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

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-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 pan-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;

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

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

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