**2. Transcription factors in hematopoietic development**

Hematopoiesis is regulated at the level of pluripotent HSCs and committed progenitors through growth and/or differentiation inducing factors (like EPO, G-CSF, GM-CSF, IL-1, IL-3) that interact with receptors and initiate signal transduction processes that culminate in the activation of new genetic programs. These external stimuli trigger intrinsic determinants of cell fate, the transcription factors which contribute to the reprogramming of HSCs into cell-

Networks Establishing Hematopoietic Stem Cell Multipotency and Self-Renewal 5

differ, presumably reflecting diverse niches that support HSC expansion and/or differentiation and intrinsic characteristics of HSCs at each stage. For instance, HSCs present

The initial wave of blood production in the mammalian yolk sac is termed ''primitive.'' The primary function for primitive hematopoiesis is the production of red blood cells that facilitate tissue oxygenation as the embryo undergoes rapid growth. The hallmark of primitive erythroid cells is expression of embryonic globin proteins. The primitive hematopoietic system is transient and rapidly replaced by adult-type hematopoiesis that is termed "definitive". In mammals, the next site of hematopoietic potential is the AGM region. Hematopoietic cells were first detected in the aorta of the developing pig more than 80 years ago. Morphological examination revealed that a sheet of lateral mesoderm migrates medially, touches endoderm, and then forms a single aorta tube. Clusters of hematopoietic cells subsequently appear in the ventral wall. Similarly, an intraembryonic source of adult HSCs in mice capable of long-term reconstitution of irradiated hosts resides in the AGM region (Muller et al., 1994). At embryonic day 10.5, little HSC activity is detectable, whereas by day 11 engrafting activity is present. Additional hematopoietic activity in the mouse embryo was detected subsequently in other sites, including the umbilical arteries and the allantois in which hematopoietic and endothelial cells are co-localized (Inman & Downs, 2007). Umbilical veins lack hematopoietic potential, suggesting that a hierarchy exists during definitive hematopoiesis in which HSCs arise predominantly during artery specification. In addition, significant numbers of HSCs are found in the mouse placenta (Gekas et al., 2005; Ottersbach & Dzierzak, 2005), nearly coincident with the appearance of HSCs in the AGM region and for several days thereafter. Placental HSCs could arise through de novo generation or colonization upon circulation, or both. The relative contribution of each of the above sites to the final pool of adult HSCs remains largely

Subsequent definitive hematopoiesis involves the colonization of the fetal liver, thymus, spleen, and ultimately the bone marrow. It is believed that none of these sites is accompanied by de novo HSC generation. Rather, their niches support expansion of populations of HSCs that migrate to these new sites. However, until very recently, there has been no evidence by fate mapping or direct visualization that HSCs from one site colonize

The AGM has been characterized largely by morphology and functional assays, but the pathways involved in HSC generation remain incompletely defined. Studies of chick embryos demonstrate that endoderm has a prominent role and secretes inducing factors. Somitic mesoderm also contributes to the dorsal aspect of the aorta, and the addition of factors such as VEGF, TGF-β, and FGF to the somitic mesoderm leads to induction of hematopoietic tissue. In contrast, TGF-α and EGF suppressed formation of hematopoietic

Signaling pathways that regulate the induction of the AGM have been uncovered in mouse and zebrafish, *Notch 1* is required for artery identity and aortic HSC production (Kumano et al., 2003). The fate decisions imposed on mesodermal progenitors within the AGM are

in the fetal liver are in cycle, whereas adult bone marrow HSCs are largely quiescent.

unknown.

subsequent sites.

**4. Pathways involved in the emergence of HSCs** 

cells (Pardanaud & Dieterlen-Lievre, 1999).

lineage restricted pathways of maturation (Zon, 2008). Although the transcription factors involved in hematopoietic development belong to all classes of DNA-binding proteins some of them are involved in the regulation of self-renewal function primarily on HSCs while the others act on MPPs and/or early committed progenitors entering the cell-lineage restricted pathways of differentiation. While transcription factors as MLL, RUNX1, TEL/ETV6, SCL/TAL1 and LMO2 are required for HSC formation and function, others are necessary as key lineage restricted factors acting at the level of early pre-committed progenitors, using key partners that act synergistically or competing to restrict cell-lineage hematopoietic differentiation. GATA-1 and PU-1, for example, physically interact and antagonize with each other to promote either myeloid or erythroid maturation (Rekhtman et al., 1999; Zhang et al., 1999), which means that suppression of GATA-1 expression favors myeloid differentiation while inhibition of PU-1 promotes erythroid maturation. Additional, antagonistic interactions with other transcription factors have also been reported as C/EBP that antagonizes FOG-1 in eosinophilic differentiation, EKLF antagonizes Fli-1 for erythroid versus megakaryocytic differentiation. Finally, repression of the *Pax-5* gene prevented Pro-B cell maturation to B cells, while promoting multi-potentiality into macrophage, T-NKs and dendritic cells (Huntly & Gilliland, 2005).

Transcription factors also interact with other proteins associated with chromatin modification and form active or repressive transcriptional complexes. Knockout of *Scl/Tal1* or *Lmo2* abrogates hematopoietic development. The precise mechanism through which such transcription activator or repressor complexes regulate the expression of several genes is critical, since the gene expression pattern regulates cell fate decision via cell-lineage restricted maturation. A critical point, however, for all these transcriptional complexes is the concentration of the transcription factor itself and its affinity to other interactive proteins.

Under normal hematopoiesis, several groups of hematopoietic and mature blood cells are generated. Hematopoiesis occurs unidirectionally and commitment from one step to the next occurs irreversibly, suggesting that transcription factors regulate cell fate along the specific cell-lineage pathways irreversibly. This occurs in such a way because intrinsic transcription factor network is coordinated with inputs resulting from external stimuli initiated within the hematopoietic cell niche. The question, however, whether one cell type of progenitors can be reprogrammed into another phenotype at the level of manipulation of transcription factor activation, is a potentially interesting one. Indeed, evidence now indicates that transfection of *Gata-1* into CMPs and/or CLPs redirects their commitment to another cell-lineage restricted pathway as megakaryocytic/erythroid. Similarly, pre-T cells can be reprogrammed to myeloid dendritic cells upon *PU-1* overexpression (Laiosa et al., 2006; Orkin & Zon, 2008).

## **3. Ontogeny of HSCs**

In vertebrates, the production of blood stem cells is accomplished by the allocation and specification of distinct embryonic cells in a variety of sites that change during development. In mammals, the sequential sites of hematopoiesis include the yolk sac; an area surrounding the dorsal aorta termed the aorta-gonad mesonephros (AGM) region, the fetal liver, and finally the bone marrow. Recently, the placenta has been recognized as an additional site that participates during the AGM to fetal liver period. The properties of HSCs in each site

lineage restricted pathways of maturation (Zon, 2008). Although the transcription factors involved in hematopoietic development belong to all classes of DNA-binding proteins some of them are involved in the regulation of self-renewal function primarily on HSCs while the others act on MPPs and/or early committed progenitors entering the cell-lineage restricted pathways of differentiation. While transcription factors as MLL, RUNX1, TEL/ETV6, SCL/TAL1 and LMO2 are required for HSC formation and function, others are necessary as key lineage restricted factors acting at the level of early pre-committed progenitors, using key partners that act synergistically or competing to restrict cell-lineage hematopoietic differentiation. GATA-1 and PU-1, for example, physically interact and antagonize with each other to promote either myeloid or erythroid maturation (Rekhtman et al., 1999; Zhang et al., 1999), which means that suppression of GATA-1 expression favors myeloid differentiation while inhibition of PU-1 promotes erythroid maturation. Additional, antagonistic interactions with other transcription factors have also been reported as C/EBP that antagonizes FOG-1 in eosinophilic differentiation, EKLF antagonizes Fli-1 for erythroid versus megakaryocytic differentiation. Finally, repression of the *Pax-5* gene prevented Pro-B cell maturation to B cells, while promoting multi-potentiality into macrophage, T-NKs and

Transcription factors also interact with other proteins associated with chromatin modification and form active or repressive transcriptional complexes. Knockout of *Scl/Tal1* or *Lmo2* abrogates hematopoietic development. The precise mechanism through which such transcription activator or repressor complexes regulate the expression of several genes is critical, since the gene expression pattern regulates cell fate decision via cell-lineage restricted maturation. A critical point, however, for all these transcriptional complexes is the concentration of the transcription factor itself and its affinity to other interactive proteins.

Under normal hematopoiesis, several groups of hematopoietic and mature blood cells are generated. Hematopoiesis occurs unidirectionally and commitment from one step to the next occurs irreversibly, suggesting that transcription factors regulate cell fate along the specific cell-lineage pathways irreversibly. This occurs in such a way because intrinsic transcription factor network is coordinated with inputs resulting from external stimuli initiated within the hematopoietic cell niche. The question, however, whether one cell type of progenitors can be reprogrammed into another phenotype at the level of manipulation of transcription factor activation, is a potentially interesting one. Indeed, evidence now indicates that transfection of *Gata-1* into CMPs and/or CLPs redirects their commitment to another cell-lineage restricted pathway as megakaryocytic/erythroid. Similarly, pre-T cells can be reprogrammed to myeloid dendritic cells upon *PU-1* overexpression (Laiosa et al.,

In vertebrates, the production of blood stem cells is accomplished by the allocation and specification of distinct embryonic cells in a variety of sites that change during development. In mammals, the sequential sites of hematopoiesis include the yolk sac; an area surrounding the dorsal aorta termed the aorta-gonad mesonephros (AGM) region, the fetal liver, and finally the bone marrow. Recently, the placenta has been recognized as an additional site that participates during the AGM to fetal liver period. The properties of HSCs in each site

dendritic cells (Huntly & Gilliland, 2005).

2006; Orkin & Zon, 2008).

**3. Ontogeny of HSCs** 

differ, presumably reflecting diverse niches that support HSC expansion and/or differentiation and intrinsic characteristics of HSCs at each stage. For instance, HSCs present in the fetal liver are in cycle, whereas adult bone marrow HSCs are largely quiescent.

The initial wave of blood production in the mammalian yolk sac is termed ''primitive.'' The primary function for primitive hematopoiesis is the production of red blood cells that facilitate tissue oxygenation as the embryo undergoes rapid growth. The hallmark of primitive erythroid cells is expression of embryonic globin proteins. The primitive hematopoietic system is transient and rapidly replaced by adult-type hematopoiesis that is termed "definitive". In mammals, the next site of hematopoietic potential is the AGM region. Hematopoietic cells were first detected in the aorta of the developing pig more than 80 years ago. Morphological examination revealed that a sheet of lateral mesoderm migrates medially, touches endoderm, and then forms a single aorta tube. Clusters of hematopoietic cells subsequently appear in the ventral wall. Similarly, an intraembryonic source of adult HSCs in mice capable of long-term reconstitution of irradiated hosts resides in the AGM region (Muller et al., 1994). At embryonic day 10.5, little HSC activity is detectable, whereas by day 11 engrafting activity is present. Additional hematopoietic activity in the mouse embryo was detected subsequently in other sites, including the umbilical arteries and the allantois in which hematopoietic and endothelial cells are co-localized (Inman & Downs, 2007). Umbilical veins lack hematopoietic potential, suggesting that a hierarchy exists during definitive hematopoiesis in which HSCs arise predominantly during artery specification. In addition, significant numbers of HSCs are found in the mouse placenta (Gekas et al., 2005; Ottersbach & Dzierzak, 2005), nearly coincident with the appearance of HSCs in the AGM region and for several days thereafter. Placental HSCs could arise through de novo generation or colonization upon circulation, or both. The relative contribution of each of the above sites to the final pool of adult HSCs remains largely unknown.

Subsequent definitive hematopoiesis involves the colonization of the fetal liver, thymus, spleen, and ultimately the bone marrow. It is believed that none of these sites is accompanied by de novo HSC generation. Rather, their niches support expansion of populations of HSCs that migrate to these new sites. However, until very recently, there has been no evidence by fate mapping or direct visualization that HSCs from one site colonize subsequent sites.

### **4. Pathways involved in the emergence of HSCs**

The AGM has been characterized largely by morphology and functional assays, but the pathways involved in HSC generation remain incompletely defined. Studies of chick embryos demonstrate that endoderm has a prominent role and secretes inducing factors. Somitic mesoderm also contributes to the dorsal aspect of the aorta, and the addition of factors such as VEGF, TGF-β, and FGF to the somitic mesoderm leads to induction of hematopoietic tissue. In contrast, TGF-α and EGF suppressed formation of hematopoietic cells (Pardanaud & Dieterlen-Lievre, 1999).

Signaling pathways that regulate the induction of the AGM have been uncovered in mouse and zebrafish, *Notch 1* is required for artery identity and aortic HSC production (Kumano et al., 2003). The fate decisions imposed on mesodermal progenitors within the AGM are

Networks Establishing Hematopoietic Stem Cell Multipotency and Self-Renewal 7

only crucial to attach HSCs to niche osteoblasts, but is also essential to maintain HSC dormancy and function. Many factors, including ligands for Notch receptors and Ncadherin, are liberated by osteoblasts, although the contribution of these to adult hematopoiesis remains to be established. The role of N-cadherin as a mediator of interactions with osteoblasts (Zhang et al., 2003), as well as the prominence of osteoblasts for HSC adherence, has been challenged (Kiel et al., 2007). Recent findings suggest that HSCs are maintained in a quiescent state through interaction with thrombopoietin-producing osteoblasts (Yoshihara et al., 2007). Thrombopoietin (TPO) is the primary cytokine that regulates megakaryocyte and platelet development. Thrombopoietin and its receptor Mpl also exert profound effects on primitive hematopoietic cells. All HSCs express Mpl; *TPO*−/− or *Mpl*−/− mice have a decreased number of repopulating HSCs (Solar et al., 1998). *In vitro* culture studies (Matsunaga et al., 1998) also indicate a role of TPO in promoting the survival of repopulating HSCs. Through study of AGM and fetal liver *Mpl*−/− HSCs, Petit-Cocault et al*.* (2007) showed that TPO contributes to both generation and expansion of HSCs during definitive hematopoiesis. An intracellular adaptor, Lnk, induces a negative signaling pathway downstream of TPO in HSCs (Buza-Vidas et al., 2006; Seita et al., 2007). Another study (Tong et al., 2007) on mice that express *Mpl* lacking the C-terminal 60 amino acids revealed a pivotal role of an unknown signal emanating from the membrane proximal region of the Mpl receptor or from JAK2 that is critical for maintenance of HSC activity.

The association of HSCs with osteoblasts is countered by other studies that place HSCs adjacent to vascular cells. The chemokine CXCL12 regulates the migration of HSCs to the vascular cells (Kiel & Morrison, 2006). Taken together, these findings suggest that HSCs reside in various sites within the marrow and that their function might depend on their precise localization. Much of the existing debate may be semantic, however, if the osteoblastic and vascular niches are intertwined and not physically separate. Alternatively, HSCs may truly reside in distinct sub-regions, which may endow them with different activities. Cellular dynamics within the niche are relevant to clinical marrow transplantation. For example, recent findings suggest that antibody-mediated clearance of host HSCs facilitates occupancy of the niche and transplantation by exogenous HSCs

The physical interactions between individual HSCs and osteoblasts may be effective in determining the stem cell number by facilitating asymmetric or symmetric divisions, which in turn enable HSCs to either self-renew themselves or give birth to early progenitors for blood cells production (Moore & Lemischka, 2006; Wilson and Trumpp, 2006). HSCs are not of static nature, but exist in a dynamic state, since they migrate from the bone marrow into the peripheral blood (frequent trafficking). Whether, or not, HSCs contribute into the repair

Many cell culture experiments have shown that HSCs respond to multiple cytokines and that the fate of a HSC self renewal, apoptosis, mobilization from the niche, formation of differentiated progeny cells depends on multiple cytokines, adhesion proteins, and other signals produced by stromal cells and likely other cells in the body. Since osteoblast (a cell derived from mesenchymal stem cells) is a key component in the HSC niche for the

of the vascular system, is still not known (Janzen & Scadden, 2006).

(Czechowicz et al., 2007).

**5.1 Signaling in the niche** 

clearly influenced by the *Notch* pathway (Burns et al., 2005). For instance, mice deficient in *RBPj* (a downstream component of the *Notch* pathway) show expanded *VE-Cadherin* and *CD31/PECAM* endothelial cell expression with concomitant loss of definitive HSCs (Robert-Moreno et al., 2005). Ablation of the COUP-TFII transcription factor in endothelial cells enabled veins to acquire arterial characteristics, including the expression of *Notch1* and the formation of ectopic HSCs (You et al., 2005). This result would favor *Notch* acting to induce HSCs from a hemogenic endothelial cell. The model in which the *Notch* pathway regulates arterial and HSC fate choice either from distinct mesodermal populations or over different developmental windows since each decision can be uncoupled *in vivo* is very attractive. The finding that both aorta and vein express HSC markers in the *Notch*-activated state with minimal change in *ephrinB2a* expression indicates that *Notch* independently regulates mesoderm–HSC and artery–vein cell fate decisions.Lateral inhibition has been proposed in the central nervous system whereby *Notch* signaling promotes non-neural fates while inhibiting neural development (Lewis, 1998). HSC fate may be established by a similar mechanism whereby *Notch* activation in an endothelial or mesenchymal cell causes downregulation of ligand production. Consequently, a cell that produces more ligand will force its neighbor to produce less, thus generating a salt-and-pepper pattern of cells containing elevated *Notch* activity. In this model, cells containing high levels of Notch Intra Cytoplasmatic Domain (NICD) would become HSCs, while those with low NICD activity would remain endothelial or mesenchymal.

#### **5. Hematopoietic niches**

Stem cells depend on their microenvironment, the niche, for regulation of self-renewal and differentiation. As the site of hematopoiesis changes during vertebrate development, the nature of the stem cell niche must also change. Mutant mice in which the *BMP* pathway is disrupted have increased numbers of osteoblasts and HSCs (Calvi et al., 2003; Zhang et al., 2003). These findings suggest that osteoblasts may represent a critical component of the bone marrow niche for HSCs. Microscopical examination revealed that HSCs appear to reside in the periosteal area of calvarium marrow, where osteoblasts represent an essential component of the bone marrow niche (Papadimitriou et al., 1994). Most recent live animal tracking experiments by using real-time imaging of individual HSCs have indicated that endosteum forms a special zone where HSCs reside (Lo Celso et al., 2009; Xie et al., 2009). The bone marrow HSC niche is constituted of mesenchymal cells type osteoblasts, extracellular matrix components and minerals (high density calcium salts), all of which contribute to the unique micro-environment (niche) (Moore & Lemischka, 2006; Wilson & Trumpp, 2006). At least two distinct hematopoietic progenitor cell supportive niches in bone marrow have been identified thus far: the osteoblastic, which is regulated by *BMP*, *osteopontin*, *angiopoietin-1*, *notch* and maybe others (Adams & Scadden, 2006; Wilson & Trumpp, 2006) and the other one, the vascular niche. The vascular niche is thought to be the site where actively dividing stem or progenitor cells is located, and osteoblastic niche is an environment promoting maintenance of quiescent HSCs (Calvi et al., 2003). Currently, how these two different niches communicate with each other is largely unknown.

The number of HSCs in the bone marrow niche is highly controlled through physical interactions among different cell types, in a way that maintains stem cell state. HSCs remain in a quiescent state through close interaction with osteoblasts where this interaction is not

clearly influenced by the *Notch* pathway (Burns et al., 2005). For instance, mice deficient in *RBPj* (a downstream component of the *Notch* pathway) show expanded *VE-Cadherin* and *CD31/PECAM* endothelial cell expression with concomitant loss of definitive HSCs (Robert-Moreno et al., 2005). Ablation of the COUP-TFII transcription factor in endothelial cells enabled veins to acquire arterial characteristics, including the expression of *Notch1* and the formation of ectopic HSCs (You et al., 2005). This result would favor *Notch* acting to induce HSCs from a hemogenic endothelial cell. The model in which the *Notch* pathway regulates arterial and HSC fate choice either from distinct mesodermal populations or over different developmental windows since each decision can be uncoupled *in vivo* is very attractive. The finding that both aorta and vein express HSC markers in the *Notch*-activated state with minimal change in *ephrinB2a* expression indicates that *Notch* independently regulates mesoderm–HSC and artery–vein cell fate decisions.Lateral inhibition has been proposed in the central nervous system whereby *Notch* signaling promotes non-neural fates while inhibiting neural development (Lewis, 1998). HSC fate may be established by a similar mechanism whereby *Notch* activation in an endothelial or mesenchymal cell causes downregulation of ligand production. Consequently, a cell that produces more ligand will force its neighbor to produce less, thus generating a salt-and-pepper pattern of cells containing elevated *Notch* activity. In this model, cells containing high levels of Notch Intra Cytoplasmatic Domain (NICD) would become HSCs, while those with low NICD activity

Stem cells depend on their microenvironment, the niche, for regulation of self-renewal and differentiation. As the site of hematopoiesis changes during vertebrate development, the nature of the stem cell niche must also change. Mutant mice in which the *BMP* pathway is disrupted have increased numbers of osteoblasts and HSCs (Calvi et al., 2003; Zhang et al., 2003). These findings suggest that osteoblasts may represent a critical component of the bone marrow niche for HSCs. Microscopical examination revealed that HSCs appear to reside in the periosteal area of calvarium marrow, where osteoblasts represent an essential component of the bone marrow niche (Papadimitriou et al., 1994). Most recent live animal tracking experiments by using real-time imaging of individual HSCs have indicated that endosteum forms a special zone where HSCs reside (Lo Celso et al., 2009; Xie et al., 2009). The bone marrow HSC niche is constituted of mesenchymal cells type osteoblasts, extracellular matrix components and minerals (high density calcium salts), all of which contribute to the unique micro-environment (niche) (Moore & Lemischka, 2006; Wilson & Trumpp, 2006). At least two distinct hematopoietic progenitor cell supportive niches in bone marrow have been identified thus far: the osteoblastic, which is regulated by *BMP*, *osteopontin*, *angiopoietin-1*, *notch* and maybe others (Adams & Scadden, 2006; Wilson & Trumpp, 2006) and the other one, the vascular niche. The vascular niche is thought to be the site where actively dividing stem or progenitor cells is located, and osteoblastic niche is an environment promoting maintenance of quiescent HSCs (Calvi et al., 2003). Currently, how

these two different niches communicate with each other is largely unknown.

The number of HSCs in the bone marrow niche is highly controlled through physical interactions among different cell types, in a way that maintains stem cell state. HSCs remain in a quiescent state through close interaction with osteoblasts where this interaction is not

would remain endothelial or mesenchymal.

**5. Hematopoietic niches** 

only crucial to attach HSCs to niche osteoblasts, but is also essential to maintain HSC dormancy and function. Many factors, including ligands for Notch receptors and Ncadherin, are liberated by osteoblasts, although the contribution of these to adult hematopoiesis remains to be established. The role of N-cadherin as a mediator of interactions with osteoblasts (Zhang et al., 2003), as well as the prominence of osteoblasts for HSC adherence, has been challenged (Kiel et al., 2007). Recent findings suggest that HSCs are maintained in a quiescent state through interaction with thrombopoietin-producing osteoblasts (Yoshihara et al., 2007). Thrombopoietin (TPO) is the primary cytokine that regulates megakaryocyte and platelet development. Thrombopoietin and its receptor Mpl also exert profound effects on primitive hematopoietic cells. All HSCs express Mpl; *TPO*−/− or *Mpl*−/− mice have a decreased number of repopulating HSCs (Solar et al., 1998). *In vitro* culture studies (Matsunaga et al., 1998) also indicate a role of TPO in promoting the survival of repopulating HSCs. Through study of AGM and fetal liver *Mpl*−/− HSCs, Petit-Cocault et al*.* (2007) showed that TPO contributes to both generation and expansion of HSCs during definitive hematopoiesis. An intracellular adaptor, Lnk, induces a negative signaling pathway downstream of TPO in HSCs (Buza-Vidas et al., 2006; Seita et al., 2007). Another study (Tong et al., 2007) on mice that express *Mpl* lacking the C-terminal 60 amino acids revealed a pivotal role of an unknown signal emanating from the membrane proximal region of the Mpl receptor or from JAK2 that is critical for maintenance of HSC activity.

The association of HSCs with osteoblasts is countered by other studies that place HSCs adjacent to vascular cells. The chemokine CXCL12 regulates the migration of HSCs to the vascular cells (Kiel & Morrison, 2006). Taken together, these findings suggest that HSCs reside in various sites within the marrow and that their function might depend on their precise localization. Much of the existing debate may be semantic, however, if the osteoblastic and vascular niches are intertwined and not physically separate. Alternatively, HSCs may truly reside in distinct sub-regions, which may endow them with different activities. Cellular dynamics within the niche are relevant to clinical marrow transplantation. For example, recent findings suggest that antibody-mediated clearance of host HSCs facilitates occupancy of the niche and transplantation by exogenous HSCs (Czechowicz et al., 2007).

The physical interactions between individual HSCs and osteoblasts may be effective in determining the stem cell number by facilitating asymmetric or symmetric divisions, which in turn enable HSCs to either self-renew themselves or give birth to early progenitors for blood cells production (Moore & Lemischka, 2006; Wilson and Trumpp, 2006). HSCs are not of static nature, but exist in a dynamic state, since they migrate from the bone marrow into the peripheral blood (frequent trafficking). Whether, or not, HSCs contribute into the repair of the vascular system, is still not known (Janzen & Scadden, 2006).

#### **5.1 Signaling in the niche**

Many cell culture experiments have shown that HSCs respond to multiple cytokines and that the fate of a HSC self renewal, apoptosis, mobilization from the niche, formation of differentiated progeny cells depends on multiple cytokines, adhesion proteins, and other signals produced by stromal cells and likely other cells in the body. Since osteoblast (a cell derived from mesenchymal stem cells) is a key component in the HSC niche for the

Networks Establishing Hematopoietic Stem Cell Multipotency and Self-Renewal 9

superfamily, play important roles in HSC specification during development. A negative role of *BMP* signaling in maintenance of mouse HSCs was shown by its control of the size of the HSC endosteal niche (Ross & Li, 2006). BMP4 supports HSC expansion in culture and partially mediates the effects of Sonic hedgehog on cultured human HSCs (Bhardwaj et al., 2001). Recently, expression characterization of TGF-β superfamily ligands, receptors, and Smads in mouse HSCs was published; primary HSCs and the Lhx2-HPC cell line express most of the proteins required to transmit signals from several TGF-β family ligands (Utsugisawa et al., 2006). In addition, Pimanda et al*.* (2007) demonstrated the integration of

All long-term repopulating bone marrow HSCs express a fibroblast growth factor (FGF) receptor (Yeoh et al., 2006); both FGF-1 and FGF-2 support HSC expansion when unfractionated mouse bone marrow cells are cultured in serum-free medium. Crcareva et al*.*  (2005) confirmed that FGF-1 stimulates *ex vivo* expansion of HSCs and showed that the expanded cells were efficiently transduced by retrovirus vectors. Conditional derivatives of FGF receptor-1 have also been used to support short-term HSC expansion and long-term HSC survival in culture (Schiedlmeier et al, 2007). However, the role of the *FGF* pathway in regulating adult HSCs or embryonic hematopoietic development is controversial as the same authors showed that the treatment of purified mouse HSCs that ectopically express *HoxB4* with the fibroblast growth factor receptor (FGFR) inhibitor SU5402 enhanced HSC repopulating activity. Similar results were obtained using primitive hematopoietic colonies derived from embryonic stem cells. These inconsistent results were obtained from different starting cell populations and under different culture conditions, suggesting that the

The WNT protein binds to a receptor complex consisting of a member of the Frizzled family of seven transmembrane proteins and the LDL receptor-related proteins LRP5 or LRP6 (Clevers, 2006). In the canonical *Wnt* pathway, receptor activation leads to stabilization of *βcatenin*, which accumulates and translocates to the nucleus where it activates target gene expression in concert with transcription factors such as TCF and LEF. Fleming et al. (2008) analyzed the role of *Wnt* signaling on HSC activity, including its effects on cell-cycle quiescence and the capacity of HSCs to reconstitute the hematopoietic system of recipient mice (whose bone marrow has been ablated by radiation). In contrast to previous studies that genetically manipulated the HSCs themselves, they analyzed the effects of blocking *Wnt* signaling in the mouse bone marrow microenvironment by overexpression of dickkopf1 (*Dkk1*), an antagonist of *Wnt/ β-catenin* signaling. Dkk1 is a soluble secreted protein that interacts with the Wnt co-receptors LRP5 and LRP6 (Kawano & Kypta, 2003). It is known that the number of osteoblasts directly affects the number of long-term repopulating HSCs (Calvi et al., 2003; Zhang et al., 2003). The overexpression of *Dkk1* in the osteoblastic lineage under the control of a 2.3 kb fragment of the *collagen 1*α promoter reduced activation of the *Tcf/Lef* transcription factors in HSCs in a non-cell-autonomous

The transgenic mice showed no significant alteration in the proportion of HSCs and common lymphoid progenitor cells under steady-state conditions. Although HSCs from the *Dkk1* transgenic mice could reconstitute the hematopoietic system of irradiated recipient mice, they lost their reconstituting capacity after repeated bone marrow transplantation, indicating that the inhibition of *Wnt* signaling in the niche results in the premature loss of

*BMP4/Smad* pathway and *Scl* and *Runx1* activity in HSC development.

crosstalk of FGF signaling with other pathways is complex.

manner.

regulation of HSC number via self-renewal (Adams & Scadden, 2006; Huang et al., 2007), modifications of osteoblast functions in co-orchestration with other niche components, would be pivotal for HSCs survival, self-renewal, differentiation and apoptosis under certain circumstances.

HSCs fate decisions is activated by external environmental stimuli and coordinated by intrinsic factors. External stimuli include hematopoietic growth factors such as SCF, BMP/TGF-, FGF, TPO, WNT proteins (WNT3A), Angiopoietin-1, IL-3, IL-6, Flt3-ligand, as well as Ca2+, hypoxia, PGE2 and retinoic acid (Wilson & Trumpp, 2006) while intrinsic factors are essentially genes controlling cell cycle, apoptosis and chromatin remodeling.
