immune system as a whole.

Predictive stem cell hemotoxicity testing is not simply the estimation of IC values so that compounds can be ranked in order of toxicity to different cell populations or species. There are several other important applications in which stem cell hemotoxicity, and indeed stem cell toxicity in general, can be used. Examples of these applications will be discussed later in this chapter. First, however, it is necessary to describe the principles, characteristics and properties of the assay that make this possible.

**Figure 4.** The Organization and Hierarchy of the Lympho-Hematopoietic System as a Model for a Definitive Continu‐

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 87

When cells proliferate or are inhibited from proliferation by drugs or other agents, the concentration of intracellular adenosine triphosphate (iATP) changes proportionately. This biochemical marker is an indicator of cellular and mitochondrial integrity and there‐ fore viability of the cells. Indeed, iATP is used as a metabolic viability assay (as opposed to a dye exclusion viability assay). Under normal conditions, stimulation of cell prolifera‐ tion requires specific growth factors and/or cytokines either alone or in combination (cocktails). For continuously proliferating systems, growth factors or cytokines need to be present continuously, albeit, in very small concentrations, in order to maintain cell sur‐ vival and production. Thus, to detect the effect of any agent on hematopoietic cells *in vi‐ tro*, the target cell population must be stimulated in order to detect changes in the cell

ously Proliferating Stem Cell System. The properties of stem cells play an integral part in predicting toxicity.

**5.1. Concepts and principles of the HALO platform**

#### **5. Materials and methods**

HALO is the acronym for Hematopoietic/Hemotoxicity Assays via Luminescence Output. This platform was originally designed and developed to provide the biopharmaceutical in‐ dustry with a high throughput, validated assay to examine the effects of virtually any com‐ pound on different cell populations of the lympho-hematopoietic system from multiple species. Initially, the assay platform was developed for fresh, primary human cells, as a sur‐ rogate assay that could be used at virtually at stage in the drug development pipeline (Fig. 3) to extrapolate to the human situation, and as an alternative to pre-clinical animal studies. The platform has since been further developed to include non-human primate, horse, pig, sheep, dog, rat and mouse, not only for toxicity studies, but also for basic research and vet‐ erinary applications.

**Figure 4.** The Organization and Hierarchy of the Lympho-Hematopoietic System as a Model for a Definitive Continu‐ ously Proliferating Stem Cell System. The properties of stem cells play an integral part in predicting toxicity.

#### **5.1. Concepts and principles of the HALO platform**

**4. Predictive stem cell hemotoxicity testing**

86 Stem Cell Biology in Normal Life and Diseases

the immune system as a whole.

of the assay that make this possible.

**5. Materials and methods**

erinary applications.

Whereas the CFU assay may be used to predict neutropenia, thrombocytopenia, anemia and the MTD indicated by the IC90 values [27-29], stem cells assays allow potential hemotoxicity to be taken to a different system-wide "global" level. The reason is provided in Fig. 2 and in more detail in Fig. 4, which shows the different lympho-hematopoietic cell populations that can be detected using a hemotoxicity screening and testing platform specifically developed for this purpose. This platform, called HALO, will be described in more detail in the next section. Figures 2 and 4 demonstrate that functionally mature cells from definitive continu‐ ously proliferating and partially proliferating cell systems, are derived from stem cells. As such, any perturbation or damage to the stem cell compartment will ultimately affect all downstream cell populations. In other words, examining the effect on stem cells allows the "global" effect on the system to be predicted. Since more is known about the organization, hierarchy and regulation of the lympho-hematopoietic system than probably any other biological system in the body, this knowledge can be used to predict and explain potentially deleterious effects to the system. Changes in the response to hematopoietic stem cells will affect all three primary hematopoietic lineages, namely the erythropoietic, myelomonocytic and magakaryopoietic lineages. Changes in the response to lympho-hematopoietic stem cells, i.e. those stem cells that can give rise to both the lymphopoietic and hematopoietic cells, will be expected to affect most, if not all cell lineages, including the T- and B-cell lineages and therefore

Predictive stem cell hemotoxicity testing is not simply the estimation of IC values so that compounds can be ranked in order of toxicity to different cell populations or species. There are several other important applications in which stem cell hemotoxicity, and indeed stem cell toxicity in general, can be used. Examples of these applications will be discussed later in this chapter. First, however, it is necessary to describe the principles, characteristics and properties

HALO is the acronym for Hematopoietic/Hemotoxicity Assays via Luminescence Output. This platform was originally designed and developed to provide the biopharmaceutical in‐ dustry with a high throughput, validated assay to examine the effects of virtually any com‐ pound on different cell populations of the lympho-hematopoietic system from multiple species. Initially, the assay platform was developed for fresh, primary human cells, as a sur‐ rogate assay that could be used at virtually at stage in the drug development pipeline (Fig. 3) to extrapolate to the human situation, and as an alternative to pre-clinical animal studies. The platform has since been further developed to include non-human primate, horse, pig, sheep, dog, rat and mouse, not only for toxicity studies, but also for basic research and vet‐ When cells proliferate or are inhibited from proliferation by drugs or other agents, the concentration of intracellular adenosine triphosphate (iATP) changes proportionately. This biochemical marker is an indicator of cellular and mitochondrial integrity and there‐ fore viability of the cells. Indeed, iATP is used as a metabolic viability assay (as opposed to a dye exclusion viability assay). Under normal conditions, stimulation of cell prolifera‐ tion requires specific growth factors and/or cytokines either alone or in combination (cocktails). For continuously proliferating systems, growth factors or cytokines need to be present continuously, albeit, in very small concentrations, in order to maintain cell sur‐ vival and production. Thus, to detect the effect of any agent on hematopoietic cells *in vi‐ tro*, the target cell population must be stimulated in order to detect changes in the cell population response to the agent. The agent is usually added in a dose-dependent man‐ ner to the target cells, which are then incubated for a specific period of time. Thereafter, the cultures are removed from the incubator and the cells lysed to release the iATP. The latter then becomes a limiting substrate for a luciferin-luciferase reaction to produce bio‐ luminescence in the form of light as shown in the equation below.

important consequences. Cell interaction reduces the time for the onset of cell prolifera‐ tion by approximately 24 hours. This means that measurement of cell proliferation can be measured within 5 to 7 days. Indeed, for all of the studies described here, human cells were incubated for 5 days. Non-human primate cells are usually incubated for the same time, but all other animal cells only require 4 days of incubation. The second con‐ sequence of allowing cell interaction to occur is the two-fold increase in assay sensitivity. As with most cell cultures, cells are incubated at 37ºC in a fully humidified atmosphere containing CO2. Incubating cells under low oxygen tension of 5% O2, which is approx. equivalent to the venous oxygen tension, reduces oxygen toxicity due to free radical pro‐ duction and improves plating efficiency [34,35] for all lympho-hematopoietic cell popula‐

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 89

Four basic controls were always included for toxicity studies. A background control included cells, but no growth factors. A vehicle control was similar to the background control, but included the vehicle used to dissolve the compound. Growth of the target cell population without any compound or vehicle constituted the growth factor control. A similar control that included the vehicle was designated the growth factor + vehicle control. Drugs and other

Prior to measuring any sample, the instrument was calibrated and the assay standardized using an external ATP standard and controls. The procedures have been described previously [32] and detailed procedures can also be obtained [36,37]. Calibration and standardization

There are other advantages for calibrating and standardizing the assay. First results can be compared over time. Second, the output of a plate luminometer is in Relative Lumi‐ nescence Units or RLU. The results are relative because different instruments demon‐ strate different ranges of RLU. These ranges may vary from 0 to 100 for one manufacturer or 0 to several million for another. This means that it would be very diffi‐ cult to directly compare results within and between laboratories using RLU values. Per‐ forming an ATP standard curve allows all the results to be interpolated from RLU

HALO was originally developed from the "classic" CFC assay because the latter was the only cell-based assay that could detect primitive hematopoietic cell populations. Since HALO is a proliferation assay, while the CFC detects differentiation of the same cells, and because proliferation occurs prior to differentiation, it follows that one assay can verify the other. Indeed, several publications have shown a direct correlation between

**5.5. Instrument calibration, assay standardization and sample processing**

were also part of the assay validation process (see Section 5.6).

values into standardized ATP concentrations (µM).

**5.6. Assay verification and validation**

the two assays [32,38,39].

tions as well as other cell types.

agents were investigated over 6 – 9 doses.

**5.4. Controls and dosing**

$$\text{LiATP} + \text{Luiciferin} + \text{O}\_2 \xrightarrow[\text{Mg}^{2+}]{\text{Lucíferase}} \text{Oxylucíferin} + \text{AMP} + \text{PP}\_i + \text{CO}\_2 + \text{LIGHT}\_i$$

The light is measured in a plate luminometer. The amount of light produced correlates directly with any change in the iATP concentration and therefore with the state of proliferation or inhibition of the cells.

#### **5.2. Cell sources**

Cells from any hematopoietic or lymphopoietic organ can be used. For most of the studies described here, fresh or cryopreserved human bone marrow or peripheral blood cells were collected with prior authorization by an Internal Review Board (IRB). Human cells were obtained from Lonza (Walkerville, MD) or Allcells (Berkely, CA). A mononuclear cell (MNC) fraction was prepared using density gradient centrifugation. A nucleated cell count was performed using a Z2 particle counter (Beckman Coulter), while dye exclusion viability was performed using 7-aminoactinoycin D (7-AAD) and flow cytometry. Metabolic viability was performed using LIVEGlo (HemoGenix, Colorado Springs, CO).

#### **5.3. Cell culture**

For all toxicity studies, MNC were diluted so that the final cell concentration was either 7,500 or 10,000 cells/well. Either 96-well or 384-well, solid white-wall plates were used and all dispensing was performed using a liquid handler (Beckman Coulter, EPICS XL-MCL). After the cell suspension was prepared, it was added to a Master Mix containing reagents including growth factors and/or cytokines to stimulate the target cell population being studied. Five different hematopoietic stem cell populations have so far been devel‐ oped for this assay, the most important being the Colony-Forming Cell – Granulocyte, Erythroid, Macrophage, Megakaryocyte or CFC-GEMM (referred to in Fig. 4 as CFC-GEMM 1). This particular stem cell population is stimulated with erythropoietin (EPO), granulocyte-macrophage and granulocyte colony stimulating factors (GM-CSF, G-CSF), Interleukins 3 and 6 (IL-3, IL-6), stem cell factor (SCF), Flt3-Ligand (Flt3-L) and thrombo‐ poietin (TPO). Compared to a "classic" CFC assay, HALO does not incorporate methyl‐ cellulose and is therefore not a clonal assay. Instead HALO uses Suspension Expansion Culture (SEC) Technology, which has several advantages over methylcellulose assays. First, SEC assays allow more accurate dispensing using liquid handlers. This is in con‐ trast to inaccurately dispensing methylcellulose with syringes and needles. Second, the use of liquid handlers allows for true high throughput capability with accurate dispens‐ ing even in 384-well plates. Third, as opposed to methylcellulose, where little or no cell interaction occurs, SEC technology allows cells to interact with each other. This has two important consequences. Cell interaction reduces the time for the onset of cell prolifera‐ tion by approximately 24 hours. This means that measurement of cell proliferation can be measured within 5 to 7 days. Indeed, for all of the studies described here, human cells were incubated for 5 days. Non-human primate cells are usually incubated for the same time, but all other animal cells only require 4 days of incubation. The second con‐ sequence of allowing cell interaction to occur is the two-fold increase in assay sensitivity. As with most cell cultures, cells are incubated at 37ºC in a fully humidified atmosphere containing CO2. Incubating cells under low oxygen tension of 5% O2, which is approx. equivalent to the venous oxygen tension, reduces oxygen toxicity due to free radical pro‐ duction and improves plating efficiency [34,35] for all lympho-hematopoietic cell popula‐ tions as well as other cell types.

#### **5.4. Controls and dosing**

population response to the agent. The agent is usually added in a dose-dependent man‐ ner to the target cells, which are then incubated for a specific period of time. Thereafter, the cultures are removed from the incubator and the cells lysed to release the iATP. The latter then becomes a limiting substrate for a luciferin-luciferase reaction to produce bio‐

The light is measured in a plate luminometer. The amount of light produced correlates directly with any change in the iATP concentration and therefore with the state of proliferation or

Cells from any hematopoietic or lymphopoietic organ can be used. For most of the studies described here, fresh or cryopreserved human bone marrow or peripheral blood cells were collected with prior authorization by an Internal Review Board (IRB). Human cells were obtained from Lonza (Walkerville, MD) or Allcells (Berkely, CA). A mononuclear cell (MNC) fraction was prepared using density gradient centrifugation. A nucleated cell count was performed using a Z2 particle counter (Beckman Coulter), while dye exclusion viability was performed using 7-aminoactinoycin D (7-AAD) and flow cytometry. Metabolic viability was

For all toxicity studies, MNC were diluted so that the final cell concentration was either 7,500 or 10,000 cells/well. Either 96-well or 384-well, solid white-wall plates were used and all dispensing was performed using a liquid handler (Beckman Coulter, EPICS XL-MCL). After the cell suspension was prepared, it was added to a Master Mix containing reagents including growth factors and/or cytokines to stimulate the target cell population being studied. Five different hematopoietic stem cell populations have so far been devel‐ oped for this assay, the most important being the Colony-Forming Cell – Granulocyte, Erythroid, Macrophage, Megakaryocyte or CFC-GEMM (referred to in Fig. 4 as CFC-GEMM 1). This particular stem cell population is stimulated with erythropoietin (EPO), granulocyte-macrophage and granulocyte colony stimulating factors (GM-CSF, G-CSF), Interleukins 3 and 6 (IL-3, IL-6), stem cell factor (SCF), Flt3-Ligand (Flt3-L) and thrombo‐ poietin (TPO). Compared to a "classic" CFC assay, HALO does not incorporate methyl‐ cellulose and is therefore not a clonal assay. Instead HALO uses Suspension Expansion Culture (SEC) Technology, which has several advantages over methylcellulose assays. First, SEC assays allow more accurate dispensing using liquid handlers. This is in con‐ trast to inaccurately dispensing methylcellulose with syringes and needles. Second, the use of liquid handlers allows for true high throughput capability with accurate dispens‐ ing even in 384-well plates. Third, as opposed to methylcellulose, where little or no cell interaction occurs, SEC technology allows cells to interact with each other. This has two

+ CO2+*LIGHT*

luminescence in the form of light as shown in the equation below.

performed using LIVEGlo (HemoGenix, Colorado Springs, CO).

Oxyluciferin + AMP + PPi

Mg2+ Luciferase

*i*ATP + Luciferin + O2 →

88 Stem Cell Biology in Normal Life and Diseases

inhibition of the cells.

**5.2. Cell sources**

**5.3. Cell culture**

Four basic controls were always included for toxicity studies. A background control included cells, but no growth factors. A vehicle control was similar to the background control, but included the vehicle used to dissolve the compound. Growth of the target cell population without any compound or vehicle constituted the growth factor control. A similar control that included the vehicle was designated the growth factor + vehicle control. Drugs and other agents were investigated over 6 – 9 doses.

#### **5.5. Instrument calibration, assay standardization and sample processing**

Prior to measuring any sample, the instrument was calibrated and the assay standardized using an external ATP standard and controls. The procedures have been described previously [32] and detailed procedures can also be obtained [36,37]. Calibration and standardization were also part of the assay validation process (see Section 5.6).

There are other advantages for calibrating and standardizing the assay. First results can be compared over time. Second, the output of a plate luminometer is in Relative Lumi‐ nescence Units or RLU. The results are relative because different instruments demon‐ strate different ranges of RLU. These ranges may vary from 0 to 100 for one manufacturer or 0 to several million for another. This means that it would be very diffi‐ cult to directly compare results within and between laboratories using RLU values. Per‐ forming an ATP standard curve allows all the results to be interpolated from RLU values into standardized ATP concentrations (µM).

#### **5.6. Assay verification and validation**

HALO was originally developed from the "classic" CFC assay because the latter was the only cell-based assay that could detect primitive hematopoietic cell populations. Since HALO is a proliferation assay, while the CFC detects differentiation of the same cells, and because proliferation occurs prior to differentiation, it follows that one assay can verify the other. Indeed, several publications have shown a direct correlation between the two assays [32,38,39].

Validation, on the other hand, is quite a different matter. Assay validation is defined as "establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes" [40]. When an assay is properly validated the accuracy (proportion of correct outcomes), sensitivity (proportion of correctly identified positive samples), selectivity (pro‐ portion of correctly identified negative samples), precision (intra and inter-laboratory varia‐ bility) and robustness (the ability of the assay to withstand changes and transferability) all combine to give the user the assurance that the results obtained are correct. The ECVAM studies described in Section 2 above were, and still are, the closest the CFC assay has come to being validated. There have been many attempts to validate the CFC assay, but all have failed. Certainly the assay has shown, from a subjective viewpoint, some of the attributes. However, since there are no standards and controls by which the CFC assay can provide documented and quantitative evidence for each of the required parameters, the assay has never been properly validated. Like many assays that have been used for decades, the CFC assay has been "grandfathered" in and used despite the problematic trustworthiness and meaning of the results obtained [33,37].

**5.7. Statistics**

All of the results provided were produced using 8 replicate wells/point. Compound dose response curves were fitted to a 4- or 5-parameter logistic curve fit using SoftMax Pro software (Molecular Devices) from results exported directed from the plate luminometer and calculated automatically. To estimate IC values, raw data were converted to a percentage of the growth factor + vehicle control. Additional statistics, curve fitting or graphing was performed using

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 91

From a practical viewpoint, stem and progenitor cells are distinguished by at least two different characteristics. First, stem and progenitor cell populations are stimulated using different cocktails of growth factors and cytokines. In this way, specific cell populations can be targeted and studied, even though the cell suspension may contain other cell types. Combined with the culture conditions, this allows detection and measurement of specific cell populations. The other distinguishing characteristic is the difference in proliferation ability and potential between stem and progenitor cells. Even within the stem cell compartment, differences in proliferation potential will indicate the primitiveness or "stemness" of populations. This characteristic is shown in Fig. 5 for normal bone marrow cells. Since the stem cells are more primitive than the progenitor cells, it would be expected that their proliferation potential would be greater. Figure 5 shows that the two stem cell populations exhibit, not only greater ATP concentration values, but also greater linear regression cell dose response slopes than the hematopoietic or lymphopoietic progenitor cells. It is the slope of the cell dose response that measures proliferation potential. The greater the slope, the higher the proliferation potential, and the more primitive the cell population. Indeed, this is the basic principle for measuring potency of hematopoietic stem cell therapeutic products for transplantation [37]. In this way, it is possible to distinguish different stem cell populations, in this case the hematopoietic stem cell, CFC-GEMM 1, from the more primitive lympho-hematopoietic stem cell, HPP-SP (high proliferative potential – stem and progenitor cell). The HPP-SP stem cell will be discussed in more detail in Section 6.4. The three cell dose response clusters showing the differences in proliferation potential in Fig. 5 for stem cells, hematopoietic progenitor cells and lympho‐ poietic progenitor cells would be expected based on the organization of the blood-forming system shown in Fig. 4. Figure 6 demonstrates the expected proliferation ability of the seven different cell populations in response to mitomycin-C, with the stem cells showing the greatest ability to proliferate followed by the three hematopoietic lineages and lymphopoietic lineages. The steepness of the linear regression slope of the cell dose response for a cell population provides a measure of the proliferation potential. Stem cells exhibit the greatest proliferation potential of all cells. Within the stem cell compartment, stem cells with different potentials for proliferation also indicate their primitiveness. Proliferation ability is measured at a single

Prism software (GraphPad) or OriginPro (OriginLab).

**6.1. Distinguishing the response of stem cells from progenitor cells**

**6. Results and discussion**

cell dose (see Fig. 6).

HALO, from the outset, was designed to be validated. The assay was developed to incorporate the range values specified in the FDA Guidance on Bioanalytical Method Validation [40]. In summary, these values are as follows:


In addition, the assay has also been validated against the Registry of Cytotoxicity Prediction Model, which will be discussed in more detail in Section 5B.

#### **5.7. Statistics**

Validation, on the other hand, is quite a different matter. Assay validation is defined as "establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes" [40]. When an assay is properly validated the accuracy (proportion of correct outcomes), sensitivity (proportion of correctly identified positive samples), selectivity (pro‐ portion of correctly identified negative samples), precision (intra and inter-laboratory varia‐ bility) and robustness (the ability of the assay to withstand changes and transferability) all combine to give the user the assurance that the results obtained are correct. The ECVAM studies described in Section 2 above were, and still are, the closest the CFC assay has come to being validated. There have been many attempts to validate the CFC assay, but all have failed. Certainly the assay has shown, from a subjective viewpoint, some of the attributes. However, since there are no standards and controls by which the CFC assay can provide documented and quantitative evidence for each of the required parameters, the assay has never been properly validated. Like many assays that have been used for decades, the CFC assay has been "grandfathered" in and used despite the problematic trustworthiness and meaning of the

HALO, from the outset, was designed to be validated. The assay was developed to incorporate the range values specified in the FDA Guidance on Bioanalytical Method Validation [40]. In

**•** Sensitivity & Selectivity by Receiver Operator Characteristics (ROC): Area Under Curve

**•** Log-log linear regression slope for ATP standard curve: 0.937 ± 15% (slope range: 0.796 –

In addition, the assay has also been validated against the Registry of Cytotoxicity Prediction

results obtained [33,37].

90 Stem Cell Biology in Normal Life and Diseases

**•** Assay linearity: => 5 logs.

**•** Accuracy: ~95%.

**•** Robustness: ~95%.

1.07)

summary, these values are as follows:

**•** Assay ATP sensitivity: ~ 0.001µM.

**•** Assay cell linearity: 1,000 - > 25,000 cells/well.

**•** Assay cell sensitivity: 20-25 cells/well, depending on cell purity).

**•** Precision: = < 15%. Lower limit of quantification (LLOQ): 20%.

**•** High throughput capability (Z-factor [57]): > 0.76.

(AUC) 0.73 – 0.752 (lowest possible value: 0.5; highest possible value, 1).

**•** Lowest ATP value indicating unsustainable cell proliferation: ~ 0.04µM.

**•** ATP value below which cells are not metabolically viable: ~0.01µM.

Model, which will be discussed in more detail in Section 5B.

All of the results provided were produced using 8 replicate wells/point. Compound dose response curves were fitted to a 4- or 5-parameter logistic curve fit using SoftMax Pro software (Molecular Devices) from results exported directed from the plate luminometer and calculated automatically. To estimate IC values, raw data were converted to a percentage of the growth factor + vehicle control. Additional statistics, curve fitting or graphing was performed using Prism software (GraphPad) or OriginPro (OriginLab).

#### **6. Results and discussion**

#### **6.1. Distinguishing the response of stem cells from progenitor cells**

From a practical viewpoint, stem and progenitor cells are distinguished by at least two different characteristics. First, stem and progenitor cell populations are stimulated using different cocktails of growth factors and cytokines. In this way, specific cell populations can be targeted and studied, even though the cell suspension may contain other cell types. Combined with the culture conditions, this allows detection and measurement of specific cell populations. The other distinguishing characteristic is the difference in proliferation ability and potential between stem and progenitor cells. Even within the stem cell compartment, differences in proliferation potential will indicate the primitiveness or "stemness" of populations. This characteristic is shown in Fig. 5 for normal bone marrow cells. Since the stem cells are more primitive than the progenitor cells, it would be expected that their proliferation potential would be greater. Figure 5 shows that the two stem cell populations exhibit, not only greater ATP concentration values, but also greater linear regression cell dose response slopes than the hematopoietic or lymphopoietic progenitor cells. It is the slope of the cell dose response that measures proliferation potential. The greater the slope, the higher the proliferation potential, and the more primitive the cell population. Indeed, this is the basic principle for measuring potency of hematopoietic stem cell therapeutic products for transplantation [37]. In this way, it is possible to distinguish different stem cell populations, in this case the hematopoietic stem cell, CFC-GEMM 1, from the more primitive lympho-hematopoietic stem cell, HPP-SP (high proliferative potential – stem and progenitor cell). The HPP-SP stem cell will be discussed in more detail in Section 6.4. The three cell dose response clusters showing the differences in proliferation potential in Fig. 5 for stem cells, hematopoietic progenitor cells and lympho‐ poietic progenitor cells would be expected based on the organization of the blood-forming system shown in Fig. 4. Figure 6 demonstrates the expected proliferation ability of the seven different cell populations in response to mitomycin-C, with the stem cells showing the greatest ability to proliferate followed by the three hematopoietic lineages and lymphopoietic lineages.

The steepness of the linear regression slope of the cell dose response for a cell population provides a measure of the proliferation potential. Stem cells exhibit the greatest proliferation potential of all cells. Within the stem cell compartment, stem cells with different potentials for proliferation also indicate their primitiveness. Proliferation ability is measured at a single cell dose (see Fig. 6).

lymphopoietic lineages.

From a practical viewpoint, stem and progenitor cells are distinguished by at least two different characteristics. First, stem and progenitor cell populations are stimulated using different cocktails of growth factors and cytokines. In this way, specific cell populations can be targeted and studied, even though the cell suspension may contain other cell types. Combined with the culture conditions, this allows detection and measurement of specific cell populations. The other distinguishing characteristic is the difference in proliferation ability and potential between stem and progenitor cells. Even within the stem cell compartment, differences in proliferation potential will indicate the primitiveness or "stemness" of populations. This characteristic is shown in Fig. 5 for normal bone marrow cells. Since the stem cells are more primitive than the progenitor cells, it would be expected that their proliferation potential would be greater. Figure 5 shows that the two stem cell populations exhibit, not only greater ATP concentration values, but also greater linear regression cell dose response slopes than the hematopoietic or lymphopoietic progenitor cells. It is the slope of the cell dose response that measures proliferation potential. The greater the slope, the higher the proliferation potential, and the more primitive the cell population. Indeed, this is the basic principle for measuring potency of hematopoietic stem cell therapeutic products for transplantation [37]. In this way, it is possible to distinguish different stem cell populations, in this case the hematopoietic stem cell, CFC-GEMM 1, from the more primitive lympho-hematopoietic stem cell, HPP-SP (high proliferative potential – stem and progenitor cell). The HPP-SP stem cell will be discussed in more detail in Section 5C. The three cell dose response clusters showing the differences in proliferation potential in Fig. 5 for stem cells, hematopoietic progenitor cells and lymphopoietic progenitor cells would be expected based on the organization of the blood-forming system

**6.2. Drug and compound screening for stem cell toxicity**

tested were also used in the ECVAM studies [26,27].

ranked, a different and more plausible picture is obtained (Fig. 8).

that could possibly result in a false interpretation and conclusion.

In its most basic form, a single drug or compound is tested in a dose dependent manner on a target cell population. If the agent is cytotoxic to the cells, a negative sigmoidal dose re‐ sponse (Fig. 6) will result from which the estimated percent inhibitory concentrations (IC) can be calculated. Figure 7 shows the dose response curves from 13 drugs and compounds tested on hematopoietic stem cells (CFC-GEMM 1) derived from fresh, human bone marrow using the MNC fraction. Although different cell types are included in this fraction, stimula‐ tion of this particular stem cell population using a specific growth factor cocktail provides the relevant information. For each of the compounds tested a 4-parameter logistic curve fit was plotted from which the IC values could be calculated. Table 1 shows all of the com‐ pounds ranked in order of IC50 (µM) value from the most to the least toxic. The IC90 value (equivalent to the maximum tolerated dose, MTD) is also provided. Many of the compounds

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 93

Table 1 shows some compounds designated as NV or NE. The term NV indicates that an IC20 values was obtained, but no IC50 or IC90 value. The term NE means "no effect" in that no IC values could be estimated. As a result, methotrexate, which is an anti-cancer agent and expected to produce a more dramatic effect on stem cells, is actually ranked near the end of the list. Furthermore, compounds that do not allow an IC value to be calculated might actually produce some effect. The problem with ranking compounds based on their IC values is that it does not take into account the "form" of the dose response curve, which can actually provide more information than the IC value alone. Figure 7 shows a large num‐ ber of different dose response curves. One of the most important parameters provided by the 4-parameter logistic curve fit is coefficient or parameter B, which describes the transition of the curve to the midpoint of the dose response. This is a measure of steepness or slope. In some cases the slope is shallow, while in other cases it is almost vertical. How can this and other parameters of the dose response curve be taken into account so that they are inde‐ pendent of the IC value? The answer lies in calculating the area under the curve (AUC) for the range of doses used. When the AUC is performed and plotted so that the compounds are

In this case, the AUC values for both stem cells (CFC-GEMM 1) and granulocyte-macro‐ phage colony-forming cells (GM-CFC) are shown. When the results for CFC-GEMM 1 are compared with those in Table 1, the results generally follow the IC50 values. Howev‐ er, the toxicity of methotrexate is significantly increased and cycloheximide is more toxic than paclitaxel. The results for the GM progenitor cells have been included to demon‐ strate that progenitor cells exhibit lower toxicities than stem cells. Unless there is evi‐ dence to demonstrate that a compound acts on a specific hematopoietic lineage, it is more prudent to analyze potential toxicity to the stem cell compartment first, rather than focusing on a particular lineage, since the latter will only provide limited information

Figure 5. Measuring Proliferation Potential of Cell Populations **Figure 5.** Measuring Proliferation Potential of Cell Populations

6).

**6.2. Drug and compound screening for stem cell toxicity** 

information that could possible result in a false interpretation and conclusion.

In its most basic form, a single drug or compound is tested in a dose dependent manner on a target cell population. If the agent is cytotoxic to the cells, a negative sigmoidal dose response (Fig. 6) will result from which the estimated percent inhibitory concentrations (IC) can be calculated. Figure 7 shows the dose response curves from 13 drugs and compounds tested on hematopoietic stem cells (CFC-GEMM 1) derived from fresh, human bone marrow using the MNC fraction. Although different cell types are included in this fraction, stimulation of this particular stem cell population using a specific growth factor cocktail provides the relevant information. For each of the compounds tested a 4-parameter logistic curve fit was plotted from which the IC values could be calculated. Table 1 shows all of the compounds ranked in order of IC50 (M) value from the most to the least toxic. The IC90 value (equivalent to the maximum tolerated dose, MTD) is also provided. Many of the compounds tested were also used

Table 1 shows some compounds designated as NV or NE. The term NV indicates that an IC20 values was obtained, but no IC50 or IC90 value. The term NE means "no effect" in that no IC values could be estimated. As a result, methotrexate, which is an anticancer agent and expected to produce a more dramatic effect on stem cells, is actually ranked near the end of the list. Furthermore, compounds that do not allow an IC value to be calculated might actually produce some effect. The problem with ranking compounds based on their IC values is that it does not take into account the "form" of the dose response curve, which can actually provide more information than the IC value alone. Figure 13 shows a large number of different dose response curves. One of the most important parameters provided by the 4-parameter logistic curve fit is coefficient or parameter B, which describes the transition of the curve to the midpoint of the dose response. This is a measure of steepness or slope. In some cases the slope is shallow, while in other cases it is almost vertical. How can this and other parameters of the dose response curve be taken into account so that they are independent of the IC value? The answer lies in calculating the area under the curve (AUC) for the range of doses used. When the AUC is performed and plotted so that the compounds are ranked, a different and more plausible picture is

In this case, the AUC values for both stem cells (CFC-GEMM 1) and granulocyte-macrophage colony-forming cells (GM-CFC) are shown. When the results for CFC-GEMM 1 are compared with those in Table 1, the results generally follow the IC50 values. However, the toxicity of methotrexate is significantly increased and cycloheximide is more toxic than paclitaxel. The results for the GM progenitor cells have been included to demonstrate that progenitor cells exhibit lower toxicities than stem cells. Unless there is evidence to demonstrate that a compound acts on a specific hematopoietic lineage, it is more prudent to analyze potential toxicity to the stem cell compartment first, rather than focusing on a particular lineage, since the latter will only provide limited

Figure 6. Demonstration of Proliferation Ability between Cell Populations **Figure 6.** Demonstration of Proliferation Ability between Cell Populations

in the ECVAM studies [26,27].

obtained (Fig. 8).

#### **6.2. Drug and compound screening for stem cell toxicity**

From a practical viewpoint, stem and progenitor cells are distinguished by at least two different characteristics. First, stem and progenitor cell populations are stimulated using different cocktails of growth factors and cytokines. In this way, specific cell populations can be targeted and studied, even though the cell suspension may contain other cell types. Combined with the culture conditions, this allows detection and measurement of specific cell populations. The other distinguishing characteristic is the difference in proliferation ability and potential between stem and progenitor cells. Even within the stem cell compartment, differences in proliferation potential will indicate the primitiveness or "stemness" of populations. This characteristic is shown in Fig. 5 for normal bone marrow cells. Since the stem cells are more primitive than the progenitor cells, it would be expected that their proliferation potential would be greater. Figure 5 shows that the two stem cell populations exhibit, not only greater ATP concentration values, but also greater linear regression cell dose response slopes than the hematopoietic or lymphopoietic progenitor cells. It is the slope of the cell dose response that measures proliferation potential. The greater the slope, the higher the proliferation potential, and the more primitive the cell population. Indeed, this is the basic principle for measuring potency of hematopoietic stem cell therapeutic products for transplantation [37]. In this way, it is possible to distinguish different stem cell populations, in this case the hematopoietic stem cell, CFC-GEMM 1, from the more primitive lympho-hematopoietic stem cell, HPP-SP (high proliferative potential – stem and progenitor cell). The HPP-SP stem cell will be discussed in more detail in Section 5C. The three cell dose response clusters showing the differences in proliferation potential in Fig. 5 for stem cells, hematopoietic progenitor cells and lymphopoietic progenitor cells would be expected based on the organization of the blood-forming system shown in Fig. 4. Figure 6 demonstrates the expected proliferation ability of the seven different cell populations in response to mitomycin-C, with the stem cells showing the greatest ability to proliferate followed by the three hematopoietic lineages and

lymphopoietic lineages.

92 Stem Cell Biology in Normal Life and Diseases

6).

Figure 5. Measuring Proliferation Potential of Cell Populations

Figure 6. Demonstration of Proliferation Ability between Cell Populations

**6.2. Drug and compound screening for stem cell toxicity** 

information that could possible result in a false interpretation and conclusion.

In its most basic form, a single drug or compound is tested in a dose dependent manner on a target cell population. If the agent is cytotoxic to the cells, a negative sigmoidal dose response (Fig. 6) will result from which the estimated percent inhibitory concentrations (IC) can be calculated. Figure 7 shows the dose response curves from 13 drugs and compounds tested on hematopoietic stem cells (CFC-GEMM 1) derived from fresh, human bone marrow using the MNC fraction. Although different cell types are included in this fraction, stimulation of this particular stem cell population using a specific growth factor cocktail provides the relevant information. For each of the compounds tested a 4-parameter logistic curve fit was plotted from which the IC values could be calculated. Table 1 shows all of the compounds ranked in order of IC50 (M) value from the most to the least toxic. The IC90 value (equivalent to the maximum tolerated dose, MTD) is also provided. Many of the compounds tested were also used

Table 1 shows some compounds designated as NV or NE. The term NV indicates that an IC20 values was obtained, but no IC50 or IC90 value. The term NE means "no effect" in that no IC values could be estimated. As a result, methotrexate, which is an anticancer agent and expected to produce a more dramatic effect on stem cells, is actually ranked near the end of the list. Furthermore, compounds that do not allow an IC value to be calculated might actually produce some effect. The problem with ranking compounds based on their IC values is that it does not take into account the "form" of the dose response curve, which can actually provide more information than the IC value alone. Figure 13 shows a large number of different dose response curves. One of the most important parameters provided by the 4-parameter logistic curve fit is coefficient or parameter B, which describes the transition of the curve to the midpoint of the dose response. This is a measure of steepness or slope. In some cases the slope is shallow, while in other cases it is almost vertical. How can this and other parameters of the dose response curve be taken into account so that they are independent of the IC value? The answer lies in calculating the area under the curve (AUC) for the range of doses used. When the AUC is performed and plotted so that the compounds are ranked, a different and more plausible picture is

In this case, the AUC values for both stem cells (CFC-GEMM 1) and granulocyte-macrophage colony-forming cells (GM-CFC) are shown. When the results for CFC-GEMM 1 are compared with those in Table 1, the results generally follow the IC50 values. However, the toxicity of methotrexate is significantly increased and cycloheximide is more toxic than paclitaxel. The results for the GM progenitor cells have been included to demonstrate that progenitor cells exhibit lower toxicities than stem cells. Unless there is evidence to demonstrate that a compound acts on a specific hematopoietic lineage, it is more prudent to analyze potential toxicity to the stem cell compartment first, rather than focusing on a particular lineage, since the latter will only provide limited

**Figure 5.** Measuring Proliferation Potential of Cell Populations

in the ECVAM studies [26,27].

**Figure 6.** Demonstration of Proliferation Ability between Cell Populations

obtained (Fig. 8).

In its most basic form, a single drug or compound is tested in a dose dependent manner on a target cell population. If the agent is cytotoxic to the cells, a negative sigmoidal dose re‐ sponse (Fig. 6) will result from which the estimated percent inhibitory concentrations (IC) can be calculated. Figure 7 shows the dose response curves from 13 drugs and compounds tested on hematopoietic stem cells (CFC-GEMM 1) derived from fresh, human bone marrow using the MNC fraction. Although different cell types are included in this fraction, stimula‐ tion of this particular stem cell population using a specific growth factor cocktail provides the relevant information. For each of the compounds tested a 4-parameter logistic curve fit was plotted from which the IC values could be calculated. Table 1 shows all of the com‐ pounds ranked in order of IC50 (µM) value from the most to the least toxic. The IC90 value (equivalent to the maximum tolerated dose, MTD) is also provided. Many of the compounds tested were also used in the ECVAM studies [26,27].

The steepness of the linear regression slope of the cell dose response for a cell population provides a measure of the proliferation potential. Stem cells exhibit the greatest proliferation potential of all cells. Within the stem cell compartment, stem cells with different potentials for proliferation also indicate their primitiveness. Proliferation ability is measure at a single cell dose (see Fig. Table 1 shows some compounds designated as NV or NE. The term NV indicates that an IC20 values was obtained, but no IC50 or IC90 value. The term NE means "no effect" in that no IC values could be estimated. As a result, methotrexate, which is an anti-cancer agent and expected to produce a more dramatic effect on stem cells, is actually ranked near the end of the list. Furthermore, compounds that do not allow an IC value to be calculated might actually produce some effect. The problem with ranking compounds based on their IC values is that it does not take into account the "form" of the dose response curve, which can actually provide more information than the IC value alone. Figure 7 shows a large num‐ ber of different dose response curves. One of the most important parameters provided by the 4-parameter logistic curve fit is coefficient or parameter B, which describes the transition of the curve to the midpoint of the dose response. This is a measure of steepness or slope. In some cases the slope is shallow, while in other cases it is almost vertical. How can this and other parameters of the dose response curve be taken into account so that they are inde‐ pendent of the IC value? The answer lies in calculating the area under the curve (AUC) for the range of doses used. When the AUC is performed and plotted so that the compounds are ranked, a different and more plausible picture is obtained (Fig. 8).

> In this case, the AUC values for both stem cells (CFC-GEMM 1) and granulocyte-macro‐ phage colony-forming cells (GM-CFC) are shown. When the results for CFC-GEMM 1 are compared with those in Table 1, the results generally follow the IC50 values. Howev‐ er, the toxicity of methotrexate is significantly increased and cycloheximide is more toxic than paclitaxel. The results for the GM progenitor cells have been included to demon‐ strate that progenitor cells exhibit lower toxicities than stem cells. Unless there is evi‐ dence to demonstrate that a compound acts on a specific hematopoietic lineage, it is more prudent to analyze potential toxicity to the stem cell compartment first, rather than focusing on a particular lineage, since the latter will only provide limited information that could possibly result in a false interpretation and conclusion.

**Compound Effect Rank IC50 (μM) IC90 (μM)** Camptothecin Anti-cancer 1 0.02 0.14 Paclitaxol Anti-cancer 2 0.18 4.81 Cycloheximide Pesticide 3 0.23 0.86 Cyclosporin A Immunosuppressant 4 2.57 8.2 5-Fluorouracil Anti-cancer 5 5.79 29.7

(Thorazine) Anti-psychotic <sup>6</sup> 5.88 7.03 Rifampicin Anti-bacterial 7 12.8 NV Zedovuidine(AZT) Anti-viral 8 30.4 NV Choramphenicol Anti-bacterial 9 94.7 NV Indomethacin Anti-inflammatory 10 394.2 947.5 Methotrexate Anti-cancer 11 NV NV Acyclovir Anti-viral 12 NE NE Warfarin Anti-coagulant 13 NE NE NV indicates No Value for these IC values. An IC20 value would have been estimated by the software program.

NE indicate No Effect. In this case, the dose response for the compound did not produce an IC values.

**Ranking of Stem and Progenitor Cell Toxicity**

Stem Cells GM Progenitor Cells

Figure 8. Ranking of Stem Cell and Granulocyte-Macrophage Progenitor (GM-CFC) Toxicity According to the Calculated Area Under the Curve

**Acyclovir**

**AZT**

**Indomethacin**

**Chloramphenicol**

Least Toxic

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 95

**Warfarin**

The Registry of Cytotoxicity (RC) is a list of 347 compounds for which the IC50 values using a neutral red uptake assay for human keratinocytes and mouse 3T3 cells and the oral LD50 values for rat or mouse are known. When validating an *in vitro* assay against the RC, a sample of reference compounds is tested. The resulting IC50 values from the *in vitro* assay are then plotted against the LD50 values for the same compounds. A linear regression should be obtained exhibiting equation constants within a specific range. If this occurs, the *in vitro* assay is considered a validated cytotoxic test. The results validating HALO against the RC Prediction Model were first reported in 2005 [33]. One of the most interesting aspects of this prediction model is that once an assay has been validated, it can be used to convert *in vitro* IC values into clinically relevant doses that can be used as starting doses for pre-clinical animal models or human clinical trials. An example of this is shown in Table 2 where the results of converting IC20, IC50 and IC90 range values derived from the effects of 18 compounds on CFC-GEMM 1 bone marrow cells is shown. The predicted doses derived from the IC values are given in both milligrams/kilogram (mg/kg) and milligrams/meter2 (mg/m2). Doses used in the clinic to treat patients are also shown in mg/kg or mg/m2 where available. With the exception of two drugs, namely acyclovir and warfarin, nearly all of the doses predicted by the *in vitro* CFC-GEMM 1 assay using ATP bioluminescence are in the same order of magnitude or very close to the doses used to treat patients. In some cases lower doses were predicted (e.g. 5-fluorouracil), while in other cases slightly higher doses were predicted (e.g. cyclosporine A, indomethacin, cisplatin and mitomycin-C). Thus these predicted starting

Figure 5 shows that the response of stem cells to toxic agents can vary dramatically. In some cases, agents cause complete eradication of all stem and progenitor cells at high doses. In other cases, there is partial cytotoxicity at which, even at high doses, stem and progenitor cells are not eradicated. This is an indication that some stem cells survive or are possibly resistant to the drug or compound. If stem cells are not noticeably affected at high doses, there is a good chance that when the drug or compound is removed, the system will reconstitute itself. If no stem cells are available, this will not occur. However, there are other aspects to

values may be used in early toxicity and efficacy studies to "bracket" the lower and higher dose ranges.

**Table 1.** Ranking of Stem Cell Toxicity According to IC50 Values

Most Toxic

(AUC) for the Dose Responses shown in Figure 7.

**Paclitaxol**

ed Area Under the Curve (AUC) for the Dose Responses shown in Figure 7.

**Cyclosporin A**

**Chlorpromazine**

**5-FU**

**Rifampicine**

**Figure 8.** Ranking of Stem Cell and Granulocyte-Macrophage Progenitor (GM-CFC) Toxicity According to the Calculat‐

**Methotrexate**

**Camptothecin**

**0**

**10,000**

**20,000**

**30,000**

**Area Under the Dose Response Curve**

**40,000**

**50,000**

**Cycloheximide**

**6.4. Residual stem cells after toxicity** 

this phenomenon that are important.

**6.3. The registry of cytotoxicity prediction model [41]** 

Chlorpromazine

**Figure 7.** The Effect of 13 Compounds on Hematopoietic CFC-GEMM Stem Cells. Diagram showing the dose response plots produced automatically by SoftMax Pro software after the data was collected by the SpectraMax L plate lumin‐ ometer. The parameters that define the 4-parameter logistic curve to which the dose responses of the compounds are fitted are as follows: Parameter A, asymptote (flat part of the curve) at low Y-values; Parameter D, asymptote at the highest Y-values; Parameter or coefficient B, the transition from the asymptotes to the center of the curve; Parameter or coefficient C, is the midpoint between parameters A and D, also called the IC50 or EC50. Data that cannot be prop‐ erly fitted will result in ambiguous results.


NV indicates No Value for these IC values. An IC20 value would have been estimated by the software program.

NE indicate No Effect. In this case, the dose response for the compound did not produce an IC values.

**Figure 7.** The Effect of 13 Compounds on Hematopoietic CFC-GEMM Stem Cells. Diagram showing the dose response plots produced automatically by SoftMax Pro software after the data was collected by the SpectraMax L plate lumin‐ ometer. The parameters that define the 4-parameter logistic curve to which the dose responses of the compounds are fitted are as follows: Parameter A, asymptote (flat part of the curve) at low Y-values; Parameter D, asymptote at the highest Y-values; Parameter or coefficient B, the transition from the asymptotes to the center of the curve; Parameter or coefficient C, is the midpoint between parameters A and D, also called the IC50 or EC50. Data that cannot be prop‐

erly fitted will result in ambiguous results.

94 Stem Cell Biology in Normal Life and Diseases

#### **Ranking of Stem and Progenitor Cell Toxicity**

Figure 8. Ranking of Stem Cell and Granulocyte-Macrophage Progenitor (GM-CFC) Toxicity According to the Calculated Area Under the Curve (AUC) for the Dose Responses shown in Figure 7. **Figure 8.** Ranking of Stem Cell and Granulocyte-Macrophage Progenitor (GM-CFC) Toxicity According to the Calculat‐ ed Area Under the Curve (AUC) for the Dose Responses shown in Figure 7.

values may be used in early toxicity and efficacy studies to "bracket" the lower and higher dose ranges.

The Registry of Cytotoxicity (RC) is a list of 347 compounds for which the IC50 values using a neutral red uptake assay for human keratinocytes and mouse 3T3 cells and the oral LD50 values for rat or mouse are known. When validating an *in vitro* assay against the RC, a sample of reference compounds is tested. The resulting IC50 values from the *in vitro* assay are then plotted against the LD50 values for the same compounds. A linear regression should be obtained exhibiting equation constants within a specific range. If this occurs, the *in vitro* assay is considered a validated cytotoxic test. The results validating HALO against the RC Prediction Model were first reported in 2005 [33]. One of the most interesting aspects of this prediction model is that once an assay has been validated, it can be used to convert *in vitro* IC values into clinically relevant doses that can be used as starting doses for pre-clinical animal models or human clinical trials. An example of this is shown in Table 2 where the results of converting IC20, IC50 and IC90 range values derived from the effects of 18 compounds on CFC-GEMM 1 bone marrow cells is shown. The predicted doses derived from the IC values are given in both milligrams/kilogram (mg/kg) and milligrams/meter2 (mg/m2). Doses used in the clinic to treat patients are also shown in mg/kg or mg/m2 where available. With the exception of two drugs, namely acyclovir and warfarin, nearly all of the doses predicted by the *in vitro* CFC-GEMM 1 assay using ATP bioluminescence are in the same order of magnitude or very close to the doses used to treat patients. In some cases lower doses were predicted (e.g. 5-fluorouracil), while in other cases slightly higher doses were predicted (e.g. cyclosporine A, indomethacin, cisplatin and mitomycin-C). Thus these predicted starting

Figure 5 shows that the response of stem cells to toxic agents can vary dramatically. In some cases, agents cause complete eradication of all stem and progenitor cells at high doses. In other cases, there is partial cytotoxicity at which, even at high doses, stem and progenitor cells are not eradicated. This is an indication that some stem cells survive or are possibly resistant to the drug or compound. If stem cells are not noticeably affected at high doses, there is a good chance that when the drug or compound is removed, the system will reconstitute itself. If no stem cells are available, this will not occur. However, there are other aspects to

**6.3. The registry of cytotoxicity prediction model [41]** 

**6.4. Residual stem cells after toxicity** 

this phenomenon that are important.

#### **6.3. The registry of cytotoxicity prediction model [41]**

The Registry of Cytotoxicity (RC) is a list of 347 compounds, for which the IC50 values using a neutral red uptake assay for human keratinocytes and mouse 3T3 cells and the oral LD50 values for rat or mouse, are known. When validating an *in vitro* assay against the RC, a sample of reference compounds is tested. The resulting IC50 values from the *in vitro* assay are then plotted against the LD50 values for the same compounds. A linear regression should be obtained exhibiting equation constants within a specific range. If this occurs, the *in vitro* assay is considered a validated cytotoxic test. The results validat‐ ing HALO against the RC Prediction Model were first reported in 2005 [33]. One of the most interesting aspects of this prediction model is that once an assay has been validat‐ ed, it can be used to convert *in vitro* IC values into clinically relevant doses that can be used as starting doses for pre-clinical animal models or human clinical trials. An exam‐ ple of this is shown in Table 2 where the results of converting IC20, IC50 and IC90 range values derived from the effects of 18 compounds on CFC-GEMM 1 bone marrow cells is shown. The predicted doses derived from the IC values are given in both milli‐ grams/kilogram (mg/kg) and milligrams/meter2 (mg/m2 ). Doses used in the clinic to treat patients are also shown in mg/kg or mg/m2 where available. With the exception of two drugs, namely acyclovir and warfarin, nearly all of the doses predicted by the *in vitro* CFC-GEMM 1 assay using ATP bioluminescence are in the same order of magnitude or very close to the doses used to treat patients. In some cases lower doses were predicted (e.g. 5-fluorouracil), while in other cases slightly higher doses were predicted (e.g. cyclo‐ sporine A, indomethacin, cisplatin and mitomycin-C). Thus these predicted starting val‐ ues may be used in early toxicity and efficacy studies to "bracket" the lower and higher dose ranges.

**Predicted Dosing Range from In Vitro Stem Cell Assay**

Drug/Compound Dose in mg/kg Dose in mg/m2 Doses or Dose Range

Cyclosporin A 14.2 – 31.2 524 – 1,155 5 – 10 Indomethacin 32 – 73 1,190 – 2,700 0.2 – 2 Zedovudine (AZT) 4.3 – 12.2 161 – 452 1 – 7.4

Acyclovir NV NV 5 – 500

Choramphenicol 16 – 24 594 – 896 12.5/30 – 50 Rifampicin 24 – 26 894 – 955 10 Warfarin NV NV 0.1 – 5

dose in mg/m2 is obtained by multiplying the dose in mg/kg by a specific factor described in [42].

Chlorpromozine (Thorazine)

Starting Doses

Doxorubicin 2.6 – 6.9 97 – 255 25/50/60/75 Daunorubicin 0.5 – 2.6 19.6 – 97 30/45/60 5-Fluorouracil 2.0 – 7.0 79 – 259 400 – 2,600 Paclitaxel 2.0 – 17.5 72 – 647 75 – 250 Imatinib (Gleevec) 3.6 – 30.5 132 – 1,125 400/600 Methotrexate 5.8 215 10 – 8,000

6.8 – 7.7 253 – 285 1 – 4.5

Camptothecin 0.36 – 1.52 13.3 – 56 25/320/470

SJG-136 0.1 – 0.3 4 – 10 6 – 40 Cisplatin 6.3 – 9.8 233 – 363 30 – 100 Mitomycin-C 1.2 – 6.0 47 – 220 6/10 – 20 The IC values obtained from the validated *in vitro* assay are entered into the equation: Y = 0.435 \* Log (IC value) + 0.625 [41]. The dose in mg/kg is then obtained by multiplying the value for Y with the molecular weight of the compound. The

**Table 2.** Using the Registry of Cytotoxicity Prediction Model to Convert *In Vitro* IC Values into Clinically Relevant

To demonstrate this, we developed an *in vitro* secondary re-plating assay for primitive stem cells called high proliferation potential – stem and progenitor cells (HPP-SP). This stem cell population, within the stem cell compartment (Fig. 4), is approximately at the divergence of the lymphopoietic and hematopoietic systems. The majority of HPP-SP stem cells are quies‐ cent. They can be induced or "primed" into proliferation with IL-3, IL-6, SCF and Fl3-L. This stem cell population is designated HPP-SP 1. Once the HPP-SP 1 cells begin proliferation, they can be expanded with a similar cocktail of growth factors and cytokines to that for CFC-GEMM 1, but with the addition of interleukins 2 and 7 (IL-2, IL-7). This fully stimulated primitive stem cell population is designated HPP-SP 2. In this two-stage assay, the HPP-SP 1, present in the MNC fraction of bone marrow are cultured in the presence of the drug or compound in a dosedependent manner. Thereafter, the cells are removed from culture, washed and re-plated in a secondary culture system in which the HPP-SP 2 population is measured. By performing a secondary re-plating step, the assay is substantiating the presence of primitive stem cells

**Published Drug Doses Used to Treat Patients**

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430

> Doses or Dose Range in mg/m2

97

in mg/kg

#### **6.4. Residual stem cells after toxicity**

Figure 7 shows that the response of stem cells to toxic agents can vary dramatically. In some cases, agents cause complete eradication of all stem and progenitor cells at high doses. In other cases, there is partial cytotoxicity at which, even at high doses, stem and progenitor cells are not eradicated. This is an indication that some stem cells survive or are possibly resistant to the drug or compound. If stem cells are not noticeably affected at high doses, there is a good chance that when the drug or compound is removed, the system will reconstitute itself. If no stem cells are available, this will not occur. However, there are other aspects to this phenom‐ enon that are important.

Primitive stem cells are usually in a quiescent state; they are not proliferating and therefore not in cell cycle. This does not mean that they cannot be affected by an agent. Small molecules can enter a cell even if it is quiescent. When required to initiate the proliferation process and begin cell division, the process may be aborted because the agent inhibits the process. This is a potential dangerous situation for two reasons. First, the "backup plan" for reconstituting the system may not function. Second, if cells do begin to proliferate and divide, they may be more sensitive to the agent. The consequence of this is that repeated administration of the drug or compound will continually reduce the proportion of residual stem cells present.


**6.3. The registry of cytotoxicity prediction model [41]**

96 Stem Cell Biology in Normal Life and Diseases

grams/kilogram (mg/kg) and milligrams/meter2 (mg/m2

dose ranges.

**6.4. Residual stem cells after toxicity**

enon that are important.

The Registry of Cytotoxicity (RC) is a list of 347 compounds, for which the IC50 values using a neutral red uptake assay for human keratinocytes and mouse 3T3 cells and the oral LD50 values for rat or mouse, are known. When validating an *in vitro* assay against the RC, a sample of reference compounds is tested. The resulting IC50 values from the *in vitro* assay are then plotted against the LD50 values for the same compounds. A linear regression should be obtained exhibiting equation constants within a specific range. If this occurs, the *in vitro* assay is considered a validated cytotoxic test. The results validat‐ ing HALO against the RC Prediction Model were first reported in 2005 [33]. One of the most interesting aspects of this prediction model is that once an assay has been validat‐ ed, it can be used to convert *in vitro* IC values into clinically relevant doses that can be used as starting doses for pre-clinical animal models or human clinical trials. An exam‐ ple of this is shown in Table 2 where the results of converting IC20, IC50 and IC90 range values derived from the effects of 18 compounds on CFC-GEMM 1 bone marrow cells is shown. The predicted doses derived from the IC values are given in both milli‐

patients are also shown in mg/kg or mg/m2 where available. With the exception of two drugs, namely acyclovir and warfarin, nearly all of the doses predicted by the *in vitro* CFC-GEMM 1 assay using ATP bioluminescence are in the same order of magnitude or very close to the doses used to treat patients. In some cases lower doses were predicted (e.g. 5-fluorouracil), while in other cases slightly higher doses were predicted (e.g. cyclo‐ sporine A, indomethacin, cisplatin and mitomycin-C). Thus these predicted starting val‐ ues may be used in early toxicity and efficacy studies to "bracket" the lower and higher

Figure 7 shows that the response of stem cells to toxic agents can vary dramatically. In some cases, agents cause complete eradication of all stem and progenitor cells at high doses. In other cases, there is partial cytotoxicity at which, even at high doses, stem and progenitor cells are not eradicated. This is an indication that some stem cells survive or are possibly resistant to the drug or compound. If stem cells are not noticeably affected at high doses, there is a good chance that when the drug or compound is removed, the system will reconstitute itself. If no stem cells are available, this will not occur. However, there are other aspects to this phenom‐

Primitive stem cells are usually in a quiescent state; they are not proliferating and therefore not in cell cycle. This does not mean that they cannot be affected by an agent. Small molecules can enter a cell even if it is quiescent. When required to initiate the proliferation process and begin cell division, the process may be aborted because the agent inhibits the process. This is a potential dangerous situation for two reasons. First, the "backup plan" for reconstituting the system may not function. Second, if cells do begin to proliferate and divide, they may be more sensitive to the agent. The consequence of this is that repeated administration of the drug or

compound will continually reduce the proportion of residual stem cells present.

). Doses used in the clinic to treat

The IC values obtained from the validated *in vitro* assay are entered into the equation: Y = 0.435 \* Log (IC value) + 0.625 [41]. The dose in mg/kg is then obtained by multiplying the value for Y with the molecular weight of the compound. The dose in mg/m2 is obtained by multiplying the dose in mg/kg by a specific factor described in [42].

**Table 2.** Using the Registry of Cytotoxicity Prediction Model to Convert *In Vitro* IC Values into Clinically Relevant Starting Doses

To demonstrate this, we developed an *in vitro* secondary re-plating assay for primitive stem cells called high proliferation potential – stem and progenitor cells (HPP-SP). This stem cell population, within the stem cell compartment (Fig. 4), is approximately at the divergence of the lymphopoietic and hematopoietic systems. The majority of HPP-SP stem cells are quies‐ cent. They can be induced or "primed" into proliferation with IL-3, IL-6, SCF and Fl3-L. This stem cell population is designated HPP-SP 1. Once the HPP-SP 1 cells begin proliferation, they can be expanded with a similar cocktail of growth factors and cytokines to that for CFC-GEMM 1, but with the addition of interleukins 2 and 7 (IL-2, IL-7). This fully stimulated primitive stem cell population is designated HPP-SP 2. In this two-stage assay, the HPP-SP 1, present in the MNC fraction of bone marrow are cultured in the presence of the drug or compound in a dosedependent manner. Thereafter, the cells are removed from culture, washed and re-plated in a secondary culture system in which the HPP-SP 2 population is measured. By performing a secondary re-plating step, the assay is substantiating the presence of primitive stem cells present in the first "priming" step of culture. The proliferation at both stages is determined using ATP bioluminescence technology. The results using busulphan and daunorubicin are shown in Figs. 9A and 9B, respectively. The effect of busulphan (Fig. 9A) on HPP-SP 1 demonstrates partial cytotoxicity to the stem cells and the presence of residual stem cells. However, when the treated cells are removed from primary culture and placed into secondary cultured to reveal their expansion potential, there are few residual cells that are available for expansion and the high doses used in the primary culture eradicated any remaining cells. There was also little change in the IC50 values. This indicates that busulphan continued to act on primitive stem cells leaving no residual stem cells (secondary culture results minus primary culture results) for possible repopulation. Daunorubicin (Fig. 9B) is highly toxic to stem cells with an IC50 value in the nanomolar range compared to the micromolar range for busulphan. At low doses of daunorubicin, residual stem cells would be available, but secondary culture demonstrates that both these and the residual cells have increased their sensitivity by approx. 3 fold, indicating that repeated drug administration would incur increased sensitivity of the stem cells to the drug.

response at the cellular level. To investigate this, we developed an assay in which drugs could

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 99

Figure 10A shows the response when verapamil is titrated against cyclosporin A, while Fig. 10B shows the effect when cyclosporin A is titrated against verapamil. Both drugs inhibit 3A4 CYP450 enzyme. Individually, both drugs are cytotoxic to CFC-GEMM 1 stem cells. However, when titrated against each other, cytotoxicity may be observed initially, but may be followed by an opposite effect at higher doses. The cells appear to overcome the inhibitory effects. In terms of DDI, this would indicate that one or other drug is present at concentrations that could cause serious harm to the patient. This unusual dose response behavior produces a U-shaped or inverted dose response curve that has been observed for many compounds, including dopamine [45] and endostatins [46]. Although often attributed to solubility, these effects appear to be pharmacologically and physiologically important, but in most cases, the mecha‐ nism is not understood. This is the first indication that DDI can occur at the stem cell level. Considering the importance of assessing toxicity to stem cells and the predictive value afforded by these cells, it is obvious that more has to be learnt before the consequences of these reactions

One of the most interesting aspects of drug treatment is the field of chronotherapy; the administration of drugs in accordance with circadian rhythms. Although studied for decades, the role of circadian rhythms to reduce toxicity and improve drug efficacy has been largely ignored by the biopharmaceutical industry. The primary reason for this is because chrono‐ therapeutic studies are difficult, time-consuming and expensive to perform. Nevertheless, many areas of chronotherapy, especially using anti-cancer drugs. have proved to be successful [47-49]. Many cellular functions are dependent upon circadian rhythms. It is not the purpose

be titrated against each other to determine potential DDI on stem cells.

**Figure 10.** Examples of Drug-Drug Interactions at the Stem Cell Level

on a stem cell system can be understood.

**6.6. Circadian rhythm and stem cells**

**Figure 9.** Assessing Residual Stem Cell Activity and Change in Stem Cell Sensitivity to Agents by Measuring the Re‐ sponse of Primitive Stem Cells in a Two-Step Secondary Re-Plating In Vitro Assay.

#### **6.5. Stem cells and drug-drug interactions**

Drug-drug interaction (DDI) can lead to dangerous consequences if not investigated properly. Traditionally, DDI are investigated using cultured hepatocytes since the liver is the organ primarily responsible for detoxification. The main enzymes investigated during DDI studies are those of the cytochrome P450 (CYP450) system present in the endoplasmic reticulum of the cells. CYP450 enzymes are present not only in hepatocytes, but in virtually all cells. There are a large number of CYP450 enzymes and assays are available for many of these. Depending on the drug or compound, one or more CYP450 enzymes can be induced or inhibited [43,44]. The response by different enzymes provides an indication as to whether an interaction between different drugs will occur. However, measurement of CYP450 activities does not indicate a

response at the cellular level. To investigate this, we developed an assay in which drugs could be titrated against each other to determine potential DDI on stem cells.

**Figure 10.** Examples of Drug-Drug Interactions at the Stem Cell Level

present in the first "priming" step of culture. The proliferation at both stages is determined using ATP bioluminescence technology. The results using busulphan and daunorubicin are shown in Figs. 9A and 9B, respectively. The effect of busulphan (Fig. 9A) on HPP-SP 1 demonstrates partial cytotoxicity to the stem cells and the presence of residual stem cells. However, when the treated cells are removed from primary culture and placed into secondary cultured to reveal their expansion potential, there are few residual cells that are available for expansion and the high doses used in the primary culture eradicated any remaining cells. There was also little change in the IC50 values. This indicates that busulphan continued to act on primitive stem cells leaving no residual stem cells (secondary culture results minus primary culture results) for possible repopulation. Daunorubicin (Fig. 9B) is highly toxic to stem cells with an IC50 value in the nanomolar range compared to the micromolar range for busulphan. At low doses of daunorubicin, residual stem cells would be available, but secondary culture demonstrates that both these and the residual cells have increased their sensitivity by approx. 3 fold, indicating that repeated drug administration would incur increased sensitivity of the

**Figure 9.** Assessing Residual Stem Cell Activity and Change in Stem Cell Sensitivity to Agents by Measuring the Re‐

Drug-drug interaction (DDI) can lead to dangerous consequences if not investigated properly. Traditionally, DDI are investigated using cultured hepatocytes since the liver is the organ primarily responsible for detoxification. The main enzymes investigated during DDI studies are those of the cytochrome P450 (CYP450) system present in the endoplasmic reticulum of the cells. CYP450 enzymes are present not only in hepatocytes, but in virtually all cells. There are a large number of CYP450 enzymes and assays are available for many of these. Depending on the drug or compound, one or more CYP450 enzymes can be induced or inhibited [43,44]. The response by different enzymes provides an indication as to whether an interaction between different drugs will occur. However, measurement of CYP450 activities does not indicate a

sponse of Primitive Stem Cells in a Two-Step Secondary Re-Plating In Vitro Assay.

**6.5. Stem cells and drug-drug interactions**

stem cells to the drug.

98 Stem Cell Biology in Normal Life and Diseases

Figure 10A shows the response when verapamil is titrated against cyclosporin A, while Fig. 10B shows the effect when cyclosporin A is titrated against verapamil. Both drugs inhibit 3A4 CYP450 enzyme. Individually, both drugs are cytotoxic to CFC-GEMM 1 stem cells. However, when titrated against each other, cytotoxicity may be observed initially, but may be followed by an opposite effect at higher doses. The cells appear to overcome the inhibitory effects. In terms of DDI, this would indicate that one or other drug is present at concentrations that could cause serious harm to the patient. This unusual dose response behavior produces a U-shaped or inverted dose response curve that has been observed for many compounds, including dopamine [45] and endostatins [46]. Although often attributed to solubility, these effects appear to be pharmacologically and physiologically important, but in most cases, the mecha‐ nism is not understood. This is the first indication that DDI can occur at the stem cell level. Considering the importance of assessing toxicity to stem cells and the predictive value afforded by these cells, it is obvious that more has to be learnt before the consequences of these reactions on a stem cell system can be understood.

#### **6.6. Circadian rhythm and stem cells**

One of the most interesting aspects of drug treatment is the field of chronotherapy; the administration of drugs in accordance with circadian rhythms. Although studied for decades, the role of circadian rhythms to reduce toxicity and improve drug efficacy has been largely ignored by the biopharmaceutical industry. The primary reason for this is because chrono‐ therapeutic studies are difficult, time-consuming and expensive to perform. Nevertheless, many areas of chronotherapy, especially using anti-cancer drugs. have proved to be successful [47-49]. Many cellular functions are dependent upon circadian rhythms. It is not the purpose of this section to describe or even summarize this field. The intention is to instead provide an example in which the circadian rhythm of cells, especially hematopoietic stem cells [50-52], can be used to predict the best time of day to administer an anti-cancer drug, which in this case, is 5-fluorouracil (5-FU) [38].

For each time point, cells were thawed and treated with 5-FU at six doses to measure the response of CFC-GEMM 1, BFU-E, GM-CFC and Mk-CFC. The slope of each negative sigmoi‐ dal dose response curve was then calculated from the 4-parameter logistic curve fit. The dose response slope values were then analyzed by cosinor analysis for each time point and for each cell population to obtain the circadian rhythms as a function of 5-FU treatment. The results are shown in Fig. 10B. Each of the hematopoietic cell populations exhibited its own circadian rhythm in response to 5-FU. When these circadian rhythms were correlated with either the continuous infusion of 5-FU that is normally used to treat patients and that of chronomodu‐ lated infusion of 5-FU as reported by Dogliotti and colleagues in 1998 [54], the results shown in Fig. 10C were obtained. For each of the administration types, the percent overall patient response rate, toxicity and tumor response are shown. These were overlaid onto the circadian rhythm for the CFC-GEMM 1 stem cell response to 5-FU and demonstrated that the lowest toxicity and highest overall and tumor response occurred when 5-FU was administered in a chronomodulated manner in the early morning hours rather than at any other time of the day. The nadir of the CFC-GEMM 1 circadian rhythm to 5-FU occurred at 14:00 hours in the afternoon. This was approximately the same time at which the highest toxicity to 5-FU was found. As expected, these results did not correlate nearly as well for the hematopoietic progenitor cells. In addition, the results clearly demonstrate that the potential for toxicity can be dramatically reduced if the circadian rhythm of the target cells is taken into account. From the brief description here, it follows that to ascertain the best time of day to administer a drug a considerable amount of work must be undertaken. The question is whether the patient response and well-being outweigh the time and cost to perform these types of studies.

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 101

To use *in vitro* stem cell assays to predict potential toxicity to the hematopoietic system, and any stem cell system for that matter, knowledge of the biology, physiology, regulation and response is required for an *in vitro* to *in vivo* concordance to be justified. This concordance plays an integral role in predicting toxicity since it allows for *in vitro* surrogate assays to be used in place of animals and therefore comply with the principle of the 3Rs (replacement, reduction and refinement) [55]. More importantly, to allows extrapolation to the human situation. Previous literature on stem cell and hematopoietic research demonstrates that *in vitro* assays show a high concordance with *in vivo* data. Using the HALO platform, Olaharski et al. demonstrated an *in vitro* to *in vivo* concordance of greater than 80% [56]. This high degree of concordance provides the basis to predict the response of the lympho-hematopoietic and other stem cell systems to potential toxic insults. This has been described previously [32], but it is worth reiterating some of these paradigms. First, virtually any compound can be toxic to stem cells. Second, toxicity to the most primitive, definitive stem cells will affect all cells of the system. Third, since stem cells only proliferate and proliferation occurs prior to differentiation, stem cell cytotoxicity will affect all downstream cell types. Fourth, if more than one cell lineage is affected by toxicity, the target is not the cells that constitute the lineages, but the stem cells producing the lineages. Finally, stem cells are more sensitive to toxicity than the progenitor

**7. Conclusions and future trends**

These studies were performed using normal peripheral blood mononuclear cells. Blood was obtained from the same donor every 4 hours over a 24 hours period. The MNCs were fractio‐ nated at each time point and cryopreserved into aliquots. Prior to cryopreservation, an aliquot of fresh cells was used to measure the proliferation ability of hematopoietic stem cells (CFC-GEMM 1), erythropoietic progenitor cells (burst-forming units – erythroid, BFU-E), GM-CFC and megakaryopoietic progenitor cells (megakaryopoietic colony-forming cells, Mk-CFC) at each time point using HALO. After collection of the cells, an aliquot from each time point was thawed and the circadian rhythms compared to fresh cells. A cosinor curve fitting analysis was performed to produce all the circadian rhythms shown in Fig. 11 [53]. The results for hematopoietic stem cells (Fig. 11A) and all progenitor cells (not shown) demonstrate that even after cryopreservation, the cell populations maintain their circadian rhythm. This was a prerequisite to use cryopreserved cells for the remainder of the study.

**Figure 11.** Using the Circadian Rhythm of Hematopoietic Stem Cells to Predict the Best Time of Day to Administer 5- Fluorouracil to Reduce Toxicity and Improve Efficacy of the Drug.

For each time point, cells were thawed and treated with 5-FU at six doses to measure the response of CFC-GEMM 1, BFU-E, GM-CFC and Mk-CFC. The slope of each negative sigmoi‐ dal dose response curve was then calculated from the 4-parameter logistic curve fit. The dose response slope values were then analyzed by cosinor analysis for each time point and for each cell population to obtain the circadian rhythms as a function of 5-FU treatment. The results are shown in Fig. 10B. Each of the hematopoietic cell populations exhibited its own circadian rhythm in response to 5-FU. When these circadian rhythms were correlated with either the continuous infusion of 5-FU that is normally used to treat patients and that of chronomodu‐ lated infusion of 5-FU as reported by Dogliotti and colleagues in 1998 [54], the results shown in Fig. 10C were obtained. For each of the administration types, the percent overall patient response rate, toxicity and tumor response are shown. These were overlaid onto the circadian rhythm for the CFC-GEMM 1 stem cell response to 5-FU and demonstrated that the lowest toxicity and highest overall and tumor response occurred when 5-FU was administered in a chronomodulated manner in the early morning hours rather than at any other time of the day. The nadir of the CFC-GEMM 1 circadian rhythm to 5-FU occurred at 14:00 hours in the afternoon. This was approximately the same time at which the highest toxicity to 5-FU was found. As expected, these results did not correlate nearly as well for the hematopoietic progenitor cells. In addition, the results clearly demonstrate that the potential for toxicity can be dramatically reduced if the circadian rhythm of the target cells is taken into account. From the brief description here, it follows that to ascertain the best time of day to administer a drug a considerable amount of work must be undertaken. The question is whether the patient response and well-being outweigh the time and cost to perform these types of studies.

#### **7. Conclusions and future trends**

of this section to describe or even summarize this field. The intention is to instead provide an example in which the circadian rhythm of cells, especially hematopoietic stem cells [50-52], can be used to predict the best time of day to administer an anti-cancer drug, which in this

These studies were performed using normal peripheral blood mononuclear cells. Blood was obtained from the same donor every 4 hours over a 24 hours period. The MNCs were fractio‐ nated at each time point and cryopreserved into aliquots. Prior to cryopreservation, an aliquot of fresh cells was used to measure the proliferation ability of hematopoietic stem cells (CFC-GEMM 1), erythropoietic progenitor cells (burst-forming units – erythroid, BFU-E), GM-CFC and megakaryopoietic progenitor cells (megakaryopoietic colony-forming cells, Mk-CFC) at each time point using HALO. After collection of the cells, an aliquot from each time point was thawed and the circadian rhythms compared to fresh cells. A cosinor curve fitting analysis was performed to produce all the circadian rhythms shown in Fig. 11 [53]. The results for hematopoietic stem cells (Fig. 11A) and all progenitor cells (not shown) demonstrate that even after cryopreservation, the cell populations maintain their circadian rhythm. This was a

**Figure 11.** Using the Circadian Rhythm of Hematopoietic Stem Cells to Predict the Best Time of Day to Administer 5-

B.

C.

prerequisite to use cryopreserved cells for the remainder of the study.

A.

Fluorouracil to Reduce Toxicity and Improve Efficacy of the Drug.

case, is 5-fluorouracil (5-FU) [38].

100 Stem Cell Biology in Normal Life and Diseases

To use *in vitro* stem cell assays to predict potential toxicity to the hematopoietic system, and any stem cell system for that matter, knowledge of the biology, physiology, regulation and response is required for an *in vitro* to *in vivo* concordance to be justified. This concordance plays an integral role in predicting toxicity since it allows for *in vitro* surrogate assays to be used in place of animals and therefore comply with the principle of the 3Rs (replacement, reduction and refinement) [55]. More importantly, to allows extrapolation to the human situation. Previous literature on stem cell and hematopoietic research demonstrates that *in vitro* assays show a high concordance with *in vivo* data. Using the HALO platform, Olaharski et al. demonstrated an *in vitro* to *in vivo* concordance of greater than 80% [56]. This high degree of concordance provides the basis to predict the response of the lympho-hematopoietic and other stem cell systems to potential toxic insults. This has been described previously [32], but it is worth reiterating some of these paradigms. First, virtually any compound can be toxic to stem cells. Second, toxicity to the most primitive, definitive stem cells will affect all cells of the system. Third, since stem cells only proliferate and proliferation occurs prior to differentiation, stem cell cytotoxicity will affect all downstream cell types. Fourth, if more than one cell lineage is affected by toxicity, the target is not the cells that constitute the lineages, but the stem cells producing the lineages. Finally, stem cells are more sensitive to toxicity than the progenitor cells. When considering using stem cells to predict potential toxicity, at least two considera‐ tions need to be taken into account. The first is the primitiveness of the stem cell population being measured, while the second is variation between human donors. The former will depend, among other things, upon the ability and sensitivity of the assay to detect specific stem cell populations and the latter will be dependent upon the state and demographics of the donors that can, in turn, affect the stem cells. Both are difficult to control, but can provide a more realistic view.

Clarification of the Nomenclature for MSC: the International Society for Cellular

Stem Cell Predictive Hemotoxicology http://dx.doi.org/10.5772/54430 103

[2] Potten, CS. Cell cycles in cell hierarchies. Int J Radiat Biol Relat Stud Phys Chem

[3] Lemischka IR. The haematopoietic stem cell and its clonal progeny: mechanism regu‐ lating the hierarchy of primitive haematopoietic cells. Cancer Surv. 1992; 15:3-18.

[4] Yahata, T, Muguruma, Y, Yumino, S, Sheng, Y, Uno, T, Matsuzawa, H, Ito, M, Kato, S, Hotta, T, Ando, K. Quiescent human hematopoietic stem cells in the bone marrow niches organize the hierarchical structure of hematopoiesis. Stem Cells. 2008; 26:

[5] Campbell, CJ, Lee, JB, Levadoux-Martin, M, Wynder, T, Xenocostas, A, Leber, B, Bha‐ tia, M. The human stem cell hierarchy is defined by a functional dependence on

[6] Levesque, JP, Winkler, IG. Hierarchy of immature hematopoietic cells related to

[7] Staal FJ, Baum C, Cowan C, Dzierzak E, Hacein-Bey-Abina S, Karlsson S, Lapidot T, Lemischka I, Mendez-Ferrer S, Mikkers H, Moore K, Moreno E, Mummery CL, Robin C, Suda T, Van Pel M, Vanden Brink G, Zwaginga JJ, Fibbe WE. Stem cell self-renew‐ al; lessons from bone marrow, gut and iPS towards clinical applications. Leukemia.

[8] Van Zant G, de Haan G, Rich IN. Alternatives to stem cell renewal from a develop‐

[9] Kola I, Landis J. Can the pharmaceutical industry reduce attrition rats? Nature Re‐

[10] Mahajan R, Gupta K. Food and drug administration's critical path iniative and inno‐ vations in drug development paradigm: Challenges, progress, and controversies. J

[11] National Cancer Institute, Division of Cancer Treatment and Diagnosis, Develop‐ mental Therapeutics Program. http://dctd.cancer.gov/ProgramPages/dtp/

[12] Yamori T. Panel of human cancer cell lines provides valuable database for drug dis‐ covery and bioinformatics. Cancer Chemother Pharmacol. 2003. 52 Suppl 1:S74-S79.

[13] Shanks N, Greek R, Greek L. Are animal models predictive for humans? Philos Ethics

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Therapy Position Statement. Cytotherapy 2005; 7:393-395.

Mcl-1 for self-renewal capacity. Blood, 2010; 116:1433-1442.

blood flow and niche. Curr. Opin. Hematol. 2011; 18: 220-225.

mental viewpoint. Exp Hematol. 1997; 25:187-192.

views: Drug Discovery. 2004; 3:711-715.

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tools\_drug\_discovery.htm.

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3228-3236.

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Based on these paradigms, it is worth briefly considering how the non-definitive stem cells systems (Fig. 1), ES and iPS cells, fit into predictive stem cell toxicity testing. At the present time, these cells are used to produce functionally, mature lineage-specific cells such as hepatocytes, cardiomyocytes and neurons. These and other cell types can be produced in larger numbers and presumably at a lower cost than their primary counterparts. Embryonic stem cells are used as an *in vitro* developmental toxicity model to predict teratogenicity. The use of ES and/or iPS cells for definitive stem cell system toxicity testing is certainly on the horizon. It should be remembered however, that even to produce functionally, mature hepatocytes, cardiomycytes and other cells, the ES and iPS cells must pass through the definitive stem cell compartment specific for the cells being produced. In other words, the ES and iPS cells should produce an organization analogous to that shown in Fig. 2. If this transpires, then the face of toxicity testing, and stem cell toxicity testing in particular, as well as many other applications, could significantly change the face of biological and toxicological research in the future.

#### **Acknowledgements**

The authors would like to thank Drs. Patricia Wood and William Hrushesky at the Dorn Research Institute of the William Jennings Bryan Dorn Veterans Affairs Medical Center in Columbia, South Carolina for sharing their insights and knowledge of circadian rhythms that led to the results shown in Section 5F.

#### **Author details**

Holli Harper and Ivan N. Rich

HemoGenix, Inc, U.S.A.

#### **References**

[1] Horwitz EM, Le Blanc K, Dominici M, Mueller I, Slaper-Cortenbach I, Marini FC, Deans RJ, Krause DS, Keating A; The International Society for Cellular Therapy: Clarification of the Nomenclature for MSC: the International Society for Cellular Therapy Position Statement. Cytotherapy 2005; 7:393-395.

[2] Potten, CS. Cell cycles in cell hierarchies. Int J Radiat Biol Relat Stud Phys Chem Med. 1986; 49:257-278.

cells. When considering using stem cells to predict potential toxicity, at least two considera‐ tions need to be taken into account. The first is the primitiveness of the stem cell population being measured, while the second is variation between human donors. The former will depend, among other things, upon the ability and sensitivity of the assay to detect specific stem cell populations and the latter will be dependent upon the state and demographics of the donors that can, in turn, affect the stem cells. Both are difficult to control, but can provide a

Based on these paradigms, it is worth briefly considering how the non-definitive stem cells systems (Fig. 1), ES and iPS cells, fit into predictive stem cell toxicity testing. At the present time, these cells are used to produce functionally, mature lineage-specific cells such as hepatocytes, cardiomyocytes and neurons. These and other cell types can be produced in larger numbers and presumably at a lower cost than their primary counterparts. Embryonic stem cells are used as an *in vitro* developmental toxicity model to predict teratogenicity. The use of ES and/or iPS cells for definitive stem cell system toxicity testing is certainly on the horizon. It should be remembered however, that even to produce functionally, mature hepatocytes, cardiomycytes and other cells, the ES and iPS cells must pass through the definitive stem cell compartment specific for the cells being produced. In other words, the ES and iPS cells should produce an organization analogous to that shown in Fig. 2. If this transpires, then the face of toxicity testing, and stem cell toxicity testing in particular, as well as many other applications, could significantly change the face of biological and toxicological research in the future.

The authors would like to thank Drs. Patricia Wood and William Hrushesky at the Dorn Research Institute of the William Jennings Bryan Dorn Veterans Affairs Medical Center in Columbia, South Carolina for sharing their insights and knowledge of circadian rhythms that

[1] Horwitz EM, Le Blanc K, Dominici M, Mueller I, Slaper-Cortenbach I, Marini FC, Deans RJ, Krause DS, Keating A; The International Society for Cellular Therapy:

more realistic view.

102 Stem Cell Biology in Normal Life and Diseases

**Acknowledgements**

**Author details**

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led to the results shown in Section 5F.

Holli Harper and Ivan N. Rich

HemoGenix, Inc, U.S.A.


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**Section 2**

**Stem Cells in Disease**

## **Stem Cells in Disease**

**Chapter 7**

**Stem Cell-Based (Auto)Grafting: From Innovative**

Bela Balint, Slobodan Obradovic, Milena Todorovic,

Mirjana Pavlovic and Biljana Mihaljevic

http://dx.doi.org/10.5772/54473

"therapeutic neovascularization" [1– 5].

unrelated SC donor is not obtainable [9–12].

**1. Introduction**

Additional information is available at the end of the chapter

**Research Toward Clinical Use in Regenerative Medicine**

In brief, adult stem cells (SCs) give rise to repopulation (engraftment) of recipient's bone marrow (BM) followed by complete and long-term reconstitution of hematopoiesis. In addition, totipotent SCs are also capable of colonizing different tissues (homing). Initial studies showed that "implantation" of autologous SCs into damaged and ischemic area induces their homing and subsequent "transdifferentiation" into the cell lineages of host organ, including collateral vessel formation. Angiogenesis growth factors – or genes en‐ coding these proteins – promote the development of collateral micro-angiogenesis or

Generally, SC transplant involves the administration of high-dose chemotherapy (condition‐ ing regimen) and (re)infusion of collected cells in order to obtain an abolition of disease, as well as to get hematopoietic reconstitution and clinical improvement of patient. SC transplant with reduced-intensity conditioning (RIC) can be offered to patients who are ineligible for high-dose conditioning because of their age or comorbidities [2]. Hematological diseases have so far been the most common indication of this treatment modality; it has been less often used for nonmalignant disorders. Nowadays BM and peripheral blood (PB) derived SC transplants are more common in adult allogeneic or autologous setting [2, 6– 8]. Umbilical cord blood (UCB) transplants have provided hopeful results in pediatric setting mainly when a matched

In clinical practice, SCs can be collected by: (a) multiple aspirations from BM; (b) harvesting PB after mobilization with chemotherapy and/or growth factors (rHuG-CSF), and (c) by specific processing from UCB. SCs collected from the stated sources can be clinically applied

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

© 2013 Balint 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 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,

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

## **Stem Cell-Based (Auto)Grafting: From Innovative Research Toward Clinical Use in Regenerative Medicine**

Bela Balint, Slobodan Obradovic, Milena Todorovic, Mirjana Pavlovic and Biljana Mihaljevic

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54473

#### **1. Introduction**

In brief, adult stem cells (SCs) give rise to repopulation (engraftment) of recipient's bone marrow (BM) followed by complete and long-term reconstitution of hematopoiesis. In addition, totipotent SCs are also capable of colonizing different tissues (homing). Initial studies showed that "implantation" of autologous SCs into damaged and ischemic area induces their homing and subsequent "transdifferentiation" into the cell lineages of host organ, including collateral vessel formation. Angiogenesis growth factors – or genes en‐ coding these proteins – promote the development of collateral micro-angiogenesis or "therapeutic neovascularization" [1– 5].

Generally, SC transplant involves the administration of high-dose chemotherapy (condition‐ ing regimen) and (re)infusion of collected cells in order to obtain an abolition of disease, as well as to get hematopoietic reconstitution and clinical improvement of patient. SC transplant with reduced-intensity conditioning (RIC) can be offered to patients who are ineligible for high-dose conditioning because of their age or comorbidities [2]. Hematological diseases have so far been the most common indication of this treatment modality; it has been less often used for nonmalignant disorders. Nowadays BM and peripheral blood (PB) derived SC transplants are more common in adult allogeneic or autologous setting [2, 6– 8]. Umbilical cord blood (UCB) transplants have provided hopeful results in pediatric setting mainly when a matched unrelated SC donor is not obtainable [9–12].

In clinical practice, SCs can be collected by: (a) multiple aspirations from BM; (b) harvesting PB after mobilization with chemotherapy and/or growth factors (rHuG-CSF), and (c) by specific processing from UCB. SCs collected from the stated sources can be clinically applied

(transplanted) immediately following harvesting (allogeneic setting) or after a long-term storage in frozen state – cryopreservation (autologous setting) [2].

only about one-third of patients have related HLA-matched donor. For that reason, some sources of allogeneic donors – including unrelated HLA-compatible individuals, have to be considered as the possible alternative. As a result, National Marrow Donor Program's registries of volunteer donors has been created and data accumulated by organizing a unique

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The late 1990s brought a new apprehension regarding the biology and related novel clinical potential of SCs. Researchers began to realize that manipulation of adult animal tissues could sometimes yield previously unsuspected cell types; for example, that some BM derived SCs could be turned into cardiomyocytes, hepatocytes or nerve and other somatic cells – phenom‐ enon known as the SC "plasticity". Finally, using SC plasticity, cell–based therapies for treatment of the ischemic heart diseases started through beginning of the new millennium and currently are in an expansion phase in the other fields of regenerative medicine [21–30].

database for potential donors (Bone Marrow Donors Worldwide – BMDW) [20].

**3. Adult stem cells: New concepts in phenotypes and functionality**

marking the cell, preferably at the single-cell level;

**4. The concept of hematopoietic stem cells**

more susceptible to crossing lineages;

newly acquired tissue;

specific organ function.

three different types of adult SCs.

To prove that SCs derived from BM and PB, including hematopoietic SCs, are indeed trans‐ differented or transformed into solid organ specific cells, several conditions must be met:

**•** The origin of the exogenous cell integrated into solid-organ time must be documented by

**•** Cell should be processed with a minimum of *ex vivo* manipulation which may make them

**•** The exogenous cells must be shown to have become an integral morphological part of the

**•** Transdifferented cells must have shown to acquire the function of the particular organ into which it has been integrated both, by expressing organ-specific proteins and by showing

Nevertheless, taking into consideration their common features described in the literature, it is very likely that various investigators have described overlapping populations of develop‐ mentally early SCs that are closely related. Our intention is to make a clear distinction between

Organ/tissue specific niche (like in BM, liver, etc) exists as a deposit (storage) of the adult SCs in a specific location [31]. These cells are circulating in a very low number in the PB. Accumu‐ lating evidence suggests that SCs may also actively migrate/circulate in the postnatal period of life. SC trafficking/circulation may be one of the crucial mechanisms that maintains the pool of SCs dispersed in SC-niches of the same tissue, that are spread throughout different ana‐

#### **2. Stem cell transplants – A short chronological consideration**

Independent SC-researchers recognized that all blood cells originate from one primitive BM cells (totipotent SC) located in marrow – space where the entire hematopoiesis takes place. Initial animal studies revealed that the BM was the organ most sensitive to the damaging effect of gamma irradiation [2]. Quickly, it became clear that (re)infusion of marrow cells or SCs could rescue lethally irradiated animals. Thomas with colleagues started on, and after that optimized (for this initial period) the BM transplant (BMT) program for humans and published in 1957 the first clinical results [13]. During the late 1950s have been also described the first syngeneic BMT in patients with leukemia [14]. Mathe and coworkers published the treatment of patients by allogeneic SC transplant after of accidental irradiation [15]. These transplants were performed before the discovery of major histocompatibility (MCH) system. In addition, it is not excluded that the observed recovery of some patients were a result of the recovery of autologous hematopoietic system [16]. The first successful BMT (allogeneic) was performed on a child with severe combined immunodeficiency disease (SCID). Cells were collected from his sister, and his immune system was restored following transplant [2]. However, in most cases transplants in humans have been unsuccessful (because of the graft rejection or expansion of the Graft versus Host Disease – GvHD).

The modern era of SC transplants – as a standard therapy – started with fundamental discovery and permanent progress in the knowledge of MHC, that is human leukocyte antigen (HLA) system [17]. These antigens give the body's immune system the ability to determine what belongs and what does not belong to the human body. Whenever the immune system does not recognize antigens expressed on a cell surface, it produces antibodies and other mediators to destroy the cells with non-recognizable antigens. In order for BMT to work, the recipient's immune system must not try to destroy the donated cells. This comprises that the HLA antigens on the donated SCs have to be identical or extremely similar to the antigens of the recipient's cells. Even with this careful HLA matching, transplant may still fail because recipient's immune system destroys transplanted cells (graft rejection) or donor's cells attempt to damage recipi‐ ent's target cells (GvHD) [2]. Thomas and coworkers almost immediately published positive results of the first allogeneic BMT in patients with hematologic malignancies – using cells from donors selected on the basis of the HLA system [18]. After all, Thomas ED was awarded the Nobel prize in medicine (1990) for his overall pioneering work on BMT topic. He was awarded the prize because of his numerous triumphant activities in both, experimental and clinical transplant setting.

In addition to marrow, PB has gained popularity as a SC source since their initial introduction in the early 1980s [19]. Over the past decades, the use of these transplants has expanded rapidly [6– 8]. Using umbilical cord blood (UCB) derived SCs, successful transplant occurred the first time in the treatment of Fanconi anemia and other disorders later than [9–12]. It is known that only about one-third of patients have related HLA-matched donor. For that reason, some sources of allogeneic donors – including unrelated HLA-compatible individuals, have to be considered as the possible alternative. As a result, National Marrow Donor Program's registries of volunteer donors has been created and data accumulated by organizing a unique database for potential donors (Bone Marrow Donors Worldwide – BMDW) [20].

(transplanted) immediately following harvesting (allogeneic setting) or after a long-term

Independent SC-researchers recognized that all blood cells originate from one primitive BM cells (totipotent SC) located in marrow – space where the entire hematopoiesis takes place. Initial animal studies revealed that the BM was the organ most sensitive to the damaging effect of gamma irradiation [2]. Quickly, it became clear that (re)infusion of marrow cells or SCs could rescue lethally irradiated animals. Thomas with colleagues started on, and after that optimized (for this initial period) the BM transplant (BMT) program for humans and published in 1957 the first clinical results [13]. During the late 1950s have been also described the first syngeneic BMT in patients with leukemia [14]. Mathe and coworkers published the treatment of patients by allogeneic SC transplant after of accidental irradiation [15]. These transplants were performed before the discovery of major histocompatibility (MCH) system. In addition, it is not excluded that the observed recovery of some patients were a result of the recovery of autologous hematopoietic system [16]. The first successful BMT (allogeneic) was performed on a child with severe combined immunodeficiency disease (SCID). Cells were collected from his sister, and his immune system was restored following transplant [2]. However, in most cases transplants in humans have been unsuccessful (because of the graft rejection or expansion

The modern era of SC transplants – as a standard therapy – started with fundamental discovery and permanent progress in the knowledge of MHC, that is human leukocyte antigen (HLA) system [17]. These antigens give the body's immune system the ability to determine what belongs and what does not belong to the human body. Whenever the immune system does not recognize antigens expressed on a cell surface, it produces antibodies and other mediators to destroy the cells with non-recognizable antigens. In order for BMT to work, the recipient's immune system must not try to destroy the donated cells. This comprises that the HLA antigens on the donated SCs have to be identical or extremely similar to the antigens of the recipient's cells. Even with this careful HLA matching, transplant may still fail because recipient's immune system destroys transplanted cells (graft rejection) or donor's cells attempt to damage recipi‐ ent's target cells (GvHD) [2]. Thomas and coworkers almost immediately published positive results of the first allogeneic BMT in patients with hematologic malignancies – using cells from donors selected on the basis of the HLA system [18]. After all, Thomas ED was awarded the Nobel prize in medicine (1990) for his overall pioneering work on BMT topic. He was awarded the prize because of his numerous triumphant activities in both, experimental and clinical

In addition to marrow, PB has gained popularity as a SC source since their initial introduction in the early 1980s [19]. Over the past decades, the use of these transplants has expanded rapidly [6– 8]. Using umbilical cord blood (UCB) derived SCs, successful transplant occurred the first time in the treatment of Fanconi anemia and other disorders later than [9–12]. It is known that

storage in frozen state – cryopreservation (autologous setting) [2].

112 Stem Cell Biology in Normal Life and Diseases

of the Graft versus Host Disease – GvHD).

transplant setting.

**2. Stem cell transplants – A short chronological consideration**

The late 1990s brought a new apprehension regarding the biology and related novel clinical potential of SCs. Researchers began to realize that manipulation of adult animal tissues could sometimes yield previously unsuspected cell types; for example, that some BM derived SCs could be turned into cardiomyocytes, hepatocytes or nerve and other somatic cells – phenom‐ enon known as the SC "plasticity". Finally, using SC plasticity, cell–based therapies for treatment of the ischemic heart diseases started through beginning of the new millennium and currently are in an expansion phase in the other fields of regenerative medicine [21–30].

#### **3. Adult stem cells: New concepts in phenotypes and functionality**

To prove that SCs derived from BM and PB, including hematopoietic SCs, are indeed trans‐ differented or transformed into solid organ specific cells, several conditions must be met:


Nevertheless, taking into consideration their common features described in the literature, it is very likely that various investigators have described overlapping populations of develop‐ mentally early SCs that are closely related. Our intention is to make a clear distinction between three different types of adult SCs.

#### **4. The concept of hematopoietic stem cells**

Organ/tissue specific niche (like in BM, liver, etc) exists as a deposit (storage) of the adult SCs in a specific location [31]. These cells are circulating in a very low number in the PB. Accumu‐ lating evidence suggests that SCs may also actively migrate/circulate in the postnatal period of life. SC trafficking/circulation may be one of the crucial mechanisms that maintains the pool of SCs dispersed in SC-niches of the same tissue, that are spread throughout different ana‐ tomical areas of the body. This phenomenon is very well described for hematopoietic SCs (HSCs), but other, already tissue committed SC or TCSC (for example, endothelial, skeletal muscle or neural SCs) are probably circulating as well. BM is the home of migrating SCs with not only HSCs within their niches, but also a small number of TCSC, which might be the reason why many authors think that HSC may transdifferentiate, although we do not have a direct proof for that. They might have plasticity, but not necessarily the "potential for transdifferen‐ tion" [32–39]. What is differentiated in the tissue of injection might be TCSC characteristic for that tissue. It has been shown that number of these cells is decreased with ageing (long living and short living mice and humans). It would be interesting to identify genes that are respon‐ sible for tissue distribution/expansion of TCSC. These genes could be involved in controlling the life length of the mammals.

SCs [38]. The newly identified cells – called "Very Small Embryonic-Like" (VSEL) SCs – have basically the same ultrastructure and protein markers as embryonic SCs [38]. Ratajczak and several other researchers from mentioned ASH meeting showed that VSEL SCs mobilize into the blood stream to help repair damaged tissue following a stroke [37]. In further research advance, Ratajczak's team also has grown VSEL cells in a lab and has stimulated them to change into nerve, heart and pancreas cells [40–42]. The differences in ultrastructure between HSC

Stem Cell-Based (Auto)Grafting: From Innovative Research Toward Clinical Use in Regenerative Medicine

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115

Along with this new concept, there is a premise that in regenerative therapy done before, with HSCs (considered to have plasticity and multipotency) the VSELs were "contaminants" that actually contributed to positive regenerative clinical outcome, since they have those capabili‐ ties. This is an interesting concept which should be seriously considered in humans. However, since VSELs have been found in human UCB and BM first [37], and then used and applied with the patients [43–44] they seem to be of a critical importance for consideration of SC transplant choice based upon the phenotype and number of SCs aimed to be transplanted

**6. The concept of mesenchymal stem cell – with dental pulp cells as an**

Many human tissues are the source of SCs responsible for tissue development and regenera‐ tion. Beside marrow (Bone Marrow Stromal Stem Cells – BMSCs), currently it is considered that dental pulp is practically the most approachable and the most important source of adult

and VSELs are shown in Figure 1.

(For picture thanks to kind permission of M. Ratajczak)

within a given clinical scenario.

mesenchymal SCs [45–47] (Figure 2).

**example**

**Figure 1.** Ultra-structural differences between mouse VSEL and HSC

Therefore, BM derived SCs are a heterogeneous population of cells with HSC and TCSC, the morphological and functional characteristics of which are different from HSC. Their number among mononuclear cells (MNCs) is very low (approximately one cell per 1 000 – 10 000 marrow MNCs) within young mammals and might play a role in healing of small injuries [31, 32].

In severe injuries (like hart infarct or stroke) they have no possibility to reveal their full therapeutic potential. The allocation of these cells to the damaged areas depends on homing signals that maybe inefficient in the presence of some other cytokines or proteolytic enzymes that are released from damaged tissue associated leukocytes and macrophages. We can envision, for example that metalloproteinases released from inflammatory cells may degrade SDF-1 locally, and thus perturb homing of CXCR4+ TCSC. There is possibility that these cells while "trapped" in BM are still in: "latent stage" – not fully functional and need the appropriate activation signals by up till now unknown factors [32–37].

These cells also, at least in some cases could be attracted to the inflammatory areas, and if not properly incorporated into the damaged tissue they may transform and initiate tu‐ mor growth. Briefly, between the pools of TCSCs, there are probably those already com‐ mitted to transdifferentiate into neural cells, or cells of tissues and organs other ten neural, but we still do not have the control over their tracking, homing and finally re‐ generative capacity in the given tissue, which is a fundamental prerequisite for success‐ ful regenerative therapy.

#### **5. The concept of "very small embryonic-like" stem cells**

In a discovery that has the potential to change the face of SC research, a University of Louisville scientist has identified cells in the adult body that seem to behave like embryonic SCs [38, 40– 44]. The cells, drawn from adult BM, look like embryonic SCs and appear to mimic their ability to multiply and develop into other kinds of cells. The finding, presented the first time at the 47th Annual Meeting of the American Society of Hematology (ASH), was announced at the society's news conference. A study by Ratajczak's team published in the journal "Leukemia" was the first to identify a type of SC in adult marrow that acts differently than other BM derived SCs [38]. The newly identified cells – called "Very Small Embryonic-Like" (VSEL) SCs – have basically the same ultrastructure and protein markers as embryonic SCs [38]. Ratajczak and several other researchers from mentioned ASH meeting showed that VSEL SCs mobilize into the blood stream to help repair damaged tissue following a stroke [37]. In further research advance, Ratajczak's team also has grown VSEL cells in a lab and has stimulated them to change into nerve, heart and pancreas cells [40–42]. The differences in ultrastructure between HSC and VSELs are shown in Figure 1.

(For picture thanks to kind permission of M. Ratajczak)

tomical areas of the body. This phenomenon is very well described for hematopoietic SCs (HSCs), but other, already tissue committed SC or TCSC (for example, endothelial, skeletal muscle or neural SCs) are probably circulating as well. BM is the home of migrating SCs with not only HSCs within their niches, but also a small number of TCSC, which might be the reason why many authors think that HSC may transdifferentiate, although we do not have a direct proof for that. They might have plasticity, but not necessarily the "potential for transdifferen‐ tion" [32–39]. What is differentiated in the tissue of injection might be TCSC characteristic for that tissue. It has been shown that number of these cells is decreased with ageing (long living and short living mice and humans). It would be interesting to identify genes that are respon‐ sible for tissue distribution/expansion of TCSC. These genes could be involved in controlling

Therefore, BM derived SCs are a heterogeneous population of cells with HSC and TCSC, the morphological and functional characteristics of which are different from HSC. Their number among mononuclear cells (MNCs) is very low (approximately one cell per 1 000 – 10 000 marrow MNCs) within young mammals and might play a role in healing of

In severe injuries (like hart infarct or stroke) they have no possibility to reveal their full therapeutic potential. The allocation of these cells to the damaged areas depends on homing signals that maybe inefficient in the presence of some other cytokines or proteolytic enzymes that are released from damaged tissue associated leukocytes and macrophages. We can envision, for example that metalloproteinases released from inflammatory cells may degrade SDF-1 locally, and thus perturb homing of CXCR4+ TCSC. There is possibility that these cells while "trapped" in BM are still in: "latent stage" – not fully functional and need the appropriate

These cells also, at least in some cases could be attracted to the inflammatory areas, and if not properly incorporated into the damaged tissue they may transform and initiate tu‐ mor growth. Briefly, between the pools of TCSCs, there are probably those already com‐ mitted to transdifferentiate into neural cells, or cells of tissues and organs other ten neural, but we still do not have the control over their tracking, homing and finally re‐ generative capacity in the given tissue, which is a fundamental prerequisite for success‐

In a discovery that has the potential to change the face of SC research, a University of Louisville scientist has identified cells in the adult body that seem to behave like embryonic SCs [38, 40– 44]. The cells, drawn from adult BM, look like embryonic SCs and appear to mimic their ability to multiply and develop into other kinds of cells. The finding, presented the first time at the 47th Annual Meeting of the American Society of Hematology (ASH), was announced at the society's news conference. A study by Ratajczak's team published in the journal "Leukemia" was the first to identify a type of SC in adult marrow that acts differently than other BM derived

activation signals by up till now unknown factors [32–37].

**5. The concept of "very small embryonic-like" stem cells**

the life length of the mammals.

114 Stem Cell Biology in Normal Life and Diseases

small injuries [31, 32].

ful regenerative therapy.

**Figure 1.** Ultra-structural differences between mouse VSEL and HSC

Along with this new concept, there is a premise that in regenerative therapy done before, with HSCs (considered to have plasticity and multipotency) the VSELs were "contaminants" that actually contributed to positive regenerative clinical outcome, since they have those capabili‐ ties. This is an interesting concept which should be seriously considered in humans. However, since VSELs have been found in human UCB and BM first [37], and then used and applied with the patients [43–44] they seem to be of a critical importance for consideration of SC transplant choice based upon the phenotype and number of SCs aimed to be transplanted within a given clinical scenario.

### **6. The concept of mesenchymal stem cell – with dental pulp cells as an example**

Many human tissues are the source of SCs responsible for tissue development and regenera‐ tion. Beside marrow (Bone Marrow Stromal Stem Cells – BMSCs), currently it is considered that dental pulp is practically the most approachable and the most important source of adult mesenchymal SCs [45–47] (Figure 2).

**7. Stem cell harvesting and** *ex vivo* **manipulation**

(autologous setting).

**8. Bone marrow derived stem cells**

**Figure 3.** Stem cell collection from bone marrow

diluted in saline (5 000 IU/500 mL) [2, 48–50].

For therapeutic use, SCs can be collected by: a) multiple aspirations from BM; b) harvesting from PB after mobilization (chemotherapy and/or growth factors – rHuG-CSF); and c) collection from UCB. Collected cells can be clinically applied (transplanted) immediately following harvesting (allogeneic setting) or after storage in frozen state or cryopreservation

Stem Cell-Based (Auto)Grafting: From Innovative Research Toward Clinical Use in Regenerative Medicine

Historically, BM was the first SC source for transplants. Cells were collected by multiple aspira‐ tions from the iliac crests, under sterile conditions, while the donor was generally anesthetized. The target volume of collected BM aspirate is 10 – 15 mL per kg of donor body mass (kgbm). In

After collection, BM aspirate should be filtered in order to remove bone and lipid particles and cell aggregates. Anticoagulation is created using citrate solution and by using of the heparin

/kgbm –

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117

order to provide required number of total nucleated cells (TNCs) – that is TNC ≥ 3x10<sup>8</sup>

around 200 aspirations are required (single aspirate volume = 2 – 5 mL) (Figure 3).

#### (With courtesy of V. Todorovic)

**Figure 2.** Mesenchymal stem cells

Within the last decade, several populations of SCs from dental pulp were isolated and characterized: a) Dental Pulp Stem Cells – DPSC; b) cells from Human Exfoliated Decidual teeth – SHED; and c) Immature Dental Pulp Cells – IDPC [46, 47]. These cells are of the ectomesenchymal origin, located in perivascular niche, highly proliferative, clonogenic, multipotent and similar to BMSCs. Within *in vitro* conditions, they can differentiate with certain intercellular differences toward odontoblasts, hondrocytes, osteoblasts, adipocytes, neuron/glial cells, smooth and skeletal muscle cells. Within *in vivo* conditions (after implan‐ tation) they show different potential for dentine formation, as well as osteogenesis; after transplant in mouse with compromised immune system, they make good grafts in different tissues, and are capable of migrating into the brain, where they survive a certain time while reaching neurogenic phenotype. DPSCs have immunomodulatory effect, as they can be involved into immune response during infection of dental pulp by NF-kB activation and by inhibiting T-cell proliferation, suggesting their immunosuppressive effect [47].

The future research should give us the complex data on the molecular and functional charac‐ teristics of dental pulp SCs, as well as differences between various populations of these cells. Such research would fundamentally contribute to the better knowledge on the dental pulp SCs, which is necessary due to their potential clinical application in *in vivo* cell transplant, tissue engineering, and gene therapy (*in vivo* or *ex vivo*). Actually, by the isolation of IDPCs, which are the most primitive, but also the most plastic (similar to embryonic SCs) they are opening the new perspectives in a potential therapeutic application of these cells not only in regener‐ ation of dentine, otherwise also the regeneration of periodontal and "bone-junction" tissue of craniofacial region, as well as in the therapy of neurotrauma, myocardial infarction and other tissue damages [46, 47].

### **7. Stem cell harvesting and** *ex vivo* **manipulation**

For therapeutic use, SCs can be collected by: a) multiple aspirations from BM; b) harvesting from PB after mobilization (chemotherapy and/or growth factors – rHuG-CSF); and c) collection from UCB. Collected cells can be clinically applied (transplanted) immediately following harvesting (allogeneic setting) or after storage in frozen state or cryopreservation (autologous setting).

#### **8. Bone marrow derived stem cells**

(With courtesy of V. Todorovic)

tissue damages [46, 47].

**Figure 2.** Mesenchymal stem cells

116 Stem Cell Biology in Normal Life and Diseases

Within the last decade, several populations of SCs from dental pulp were isolated and characterized: a) Dental Pulp Stem Cells – DPSC; b) cells from Human Exfoliated Decidual teeth – SHED; and c) Immature Dental Pulp Cells – IDPC [46, 47]. These cells are of the ectomesenchymal origin, located in perivascular niche, highly proliferative, clonogenic, multipotent and similar to BMSCs. Within *in vitro* conditions, they can differentiate with certain intercellular differences toward odontoblasts, hondrocytes, osteoblasts, adipocytes, neuron/glial cells, smooth and skeletal muscle cells. Within *in vivo* conditions (after implan‐ tation) they show different potential for dentine formation, as well as osteogenesis; after transplant in mouse with compromised immune system, they make good grafts in different tissues, and are capable of migrating into the brain, where they survive a certain time while reaching neurogenic phenotype. DPSCs have immunomodulatory effect, as they can be involved into immune response during infection of dental pulp by NF-kB activation and by

inhibiting T-cell proliferation, suggesting their immunosuppressive effect [47].

The future research should give us the complex data on the molecular and functional charac‐ teristics of dental pulp SCs, as well as differences between various populations of these cells. Such research would fundamentally contribute to the better knowledge on the dental pulp SCs, which is necessary due to their potential clinical application in *in vivo* cell transplant, tissue engineering, and gene therapy (*in vivo* or *ex vivo*). Actually, by the isolation of IDPCs, which are the most primitive, but also the most plastic (similar to embryonic SCs) they are opening the new perspectives in a potential therapeutic application of these cells not only in regener‐ ation of dentine, otherwise also the regeneration of periodontal and "bone-junction" tissue of craniofacial region, as well as in the therapy of neurotrauma, myocardial infarction and other

Historically, BM was the first SC source for transplants. Cells were collected by multiple aspira‐ tions from the iliac crests, under sterile conditions, while the donor was generally anesthetized. The target volume of collected BM aspirate is 10 – 15 mL per kg of donor body mass (kgbm). In order to provide required number of total nucleated cells (TNCs) – that is TNC ≥ 3x10<sup>8</sup> /kgbm – around 200 aspirations are required (single aspirate volume = 2 – 5 mL) (Figure 3).

**Figure 3.** Stem cell collection from bone marrow

After collection, BM aspirate should be filtered in order to remove bone and lipid particles and cell aggregates. Anticoagulation is created using citrate solution and by using of the heparin diluted in saline (5 000 IU/500 mL) [2, 48–50].

BM aspirate volume – precisely red blood cell count or plasma quantity – reduction is required (processing), especially for ABO incompatible transplants. Depletion of T-cells in cell suspen‐ sion is achieved using *ex vivo* purging (by immunomagnetic technique). These SC purification procedures (processing and purging) enable reduction of red cell for around 80 – 90% and depletion of T-cells with 3 – 4 Log10 [50].

after 3rd day, and peaks on the 5th day of rHuG-CSF administration. In autologous setting, patients are given higher rHuG-CSF dose (12-16 µg/kgbm or more daily) combined with

Stem Cell-Based (Auto)Grafting: From Innovative Research Toward Clinical Use in Regenerative Medicine

In allogeneic setting, the first SC collection is performed typically on the 5th day (Figure 4.). The optimized timing of an autologous SC harvesting is more complex and controversial. The leukocyte count commonly does not correlate with the circulating CD34+ number. The count

to expect successful transplant. Recent data support a benefit associated with greater CD34+

Patient's request for SCs have only in ≤ 30% (related) and ≤ 85% (unrelated) possibility of finding an adult allogeneic donor [20]. Because of the limited availability of donors, attention has turned to alternative sources of HLA-typed SCs. In recent years, UCB has emerged as a

SCs obtained from the UCB immediately after birth are usually referred to as neonatal SCs. These cells are less mature than those in BM. The advantage of the use of UCB is painless and non–invasive collection. UCB has advantage that – despite its high content of immune cells – it does not produce severe GvHD. Precisely, the "naive" nature of UCB lymphocytes permits the use of partly HLA-mismatched grafts without higher risk for severe GvHD relative to BM transplant from a full matched unrelated donor. Thus, UCB grafts do not need to be as

On the contrary, the major disadvantage of this cell source is the limited number of SCs. UCB

 per unit. Thus, UCB is an accepted cell source for pediatric patients and for whom a matched unrelated BM or PB cell donor is unavailable. However, a higher risk of graft failure was noticed in children weighing ≥ 45 kg. Since the number of SCs in UCB is limited and the collection can occur only in a single occasion – its use in adult patients can be more problematic. Finally, since SCs in the UCB are "more primitive", the engraftment process takes longer with UCB, leaving the patient vulnerable to posttransplant infections or bleeding. However, "more primitive" SCs in UCB have the potential to give rise to non-hematopoietic cells (myocardial,

/kgbm) in autologous setting [20]. Finally, results obtained in our SC transplant center

/kgbm) compared to the minimum required cell quantity for engraftment (≥ 1 x

count ≅ 10/µL, expected SC yield ≅ 1x106

≥ 20-40/µL of PB the possibility of the CD34+

The target CD34+ cells should be 330x106 per unit or ≥ 2-4x10<sup>6</sup>

**10. Umbilical cord blood derived stem cells**

feasible alternative source of transplantable CD34+

confirmed that large-volume apheresis is efficient (CD34+ ≥ 5x106

count was around 40-60/µL after mobilizing regiment [8].

patients who lack HLA-matched donors of BM or PB derived SCs [9–1 2].

"rigorously" matched to a recipient as BM or PB grafts [10–12].

neural and endothelial cells, etc) by transdifferentiation [2, 9–12].

volume is typically 80 – 200 mL, with a TNC count ≈1x10<sup>9</sup>

one standard apheresis, and 60% after one large-volume SC harvesting [6–8, 49].

cells evidently correlate with the superior CD34+ yield in harvest.

per kgbm. It is also presented

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119

≥ 2.5x10<sup>6</sup> per kgbm is 15% using

/kgbm of the recipient in order

cells for allogeneic transplant, mainly in

and approximately CD34+ count

/kgbm) if the circulating

chemotherapy [8, 48–51].

of circulating CD34+

that for a CD34+

yield (≥ 5x10<sup>6</sup>

106

CD34+

≈3x10<sup>6</sup>

Generally, at PB CD34+

### **9. Peripheral blood derived stem cells**

The PB derived SC transplants are characterized by: a) less invasive cell collection; b) lack of the risks of general anesthesia; c) rapid hematopoietic reconstruction; d) low harvest volume (200 – 300 mL), and e) inferior transplant-related morbidity. Thus, the number of patients treated by PB derived SCs is ever increasing, especially in autologous transplant setting [6–8, 48–51].

In steady state hematopoiesis SCs, that is CD34+ cells are in very low proportion (0.01% to 0.1% compared to MNCs) in PB, but they can be mobilized from BM. Allogeneic donors are given rHuG-CSF 5 (10 µg/kgbm per day). The count of CD34+ cells in the circulation begins to rise after 3rd day, and peaks on the 5th day of rHuG-CSF administration. In autologous setting, patients are given higher rHuG-CSF dose (12-16 µg/kgbm or more daily) combined with chemotherapy [8, 48–51].

In allogeneic setting, the first SC collection is performed typically on the 5th day (Figure 4.). The optimized timing of an autologous SC harvesting is more complex and controversial. The leukocyte count commonly does not correlate with the circulating CD34+ number. The count of circulating CD34+ cells evidently correlate with the superior CD34+ yield in harvest. Generally, at PB CD34+ count ≅ 10/µL, expected SC yield ≅ 1x106 per kgbm. It is also presented that for a CD34+ ≥ 20-40/µL of PB the possibility of the CD34+ ≥ 2.5x10<sup>6</sup> per kgbm is 15% using one standard apheresis, and 60% after one large-volume SC harvesting [6–8, 49].

The target CD34+ cells should be 330x106 per unit or ≥ 2-4x10<sup>6</sup> /kgbm of the recipient in order to expect successful transplant. Recent data support a benefit associated with greater CD34+ yield (≥ 5x10<sup>6</sup> /kgbm) compared to the minimum required cell quantity for engraftment (≥ 1 x 106 /kgbm) in autologous setting [20]. Finally, results obtained in our SC transplant center confirmed that large-volume apheresis is efficient (CD34+ ≥ 5x106 /kgbm) if the circulating CD34+ count was around 40-60/µL after mobilizing regiment [8].

### **10. Umbilical cord blood derived stem cells**

BM aspirate volume – precisely red blood cell count or plasma quantity – reduction is required (processing), especially for ABO incompatible transplants. Depletion of T-cells in cell suspen‐ sion is achieved using *ex vivo* purging (by immunomagnetic technique). These SC purification procedures (processing and purging) enable reduction of red cell for around 80 – 90% and

The PB derived SC transplants are characterized by: a) less invasive cell collection; b) lack of the risks of general anesthesia; c) rapid hematopoietic reconstruction; d) low harvest volume (200 – 300 mL), and e) inferior transplant-related morbidity. Thus, the number of patients treated by PB derived SCs is ever increasing, especially in autologous transplant setting [6–8, 48–51].

compared to MNCs) in PB, but they can be mobilized from BM. Allogeneic donors are given rHuG-CSF 5 (10 µg/kgbm per day). The count of CD34+ cells in the circulation begins to rise

cells are in very low proportion (0.01% to 0.1%

depletion of T-cells with 3 – 4 Log10 [50].

118 Stem Cell Biology in Normal Life and Diseases

**Figure 4.** Stem cell harvesting from peripheral blood

In steady state hematopoiesis SCs, that is CD34+

**9. Peripheral blood derived stem cells**

Patient's request for SCs have only in ≤ 30% (related) and ≤ 85% (unrelated) possibility of finding an adult allogeneic donor [20]. Because of the limited availability of donors, attention has turned to alternative sources of HLA-typed SCs. In recent years, UCB has emerged as a feasible alternative source of transplantable CD34+ cells for allogeneic transplant, mainly in patients who lack HLA-matched donors of BM or PB derived SCs [9–1 2].

SCs obtained from the UCB immediately after birth are usually referred to as neonatal SCs. These cells are less mature than those in BM. The advantage of the use of UCB is painless and non–invasive collection. UCB has advantage that – despite its high content of immune cells – it does not produce severe GvHD. Precisely, the "naive" nature of UCB lymphocytes permits the use of partly HLA-mismatched grafts without higher risk for severe GvHD relative to BM transplant from a full matched unrelated donor. Thus, UCB grafts do not need to be as "rigorously" matched to a recipient as BM or PB grafts [10–12].

On the contrary, the major disadvantage of this cell source is the limited number of SCs. UCB volume is typically 80 – 200 mL, with a TNC count ≈1x10<sup>9</sup> and approximately CD34+ count ≈3x10<sup>6</sup> per unit. Thus, UCB is an accepted cell source for pediatric patients and for whom a matched unrelated BM or PB cell donor is unavailable. However, a higher risk of graft failure was noticed in children weighing ≥ 45 kg. Since the number of SCs in UCB is limited and the collection can occur only in a single occasion – its use in adult patients can be more problematic. Finally, since SCs in the UCB are "more primitive", the engraftment process takes longer with UCB, leaving the patient vulnerable to posttransplant infections or bleeding. However, "more primitive" SCs in UCB have the potential to give rise to non-hematopoietic cells (myocardial, neural and endothelial cells, etc) by transdifferentiation [2, 9–12].

#### **11. Stem cell cryopreservation practice**

Efficient transplant program requires both, high-quality harvesting and cryopreservation techniques for obtaining adequate yield and recovery of the SCs. In practice, SC cryopreser‐ vation consists of the following steps: a) aspirate processing; b) equilibration (cell exposure to cryoprotective agent) and freezing; c) storage at temperature –80±5°C (mechanical freezer), at –140±5°C (mechanical freezer or steam of nitrogen) or at -196°C (liquid nitrogen); and d) cell thawing in a water bath at 37±3°C. There are several cryopreservation protocols, using primarily DMSO in autologous plasma. The optimal cooling velocity in controlled-rate cryopreservation setting is –1°C. The transition from liquid to solid phase is also critical period – because of released fusion heat – since a significant reduction in cell recovery and viability has been observed when this period is prolonged. The compensation of the released fusion heat is required, using elevated cooling rate (–2 °C per minute) during transition period. Finally, there is data showing that uncontrolled-rate freezing is also useful in SC cryopreser‐ vation [48–52].

therapy of hematological malignancies has long been known, but only recently have systems been developed to separate the GvL effect from GvHD. Using donor-specific lymphocytes, the best results were obtained in chronic myelogenous leukemia. Moderate successes have been reported in relapsing acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma

Stem Cell-Based (Auto)Grafting: From Innovative Research Toward Clinical Use in Regenerative Medicine

http://dx.doi.org/10.5772/54473

121

and some responses were obtained for acute lymphoid leukemia [2, 8, 50].

**BM malignant or dysplastic disorders**

Hodgkin's and non-Hodgkin's lymphoma

Myelodysplastic/myeloproliferative disorders

**Benign immune mediated disorders** Severe combined immunodeficiency disease

Marrow failure syndromes Severe aplastic anemia Autoimmune disorders

Congenital Immune deficiencies

**Table 1.** Current indications and relative suggestions for SC transplant

Although SC transplant-related mortality and morbidity have reduced, SC transplants continue to pose numerous potential complications. The most frequent complications are even now engrafting failure, virus or opportunistic infections and acute or chronic GvHD. Less toxic transplants, in the form of non-myeloablative conditioning regimens, are being actively investigated, with the promise of expanding indications for allogeneic transplant. In addition, SC transplant with RIC can be offered to patients who are disqualified for high-dose condi‐ tioning because of their age or comorbidities. A careful proactive assessment to identify, treat,

and, hopefully, prevent adverse events is essential to a successful transplant [2].

Leukemias

Thalassemia

**Solid tumors** Breast cancer Ovarian cancer Testicular cancer Wilm's tumor Neuroblastoma Rhabdomyosarcoma Ewing sarcoma

Multiple myeloma

Cryopreserved SCs are thawed rapidly in a water bath at 38±2°C at the patient's bedside and infused immediately through a central vein catheter. Generally, patients tolerate the infusion of unprocessed SCs well, with no side effects. The grade of the potential reinfusion−related toxicity is associated with total DMSO quantity in the thawed cell concentrate. Alternatively, cryoprotectant can be removed by washing after thawing, but this procedure results in substantial cell loss.

#### **12. Conventional stem cell transplants – A synopsis of the clinical practice**

Generally, SC transplants include the use of high-dose chemotherapy in order to obtain disease eradication and (re)infusion (allogeneic transplant or autologous SC support) of cells collected to get hematopoietic and clinical renewal. SC transplant with reduced-intensity conditioning (RIC) can be offered to patients who are disqualified for high-dose conditioning because of their age or comorbidities. Malignant hematological diseases and some immune-mediated dis‐ orders (Table 1) are the most common indication of this therapeutic approach using SCs [2, 50].

The efficacy of transplants depends on the type of disease, its stage and sensitivity for chemotherapy, patients' age and general condition, as well as degree of the HLA-matching. In general, survival rates are around 30 – 60% for otherwise fatal diseases. Details of the SC clinical use – that is optimized treatment timing and efficacy, peritransplant complications, etc. – of the transplants in presented hematological disorders will not discussed in this paper.

Briefly, autoimmune diseases, which do not respond to standard immunosuppression, could benefit from immunoablative therapy. The idea of treatment of immune-mediated disorders (e.g. multiple sclerosis) by autologous SC transplant is based on the hypothesis that immu‐ noablative treatment can destroy the patients "anti-self-lymphocytes" (i.e. an "immuneresetting" process). The beneficial immunomodulating effect of allogeneic SC transplant in therapy of hematological malignancies has long been known, but only recently have systems been developed to separate the GvL effect from GvHD. Using donor-specific lymphocytes, the best results were obtained in chronic myelogenous leukemia. Moderate successes have been reported in relapsing acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma and some responses were obtained for acute lymphoid leukemia [2, 8, 50].

**11. Stem cell cryopreservation practice**

120 Stem Cell Biology in Normal Life and Diseases

vation [48–52].

substantial cell loss.

Efficient transplant program requires both, high-quality harvesting and cryopreservation techniques for obtaining adequate yield and recovery of the SCs. In practice, SC cryopreser‐ vation consists of the following steps: a) aspirate processing; b) equilibration (cell exposure to cryoprotective agent) and freezing; c) storage at temperature –80±5°C (mechanical freezer), at –140±5°C (mechanical freezer or steam of nitrogen) or at -196°C (liquid nitrogen); and d) cell thawing in a water bath at 37±3°C. There are several cryopreservation protocols, using primarily DMSO in autologous plasma. The optimal cooling velocity in controlled-rate cryopreservation setting is –1°C. The transition from liquid to solid phase is also critical period – because of released fusion heat – since a significant reduction in cell recovery and viability has been observed when this period is prolonged. The compensation of the released fusion heat is required, using elevated cooling rate (–2 °C per minute) during transition period. Finally, there is data showing that uncontrolled-rate freezing is also useful in SC cryopreser‐

Cryopreserved SCs are thawed rapidly in a water bath at 38±2°C at the patient's bedside and infused immediately through a central vein catheter. Generally, patients tolerate the infusion of unprocessed SCs well, with no side effects. The grade of the potential reinfusion−related toxicity is associated with total DMSO quantity in the thawed cell concentrate. Alternatively, cryoprotectant can be removed by washing after thawing, but this procedure results in

**12. Conventional stem cell transplants – A synopsis of the clinical practice**

Generally, SC transplants include the use of high-dose chemotherapy in order to obtain disease eradication and (re)infusion (allogeneic transplant or autologous SC support) of cells collected to get hematopoietic and clinical renewal. SC transplant with reduced-intensity conditioning (RIC) can be offered to patients who are disqualified for high-dose conditioning because of their age or comorbidities. Malignant hematological diseases and some immune-mediated dis‐ orders (Table 1) are the most common indication of this therapeutic approach using SCs [2, 50].

The efficacy of transplants depends on the type of disease, its stage and sensitivity for chemotherapy, patients' age and general condition, as well as degree of the HLA-matching. In general, survival rates are around 30 – 60% for otherwise fatal diseases. Details of the SC clinical use – that is optimized treatment timing and efficacy, peritransplant complications, etc. – of

Briefly, autoimmune diseases, which do not respond to standard immunosuppression, could benefit from immunoablative therapy. The idea of treatment of immune-mediated disorders (e.g. multiple sclerosis) by autologous SC transplant is based on the hypothesis that immu‐ noablative treatment can destroy the patients "anti-self-lymphocytes" (i.e. an "immuneresetting" process). The beneficial immunomodulating effect of allogeneic SC transplant in

the transplants in presented hematological disorders will not discussed in this paper.


**Table 1.** Current indications and relative suggestions for SC transplant

Although SC transplant-related mortality and morbidity have reduced, SC transplants continue to pose numerous potential complications. The most frequent complications are even now engrafting failure, virus or opportunistic infections and acute or chronic GvHD. Less toxic transplants, in the form of non-myeloablative conditioning regimens, are being actively investigated, with the promise of expanding indications for allogeneic transplant. In addition, SC transplant with RIC can be offered to patients who are disqualified for high-dose condi‐ tioning because of their age or comorbidities. A careful proactive assessment to identify, treat, and, hopefully, prevent adverse events is essential to a successful transplant [2].

We have previously analyzed our results of PB vs. BM derived SC transplants based on the hematopoietic reconstitution. Transplants were used for the treatment of patients with severe aplastic anemia, acute lymphoblastic leukemia, acute non–lymphoblastic leukemia, chronic myeloid leukemia, multiple myeloma, Hodgkin's and non–Hodgkin's lymphoma, breast and ovarian cancer, extragonadal non–seminal germ cell tumor, and severe multiple sclerosis. The CD34+ yields for allogeneic and autologous transplants were eminent: 16.7±9.8 × 106 /kgbm and 11.8±6.1 × 106 /kgbm, respectively [8, 26, 27]. A typical histogram with high–level of the CD34+ ratio in obtained PB harvest is presented in Figure 5.

Several data related to our immature vs. mature CD34–

CD34 PE / CD90 FITC [%] 1,72±1,47 1,25±0,82 2,72±2,06 CD34 PE / CD38 FITC [%] 2,02±1,18 2,02±1,18 2,3±1,16 CD34 PE / HLA–DR FITC [%] 2,01±0,92 2,0±0,92 2,0±0,88 CD34 PE / CD33 FITC [%] 1,90±1,23 1,9±1,23 2,75±1,12

Stem Cell-Based (Auto)Grafting: From Innovative Research Toward Clinical Use in Regenerative Medicine

PB–SCs I = stem cell collected from peripheral blood after mobilization with chemotherapy and rHuG–CSF; PB–SCs II = stem cell collected from peripheral blood after mobilization with rHuG–CSF alone; BM–SCs = stem cell collected from

Finally, we found that the use of large volume vs. conventional (repetitive) apheresis resulted

of higher ratio of immature vs. mature CD34–subsets correlated with BM repopulation ability (complete and long–term engraftment) and rapid hematopoietic reconstitution, as well as

SCs are considered optimal targets for gene transduction due to their ability to renew them‐ selves and differentiate into progeny cells and generate a self-perpetuating cell population that contains the transduced gene for the lifetime of the patient. Specific diseases that could be candidates for SC gene therapy include thalassemia, sickle cell anemia, Fanconi anemia, severe combined immune deficiency secondary to adenosine deaminase deficiency or purine nucleoside phosphorylase deficiency, chronic granulomatous disease, leukocyte adhesion deficiency, Gaucher's disease, and a variety of other metabolic/storage deficiencies. UCB derived SCs potentially could be used to correct genetic deficiencies at birth after successful

**13. Stem cells in regenerative medicine – A rapid consideration**

therapy is a new and promising manner of an infarcted heart healing.

Cardiac repair following SC application.The occurrence of heart failure following acute myocardial infarction – during the hospital stay and during the next few months or years – is high (up to 50%). Patients' mortality with heart failure after infarction is also considerable. The left ventricle dilatation occurs in even approximately one third of the patients, reperfused effectively with primary angioplasty. The incidence of heart failure after infarction has increased and mortality decreased with better reperfusion therapy [2, 3, 28]. Consequently, it is imperative to develop a curative approach to prevent of myocardium remodeling. The SC

yield and viability (7–AAD flow cytometric assay) [2, 8, 50]. The harvesting

from different sources are presented in Table 2.

**Table 2.** Ratio of CD34 cell markers using double staining

superior organ repair or SC regenerative potential.

gene transduction and autologous transplant [2, 50].

bone marrow.

in improved CD34+

subset investigations of cells collected

http://dx.doi.org/10.5772/54473

123

**PB–SCs I PB–SCs II BM–SCs**

**Figure 5.** The ratio of the CD45/CD34+ cells in peripheral blood harvest

For autologous SC transplants, the use of the best freezing process and choice of the most appropriate cryoprotective agent is required (optimized cryobiosystem). Nowadays a variety of protocols are used in blood–derived cell freezing practice. Generally, microprocessor– controlled (controlled–rate) freezing is more efficient than uncontrolled–rate (without programmed cooling) procedure due to better cell recovery. Our earlier results obtained for cryopreserved bone marrow cells and peripheral blood mononuclear cells were in agreement with these findings [8, 52]. These results imply a different "cryobiological request" of MRA cells in comparison with the mature progenitors. Moreover, our clinical studies showed that therapeutic use of the SCs – cryopreserved by our own controlled-rate system resulted with high cell recovery (91%) and rapid posttransplant hematopoietic reconstitution – on the 11th day in average [2, 8].

We have also investigated SC–harvesting protocols with optimized cell source, collection time– point and processed blood volume, CD34–threshold dose (calculed by ideal body mass), as well as immature (CD34+ /CD33– , CD34+ /CD38– , CD34+ /DR– , CD34+ /CD90+ ) vs. mature (CD34+ / CD33+ , CD34+ /CD38+ , CD34+ /DR+ , CD34+ /CD90– ) CD34–subset ratio in harvest [8, 26, 28, 50].


Several data related to our immature vs. mature CD34– subset investigations of cells collected from different sources are presented in Table 2.

PB–SCs I = stem cell collected from peripheral blood after mobilization with chemotherapy and rHuG–CSF; PB–SCs II = stem cell collected from peripheral blood after mobilization with rHuG–CSF alone; BM–SCs = stem cell collected from bone marrow.

**Table 2.** Ratio of CD34 cell markers using double staining

We have previously analyzed our results of PB vs. BM derived SC transplants based on the hematopoietic reconstitution. Transplants were used for the treatment of patients with severe aplastic anemia, acute lymphoblastic leukemia, acute non–lymphoblastic leukemia, chronic myeloid leukemia, multiple myeloma, Hodgkin's and non–Hodgkin's lymphoma, breast and ovarian cancer, extragonadal non–seminal germ cell tumor, and severe multiple sclerosis. The

yields for allogeneic and autologous transplants were eminent: 16.7±9.8 × 106

For autologous SC transplants, the use of the best freezing process and choice of the most appropriate cryoprotective agent is required (optimized cryobiosystem). Nowadays a variety of protocols are used in blood–derived cell freezing practice. Generally, microprocessor– controlled (controlled–rate) freezing is more efficient than uncontrolled–rate (without programmed cooling) procedure due to better cell recovery. Our earlier results obtained for cryopreserved bone marrow cells and peripheral blood mononuclear cells were in agreement with these findings [8, 52]. These results imply a different "cryobiological request" of MRA cells in comparison with the mature progenitors. Moreover, our clinical studies showed that therapeutic use of the SCs – cryopreserved by our own controlled-rate system resulted with high cell recovery (91%) and rapid posttransplant hematopoietic reconstitution – on the 11th

We have also investigated SC–harvesting protocols with optimized cell source, collection time– point and processed blood volume, CD34–threshold dose (calculed by ideal body mass), as

, CD34+

/DR–

, CD34+

/CD90+

) CD34–subset ratio in harvest [8, 26, 28, 50].

) vs. mature (CD34+

/

/CD38–

/CD90–

ratio in obtained PB harvest is presented in Figure 5.

**Figure 5.** The ratio of the CD45/CD34+ cells in peripheral blood harvest

/CD33–

/DR+

, CD34+

, CD34+

, CD34+

/kgbm, respectively [8, 26, 27]. A typical histogram with high–level of the

/kgbm and

CD34+

CD34+

11.8±6.1 × 106

122 Stem Cell Biology in Normal Life and Diseases

day in average [2, 8].

well as immature (CD34+

/CD38+

, CD34+

CD33+

Finally, we found that the use of large volume vs. conventional (repetitive) apheresis resulted in improved CD34+ yield and viability (7–AAD flow cytometric assay) [2, 8, 50]. The harvesting of higher ratio of immature vs. mature CD34–subsets correlated with BM repopulation ability (complete and long–term engraftment) and rapid hematopoietic reconstitution, as well as superior organ repair or SC regenerative potential.

SCs are considered optimal targets for gene transduction due to their ability to renew them‐ selves and differentiate into progeny cells and generate a self-perpetuating cell population that contains the transduced gene for the lifetime of the patient. Specific diseases that could be candidates for SC gene therapy include thalassemia, sickle cell anemia, Fanconi anemia, severe combined immune deficiency secondary to adenosine deaminase deficiency or purine nucleoside phosphorylase deficiency, chronic granulomatous disease, leukocyte adhesion deficiency, Gaucher's disease, and a variety of other metabolic/storage deficiencies. UCB derived SCs potentially could be used to correct genetic deficiencies at birth after successful gene transduction and autologous transplant [2, 50].

#### **13. Stem cells in regenerative medicine – A rapid consideration**

Cardiac repair following SC application.The occurrence of heart failure following acute myocardial infarction – during the hospital stay and during the next few months or years – is high (up to 50%). Patients' mortality with heart failure after infarction is also considerable. The left ventricle dilatation occurs in even approximately one third of the patients, reperfused effectively with primary angioplasty. The incidence of heart failure after infarction has increased and mortality decreased with better reperfusion therapy [2, 3, 28]. Consequently, it is imperative to develop a curative approach to prevent of myocardium remodeling. The SC therapy is a new and promising manner of an infarcted heart healing.

It is generally accepted that adult SCs from different tissues may transdifferentiate in special situations into cardiomyocytes or endothelial cells. In addition, the presence of an important quantity of cardiac cells is confirmed in proliferative state in peri-infarction region. The first source of these "regenerative cells" is maybe cardiac SCs – which are in the inactive stage in intact (undamaged) myocardium – but following infarction they differentiate into cardiomyo‐ cytes, smooth muscle cells and endothelial cells [2, 3]. The next discovery is that for the period of infarction, myocardial ischemia initiates release of some cytokines, growth-factors and chemokines – which induce SC mobilization from other niches and their homing into the damaged myocardium. The knowledge of these processes is important because the treatment efficacy depends on artificial *ex vivo* and/or *in vivo* intensification of some "steps" in order to make regenerative process more beneficial.

The final purpose of SC therapy, organ repopulation strategies and tissue engineering is to regenerate tissues/organs or to produce new grafts/organs for transplant. With the ex‐ pansion of complete organ "decellularization" methods the equation of organ shortage could be radically altered in the future. Decellularized organs provide the ideal trans‐ plantable platform with all the essential microstructure and extra-cellular signals for cell connection (homing), transdifferentiation, tissue vascularization, etc. Novel systems to reengineer organs may have key connotations for the fields of regenerative biology and ul‐

Stem Cell-Based (Auto)Grafting: From Innovative Research Toward Clinical Use in Regenerative Medicine

Currently available β-cell replacement therapies for patients with Type 1 diabetes (T1D), including islet and pancreas transplant, are largely successful in restoring normal glucose metabolism. However, there are data concerning the use of SCs to generate β-cells for islet transplant, indicating the need for improved protocols for their derivation and full maturation. Researchers also considered evidence indicating that adult SCs may affect islet transplant by improving the viability of engrafted islets and controlling immune-related reactions to islet antigens. A novel SC-based applications or regeneration-type approaches include stimulation of endogenous regenerative mechanisms or inducing reprogramming of non-β cells into β cells. Because these strategies would finally generate allogeneic or syngeneic β cells, the control of alloimmunity or autoimmunity in addition to replacing lost β cells will be of the greatest

For the SC treatment of our patients with liver failure (n = 8) and T1D (n = 4), cells were harvested from PB following mobilization (rHuG-CSF 10µg/day; 5 days). The mean volume of processed blood was 15.2±1.6 L (ratio: 12.8 – 18.4 L). The total count of MNC and CD34+

The use of SCs in neurology/neurosurgery. Alzheimer's, Parkinson's and Huntington's diseases, amyotrophic lateral sclerosis (ALS), and Friedreich's ataxia are the most common human neurodegenerative diseases – pathologically characterized by a progressive and specific loss of certain neuronal populations. Currently there are no effective clinical therapies for many of these diseases. The recently acquired ability to reprogram human adult somatic cells to "induced pluripotent SCs" (iPSCs) in culture may provide a powerful tool for *in vitro* neurodegenerative disease modeling and an unlimited source for cell replacement therapy. Reprogramming of somatic cells into iPSCs ushered in a new era of regenerative medicine. Human iPSCs give potent new approaches for disease modeling, drug testing, developmental

We earlier largely described the specific therapeutic actions and clinical use of SCs in neurol‐ ogy/neurosurgery [2]. Cerebral tumors, stroke, neurodegenerative diseases, brain and spinal injuries are used as examples with different limitations and possibilities to be approached with adult SC and/or different regenerative treatments. It is clear, that despite a spectrum of successful approaches, there are current limitations in this field of therapeutic interventions, which makes the research more intriguing and opens the new avenues for the development

magnetic selection and *ex vivo* transdifferentiation and expansion across catheter [2, 50].

, respectively. Cells were applied after immuno‐

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125

and 1.6±08x107

timately organ transplant [2, 50].

importance [2, 50].

collected cells were 6.4±3.1x09

studies, and therapeutic applications.

of novel concepts, their future prove, and possible application.

The exact mechanism by which SCs create a protective effect resulting in tissue/organ repair and heart function improvement is also a matter of debate. A number of possible mechanisms have been proposed: a) SC transdifferentiation into cells of other lineages (cardiomyocytes or endothelial cells) resulting in formation of new tissue; b) mobilization of tissue specific SCs from the BM that home to the damaged tissue and participate in tissue regeneration; c) fusion of the SCs with cells of the target tissue giving rise to new cells; and d) creation of a milieu (perhaps by releasing growth factors) that enhances regeneration of endogenous cells [2, 3, 50].

At present, BM is the most frequent source of cells used for clinical cardiac repair. It contains a complex mixture of progenitor cells – including SCs; so-called side population (SP) cells, which account for most if not all long-term self-renewal and reconstitute the full panoply of hematopoietic lineages after single-cell grafting; a subset of mesenchymal or stromal cells (MSCs), which are already defined; multipotent adult non-hematopoietic progenitor cells (MAPCs – for example, VSEL cells), which can differentiate into all possible lineages, and a fraction of TCSC discovered recently by Ratajczak et coworkers [2, 38–44]. These TCSCs circulate at the highest level and thus accumulate in BM during rapid body growth and become a reserve pool of SCs for tissue/organ regeneration. They are chemo-attracted from PB to injured organs by signaling proteins, such as stromal cell-derived factor-1, which become highly expressed in damaged heart tissue. For therapeutic purposes, marrow is aspirated, the entire MNC fraction is obtained – a mixed combination of mentioned cells – or specific subpopulations are purified and isolated cells are injected into the heart without need of further *ex vivo* manipulation/expansion.

In conclusion, current challenges for cell-based therapy in cardiac repair include identifying the origins of the novel cardiac SCs found inside heart, pinpointing biologically active cells from BM and other cell populations, optimizing cell mobilization and homing, increase of survival of grafted SCs, and exploiting cell therapy as a platform for secretor signals. Thus, we need a lot of basic research and randomized clinical trials to define the exact role of this probably revolutionary therapy for ischemic heart disease.

Application of SCs for liver and pancreas regeneration. The growing donor organ shortage requires consideration of alternative emerging technologies. Regenerative medicine may offer novel strategies to treat patients with end-stage or severe organ failure.

The final purpose of SC therapy, organ repopulation strategies and tissue engineering is to regenerate tissues/organs or to produce new grafts/organs for transplant. With the ex‐ pansion of complete organ "decellularization" methods the equation of organ shortage could be radically altered in the future. Decellularized organs provide the ideal trans‐ plantable platform with all the essential microstructure and extra-cellular signals for cell connection (homing), transdifferentiation, tissue vascularization, etc. Novel systems to reengineer organs may have key connotations for the fields of regenerative biology and ul‐ timately organ transplant [2, 50].

It is generally accepted that adult SCs from different tissues may transdifferentiate in special situations into cardiomyocytes or endothelial cells. In addition, the presence of an important quantity of cardiac cells is confirmed in proliferative state in peri-infarction region. The first source of these "regenerative cells" is maybe cardiac SCs – which are in the inactive stage in intact (undamaged) myocardium – but following infarction they differentiate into cardiomyo‐ cytes, smooth muscle cells and endothelial cells [2, 3]. The next discovery is that for the period of infarction, myocardial ischemia initiates release of some cytokines, growth-factors and chemokines – which induce SC mobilization from other niches and their homing into the damaged myocardium. The knowledge of these processes is important because the treatment efficacy depends on artificial *ex vivo* and/or *in vivo* intensification of some "steps" in order to

The exact mechanism by which SCs create a protective effect resulting in tissue/organ repair and heart function improvement is also a matter of debate. A number of possible mechanisms have been proposed: a) SC transdifferentiation into cells of other lineages (cardiomyocytes or endothelial cells) resulting in formation of new tissue; b) mobilization of tissue specific SCs from the BM that home to the damaged tissue and participate in tissue regeneration; c) fusion of the SCs with cells of the target tissue giving rise to new cells; and d) creation of a milieu (perhaps by releasing growth factors) that enhances regeneration of endogenous cells [2, 3, 50].

At present, BM is the most frequent source of cells used for clinical cardiac repair. It contains a complex mixture of progenitor cells – including SCs; so-called side population (SP) cells, which account for most if not all long-term self-renewal and reconstitute the full panoply of hematopoietic lineages after single-cell grafting; a subset of mesenchymal or stromal cells (MSCs), which are already defined; multipotent adult non-hematopoietic progenitor cells (MAPCs – for example, VSEL cells), which can differentiate into all possible lineages, and a fraction of TCSC discovered recently by Ratajczak et coworkers [2, 38–44]. These TCSCs circulate at the highest level and thus accumulate in BM during rapid body growth and become a reserve pool of SCs for tissue/organ regeneration. They are chemo-attracted from PB to injured organs by signaling proteins, such as stromal cell-derived factor-1, which become highly expressed in damaged heart tissue. For therapeutic purposes, marrow is aspirated, the entire MNC fraction is obtained – a mixed combination of mentioned cells – or specific subpopulations are purified and isolated cells are injected into the heart without need of further

In conclusion, current challenges for cell-based therapy in cardiac repair include identifying the origins of the novel cardiac SCs found inside heart, pinpointing biologically active cells from BM and other cell populations, optimizing cell mobilization and homing, increase of survival of grafted SCs, and exploiting cell therapy as a platform for secretor signals. Thus, we need a lot of basic research and randomized clinical trials to define the exact role of this

Application of SCs for liver and pancreas regeneration. The growing donor organ shortage requires consideration of alternative emerging technologies. Regenerative medicine may offer

make regenerative process more beneficial.

124 Stem Cell Biology in Normal Life and Diseases

*ex vivo* manipulation/expansion.

probably revolutionary therapy for ischemic heart disease.

novel strategies to treat patients with end-stage or severe organ failure.

Currently available β-cell replacement therapies for patients with Type 1 diabetes (T1D), including islet and pancreas transplant, are largely successful in restoring normal glucose metabolism. However, there are data concerning the use of SCs to generate β-cells for islet transplant, indicating the need for improved protocols for their derivation and full maturation. Researchers also considered evidence indicating that adult SCs may affect islet transplant by improving the viability of engrafted islets and controlling immune-related reactions to islet antigens. A novel SC-based applications or regeneration-type approaches include stimulation of endogenous regenerative mechanisms or inducing reprogramming of non-β cells into β cells. Because these strategies would finally generate allogeneic or syngeneic β cells, the control of alloimmunity or autoimmunity in addition to replacing lost β cells will be of the greatest importance [2, 50].

For the SC treatment of our patients with liver failure (n = 8) and T1D (n = 4), cells were harvested from PB following mobilization (rHuG-CSF 10µg/day; 5 days). The mean volume of processed blood was 15.2±1.6 L (ratio: 12.8 – 18.4 L). The total count of MNC and CD34+ collected cells were 6.4±3.1x09 and 1.6±08x107 , respectively. Cells were applied after immuno‐ magnetic selection and *ex vivo* transdifferentiation and expansion across catheter [2, 50].

The use of SCs in neurology/neurosurgery. Alzheimer's, Parkinson's and Huntington's diseases, amyotrophic lateral sclerosis (ALS), and Friedreich's ataxia are the most common human neurodegenerative diseases – pathologically characterized by a progressive and specific loss of certain neuronal populations. Currently there are no effective clinical therapies for many of these diseases. The recently acquired ability to reprogram human adult somatic cells to "induced pluripotent SCs" (iPSCs) in culture may provide a powerful tool for *in vitro* neurodegenerative disease modeling and an unlimited source for cell replacement therapy. Reprogramming of somatic cells into iPSCs ushered in a new era of regenerative medicine. Human iPSCs give potent new approaches for disease modeling, drug testing, developmental studies, and therapeutic applications.

We earlier largely described the specific therapeutic actions and clinical use of SCs in neurol‐ ogy/neurosurgery [2]. Cerebral tumors, stroke, neurodegenerative diseases, brain and spinal injuries are used as examples with different limitations and possibilities to be approached with adult SC and/or different regenerative treatments. It is clear, that despite a spectrum of successful approaches, there are current limitations in this field of therapeutic interventions, which makes the research more intriguing and opens the new avenues for the development of novel concepts, their future prove, and possible application.

Finally, in our center SC are applied the first time in the treatment of ALS female patients (intratecal application of non-manipulated BM derived cells; two repeated treatments) and another female patient after brain infarction (intra-arterial cell injection using percutaneous catheter). Logically, preliminary or particularly definitive conclusions can only be drawn from larger, randomized, controlled clinical trials.

In our Center for regenerative medicine, total of 60 patients were treated by non-manip‐ ulated or *ex vivo* cytokine stimulated BM derived cells (collected in steady state hemato‐ poiesis or after priming of the marrow). Cells were applied across percutaneous catheter intracoronarly or directly into the myocardium (transpericardial approach). In the most recent stage, we investigated the effects of rHuG-CSF facilitated BM (primed BM) SC therapy on improvement of the global and regional function of left ventricle after large

Stem Cell-Based (Auto)Grafting: From Innovative Research Toward Clinical Use in Regenerative Medicine

The main inclusion criteria for the enrollment in the study are: patients with the first ST segment elevation myocardial infarction; age younger than 71 years old; successful percuta‐ neous coronary intervention on the infarction artery inside the 24 hours from the onset of pain and with the left ventricle ejection fraction lower than 41% at the fifth day estimated by the transthoracic echocardiography. The main exclusion criteria are the presence of other serious illness, any pre-infarction significant damage of the heart, allergy to aspirin and resistance to clopidogrel, and the presence of the symptoms and signs of heart failure five days after infarction. The local Ethical Committee approved the study and all patients were given written

**Pre-transplant examination**. At the fifth day of infarction, global and regional left ventricle systolic function together with the end-diastolic and end-systolic volumes are measured by the transthoracic echocardiography. Infarction size is estimated by the Technetium-sestamibi

**Stem cell harvesting and application**. The day before BM harvest, patients receive 5 – 10 µg/ kgbm of rHuG-CSF. Between 7 – 12 days from the infarction in the general anesthesia 300 mL of the BM is harvested from the posterior iliac crests. After that BM was filtered and processed to the final volume of 30 – 50 mL of concentrated mono-nuclear cell suspension. Boluses of 10 mL are injected through the diagnostic catheter into the infarction related coronary artery.

**Control groups**. There are two control groups with the same inclusion and exclusion criteria. The first are patients who did not submit to any SC procedure. And the second represents the patients who were treated with the autologous intracoronary, BM derived SC therapy without

**The follow-up**. Clinical examination is scheduled for the one, fourth and sixth months after infarction and every 6 months after that. Echocardiography measurement of global and left ventricle ejection fraction and volumes is planned after 4 months and every year after infarction. Myocardial scintigraphy is planned after 4 months and after two years

**End points**. The main end points are comparison of the 4 months and 2 years change in left

ventricle ejection fraction, volumes and infarction size between three groups.

of MNCs and patients without

http://dx.doi.org/10.5772/54473

127

myocardial scintigraphy between the 5-8 days from the infarction.

Patients with rHuG-CSF facilitated SC therapy received 18.4x108

of the MNCs.

myocardial infarction.

**14.2. Methodology**

informed consent.

rHuG-CSF received 7.9x108

rHuG-CSF.

from the infarction.

### **14. The treatment of large myocardial infarction by intracoronary applied rHuG-CSF facilitated BM derived SCs – Our experience**

#### **14.1. Introduction**

Intracoronary autologous, BM derived SC transplant for the treatment of myocardial infarction went through the three important steps during the last decade. In the first step, small nonrandomized trials of Strauer [53] and TOPCARE study [54], established the basic methodology of SC harvesting, cell processing and intracoronary delivery and confirmed the safety and regenerative potential of SCs for the improvement of myocardial viability and function after infarction.

The next step representing two landmark studies has brought controversial results. The REPAIR-AMI [55] has showed that intracoronary delivery of BM derived SCs led to the improvement of six months global ejection fraction and lowers the major cardiac adverse events. On the contrary, the ASTAMI trial [56] has failed to prove any benefit of early intra‐ coronary application of BM derived SCs to the global ejection fraction and left ventricle remodeling measured by magnetic resonance imaging at baseline and after four months of anterior infarction. However, those studies suggested several important issues. The first of all were that choice of patients with more severe damaged myocardium and the delayed SC delivery for at least five days after infarction resulted to better results of SC regenerative capacity. The second key conclusion is that SC processing process might be essential for successful SC therapy after myocardial infarction.

In the third step, two relatively large randomized studies tried to define the efficacy of different SC population for treatment of myocardial infarction. The REGENT study [57] used selection of CD34+ /CD-CXC4+ cells, and the HEBE trial [58] examine selection of PB derived MNCs and compare the results with two control groups, non-selected BM origin MNCs and controls. These trials didn't show any usefulness of cell selection and suggested again that patients with more damaged myocardium had better improvement with intracoronary SC delivery. On the other hand, our study has showed that there is a limit for the amount of myocardial necrosis in which we can achieve improvement of myocardial function after intracoronary SC delivery and the patients with the huge loss of myocardium has no any benefit from the cell therapy [26–28]. However, improvement of global and regional left ventricle function was modest and faraway from the expected in all studies. The next steps in SC therapy are *ex vivo* expansion of the number and regenerative capacity of harvested SCs, *in vivo* facilitation of that, and improvement of methods of SC delivery.

In our Center for regenerative medicine, total of 60 patients were treated by non-manip‐ ulated or *ex vivo* cytokine stimulated BM derived cells (collected in steady state hemato‐ poiesis or after priming of the marrow). Cells were applied across percutaneous catheter intracoronarly or directly into the myocardium (transpericardial approach). In the most recent stage, we investigated the effects of rHuG-CSF facilitated BM (primed BM) SC therapy on improvement of the global and regional function of left ventricle after large myocardial infarction.

#### **14.2. Methodology**

Finally, in our center SC are applied the first time in the treatment of ALS female patients (intratecal application of non-manipulated BM derived cells; two repeated treatments) and another female patient after brain infarction (intra-arterial cell injection using percutaneous catheter). Logically, preliminary or particularly definitive conclusions can only be drawn from

**14. The treatment of large myocardial infarction by intracoronary applied**

Intracoronary autologous, BM derived SC transplant for the treatment of myocardial infarction went through the three important steps during the last decade. In the first step, small nonrandomized trials of Strauer [53] and TOPCARE study [54], established the basic methodology of SC harvesting, cell processing and intracoronary delivery and confirmed the safety and regenerative potential of SCs for the improvement of myocardial viability and function after

The next step representing two landmark studies has brought controversial results. The REPAIR-AMI [55] has showed that intracoronary delivery of BM derived SCs led to the improvement of six months global ejection fraction and lowers the major cardiac adverse events. On the contrary, the ASTAMI trial [56] has failed to prove any benefit of early intra‐ coronary application of BM derived SCs to the global ejection fraction and left ventricle remodeling measured by magnetic resonance imaging at baseline and after four months of anterior infarction. However, those studies suggested several important issues. The first of all were that choice of patients with more severe damaged myocardium and the delayed SC delivery for at least five days after infarction resulted to better results of SC regenerative capacity. The second key conclusion is that SC processing process might be essential for

In the third step, two relatively large randomized studies tried to define the efficacy of different SC population for treatment of myocardial infarction. The REGENT study [57] used selection

compare the results with two control groups, non-selected BM origin MNCs and controls. These trials didn't show any usefulness of cell selection and suggested again that patients with more damaged myocardium had better improvement with intracoronary SC delivery. On the other hand, our study has showed that there is a limit for the amount of myocardial necrosis in which we can achieve improvement of myocardial function after intracoronary SC delivery and the patients with the huge loss of myocardium has no any benefit from the cell therapy [26–28]. However, improvement of global and regional left ventricle function was modest and faraway from the expected in all studies. The next steps in SC therapy are *ex vivo* expansion of the number and regenerative capacity of harvested SCs, *in vivo* facilitation of that, and

/CD-CXC4+ cells, and the HEBE trial [58] examine selection of PB derived MNCs and

**rHuG-CSF facilitated BM derived SCs – Our experience**

larger, randomized, controlled clinical trials.

126 Stem Cell Biology in Normal Life and Diseases

successful SC therapy after myocardial infarction.

improvement of methods of SC delivery.

**14.1. Introduction**

infarction.

of CD34+

The main inclusion criteria for the enrollment in the study are: patients with the first ST segment elevation myocardial infarction; age younger than 71 years old; successful percuta‐ neous coronary intervention on the infarction artery inside the 24 hours from the onset of pain and with the left ventricle ejection fraction lower than 41% at the fifth day estimated by the transthoracic echocardiography. The main exclusion criteria are the presence of other serious illness, any pre-infarction significant damage of the heart, allergy to aspirin and resistance to clopidogrel, and the presence of the symptoms and signs of heart failure five days after infarction. The local Ethical Committee approved the study and all patients were given written informed consent.

**Pre-transplant examination**. At the fifth day of infarction, global and regional left ventricle systolic function together with the end-diastolic and end-systolic volumes are measured by the transthoracic echocardiography. Infarction size is estimated by the Technetium-sestamibi myocardial scintigraphy between the 5-8 days from the infarction.

**Stem cell harvesting and application**. The day before BM harvest, patients receive 5 – 10 µg/ kgbm of rHuG-CSF. Between 7 – 12 days from the infarction in the general anesthesia 300 mL of the BM is harvested from the posterior iliac crests. After that BM was filtered and processed to the final volume of 30 – 50 mL of concentrated mono-nuclear cell suspension. Boluses of 10 mL are injected through the diagnostic catheter into the infarction related coronary artery. Patients with rHuG-CSF facilitated SC therapy received 18.4x108 of MNCs and patients without rHuG-CSF received 7.9x108 of the MNCs.

**Control groups**. There are two control groups with the same inclusion and exclusion criteria. The first are patients who did not submit to any SC procedure. And the second represents the patients who were treated with the autologous intracoronary, BM derived SC therapy without rHuG-CSF.

**The follow-up**. Clinical examination is scheduled for the one, fourth and sixth months after infarction and every 6 months after that. Echocardiography measurement of global and left ventricle ejection fraction and volumes is planned after 4 months and every year after infarction. Myocardial scintigraphy is planned after 4 months and after two years from the infarction.

**End points**. The main end points are comparison of the 4 months and 2 years change in left ventricle ejection fraction, volumes and infarction size between three groups.

#### **14.3. Results**

The baseline characteristics of three groups of patients were similar except that control group were slightly older than patients in both SC groups and have more often multivessel disease. Gender and risk factors distribution were similar between groups. There is also no difference in ischemic time in three groups. Baseline global left ventricle ejection fraction and infarction size were similar in all three groups. However, both end-diastolic and end-systolic cardiac indices were lower in patients with rHuG-CSF facilitated SC therapy.

Difference between baseline and 4-months infarction size is significant only in patients with rHuG-CSF facilitated SC therapy (Figure 6). There was no significant difference between the

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129

**Figure 6.** Change in infarction size between baseline and 4-months among three groups of patients

**Figure 7.** Change in left ventricle ejection fraction: baseline vs. 4-months among three groups of patients

change of LVEF at baseline and after 4 months (Figure 6).

After 4 months left ventricle ejection fraction has improved in group of patients treated with SCs and in control group but did not reach statistical significance in the group treated with rHuG-CSF facilitated SC therapy because of small number of patients in that group (Table 1). Infarction size has the same pattern (Table 3). End-diastolic and end-systolic volumes increased in all groups but also did not get to significant statistical difference in the rHuG-CSF facilitated SC group.


**Table 3.** Infarction size end left ventricle systolic function and volumes at baseline and after 4 months.

Difference between baseline and 4-months infarction size is significant only in patients with rHuG-CSF facilitated SC therapy (Figure 6). There was no significant difference between the change of LVEF at baseline and after 4 months (Figure 6).

**14.3. Results**

128 Stem Cell Biology in Normal Life and Diseases

SC group.

**Parameters**

LV%±SD

LVEF at baseline

LVEF after 4 months

EDVCI at baseline

ESVCI at baseline

ESVCI at 4 months

EDVCI after 4 months

Infarction size at baseline

Infarction size after 6 months

The baseline characteristics of three groups of patients were similar except that control group were slightly older than patients in both SC groups and have more often multivessel disease. Gender and risk factors distribution were similar between groups. There is also no difference in ischemic time in three groups. Baseline global left ventricle ejection fraction and infarction size were similar in all three groups. However, both end-diastolic and end-systolic cardiac

After 4 months left ventricle ejection fraction has improved in group of patients treated with SCs and in control group but did not reach statistical significance in the group treated with rHuG-CSF facilitated SC therapy because of small number of patients in that group (Table 1). Infarction size has the same pattern (Table 3). End-diastolic and end-systolic volumes increased in all groups but also did not get to significant statistical difference in the rHuG-CSF facilitated

LV%±SD 28,4±11,3 35,6±8,0 31,4±12,8 ns

% ± SD 32,9±4,1 36,4±3,0 34,3±5,2 ns

% ± SD 37,0±9,0 43,8±3,0 36,9±8,2 0,01

ml/m2 ± SD 68,3±11,3 46,1±10,0 67,8±17,6 0,01

ml/m2 ± SD 75,7±15,7 54,3±6,1 73,8±20,4 0,053

ml/m2 ± SD 44,8±9,8 28,0±4,4 45,0±15,1 0.02

ml/m2 ± SD 47,8±14,3 30,3±2,6 47,5±17,6 0,07

**G-CSF facilitated SC therapy in AMI n=5**

25.2±12.6 25.2±8.6 27.9±10.7 ns

**Control group**

**n=17 <sup>p</sup>**

indices were lower in patients with rHuG-CSF facilitated SC therapy.

**SC therapy in AMI n=19**

p 0.001 0.068 0.001

p 0.004 0.313 0.004

p 0.024 0.161 0.004

p 0.001 0.142 0.002

**Table 3.** Infarction size end left ventricle systolic function and volumes at baseline and after 4 months.

**Figure 6.** Change in infarction size between baseline and 4-months among three groups of patients

**Figure 7.** Change in left ventricle ejection fraction: baseline vs. 4-months among three groups of patients

#### **15. Conclusion**

Our preliminary results have shown that rHuG-CSF facilitated autologous, intracoronary SC transfer was safe; between two-three times higher number of MNCs were given and there was a trend toward larger increase of 4-months ejection fraction and greater decrease of the infarction size than the control groups. Any procedure that increases the left ventricle ejection fraction for more than 5% after a several months follow-up would be of great clinical and economic value. Autologous BM derived, intracoronary SC transfer in the second week of large myocardial infarction very probably improved the global left ventricle ejection fraction by 3 – 5% although results of the published trial are controversial. Granulocyte colony stimulating factor given alone for several days after myocardial infarction did not improve significantly global ejection fraction in the several trials, but it seems that its early application in patients with larger infarction could be useful. Further investigation is needed for the justification of rHuG-CSF facilitated, BM derived SC therapy in the early phase of acute ST elevation myo‐ cardial infarction.

[2] Pavlovic M, Balint B. Stem Cells and Tissue Engineering. New York: Springer. In press

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131

[3] Obradovic S, Balint B, Trifunovic Z. Stem Cell Therapy in Myocardial Infarction Clinical Point of View and the Results of the REANIMA Study. In: Gholamrezanezhad A. (Ed).

[4] Janssens S, Dubois C, Bogaert J, Theunissen K, Deroose C, Desmet W, et al. Autologous bone marrow-derived stem-cell transfer in patient with ST-segment elevation myocar‐ dial infarction: double-blind, randomized controlled trial. Lancet 2006; 367: 113−21.

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2012.

#### **Author details**

Bela Balint1,2,3\*, Slobodan Obradovic3,4, Milena Todorovic5,6, Mirjana Pavlovic7 and Biljana Mihaljevic5,6


7 Department of Computer and Electrical Engineering and Computer Science, FAU, Boca Raton, FL, USA

#### **References**

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[2] Pavlovic M, Balint B. Stem Cells and Tissue Engineering. New York: Springer. In press 2012.

**15. Conclusion**

130 Stem Cell Biology in Normal Life and Diseases

cardial infarction.

**Author details**

Biljana Mihaljevic5,6

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Our preliminary results have shown that rHuG-CSF facilitated autologous, intracoronary SC transfer was safe; between two-three times higher number of MNCs were given and there was a trend toward larger increase of 4-months ejection fraction and greater decrease of the infarction size than the control groups. Any procedure that increases the left ventricle ejection fraction for more than 5% after a several months follow-up would be of great clinical and economic value. Autologous BM derived, intracoronary SC transfer in the second week of large myocardial infarction very probably improved the global left ventricle ejection fraction by 3 – 5% although results of the published trial are controversial. Granulocyte colony stimulating factor given alone for several days after myocardial infarction did not improve significantly global ejection fraction in the several trials, but it seems that its early application in patients with larger infarction could be useful. Further investigation is needed for the justification of rHuG-CSF facilitated, BM derived SC therapy in the early phase of acute ST elevation myo‐

Bela Balint1,2,3\*, Slobodan Obradovic3,4, Milena Todorovic5,6, Mirjana Pavlovic7

2 Institute for Transfusiology and Hemobiology of MMA, Belgrade, Serbia

7 Department of Computer and Electrical Engineering and Computer Science, FAU, Boca

[1] Mayorga M, Finan A, Penn M. Pre–transplantation specification of stem cells to cardiac lineage for regeneration of cardiac tissue. Stem Cell Rev Rep 2009; 5 (1): 51–60.

\*Address all correspondence to: beciusb@yahoo.com

1 Institute for Medical Research, University of Belgrade, Serbia

3 Faculty of Medicine MMA, University of Defense, Serbia

4 Clinic of Emergency Medicine of MMA, Belgrade, Serbia

6 Faculty of Medicine, University of Belgrade, Serbia

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[56] Lunde K, Solheim S, Aakhus S, Arnesen H, Abdelnoor M, Egeland T, et al. Intracoro‐ nary injection of mononuclear bone marrow cells in acute myocardial infarction. N Engl

[57] Tendera M, Wojakowski W, Ruzytto W, Chojnowska L, Kepka C, Tracz W, et al. Intracoronary infusion of bone-marrow derived selected CD34+CXCR4+ cells and nonselected MNCs in patients with acute STEMI and reduced left ventricular ejection fraction: results of randomized, multicentre Myocardial Regeneration by Intracoronary Infusion of Selected Population of STEM Cells in Acute Myocardial Infarction (RE‐

[58] Hirsch A., Nijveldt R., Vleuten P.A., Tijssen J., Giessen W., Tio R. et al. Intracoronary infusion of MNCs from bone marrow or peripheral blood compared with standard therapy in patients after acute myocardial infarction treated by primary percutaneous coronary intervention: results of the randomized controlled HEBE trial. Eur Heart J

REPAIR-AMI trial. Eur Heart J 2006; 27: 2775–83.

GENT) Trial. Eur Heart J 2009; 30: 1313–21.

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2010


derived progenitor cells in acute myocardial infarction: final 1-year results of the REPAIR-AMI trial. Eur Heart J 2006; 27: 2775–83.

[56] Lunde K, Solheim S, Aakhus S, Arnesen H, Abdelnoor M, Egeland T, et al. Intracoro‐ nary injection of mononuclear bone marrow cells in acute myocardial infarction. N Engl J Med 2006; 355: 1199–209.

[42] Kucia M, Reca R, Campbell FR, Surma–Zuba E, Majka M, Ratajczak M, and Ratajczak MZ A population of very small embryonic – like (VSEL) CXR4+SSEA–1+Oct4+ stem

[43] Ratajczak MZ, Zuba–Surma E, Kucia M, Poniewierska A, Suszynska M, Ratajczak J. Pluripotent and multipotent stem cells in aduld tissue. Adv Med Sci 2012; 19: 1–17. [44] Ratajczak MZ, Suszynska M, Pedziwiatr D, Mierzejewska K, Greco NJ. Umbilical cord blood–derived very small embryonic like stem cells (VSELs) as a source of pluripotent

stem cells for regenerative medicine. Pediatr Endocrinol Rev 2012; 9: 639–43.

[45] Arthur A, Rychkov G, Shi S, Koblar SA, GronthosS. Adult human dental pulp stem cellsdifferentiate toward functionally active neuronsunder appropriateenvironmental

[46] Todorovic V, Markovic D, Milosevic–Jovcic N, Petakov M, Balint B, Colic M. Dental pulp stem cells – potential significancein regenerative medicine. Stom Glas 2008; 55:

[47] Morsczeck K, Frerich B, Driemel O. Dental Stem Cell Patents. Clin Oral Invest 2008; 12:

[48] Balint B. Coexistent cryopreservation strategies: microprocessor−restricted vs. uncon‐ trolled–rate freezing of the "blood−derived" progenitors/cells. Blood Banking Transf

[49] Balint B. Stem and progenitor cell harvesting, extracorporeal "graft engineering" and clinical use – initial expansion vs. current dillemas. Clin Appl Immunol 2006; 5 (1): 518–

[50] Balint B, Todorovic M, Jocic M, Stamatovic D. Haemo-therapeutic approach to cell and

[51] Balint B, Stamatovic D, Todorovic M, Elez M, Vojvodic D, Pavlovic M, Cucuz–Jokic M.

[52] Balint B, Ivanovic Z, Petakov M, Taseski J, Jovcic G, Stojanovic N, et al. The cryopre‐ servation protocol optimal for progenitor recovery is not optimal for preservation of

[53] Strauer BE., Brehm M., Zeus T., Köstering M., Hernandez A., Sorg RV, et al. (). Repair of infracted myocardium by autologous intracoronary mononuclear bone marrow cell

[54] Assmus B., Rolf A., Erbs S., Elsässer A., Haberbosch W., Hambrecht R., et al. Clinical outcome 2 years after intracoronary administration of bone marrow-derived progenitor

[55] Schächinger V, Erbs S, Elsässer A, Haberbosch W, Hambrecht R, Hölschermann H, et al. Improved clinical outcome after intracoronary administration of bone-marrow-

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**Chapter 8**

**Hematopoietic Stem Cells in Chronic Myeloid Leukemia**

Chronic Myeloid Leukemia (CML) is a clonal disease, originated at the level of Hematopoietic Stem Cells (HSC) and characterized by the presence of the *Philadelphia* (Ph) chromosome and its oncogenic product p210Bcr-Abl. Such a protein has been shown to be essential for malignant transformation, since it is capable of altering cell adhesion, proliferation and apoptosis.

Current treatment options in CML include tyrosine kinase inhibitors (Imatinib, Nilotinib and Dasatinib), compounds that inhibit the activity of the BCR-ABL protein. However some patients will develop resistance or intolerance to these drugs and resistance has been associated with different mechanism including the quiescence of leukemic stem cells and Pgp or Src

In this chapter we focus on the basic biology of hematopoietic stem and progenitor cells from

Chronic myeloid leukemia (CML) is a lethal hematological malignancy characterized by the abnormal amplification of the myeloid (mainly granulocityc) compartment of the hemato‐ poietic system. It originates from the transformation of a primitive hematopoietic cell that suffers a t(9;22) (q34; q11) balanced reciprocal translocation that results in the generation of the Philadelphia chromosome (Ph). Ph produces BCR-ABL, a constitutively active tyrosine

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

© 2013 Chávez-González 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 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,

CML and analyze the most relevant and current concepts in this area.

kinase that drives a wide variety of physiological alterations [1].

Antonieta Chávez-González, Sócrates Avilés-Vázquez,

http://dx.doi.org/10.5772/54651

**1. Introduction**

kinase overexpression.

**2. Chronic myeloid leukemia**

Dafne Moreno-Lorenzana and Héctor Mayani

Additional information is available at the end of the chapter

## **Hematopoietic Stem Cells in Chronic Myeloid Leukemia**

Antonieta Chávez-González, Sócrates Avilés-Vázquez, Dafne Moreno-Lorenzana and Héctor Mayani

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54651

#### **1. Introduction**

Chronic Myeloid Leukemia (CML) is a clonal disease, originated at the level of Hematopoietic Stem Cells (HSC) and characterized by the presence of the *Philadelphia* (Ph) chromosome and its oncogenic product p210Bcr-Abl. Such a protein has been shown to be essential for malignant transformation, since it is capable of altering cell adhesion, proliferation and apoptosis.

Current treatment options in CML include tyrosine kinase inhibitors (Imatinib, Nilotinib and Dasatinib), compounds that inhibit the activity of the BCR-ABL protein. However some patients will develop resistance or intolerance to these drugs and resistance has been associated with different mechanism including the quiescence of leukemic stem cells and Pgp or Src kinase overexpression.

In this chapter we focus on the basic biology of hematopoietic stem and progenitor cells from CML and analyze the most relevant and current concepts in this area.

#### **2. Chronic myeloid leukemia**

Chronic myeloid leukemia (CML) is a lethal hematological malignancy characterized by the abnormal amplification of the myeloid (mainly granulocityc) compartment of the hemato‐ poietic system. It originates from the transformation of a primitive hematopoietic cell that suffers a t(9;22) (q34; q11) balanced reciprocal translocation that results in the generation of the Philadelphia chromosome (Ph). Ph produces BCR-ABL, a constitutively active tyrosine kinase that drives a wide variety of physiological alterations [1].

© 2013 Chávez-González 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 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.

CML was initially described in 1845 by John Hughes Bennett, who reported the case of a patient with "milky" blood and suggested that it was an infectious disease that caused hypertrophy of the liver and spleen, leading to the patient's death. A few weeks later, Rudolf Virchow reported a similar case, but, in contrast to Bennett, he suggested that the disease was not infectious and implied an increase in the number of blood cells. He coined the term leukemia (from the Greek *leukos*, white, and "Aemia", blood). In 1870, Neumann described that leukemia cells originate in the bone marrow; almost one hundred years later, in 1960, Nowel and Hungerford reported that in all cases of this malignancy there was a small, abnormal chro‐ mosome 22. However, was until 1973 that Janet Rowley described that the abnormal chromo‐ some was caused by a reciprocal translocation between the long arms of chromosomes 9 and 22, designating the name of Philadelphia (Ph) chromosome[2, 3].

The normal human ABL gene encodes for a non-receptor tyrosine kinase that is ubiquitously expressed. Such a 145 kDa protein is involved in the regulation of the cell cycle, the response to genotoxic stress, and intracellular signaling mediated by the integrin family [9]. There are three isoforms of the BCR-ABL fusion protein all of which encode the same portion of the ABL tyrosine kinase, but differ in the length of the BCR sequence at the N-terminus. p185/p190 BCR-ABL is expressed in Acute Lypmphoblastic Leukaemia (ALL), p210 BCR-ABL is characteristic of Chronic Myeloid Leukemia, and p230 BCR-ABL has been associated with a subgroup of

**Figure 1.** Structure of the Bcr-Abl gene. It is formed by a reciprocal translocation between chromosomes 22 (Bcr gene) and 9 (Abl gene). Ther M-BCR breakpoint resulting in a P210 BCR/ABL fusion transcripts b2a2 or b3a2 and they encode a pro‐

BCR-ABL fusion protein localizes in the cytoplasm and shows an increased and constitutive tyrosine kinase activity as a result of oligomerization of its coiled region and deletion of the SH domain of ABL. It activates a number of cytoplasmic and nuclear signal-transduction pathways involved in cell adherence, migration, inhibition of apoptosis, and induction of cell proliferation through activation of signaling proteins such as p21RAS, c-Myc, lipid kinasse PI3k, MAPk (mitogen-activated protein kinase family), tyrosine phosphatases, and signal

There is an increasing body of evidence indicating that, similar to normal hematopoiesis, a

of CML patients. Such Leukemic Stem Cells (LSC) seem to be the ones driving CML progres‐ sion, following a similar pattern to the one observed in normal hematopoiesis. That is to say,

Just like normal hematopoietic stem cells (HSC), CML stem cells express high levels of CD34, and lack the cell surface markers CD38, CD45RA, or CD71, as well as lineage-specific markers.

LSC give rise to CML progenitor cells, which, in turn, give rise to more mature cells.

cell compartment- exists in the bone marrow

Hematopoietic Stem Cells in Chronic Myeloid Leukemia

http://dx.doi.org/10.5772/54651

139

tein of 210 kDa (BCR-ABLp210) present in almost all patients with Chronic Mieloid Leukemia (modified to [9]).

transducer and activator of transcription (STATs) factors [9, 10].

**2.3. Leukemic Stem Cells in chronic myeloid leukemia**

quiescent stem cell population -within the CD34+

CML patients with a more indolent disease (Figure 1) [4].

#### **2.1. Epidemiology and clinical characteristics**

Chronic myelogenous leukemia has a worldwide incidence of 1-2 cases per 100,000 individuals [4]. The average age at diagnosis is 60 years; it occurs less frequently in young people and a tendency to increase exponentially with age has been observed. There is no geographic or genetic predisposition to acquire this condition, although some authors have associated it with exposure to high doses of ionizing radiation. The current CML prevalence of 24,000 affected patients in the United Sates is relatively low; it is expected to increase significantly over the next 20 years as a result of widespread use of BCR-ABL tyrosine kinase inhibitor therapy [5]. In Mexico, there are no official data on the incidence of such a disease, however, it has been estimated that there are about 80,000 cases of leukemia and 10% corresponds to CML [6].

The clinical presentation often includes granulocytosis, spenomegaly and marrow hypercel‐ lularity; however about 40% of patients are asymptomatic and their diagnosis is based on abnormal blood cell counts [1]. The natural course of the disease involves three sequential phases, namely chronic, accelerated and blast crises. Ninety percent of patients are diagnosed in chronic phase and they remain in it for 3 to 8 years. In this phase, the blood cells retain their ability to differentiate until the illness progresses to the accelerated phase, which is character‐ ized by the egress of immature cells into the bloodstream. Finally, the disease progresses to the blast crisis, defined by the presence of 30 percent or more leukemic cells in peripheral blood or marrow or extramedullary infiltrates of blast. During this phase the survival of patients is reduced to months and even weeks [7].

#### **2.2. Molecular events (Bcr-Abl oncogene)**

As mentioned before, the Philadelphia chromosome, which defines CML, is a shortened chromosome 22 originated from the reciprocal translocation between the long arms of chromosomes 9 and 22 [t (9; 22)] and involves addition of 3' segments of the *abl* gene (9q34) to 5' segments of the *bcr* gene (22q11) given rise to a *bcr-abl* fusion gene that transcribes a chimeric mRNA of 8.5 kb that, in turn, gives rise to a BCR-ABL fusion protein [7]. t(9;22) is evident in more than 95% of CML patients; between 5% and 10% of CML patients also present complex rearrangements that may involve one or more chromosomes in addition to 9 and 22 [8].

The normal human ABL gene encodes for a non-receptor tyrosine kinase that is ubiquitously expressed. Such a 145 kDa protein is involved in the regulation of the cell cycle, the response to genotoxic stress, and intracellular signaling mediated by the integrin family [9]. There are three isoforms of the BCR-ABL fusion protein all of which encode the same portion of the ABL tyrosine kinase, but differ in the length of the BCR sequence at the N-terminus. p185/p190 BCR-ABL is expressed in Acute Lypmphoblastic Leukaemia (ALL), p210 BCR-ABL is characteristic of Chronic Myeloid Leukemia, and p230 BCR-ABL has been associated with a subgroup of CML patients with a more indolent disease (Figure 1) [4].

CML was initially described in 1845 by John Hughes Bennett, who reported the case of a patient with "milky" blood and suggested that it was an infectious disease that caused hypertrophy of the liver and spleen, leading to the patient's death. A few weeks later, Rudolf Virchow reported a similar case, but, in contrast to Bennett, he suggested that the disease was not infectious and implied an increase in the number of blood cells. He coined the term leukemia (from the Greek *leukos*, white, and "Aemia", blood). In 1870, Neumann described that leukemia cells originate in the bone marrow; almost one hundred years later, in 1960, Nowel and Hungerford reported that in all cases of this malignancy there was a small, abnormal chro‐ mosome 22. However, was until 1973 that Janet Rowley described that the abnormal chromo‐ some was caused by a reciprocal translocation between the long arms of chromosomes 9 and

Chronic myelogenous leukemia has a worldwide incidence of 1-2 cases per 100,000 individuals [4]. The average age at diagnosis is 60 years; it occurs less frequently in young people and a tendency to increase exponentially with age has been observed. There is no geographic or genetic predisposition to acquire this condition, although some authors have associated it with exposure to high doses of ionizing radiation. The current CML prevalence of 24,000 affected patients in the United Sates is relatively low; it is expected to increase significantly over the next 20 years as a result of widespread use of BCR-ABL tyrosine kinase inhibitor therapy [5]. In Mexico, there are no official data on the incidence of such a disease, however, it has been estimated that there are about 80,000 cases of leukemia and 10% corresponds to CML [6].

The clinical presentation often includes granulocytosis, spenomegaly and marrow hypercel‐ lularity; however about 40% of patients are asymptomatic and their diagnosis is based on abnormal blood cell counts [1]. The natural course of the disease involves three sequential phases, namely chronic, accelerated and blast crises. Ninety percent of patients are diagnosed in chronic phase and they remain in it for 3 to 8 years. In this phase, the blood cells retain their ability to differentiate until the illness progresses to the accelerated phase, which is character‐ ized by the egress of immature cells into the bloodstream. Finally, the disease progresses to the blast crisis, defined by the presence of 30 percent or more leukemic cells in peripheral blood or marrow or extramedullary infiltrates of blast. During this phase the survival of patients is

As mentioned before, the Philadelphia chromosome, which defines CML, is a shortened chromosome 22 originated from the reciprocal translocation between the long arms of chromosomes 9 and 22 [t (9; 22)] and involves addition of 3' segments of the *abl* gene (9q34) to 5' segments of the *bcr* gene (22q11) given rise to a *bcr-abl* fusion gene that transcribes a chimeric mRNA of 8.5 kb that, in turn, gives rise to a BCR-ABL fusion protein [7]. t(9;22) is evident in more than 95% of CML patients; between 5% and 10% of CML patients also present complex rearrangements that may involve one or more chromosomes in addition to 9 and 22 [8].

22, designating the name of Philadelphia (Ph) chromosome[2, 3].

**2.1. Epidemiology and clinical characteristics**

138 Stem Cell Biology in Normal Life and Diseases

reduced to months and even weeks [7].

**2.2. Molecular events (Bcr-Abl oncogene)**

**Figure 1.** Structure of the Bcr-Abl gene. It is formed by a reciprocal translocation between chromosomes 22 (Bcr gene) and 9 (Abl gene). Ther M-BCR breakpoint resulting in a P210 BCR/ABL fusion transcripts b2a2 or b3a2 and they encode a pro‐ tein of 210 kDa (BCR-ABLp210) present in almost all patients with Chronic Mieloid Leukemia (modified to [9]).

BCR-ABL fusion protein localizes in the cytoplasm and shows an increased and constitutive tyrosine kinase activity as a result of oligomerization of its coiled region and deletion of the SH domain of ABL. It activates a number of cytoplasmic and nuclear signal-transduction pathways involved in cell adherence, migration, inhibition of apoptosis, and induction of cell proliferation through activation of signaling proteins such as p21RAS, c-Myc, lipid kinasse PI3k, MAPk (mitogen-activated protein kinase family), tyrosine phosphatases, and signal transducer and activator of transcription (STATs) factors [9, 10].

#### **2.3. Leukemic Stem Cells in chronic myeloid leukemia**

There is an increasing body of evidence indicating that, similar to normal hematopoiesis, a quiescent stem cell population -within the CD34+ cell compartment- exists in the bone marrow of CML patients. Such Leukemic Stem Cells (LSC) seem to be the ones driving CML progres‐ sion, following a similar pattern to the one observed in normal hematopoiesis. That is to say, LSC give rise to CML progenitor cells, which, in turn, give rise to more mature cells.

Just like normal hematopoietic stem cells (HSC), CML stem cells express high levels of CD34, and lack the cell surface markers CD38, CD45RA, or CD71, as well as lineage-specific markers. However, LSC are Ph+ /BCR-ABL+ , which is not present in their normal counterparts. Interest‐ ingly, it has recently been shown that a novel population of lineage-negative, CD34-negative hematopoietic stem cells from CML patients also correspond to BCR-ABL+ leukemic stem cells capable to engraft immunodeficient mice [13]. Thus, it seems that most LSC are CD34+ but a subpopulation may be CD34- . Importantly, despite the predominance of LSC in CML, a residual population of normal hematopoietic stem cells (BCR-ABL- CD34+ ) persists in the marrow's patient, which seems to be responsible for hematopoietic recovery after a successful treatment using Tyrosine Kinase Inhibitors (TKIs).

the two major STATs phosphorilated by Bcr-Abl. STAT5 has pleiotropic physiologic functions, and its main effect in Bcr-Abl-transformed cells appears to be primarily anti-apoptotic, involving transcriptional activation of Bcl-xL [25]. Also, in some experimental systems there is evidence that Bcr-Abl induces an IL-3 and G-CSF autocrine loop in early progenitor cells [12].

Hematopoietic Stem Cells in Chronic Myeloid Leukemia

http://dx.doi.org/10.5772/54651

141

Leukemic Stem Cells acquire the ability for long-term survival primarily by deregulation of apoptosis. In CML, blocking of apoptosis is mediated by Bcr-Abl. Bcr-Abl may block the release of cytochrome C from mitochondria and thus activation of caspases. This ef‐ fect upstream of caspase activation might be mediated by the Bcl-2 family of proteins [26]. Bcr-Abl has been shown to up-regulate anti-apoptotic protein Bcl-xL in a STAT5-de‐ pend manner, as mention above [27]. Another link between Bcr-Abl and the inhibition of apoptosis might be the phosphorylation of the pro-apoptotic protein Bad through PI3k pathway. Bcr-Abl forms multimeric complexes with PI3 kinase, Cbl, and the adapter molecules Crk and Crkl, in which PI3 kinase is activated. The next substrate in this cas‐ cade appears to be the serine-threonine kinase Akt. This kinase had previously been im‐ plicated in antiapoptotic signaling and protein Bad as a key substrate of Akt (Figure 1). Phosphorylated Bad is inactive because it is no longer able to bind anti-apoptotic pro‐

In CML, progenitor cells exhibit decreased adhesion to bone marrow stroma cells and extracellular matrix. From this point of view, adhesion to stroma negatively regulates cell proliferation, and CML cells escape this regulation by virtue of their perturbed adhesion properties. Bcr-Abl directly phosphorylates Crkl, a protein involved in the regulation of cell motility and in integrin-mediated cell adhesion by association with other focal adhesion proteins such as paxillin, the focal adhesion kinase Fak, p130 Cas and Hef1 [29, 30] (Figure 1). In addition to this, it has been demonstrated that the activity of Bcr-Abl promotes expression of integrin β1, a variant not found in the normal counterpart that inhibits adhesion to stroma and cell matrix, together with the effect of expansion and premature exit of myeloid progen‐

Deregulation of self-renewal has been recognized as an important event in disease progression. In normal hematopoietic stem cells, self-renewal capacity involves several signaling pathways:

Notch receptors are an evolutionarily conserved family of trans-membrane receptors that are known to be expressed and activated in normal HSC. Binding to their physiological ligands, which are part of the Delta and Serrata families, leads to separation of an intracellular portion of Notch. This fragment is capable of entering the nucleus where it binds transcriptional

teins such as Bcl-xL and it is trapped by cytoplasmic 14-3-3 proteins [28].

**3.2. Inhibition of apoptosis**

**3.3. Altered adhesion properties**

itors and precursors to bloodstream [31].

Notch, Wnt, Sonic Hedgehog (Shh), FoxO and Alox5 [32-34].

**3.4. Self-renewal**

**Notch pathway**

As mentioned before, LSC are in a quiescent state, however, they can spontaneously exit G0 to enter a proliferating state and are capable of engrafting inmmunodeficient mice [11]. In this regard, several studies have shown that TKIs, like Imatinib, Nilotinib, Dasatinib, Bosutinib, and Lonafarnib, have antiproliferative or apoptotic effects in almost all dividing CML cells; however, the population of stem cells remains viable in a quiescent state [16-21].

In vitro studies indicate that LSCs are capable of surviving for several weeks in the absence of added growth factors due to autocrine mechanisms involving production of granulocyte colony-stimulating factor (G-CSF) and Interleukin 3 (IL-3) [12]. This, in fact, is an important difference between normal and CML HSC, since the former depends on the presence of exogenous cytokines for their growth, whereas the latter, as just mentioned, can utilize autocrine mechanisms. Although there is strong evidence that Bcr-Abl is sufficient to induce CML-like disease in transduction and transgenic murine models [14], it is still unclear whether Bcr-Abl is always the first hit in CML, since in some patients with a complete cytogenetic response after treatment, BCR-ABL transcripts are still detectable by RT-PCR, which indicates that leukemic cells persist even when the disease is reduced below detectable limits [15].

#### **3. Functional characteristic of leukemic stem cells in CML**

#### **3.1. Proliferation**

Proliferation of leukemic stem and progenitor cells is regulated by Bcr-Abl. Such a tyrosine kinase activates the Ras/Raf/MEK/ERK and JAK/STAT signal transduction pathways, and this results in an amplified proliferative state [22]. Bcr-Abl causes hyperactivity of Ras, Raf and JAK/STAT, which can occur by multiple mechanisms; i.e., by Bcr-Abl activating these path‐ ways directly, or by the induction of autocrine cytokines, which in turn activate these pathways [23]. Bcr-Abl autophosphorylation of tyrosine 177 provides a docking site for the adapter molecule Grb-2. Grb-2, after binding to the Sos protein, stabilizes Ras in its active GTP-bound form. Two other adapter molecules, Shc and Crkl, can also activate Ras [9, 24]. Ras activates Raf, and finally, Raf initiates a signaling cascade through the serine–threonine kinases Mek1/ Mek2 and Erk, which ultimately leads to the transcription of genes involved in cell proliferation and survival (Figure 1), such as c-Myc, Cyclin D, Cyclin A, Bcl-2, cytokines, etc [22].

The JAK/STAT pathway has been demonstrated to be constitutively activated In CML. Among all the molecules participating in these pathways, STAT1 and STAT5 have been found to be the two major STATs phosphorilated by Bcr-Abl. STAT5 has pleiotropic physiologic functions, and its main effect in Bcr-Abl-transformed cells appears to be primarily anti-apoptotic, involving transcriptional activation of Bcl-xL [25]. Also, in some experimental systems there is evidence that Bcr-Abl induces an IL-3 and G-CSF autocrine loop in early progenitor cells [12].

#### **3.2. Inhibition of apoptosis**

However, LSC are Ph+

**3.1. Proliferation**

subpopulation may be CD34-

140 Stem Cell Biology in Normal Life and Diseases

/BCR-ABL+

treatment using Tyrosine Kinase Inhibitors (TKIs).

, which is not present in their normal counterparts. Interest‐

. Importantly, despite the predominance of LSC in CML, a

but a

) persists in the

ingly, it has recently been shown that a novel population of lineage-negative, CD34-negative hematopoietic stem cells from CML patients also correspond to BCR-ABL+ leukemic stem cells capable to engraft immunodeficient mice [13]. Thus, it seems that most LSC are CD34+

marrow's patient, which seems to be responsible for hematopoietic recovery after a successful

As mentioned before, LSC are in a quiescent state, however, they can spontaneously exit G0 to enter a proliferating state and are capable of engrafting inmmunodeficient mice [11]. In this regard, several studies have shown that TKIs, like Imatinib, Nilotinib, Dasatinib, Bosutinib, and Lonafarnib, have antiproliferative or apoptotic effects in almost all dividing CML cells;

In vitro studies indicate that LSCs are capable of surviving for several weeks in the absence of added growth factors due to autocrine mechanisms involving production of granulocyte colony-stimulating factor (G-CSF) and Interleukin 3 (IL-3) [12]. This, in fact, is an important difference between normal and CML HSC, since the former depends on the presence of exogenous cytokines for their growth, whereas the latter, as just mentioned, can utilize autocrine mechanisms. Although there is strong evidence that Bcr-Abl is sufficient to induce CML-like disease in transduction and transgenic murine models [14], it is still unclear whether Bcr-Abl is always the first hit in CML, since in some patients with a complete cytogenetic response after treatment, BCR-ABL transcripts are still detectable by RT-PCR, which indicates that leukemic cells persist even when the disease is reduced below detectable limits [15].

Proliferation of leukemic stem and progenitor cells is regulated by Bcr-Abl. Such a tyrosine kinase activates the Ras/Raf/MEK/ERK and JAK/STAT signal transduction pathways, and this results in an amplified proliferative state [22]. Bcr-Abl causes hyperactivity of Ras, Raf and JAK/STAT, which can occur by multiple mechanisms; i.e., by Bcr-Abl activating these path‐ ways directly, or by the induction of autocrine cytokines, which in turn activate these pathways [23]. Bcr-Abl autophosphorylation of tyrosine 177 provides a docking site for the adapter molecule Grb-2. Grb-2, after binding to the Sos protein, stabilizes Ras in its active GTP-bound form. Two other adapter molecules, Shc and Crkl, can also activate Ras [9, 24]. Ras activates Raf, and finally, Raf initiates a signaling cascade through the serine–threonine kinases Mek1/ Mek2 and Erk, which ultimately leads to the transcription of genes involved in cell proliferation

and survival (Figure 1), such as c-Myc, Cyclin D, Cyclin A, Bcl-2, cytokines, etc [22].

The JAK/STAT pathway has been demonstrated to be constitutively activated In CML. Among all the molecules participating in these pathways, STAT1 and STAT5 have been found to be

residual population of normal hematopoietic stem cells (BCR-ABL- CD34+

however, the population of stem cells remains viable in a quiescent state [16-21].

**3. Functional characteristic of leukemic stem cells in CML**

Leukemic Stem Cells acquire the ability for long-term survival primarily by deregulation of apoptosis. In CML, blocking of apoptosis is mediated by Bcr-Abl. Bcr-Abl may block the release of cytochrome C from mitochondria and thus activation of caspases. This ef‐ fect upstream of caspase activation might be mediated by the Bcl-2 family of proteins [26]. Bcr-Abl has been shown to up-regulate anti-apoptotic protein Bcl-xL in a STAT5-de‐ pend manner, as mention above [27]. Another link between Bcr-Abl and the inhibition of apoptosis might be the phosphorylation of the pro-apoptotic protein Bad through PI3k pathway. Bcr-Abl forms multimeric complexes with PI3 kinase, Cbl, and the adapter molecules Crk and Crkl, in which PI3 kinase is activated. The next substrate in this cas‐ cade appears to be the serine-threonine kinase Akt. This kinase had previously been im‐ plicated in antiapoptotic signaling and protein Bad as a key substrate of Akt (Figure 1). Phosphorylated Bad is inactive because it is no longer able to bind anti-apoptotic pro‐ teins such as Bcl-xL and it is trapped by cytoplasmic 14-3-3 proteins [28].

#### **3.3. Altered adhesion properties**

In CML, progenitor cells exhibit decreased adhesion to bone marrow stroma cells and extracellular matrix. From this point of view, adhesion to stroma negatively regulates cell proliferation, and CML cells escape this regulation by virtue of their perturbed adhesion properties. Bcr-Abl directly phosphorylates Crkl, a protein involved in the regulation of cell motility and in integrin-mediated cell adhesion by association with other focal adhesion proteins such as paxillin, the focal adhesion kinase Fak, p130 Cas and Hef1 [29, 30] (Figure 1). In addition to this, it has been demonstrated that the activity of Bcr-Abl promotes expression of integrin β1, a variant not found in the normal counterpart that inhibits adhesion to stroma and cell matrix, together with the effect of expansion and premature exit of myeloid progen‐ itors and precursors to bloodstream [31].

#### **3.4. Self-renewal**

Deregulation of self-renewal has been recognized as an important event in disease progression. In normal hematopoietic stem cells, self-renewal capacity involves several signaling pathways: Notch, Wnt, Sonic Hedgehog (Shh), FoxO and Alox5 [32-34].

#### **Notch pathway**

Notch receptors are an evolutionarily conserved family of trans-membrane receptors that are known to be expressed and activated in normal HSC. Binding to their physiological ligands, which are part of the Delta and Serrata families, leads to separation of an intracellular portion of Notch. This fragment is capable of entering the nucleus where it binds transcriptional repressor CBF-1. Interconnection of Notch, CBF-1 and the co-factor MAML-1 (mastermindlike-1) leads to transcriptional activation of target genes [35]. Constitutively active Notch is able to mediate multilineage potential *in vivo*. Differentiation of cells leads conversely to downregulation of Notch [36].

more, a study of zebrafish showed that the mutations of the Hh pathway members or in‐ hibition of the Hh pathway with the Hh inhibitor cyclopamine can cause a developmental defect in adult HSC [45]. In addition, activation of Hh pathway has been observed in different human cancers. In CML patients, more than four-fold induction of the transcript levels of Gli1 and Ptch was observed in CD34+ cells in both chronic phase and blast crisis. In two studies using a CML mouse model, recipients of the Bcr-Abl transduced bone marrow cells from Smo-/- donor mice developed CML significantly slow‐ er than recipients of Bcr-Abl transduced bone marrow cells from wild-type donor mice. When the frequency and function of the LSCs were examined, Smo deletion caused a significant reduction of the percentage or LSCs [46]. By contrast, over expression of Smo led to an increased percentage of LSC and accelerated the progression of CML [47].

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The FoxO (Forkhead-O) subfamily of transcription factors regulate cell cycle, stress resistance, differentiation, and long-term regenerative potential of HSC [48], and protect integrity of the stem cell pool. There are four members (FoxO1, FoxO3, FoxO4 and FoxO6) and are known to be effectors of the PI3k/AKT pathway, which is frequently mutated or hyperactivated in hematologic malignancies, and are abundantly expressed in the hematopoietic system. Akt directly phosporylates the FoxO members from the nucleus and promotes its degradation in the cytoplasm. FoxO members localize to the nucleus and regulate apoptosis, cell cycle progression and oxidative stress responses [49]. In a model of deficient FoxO mice it was shown a defect in the long-term expansion capacity of the HSC pool. Such a defect has been correlated

FoxO transcription factors have also been shown to have essential roles in the maintenance of CML LSCs [50]. FoxO3 localizes to the cell nucleus and it causes a decrease in Akt phosphor‐ ylation in the LSC population. In addition, serial CML transplantation showed that FoxO3 deficiency severely impairs the ability of LSCs to induce CML. Furthermore, transforming growth factor-β (TGF-β) is a crucial regulator of Akt activation and controls FoxO3 localization in LSCs of CML. A combination strategy of TGF-β inhibition, FoxO3 deficiency and Bcr-Abl kinase inhibition results in efficient LSCs depletion and suppression of CML development [51].

The Alox5 pathway is the only one signaling pathway not shared by LSC with normal HSC. The *Alox5* gene encoding arachidonate 5-lipoxygenase (5-LO) is involved in numerous physiological and pathological processes, including oxidative stress response, inflammation and cancer [52]. 5-LO is responsible for producing leukotrienes, a group of inflammatory substances that cause human asthma [53]. Altered arachidonate metabolism by leukocytes and platelets was reported in association with myeloproliferative disorders [54]. Several selective 5-LO inhibitors were found to reduce proliferation and induce apoptosis of CML cells in vitro [55]. Recently, human CML microarray studies have shown that Alox5 is differentially

the function of Alox5 in LSCs needs to be tested. Other microarray analysis of gene expression in LSCs in CML mice showed that the ALox5 gene was up-regulated by Bcr-Abl and that this

CML cells suggesting a role for Alox5 in human CML stem cells. However,

**FoxO pathway**

**Alox5 pathway**

expressed in CD34+

with increased cell division and apoptosis of HSCs.

Notch signaling may also be important in advanced stages of CML. Hes1, a key Notch target gene, was found to be highly expressed in 8 out of 20 patients with CML in blast crisis, but was not seen in the chronic phase. In mice, the combination of Hes1 and BCR-ABL expression in myeloid lineage progenitor cells resulted in an acute leukemia resem‐ bling blast crisis CML [37]. This suggests that Notch inhibitors may be useful in strategies aimed at eradicating CML LSC.

#### **Wnt pathway**

In normal hematopoiesis, Wnt pathway activity is required in the bone marrow niche to regulate HSC proliferation and to preserve self-renewal capacity [38]. Activation of the canonical Wnt/β-catenin pathway consists of binding of Wnt proteins to members of the Frizzled and low-density lipoprotein receptor related (LPR) families on the cell surface. In the absence of Wnt signals, β-catenin is associated with a large multiprotein complex that includes Axin, APC, and glycogen synthase kinase 3β (GSK3β), among others. Through a mechanism not entirely understood, when Wnt proteins bind to their target, Axin facilitates phosphorylation of β-catenin by GSK3β. Phosphorylation, in turn, results in ubiquitination, targeting β-catenin for degradation. Thus, axin serves as an inhibitor of β-catenin activity. Binding of Wnt proteins to their receptors leads to activation of Dis‐ shevled (Dsh), which inhibits phosphorylation of β-catenin by GSKβ, so it accumulates in the cytoplasm and translocates to the nucleus, where it activates transcription factors, such as LEF/TEF and allows expression of target genes [39].

This pathway has been implicated in CML. Indeed, in blast crisis CML, the LSC, which resemble granulocyte-macrophage progenitor cells (GMP), have aberrant activation of βcatenin via the canonical Wnt signaling pathway. In a proportion of these cases, the pathway is activated through abnormal missplicing of GSK3β [40].

#### **Sonic Hedgehog (Shh) pathway**

The Hedgehog (Hh) pathway is a highly conserved developmental pathway, which regulates the proliferation, migration and differentiation of cells during development [41]. It is typically active during development, but silenced in adult tissues, except during tissue regeneration and injury repair [42]. Three distinct ligands, i.e., Sonic (Shh), Indian (Ihh) and Desert (Dhh) Hedgehog exist in humans. Upon ligand binding to the receptor patched (Ptch), inhibition of smoothened (Smo) receptor is relieved. Smo then activates members of the Gli family of zincfinger transcription factors, which translocate to the nucleus to regulate the transcription of Hh target genes, including Gli1, Gli2, Ptch and regulators of cell proliferation and survival [43].

Based on murine embryonic stem cell studies, it has been found that Hh signaling plays major roles during primitive hematopoiesis. Ihh is a primitive endoderm-secreted signal and is sufficient to activate embryonic hematopoiesis and vasculogenesis [44]. Further‐ more, a study of zebrafish showed that the mutations of the Hh pathway members or in‐ hibition of the Hh pathway with the Hh inhibitor cyclopamine can cause a developmental defect in adult HSC [45]. In addition, activation of Hh pathway has been observed in different human cancers. In CML patients, more than four-fold induction of the transcript levels of Gli1 and Ptch was observed in CD34+ cells in both chronic phase and blast crisis. In two studies using a CML mouse model, recipients of the Bcr-Abl transduced bone marrow cells from Smo-/- donor mice developed CML significantly slow‐ er than recipients of Bcr-Abl transduced bone marrow cells from wild-type donor mice. When the frequency and function of the LSCs were examined, Smo deletion caused a significant reduction of the percentage or LSCs [46]. By contrast, over expression of Smo led to an increased percentage of LSC and accelerated the progression of CML [47].

#### **FoxO pathway**

repressor CBF-1. Interconnection of Notch, CBF-1 and the co-factor MAML-1 (mastermindlike-1) leads to transcriptional activation of target genes [35]. Constitutively active Notch is able to mediate multilineage potential *in vivo*. Differentiation of cells leads conversely to

Notch signaling may also be important in advanced stages of CML. Hes1, a key Notch target gene, was found to be highly expressed in 8 out of 20 patients with CML in blast crisis, but was not seen in the chronic phase. In mice, the combination of Hes1 and BCR-ABL expression in myeloid lineage progenitor cells resulted in an acute leukemia resem‐ bling blast crisis CML [37]. This suggests that Notch inhibitors may be useful in

In normal hematopoiesis, Wnt pathway activity is required in the bone marrow niche to regulate HSC proliferation and to preserve self-renewal capacity [38]. Activation of the canonical Wnt/β-catenin pathway consists of binding of Wnt proteins to members of the Frizzled and low-density lipoprotein receptor related (LPR) families on the cell surface. In the absence of Wnt signals, β-catenin is associated with a large multiprotein complex that includes Axin, APC, and glycogen synthase kinase 3β (GSK3β), among others. Through a mechanism not entirely understood, when Wnt proteins bind to their target, Axin facilitates phosphorylation of β-catenin by GSK3β. Phosphorylation, in turn, results in ubiquitination, targeting β-catenin for degradation. Thus, axin serves as an inhibitor of β-catenin activity. Binding of Wnt proteins to their receptors leads to activation of Dis‐ shevled (Dsh), which inhibits phosphorylation of β-catenin by GSKβ, so it accumulates in the cytoplasm and translocates to the nucleus, where it activates transcription factors,

This pathway has been implicated in CML. Indeed, in blast crisis CML, the LSC, which resemble granulocyte-macrophage progenitor cells (GMP), have aberrant activation of βcatenin via the canonical Wnt signaling pathway. In a proportion of these cases, the pathway

The Hedgehog (Hh) pathway is a highly conserved developmental pathway, which regulates the proliferation, migration and differentiation of cells during development [41]. It is typically active during development, but silenced in adult tissues, except during tissue regeneration and injury repair [42]. Three distinct ligands, i.e., Sonic (Shh), Indian (Ihh) and Desert (Dhh) Hedgehog exist in humans. Upon ligand binding to the receptor patched (Ptch), inhibition of smoothened (Smo) receptor is relieved. Smo then activates members of the Gli family of zincfinger transcription factors, which translocate to the nucleus to regulate the transcription of Hh target genes, including Gli1, Gli2, Ptch and regulators of cell proliferation and survival [43].

Based on murine embryonic stem cell studies, it has been found that Hh signaling plays major roles during primitive hematopoiesis. Ihh is a primitive endoderm-secreted signal and is sufficient to activate embryonic hematopoiesis and vasculogenesis [44]. Further‐

downregulation of Notch [36].

142 Stem Cell Biology in Normal Life and Diseases

**Wnt pathway**

strategies aimed at eradicating CML LSC.

such as LEF/TEF and allows expression of target genes [39].

is activated through abnormal missplicing of GSK3β [40].

**Sonic Hedgehog (Shh) pathway**

The FoxO (Forkhead-O) subfamily of transcription factors regulate cell cycle, stress resistance, differentiation, and long-term regenerative potential of HSC [48], and protect integrity of the stem cell pool. There are four members (FoxO1, FoxO3, FoxO4 and FoxO6) and are known to be effectors of the PI3k/AKT pathway, which is frequently mutated or hyperactivated in hematologic malignancies, and are abundantly expressed in the hematopoietic system. Akt directly phosporylates the FoxO members from the nucleus and promotes its degradation in the cytoplasm. FoxO members localize to the nucleus and regulate apoptosis, cell cycle progression and oxidative stress responses [49]. In a model of deficient FoxO mice it was shown a defect in the long-term expansion capacity of the HSC pool. Such a defect has been correlated with increased cell division and apoptosis of HSCs.

FoxO transcription factors have also been shown to have essential roles in the maintenance of CML LSCs [50]. FoxO3 localizes to the cell nucleus and it causes a decrease in Akt phosphor‐ ylation in the LSC population. In addition, serial CML transplantation showed that FoxO3 deficiency severely impairs the ability of LSCs to induce CML. Furthermore, transforming growth factor-β (TGF-β) is a crucial regulator of Akt activation and controls FoxO3 localization in LSCs of CML. A combination strategy of TGF-β inhibition, FoxO3 deficiency and Bcr-Abl kinase inhibition results in efficient LSCs depletion and suppression of CML development [51].

#### **Alox5 pathway**

The Alox5 pathway is the only one signaling pathway not shared by LSC with normal HSC. The *Alox5* gene encoding arachidonate 5-lipoxygenase (5-LO) is involved in numerous physiological and pathological processes, including oxidative stress response, inflammation and cancer [52]. 5-LO is responsible for producing leukotrienes, a group of inflammatory substances that cause human asthma [53]. Altered arachidonate metabolism by leukocytes and platelets was reported in association with myeloproliferative disorders [54]. Several selective 5-LO inhibitors were found to reduce proliferation and induce apoptosis of CML cells in vitro [55]. Recently, human CML microarray studies have shown that Alox5 is differentially expressed in CD34+ CML cells suggesting a role for Alox5 in human CML stem cells. However, the function of Alox5 in LSCs needs to be tested. Other microarray analysis of gene expression in LSCs in CML mice showed that the ALox5 gene was up-regulated by Bcr-Abl and that this up-regulation was not inhibited by Imatinib treatment, providing a possible explanation of why LSCs are not sensitive to inhibition by Bcr-Abl kinase inhibitors [56].

**4. Current therapies**

**4.1. Interferon-α**

with a median survival of 89 months [58].

β2-Integrin, L-selectin, ICAM-1 and ICAM352 [58, 62].

**4.2. Tyrosine kinase inhibitors**

The first effective treatment for CML was the solution of Fowler's, which contained arsenic as active component and was used in the early 20th century. Later between 1920 y1930 irradiation to the spleen was the main therapeutic option, since it offered patients the decrease of symptoms, although it did not prolong their lives. In 1953, busulfan was included in CML treatment. This compound provided benefit in terms of survival, although it was shown to be extremely toxic for hematopoietic progenitor cells. The next drugs effective in the treatment of CML were hydroxyurea and cytosine arabinoside, both less toxic than busulfan and able to block proliferation of cells, but unable to induce specific damage to leukemic cells; thus,

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Interferon-α (IFNα) was the first drug capable of extending the chronic phase of the disease and retarding the evolution to the accelerated phase. IFNα is a nonspecific stimulant of the immune system that regulates T-cell activity and produces a complete hematologic response (CHR) in 40-80% of patients, and a complete cytogenetic response (CCR) in 6-10% of patients

In vitro studies have indicated that IFN*α* might function via selective toxicity against the leukemic clone, since it is able to inhibit long-term cultures from patients with CML in chronic phase and reduces the percentage of Ph+ cells [59]. It also inhibits CML myeloid progenitors while sparing normal myeloid progenitors [60]. In vivo, IFNα enhances immune regulation through the activation of dendritic, natural killer, and cytotoxic T cells, all of them capable of generating anti-tumor responses. In Bcr-Abl+ cells, IFNα induces a state of tumor dormancy and delays progression to advanced phase [61], and is able to modulate hematopoiesis through enhanced adhesion of CML progenitor cells to stromal cells, whereas adhesion of normal progenitors was unaffected. This enhanced adhesion by CML progenitor cells has been associated with a reduction in neuraminic acid levels and by enhanced hematopoietic cellmicroenvironmental cell interactions, which is achieved by the induction of molecules such as

Because IFNα is a nonspecific immunostimulant, it produces secondary symptoms and toxicities and many patients discontinue therapy. However there are evidence that a significant proportion of IFNα-treated patients in prolonged CCR were able to discontinue treatment without disease relapse [63], and it was recently reported that in a specific group of patients treated with monotherapy there are increased numbers of NK cells and clonal γδ T cells [64].

Having identified that tyrosine kinase activity of Bcr-Abl is a major factor in the pathophysi‐ ology of CML, it was clear that such a molecule was an attractive target for designing a selective kinase inhibitor. In 1996, Buchdunger et al, synthesized several compounds that inhibit the activity of platelet-derived growth factor receptor (PDGF-R) and ABL kinase. One of these was

patients usually progressed to the accelerated and blast crisis phases [57].

**Figure 2.** Signaling pathways involved in the signaling of BCR-ABL. A) Schematic representation of principal molecules that participate in proliferation, adhesion and apoptosis. B) Pathways involved in self-renewal.

#### **4. Current therapies**

up-regulation was not inhibited by Imatinib treatment, providing a possible explanation of

**Figure 2.** Signaling pathways involved in the signaling of BCR-ABL. A) Schematic representation of principal molecules

that participate in proliferation, adhesion and apoptosis. B) Pathways involved in self-renewal.

why LSCs are not sensitive to inhibition by Bcr-Abl kinase inhibitors [56].

144 Stem Cell Biology in Normal Life and Diseases

The first effective treatment for CML was the solution of Fowler's, which contained arsenic as active component and was used in the early 20th century. Later between 1920 y1930 irradiation to the spleen was the main therapeutic option, since it offered patients the decrease of symptoms, although it did not prolong their lives. In 1953, busulfan was included in CML treatment. This compound provided benefit in terms of survival, although it was shown to be extremely toxic for hematopoietic progenitor cells. The next drugs effective in the treatment of CML were hydroxyurea and cytosine arabinoside, both less toxic than busulfan and able to block proliferation of cells, but unable to induce specific damage to leukemic cells; thus, patients usually progressed to the accelerated and blast crisis phases [57].

#### **4.1. Interferon-α**

(b)

(a)

)

Interferon-α (IFNα) was the first drug capable of extending the chronic phase of the disease and retarding the evolution to the accelerated phase. IFNα is a nonspecific stimulant of the immune system that regulates T-cell activity and produces a complete hematologic response (CHR) in 40-80% of patients, and a complete cytogenetic response (CCR) in 6-10% of patients with a median survival of 89 months [58].

In vitro studies have indicated that IFN*α* might function via selective toxicity against the leukemic clone, since it is able to inhibit long-term cultures from patients with CML in chronic phase and reduces the percentage of Ph+ cells [59]. It also inhibits CML myeloid progenitors while sparing normal myeloid progenitors [60]. In vivo, IFNα enhances immune regulation through the activation of dendritic, natural killer, and cytotoxic T cells, all of them capable of generating anti-tumor responses. In Bcr-Abl+ cells, IFNα induces a state of tumor dormancy and delays progression to advanced phase [61], and is able to modulate hematopoiesis through enhanced adhesion of CML progenitor cells to stromal cells, whereas adhesion of normal progenitors was unaffected. This enhanced adhesion by CML progenitor cells has been associated with a reduction in neuraminic acid levels and by enhanced hematopoietic cellmicroenvironmental cell interactions, which is achieved by the induction of molecules such as β2-Integrin, L-selectin, ICAM-1 and ICAM352 [58, 62].

Because IFNα is a nonspecific immunostimulant, it produces secondary symptoms and toxicities and many patients discontinue therapy. However there are evidence that a significant proportion of IFNα-treated patients in prolonged CCR were able to discontinue treatment without disease relapse [63], and it was recently reported that in a specific group of patients treated with monotherapy there are increased numbers of NK cells and clonal γδ T cells [64].

#### **4.2. Tyrosine kinase inhibitors**

Having identified that tyrosine kinase activity of Bcr-Abl is a major factor in the pathophysi‐ ology of CML, it was clear that such a molecule was an attractive target for designing a selective kinase inhibitor. In 1996, Buchdunger et al, synthesized several compounds that inhibit the activity of platelet-derived growth factor receptor (PDGF-R) and ABL kinase. One of these was the 2- phenylaminopyrimidine, which served as a starting point for the development of other related compounds [65]. The activity of the 2-phenylaminopyrimidine series was optimized and gave rise to STI571 (also named imatinib mesylate, CGP57148B or Gleevec®, Novartis Pharmaceuticals).

Imatinib-induced apoptosis is preceded by Bim accumulation; this effect was decreased when cells were cultured in a cytokine-containing medium [78]. In contrast to Imatinib, whose main effect on CML cells seems to be induction of apoptosis, the predominant effect of nilotinib seems to be antiproliferative -rather than apoptotic [17]. Indeed, it has been suggested that Nilotinib can induce a G0/G1 cell cycle blockade in cells expressing wild type Bcr-Abl, which

Dasatinib (Sprycel, Bristol-Myers Squibb) is a potent, orally bioavailable thiazolecarboxamide. It is structurally unrelated to imatinib; it has the ability to bind to multiple conformations of the Abl kinase domain and it also inhibits SRC family kinases. In vitro, Dasatinib demonstrated 325-fold greater activity against native Bcr-Abl, as compared with imatinib, and it has shown efficacy against all imatinib-resistant Bcr-Abl mutants with the exception of T351I. Dasatinib

Dasatinib is very effective at inducing apoptosis in CML cells –either, in the presence or absence of added growth factors- and in contrast to Imatinib, that kills those cells destined to move from G0/G1 cell cycle phases, but is unable to act on those cells destined to remain quiescent in

mode of action, it has selective cytotoxic activity for leukemic cells over normal cells [80].

Several TKIs have been developed that exhibit a target spectrum similar to the approved drugs,

Bosutinib (Wyeth) is a 4 anilino-3-quinolinecarbonitrile dual inhibitor of Src and Abl kinases without effect in c-Kit or PDGFR. It has 200-fold grater potency for Bcr-Abl than imatinib and has activity against a number of mutations, but not T315I [76]. In clinical trials, Bosutinib induced 73% of complete hematological response in patients pretreated with Imatinib followed by Dasatinib [82]. In vitro, Bosutinib effectively inhibits Bcr-Abl kinase activity and Src phosphorylation, and reduces the proliferation and CFC growth in CML CD34+ cells; however,

Ponatinib, is a mulitargeted kinase inhibitor that is active against all BCR-ABL mutants, including T315I. This drug also inhibits FLT3, FGFR, VEGFR, c-Kit, and PDGFR and is able to reduce the proliferation of different cell lines and prolong survival of mice that have been injected intravenously with BCR-ABL. Ponatinib showed significant activity in a phase I study

Although molecular therapy for CML is highly effective and generally non-toxic, it is unclear whether long-term outcomes with the different therapies (IFNα or TKIs) will be equivalent to

cells. As expected, based on its structure and

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is also active against PDGFR, C-Kit and ephrin A receptor [75, 76].

could result in disease persistence [79].

culture, Dasatinib can act on quiescent CD34+

although they are distinct in terms of off-target effects [81].

of patients with Ph+ cells who had failed to other TKIs [81, 83].

**Other tyrosine kinase inhibitors**

it does not seem to induce apoptosis [19].

**4.3. Hematopoietic cell transplant**

**SKI-606 (Bosutinib)**

**AP24534 (Ponatinib)**

**Dasatinib**

#### **Imatinib**

Imatinib is a highly selective inhibitor of the protein tyrosine kinase family, which includes BCR-ABL protein, PDGF-R and the c-kit receptor. It competitively binds to the ATP-binding site of BCR.ABL and inhibits protein tyrosine phosphorylation *in vitro* and *in vivo* [66]. In vitro studies had shown that Imatinib is capable to inhibit cell proliferation of cell lines expressing Bcr-Abl [67-69], effect accomplished through JAK5-STAT and PI3 kinase signaling inhibition [70, 71]. It has also been shown that STI571 can inhibit CML MNC, obtained both in chronic phase and blast crisis [71] and reduces the colony forming cells from Mobilized Peripheral Blood (MPB) and Bone Marrow from patients with CML in chronic phase [60]. Furthermore, Imatinib inhibits proliferation and cell cycle of stem (CD34+CD38-) and progenitor (CD34+ CD38+ ) cells without altering the behavior of normal cells [72].

Studies in CML marrow by Holyoake and her colleagues have demonstrated the presence of a rare, highly quiescent, CD34+ cell subpopulation in which most of the cells are Ph+ with the ability to proliferate upon specific induction [11]. These cells are insensitive to the effects of STI571 and remain quiescent and viable even in the presence of growth factors [16]. This tumor resistance feature was also reported by Bathia, who mention that STI571 suppressed but does not eliminate primitive cells even after patients remain in CCR [73]. These primitive Ph+ cells could not be detected by nested PCR, when they are obtained from Imatinib-treated patients; however, when the cells are cultured in liquid cultures for a couple of weeks, the Ph+ popu‐ lation becomes detectable, indicating that they were able to remain even after Imatinib treatment [74].

In clinical trials, Imatinib has been shown remarkably effective as a single agent in IFNαresistant CML chronic phase patients. It induces complete cytogenetic responses in more than 80% of newly diagnosed patients; however, the persistence of detectable leukemic cells in a quiescent state and the presence of patients with resistance or intolerance to Imatinib, lead to the development of a second generation of Tyrosine Kinase Inhibithors.

#### **Nilotinib**

Nilotinib (Tasigna, Novartis Pharmaceutical), is an oral aminopyrimidine that is a structural derivative of Imatinib. It was designed to be more selective against the Bcr-Abl tyrosine kinase than imatinib. Like imatinib, it acts through competitive inhibition of the ATP site in the kinase domain [75]. Clinically Nilotinib showed activity in imatinib-resistant patients in all phases of the disease. In chronic phase, it induced 92% of CHR and in accelerated phase and blast crisis the hematological responses were achieved in 72% of cases [76].

In vitro, Nilotinib is 20 times more potent than imatinib against cells expressing wild type Bcr-Abl, and similar results have been observed in studies of mutants cell lines, with the exception of the T315I mutation, which is resistant to both TKIs [77]. In primary CML CD34+ cells, Imatinib-induced apoptosis is preceded by Bim accumulation; this effect was decreased when cells were cultured in a cytokine-containing medium [78]. In contrast to Imatinib, whose main effect on CML cells seems to be induction of apoptosis, the predominant effect of nilotinib seems to be antiproliferative -rather than apoptotic [17]. Indeed, it has been suggested that Nilotinib can induce a G0/G1 cell cycle blockade in cells expressing wild type Bcr-Abl, which could result in disease persistence [79].

#### **Dasatinib**

the 2- phenylaminopyrimidine, which served as a starting point for the development of other related compounds [65]. The activity of the 2-phenylaminopyrimidine series was optimized and gave rise to STI571 (also named imatinib mesylate, CGP57148B or Gleevec®, Novartis

Imatinib is a highly selective inhibitor of the protein tyrosine kinase family, which includes BCR-ABL protein, PDGF-R and the c-kit receptor. It competitively binds to the ATP-binding site of BCR.ABL and inhibits protein tyrosine phosphorylation *in vitro* and *in vivo* [66]. In vitro studies had shown that Imatinib is capable to inhibit cell proliferation of cell lines expressing Bcr-Abl [67-69], effect accomplished through JAK5-STAT and PI3 kinase signaling inhibition [70, 71]. It has also been shown that STI571 can inhibit CML MNC, obtained both in chronic phase and blast crisis [71] and reduces the colony forming cells from Mobilized Peripheral Blood (MPB) and Bone Marrow from patients with CML in chronic phase [60]. Furthermore, Imatinib inhibits proliferation and cell cycle of stem (CD34+CD38-) and progenitor

) cells without altering the behavior of normal cells [72].

Studies in CML marrow by Holyoake and her colleagues have demonstrated the presence of a rare, highly quiescent, CD34+ cell subpopulation in which most of the cells are Ph+ with the ability to proliferate upon specific induction [11]. These cells are insensitive to the effects of STI571 and remain quiescent and viable even in the presence of growth factors [16]. This tumor resistance feature was also reported by Bathia, who mention that STI571 suppressed but does not eliminate primitive cells even after patients remain in CCR [73]. These primitive Ph+ cells could not be detected by nested PCR, when they are obtained from Imatinib-treated patients; however, when the cells are cultured in liquid cultures for a couple of weeks, the Ph+ popu‐ lation becomes detectable, indicating that they were able to remain even after Imatinib

In clinical trials, Imatinib has been shown remarkably effective as a single agent in IFNαresistant CML chronic phase patients. It induces complete cytogenetic responses in more than 80% of newly diagnosed patients; however, the persistence of detectable leukemic cells in a quiescent state and the presence of patients with resistance or intolerance to Imatinib, lead to

Nilotinib (Tasigna, Novartis Pharmaceutical), is an oral aminopyrimidine that is a structural derivative of Imatinib. It was designed to be more selective against the Bcr-Abl tyrosine kinase than imatinib. Like imatinib, it acts through competitive inhibition of the ATP site in the kinase domain [75]. Clinically Nilotinib showed activity in imatinib-resistant patients in all phases of the disease. In chronic phase, it induced 92% of CHR and in accelerated phase and blast crisis

In vitro, Nilotinib is 20 times more potent than imatinib against cells expressing wild type Bcr-Abl, and similar results have been observed in studies of mutants cell lines, with the exception of the T315I mutation, which is resistant to both TKIs [77]. In primary CML CD34+ cells,

the development of a second generation of Tyrosine Kinase Inhibithors.

the hematological responses were achieved in 72% of cases [76].

Pharmaceuticals).

146 Stem Cell Biology in Normal Life and Diseases

**Imatinib**

(CD34+

CD38+

treatment [74].

**Nilotinib**

Dasatinib (Sprycel, Bristol-Myers Squibb) is a potent, orally bioavailable thiazolecarboxamide. It is structurally unrelated to imatinib; it has the ability to bind to multiple conformations of the Abl kinase domain and it also inhibits SRC family kinases. In vitro, Dasatinib demonstrated 325-fold greater activity against native Bcr-Abl, as compared with imatinib, and it has shown efficacy against all imatinib-resistant Bcr-Abl mutants with the exception of T351I. Dasatinib is also active against PDGFR, C-Kit and ephrin A receptor [75, 76].

Dasatinib is very effective at inducing apoptosis in CML cells –either, in the presence or absence of added growth factors- and in contrast to Imatinib, that kills those cells destined to move from G0/G1 cell cycle phases, but is unable to act on those cells destined to remain quiescent in culture, Dasatinib can act on quiescent CD34+ cells. As expected, based on its structure and mode of action, it has selective cytotoxic activity for leukemic cells over normal cells [80].

#### **Other tyrosine kinase inhibitors**

Several TKIs have been developed that exhibit a target spectrum similar to the approved drugs, although they are distinct in terms of off-target effects [81].

#### **SKI-606 (Bosutinib)**

Bosutinib (Wyeth) is a 4 anilino-3-quinolinecarbonitrile dual inhibitor of Src and Abl kinases without effect in c-Kit or PDGFR. It has 200-fold grater potency for Bcr-Abl than imatinib and has activity against a number of mutations, but not T315I [76]. In clinical trials, Bosutinib induced 73% of complete hematological response in patients pretreated with Imatinib followed by Dasatinib [82]. In vitro, Bosutinib effectively inhibits Bcr-Abl kinase activity and Src phosphorylation, and reduces the proliferation and CFC growth in CML CD34+ cells; however, it does not seem to induce apoptosis [19].

#### **AP24534 (Ponatinib)**

Ponatinib, is a mulitargeted kinase inhibitor that is active against all BCR-ABL mutants, including T315I. This drug also inhibits FLT3, FGFR, VEGFR, c-Kit, and PDGFR and is able to reduce the proliferation of different cell lines and prolong survival of mice that have been injected intravenously with BCR-ABL. Ponatinib showed significant activity in a phase I study of patients with Ph+ cells who had failed to other TKIs [81, 83].

#### **4.3. Hematopoietic cell transplant**

Although molecular therapy for CML is highly effective and generally non-toxic, it is unclear whether long-term outcomes with the different therapies (IFNα or TKIs) will be equivalent to cases treated with allogeneic stem cell transplantation, which has shown the highest percent‐ age of long-term disease-free survival of any therapy [75].

In CML, the criteria for successful response to treatment, as established by the European consortium LeukemiaNet and subsequently adopted by the National Comprehensive Cancer Network (NCCN) [88], include: complete hematologic remission (CHR), that is to say, a normal blood cell count and complete disappearance of signs and symptoms of the disease; complete cytogenetic response (CCR), which means the total absence of Ph+ metaphases; and complete molecular response, in which transcripts for BCR-ABL are no longer detectable. Using these response criteria, drug resistance is defined as the inability to achieve any of the following: a complete hematologic response (CHR) at 3 months, any cytogenetic response (CyR) at 6 months, partial cytogenetic response (PCyR) at 12 months, or a complete cytogenetic response

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Two types of resistance mechanisms to TKIs have been described: 1) Primary resistance, which occurs in less than 10% of cases and is defined as the failure of therapeutic effect during the chronic phase of CML without changing clones; and 2) secondary resistance, defined as the loss of the response initially obtained, and commonly occurs in accelerated phase (40-50%) and

It is estimated that the probability of an individual to stay in CCR for 5 years after diagnosis, after treatment with Imatinib is approximately 63%; however, this percentage may represent a sub-estimation since in a significant proportion of cases there is discontinuation of treatment

The molecular mechanisms of acquired drug resistance can be divided into two categories:

The inhibition of the activity of tyrosine kinase turned out to be an ideal target for molecular therapy in CML [67]. However, shortly after the introduction of Imatinib, in vitro studies demonstrated that some cell lines became refractory to the drug, suggesting a possible inherent or acquired resistance to therapy [92]. This was quickly followed by the clinical description of

The most common mechanism against TKIs therapy are point mutations within the kinase domain, which make conformational changes that decrease the affinity of the TKIs to BCR-ABL kinase domain. These point mutations in the *BCR-ABL* kinase domain are a major cause of Imatinib resistance, and may be identified in approximately 50% or more of the cases. Many more than 100 different mutations affecting more than 70 amino acids have so far been

The first point mutation reported in TKI resistance was in the region coding for the ATP-binding site of the ABL kinase domain resulting in a threonine to isoleucine substi‐ tution at amino acid 315 (Th315→Ile315; T315I) preventing the formation of a hydrogen bond between the oxygen atom provided by the side chain of threonine 315 and the sec‐ ondary amino group of Imatinib. Moreover, isoleucine contains an extra hydrocarbon

(CCR) at 18 months of treatment with Imatinib [89].

BCR-ABL-dependent and BCR-ABL-independent.

**5.1. Bcr-Abl-dependent resistance mechanisms**

identified, with varying degrees of clinical relevance [93].

patients resistant to Imatinib.

**BCR-ABL mutations**

and this, of course, may underestimate the efficacy of the drug [91].

blast (80%) [90]

In patients younger than 50 years of age and who receive a transplant before 1 year after diagnosis, 5 years survival rates superior to 70% have been attained. However, the application of this procedure is limited by the availability of matched donors and by the toxicity of the procedure in older patients. Moreover, outcomes deteriorate with disease duration [76]. This information associated with the knowledge that quiescent leukemic stem cells remain in patients after treatment, several other agents has been reported.

#### **4.4. Other agents**

Danusertib (PHA 739358) is a small molecule with activity against BCR-ABL and aurora kinases and it is able to block the proliferation of leukemia cell lines as well as CD34+ cells from newly diagnosed CML patients including the mutation T315I. However, similarly to other tyrosine kinase inhibitors, no induction of apoptosis in quiescent hematopoietic stem cells could be achieved and resistant BCR-ABL positive clones emerged in the course of Danusertib treatment. This latter observation is related to Abcg2 proteins over-expression [84].

Lonafarnib (SCH66336) is an orally bioavailable non peptidomimetic farnesyl trransferase inhibitor with significant activity against Bcr-Abl+ cell lines and primary CML cells. It can enhance the toxicity of Imatinib in K562 cell line and can inhibit the proliferation of imatinibresistant cells and increases imatinib-induced apoptosis. However it is unable to kill quiescent CD34+ leukemic cells [20]. In a clinical phase 1 study, it was shown that the combination of Lonafarnib and Imatinib is well tolerated in patients with CML who failed Imatinib, with some patients achieving a complete hematologic response and a complete cytogenetic response [85].

INNO 406 is a 2 phenylaminopyrimidine Bcr-Abl inhibitor with activity against PDGF, c-kit and Lyn that have shown to be 25-55 times more potent than Imatinib in Bcr-Abl+ cell lines. In contrast to other molecules INNO406 does not inhibit all SRC kinases, but it induces programmed cell death in chronic myelogenous leukemia (CML) cell lines through both caspase-mediated and caspase-independent pathways [86].

MK0457 is an aurora kinase inhibitor with activity against Bcr-Abl. This agent was observed to inhibit autophosphorylation of T315I mutant and demonstrate antiproliferative effects in CML cells derived from patients with this mutation, an event that may lead to its use as a combination partner with the approved and established TKI [76].

#### **5. TKI resistance mechanisms**

The knowledge of the central role of BCR-ABL in the pathogenesis of CML has allowed the development of several drugs that inhibit the constitutive activity of such an ABL tyrosine kinase. However, although the treatment with tyrosine kinase inhibitors has proven effective in about 80% of CML patients at any stage, the remaining 20% can't respond to it [87].

In CML, the criteria for successful response to treatment, as established by the European consortium LeukemiaNet and subsequently adopted by the National Comprehensive Cancer Network (NCCN) [88], include: complete hematologic remission (CHR), that is to say, a normal blood cell count and complete disappearance of signs and symptoms of the disease; complete cytogenetic response (CCR), which means the total absence of Ph+ metaphases; and complete molecular response, in which transcripts for BCR-ABL are no longer detectable. Using these response criteria, drug resistance is defined as the inability to achieve any of the following: a complete hematologic response (CHR) at 3 months, any cytogenetic response (CyR) at 6 months, partial cytogenetic response (PCyR) at 12 months, or a complete cytogenetic response (CCR) at 18 months of treatment with Imatinib [89].

Two types of resistance mechanisms to TKIs have been described: 1) Primary resistance, which occurs in less than 10% of cases and is defined as the failure of therapeutic effect during the chronic phase of CML without changing clones; and 2) secondary resistance, defined as the loss of the response initially obtained, and commonly occurs in accelerated phase (40-50%) and blast (80%) [90]

It is estimated that the probability of an individual to stay in CCR for 5 years after diagnosis, after treatment with Imatinib is approximately 63%; however, this percentage may represent a sub-estimation since in a significant proportion of cases there is discontinuation of treatment and this, of course, may underestimate the efficacy of the drug [91].

The molecular mechanisms of acquired drug resistance can be divided into two categories: BCR-ABL-dependent and BCR-ABL-independent.

#### **5.1. Bcr-Abl-dependent resistance mechanisms**

The inhibition of the activity of tyrosine kinase turned out to be an ideal target for molecular therapy in CML [67]. However, shortly after the introduction of Imatinib, in vitro studies demonstrated that some cell lines became refractory to the drug, suggesting a possible inherent or acquired resistance to therapy [92]. This was quickly followed by the clinical description of patients resistant to Imatinib.

#### **BCR-ABL mutations**

cases treated with allogeneic stem cell transplantation, which has shown the highest percent‐

In patients younger than 50 years of age and who receive a transplant before 1 year after diagnosis, 5 years survival rates superior to 70% have been attained. However, the application of this procedure is limited by the availability of matched donors and by the toxicity of the procedure in older patients. Moreover, outcomes deteriorate with disease duration [76]. This information associated with the knowledge that quiescent leukemic stem cells remain in

Danusertib (PHA 739358) is a small molecule with activity against BCR-ABL and aurora kinases and it is able to block the proliferation of leukemia cell lines as well as CD34+ cells from newly diagnosed CML patients including the mutation T315I. However, similarly to other tyrosine kinase inhibitors, no induction of apoptosis in quiescent hematopoietic stem cells could be achieved and resistant BCR-ABL positive clones emerged in the course of Danusertib treatment. This latter observation is related to Abcg2 proteins over-expression [84].

Lonafarnib (SCH66336) is an orally bioavailable non peptidomimetic farnesyl trransferase inhibitor with significant activity against Bcr-Abl+ cell lines and primary CML cells. It can enhance the toxicity of Imatinib in K562 cell line and can inhibit the proliferation of imatinibresistant cells and increases imatinib-induced apoptosis. However it is unable to kill quiescent CD34+ leukemic cells [20]. In a clinical phase 1 study, it was shown that the combination of Lonafarnib and Imatinib is well tolerated in patients with CML who failed Imatinib, with some patients achieving a complete hematologic response and a complete cytogenetic response [85].

INNO 406 is a 2 phenylaminopyrimidine Bcr-Abl inhibitor with activity against PDGF, c-kit and Lyn that have shown to be 25-55 times more potent than Imatinib in Bcr-Abl+ cell lines. In contrast to other molecules INNO406 does not inhibit all SRC kinases, but it induces programmed cell death in chronic myelogenous leukemia (CML) cell lines through both

MK0457 is an aurora kinase inhibitor with activity against Bcr-Abl. This agent was observed to inhibit autophosphorylation of T315I mutant and demonstrate antiproliferative effects in CML cells derived from patients with this mutation, an event that may lead to its use as a

The knowledge of the central role of BCR-ABL in the pathogenesis of CML has allowed the development of several drugs that inhibit the constitutive activity of such an ABL tyrosine kinase. However, although the treatment with tyrosine kinase inhibitors has proven effective in about 80% of CML patients at any stage, the remaining 20% can't respond to it [87].

age of long-term disease-free survival of any therapy [75].

patients after treatment, several other agents has been reported.

caspase-mediated and caspase-independent pathways [86].

**5. TKI resistance mechanisms**

combination partner with the approved and established TKI [76].

**4.4. Other agents**

148 Stem Cell Biology in Normal Life and Diseases

The most common mechanism against TKIs therapy are point mutations within the kinase domain, which make conformational changes that decrease the affinity of the TKIs to BCR-ABL kinase domain. These point mutations in the *BCR-ABL* kinase domain are a major cause of Imatinib resistance, and may be identified in approximately 50% or more of the cases. Many more than 100 different mutations affecting more than 70 amino acids have so far been identified, with varying degrees of clinical relevance [93].

The first point mutation reported in TKI resistance was in the region coding for the ATP-binding site of the ABL kinase domain resulting in a threonine to isoleucine substi‐ tution at amino acid 315 (Th315→Ile315; T315I) preventing the formation of a hydrogen bond between the oxygen atom provided by the side chain of threonine 315 and the sec‐ ondary amino group of Imatinib. Moreover, isoleucine contains an extra hydrocarbon group on its side chain, and this inhibits the binding of Imatinib [94]. T315I confers re‐ sistance to all currently approved BCR–ABL kinase inhibitors. Recent reports have shown that T315I mutation can be found in approximately 15% of patients after failure of imatinib therapy [85].

**5.2. BCR-ABL-independent resistance mechanisms**

readily restored imatinib cytotoxicity in these cells [110].

of the fluorescent dyes in HSCs [106].

pressed in CML stem cells [111, 112].

compared with high-dose Imatinib [117].

**Drug intake**

HSC are characterized by their ability to pump-out fluorescent dyes, and this led to isolation of stem cells based on this property. In fact, such an efflux capacity has become one of the most efficient methods to purify stem cells from different sources [105]. In this regard, ATP-binding cassette (ABC) transmembrane transporters have shown to be responsible for most of the efflux

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In cancer cell lines, multidrug resistance is often associated with an ATP-dependent decrease in cellular drug accumulation, which is attributed to the overexpression of ABC transporter proteins [107]. The first studies on imatinib-resistance showed increased levels of the multi‐ drug resistance protein MDR1 (ABCB1) in Imatinib resistant BCR-ABL+ cell lines [108]. Later on, it was confirmed that Imatinib is a substrate of membrane ABC transporters, such as ABCB1 (MDR1, P-gp), and that variations in the activity or expression of P-gp affects the pharmaco‐ kinetics of Imatinib, reducing or increasing its bioavailability [109]. P-gp-positive leukemic cells have low intracellular levels of Imatinib; decreased Imatinib levels, in turn, were associ‐ ated with a retained phosphorylation pattern of the Bcr-Abl target Crkl and loss of effect of Imatinib on cellular proliferation and apoptosis. The modulation of P-gp by Ciclosporin A

Another drug efflux pump, the breast cancer resistance protein BRCP encoded by ABCG2, has also been implicated in Imatinib resistance. Imatinib has been variably re‐ ported to be a substrate and/or an inhibitor for the BCRP/ABCG2 drug efflux pump, which is overexpressed in many human tumors and also found to be functionally ex‐

CML stem cells have been shown to express the ATP dependent transporter cassette protein ABCG2, which could decrease the intracellular accumulation of Imatinib in CML LSC [103]. Thus, overexpression of ABC transporters gives protection to tumor cells from TKIs [114].

Inversely to the drug efflux pump proteins, the human organic cation transporter 1 (OCT1) mediates the active transport of Imatinib into cells, and inhibition of OCT1 decreases the intracellular concentration of Imatinib [115]. OCT1 was also found to be expressed in signifi‐ cantly higher levels in patients who achieved a CCR to Imatinib than in those who were more than 65% Ph chromosome positive after 10 months of treatment [116]. Tyrosine Kinase Inhibitor Optimization and Selectivity (TOPS) trial suggested that patients with lower hOCT1 levels had reduced MMR rates at 12 months when receiving the standard dose of Imatinib,

Recently Engler and cols. found that the intracellular uptake and retention (IUR) of imatinib, OCT-1 activity and OCT-1 mRNA expression are all significantly lower in CML CD34+

However, no differences in IUR or OCT-1 activity were observed between these subsets in healthy donors. Low Imatinib accumulation in primitive CML cells, mediated through reduced

OCT-1 activity may be a critical determinant of long-term disease persistence [118].

cells.

**Drug efflux**

Other important TKI's resistant mutations are frequently mapped to the P-loop region (residues 244 to 256) of the kinase domain, which serves as a docking site for phosphate moieties of ATP and interacts with imatinib through hydrogen and van der Waals bonds. These mutations modify the flexibility of the P-loop and destabilize the conformation required for Imatibib binding [95]. Clinical relevance of P-loop mutations is that imatinib treated patients who harbor them have been suggested to have a worse prognosis than those with non-P-loop mutations [96]. Another study identified BCR/ABL mutations in CD34+ cells from CML patients in CCR following Imatinib treatment and suggested that these mutations could lead to imatinib resistance in a small population of progenitors, which consequently could expand and cause the relapse [97].

Several additional mutations that disrupt the interaction between TKIs and BCR-ABL have been characterized, including the P-loop, C-helix, SH2 domain, substrate binding site, A-loop, and C-terminal lobe, some even prior to the initiation of therapy [98]. Most of the reported mutants are rare, however seven mutated sites constitute two thirds of all detected mutations: G250, Y253, E255 (P loop), T315I (gatekeeper), M351, F359, and H396 (activation loop or activation loop backbone) and are frequently evident in the lat‐ er disease stages [99]. Recently a pan-BCR-ABL inhibitor active against the native en‐ zyme and all tested resistant mutants, including the uniformly resistant T315I mutation has been developed [100].

BCR-ABL kinase domain mutations are not induced by the drug, but rather, just like antibioticresistance in bacteria, arise through a process whereby rare pre-existing mutant clones are selfselected due to their capacity to survive and expand in the presence of the drug thus gradually outgrowing drug-sensitive cells [101].

#### **BCR-ABL gene amplification**

Overexpression of Bcr-Abl leads to resistance by increasing the amount of target protein needed to be inhibited by the therapeutic dose of the drug. Amplification of the BCR–ABL gene was first described in resistant CML cell lines generated by serial passage of the cells in Imatinib containing media and demonstrated elevated Abl kinase activity due to a genetic amplification of the Bcr–Abl sequence [102, 103].

Cells expressing high amounts of Bcr-Abl in CD34+ CML cells, as in blast crisis, are much less sensitive to Imatinib and, more significantly, take a substantially shorter time for yielding a mutant subclone resistant to the inhibitor than cells with low expression levels, as in chronic phase [104]. However overexpression and amplification of the *BCR-ABL* gene itself accounts for Imatinib failure in a smaller percentage of patients with an overall percentage of 18% [94].

#### **5.2. BCR-ABL-independent resistance mechanisms**

#### **Drug efflux**

group on its side chain, and this inhibits the binding of Imatinib [94]. T315I confers re‐ sistance to all currently approved BCR–ABL kinase inhibitors. Recent reports have shown that T315I mutation can be found in approximately 15% of patients after failure

Other important TKI's resistant mutations are frequently mapped to the P-loop region (residues 244 to 256) of the kinase domain, which serves as a docking site for phosphate moieties of ATP and interacts with imatinib through hydrogen and van der Waals bonds. These mutations modify the flexibility of the P-loop and destabilize the conformation required for Imatibib binding [95]. Clinical relevance of P-loop mutations is that imatinib treated patients who harbor them have been suggested to have a worse prognosis than those with non-P-loop mutations [96]. Another study identified BCR/ABL mutations in CD34+ cells from CML patients in CCR following Imatinib treatment and suggested that these mutations could lead to imatinib resistance in a small population of progenitors, which consequently could expand

Several additional mutations that disrupt the interaction between TKIs and BCR-ABL have been characterized, including the P-loop, C-helix, SH2 domain, substrate binding site, A-loop, and C-terminal lobe, some even prior to the initiation of therapy [98]. Most of the reported mutants are rare, however seven mutated sites constitute two thirds of all detected mutations: G250, Y253, E255 (P loop), T315I (gatekeeper), M351, F359, and H396 (activation loop or activation loop backbone) and are frequently evident in the lat‐ er disease stages [99]. Recently a pan-BCR-ABL inhibitor active against the native en‐ zyme and all tested resistant mutants, including the uniformly resistant T315I mutation

BCR-ABL kinase domain mutations are not induced by the drug, but rather, just like antibioticresistance in bacteria, arise through a process whereby rare pre-existing mutant clones are selfselected due to their capacity to survive and expand in the presence of the drug thus gradually

Overexpression of Bcr-Abl leads to resistance by increasing the amount of target protein needed to be inhibited by the therapeutic dose of the drug. Amplification of the BCR–ABL gene was first described in resistant CML cell lines generated by serial passage of the cells in Imatinib containing media and demonstrated elevated Abl kinase activity due to a genetic

Cells expressing high amounts of Bcr-Abl in CD34+ CML cells, as in blast crisis, are much less sensitive to Imatinib and, more significantly, take a substantially shorter time for yielding a mutant subclone resistant to the inhibitor than cells with low expression levels, as in chronic phase [104]. However overexpression and amplification of the *BCR-ABL* gene itself accounts for Imatinib failure in a smaller percentage of patients with an

of imatinib therapy [85].

150 Stem Cell Biology in Normal Life and Diseases

and cause the relapse [97].

has been developed [100].

outgrowing drug-sensitive cells [101].

amplification of the Bcr–Abl sequence [102, 103].

**BCR-ABL gene amplification**

overall percentage of 18% [94].

HSC are characterized by their ability to pump-out fluorescent dyes, and this led to isolation of stem cells based on this property. In fact, such an efflux capacity has become one of the most efficient methods to purify stem cells from different sources [105]. In this regard, ATP-binding cassette (ABC) transmembrane transporters have shown to be responsible for most of the efflux of the fluorescent dyes in HSCs [106].

In cancer cell lines, multidrug resistance is often associated with an ATP-dependent decrease in cellular drug accumulation, which is attributed to the overexpression of ABC transporter proteins [107]. The first studies on imatinib-resistance showed increased levels of the multi‐ drug resistance protein MDR1 (ABCB1) in Imatinib resistant BCR-ABL+ cell lines [108]. Later on, it was confirmed that Imatinib is a substrate of membrane ABC transporters, such as ABCB1 (MDR1, P-gp), and that variations in the activity or expression of P-gp affects the pharmaco‐ kinetics of Imatinib, reducing or increasing its bioavailability [109]. P-gp-positive leukemic cells have low intracellular levels of Imatinib; decreased Imatinib levels, in turn, were associ‐ ated with a retained phosphorylation pattern of the Bcr-Abl target Crkl and loss of effect of Imatinib on cellular proliferation and apoptosis. The modulation of P-gp by Ciclosporin A readily restored imatinib cytotoxicity in these cells [110].

Another drug efflux pump, the breast cancer resistance protein BRCP encoded by ABCG2, has also been implicated in Imatinib resistance. Imatinib has been variably re‐ ported to be a substrate and/or an inhibitor for the BCRP/ABCG2 drug efflux pump, which is overexpressed in many human tumors and also found to be functionally ex‐ pressed in CML stem cells [111, 112].

CML stem cells have been shown to express the ATP dependent transporter cassette protein ABCG2, which could decrease the intracellular accumulation of Imatinib in CML LSC [103]. Thus, overexpression of ABC transporters gives protection to tumor cells from TKIs [114].

#### **Drug intake**

Inversely to the drug efflux pump proteins, the human organic cation transporter 1 (OCT1) mediates the active transport of Imatinib into cells, and inhibition of OCT1 decreases the intracellular concentration of Imatinib [115]. OCT1 was also found to be expressed in signifi‐ cantly higher levels in patients who achieved a CCR to Imatinib than in those who were more than 65% Ph chromosome positive after 10 months of treatment [116]. Tyrosine Kinase Inhibitor Optimization and Selectivity (TOPS) trial suggested that patients with lower hOCT1 levels had reduced MMR rates at 12 months when receiving the standard dose of Imatinib, compared with high-dose Imatinib [117].

Recently Engler and cols. found that the intracellular uptake and retention (IUR) of imatinib, OCT-1 activity and OCT-1 mRNA expression are all significantly lower in CML CD34+ cells. However, no differences in IUR or OCT-1 activity were observed between these subsets in healthy donors. Low Imatinib accumulation in primitive CML cells, mediated through reduced OCT-1 activity may be a critical determinant of long-term disease persistence [118].

Differential interactions between drug efflux/influx pumps and kinase inhibitors might be a possible means to tailor drug selection for individual patients, because OCT-1 expression is a key determinant of intracellular availability of Imatinib but not of Nilotinib [119]. Other TKIs, such as Dasatinib and, as just mentioned, Nilotinib, do not appear to be substrates for hOCT1, but whether this difference alone will lead to reduced resistance rates with these secondgeneration TKIs remains unknown [120]. An adequate balance between influx (hOCT1) and efflux (MDR1, ABCG2) transporters may be a critical determinant of intracellular drug levels and, hence, resistance to Imatinib.

and the SH2 domain of ABL1 [130]. Bcr-Abl protein is able to activate the Ras/Raf/Mek kinase pathway and the phosphatidylinositol 3′ kinase (PI3K)/Erk pathways through GRB2 [131, 132]. Autocrine loops could contribute to resistance. It has been demonstrated that IL-3 and granulocyte-colony G-CSF are produced within primitive CD34+ cells from patients with CML-CP, both of these cytokines stimulate cellular proliferation in an autocrine manner and

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**Figure 3.** Resistance mechanism in Chronic Myeloid Leukemia. Principal mechanisms involved in dependent and inde‐

The presence of a rare population of cells capable of initiating and sustaining leukemia in CML (LSC) has major implications for the biology of the disease and the development of new and more effective treatments. As recognized by several investigators, LSC are key players in the origin and progression of CML, as well as in the reappearance of the disease after treatment. Thus, it is evident that novel therapies must be directed towards the elimination of such cells. However, since their numbers within the marrow microenvironment are extremely low, as

protect cells from Imatinib-induced apoptosis [122].

pendent BCR-ABL mechanisms are shown (modified to [99]).

**6. Concluding remarks**

#### **Quiescence**

One feature of CML is the presence of a population of highly quiescent primitive cells [11], which, as their normal counterparts, is capable of regenerating hematopoiesis and reconsti‐ tutes the disease in immunocompromised mice [121]. These stem cells are Ph+, express high levels of CD34 and do not express CD38, CD45RA and CD71, and may spontaneously exit the G0 phase and enter a state of constant proliferation [122]. Several reports have documented that quiescent cells from CML patients are insensitive to in vitro treatment with Imatinib and Dasatinib [16, 123].

A possible cause of insensitivity to TKIs is that BCR-ABL mRNA transcript levels are 300-fold higher in the most primitive CD34+ CD38- Lin- population than in terminally differentiating CD34- Lin+ CML cells [124]. It has been reported that elevated levels of Bcr-Abl confer reduced sensitivity to Imatinib [125]. Moreover, the quiescent state of CML stem cells allows them to evade chemotherapy treatments, which are designed to eliminate metabolically active cell population as well as targeted therapies, thus contributing to relapse when treatment with tyrosine kinase inhibitors is discontinued.

#### **Activation of BCR-ABL alternative signaling**

BCR-ABL activates different signaling pathways that promote the growth and survival of hematopoietic cells, thus inducing cell transformation. These pathways include Ras, mitogen activated protein kinase (MAPK), c-jun N-terminal kinase (JNK), stress-activated protein kinase (SAPK), nuclear factor kappa B(NF-kB), signal transducers and activators of transcrip‐ tion (STAT), phosphoinositide 3- (PI-3) kinase, and c-Myc [126]. A well characterized pathway involves the Src Family Kinases (SFKs), which are activated by BCR-ABL and the subsequent inhibition of BCR-ABL by Imatinib may not result in the complete inhibition of Src family kinases elucidating a Bcr-Abl independent mechanism of imatinib resistance [127]. Phosphor‐ ylation of the Bcr-Abl SH2 and SH3 domains by the SFK may increase the activity of the Abl kinase and may alter its susceptibility to Imatinib [128].

Activation of the Janus kinase (Jak) and subsequent phosphorylation of several Signal Transducer and Activator of Transcription (STAT) family members has been identified in both Bcr–Abl–positive cell lines and in primary CML cells and may contribute to the transforming ability of Bcr–Abl [129].

The tyrosine residue at position 177 within the BCR portion is essential for the binding of adaptor proteins, including Growth Factor Receptor-Bound Protein 2 (GRB2) GRB10, 14-3-3, and the SH2 domain of ABL1 [130]. Bcr-Abl protein is able to activate the Ras/Raf/Mek kinase pathway and the phosphatidylinositol 3′ kinase (PI3K)/Erk pathways through GRB2 [131, 132].

Autocrine loops could contribute to resistance. It has been demonstrated that IL-3 and granulocyte-colony G-CSF are produced within primitive CD34+ cells from patients with CML-CP, both of these cytokines stimulate cellular proliferation in an autocrine manner and protect cells from Imatinib-induced apoptosis [122].

**Figure 3.** Resistance mechanism in Chronic Myeloid Leukemia. Principal mechanisms involved in dependent and inde‐ pendent BCR-ABL mechanisms are shown (modified to [99]).

#### **6. Concluding remarks**

Differential interactions between drug efflux/influx pumps and kinase inhibitors might be a possible means to tailor drug selection for individual patients, because OCT-1 expression is a key determinant of intracellular availability of Imatinib but not of Nilotinib [119]. Other TKIs, such as Dasatinib and, as just mentioned, Nilotinib, do not appear to be substrates for hOCT1, but whether this difference alone will lead to reduced resistance rates with these secondgeneration TKIs remains unknown [120]. An adequate balance between influx (hOCT1) and efflux (MDR1, ABCG2) transporters may be a critical determinant of intracellular drug levels

One feature of CML is the presence of a population of highly quiescent primitive cells [11], which, as their normal counterparts, is capable of regenerating hematopoiesis and reconsti‐ tutes the disease in immunocompromised mice [121]. These stem cells are Ph+, express high levels of CD34 and do not express CD38, CD45RA and CD71, and may spontaneously exit the G0 phase and enter a state of constant proliferation [122]. Several reports have documented that quiescent cells from CML patients are insensitive to in vitro treatment with Imatinib and

A possible cause of insensitivity to TKIs is that BCR-ABL mRNA transcript levels are 300-fold

BCR-ABL activates different signaling pathways that promote the growth and survival of hematopoietic cells, thus inducing cell transformation. These pathways include Ras, mitogen activated protein kinase (MAPK), c-jun N-terminal kinase (JNK), stress-activated protein kinase (SAPK), nuclear factor kappa B(NF-kB), signal transducers and activators of transcrip‐ tion (STAT), phosphoinositide 3- (PI-3) kinase, and c-Myc [126]. A well characterized pathway involves the Src Family Kinases (SFKs), which are activated by BCR-ABL and the subsequent inhibition of BCR-ABL by Imatinib may not result in the complete inhibition of Src family kinases elucidating a Bcr-Abl independent mechanism of imatinib resistance [127]. Phosphor‐ ylation of the Bcr-Abl SH2 and SH3 domains by the SFK may increase the activity of the Abl

Activation of the Janus kinase (Jak) and subsequent phosphorylation of several Signal Transducer and Activator of Transcription (STAT) family members has been identified in both Bcr–Abl–positive cell lines and in primary CML cells and may contribute to the transforming

The tyrosine residue at position 177 within the BCR portion is essential for the binding of adaptor proteins, including Growth Factor Receptor-Bound Protein 2 (GRB2) GRB10, 14-3-3,

Lin+ CML cells [124]. It has been reported that elevated levels of Bcr-Abl confer reduced sensitivity to Imatinib [125]. Moreover, the quiescent state of CML stem cells allows them to evade chemotherapy treatments, which are designed to eliminate metabolically active cell population as well as targeted therapies, thus contributing to relapse when treatment with

Lin- population than in terminally differentiating

CD38-

and, hence, resistance to Imatinib.

152 Stem Cell Biology in Normal Life and Diseases

higher in the most primitive CD34+

tyrosine kinase inhibitors is discontinued.

**Activation of BCR-ABL alternative signaling**

kinase and may alter its susceptibility to Imatinib [128].

**Quiescence**

Dasatinib [16, 123].

ability of Bcr–Abl [129].

CD34-

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#### **Acknowledgements**

Antonieta Chàvez-González is recipient of funding from the National Council of Science and Technology CONACYT (grant CB 2008-01-105994) and from the Mexican Institute for Social Security IMSS (grant IMSS/PROT/G11/946). Dafne Moreno-Lorenzana and Socrates Avilés-Vazquez are scholarship holders from CONACYT and IMSS. Héctor Mayani is a scholar of FUNDACION IMSS (Mexico) and his research is supported by grants from the National Council of Science and Technology-CONACYT (grant SALUD-69664).

#### **Author details**

Antonieta Chávez-González\* , Sócrates Avilés-Vázquez, Dafne Moreno-Lorenzana and Héctor Mayani

\*Address all correspondence to: achavez\_g@yahoo.com.mx

Oncology Research Unit, Oncology Hospital, Mexican Institute for Social Security, Mexico City, Mexico

#### **References**


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compared to the bulk of the malignant cells, and their biology is quite different from that of the rest of the CML cells, the task of finding solutions to this problem is a rather difficult one. It is a great challenge, but significant advances will surely be achieved in the years to come.

Antonieta Chàvez-González is recipient of funding from the National Council of Science and Technology CONACYT (grant CB 2008-01-105994) and from the Mexican Institute for Social Security IMSS (grant IMSS/PROT/G11/946). Dafne Moreno-Lorenzana and Socrates Avilés-Vazquez are scholarship holders from CONACYT and IMSS. Héctor Mayani is a scholar of FUNDACION IMSS (Mexico) and his research is supported by grants from the National

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[127] Wu, J, Meng, F, Kong, L, et al. Association between imatinib-resistant BCR-ABL mutation negative leukemia and persistent activation of LYN kinase. Journal of the

[128] Mein, M. A. rd, Wilson M, Abdi F, et al. Src family kinases phosphorylate the Bcr-Abl SH3-SH2 region and modulate Bcr-Abl transforming activity. Journal of Biological

[129] De Groot, R, Raaijmakers, J, Lammers, J, et al. STAT5 activation by BCR-Abl contributes

to transformation of K562 leukemia cells. Blood (1999). , 94, 1108-1112.

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quiescent fraction. Blood (2006). , 107, 4532-4539.

mesylate resistant differentiated progeny. Blood (2004). a.

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3881-3888.

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[117] White, D, Saunders, V, & Dang, P. CML patients with low OCT-1 activity achieve better molecular responses on high dose imatinib than on standard dose. those with high OCT-1 activity have excellent responses on either dose: a TOPS correlative study. Blood (2008).

[104] Barnes, D, Palaiologou, D, Panousopoulou, E, et al. Bcr-Abl expression levels determine the rate of development of resistance to imatinib mesylate in chronic myeloid leukemia.

[105] Uchida, N, Combs, J, Chen, S, et al. Primitive human hematopoietic cells displaying differential efflux of the rhodamine 123 dye have distinct biological activities. Blood

[106] Chaudhary, P, & Roninson, I. Expression and activity of P glycoprotein, a multidrug

[107] Leslie, E, Deeley, R, & Cole, S. Multidrug resistance proteins: role of P glycoprotein, MRP1, MRP2 and BCRP(ABCG2) in tissue defense. Toxicology and Applied Pharma‐

[108] Mahon, F. X, Deininger, M. W, Schultheis, B, et al. Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor

[109] Hamada, A, Miyano, H, Watanabe, H, et al. Interaction of imatinib mesilate with human P-glycoprotein. Journal of Pharmacology and Experimental Therapeutics (2003). , 307,

[110] Illmer, T, Schaich, M, Platzbecker, U, et al. P-Glycoprotein-mediated drug efflux is a resistance mechanism of chronic myelogenous leukemia cells to treatment with

[111] Burger, H, Van Tol, H, & Brok, M. Chronic imatinib mesylate exposure leads to reduced intracellular drug accumulation by induction of the ABCG2 (BCRP) and ABCB1 (MDR1) drug transport pumps. Cancer Biology and Therapy (2005). , 4, 747-752.

[112] Jordanides, N. E, Jorgensen, H. G, Holyoake, T. L, et al. Functional ABCG2 is over expressed on primary CML CD34+ cells and is inhibited by imatinib mesylate. Blood

[113] Burger, H, & Nooter, K. Pharmacokinetic resistance to imatinib mesylate: role of the ABC drug pumps ABCG2 (BCRP) and ABCB1 (MDR1) in the oral bioavailability of

[114] Brozik, A, Hegedus, C, Erdei, Z, Hegedus, T, Ozvegy-laczka, C, & Szakacs, G. Tyrosine kinase inhibitors as modulators of ATP binding cassette multidrug transporters: substrates, chemo-sensitizers or inducers of acquired multidrug resistance?. Expert

[115] Thomas, J, Wang, L, Clark, R. E, et al. Active transport of imatinib into and out of cells:

[116] Crossman, L, Druker, B, & Deininger, M. hOCT 1 and resistance to imatinib. Blood

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implications for drug resistance. Blood (2004). , 104, 3739-45.

STI571: diverse mechanisms of resistance. Blood (2000). , 96, 1070-1079.

efflux pump, in human hematopoietic stem cells. Cell (1991).

imatinib mesylate. Leukemia (2004). , 18, 401-408.

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[130] Quintas-cardama, A, & Cortes, J. Molecular biology of bcr-abl1-positive chronic myeloid leukemia. Blood (2009). , 113, 1619-30.

**Chapter 9**

**Role of Bone Marrow Derived**

**of Graft Versus Host Disease**

Additional information is available at the end of the chapter

renewal and differentiation into multiple cell lineages [3-6].

Aisha Nasef

**1. Introduction**

current research finding.

http://dx.doi.org/10.5772/55013

**Mesenchymal Stem Cells in Management**

Human bone marrow stromal cells, referred as mesenchymal stem cells (MSC) are multipotent unspecialized cells localized in the medullary stroma [1,2]. They have a capacity for self-

Mesenchymal and tissue stem cell committee of the International society for cellular therapy proposes minimal criteria to define human MSCs. First, MSCs must be plastic-adherent when maintained in standard culture conditions. Second, MSCs must express CD105, CD73 and CD90, and lack the expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules. Third, MSCs must be capable to differentiate into osteoblasts, adipo‐ cytes and chondroblasts in vitro [7,8]. In the hand of Delorme B and Charbord P, MSCs can be defined phenotypically with a minimal set of markers as CD31-, CD34-, and CD45-negative cells and CD13, CD29, CD73, CD90, CD105, and CD166 positive cells [9]. MSCs have been used in cell-based therapy in various disease conditions. Experimental evidence and preliminary clinical studies have demonstrated that MSCs, have an important immunomodulatory function in the management of allogeneic hematopoietic stem cell (HSC) transplantation [10,11]. These immunomodulatory effects have been demonstrated in various species, including humans, rodents, and primates and show clinical promise for the treatment of graft versus host disease (GVHD) and graft failure management. In this chapter we will discuss

> © 2013 Nasef; 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 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,

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

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


**Chapter 9**

## **Role of Bone Marrow Derived Mesenchymal Stem Cells in Management of Graft Versus Host Disease**

Aisha Nasef

[130] Quintas-cardama, A, & Cortes, J. Molecular biology of bcr-abl1-positive chronic

[131] Pendergast, A, Quilliam, L, Cripe, L, et al. BCR/ABL- induced oncogenesis is mediated by direct interaction with the SH2 domain of the GRB-2 adaptor protein. Cell (1993). ,

[132] Sattler, M, Mohi, M, Pride, Y, et al. Critical role for Gab2 in transformation by BCR/

myeloid leukemia. Blood (2009). , 113, 1619-30.

ABL. Cancer Cell (2002). , 1, 479-492.

75, 175-185.

164 Stem Cell Biology in Normal Life and Diseases

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55013

#### **1. Introduction**

Human bone marrow stromal cells, referred as mesenchymal stem cells (MSC) are multipotent unspecialized cells localized in the medullary stroma [1,2]. They have a capacity for selfrenewal and differentiation into multiple cell lineages [3-6].

Mesenchymal and tissue stem cell committee of the International society for cellular therapy proposes minimal criteria to define human MSCs. First, MSCs must be plastic-adherent when maintained in standard culture conditions. Second, MSCs must express CD105, CD73 and CD90, and lack the expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules. Third, MSCs must be capable to differentiate into osteoblasts, adipo‐ cytes and chondroblasts in vitro [7,8]. In the hand of Delorme B and Charbord P, MSCs can be defined phenotypically with a minimal set of markers as CD31-, CD34-, and CD45-negative cells and CD13, CD29, CD73, CD90, CD105, and CD166 positive cells [9]. MSCs have been used in cell-based therapy in various disease conditions. Experimental evidence and preliminary clinical studies have demonstrated that MSCs, have an important immunomodulatory function in the management of allogeneic hematopoietic stem cell (HSC) transplantation [10,11]. These immunomodulatory effects have been demonstrated in various species, including humans, rodents, and primates and show clinical promise for the treatment of graft versus host disease (GVHD) and graft failure management. In this chapter we will discuss current research finding.

© 2013 Nasef; 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 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.

### **2. General background**

#### **2.1. MSC administration**

#### *2.1.1. Safety and therapeutic methods of MSC administration*

The extensive capacity for expansion in vitro at clinical scale has recently facilitated the development of clinical trials designed to assess safety, feasibility, and efficacy of transplanting MSCs for a variety of diseases [11]. There was no toxicities related to the infusion of expanded autologus MSCs into patients with advanced breast cancer or with haematological malignancy, into hurler syndrome patients and into patients of metachromatic leukodystrophy [12,13].

route of administration nor by the difference in conditioning protocols [32]. Clinically, both autologous or allogeneic MSCs were given to patients [33, 34]. Allogeneic HLA-mismatched male fetal MSCs injected into HLA-incompatible female fetus with osteogenesis imperfecta (OI) engrafted and differentiated into bone [35]. Haploidentical MSCs had a low level of engrafment in a patient with aplastic anemia, however there was a partial restoration of bone marrow microenvironement [36]. In contrast, Infused allogeneic MSCs did not expand significantly in patients and they originate from the host [34, 37]. It is of interest to know into which organs MSCs home. Allogeneic and autologus MSCs distributed to a wide range of

Role of Bone Marrow Derived Mesenchymal Stem Cells in Management of Graft Versus Host Disease

http://dx.doi.org/10.5772/55013

167

MSCs transformed in vitro. The transformation of MSCs is associated with chromosomal abnormalities, increased levels of telomerase activity and c-myc expression [38]. Human MSCs isolated from adipose tissue undergo spontaneous transformation after long-term culture (4-5months) [39]. Others found that short-term culture was sufficient for the transformation of murine MSCs into a cell population with autonomous growth and biological characteristics of osteosarcoma [40]. In contrast, even long term cultures could not induce MSC transforma‐ tion [41]. Previous study reported that gastric cancer could originate from BM-derived cells, presumably MSCs [42]. Human BM-MSCs cultured extensively, with a high Telomerase

MSCs could migrate towards primary tumors and metastatic sites. Chemokines secreted by MSCs have been shown to enhance the emergence of pulmonary metastases and such secretion has a strong interaction between breast cancer cells and MSCs. In addition, MSCs have also been found to play a role in drug resistance in various cancer cells. MSCs protect chronic lymphocytic leukemic cells from fludarabine-, dexamethosone-, and cyclophosphamideinduced apoptosis. MSC non-selectively protected chronic myeloid leukemia cells from

Indeed, the transformation potential in cultured MSCs must be documented before consider‐ ing infusion of these cells into patients. However, this issue remains controversial, as other

In conclusion, it is possible that the way MSCs are expanded and long term culture lead to transformation. The safety of using MSCs in humans remains open. The use of MSCs in patients should definitely require precise and limited procedures of expansion to avoid the risk of injecting transformed cells. These finding emphasize the need for accurate studies aimed at

So far, very few studies have addressed the question of the effects of MSCs on metastasis. A few studies have supported that MSCs may suppress tumor growth while others believe that MSCs may contribute to tumor protection via antiapoptotic effect, tumor progression,

activity is capable of forming solid tumors in multiple organs in mice [43].

studies did not observe transformation of human BM-MSCs.

tissues in baboons [22].

**2.4. Transformation of MSCs**

imatinib-induced apoptosis. [44].

investigating the bio safety of these cells

metastasis, and drug-resistance of cancer cells.

**2.5. MSCs and metastasis**

MSC could be delivered systematically and locally. Systemic delivery circumvent the problems such as tissue damage and unsuitability of delivering multiple doses. Site specific delivery has the advantage of delivering large numbers of cell directly to the required sites. Majority of studies showed that MSC infused systematically homed to injured tissues [14,15].

Successful systemic delivery of MSC is dependent upon efficient homing of the cells to the required sites. In this respect, migration of MSC from the circulation into damaged tissues leading to therapeutic effect s has been documented [16]. Ability of MSC to home to bone marrow (BM) can be affected by in vitro culture, which could be due to decrease of adhesion molecules and Chemokine receptors [17].

#### **2.2. Mechanism of homing of MSC**

Homing of leukocytes to inflammatory sites involve selectin, chemokines, integrins and other adhesion molecules [18]. Hemopoietic stem cells (HSC) recruited from blood vessels on similar process to that of leucocytes [19]. MSC recruited to damaged tissues. It is a fair assumption to suppose that they utilize comparable mechanisms of recruitment of MSC. P-selectin play an important role in the trafficking of MSC [20]. MSC roll upon endothelial cells as the first stage in their recruitment. Chemokine receptors are expressed on the cell surface of MSC [21] and their stimulation has been shown to induce cell migration and directing MSC.

#### **2.3. Transplant ability, engraftment and tracking of MSC**

Numerous studies have demonstrated migration and multiorgan engraftment of MSCs both in animal models and in human clinical trials. [22-24].

Direct injection of human marrow stromal cells into the corpus striatum of rat brain showed engraftment of 20% of the infused cells [25]. Rat bone marrow stromal cells infused into briefly distally occluded ascending aorta migrated after 8 weeks in the scar and periscar tissue [26]. MSCs injected intravenously into irradiated primate had the ability of engraftment in different injured tissues as the bone marrow, skin, digestive tract and muscle [9,11]. In rat model, MSC engrafted in multiple organs such as lung, liver, kidneys and spleen.

Human MSCs engrafted and demonstrated site-specific differentiation in sheep [27,28] and in murine models [29-31]. The capacity of engrafment of MSCs was not influenced neither by the route of administration nor by the difference in conditioning protocols [32]. Clinically, both autologous or allogeneic MSCs were given to patients [33, 34]. Allogeneic HLA-mismatched male fetal MSCs injected into HLA-incompatible female fetus with osteogenesis imperfecta (OI) engrafted and differentiated into bone [35]. Haploidentical MSCs had a low level of engrafment in a patient with aplastic anemia, however there was a partial restoration of bone marrow microenvironement [36]. In contrast, Infused allogeneic MSCs did not expand significantly in patients and they originate from the host [34, 37]. It is of interest to know into which organs MSCs home. Allogeneic and autologus MSCs distributed to a wide range of tissues in baboons [22].

#### **2.4. Transformation of MSCs**

**2. General background**

166 Stem Cell Biology in Normal Life and Diseases

*2.1.1. Safety and therapeutic methods of MSC administration*

molecules and Chemokine receptors [17].

**2.2. Mechanism of homing of MSC**

The extensive capacity for expansion in vitro at clinical scale has recently facilitated the development of clinical trials designed to assess safety, feasibility, and efficacy of transplanting MSCs for a variety of diseases [11]. There was no toxicities related to the infusion of expanded autologus MSCs into patients with advanced breast cancer or with haematological malignancy, into hurler syndrome patients and into patients of metachromatic leukodystrophy [12,13]. MSC could be delivered systematically and locally. Systemic delivery circumvent the problems such as tissue damage and unsuitability of delivering multiple doses. Site specific delivery has the advantage of delivering large numbers of cell directly to the required sites. Majority of

Successful systemic delivery of MSC is dependent upon efficient homing of the cells to the required sites. In this respect, migration of MSC from the circulation into damaged tissues leading to therapeutic effect s has been documented [16]. Ability of MSC to home to bone marrow (BM) can be affected by in vitro culture, which could be due to decrease of adhesion

Homing of leukocytes to inflammatory sites involve selectin, chemokines, integrins and other adhesion molecules [18]. Hemopoietic stem cells (HSC) recruited from blood vessels on similar process to that of leucocytes [19]. MSC recruited to damaged tissues. It is a fair assumption to suppose that they utilize comparable mechanisms of recruitment of MSC. P-selectin play an important role in the trafficking of MSC [20]. MSC roll upon endothelial cells as the first stage in their recruitment. Chemokine receptors are expressed on the cell surface of MSC [21] and

Numerous studies have demonstrated migration and multiorgan engraftment of MSCs both

Direct injection of human marrow stromal cells into the corpus striatum of rat brain showed engraftment of 20% of the infused cells [25]. Rat bone marrow stromal cells infused into briefly distally occluded ascending aorta migrated after 8 weeks in the scar and periscar tissue [26]. MSCs injected intravenously into irradiated primate had the ability of engraftment in different injured tissues as the bone marrow, skin, digestive tract and muscle [9,11]. In rat model, MSC

Human MSCs engrafted and demonstrated site-specific differentiation in sheep [27,28] and in murine models [29-31]. The capacity of engrafment of MSCs was not influenced neither by the

studies showed that MSC infused systematically homed to injured tissues [14,15].

their stimulation has been shown to induce cell migration and directing MSC.

**2.3. Transplant ability, engraftment and tracking of MSC**

engrafted in multiple organs such as lung, liver, kidneys and spleen.

in animal models and in human clinical trials. [22-24].

**2.1. MSC administration**

MSCs transformed in vitro. The transformation of MSCs is associated with chromosomal abnormalities, increased levels of telomerase activity and c-myc expression [38]. Human MSCs isolated from adipose tissue undergo spontaneous transformation after long-term culture (4-5months) [39]. Others found that short-term culture was sufficient for the transformation of murine MSCs into a cell population with autonomous growth and biological characteristics of osteosarcoma [40]. In contrast, even long term cultures could not induce MSC transforma‐ tion [41]. Previous study reported that gastric cancer could originate from BM-derived cells, presumably MSCs [42]. Human BM-MSCs cultured extensively, with a high Telomerase activity is capable of forming solid tumors in multiple organs in mice [43].

MSCs could migrate towards primary tumors and metastatic sites. Chemokines secreted by MSCs have been shown to enhance the emergence of pulmonary metastases and such secretion has a strong interaction between breast cancer cells and MSCs. In addition, MSCs have also been found to play a role in drug resistance in various cancer cells. MSCs protect chronic lymphocytic leukemic cells from fludarabine-, dexamethosone-, and cyclophosphamideinduced apoptosis. MSC non-selectively protected chronic myeloid leukemia cells from imatinib-induced apoptosis. [44].

Indeed, the transformation potential in cultured MSCs must be documented before consider‐ ing infusion of these cells into patients. However, this issue remains controversial, as other studies did not observe transformation of human BM-MSCs.

In conclusion, it is possible that the way MSCs are expanded and long term culture lead to transformation. The safety of using MSCs in humans remains open. The use of MSCs in patients should definitely require precise and limited procedures of expansion to avoid the risk of injecting transformed cells. These finding emphasize the need for accurate studies aimed at investigating the bio safety of these cells

#### **2.5. MSCs and metastasis**

So far, very few studies have addressed the question of the effects of MSCs on metastasis. A few studies have supported that MSCs may suppress tumor growth while others believe that MSCs may contribute to tumor protection via antiapoptotic effect, tumor progression, metastasis, and drug-resistance of cancer cells.

Human MSCs played a dual role in tumor cell growth *in vitro* and *in vivo*. It was found that human MSCs inhibited the proliferation of cancer cell lines and caused G1 phase cell cycle arrest and apoptosis *in vitro*. However they enhance tumor formation and growth *in vivo*. MSCs have also been found to prevent apoptosis of acute myeloid leukemia cells by up-regulation of antiapoptotic proteins [44].

*3.1.1. MSCs immunosuppressive effect in experimental animal models*

In the field of HSC transplantation, there are two applications of MSCs:

**•** Treatment of severe GVHD based on the immunomodulatory ability.

hematopoesis and results in rapid hematopoietic recovery [64].

*3.1.2.2. Role of MSCs in management of graft versus host disease (GVHD)*

mediated through several inducible soluble factors [71].

*3.1.2. MSCs immunosuppressive effect in humans*

*3.1.2.1. Role of MSCs in support of hematopoiesis*

poietic recovery.

based on the hematopoesis-supporting ability.

Various animal models have been used to study the immunomodulatory effects of MSCs in treatment of GVHD, autoimmune disorders and tumor immunity. It seems to be that early and repeated injection of MSCs following HSCs transplantation is primordial to control GVHD [55, 56]. In autoimmunity model only early and re-injection of MSCs at the peak of the disease were effective as compared to injection after disease stabilization [57]. MSCs do not elicit immuno‐ logical reaction in recipients [49, 56]. MSCs play tolerogeneic effect in recipients [58-60]. How‐ ever, MSCs had been rejected by mismatched recipients [61] and failed to prevent GVHD [56].

Role of Bone Marrow Derived Mesenchymal Stem Cells in Management of Graft Versus Host Disease

http://dx.doi.org/10.5772/55013

169

**•** Improvement of stem cell engrafting and the acceleration of hematopoietic reconstitution

MSCs constitute the functional and structural support of medullary hematopoiesis by providing growth factors and extracellular matrix [62, 63]. MSCs have a positive impact on

Co transplantation of HSCs and haplotype-mismatched MSCs to patients with high-risk acute myelogenous leukemia result in rapid engraftment [65]. Co transplantation of allogeneic MSCs and multidonor umbilical cord blood (UCB) correlated with a higher overall level of engraft‐ ment into NOD/SCID mice [66]. In European phase I-II study co-infusion of haploidentical HSCs and MSCs accelerated leukocyte recovery as compared to control group [67]. In multi‐ centre clinical trial, co transplantation of MSCs with an HLA-identical sibling HSCs after a myeloablative conditioning regimen induced hematopoietic recovery of peripheral mono nuclear cells (MNC) and platelets [68, 69] and resulted in fast engraftment of absolute neutro‐ phils count and 100% donor chimerism [69]. Transplantation of MSCs into immunosupressed patients generated neither alloantibody against MSC nor against fetal calf serum (FCS) [70].

Injection of MSCs could cure severe graft versus host disease (GVHD) and promote hemato‐

MSC-mediated inhibition of immune response is a complex mechanism involving changes in the maturation of antigen-presenting cells and in cytokine secretion profiles as well as the suppression of monocyte differentiation into dendritic cells [71]. They exert profound immu‐ nosuppression by inhibiting T-cell proliferation in response to various stimuli in vitro. They induce regulatory immunosuppressive lymphocytes and CD8 apoptosis. MSCs inhibit cell cycle progression and CD4 allo-proliferation. This immunosuppressive effect of MSCs is

The main adverse role of MSCs was its pro-invasive potential [45]. It is likely that other molecules participate to the enhancement of metastasis by MSCs and this will be the challenge of future studies.

To use MSCs in anti-cancer therapies, it will be essential to identify the factors produced by MSCs cells responsible for the inhibition or the enhancement of tumor growth and those governing the response of tumor cells.

Evaluation of the potential use of MSCs in cell-based anti-cancer therapies is just starting. These cells have shown some promise as several studies have reported that a portion (which remains to be defined) of MSCs is able to migrate to the tumor site. However, this homing of MSCs is not selective and it will be important to evaluate possible side effects in organs, which are not affected by the disease. Overall, MSCs represent great hope for cancer therapies, but a thorough evaluation of their potential risk will be pre-required step [46].

### **3. Immunological characteristics of MSCs**

Studies in animal models and in humans demonstrated that co-transplantation of HSCs along with MSCs obtained by ex-vivo expansion enhance hematopoietic reconstitution [47,48]. Experimental and clinical studies demonstrated the safety and immunosuppressive role of MSCs infusion [49, 50].

#### **3.1. Immunological characteristics of MSCs** *in vivo*

It has been demonstrated since 1984 that reconstitution of irradiated host with T-cell de‐ pleted bone marrow containing both host (syngeneic) and donor (allogeneic or Xenoge‐ neic) components leads to long-term survival of the reconstituted animals and specific prolongation of subsequent skin grafts of donor type. However these animals are fully reactive to third-party allograft and do not appear to manifest signs of graft-versus-host disease (GVHD) [51]. The possibility of the presence of immunomodulatory cells in bone has been noticed after donor specific long-term hypo responsive status obtained by trans‐ plantation of bone and HSCs in murine models [52, 53]. MSCs constitute the stromal scaffold which is close to the endostenum and interact tightly with HSCs [54]. They con‐ tribute to the formation of HSCs niche, support HSCs engraftment and survival of blood cells in vivo [11]. This indicate the presence of underlying immunomodulatory effect of MSCs.

#### *3.1.1. MSCs immunosuppressive effect in experimental animal models*

Various animal models have been used to study the immunomodulatory effects of MSCs in treatment of GVHD, autoimmune disorders and tumor immunity. It seems to be that early and repeated injection of MSCs following HSCs transplantation is primordial to control GVHD [55, 56]. In autoimmunity model only early and re-injection of MSCs at the peak of the disease were effective as compared to injection after disease stabilization [57]. MSCs do not elicit immuno‐ logical reaction in recipients [49, 56]. MSCs play tolerogeneic effect in recipients [58-60]. How‐ ever, MSCs had been rejected by mismatched recipients [61] and failed to prevent GVHD [56].

#### *3.1.2. MSCs immunosuppressive effect in humans*

Human MSCs played a dual role in tumor cell growth *in vitro* and *in vivo*. It was found that human MSCs inhibited the proliferation of cancer cell lines and caused G1 phase cell cycle arrest and apoptosis *in vitro*. However they enhance tumor formation and growth *in vivo*. MSCs have also been found to prevent apoptosis of acute myeloid leukemia cells by up-regulation

The main adverse role of MSCs was its pro-invasive potential [45]. It is likely that other molecules participate to the enhancement of metastasis by MSCs and this will be the challenge

To use MSCs in anti-cancer therapies, it will be essential to identify the factors produced by MSCs cells responsible for the inhibition or the enhancement of tumor growth and those

Evaluation of the potential use of MSCs in cell-based anti-cancer therapies is just starting. These cells have shown some promise as several studies have reported that a portion (which remains to be defined) of MSCs is able to migrate to the tumor site. However, this homing of MSCs is not selective and it will be important to evaluate possible side effects in organs, which are not affected by the disease. Overall, MSCs represent great hope for cancer therapies, but a thorough

Studies in animal models and in humans demonstrated that co-transplantation of HSCs along with MSCs obtained by ex-vivo expansion enhance hematopoietic reconstitution [47,48]. Experimental and clinical studies demonstrated the safety and immunosuppressive role of

It has been demonstrated since 1984 that reconstitution of irradiated host with T-cell de‐ pleted bone marrow containing both host (syngeneic) and donor (allogeneic or Xenoge‐ neic) components leads to long-term survival of the reconstituted animals and specific prolongation of subsequent skin grafts of donor type. However these animals are fully reactive to third-party allograft and do not appear to manifest signs of graft-versus-host disease (GVHD) [51]. The possibility of the presence of immunomodulatory cells in bone has been noticed after donor specific long-term hypo responsive status obtained by trans‐ plantation of bone and HSCs in murine models [52, 53]. MSCs constitute the stromal scaffold which is close to the endostenum and interact tightly with HSCs [54]. They con‐ tribute to the formation of HSCs niche, support HSCs engraftment and survival of blood cells in vivo [11]. This indicate the presence of underlying immunomodulatory effect of

of antiapoptotic proteins [44].

168 Stem Cell Biology in Normal Life and Diseases

governing the response of tumor cells.

evaluation of their potential risk will be pre-required step [46].

**3. Immunological characteristics of MSCs**

**3.1. Immunological characteristics of MSCs** *in vivo*

of future studies.

MSCs infusion [49, 50].

MSCs.

In the field of HSC transplantation, there are two applications of MSCs:


#### *3.1.2.1. Role of MSCs in support of hematopoiesis*

MSCs constitute the functional and structural support of medullary hematopoiesis by providing growth factors and extracellular matrix [62, 63]. MSCs have a positive impact on hematopoesis and results in rapid hematopoietic recovery [64].

Co transplantation of HSCs and haplotype-mismatched MSCs to patients with high-risk acute myelogenous leukemia result in rapid engraftment [65]. Co transplantation of allogeneic MSCs and multidonor umbilical cord blood (UCB) correlated with a higher overall level of engraft‐ ment into NOD/SCID mice [66]. In European phase I-II study co-infusion of haploidentical HSCs and MSCs accelerated leukocyte recovery as compared to control group [67]. In multi‐ centre clinical trial, co transplantation of MSCs with an HLA-identical sibling HSCs after a myeloablative conditioning regimen induced hematopoietic recovery of peripheral mono nuclear cells (MNC) and platelets [68, 69] and resulted in fast engraftment of absolute neutro‐ phils count and 100% donor chimerism [69]. Transplantation of MSCs into immunosupressed patients generated neither alloantibody against MSC nor against fetal calf serum (FCS) [70].

#### *3.1.2.2. Role of MSCs in management of graft versus host disease (GVHD)*

Injection of MSCs could cure severe graft versus host disease (GVHD) and promote hemato‐ poietic recovery.

MSC-mediated inhibition of immune response is a complex mechanism involving changes in the maturation of antigen-presenting cells and in cytokine secretion profiles as well as the suppression of monocyte differentiation into dendritic cells [71]. They exert profound immu‐ nosuppression by inhibiting T-cell proliferation in response to various stimuli in vitro. They induce regulatory immunosuppressive lymphocytes and CD8 apoptosis. MSCs inhibit cell cycle progression and CD4 allo-proliferation. This immunosuppressive effect of MSCs is mediated through several inducible soluble factors [71].

#### *3.1.2.3 Clinical trials of GVHD management by MSCs*

Patients treated with MSCs had less GVHD and less toxicity in a retrospective study comparing allograft of geno-identical HSCs with and without MSCs [72]. No acute side effects occurred after the infusion of haploidentical and mismatched MSCs from unrelated donors into patients suffering from GVHD [50, 73]. Haploidentical MSCs was used to treat grade IV GVHD of the gut and liver that was resistant to conventional treatment. The aim was to use the tissue repair effect shown in vivo in animal models, and the immunomodulatory effects seen in vitro on human lymphocytes. The clinical response was striking with normalization of stool and bilirubin on two occasions. They leads to healing of damaged bowel epithelia [50]. The patient was highly immunosupressed. However, his lymphocytes continued to proliferate in response to lymphocytes of MSCs donor in vitro in several occasions. This could indicate an immuno‐ suppressive effect of MSCs rather than tolerance induction. We would be able to prevent GVHD after MSCs infusion and maintaining in the same time the response of host lymphocytes against alloantigens. Infusion of MSCs into 8 patients with steroid refractory GVHD cause dramatical disappearance of all symptoms and repaired gut in six patients and liver in one. The survival curve was better than that of 16 patients with steroid-resistant biopsy-proven gastrointestinal GVHD, not treated with MSCs [245]. MSCs were used to treat 40 patients with acute and chronic GVHD. More than forty-seven (47.5 %) showed complete response, 22.5 % showed improvement, 10 % had stable disease and 17.5 % had no response. Between 6 weeks up to 3.5 years after transplantation more than half of patients are alive [73].

In a phase I/II clinical trial, ten percent of patients treated for acute refractory GVHD obtained a complete response, 60 % had a partial response and 30 % did not respond. They found that 50% of patients with chronic refractory GVHD did not respond, 12.5 % had complete remission and 37.5% had partial response [77]. Transplantation of MSCs into 15 patients with haemato‐

Role of Bone Marrow Derived Mesenchymal Stem Cells in Management of Graft Versus Host Disease

http://dx.doi.org/10.5772/55013

171

These preliminary data suggest that MSCs may also play a role in repairing severe tissue toxicity. It seems to be that there is beneficial effect of MSCs infusion in humans. However, The optimal MSCs dose and frequency of administration needed to be evaluated to control

The underlying molecular mechanisms of immunosuppression in vivo are unknown. It has been demonstrated that MSCs engrafted to injured tissues rich on inflammatory cytokines [107]. MSCs inhibitory effect is inducible [79] and presence of MSCs in such media could explain partially the beneficial effect of MSCs infusion in the recipients, despite non detectable

Co-culture of MSCs with allogeneic lymphocytes failed to stimulate their proliferation, indicating that these cells are innately not immunogeneic. Recent reports suggest that MSCs have immunomodulatory properties and can inhibit lymphocyte antigen presenting cells, natural killer cells, and cytotoxic lymphocyte proliferation in mixed-lymphocyte reactions (MLR). MSCs inhibit CD2, CD4 and CD8 subsets of T lymphocytes. The immunosuppressive effect of human MSCs was higher in cultures with cell contact than in cultures without contact (transwell), and the difference was statistically significant [80, 81]. MSCs produce a variety of growth factors that are likely to play a role in immunomodulation. Human and murine MSCs do inhibit the proliferation of lymphocytes in MLR by soluble factors [80-83]. Indoleamine 2,3 dioxygenase (IDO ) inhibit the alloreactivity induced by antigen presenting cells (APC) [84]. The inhibitory effect of MSCs on alloreactivity seems to be due to other mechanisms rather than apoptosis of lymphocytes. Several studies demonstrated reversibility of MSCs inhibitory

Cell anergy is a state of immune unresponsiveness, defines the inability of an immune cell to mount a complete response against its target. MSCs induce anergy due to divisional arrest of T cells. IFN-γ production but not proliferation of murine cells was restored [87]. Restoration of CD4+ cell proliferation but not CD8+ cells after the removal of human MSCs was observed. For the time being we could assume that MSCs do induce anergy [88]. MSCs induced regula‐ tory cells [88]. Regulatory cells have been involved in the regulation of immune response.

Despite the expression of human leukocyte antigen (HLA) by MSCs, they were well tolerated without side effects in allogeneic hosts. Major Histocomaptability Complex (MHC) had no role in MSCs inhibitory effect as autologus and allogenic human [81], murine [89] and baboon [49]. MSCs were capable of inhibiting the proliferation of lymphocytes. In addition, Xenogeneic murine MSCs inhibited the proliferation of human cells [79]. However, one study showed that

logical malignancies was safe and induced complete remission [78].

**3.2. Suggested mechanisms for MSC immunomodulatory effect**

GVHD.

MSCs in recipients BM.

effect. [81, 85, 86].

At the same time of hematopoietic stem cell transplantation (HSCT), 46 patients received culture-expanded MSCs from their HLA-identical sibling donors [74]. Moderate to severe acute GVHD was observed in 13 (28%) of 46 patients. Chronic GVHD was observed in 22 (61%) of 36 patients and 2-year survival was 53%. No MSC-associated toxicities were seen. Stromal cell chimerism was demonstrated in 2 of 19 examined patients at 6 and 18 months after transplantation. MSCs are safe to give, but are difficult to detect after infusion, even in immunocompromised patients who have undergone HSCT [75].

Ten patients undergoing HSCT were treated with MSCs due to tissue toxicity. In five patients, severe hemorrhagic cystitis cleared after MSC infusion. Gross hematuria disappeared after a median of 3 days. Two patients with grade five hemorrhagic cystitis had reduced transfusion requirements after MSC infusions, but both died of multi-organ failure. MSC donor DNA was demonstrated in the urinary bladder in one of them. Two patients were treated for pneumo‐ mediastinum, which disappeared after MSC infusion. A patient with steroid-resistant GVHD of the gut experienced perforated diverticulitis and peritonitis that was dramatically reversed twice after infusion of MSCs[76].

In Europe, 55 patients have been treated for steroid-resistant acute GVHD with an overall response rate of 69%. Non responders have died of progressive GVHD and several responders have died from infections with an overall survival of 23 of 55 (42%) from 2 months to 5 years. Although the experience is limited, MSCs seems a promising treatment for severe steroidresistant acute GVHD [73].

In a phase I/II clinical trial, ten percent of patients treated for acute refractory GVHD obtained a complete response, 60 % had a partial response and 30 % did not respond. They found that 50% of patients with chronic refractory GVHD did not respond, 12.5 % had complete remission and 37.5% had partial response [77]. Transplantation of MSCs into 15 patients with haemato‐ logical malignancies was safe and induced complete remission [78].

These preliminary data suggest that MSCs may also play a role in repairing severe tissue toxicity. It seems to be that there is beneficial effect of MSCs infusion in humans. However, The optimal MSCs dose and frequency of administration needed to be evaluated to control GVHD.

The underlying molecular mechanisms of immunosuppression in vivo are unknown. It has been demonstrated that MSCs engrafted to injured tissues rich on inflammatory cytokines [107]. MSCs inhibitory effect is inducible [79] and presence of MSCs in such media could explain partially the beneficial effect of MSCs infusion in the recipients, despite non detectable MSCs in recipients BM.

#### **3.2. Suggested mechanisms for MSC immunomodulatory effect**

*3.1.2.3 Clinical trials of GVHD management by MSCs*

170 Stem Cell Biology in Normal Life and Diseases

Patients treated with MSCs had less GVHD and less toxicity in a retrospective study comparing allograft of geno-identical HSCs with and without MSCs [72]. No acute side effects occurred after the infusion of haploidentical and mismatched MSCs from unrelated donors into patients suffering from GVHD [50, 73]. Haploidentical MSCs was used to treat grade IV GVHD of the gut and liver that was resistant to conventional treatment. The aim was to use the tissue repair effect shown in vivo in animal models, and the immunomodulatory effects seen in vitro on human lymphocytes. The clinical response was striking with normalization of stool and bilirubin on two occasions. They leads to healing of damaged bowel epithelia [50]. The patient was highly immunosupressed. However, his lymphocytes continued to proliferate in response to lymphocytes of MSCs donor in vitro in several occasions. This could indicate an immuno‐ suppressive effect of MSCs rather than tolerance induction. We would be able to prevent GVHD after MSCs infusion and maintaining in the same time the response of host lymphocytes against alloantigens. Infusion of MSCs into 8 patients with steroid refractory GVHD cause dramatical disappearance of all symptoms and repaired gut in six patients and liver in one. The survival curve was better than that of 16 patients with steroid-resistant biopsy-proven gastrointestinal GVHD, not treated with MSCs [245]. MSCs were used to treat 40 patients with acute and chronic GVHD. More than forty-seven (47.5 %) showed complete response, 22.5 % showed improvement, 10 % had stable disease and 17.5 % had no response. Between 6 weeks

up to 3.5 years after transplantation more than half of patients are alive [73].

immunocompromised patients who have undergone HSCT [75].

twice after infusion of MSCs[76].

resistant acute GVHD [73].

At the same time of hematopoietic stem cell transplantation (HSCT), 46 patients received culture-expanded MSCs from their HLA-identical sibling donors [74]. Moderate to severe acute GVHD was observed in 13 (28%) of 46 patients. Chronic GVHD was observed in 22 (61%) of 36 patients and 2-year survival was 53%. No MSC-associated toxicities were seen. Stromal cell chimerism was demonstrated in 2 of 19 examined patients at 6 and 18 months after transplantation. MSCs are safe to give, but are difficult to detect after infusion, even in

Ten patients undergoing HSCT were treated with MSCs due to tissue toxicity. In five patients, severe hemorrhagic cystitis cleared after MSC infusion. Gross hematuria disappeared after a median of 3 days. Two patients with grade five hemorrhagic cystitis had reduced transfusion requirements after MSC infusions, but both died of multi-organ failure. MSC donor DNA was demonstrated in the urinary bladder in one of them. Two patients were treated for pneumo‐ mediastinum, which disappeared after MSC infusion. A patient with steroid-resistant GVHD of the gut experienced perforated diverticulitis and peritonitis that was dramatically reversed

In Europe, 55 patients have been treated for steroid-resistant acute GVHD with an overall response rate of 69%. Non responders have died of progressive GVHD and several responders have died from infections with an overall survival of 23 of 55 (42%) from 2 months to 5 years. Although the experience is limited, MSCs seems a promising treatment for severe steroidCo-culture of MSCs with allogeneic lymphocytes failed to stimulate their proliferation, indicating that these cells are innately not immunogeneic. Recent reports suggest that MSCs have immunomodulatory properties and can inhibit lymphocyte antigen presenting cells, natural killer cells, and cytotoxic lymphocyte proliferation in mixed-lymphocyte reactions (MLR). MSCs inhibit CD2, CD4 and CD8 subsets of T lymphocytes. The immunosuppressive effect of human MSCs was higher in cultures with cell contact than in cultures without contact (transwell), and the difference was statistically significant [80, 81]. MSCs produce a variety of growth factors that are likely to play a role in immunomodulation. Human and murine MSCs do inhibit the proliferation of lymphocytes in MLR by soluble factors [80-83]. Indoleamine 2,3 dioxygenase (IDO ) inhibit the alloreactivity induced by antigen presenting cells (APC) [84]. The inhibitory effect of MSCs on alloreactivity seems to be due to other mechanisms rather than apoptosis of lymphocytes. Several studies demonstrated reversibility of MSCs inhibitory effect. [81, 85, 86].

Cell anergy is a state of immune unresponsiveness, defines the inability of an immune cell to mount a complete response against its target. MSCs induce anergy due to divisional arrest of T cells. IFN-γ production but not proliferation of murine cells was restored [87]. Restoration of CD4+ cell proliferation but not CD8+ cells after the removal of human MSCs was observed. For the time being we could assume that MSCs do induce anergy [88]. MSCs induced regula‐ tory cells [88]. Regulatory cells have been involved in the regulation of immune response.

Despite the expression of human leukocyte antigen (HLA) by MSCs, they were well tolerated without side effects in allogeneic hosts. Major Histocomaptability Complex (MHC) had no role in MSCs inhibitory effect as autologus and allogenic human [81], murine [89] and baboon [49]. MSCs were capable of inhibiting the proliferation of lymphocytes. In addition, Xenogeneic murine MSCs inhibited the proliferation of human cells [79]. However, one study showed that transplantation of allogeneic MSCs resulted in rejection by class I and class II mismatched recipients in murine model [61].

inhibit T-cell activation and, on the contrary, MSC supernatant had a stimulatory effect on MLR. Surprisingly, cell-free supernatant obtained from MLR had an inhibitory effect similar to that seen with the addition of MSCs [58]. In agreement Djouad et al. found that only MSC conditioned supernatant by lymphocytes was capable to inhibit secondary MLR but less efficiently than the co-culture of cell partners, in indicating that the immunosuppressive properties of MSCs are mediated by inducible soluble factors and the interaction between the

Role of Bone Marrow Derived Mesenchymal Stem Cells in Management of Graft Versus Host Disease

http://dx.doi.org/10.5772/55013

173

In addition, an enhancement of the MSC immunosuppressive effect was observed after addition of irradiated third party MNC to MSC culture, indicating that the physical interaction between MSC and PBMC increase the suppressive activity [98]. Human MSCs do not consti‐ tutively express IDO. But activation by IFN-γ secreted by allogeneic lymphocytes in a dose

It has been demonstrated that auto reactive cells may induce the transdifferentiation of MSCs to neural stem cells. This phenomenon could be due to stimulation of cytokine production and generation of suitable environment that results in differentiation into neural stem cells [100]. All these data could confirm the interaction between MSCs and lymphocytes. Physical interaction between MSCs and T-cells increases the suppressive activity as there was an increased expression of IL-10 and TGF-β genes [80] as compared to non contact cultures. All these data could complicate the issue. Is MSC mediated inhibitory effect is a consequence of MSC activation by IFN-γ secreted by two allogeneic populations of lymphocytes in MLR?.

Are there two separate effector mechanisms, one to escape the immunological recognition and the other to inhibit the alloreactivity? Is the constitutive expression of TGF-β and HLA-G by

Maccario et al. had demonstrated the absence of regulatory cell in co-culture of MSCs with single allogeneic PBMCs as compared to the presence of MSCs in MLR [88]. This could indicate that allogeneic lymphocytes initiate the first signals to stimulate the production of molecules

It has been found that CD14+ monocyte are the PBMC subpopulation, being responsible for MSC activation through IL-B secretion [101]. MSC inhibitory effect was mediated through CD8+ in human and murine model [97, 88]. CD8+ cells are the executive cells for MSC inhibitory effect rather than being the inducers of MSC inhibitory effect. It has been demon‐ strated that MSC inhibitory mechanisms differ depending on nature of stimulus [102]. This could indicate that alloantigens or mitogen are responsible for the first stimulating signals in MLR, or at least they are responsible for later on modification of these signals. It has been found that lymphocytes and MSCs are mutually inhibitory on their respective proliferation and indicate the bi-directional interaction and cross talk between lymphocytes and MSCs [103, 104]. More recently, it has been found that IFNγ and concomitant presence of TNFα or IL-1α or IL-1β induce the expression of several chemokine and inducible Nitric oxide synthase by

MSCs and lymphocytes is a pre-requirement for MSC mediated inhibitory effect [79].

dependent manner is required for induction of functional IDO activity [99].

Why there is no stimulation of single allogeneic PBL by allogeneic MSCs?

the MSCs play a role in immunological escape?

that induce the production of regulatory cells.

MSCs [105].

The major effectors of the innate immunity are natural killer (NK) cells. They eliminate malignant and virus infected cells. MSCs alter the phenotype of NK cells and suppress proliferation and cytotoxicity against HLA-class I-expressing targets in time and dose de‐ pendent manner in cell contact or transwell cultures [90, 91].

Veto activity was defined by Miller [92] as the ability to induce specific suppression of cytotoxic T lymphocyte precursors (CTL-Ps) against antigens present on veto cell surface, but not against those on third-party allogeneic cells. MSCs had veto activity [93]. This is contradictory to all the present data where MSCs were able to inhibit the allo-response to allogeneic lymphocytes in MLR in different species [79, 81]. Even more, veto property contrasts with the proliferation of allogeneic lymphocytes observed in the presence of MSCs by the same team [93].

There was drastic reduction of the recipient's CTL response against injected class I antigens due to veto phenomenon [94]. These findings still keep on with Miller's definition [92]. Veto mechanism could play a role in low immunogenicity of MSCs, but certainly doesn't explain the suppression of allogenic lymphocytes by MSCs [71]. MSCs have no effect on the lympho‐ cyte response to recall antigen [93].

Human MSC inhibited the pro--inflammatory Th1 cytokines (IFN-γ, TNF- α, IL-1α and IL-β) whereas the anti-inflammatory Th2 cytokines IL-3, IL-5, IL-10, IL-4 and IL-13 and the Th2 Chemokine I-309 (a chemo attractant for regulatory T cells) were increased [71, 95]. This could indicate a shift from the prominence of pro-inflammatory Th1 cells towards an increase in antiinflammatory TH2 cells and support MSC inhibitory properties. MSCs can skew the dendritic cells (DCs) function, thus biasing the immune system toward Th2 and away from Th1 responses [96]. MSC had no effect on neutrophils phagocytosis, expression of adhesion molecules and chemotaxis [97]. This is an interesting finding, as MSCs could not interfere with neutrophils function. Summary of MSCs inhibitory effect is illustrated in figure-1. For further reading, about detailed description of possible MSCs inhibitory mechanisms, refer to this article [10].

#### *3.2.1. Inhibitory effect of autogenic and allogenic MSCs*

Allogeneic MSCs have stronger immunosuppressive effects than autologus MSCs [88]. Others found comparable and significant inhibition was elicited by autologus or allogeneic MSCs [81,85]. This could help in management of GVHD in cases where non-matched HSCs are used

### **4. What starts the first step in induction of inhibitory effect in MSC/Mixed lymphocyte reaction MLR?**

It is not known for the time being whether MSCs or lymphocytes do start the first signals to activate the other one to induce the immunosuppressive effect. MSC supernatant failed to inhibit T-cell activation and, on the contrary, MSC supernatant had a stimulatory effect on MLR. Surprisingly, cell-free supernatant obtained from MLR had an inhibitory effect similar to that seen with the addition of MSCs [58]. In agreement Djouad et al. found that only MSC conditioned supernatant by lymphocytes was capable to inhibit secondary MLR but less efficiently than the co-culture of cell partners, in indicating that the immunosuppressive properties of MSCs are mediated by inducible soluble factors and the interaction between the MSCs and lymphocytes is a pre-requirement for MSC mediated inhibitory effect [79].

transplantation of allogeneic MSCs resulted in rejection by class I and class II mismatched

The major effectors of the innate immunity are natural killer (NK) cells. They eliminate malignant and virus infected cells. MSCs alter the phenotype of NK cells and suppress proliferation and cytotoxicity against HLA-class I-expressing targets in time and dose de‐

Veto activity was defined by Miller [92] as the ability to induce specific suppression of cytotoxic T lymphocyte precursors (CTL-Ps) against antigens present on veto cell surface, but not against those on third-party allogeneic cells. MSCs had veto activity [93]. This is contradictory to all the present data where MSCs were able to inhibit the allo-response to allogeneic lymphocytes in MLR in different species [79, 81]. Even more, veto property contrasts with the proliferation

There was drastic reduction of the recipient's CTL response against injected class I antigens due to veto phenomenon [94]. These findings still keep on with Miller's definition [92]. Veto mechanism could play a role in low immunogenicity of MSCs, but certainly doesn't explain the suppression of allogenic lymphocytes by MSCs [71]. MSCs have no effect on the lympho‐

Human MSC inhibited the pro--inflammatory Th1 cytokines (IFN-γ, TNF- α, IL-1α and IL-β) whereas the anti-inflammatory Th2 cytokines IL-3, IL-5, IL-10, IL-4 and IL-13 and the Th2 Chemokine I-309 (a chemo attractant for regulatory T cells) were increased [71, 95]. This could indicate a shift from the prominence of pro-inflammatory Th1 cells towards an increase in antiinflammatory TH2 cells and support MSC inhibitory properties. MSCs can skew the dendritic cells (DCs) function, thus biasing the immune system toward Th2 and away from Th1 responses [96]. MSC had no effect on neutrophils phagocytosis, expression of adhesion molecules and chemotaxis [97]. This is an interesting finding, as MSCs could not interfere with neutrophils function. Summary of MSCs inhibitory effect is illustrated in figure-1. For further reading, about detailed description of possible MSCs inhibitory mechanisms, refer to this

Allogeneic MSCs have stronger immunosuppressive effects than autologus MSCs [88]. Others found comparable and significant inhibition was elicited by autologus or allogeneic MSCs [81,85]. This could help in management of GVHD in cases where non-matched HSCs are used

**4. What starts the first step in induction of inhibitory effect in MSC/Mixed**

It is not known for the time being whether MSCs or lymphocytes do start the first signals to activate the other one to induce the immunosuppressive effect. MSC supernatant failed to

of allogeneic lymphocytes observed in the presence of MSCs by the same team [93].

recipients in murine model [61].

172 Stem Cell Biology in Normal Life and Diseases

cyte response to recall antigen [93].

*3.2.1. Inhibitory effect of autogenic and allogenic MSCs*

**lymphocyte reaction MLR?**

article [10].

pendent manner in cell contact or transwell cultures [90, 91].

In addition, an enhancement of the MSC immunosuppressive effect was observed after addition of irradiated third party MNC to MSC culture, indicating that the physical interaction between MSC and PBMC increase the suppressive activity [98]. Human MSCs do not consti‐ tutively express IDO. But activation by IFN-γ secreted by allogeneic lymphocytes in a dose dependent manner is required for induction of functional IDO activity [99].

It has been demonstrated that auto reactive cells may induce the transdifferentiation of MSCs to neural stem cells. This phenomenon could be due to stimulation of cytokine production and generation of suitable environment that results in differentiation into neural stem cells [100]. All these data could confirm the interaction between MSCs and lymphocytes. Physical interaction between MSCs and T-cells increases the suppressive activity as there was an increased expression of IL-10 and TGF-β genes [80] as compared to non contact cultures. All these data could complicate the issue. Is MSC mediated inhibitory effect is a consequence of MSC activation by IFN-γ secreted by two allogeneic populations of lymphocytes in MLR?. Why there is no stimulation of single allogeneic PBL by allogeneic MSCs?

Are there two separate effector mechanisms, one to escape the immunological recognition and the other to inhibit the alloreactivity? Is the constitutive expression of TGF-β and HLA-G by the MSCs play a role in immunological escape?

Maccario et al. had demonstrated the absence of regulatory cell in co-culture of MSCs with single allogeneic PBMCs as compared to the presence of MSCs in MLR [88]. This could indicate that allogeneic lymphocytes initiate the first signals to stimulate the production of molecules that induce the production of regulatory cells.

It has been found that CD14+ monocyte are the PBMC subpopulation, being responsible for MSC activation through IL-B secretion [101]. MSC inhibitory effect was mediated through CD8+ in human and murine model [97, 88]. CD8+ cells are the executive cells for MSC inhibitory effect rather than being the inducers of MSC inhibitory effect. It has been demon‐ strated that MSC inhibitory mechanisms differ depending on nature of stimulus [102]. This could indicate that alloantigens or mitogen are responsible for the first stimulating signals in MLR, or at least they are responsible for later on modification of these signals. It has been found that lymphocytes and MSCs are mutually inhibitory on their respective proliferation and indicate the bi-directional interaction and cross talk between lymphocytes and MSCs [103, 104]. More recently, it has been found that IFNγ and concomitant presence of TNFα or IL-1α or IL-1β induce the expression of several chemokine and inducible Nitric oxide synthase by MSCs [105].

This could indicate that lymphocytes start the first signals for MSCs activation, however cross talk between both of them are essential to have the full immunological effects.

through IDO. MSCs inhibit lymphocytes T proliferation and cytotoxicity through IDO, PGE2,

Role of Bone Marrow Derived Mesenchymal Stem Cells in Management of Graft Versus Host Disease

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175

[1] Friedenstein, A. J, Chailakhyan, R. K, Latsinik, N. V, Panasyuk, A. F, & Keiliss-borok, I. V. Stromal cells responsible for transferring the microenvironment of the hemo‐ poietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation

[2] Friedenstein, A. J. Precursor cells of mechanocytes. Int Rev Cytol (1976). , 47(1976),

[3] Friedenstein, A. J, Petrakova, K. V, Kurolesova, A. I, & Frolova, G. P. Heterotopic of bone marrow.Analysis of precursor cells for osteogenic and hematopoietic tissues.

[4] Friedenstein, A. J, Chailakhyan, R. K, & Gerasimov, U. V. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue

[5] Owen, M, & Friedenstein, A. J. Stromal stem cells: marrow-derived osteogenic pre‐

[6] Prockop, D. J. Marrow stromal cells as stem cells for nonhematopoietic tissues. Sci‐

[7] Horwitz, E. M, Le, K, Blanc, M, Dominici, I, Mueller, I, Slaper-cortenbach, F. C, Mari‐ ni, R. J, & Deans, D. S. Krause, and A. Keating, Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy

[8] Dominici, M, Le, K, Blanc, I, Mueller, I, Slaper-cortenbach, F, Marini, D, Krause, R, Deans, A, & Keating, D. Prockop, and E. Horwitz, Minimal criteria for defining mul‐ tipotent mesenchymal stromal cells. The International Society for Cellular Therapy

HLA-G, TGF-b, HGF, Il-10 and IL-1 b.

Address all correspondence to: Nasef@doctor.com

National Medical Research Centre NMRC, Libya

(1974). , 17(1974), 331-40.

Transplantation (1968). , 6(1968), 230-47.

cursors. Ciba Found Symp (1988). , 136(1988), 42-60.

position statement. Cytotherapy (2006). , 8(2006), 315-7.

Kinet (1987). , 20(1987), 263-72.

ence (1997). , 276(1997), 71-4.

(2005). , 7(2005), 393-5.

**Author details**

Aisha Nasef\*

**References**

327-59.

In conclusion, MSC must be handled with extreme caution before a large scale clinical trial is performed. it has been found in a pilot clinical study, that co transplantation of MSC and HSC prevent GVHD, but caused a higher relapse rate in hematologic malignancy patients as compared to control [106].

**Figure 1.** Mechanisms of action of MSCs upon dendrietic, lymphocytes T, lymphocytes B and natural killer cells.

MSCs inhibit monocytes (MO) differentiation into immature DCs (IDC) and skewed them toward macrophage. MSCs reduced percentage of CD34(+) derived IDC and increased plasmacytoid DC (pDC). They inhibit maturation IDC into mature DCs. They inhibit the regulation of HLA-DR, CD80, CD86, CD1a, CD40, CD14 and CD83 antigen on DCs surface through IL-6, M-CSF, PGE2, IL-10 and TGF- b secretion. MSCs increased production of IL-10 and decreased production of Il-12, TNF-a and INF-g by DCs. MSCs induced DC that exhibit a suppressor phenotype (supp APC) and generated alloantigen-specific regulatory cells (Reg cells). MSCs inhibit NK proliferation and change cytokines secretion pattern through IDO, TGF-b, PGE2. MSCs inhibit lymphocytes B Proliferation & differentiation to plasma cells through IDO. MSCs inhibit lymphocytes T proliferation and cytotoxicity through IDO, PGE2, HLA-G, TGF-b, HGF, Il-10 and IL-1 b.

#### **Author details**

Aisha Nasef\*

This could indicate that lymphocytes start the first signals for MSCs activation, however cross

In conclusion, MSC must be handled with extreme caution before a large scale clinical trial is performed. it has been found in a pilot clinical study, that co transplantation of MSC and HSC prevent GVHD, but caused a higher relapse rate in hematologic malignancy patients as

**Figure 1.** Mechanisms of action of MSCs upon dendrietic, lymphocytes T, lymphocytes B and natural killer cells.

MSCs inhibit monocytes (MO) differentiation into immature DCs (IDC) and skewed them toward macrophage. MSCs reduced percentage of CD34(+) derived IDC and increased plasmacytoid DC (pDC). They inhibit maturation IDC into mature DCs. They inhibit the regulation of HLA-DR, CD80, CD86, CD1a, CD40, CD14 and CD83 antigen on DCs surface through IL-6, M-CSF, PGE2, IL-10 and TGF- b secretion. MSCs increased production of IL-10 and decreased production of Il-12, TNF-a and INF-g by DCs. MSCs induced DC that exhibit a suppressor phenotype (supp APC) and generated alloantigen-specific regulatory cells (Reg cells). MSCs inhibit NK proliferation and change cytokines secretion pattern through IDO, TGF-b, PGE2. MSCs inhibit lymphocytes B Proliferation & differentiation to plasma cells

talk between both of them are essential to have the full immunological effects.

compared to control [106].

174 Stem Cell Biology in Normal Life and Diseases

Address all correspondence to: Nasef@doctor.com

National Medical Research Centre NMRC, Libya

#### **References**


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### *Edited by Kamran Alimoghaddam*

Stem cells have a prominent role in normal life and also in pathogenesis of disorders. Today, these cells are clinically applicable in hematopoietic stem cell transplantation but expansion of their application in many more disorders needs more work. For safe and effective application of these cells, we need better knowledge of their biology, their interaction with other cells (especially supporting niche cells), growth, maturation and also immigration of stem cells through body in normal and abnormal conditions. Also for clinical application we need to understand better, their separation methods and safe manipulation. This book is written to clarify some aspects of stem cell biology, their characteristics, assessment of damage to cells during ex vivo manipulation and also their role in a model of cancers (chronic myeloid leukemia).

Photo by Gio\_tto / iStock

Stem Cell Biology in Normal Life and Diseases

Stem Cell Biology in

Normal Life and Diseases

*Edited by Kamran Alimoghaddam*