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Human multipotent mesenchymal stromal cells and their potential to differentiate into hepatocytes and beta cells

BAERTSCHIGER, Reto Marc

Abstract

Dans le domaine de la médecine régénérative, la transplantation de cellules souches a gagné de l'intérêt pour le traitement de pathologies terminales de certains organes (coeur, foie, pancréas, poumons, reins). Actuellement, la transplantation hépatique est le seul traitement possible lors d'atteinte irréversible du foie. Le développement d'alternatives thérapeutiques lors de pathologies hépatiques est par conséquent essentiel, et des traitements basés sur la thérapie cellulaire en utilisant des cellules souches est une approche novatrice...

BAERTSCHIGER, Reto Marc. Human multipotent mesenchymal stromal cells and their potential to differentiate into hepatocytes and beta cells. Thèse de doctorat : Univ.

Genève, 2009, no. Sc. 4079

URN : urn:nbn:ch:unige-24465

DOI : 10.13097/archive-ouverte/unige:2446

Available at:

http://archive-ouverte.unige.ch/unige:2446

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITE DE GENEVE

Département de Biologie Cellulaire FACULTE DES SCIENCES

Professeur Jean-Claude Martinou

Département de Chirurgie FACULTE DE MEDECINE

Professeur Philippe Morel Professeur Léo H. Bühler

Human multipotent mesenchymal stromal cells and their potential to differentiate into hepatocytes and beta cells

THESE

présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès Sciences, mention Biologie

par

Reto Marc BAERTSCHIGER de

Murgenthal (AG) Thèse n° 4079

Centre Editions des Hôpitaux Universitaires de Genève Genève

2009

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A Alphonse-Alphonsette qui est devenue Salomé

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TABLE OF CONTENT

ABBREVIATIONS ... 4

SUMMARY ... 6

RESUME EN FRANÇAIS... 9

1: INTRODUCTION ... 12

1.1. Stem cells ... 12

1.2. Embryonic stem cells ... 14

1.3. Induced pluripotent stem cells (IPS) ... 16

1.4. Adult stem cells ... 17

1.4.1. Hair follicle stem cells... 18

1.4.2. Neural stem cells ... 19

1.4.3. Intestinal stem cells ... 20

1.4.4. Liver stem cells / oval cells ... 22

1.4.5. Hematopoietic stem cells ... 24

1.4.6. Multipotent adult progenitor cells (MAPC) / marrow-isolated adult multilineage inducible cells (MIAMI) / unrestricted somatic stem cell (USSC) ... 29

1.5. Mesenchymal stem cells / multipotent marrow stromal cells ... 30

1.5.1. Isolation and tissue distribution ... 30

1.5.2. Characterization ... 31

Established protocols for differentiation of MSC into osteoblasts, chondrocytes and adipocytes... 33

1.5.3. Stem cell marker expression and cloning ability... 34

1.5.4. Plasticity of MSC ... 36

1.5.4.1. Neural differentiation... 36

1.5.4.2. Cardiac differentiation ... 38

1.5.4.3. Hepatic differentiation ... 40

1.5.4.4. Pancreatic differentiation (beta-like cells) of MSC... 44

1.5.5. Influence of donor age on expansion and differentiation ... 49

1.5.6. Immunomodulation and present clinical applications ... 52

1.5.7. Clinical applications of multipotent mesenchymal stromal cells and future perspectives ... 58

1.6. The liver, anatomy, physiology, development and functions ... 60

1.6.1. Anatomy and basic physiology... 60

1.6.2. Liver embryology... 61

1.6.3. Liver diseases... 62

1.6.3.1. Fulminant - acute - chronic hepatic failures and liver regeneration ... 62

1.6.3.2. Rodent models of liver diseases and regeneration ... 64

1.7. The pancreas, anatomy, physiology, development and functions ... 68

1.7.1. Anatomy and basic physiology... 68

1.7.1.1. The exocrine pancreas ... 68

1.7.1.2. The endocrine pancreas ... 68

1.7.1.3. The beta cell ... 69

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1.7.2. Pancreas development ... 71

1.7.3. Diabetes ... 73

1.7.4. Pancreatic and beta cell regeneration/proliferation ... 74

2. RESULTS, EXPERIMENTAL DATA AND ARTICLES ... 76

2.1. Aim of the study ... 76

2.2. Article 1: Fibrogenic potential of human multipotent mesenchymal stromal cells in injured liver... 76

2.2.1. Introduction and specific aims... 76

2.2.2. Submitted article ... 78

2.3. Article 2: Mesenchymal stem cells derived from human exocrine pancreas express transcription factors implicated in beta-cell development. ... 114

2.3.1. Introduction and specific aims... 114

2.3.2. Second article ... 116

3. MAJOR FINDINGS AND FUTURE PERSPECTIVES ... 126

3.1. First publication: discussion ... 126

3.2. Future perspectives of MSC in hepatic replacement ... 128

3.3. Second publication: discussion ... 129

3.4. Future perspectives of pancreatic MSC... 130

4. CONCLUSION ... 132

5. ACKNOWLEDGMENTS... 134

6. REMERCIEMENTS ... 136

7. REFERENCES ... 138

8. ADDENDUM 1: COMPOSITION OF THE THESIS JURY: ... 179

9. ADDENDUM 2: PUBLICATIONS BY THE AUTHOR, NOT RELATED TO THIS WORK ... 180

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ABBREVIATIONS

AFP alpha fetoprotein

AGM aorta, gonad, mesonephros

API alpha1 anti-trypsin

5-AZA 5-azacytidine

BMP bone morphogenic protein

cAMP cyclic adenosine monophosphate CD cluster of differentiation

CFU colony forming unit

CK cytokeratin

DKK-1 Dickkopf-1

EGF epidermal growth factor

EMT epithelial mesenchymal transition

ESC embryonic stem cells

FACS fluorescent activated cell sorting

FAH-/- fumaryl aceto-acetate hydrolase deficient FGF fibroblast growth factor

FoxA2 forkhead box A2

GFAP glial fibrillary acid protein GLP-1 glucagon like peptide 1

GM-CSF granulocyte-macrophage colony stimulating factor GvHD graft versus host disease

HGF hepatocyte growth factor

HDL high density lipoprotein

HLA human leukocyte antigen

HNF hepatocyte nuclear factor

HO-1 heme-oxygenase-1

HSC hematopoietic stem cells

IBMX isobutyl methyl xanthine

IDO indoleamine 2,3-dioxygenase

Ifn interferon

IGF-1 insulin-like growth factor-1

Il interleukin

iNOS inducible nitric oxide synthase

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IPS induced pluripotent stem cell

KO knock-out

KGF keratinocyte growth factor

MAPC multipotent adult progenitor cells MHC major histocompatibility complex

MIAMI marrow-isolated adult multilineage inducible cells

MMP matrix metalloproteinase

MSC multipotent mesenchymal stromal cells

Ngn3 neurogenin 3

NIH national institute of health

NK natural killer

NOD/SCID non-obese-diabetic/severe combined immunodeficient NTBC 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3-cyclohexanedione Oct-3/4 octamer-binding-transcription-factor-3/4

PPAR peroxysome proliferation-activated receptor Pdx1 pancreatic and duodenal homeobox 1

SCF stem cell factor

SCID severe combined immunodeficient SDF-1 stromal-derived factor-1

SHH sonic hedgehog

Sox-2 SRY-related high-mobility-group-box protein 2 TGF transforming growth factor

TLR toll-like receptor

TNF tumor necrosis factor

UCB umbilical cord blood

USSC unrestricted somatic stem cells VEGF vascular endothelial growth factor

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SUMMARY

In the field of regenerative medicine, there is an increasing interest in stem cell transplantation for treatment of organ failure. Currently, liver transplantation is the only available therapy for end-stage liver disease. Due to chronic lack or organs, developing an alternative method for liver transplantation is crucial, and a stem cell- based therapy for liver regeneration is a promising approach.

Type 1 diabetes is a disease characterized by the destruction of insulin producing cells (called beta cells) and subsequent impaired control of blood sugar, resulting in hyperglycemia. Insulin injections restore glycemic control but beta cell replacement would be a more physiologic treatment. Pancreas and islet transplantation are currently performed, but discrepancy between numbers of patients suffering from type 1 diabetes and available organs stresses the need to find alternative tissue sources, such as stem cell based beta cell replacement.

Recently, several studies have shown that adult stem cells from the bone marrow can differentiate into cells of other organs, a characteristic called plasticity. Adult multipotent mesenchymal stromal cells (MSC), isolated from the bone marrow can differentiate in vitro into mesodermal tissues such as muscle, bone, cartilage, fat, but some evidence exist that they may also differentiate towards endoderm, such as hepatocytes or beta cells, or neurectoderm, such as neurons.

The present thesis is composed of a general introduction on stem cell biology, liver development and diseases as well as pancreas development and diabetes. The second section is subdivided into two parts, with two articles both investigating the potential of MSC to differentiate towards cells of the endoderm.

Differentiation of MSC towards endodermal lineages, including hepatocytes, remains controversial. To address this question we studied MSC derived from human bone marrow and their potential to differentiate into hepatocytes in vitro and in vivo. As plasticity of MSC might be dependent on donor age, we isolated MSC from adult and pediatric bone marrow. After characterization, adult and pediatric MSC were co- cultured with a human hepatoma cell line (Huh7) in medium containing hepatocyte growth factor (HGF), fibroblast growth factor (FGF)-4 and oncostatin M. These co- cultures with Huh7 cells induced expression of albumin in adult MSC (2/10 experiments) and pediatric MSC (5/8 experiments). However, when cultured in Huh7- conditioned medium, MSC rapidly expressed α-smooth muscle actin and demonstrated a myofibroblast differentiation potential. In vivo, MSC were

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transplanted into spleen or liver of immunodeficient mice undergoing partial hepatectomy. We analyzed the expression of mesenchymal, fibrogenic and hepatic markers by RT-PCR, Western blot and immunohistochemistry. After intrasplenic injection, MSC were detected in the spleen but few MSC migrated to the liver. After intrahepatic injection, MSC maintained mesenchymal morphology and expressed vimentin and α-smooth muscle actin, but no hepatic markers. Therefore, in this first article we showed that, despite more frequent induction of albumin expression by pediatric MSC in vitro, adult and pediatric MSC implanted in regenerating liver parenchyma did not differentiate into hepatocytes. In vitro and in vivo, MSC expressed α-smooth muscle actin demonstrating myofibroblast differentiation. A fibrogenic potential of human MSC should thus be considered when transplanted in a regenerating liver environment.

Transplantation of in vitro generated islets or insulin producing cells represents an attractive option to overcome organ shortage, for the treatment of type 1 diabetes. In the second publication we isolated, expanded, and characterized cells from human exocrine pancreas and analyzed their potential to differentiate into beta cells.

Fibroblast-like cells emerged from 14/18 human pancreatic exocrine fractions and were expanded up to 40 population doublings. These cells displayed surface antigens similar to MSC from bone marrow. Culture of these cells in adipogenic and chondrogenic differentiation media allowed differentiation into adipocyte- and chondrocyte-like cells. During expansion, these cells expressed transcription factors implicated in islet development such as Isl1, Nkx2.2, Nkx6.1, Ngn3, Pdx1, and NeuroD, as well as nestin, a marker for beta cells and neural progenitors. To study their ability to differentiate towards endocrine lineage, pancreas-derived MSC were cultured in medium containing Activin A and hepatocyte growth factor. We detected low levels of expression of insulin, glucagon, and glucokinase. Therefore proliferating cells with characteristics of MSC and endocrine progenitors were isolated from exocrine tissue. These cells expressed little insulin, and are therefore considered as potential beta cell progenitors. Further improvement of differentiation conditions are required to demonstrate their potential use in clinical settings.

In both studies we have performed, MSC were isolated and partially differentiated towards endodermal cells. Despite the expression of endodermal markers such as albumin and insulin by MSC, in our experiments, their differentiation potential seems limited, in vitro and in vivo. Therefore, we believe that MSC will probably be used in research and clinical treatments for their immunomodulatory characteristics rather

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than to replace hepatocytes and beta cells. Indeed, the insufficient engraftment, questionable function after differentiation and potential to induce fibrosis will prevent them to support acutely injured liver or destroyed islets in hepatic failure and diabetes, respectively.

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RESUME EN FRANÇAIS

Dans le domaine de la médecine régénérative, la transplantation de cellules souches a gagné de l’intérêt pour le traitement de pathologies terminales de certains organes (cœur, foie, pancréas, poumons, reins). Actuellement, la transplantation hépatique est le seul traitement possible lors d’atteinte irréversible du foie. Le développement d’alternatives thérapeutiques lors de pathologies hépatiques est par conséquent essentiel, et des traitements basés sur la thérapie cellulaire en utilisant des cellules souches est une approche novatrice.

Le diabète de type 1 est une maladie caractérisée par une destruction des cellules produisant et sécrétant l’insuline (cellules bêta du pancréas) en réponse au glucose.

La destruction des cellules bêta altère l’homéostasie du glucose dans le sang et a pour conséquence une hyperglycémie avec des complications immédiates et au long court. L’injection d’insuline exogène permet de contrôler la glycémie, mais le remplacement de cellules bêta serait un traitement « plus physiologique ». La greffe de pancréas entier ou d’îlots de Langerhans (contenant des cellules bêta) se font actuellement en clinique, mais la différence entre le nombre de patients diabétiques et le nombre d’organes disponibles implique la nécessité de trouver des sources alternatives, par exemple des cellules bêta dérivées de cellules souches.

Récemment, plusieurs études ont montré que des cellules souches de la moelle osseuse peuvent se différencier en cellules d’autres organes, une caractéristique nommée plasticité. Les cellules multipotentes stromales mésenchymateuses (MSC), isolées à partir de moelle osseuse peuvent se différencier in vitro en tissus d’origine mésodermique (muscle, tissu osseux, cartilage, tissu adipeux). Certaines études récentes suggèrent également la capacité des MSC de se différencier en endoderme (hépatocytes, cellules bêta) ou en neuroectoderme (neurones).

Cette thèse se compose d’une introduction générale décrivant la biologie des cellules souches, le foie, son développement et certaines pathologies, ainsi que le pancréas, son développement et le diabète de type 1. La seconde partie est divisée en deux chapitres, avec deux publications décrivant le potentiel de différentiation des MSC en endoderme, en hépatocytes et cellules bêta, respectivement.

Les MSC sont actuellement évaluées comme thérapies cellulaires dans le traitement de pathologies hépatiques. La différentiation des MSC en cellules épithéliales hépatocytaires reste cependant controversée dans la littérature scientifique. Pour aborder cette question, nous avons étudié le potentiel de différentiation en

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hépatocytes in vitro et in vivo des MSC isolées de moelle osseuse humaine. Comme la plasticité et par conséquent le potentiel de différentiation des MSC pourraient être dépendants de l’âge du donneur, nous avons isolé des MSC de moelle osseuse adulte et pédiatrique.

Après amplification et caractérisation, nous avons co-cultivé les MSC humaines adultes et pédiatriques avec une lignée de cellules hépatocytaires (Huh7) dans du milieu contenant les facteurs de croissance HGF, FGF-4 et oncostatin M. La co- culture avec les cellules Huh7 a induit l’expression de marqueurs hépatiques par les MSC adultes (2/10 expériences indépendantes) et pédiatriques (5/8 expériences indépendantes). Cependant, lors de culture avec des milieux conditionnés par les cellules Huh7, les MSC expriment un marqueur myofibroblastique: l’actine de la musculature lisse (alpha-smooth muscle actin). In vivo, des souris NOD/SCID ayant subi une hépatectomie partielle ont été transplantées avec des MSC soit dans la rate, soit directement dans le parenchyme hépatique restant. Nous avons analysé l’expression de marqueurs mésenchymateux, fibrogènes et hépatiques par RT-PCR, Western blot et immunohistochimie. Après injection intrasplénique, seules quelques MSC migrent dans le foie, la majorité étant restée dans la rate, sans exprimer des marqueurs hépatiques. Lors d’injection intraparenchymateuse dans le foie, les MSC n’expriment pas de marqueurs hépatiques, mais maintiennent l’expression de vimentine et d’alpha-smooth muscle actin. Dans cette première publication nous avons donc démontré que, malgré l’expression plus fréquente de marqueurs hépatiques par les MSC pédiatriques lors de co-culture in vitro, les MSC adultes et pédiatriques implantées dans un foie en voie de régénération ne se différencient pas en hépatocytes mais expriment des marqueurs de myofibroblastes. Par conséquent, les MSC humaines transplantées dans un foie en voie de régénération ont un potentiel fibrogène et risquent d’aggraver la pathologie hépatique pour laquelle une transplantation était envisagée.

La transplantation de cellules produisant de l’insuline ou d’îlots développés in vitro représente une alternative thérapeutique intéressante pour pallier au manque chronique d’organes dans le traitement du diabète de type 1. Le second article expose la présence de MSC dans la fraction exocrine du pancréas humain, leur potentiel d’expansion, leur expression de facteurs de transcription importants pour le développement des cellules endocrines du pancréas et leur potentiel de différentiation vers la cellule bêta. Sur 18 mises en culture de tissu exocrine de pancréas humains, 14 ont permis d’isoler des cellules fibroblastiques. Ces cellules

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ont pu être cultivées jusqu’à 40 divisions (doublements de population). Ces cellules expriment des marqueurs de surface similaires aux MSC isolées de la moelle osseuse. Elles ont pu être différenciées en adipocytes et chondrocytes, après culture dans un milieu adipogène et chondrogène, respectivement. En phase de prolifération, ces cellules expriment les facteurs de transcription endocrines Isl1, Nkx2.2, Nkx6.1, Ngn3, Pdx1 et NeuroD ainsi que la nestin, un filament intermédiaire du cytosquelette, caractéristique des précurseurs bêta et neuronaux. Lorsque le milieu de culture est supplémenté par de l’Activin A et du HGF, les cellules expriment des marqueurs endocrines, tels que insuline, glucagon et glucokinase. Nous avons donc isolé des cellules ayant des caractéristiques de MSC à partir de tissu exocrine humain. Ces cellules expriment des facteurs de transcription présents lors du développement des cellules bêta. Elles expriment de faibles quantités d’insuline et sont par conséquent considérées comme potentiels progéniteurs de cellules bêta.

Pour être utilisées dans un contexte clinique, les protocoles de différentiation doivent être améliorés, afin d’augmenter l’expression d’insuline et d’induire une sécrétion régulée par le glucose.

En conclusion, nos résultats démontrent la différenciation partielle des MSC vers des cellules épithéliales, classiquement d’origine endodermique. Malgré l’expression d’albumine et d’insuline in vitro, ces cellules ont un potentiel de différentiation limité dans nos conditions, aussi bien in vitro et in vivo. Dans le future, les MSC vont probablement être utilisées en recherche et lors de traitements cliniques pour leurs capacités immunomodulatrices, plutôt que pour leur potentiel de différentiation en endoderme. En effet, la prise de greffe souvent insuffisante, une fonction limitée après différenciation et le risque d’induire une fibrose après transplantation ne permettront pas aux MSC d’être utilisée en clinique, lors d’atteinte hépatique aigüe ou lors de destruction des cellules beta chez les patients diabétiques, respectivement.

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1: INTRODUCTION 1.1. Stem cells

Stem cells are defined as cells able to proliferate, self-renew, and differentiate into functional and specialized daughter cells. These characteristics enable stem cells to generate or regenerate tissues and organs (Mimeault, 2006; Weiner, 2008). They have therefore been attracting much interest within the last decades for cellular therapy and tissue regeneration and transplantation.

Stem cells are divided into different categories according to their origin or potency.

A fertilized ovocyte, called zygote, defines totipotent stem cells, meaning cells able to form a whole embryo with its three layers and extra-embryonic structures such as the placenta.

Totipotent stem cells specialize into pluripotent cells that can give rise to most, but not all, of the tissues necessary for fetal development, especially not to extra- embryonic tissues. Embryonic stem cells are the example of pluripotent stem cells per se. Embryonic stem cells can differentiate into cells of all three embryonic layers, endoderm, mesoderm and ectoderm, but also germline cells. They undergo further specialization into multipotent cells that are committed to give rise to cells from one embryonic layer, i.e. mesenchymal stem cells, able to differentiate into osteoblasts, chondrocytes and adipocytes, or hematopoietic stem cells able to form all types of blood cells. This stem cell hierarchy is illustrated in figure 1.

Self renewal and multilineage differential potential are characteristics that are found in embryonic stem cells, induced pluripotent stem cells and most of adult stem cells.

Self renewal is mandatory for stem cells to be able to keep their undifferentiated state and expand. How self renewal is regulated and controlled has been studied intensively in recent years. Self renewal and pluripotency are closely related. Stem cells that self-renew have to inhibit differentiation and thus keep their potential (Zhang, 2006a). Both transcriptional and epigenetic regulations are two essential mechanisms underlying pluripotency. In undifferentiated embryonic stem cells, pluripotency factors (developed below) work together with epigenetic regulators to activate genes involved in pluripotency maintenance and to suppress differentiation- related genes (Yeo, 2007). Although genes controlling differentiation are transcriptionally inactive, they are nevertheless maintained in a potent state for transcriptional activation, to be activated once differentiation signals have reached

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them. Chromatin structure also influences directly pluripotency and self-renewal. In embryonic stem cells this structure is hyperdynamic and can be readily activated.

Upon differentiation, a diminution of pluripotency factors facilitated by miRNAs leads to dramatic changes in the transcriptional profile, where genes required for pluripotency maintenance (Oct-4, Sox-2, Nanog) become silenced (Stadler, 2008;

Wang, 2007). The chromatin in differentiated cells becomes more compact, which correlates with decreased differentiation potential and lineage restriction (Bibikova, 2008).

To further understand the molecular mechanisms of pluripotency, studies on how the chromatin remodeling and histone modifying complexes modulate the global and regional chromatin structure in pluripotent cells are required.

Figure 1: Stem cells hierarchy and differentiation potential.

ICM: inner cell mass, EE: extra-embryonic tissue. From Eckfeldt C.E. et al, Nature Reviews Molecular Cell Biology, 2005 (Eckfeldt, 2005).

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1.2. Embryonic stem cells

Embryonic stem cells (ESC) are derived from the inner cells mass of the blastocyst (Bibikova, 2008). Within the developing embryo they can give rise to billions of cells through symmetric divisions, making two identical daughter cells, or to one differentiated and one undifferentiated daughter cell by asymmetrical divisions (Morrison, 2006). ESC can be isolated in vitro, maintained and expanded in undifferentiated state in culture, thus becoming ESC lines. The derivation and use of ESC lines have first been described for mouse embryonic stem cells in the 1980’s (Evans, 1981; Kaufman, 1983). These cells have been used in hundreds of studies since then and have been thoroughly characterized. Human ESC lines were isolated, derived and expanded later during the end of the 1990’s (Reubinoff, 2000; Thomson, 1998), using similar protocols as for primate ESC lines (Thomson, 1995; Thomson, 1996). To date, ESC have been derived for mice (Evans, 1981), humans (Thomson, 1998), sheep (Notarianni, 1991), rabbit (Graves, 1993), cattle (Mitalipova, 2001), horse (Saito, 2002), pigs (Li, 2003), rats (Ueda, 2008), dogs (Hayes, 2008), and rhesus monkeys (Wianny, 2008).

ESC express high levels of telomerase and alkaline phosphatase, proliferate almost indefinitely and are able to differentiate into cells from all three embryonic layers, i.e.

endoderm, mesoderm and ectoderm (Amit, 2000; Itskovitz-Eldor, 2000). Human ESC keep stable karyotype in culture, whereas mouse ESC are more at risk for the development of aneuploidy (Rebuzzini, 2008). Mouse and human ESC lines have been used in vitro to study differentiation into many different cells types, such as dopaminergic neurons (Chiba, 2008; Cho, 2008), motoneurons (Lee, 2007; Li, 2008c), hepatocytes (Hay, 2008a; Hay, 2008b; Jones, 2002; Yamada, 2002), beta cells of the pancreas (Boyd, 2008; Kroon, 2008; Liew, 2008), cardiomyocytes (He, 2003; Kehat, 2001; Sartiani, 2007; Xu, 2002), chondrocytes (Hwang, 2008), osteocytes (Jukes, 2008), and hematopoietic stem cells (Bowles, 2006; Wang, 2005b). This wide differentiation potential in vitro has focused hope and major medical and scientific interest on ESC as potential tools for cell therapy. Many studies have also shown possible application of in vitro differentiated ESC after transplantation in animals, in vivo (Chiba, 2008; Kroon, 2008).

ESC have been characterized in details. They express markers of pluripotency, such as transcription factors Octamer-binding-transcription-factor-4 (Oct-4), Nanog, SRY- related high-mobility-group-box protein 2 (Sox-2), Rex1 (an acidic zinc finger gene also called Zfp-42), and c-Myc (Babaie, 2007). The expression of these factors will

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be discussed further for induced pluripotent stem cells (IPS) and for adult stem cells.

These transcription factors can be divided into two classes, those that bind to c-Myc promoter and those which do not (Kim, 2008a). These specific transcription factors act on two classes of target genes: 1) promoters bound by few factors which tend to be inactive, 2) promoters bound by more than 4 factors which are active in the pluripotent state and become repressed when ESC undergo differentiation (Kim, 2008a).

Unfortunately, ESC and ESC lines have major risks to induce tumors, more specifically teratomas (Klimanskaya, 2005). These characteristics demonstrate their pluripotency, but harbor the risk to induce tumors in potential recipients. Even after differentiation of ESC towards functional cells, the risk of teratoma is still present, as only few remaining undifferentiated cells are sufficient to induce teratomas (Brederlau, 2006; Fujikawa, 2005). Presently, strategies to obtain pure cells populations after in vitro differentiation are under evaluation, such as fluorescence activated cell sorting (FACS) for a marker, the expression of which is under the control of a differentiation marker (Hedlund, 2007).

The use of ESC, especially human ESC and their derivation from pre-implantation blastocysts after in vitro fertilization has raised important ethical questions (Sugarman, 2008; Vogel, 2008). These ethical questions have found different answers around the world and are widely related to religious beliefs and to moral, social and political sensitivities. In the United Kingdom, the use of human ESC is approved and the approach very liberal, whereas it is very controlled in the USA with limited or even absent National Institute of Health (NIH) funding for derivation of new ESC lines (Gottweis, 2006a). In Germany, most research on human ESC is forbidden whereas in Switzerland new derivation of human ESC lines from donated zygotes after in vitro fertilization was approved by the population, but is nevertheless strictly controlled by the Swiss Federal Health Office (Feki, 2008). The South Korean debacle and the proven fraud of Hwang et al (Hwang, 2005), claiming the first cloned human embryonic stem cell line derivation has raised negative concerns on the developing field of regenerative medicine and use of human ESC (Gottweis, 2006b).

Very recently, the International Society for Stem Cell Research (ISSCR) has edited guidelines concerning the procurement of research materials, the derivation of stem cells, and banking, distribution and use of ESC and ESC lines derived from pre- implantation human embryos; these guidelines were defined by researchers, ethicists and lawyers (Dickens, 2008). The use of mouse and human ESC in regenerative

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medicine remains ethically debatable. Further developments of techniques for their purification after differentiation are also required. Adult stem cells seemed therefore a more suitable approach to us, and, as opposed to ESC, did not imply major ethical and legal debates.

1.3. Induced pluripotent stem cells (IPS)

Recently, Takahashi et al identified the four transcription factors necessary and sufficient for pluripotency and reprogramming of somatic cells (Takahashi, 2006).

Using mouse fibroblasts, cultured in ESC-conditions and using previous knowledge on transcription regulation of ESC, they demonstrated that retroviral-mediated introduction of Oct-4, Sox-2, c-Myc and Kruppel-like-factor-4 (Klf4) was able to induce pluripotency. Mouse fibroblasts thus behaved like ESC, with teratoma formation, multilineage differentiation potential (endoderm, mesoderm, ectoderm) and ability to contribute to mouse embryonic development after injection into blastocysts (Takahashi, 2006). The same technique has been applied to other mouse somatic cells and to human fibroblasts with the same results (Meissner, 2007; Park, 2008; Takahashi, 2007b; Wernig, 2007). These cells were totally reprogrammed and behaved like ESC in vitro and in vivo. Notably, mouse IPS formed viable chimaeras and contributed to the germ line (Okita, 2007; Wernig, 2007). In vitro, the biological potency and epigenetic state of induced pluripotent stem cells were indistinguishable from those of ESC, for mice and human cells. These results have been obtained in different laboratories, but the detailed mechanisms for reprogramming remain unknown and are currently under intense investigations. Genetic stability and telomere length of IPS need to be analyzed further to exclude potential risks of transformation and degeneration.

IPS have attracted also clinical interests, as a simple biopsy could be used to extract fibroblasts or any other somatic cell, amplified, reprogrammed and then banked for future use. As these cells could be derived from potential patients (Park, 2008), autologous transplantation could be performed without the need for immunosuppression. However, besides the problems already encountered for ESC (teratoma formation), the retroviral-mediated introduction of transcription factors and oncogenes such as Myc also harbor risks for the recipients, as transgenes may integrate randomly and the oncogene Myc, even though not totally necessary for reprogramming (Nakagawa, 2008; Yu, 2007), may induce uncontrolled behavior and cell cycle.

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In summary, IPS focused the attention of the “Stem cell community” because they have almost identical characteristics to ESC, and do not raise ethical and legal concerns. It is, however, too early to consider these cells as safe alternative for cell replacement therapies. The risk of teratoma formation and tumor degeneration is presently one of the major concerns with both these cell types, together with biosaftey aspects of lentiviral-infected cell transplantation (Blum, 2008; Yamanaka, 2008). More precisely on the biosafety aspects of lentivirus infection, it seems to be avoidable, as Okita et al from the Yamanaka group were able to transfect cells with a single plasmid containing the cDNAs of Oct-3/4, Sox-2, and Klf4, together with a c- Myc expression plasmid, into mice embryonic fibroblasts, which resulted in the production of induced pluripotent stem cells of mice (Okita, 2008). Thus the production of virus-free induced pluripotent stem cells is feasible but more safety data are required to use these cells in regenerative medicine.

1.4. Adult stem cells

Adult stem cells are defined as cells isolated after birth (postnatal, as opposed to embryonic or fetal) from certain tissues, able to self renew within a given individual or in vitro, and with the ability to differentiate into at least two functional cell types (Korbling, 2003). The presence of stem cells in postnatal vertebrates seems evident from the observed continuation of tissue and organ growth, development and differentiation, which seem to be an extension of prenatal gestation, at least for mammals. For adult vertebrates, following sexual and skeletal maturation, the presence of stem cells seems less obvious at first sight. In the adult male reproductive tract, the production of million of spermatozoids, i.e. germ cells, which occurs throughout life, represents one easily understandable argument for the presence of stem cells within adult organism. In spermatogenesis, a self-renewing population of premeiotic stem cells (spermatogonial stem cells) seems to persist throughout life, and remains able to fertilize ovocytes and induce the formation of zygotes and embryos. These cells are derived from primordial germ cells migrating to the urogenital ridges in the embryo. They differentiate towards gonocytes, the precursors of spermatogonial stem cells (Oatley, 2008).

In contrast, many somatic tissues do not appear to be expanding or further developing or regenerating. The identification of adult stem cells within these somatic tissues, i.e. bone marrow (Spangrude, 1988), hair follicle (Cotsarelis, 1990), intestine (Bjerknes, 1981b; Cheng, 1974a; Potten, 1977) has therefore taken extensive time of

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intensive research and the accumulation of considerable experimental evidence has finally identified the presence of various types of adult stem cells within a given organism. The ancient Greek mythology already anticipated the presence of self- renewing or at least regenerating tissues such as the liver, which regenerating characteristics were elegantly described in the myth of Prometheus, by the poet Hesiod (20th century B.C.) (Pahlavan, 2006; Power, 2008).

We will focus here on the present knowledge of some adult stem cell types present in mammals, from hair follicle stem cells, neural stem cells and intestinal stem cells to hematopoietic stem cells, which were the first to be used in the clinical setting.

1.4.1. Hair follicle stem cells

Mammalian organisms contain hair follicle stem cells in their skin, which reside in a relatively quiescent state within a well defined, anatomically distinct region of the hair follicle called the bulge. Hair follicle stem cells participate in homeostasis of skin and hair regeneration, but they also ensure the repair of skin after injuries. In mice, hair follicle stem cells can contribute to all three epithelial lineages, such as sebaceous glands and hair follicles, but also to keratinocytes in the epidermis (Blanpain, 2004;

Morris, 2004; Oshima, 2001). Precise localization of hair follicle stem cells has been a matter of debate for a long time (Cotsarelis, 1990; Hardy, 1992; Reynolds, 1991). It has now been demonstrated that epithelial hair follicle stem cells reside in the bulge, as shown by pulse-chase experiments with labeled nucleotides or transgenic expression of a fluorescent histone protein (Cotsarelis, 1990; Fuchs, 2004; Morris, 1999; Taylor, 2000; Tumbar, 2004). These cells are able to adapt after skin injuries and stem cells residing in the bulge move towards the skin surface, whereas mesenchymal-epithelial interactions, related to the hair cycle, stimulate downward movements (Morris, 2004; Taylor, 2000; Tumbar, 2004). These bulge stem cells can be isolated and expanded in vitro. They form larger colonies than other cells isolated from the skin, and express specific markers such as CD34, keratin 15 (Blanpain, 2004; Liu, 2003; Trempus, 2003) and upregulate transcription factors such as Sox9, Lhx2, Tcf3, and Nftac1 (Blanpain, 2004; Morris, 2004; Trempus, 2003; Tumbar, 2004). A recent study showed that these hair follicle stem cells residing within the bulge are present early during embryogenesis, at initial stages of hair follicle morphogenesis and that primary stem cells specification is dependent on Sox9 expression. Importantly, if these stem cells are absent, hair follicle and sebaceous gland development is dysmorphic and epidermal wound repair is compromised

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(Nowak, 2008). Thus, hair follicle stem cells have been characterized, and their early embryologic origin defined. They can be expanded in vitro and may be used in the future for skin replacement therapies. Furthermore, signaling within the niche and with different components of the niche has been described and will be of help for the understanding of a wide variety of human skin diseases, including cancers and genetic mutations affecting skin development (Blanpain, 2004), reviewed in (Fuchs, 2008).

1.4.2. Neural stem cells

Until recently, the paradigm of senescence of neurons and the absence of neural stem cells within adult brains was thought to be true and unchanging. During the last decades however, the view of the regenerative capacity of the adult mammalian brain has evolved dramatically. Few reports in the 1960’s already described proliferating cells and neurogenesis in the post-natal rodent brain, by 3H-thymidine incorporation (Altman, 1965; Messier, 1958). These proliferating cells are located in the subventricular zone of the lateral ventricles and the subcallosal zone at the border between the corpus callosum and the hippocampus. Some controversy exists about the presence of neural stem cells within the subgranular zone of the dentate gyrus, and the cerebellum, at the border between the internal granular layer and the white matter (Sutter, 2007). These neural stem cells are able to self-renew and differentiate into neurons, oligodendrocytes and astrocytes in vitro (Doetsch, 1999;

Haubensak, 2004; Johansson, 1999). Interestingly these neural stem cells are characterized as radial glia and neuroectodermal cells, which self-renew, express nestin (an intermediate filament) and have a mitotic spindle that is apically located (Gotz, 2005). Neural stem cell populations in adult organisms persist by asymmetric cell divisions, giving one neural stem cell and one daughter, transient amplifying cell, also called neurogenic astrocyte, sharing expression of glial fibrillary acid protein (GFAP) with astrocytes (Alvarez-Buylla, 2004; Garcia, 2004; Merkle, 2004; Sanai, 2004). Neural stem cells can be isolated from these subventricular regions of the brain by cell sorting for stem cell markers such as CD133 (Coskun, 2008; Lee, 2005).

They can be cultured in media containing mitogens and form heterogeneous cell clusters called neurospheres. Cells within neurospheres can be expanded and passaged in vitro, before differentiation. Whether neural stem cells from different regions in the brain can differentiate into all types of neurons or whether there is an anatomical restriction and correlation between isolation site of neural stem cells and

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neuronal type is currently studied. As neural spheres seem heterogeneous in composition, it was difficult to fully characterize gene expression profiles of neural stem cells (Karsten, 2003; Suslov, 2002). Further clonal analysis of gene expression profiles of single site neural stem cells need to be undertaken to fully characterize genetic patterns of neural stem cells. Merkle et al showed some evidence that not all neural stem cells can differentiate into all types of neurons (Merkle, 2007). In labeling and neural stem cell transplantation experiments, they showed that the potential of postnatal neural stem cells is determined by a spatial code, probably in the form of specific expression of transcription factors. Therefore, neural stem cells seem not to be as multipotent as expected.

Neural stem cells have also been isolated from adult human brains, from fetal brain (Uchida, 2000) and from biopsies of patients suffering from epilepsy (Ayuso-Sacido, 2008; Walton, 2006). Whether human cells are also patterned to differentiate into a specific neuronal type has not been demonstrated so far. For future clinical applications, dopamine expressing neurons would be of particular interest for the treatment of Parkinson’s disease, and major work is currently addressing these challenges.

1.4.3. Intestinal stem cells

The adult mammalian intestinal epithelium is one of the most actively proliferating tissues in the organism. It is in close contact with the underlying submucosal mesenchyme, to control self-renewal, proliferation, differentiation into various cell types, migration towards the top of the villi or towards the surface of the crypts, and apoptosis, once cells reached their final location. From a histological and anatomical point of view, the intestinal epithelium contains 4 differentiated cell types: absorptive- also called enterocytes, mucosecreting-, enteroendocrine and Paneth cells (Sancho, 2003). These 4 cell-types develop all from one type of multipotent stem cells, called intestinal stem cells. The exact identity of the intestinal stem cell has remained controversial for the last three decades, with two opposing models presented by experts. In the late 1950’s, the first model called the +4 model was described: the stem cell was identified at position +4 relative to the bottom of the crypt while the three first positions were occupied by Paneth cells. This model was supported by experimental data by Potten et al in the 1970’s (Potten, 1977; Potten, 1974). The +4 cells are actively proliferating. One characteristic of these stem cells, also described for hair follicle stem cells, is label-retaining capacity. Label retaining capacity might

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result from asymmetric segregation of old and new DNA strands (Potten, 1977;

Potten, 2002). It has however never been demonstrated in vivo that the progeny of +4 stem cells constitutes all 4 differentiated intestinal epithelial cells.

The second model was proposed in the late 1970’s by Cheng et al and was called the “stem cell zone” model (Cheng, 1974a, b). In this model, the stem cell is located within the bottom of the crypt, between the Paneth cells. It is described as small, rapidly cycling, undifferentiated and termed “crypt base columnar cells”. Based on morphological studies but also more recently on the Dlb-1-based clonal labeling technique, various groups have shown that crypt base columnar cells have stem cell characteristics and can give rise to all four cell types. Paneth cells migrate towards the bottom of the crypt after differentiation whereas enterocytes, mucosecreting-, and enteroendocrine cells migrate upwards to the villus (Bjerknes, 1981a, b, 1999;

Cheng, 1974a, b; Stappenbeck, 2003).

Wnt signaling is also important in intestinal epithelial cells, as multiple Wnt factors are secreted at the bottom of crypts (Gregorieff, 2005), and create a gradient of Wnt factors on the crypt-villus axis. Furthermore, Wnt activity at the bottom of crypts was demonstrated by accumulation of nuclear beta-catenin (a molecule which is activated upon Wnt signaling) (van de Wetering, 2002). Wnt signaling also interferes with the cell cycle in stem cells at the bottom of the crypts (Barker, 2008), and is important for differentiation towards Paneth cells. By analyzing the expression of Wnt target genes, Barker et al were able to identify one gene that was specifically expressed within the crypts: Lgr5/Gpr49 gene, encoding an orphan G protein coupled receptor, which is closely related to receptors such as TSH, FSH and LH receptors (Barker, 2007). Lgr5/Gpr49 is also expressed in the hair follicle stem cells (Morris, 2004).

Barker et al demonstrated that Lgr5/Gpr49 expressing cells were the actively cycling crypt base columnar cells, supposed stem cells described earlier (Barker, 2007;

Cheng, 1974a, b). Lineage tracing in transgenic mice models, integrating enhance green fluorescent protein into the Lgr5/Gpr49 gene showed that crypt base columnar cells can give rise to all four cell types. They self-renew, are actively cycling and therefore fulfill the definition of stem cells. Interestingly, these cells appear never to be quiescent. Thus telomere length maintenance, protection of genomic integrity and prevention of cellular transformation are mechanisms that must be highly regulated and conserved. Bmi1 was recently identified as another marker of intestinal stem cells (Sangiorgi, 2008). Bmi1 gene encodes a component of a polycomb repressing complex 1, important for maintenance of chromatin silencing (Widschwendter, 2007),

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but also for self-renewal of neuronal and hematopoietic cells (Lessard, 2003;

Molofsky, 2003). In their experiments, Sangiorgi et al demonstrated that Bmi1 expressing cells are able to self-renew, to differentiate into all four cell types of the intestinal epithelium, and to induce cancer if beta-catenin is stabilized in these cells.

If Bmi1 allele is ablated, crypts disappear demonstrating the necessity of Bmi1 expression for intestinal morphology maintenance and epithelium self-renewal. Worth noting is that Bmi1 expressing cells are found at the +4 position described by Cheng et al (Cheng, 1974a, b; Sangiorgi, 2008). In summary, two markers for intestinal stem cells have been described recently but they have not been used together to stain cells in intestinal crypts. Thus, definitive proof for either model has not been yet demonstrated, but these markers will help to better understand the biology of intestinal stem cells in the future (Barker, 2008; Sancho, 2003; van der Flier, 2008).

1.4.4. Liver stem cells / oval cells

As mentioned previously, the ability of the liver to regenerate was known by the ancient Greeks and illustrated in the legend of Prometheus. In animal models, survival after partial liver resections of up to 80 or 90% of liver mass shows this tremendous ability of regeneration (Higgins, 1931). This process can also be repeated without reduction of the regenerative capacity. These characteristics are almost unique in the mammalian organism, as opposed to a limited regeneration of pancreas or kidney (Stocker, 1973). The regeneration process of liver after partial hepatectomy is not dependent on stem cells and is regulated as in normal liver turnover. The molecular regulation of regeneration has been extensively described and is dependent on various cytokines such as tumor necrosis factor (TNF) alpha, interleukin (IL)-6, Hepatocyte growth factor (HGF) and transforming growth factor (TGF) beta1 (Cressman, 1996; Jochheim-Richter, 2006; Michalopoulos, 1997;

Monga, 2001; Yamada, 1997). This process is usually completed within one week.

During regeneration, liver size is well controlled to prevent organ overgrowth. The molecular control of size and the halt of regeneration have not been totally understood, but removal of regenerative signals such as reduction of HGF stops liver overgrowth. Overexpression of HGF through a plasmid in the liver of adult mice induces hepatomegaly, demonstrating the importance of HGF reduction at the end of regeneration (Apte, 2006).

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Although “physiological” hepatocyte turnover and liver regeneration are not dependent on stem cells, certain liver injuries require stem cells for hepatocyte replacement. When treating rats with carcinogenic agents such as ethionine, 2- acetylaminofluorene and 3-methyl-4-dimethyl aminobenzene, Farber observed the appearance of non-parenchymal cells with specific morphologic characteristics, first beginning within the portal areas (Farber, 1956). These cells were described as small oval cells, with high nuclear/cytoplasmic ratio, pale blue nuclei and scant basophilic cytoplasm, probably related to bile duct epithelial cells. These cells were proliferating and progressively invaded most liver lobules (Farber, 1956). These cells, which will become differentiated hepatocytes, were termed oval cells because of their morphology. It was first unclear whether oval cells could differentiate towards hepatocytes (Tatematsu, 1984), but using 3H-thymidine labeling and treating rats with 2-acetylaminofluorene, Evarts et al showed the appearance of 3H labeled basophilic hepatocytes as clusters within liver lobules (Evarts, 1987). Oval cells express bile duct epithelial markers (Cytokeratin (CK)-7 and CK-19), hepatocyte markers (albumin and alphafetoprotein (AFP)) and hepatic transcription factors (HNF1alpha and HNF1beta, HNF3gamma) (Evarts, 1989; Nagy, 1994). HNF4 is only expressed once oval cells start to differentiate into basophilic hepatocytes. The expression of stem cell markers, especially c-kit, CD34, flk-1 and leukemia inhibitory factor (Cano) by oval cells during proliferation suggested that they harbor stem cell properties (Fujio, 1994; Omori, 1997a; Omori, 1996; Omori, 1997b; Tsuchiya, 2007). Oval cells are not derived from hepatocytes, but their origin remains controversial. They were first thought to originate from ductular cells in the canal of Hering (Factor, 1994), but later hematopoietic stem cells were suggested as origin (Oh, 2007; Petersen, 1999;

Petersen, 1998; Petersen, 2003). Yet, this fact is still controversial in the literature (Menthena, 2004; Wang, 2003a). Oval cells are therefore facultative stem cells, which proliferate in vivo only in certain conditions such as treatments with carcinogens. They can be isolated from the liver, expanded in vitro and transplanted to repopulate the liver of recipients. Oval cell transplantation is however hampered by some limitations also encountered for hepatocyte transplantation: 1) lack of organ donors, 2) limited capacity to repopulate diseased liver parenchyma, 3) need to pre- treat the donor to increase oval cell numbers, 4) risk to become potential liver cancer stem cells (Sell, 2008; Song, 2004; Theise, 2003; Wang, 2003a). Recently, Dorrel et al developed a panel of monoclonal antibodies for surface markers of murine oval cells, antibodies which will certainly improve their isolation (Dorrell, 2008).

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Another type of hepatic stem cells suitable for transplantation would be fetal hepatoblasts, isolated from hepatic tissue of fetuses. These cells are able to proliferate and expand more than adult hepatocytes, give rise to cholangiocytes and hepatocytes and repopulate liver tissue extensively (Mahieu-Caputo, 2004; Malhi, 2002). Their clinical use may however be ethically questionable, as they are of fetal origin.

1.4.5. Hematopoietic stem cells

Hematopoietic stem cells (HSC) are self-renewing, multipotent progenitors that give rise to all types of mature blood cells (Spangrude, 1988). They were the first to be identified in the 1960’s as clonogenic bone marrow precursors that give rise to multilineage hematopoietic colonies in the spleen, and were able to repopulate the hematopoietic system of mice after lethal irradiation. They were called HSC (Becker, 1963; Siminovitch, 1963; Till, 1961). HSC were then extensively studied in vitro and in vivo and characterized during the end of the 1980’s (Baum, 1992; Spangrude, 1988; Uchida, 1992). They reside in the bone marrow during adult life, from where they could be isolated and purified. They were the first cells to be successfully transplanted and could restore function in patients after myeloablation or in children in whom hematopoiesis was deficient (Cline, 1977; Radl, 1972).

From a biological point of view, HSC fulfill two major stem characteristics: they self- renew, and they are able to undergo differentiation to progenitor cells that become variously committed to different hematopoietic lineages. During the last decade, origin, characterization, regulation, plasticity and developmental potential of HSC have been studied extensively. Identification and characterization of surface antigens further enabled researchers to better isolate and purify them using monoclonal antibodies (Gerrits, 2008; Kiel, 2008; Weissman, 2000). These monoclonal antibodies allowed identification but also isolation of mouse and human HSC to near homogeneity (Krause, 2001; Osawa, 1996). Using these techniques on mice and human cells, it was shown that CD34+ and very rare CD34-, c-kit+, sca-1+ (in mice), thy-1+ (human), lineage negative phenotype (lin-) cells have characteristics of long- term repopulating HSC. Interestingly, CD34 was thought be a specific stem cell marker, but some subpopulations of CD34- cells were shown to be able to repopulate bone marrow and restore hematopoiesis in mice (Osawa, 1996). Furthermore Lin-, CD34-, CD38- human HSC showed to be a biologically distinct population, which was

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more potent for severe combined immunodeficient mice repopulation (Bhatia, 1998).

The exact role of CD34 has not been fully established to date. CD34 could function as adhesion molecule with a role in early hematopoiesis. It may mediate the attachment of HSC to the bone marrow extracellular matrix, or directly to stromal cells, or it may act as a scaffold for the attachment of lineage specific glycans (Gangenahalli, 2005; Gangenahalli, 2006; Krause, 1996; Simmons, 1992). HSC also extrude cellular dyes such as Hoechst fluorescent- and Rhodamine fluorescent dyes, similarly to hair follicle stem cells and intestinal stem cells (Bhatia, 1998).

The description of different populations of HSC (i.e. CD34+, CD34-) hampered precise and direct molecular characterization during the 1990’s (Bhatia, 1998;

Osawa, 1996).

Hematopoietic malignancies allowed the description of many cell-intrinsic genes which define HSC. The identification of SCL/tal1, Lmo2 or Notch1 and their importance in HSC proliferation were described for acute T-cell leukemia related chromosomal translocation (Begley, 1989; Ellisen, 1991; Finger, 1989) and reviewed in (Begley, 1999). The importance of these factors, and others such as Hoxb4, Bmi1, Bone morphogenic protein (BMP)-4 and sonic hedgehog (SHH), for HSC self- renewal, maintenance, proliferation, and terminal differentiation were described later (Aoyama, 2007; Bhardwaj, 2001; Bhatia, 1999; Brunet de la Grange, 2006; Delaney, 2005; Hansson, 2007; Henning, 2008; Hosen, 2007; Hutton, 2006; Iacovino, 2008;

Pimanda, 2007; Rizo, 2008; Sadlon, 2004; Schiedlmeier, 2007; Zhou, 2008a) and reviewed in (Chiba, 2006) and (Loose, 2007).

HSC are an embryologic derivative of mesodermal origin. In mammals, during embryogenesis, hematopoiesis develops in successive anatomical sites, from the yolk sac, outside the embryo, to the fetal liver and spleen and finally HSC enter the bone marrow of the fetus, where normal hematopoiesis takes place throughout adult life (Godin, 2005). Before 1975, it was believed that embryonic hematopoiesis occurred only outside the embryo, within blood islands of the yolk sac. In 1975, Dieterlen-Lièvre published a study that changed the understanding of HSC development. This study showed that HSC developed also within the liver, spleen and thymus of chick and quail embryos, using yolk sac chimeras by grafting quail embryos on extraembryonic area of a chick blastodisc (Dieterlen-Lievre, 1975;

Dzierzak, 2008). More recently, another transitory embryonic site of hematopoiesis was described within in mice embryos located in the para-aortic planchnopleura and called the aorta, gonad, mesonephros (AGM) region (Cumano, 2000). The AGM-

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derived HSC can reconstitute irradiated recipients and showed multilineage differentiation in vivo. Understanding the embryonic anatomical sites of hematopoiesis may be important to better characterize the signaling cascades of HSC induction in the embryo and to subsequently apply these signals to ESC culture to induce differentiation into HSC. Very recently, Taoudi et al showed that HSC from the AGM region represent a major source for definitive HSC in the post-natal organism (Taoudi, 2008). AGM-derived HSC have a strong repopulation potential, especially the subpopulations negative for CD34 and expressing CD45, a pan- leukocyte marker, also called leukocyte common antigen, and vascular-endothelial cadherin, (CD45+, VE-cadherin+, CD34-) (Taoudi, 2008). It has been speculated that CD45 and VE-cadherin expressing AGM-derived HSC can migrate through vessels to the bone marrow and thus constitute the major source of adult HSC. How this migration occurs remains unknown (Saito, 2008). Co-culture of mouse AGM-derived HSC with human ESC induced the differentiation of ESC into early hematopoietic progenitors, expressing CD34 (Ledran, 2008). These ESC-derived HSC could engraft at high levels in immunodeficient mice (Ledran, 2008).

In the bone marrow, HSC self-renew and partially differentiate within a niche. The concept of niche is recurrent in stem cell biology, for most adult stem cells. The interaction of adult stem cells with various and different cellular partners and matrices allows them to maintain their stemness but also to differentiate towards progenitors and functional effectors. David T Scadden defined the niche as “specific anatomic location that regulates how stem cells participate in tissue generation, maintenance and repair. The niche saves stem cells from depletion, while protecting the host from over-exuberant stem-cell proliferation. It constitutes a basic unit of tissue physiology, integrating signals that mediate the balanced response of stem cells to the needs of organisms” (Scadden, 2006). In the bone marrow, the HSC niche is located at the endosteum mainly composed of endosteal cells, which promote HSC maintenance (Adams, 2006; Kiel, 2008). The endosteum is a highly vascularized zone between the bone and bone marrow, lined by osteoclasts and bone progenitors, but probably also multipotent marrow stromal cells (MSC) (Le Blanc, 2007b). The highly vascularized structure, with its endothelial and perivascular cells defines perivascular niches where self-renewal and differentiation are regulated (Kiel, 2007; Kiel, 2005;

Sacchetti, 2007; Sugiyama, 2006). Within these sites, the interaction of HSC with osteoblasts, perivascular mesenchymal progenitors and endothelial cells, through various signals, such as CXCL12-CXCR4 interaction (Dar, 2006; Kollet, 2006;

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Sugiyama, 2006), and secretion of thrombopoeitin and angiopoeitin (Arai, 2004;

Sacchetti, 2007; Yoshihara, 2007) maintain pluripotency of HSC. Pluripotency may also depend on homotypic N-cadherin interactions and Notch-Jagged-1 signaling between HSC and the niche cells (Calvi, 2003; Ganapati, 2007; Haug, 2008; Kiel, 2007; Mancini, 2005; Wilson, 2004). Cytokines secreted by niche cells also maintain HSC in their microenvironment and enable them to proliferate and self-renew (Zhang, 2008).

For therapeutic purposes, in allo- and autografts, HSC were first transplanted using whole bone marrow cells. Then, HSC were isolated from the bone marrow by positive selection for CD34 and depletion of differentiated cells (Kasow, 2007; Koca, 2008).

More recently, CD34+ cells were isolated from the peripheral blood of donors, after stimulation by granulocyte colony-stimulating factor (G-CSF) and CXCR4 antagonists. These cells were able to repopulate recipient patients, in an allogeneic setting in bone marrow failure but also in autologous stem cell transplantation, after myelotoxic chemotherapy for various cancers (Filipovich, 2008; Pelus, 2008). One other source for HSC is the umbilical cord blood (UCB) (Brown, 2008). After transplantation of HSC from bone marrow, complications may occur, such as infections or graft/marrow failure. In an allogeneic setting graft versus host disease (GvHD), might occur. In this disease, mature alloreactive T helper cells from the donor, contaminating the graft, are activated by major histocompatibility complex (MHC) class1 and class2 of the recipient. This results in aggressive immune responses against the skin and gut of the recipient (Ball, 2008; Messina, 2008). In this context, umbilical cord blood (UCB) gained much interest as an alternative source of HSC for transplantation. In the early 1970’s already, Knudtzon et al showed granulocytic colony-forming cells grown from UCB-derived HSC (Knudtzon, 1974).

Later, several studies showed that HSC can be isolated from UCB and used for autologous or allogeneic hematopoietic transplantation (Broxmeyer, 1989; Gluckman, 1989). UCB contains fewer HSC than bone marrow. UCB-derived HSC transplantation was therefore first addressed to pediatric patients. UCB-derived HSC take longer to reconstitute hematopoiesis in the recipients, who are thus more prone to infection-related complications. UCB-derived HSC also contain more regulatory T- cells than adult bone marrow-derived or blood-mobilized HSC, and have therefore a stronger suppressor activity. In the hematologic malignancy setting, UCB-derived HSC have shown a strong graft versus leukemia effect, despite their fewer numbers

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and long time to reconstitution (Barker, 2001; Laughlin, 2004; Rocha, 2001; Rocha, 2004). In summary, UCB-derived HSC can be an alternative to bone marrow-derived HSC, but their fewer numbers and their delayed function puts the recipients at risk.

Application in the adult setting of hematopoietic transplantation seems to have various hurdles to overcome, such as higher numbers of CD34+ cells and shorter time to reconstitution, to prevent infections (Brown, 2008). Nevertheless, UCB- derived HSC are cells that are easier to obtain for HSC-related basic research. UCB also contains other types of stem cells, such as multipotent marrow stromal stem cells, subject developed later in this thesis.

HSC have gained even more attention when they showed to have a certain plasticity and differentiation potential outside the hematologic hierarchy and maturation algorithms. By plasticity also known as transdifferentiation, one understands the fact that under special conditions, tissue-specific adult stem cells can generate a whole spectrum of cell types of other tissues, even crossing germ layers (Filip, 2004; Rovo, 2008; Togel, 2007). Many different studies have shown that HSC can differentiate into various different epithelial, muscle or neural cells (Rovo, 2008). At the end of the 1990’s, HSC were shown to differentiate into muscle (Gussoni, 1999). In the early 2000, a study on humans showed that, after peripheral blood-derived HSC transplantation, donor-derived cells can be found within the skin and liver of recipients (Korbling, 2002). In mice models, Lagasse et al also showed potential of HSC to differentiate into hepatocytes in vivo and correct genetic diseases (Lagasse, 2000). Subsequent studies raised the question whether donor-derived HSC could fuse with recipient cells. Many controversial data were reported in favor of fusion of donor HSC and recipient hepatocytes (Camargo, 2004; Fujino, 2007; Vassilopoulos, 2003; Wang, 2003b; Willenbring, 2004) or against (Jang, 2004; Khurana, 2007;

Newsome, 2003). This question remains a matter of debate today, even after in vivo tracing of green fluorescent protein expressing HSC. The type of liver or other organ injury may influence the outcome of transplanted cells (Fujino, 2007; Khurana, 2007).

In summary, HSC can be isolated from the bone marrow, umbilical cord and peripheral blood after donor stimulation with G-CSF and other cytokines. They are characterized by their capacity to repopulate recipient bone marrow and differentiate into all hematopoietic lineages. Clinically, they are selected by depletion of lineage markers and positive selection for CD34, even though some HSC populations have

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been shown not to express CD34. HSC transplantation is a widely used treatment to reconstitute bone marrow after major chemotherapy for leukemia or other malignancies, but also for some genetic diseases affecting the bone marrow.

Recently, HSC were shown to possibly participate in regeneration of epithelial organs and muscle, but whether this happens by fusion or primary differentiation has not been fully elucidated so far.

1.4.6. Multipotent adult progenitor cells (MAPC) / marrow-isolated adult multilineage inducible cells (MIAMI) / unrestricted somatic stem cell (USSC) Different groups have isolated stem cells from bone marrow and the umbilical cord blood that display pluripotency. Catherine Verfaillie’s group isolated the so-called

“Multipotent Adult Progenitor Cells” (MAPC) from bone marrow of rodents and humans (Jiang, 2002; Reyes, 2001a; Reyes, 2001b; Schwartz, 2002). D’Ippolito et al isolated the so-called “Marrow-isolated Adult Multilineage Inducible Cells” (MIAMI), also from the bone marrow (D'Ippolito, 2004; D'Ippolito, 2006). Kogler et al isolated the “Unrestricted Somatic Stem Cells” (USSC) from umbilical cord blood (Kogler, 2004). These three cell types share similarities, such as the absence of CD34, CD45 and CD117 expression, the presence of CD13, CD29, CD44 (low), CD49, and CD90, and expansion potential of at least 45 population doublings. They were able to differentiate in vitro and in vivo into cells of all three embryonic lineages, endoderm, mesoderm and neuro-ectoderm. After injection into blastocysts, mouse MAPC participated in most organs within the embryo (Jiang, 2002). When injected into fetal sheep, USSC differentiated into hepatocytes, cardiomyocytes, and neurons (Kogler, 2004). MIAMI cells were not injected in vivo, but differentiated in vitro into cells from all three germ layers, such as osteoblasts, chondrocytes, adipocytes, neural cells and pancreatic islet-like structures, expressing insulin but not tested for their response to glucose. MAPC and MIAMI cells were shown to express stem cell markers such as Oct-3/4 (Serafini, 2007) and SSEA4 (D'Ippolito, 2006). USSC were not analyzed for markers of stemness, but several recent studies have demonstrated their engraftment and differentiation into various cell types with in vivo function (Ghodsizad, 2008; Greschat, 2008; Kogler, 2005; Kogler, 2006; Sensken, 2007).

Small sized cells, called very small embryonic-like cells expressing embryonic stem cells markers such as Oct-3/4, Rex1, SSEA1, Nanog, and CD34 were isolated from the bone marrow and umbilical cord blood (Kucia, 2007a; Kucia, 2006; Kucia, 2007b;

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