**6. Quiescence or self-renewal**

16 Advances in Hematopoietic Stem Cell Research

Bone marrow is a very low oxygen tension environment that would protect cells from exposure to oxidative stress. Various intrinsic factors have also been identified to function in maintaining low oxidant levels in HSCs. ATM, a cell-cycle checkpoint regulator activated after DNA damage, is shown to regulate oxidant levels in HSCs (Ito et al., 2006). ATM deficiency-induced ROS elevation in HSCs specifically activates the p38 mitogen-activated protein kinase (MAPK) pathway, a signaling pathway responding to diverse cellular stresses, leading to a defect in the maintenance of HSC quiescence (Ito et al., 2004). *ATM*−/<sup>−</sup> mice over the age of 24 weeks show progressive bone marrow failure due to a defect in HSC function associated with elevated levels of ROS. Treatment with anti-oxidative reagents, Nacetyl cysteine or with a MAPK inhibitor restores reconstitutive capacity and quiescence of

Members of the FoxO subfamily of forkhead transcription factors have been shown to protect HSCs from oxidative stress by up-regulating genes involved in their detoxification. Triple knockout mice of *FoxO1*, *FoxO3*, and *FoxO4* exhibited defective long-term repopulating activity of HSCs, which correlated with increased cycling and apoptosis of HSCs, as well as increased levels of ROS in HSCs (Tothova et al., 2007). Similarly, the HSC compartment in *FoxO3a* null mice suffers from augmented levels of ROS and subsequent bone marrow failure (Miyamoto et al., 2007). The HSC defect resulting from loss of *FoxOs*

It is conceivable that both the hypoxic environment in which the HSCs reside and the intrinsic factors in HSCs serve to protect HSCs from oxygen radicals, keeping HSCs'

The *JAK–STAT* (Janus family kinase–signal transducer and activator of transcription) pathway is a common downstream pathway of cytokine signaling that promotes hematopoiesis. Constitutive activation of the transcription factors of the *Stat* family, particularly *Stat3* and *Stat5*, are frequently detected in leukemias, lymphomas and solid tumors. In order to evaluate their role in HSCs, constitutively active *Stat* mutants were used to activate signaling in HSCs. Activation of *Stat5* in HSCs led to the dramatic expansion of multipotent progenitors and promoted HSC self-renewal *ex vivo* (Kato et al., 2005). Deletion of *Stat5* resulted in profound defects in hematopoiesis and markedly reduced ability of the mutant cells to repopulate the bone marrow of lethally irradiated mice (Snow et al., 2002). In a mouse model of myeloproliferative disease (MPD), sustained *Stat5* activation in HSCs and not multipotent progenitors induced fatal MPD, suggesting that the capacity of *Stat5* to promote self-renewal of hematopoietic stem cells is crucial for MPD development. Another group showed that transduction of adult mouse bone marrow cells with a constitutively activated form of *Stat3* increased their regenerative activity in lethally irradiated recipients, whereas the transduction of these cells with a dominant negative form of *Stat3* suppressed their regenerative activity (Chung et al., 2006). These studies suggest that Stat proteins play a role in HSC self-renewal and potentially in other tissues; owing to the wide range of solid tissue and blood

Studies using transgenic mice constitutively expressing *BCL2* (*B-cell lymphoma 2*) in all hematopoietic tissues provide evidence directly supporting this theory. The forced

could also be rescued by administration of the antioxidant N-acetyl cysteine.

malignancies that harbor constitutively activated Stats.

*ATM*−/− HSCs.

quiescent status.

In order to both maintain a supply of mature blood cells and not exhaust HSCs throughout the lifetime of an individual, under steady state, most HSCs remain quiescent and only a small number enter the cell cycle. However, in response to hematopoietic stress such as blood loss, HSCs exit quiescence and rapidly expand and differentiate to repopulate the peripheral hematopoietic compartments. When quiescence is disrupted, HSCs displayed defective maintenance in G0 phase of cell cycle, leading to premature exhaustion of the stem cell pool under conditions of hematopoietic stress, impaired self-renewal, and loss of competitive repopulating capacity, eventually causing hematological failure.

Quiescence of HSCs is not only critical for protecting the stem cell compartment and sustaining stem cell pools over long periods, but it is also critical for protecting stem cells by minimizing their accumulation of replication-associated mutations. The balance between quiescence and proliferation is tightly controlled by both HSC-intrinsic and -extrinsic mechanisms. Understanding quiescence regulation in HSC is of great importance not only for understanding the physiological foundation of HSCs, but also for understanding the pathophysiological origins of many related disorders.

In steady state conditions HSCs are in a slowly dividing state, termed relative quiescence, with a cell division cycle in the mouse in the range of 2–4 weeks, localized in close contact with stromal cells, including osteoblasts (Calvi et al., 2003; Zhang et al., 2003). This is in contrast to the rapidly cycling hematopoietic progenitor cells, which are more committed to differentiation than HSCs. The balance between quiescent and cycling stem cells was proposed to rely on the amount of soluble cytokines, which result in HSCs relocating from the osteoblastic to the vascular niche (Heissig et al., 2002). However new results indicate that it depends on a complex network of signals.

Fig. 1. Networks interaction model for: A) quiescence, B) self-renewal and C) survival.

Networks Establishing Hematopoietic Stem Cell Multipotency and Self-Renewal 19

Li (2008) proposed that HSC quiescence is maintained through several signaling pathways including positive and negative regulators from extrinsic and intrinsic factors already described. In this context the *Tie-2/Ang-1* signaling pathway plays a critical role in the maintenance of HSCs in a quiescent state in the bone marrow niche (Adams et al., 2006). HSCs express the receptor tyrosine kinase Tie-2 and osteoblasts are the source of the Ang-1 ligand for Tie-2. *Tie-2/Ang-1* signaling activates its key downstream targets, *β1-integrin* and *N-cadherin* in lineage-negative, *Sca-1*, *C-kit* double-positive (LSK), and *Tie-2*-positive cells, and promotes HSC interactions with extracellular matrix and cellular components of the niche. This interaction is sufficient to maintain the quiescence and enhanced survival of HSCs by preventing cell division (Arai et al., 2007). *Ang-1/Tie-2* signaling also activates the phosphatidylinositol 3-kinase/Akt signaling pathway (Visnjic et al., 2004). Phosphatidylinositol 3-kinase/Akt signaling regulates several cell-cycle regulators, such as

Other pathway that has been proven to enhance quiescence is TPO/MPL. TPO is secreted by osteoblast while MPL is expressed in the membrane of HSCs. Interaction of these two proteins maintain HSCs attached to osteoblasts by activation of a pathway that results in the expression of their adhesion molecules targets and at the same time activate genetic programs which will control entry in cell cycle and survival of HSC. How these positive regulators interact with other positive and negative regulators is not completely understood. Moreover, which signaling pathways are being activated and which genes have their

A recent study by Wang et al. (2009) recently identified STAT5, a downstream target of MPL, as a positive regulator of HSC quiescence by analyzing *STAT5*−/− mice. Expression of quiescence regulators including *Tie-2* and *p57* are decreased in *STAT5*−/− HSCs. This study demonstrated that *STAT5* might mediate *MPL* effects in maintaining HSC quiescence during steady state hematopoiesis and that the same pathway directly or indirectly regulates *Tie-2* and *p57*. Interestingly, up-regulation of *p57* is essential for TGF-β−induced

Two other signaling pathways that act as positive and negative regulators of quiescence deserve more discussion, the Hypoxia induced factor (HIF) and Osteopontina signaling pathway. Hypoxia microenvironment seems to be important for maintaining HSC quiescence The molecular mechanisms for this involve the hypoxia-inducible factor-1a (HIF-1a) regulated gene expressions in stromal cells. Two genes known to be targets of the HIF pathway are *c-Kit* and stromal cell derived factor−1 (*SDF-1* or *CXCL12*) that both have

Osteopontina (*OPN*) is a negative regulator of HSC quiescence as an OPN-null microenvironment is sufficient to increase the number of stem cells associated with increased stromal *Jagged-1* and *Ang-1* expression and reduced primitive hematopoietic cell apoptosis. OPN seems to function by preventing HSC cycling. It is interesting to note that the release of this inhibition occurs in parallel with the possible activation of the *Notch*

As we can see there is many connections between intrinsic factors and extrinsic cues and between different intrinsic factors or different extrinsic factors. Some intrinsic factors function through affecting extrinsic factors, such as *c-Myc*, which negatively regulates HSC

cell-cycle arrest. How these pathways are connected awaits more investigation.

the CDK inhibitor, p21, which in turn leads to HSC quiescence.

expression changed waits to be clarified.

proven to be important to HSC maintenance.

pathway.

In part, the dramatic contrast in cell cycle status between stem and progenitor cells has led to the hypothesis that cell cycle regulation plays a fundamentally important role in stem cell fate determination. This hypothesis is supported by recent data demonstrating a slower rate of division in Hoxb4hiPbx1lo cells, which extensively self-renewal *in vitro*, compared to control cells (Cellot et al., 2007). It is essential for an HSC to undergo cell division if it is to self-renew, but how the cell division cycle is integrated into the process of self-renewal is unclear. It is also unknown as to whether cell cycle regulation represents an intrinsic or extrinsic modifier of HSC fate.

#### **6.1 How HSC maintain quiescence**

Negative regulators of both *Cdk2* and *Cdk4/6* activity, and therefore *Rb* function, have been demonstrated to have roles in regulating HSCs (Cheng et al., 2000; Janzen et al., 2006; Stepanova & Sorrentino, 2005; Van Os et al., 2007; Walkley et al., 2005). For the most part however these phenotypes have been relatively subtle, particularly when compared to hematopoietic phenotypes apparent after disruption of transcription factors such as C/EBPα (Hock et al., 2004) and Tel (Zhang et al., 2004) amongst others, and are often apparent only after serial transplantation. The "Rb pathway" has also been implicated in phenotypes observed in both the *Bmi1*-/- and *ATM*-/- HSCs (Ito et al., 2004; Lessard & Sauvageau, 2003; Park et al., 2003). The interaction of cell cycle regulators with other factors such as Hoxb4 or telomerase deficiency has produced much more striking phenotypes than that observed for the cell cycle mutants in isolation (Choudhury et al., 2007; Miyake et al., 2006). While clearly demonstrating that cell cycle modifiers have roles in regulating stem cells, particularly HSCs, the aforementioned studies have not been able to clearly discriminate between intrinsic or extrinsic contributions to HSC fate as all studies to date had utilized nonhematopoietic restricted mutant alleles. A study demonstrating that the *p27Kip1-/*  microenvironment mediates the myelo-lymphoid expansion observed in the *p27Kip1-/* animals raises the possibility that the HSC expansion observed in *p27Kip1-/-* bone marrow is extrinsic in nature (Chien et al., 2006; Walkley et al, 2005). This result suggested that cell cycle regulators might play a role in regulating the competence of the hematopoietic niche, in addition to having potential intrinsic roles in HSC fate determination. Moreover Daria et al (2007) observed a requirement for *Rb* in the stress response of HSCs and this has also previously been suggested in the context of the role of Rb in erythropoiesis (Spike et al., 2004; Spike & Macleod, 2005).

Also of note is that the cell division dynamics of HSCs change during development, from rapidly cycling and dividing cells during the fetal liver and early stages of life to relatively quiescent and more slowly cycling in the adult context (Bowie et al., 2007; Bradford et al., 1997; Ito et al, 2000; Kiel et al., 2007; Sato et al., 1999). Thus the role for *Rb* may be context dependent, both in terms of stress response and developmentally in the regulation of HSC fate.

One important point that is becoming clearer recently is how some HSCs are maintained quiescent while others enter self-renewal program. Although bone–lining cells in the endosteal surface are often described as osteoblasts in the literature, they are heterogeneous in their degree of differentiation, and only a minority of these cells are actually bone synthesizing osteoblasts. So a good hypothesis is that in the endosteal niche some cells are in contact with true osteoblasts that expresses the necessary factors to maintain quiescence while others are not receiving the same signalization so will follow other fate.

In part, the dramatic contrast in cell cycle status between stem and progenitor cells has led to the hypothesis that cell cycle regulation plays a fundamentally important role in stem cell fate determination. This hypothesis is supported by recent data demonstrating a slower rate of division in Hoxb4hiPbx1lo cells, which extensively self-renewal *in vitro*, compared to control cells (Cellot et al., 2007). It is essential for an HSC to undergo cell division if it is to self-renew, but how the cell division cycle is integrated into the process of self-renewal is unclear. It is also unknown as to whether cell cycle regulation represents an intrinsic or

Negative regulators of both *Cdk2* and *Cdk4/6* activity, and therefore *Rb* function, have been demonstrated to have roles in regulating HSCs (Cheng et al., 2000; Janzen et al., 2006; Stepanova & Sorrentino, 2005; Van Os et al., 2007; Walkley et al., 2005). For the most part however these phenotypes have been relatively subtle, particularly when compared to hematopoietic phenotypes apparent after disruption of transcription factors such as C/EBPα (Hock et al., 2004) and Tel (Zhang et al., 2004) amongst others, and are often apparent only after serial transplantation. The "Rb pathway" has also been implicated in phenotypes observed in both the *Bmi1*-/- and *ATM*-/- HSCs (Ito et al., 2004; Lessard & Sauvageau, 2003; Park et al., 2003). The interaction of cell cycle regulators with other factors such as Hoxb4 or telomerase deficiency has produced much more striking phenotypes than that observed for the cell cycle mutants in isolation (Choudhury et al., 2007; Miyake et al., 2006). While clearly demonstrating that cell cycle modifiers have roles in regulating stem cells, particularly HSCs, the aforementioned studies have not been able to clearly discriminate between intrinsic or extrinsic contributions to HSC fate as all studies to date had utilized nonhematopoietic restricted mutant alleles. A study demonstrating that the *p27Kip1-/*  microenvironment mediates the myelo-lymphoid expansion observed in the *p27Kip1-/* animals raises the possibility that the HSC expansion observed in *p27Kip1-/-* bone marrow is extrinsic in nature (Chien et al., 2006; Walkley et al, 2005). This result suggested that cell cycle regulators might play a role in regulating the competence of the hematopoietic niche, in addition to having potential intrinsic roles in HSC fate determination. Moreover Daria et al (2007) observed a requirement for *Rb* in the stress response of HSCs and this has also previously been suggested in the context of the role of Rb in erythropoiesis (Spike et al.,

Also of note is that the cell division dynamics of HSCs change during development, from rapidly cycling and dividing cells during the fetal liver and early stages of life to relatively quiescent and more slowly cycling in the adult context (Bowie et al., 2007; Bradford et al., 1997; Ito et al, 2000; Kiel et al., 2007; Sato et al., 1999). Thus the role for *Rb* may be context dependent,

One important point that is becoming clearer recently is how some HSCs are maintained quiescent while others enter self-renewal program. Although bone–lining cells in the endosteal surface are often described as osteoblasts in the literature, they are heterogeneous in their degree of differentiation, and only a minority of these cells are actually bone synthesizing osteoblasts. So a good hypothesis is that in the endosteal niche some cells are in contact with true osteoblasts that expresses the necessary factors to maintain quiescence

both in terms of stress response and developmentally in the regulation of HSC fate.

while others are not receiving the same signalization so will follow other fate.

extrinsic modifier of HSC fate.

2004; Spike & Macleod, 2005).

**6.1 How HSC maintain quiescence** 

Li (2008) proposed that HSC quiescence is maintained through several signaling pathways including positive and negative regulators from extrinsic and intrinsic factors already described. In this context the *Tie-2/Ang-1* signaling pathway plays a critical role in the maintenance of HSCs in a quiescent state in the bone marrow niche (Adams et al., 2006). HSCs express the receptor tyrosine kinase Tie-2 and osteoblasts are the source of the Ang-1 ligand for Tie-2. *Tie-2/Ang-1* signaling activates its key downstream targets, *β1-integrin* and *N-cadherin* in lineage-negative, *Sca-1*, *C-kit* double-positive (LSK), and *Tie-2*-positive cells, and promotes HSC interactions with extracellular matrix and cellular components of the niche. This interaction is sufficient to maintain the quiescence and enhanced survival of HSCs by preventing cell division (Arai et al., 2007). *Ang-1/Tie-2* signaling also activates the phosphatidylinositol 3-kinase/Akt signaling pathway (Visnjic et al., 2004). Phosphatidylinositol 3-kinase/Akt signaling regulates several cell-cycle regulators, such as the CDK inhibitor, p21, which in turn leads to HSC quiescence.

Other pathway that has been proven to enhance quiescence is TPO/MPL. TPO is secreted by osteoblast while MPL is expressed in the membrane of HSCs. Interaction of these two proteins maintain HSCs attached to osteoblasts by activation of a pathway that results in the expression of their adhesion molecules targets and at the same time activate genetic programs which will control entry in cell cycle and survival of HSC. How these positive regulators interact with other positive and negative regulators is not completely understood. Moreover, which signaling pathways are being activated and which genes have their expression changed waits to be clarified.

A recent study by Wang et al. (2009) recently identified STAT5, a downstream target of MPL, as a positive regulator of HSC quiescence by analyzing *STAT5*−/− mice. Expression of quiescence regulators including *Tie-2* and *p57* are decreased in *STAT5*−/− HSCs. This study demonstrated that *STAT5* might mediate *MPL* effects in maintaining HSC quiescence during steady state hematopoiesis and that the same pathway directly or indirectly regulates *Tie-2* and *p57*. Interestingly, up-regulation of *p57* is essential for TGF-β−induced cell-cycle arrest. How these pathways are connected awaits more investigation.

Two other signaling pathways that act as positive and negative regulators of quiescence deserve more discussion, the Hypoxia induced factor (HIF) and Osteopontina signaling pathway. Hypoxia microenvironment seems to be important for maintaining HSC quiescence The molecular mechanisms for this involve the hypoxia-inducible factor-1a (HIF-1a) regulated gene expressions in stromal cells. Two genes known to be targets of the HIF pathway are *c-Kit* and stromal cell derived factor−1 (*SDF-1* or *CXCL12*) that both have proven to be important to HSC maintenance.

Osteopontina (*OPN*) is a negative regulator of HSC quiescence as an OPN-null microenvironment is sufficient to increase the number of stem cells associated with increased stromal *Jagged-1* and *Ang-1* expression and reduced primitive hematopoietic cell apoptosis. OPN seems to function by preventing HSC cycling. It is interesting to note that the release of this inhibition occurs in parallel with the possible activation of the *Notch* pathway.

As we can see there is many connections between intrinsic factors and extrinsic cues and between different intrinsic factors or different extrinsic factors. Some intrinsic factors function through affecting extrinsic factors, such as *c-Myc*, which negatively regulates HSC

Networks Establishing Hematopoietic Stem Cell Multipotency and Self-Renewal 21

Taken together, the existing studies suggest that canonical Wnt signaling may not be strictly required for HSC function, but that canonical Wnt signaling may affect self-renewal and differentiation of HSCs depending on the extent of canonical Wnt signaling and on the context of expression of additional genes. Non-canonical Wnt signaling and/or other signaling pathways may also compensate for the absence of canonical Wnt signaling in

 To exactly control the fine tune of *Wnt* in HSC it is likely that the numerous Wnt-signaling inhibitors (Dickkopf homolog (Dkk), Wnt inhibitory factor (Wif) or secreted frizzled-related protein (Sfrp), or other Wnt antagonists, such as Kremen, Ctgf, Cyr61, Sost and Sostdc1) have to be the correctly expressed. Interestingly, some of these molecules also directly stimulate certain Fzds independent of Wnt factors. For example, Sfrp1 directly activates Fzd2, as well as Fzd4, and Fzd7 but can also interact with Wnt5a (Rodriguez et al., 2005; Dufourc et al, 2008; Matsuyama et al, 2009 & Kirstetter et al., 2006). This balance and feedback mechanisms between canonical and non-canonical Wnt signaling, suggests that

*catenin* is the primary regulatory target of Wnt signaling. However, overexpression or

of myelopoiesis is due to a differentiation block (Renstrom et al., 2010), suggesting that b-

Conversely, Wnt signaling also induces increased expression of *HOXB4, Bmi1* and targets of *Notch-1*, genes that are implicated in self-renewal of HSCs . Transcription factors of homeodomain family (HOX family) have been found to regulate HSC self-renewal and downregulate differentiation. Disruption of HOX genes in mice led to abnormalities in multiple hematopoietic cell lineages. Moreover, overexpression of HOX genes (like HOXB4) has been associated with HSCs ex vivo expansion and HOX gene mutation with acute leukemia. *Bmi1,*a polycomb gene, seems to have a repressor role over *p16* inhibiting apoptosis of HSCs and thus contributing to its maintenance . So the correct Wnt signaling seems to be essential to integrate the intracellular response in the decision to self-renew or

The investigation of the interactions between Bmi1 and Hoxb4, showed that Bmi1 is not required for the in vivo expansion of fetal HSCs but is essential for the long-term maintenance of adult HSCs. Moreover, Hoxb4 overexpression induces an expansion of Bmi1-/- STR-HSCs leading to a rescue of their repopulation defect. Together, these results support the emerging concept that fate and sustainability of this fate are two critical

 Moreover Polycomb group (PcG) proteins play a role in the transcriptional repression of genes through histone modifications. Recent studies have clearly demonstrated that PcG proteins are required for the maintenance of embryonic as well as a broad range of adult stem cells, including hematopoietic stem cells (HSCs). PcG proteins maintain the self-renewal capacity of HSCs by repressing tumor suppressor genes and keep differentiation programs poised for activation in HSCs by repressing a cohort of hematopoietic developmental regulator genes via bivalent chromatin domains. Enforced expression of one of the PcG genes, Bmi1, augments the self-renewal capacity of HSCs. PcG proteins also maintain redox homeostasis to prevent premature loss of HSCs. These findings established PcG proteins as essential regulators of HSCs and underscored epigenetics as a new field of HSC research (Li et al., 2010; Komuna, 2010).

*-catenin* results in expansion of the HSC pool, but, at the same time, the loss

*-*

maintaining the self-renewal of HSCs (Huang, 2007).

catenin promotes self-renewal and/or inhibits differentiation.

components of self-renewal in adult stem cells such as HSCs.

stabilization of

differentiate (Reya et al., 2003).

quiescence by controlling *N-cadherin* expression level, reducing the *N-cadherin*−mediated interaction between HSCs and niche. Some extrinsic cues function through certain intrinsic factors, such as *STAT5*, which may serve as a component of *MPL*−induced signaling pathway, mediating MPL's effects in maintaining HSC quiescence.

Interestingly, among those HSC quiescence regulators that have been identified so far, the majority are positive regulators; few are negative for the maintenance of HSCs quiescence. This is consistent with the idea showing that the bias toward reduced gene expression that actively maintains HSC quiescence is an important mechanism of HSC proliferation, suggesting that various positive regulators of HSC quiescence are actively restricting proliferation of HSCs, and that there may exist signals in the environment to promote HSC proliferation.

### **6.2 Deciding for self-renewal**

Many signaling pathways are thought to contribute to stem cell self-renewal in the marrow niche including *Notch* (Maillard et al., 2003), Wnt (Duncan et al. 2005; Reya et al., 2003; Willert et al., 2003) and *Hedgehog* (Baron, 2001 ; Bhardwaj et al., 2001; Gering & Patient 2005).

Activated *Notch* expands the stem and progenitor cell compartment by either influencing undifferentiated cells to adopt a HSC fate or by causing a G0 HSC population to up-regulate *runx1*-dependent gene expression. Findings that the stem cell markers *runx1*, *scl*, and *lmo2*  were transcriptionally increased in response to NICD (Notch Intra-Cytoplamatic Domain) indicate that stem and progenitor cells were expanded in the adult marrow, possibly by increasing stem cell self-renewal. A conditional allele of *runx1* was generated in the mouse to study the loss of *Runx1* function during adult hematopoiesis (Growney et al., 2005; Ichikawa et al., 2004). In transplantation studies, *Runx1*-excised marrow cells showed a reduced competitive repopulating ability in long-term engraftment assays (Growney et al., 2005), demonstrating that Runx1 is essential for normal stem cell function.

The *Wnt/-catenin* signaling pathway also plays a crucial role during self-renewal of HSCs (Nemeth & Bodine, 2007). Deregulation of this pathway has been implicated in the formation of solid tumors, like lung epidermal adenocarcinomas, breast carcinomas and intestinal colorectal tumors just to mention a few (Reya & Clevers, 2005). Although several *Wnt* genes are expressed in bone marrow, the precise role of *Wnt* signaling pathway in HSCs and its mechanism(s) of action remained unclear until very recently.

There is a multitude of *Wnt* signaling cascades some of them regulating one another. Using different receptors, Wnt proteins can trigger at least three intracellular signaling pathways: the canonical b-catenin pathway, the non-canonical calcium pathway and the c-Jun Nterminal kinase pathway (Zeng et al., 2004). Several components of the *Wnt* signaling machinery have been shown to play a role in HSC self-renewal. Both canonical as well as non-canonical pathways seem to be involved, since the canonical ligand *Wnt3a* intrinsically promotes self-renewal (Luis et al., 2009). On the other hand, the non-canonical ligand *Wnt5a* has been shown to extrinsically promote self-renewal by inhibiting canonical signaling (Murdoch et al., 2003). The mechanistic basis for the balance between canonical and noncanonical pathways is not fully understood. It is likely that numerous *Wnt* inhibitors or antagonists are modulating *Wnt* signaling.

quiescence by controlling *N-cadherin* expression level, reducing the *N-cadherin*−mediated interaction between HSCs and niche. Some extrinsic cues function through certain intrinsic factors, such as *STAT5*, which may serve as a component of *MPL*−induced signaling

Interestingly, among those HSC quiescence regulators that have been identified so far, the majority are positive regulators; few are negative for the maintenance of HSCs quiescence. This is consistent with the idea showing that the bias toward reduced gene expression that actively maintains HSC quiescence is an important mechanism of HSC proliferation, suggesting that various positive regulators of HSC quiescence are actively restricting proliferation of HSCs, and that there may exist signals in the environment to promote HSC

Many signaling pathways are thought to contribute to stem cell self-renewal in the marrow niche including *Notch* (Maillard et al., 2003), Wnt (Duncan et al. 2005; Reya et al., 2003; Willert et al., 2003) and *Hedgehog* (Baron, 2001 ; Bhardwaj et al., 2001; Gering & Patient 2005). Activated *Notch* expands the stem and progenitor cell compartment by either influencing undifferentiated cells to adopt a HSC fate or by causing a G0 HSC population to up-regulate *runx1*-dependent gene expression. Findings that the stem cell markers *runx1*, *scl*, and *lmo2*  were transcriptionally increased in response to NICD (Notch Intra-Cytoplamatic Domain) indicate that stem and progenitor cells were expanded in the adult marrow, possibly by increasing stem cell self-renewal. A conditional allele of *runx1* was generated in the mouse to study the loss of *Runx1* function during adult hematopoiesis (Growney et al., 2005; Ichikawa et al., 2004). In transplantation studies, *Runx1*-excised marrow cells showed a reduced competitive repopulating ability in long-term engraftment assays (Growney et al.,

*-catenin* signaling pathway also plays a crucial role during self-renewal of HSCs

(Nemeth & Bodine, 2007). Deregulation of this pathway has been implicated in the formation of solid tumors, like lung epidermal adenocarcinomas, breast carcinomas and intestinal colorectal tumors just to mention a few (Reya & Clevers, 2005). Although several *Wnt* genes are expressed in bone marrow, the precise role of *Wnt* signaling pathway in

There is a multitude of *Wnt* signaling cascades some of them regulating one another. Using different receptors, Wnt proteins can trigger at least three intracellular signaling pathways: the canonical b-catenin pathway, the non-canonical calcium pathway and the c-Jun Nterminal kinase pathway (Zeng et al., 2004). Several components of the *Wnt* signaling machinery have been shown to play a role in HSC self-renewal. Both canonical as well as non-canonical pathways seem to be involved, since the canonical ligand *Wnt3a* intrinsically promotes self-renewal (Luis et al., 2009). On the other hand, the non-canonical ligand *Wnt5a* has been shown to extrinsically promote self-renewal by inhibiting canonical signaling (Murdoch et al., 2003). The mechanistic basis for the balance between canonical and noncanonical pathways is not fully understood. It is likely that numerous *Wnt* inhibitors or

2005), demonstrating that Runx1 is essential for normal stem cell function.

HSCs and its mechanism(s) of action remained unclear until very recently.

pathway, mediating MPL's effects in maintaining HSC quiescence.

proliferation.

The *Wnt/*

antagonists are modulating *Wnt* signaling.

**6.2 Deciding for self-renewal** 

Taken together, the existing studies suggest that canonical Wnt signaling may not be strictly required for HSC function, but that canonical Wnt signaling may affect self-renewal and differentiation of HSCs depending on the extent of canonical Wnt signaling and on the context of expression of additional genes. Non-canonical Wnt signaling and/or other signaling pathways may also compensate for the absence of canonical Wnt signaling in maintaining the self-renewal of HSCs (Huang, 2007).

 To exactly control the fine tune of *Wnt* in HSC it is likely that the numerous Wnt-signaling inhibitors (Dickkopf homolog (Dkk), Wnt inhibitory factor (Wif) or secreted frizzled-related protein (Sfrp), or other Wnt antagonists, such as Kremen, Ctgf, Cyr61, Sost and Sostdc1) have to be the correctly expressed. Interestingly, some of these molecules also directly stimulate certain Fzds independent of Wnt factors. For example, Sfrp1 directly activates Fzd2, as well as Fzd4, and Fzd7 but can also interact with Wnt5a (Rodriguez et al., 2005; Dufourc et al, 2008; Matsuyama et al, 2009 & Kirstetter et al., 2006). This balance and feedback mechanisms between canonical and non-canonical Wnt signaling, suggests that  *catenin* is the primary regulatory target of Wnt signaling. However, overexpression or stabilization of *-catenin* results in expansion of the HSC pool, but, at the same time, the loss of myelopoiesis is due to a differentiation block (Renstrom et al., 2010), suggesting that bcatenin promotes self-renewal and/or inhibits differentiation.

Conversely, Wnt signaling also induces increased expression of *HOXB4, Bmi1* and targets of *Notch-1*, genes that are implicated in self-renewal of HSCs . Transcription factors of homeodomain family (HOX family) have been found to regulate HSC self-renewal and downregulate differentiation. Disruption of HOX genes in mice led to abnormalities in multiple hematopoietic cell lineages. Moreover, overexpression of HOX genes (like HOXB4) has been associated with HSCs ex vivo expansion and HOX gene mutation with acute leukemia. *Bmi1,*a polycomb gene, seems to have a repressor role over *p16* inhibiting apoptosis of HSCs and thus contributing to its maintenance . So the correct Wnt signaling seems to be essential to integrate the intracellular response in the decision to self-renew or differentiate (Reya et al., 2003).

The investigation of the interactions between Bmi1 and Hoxb4, showed that Bmi1 is not required for the in vivo expansion of fetal HSCs but is essential for the long-term maintenance of adult HSCs. Moreover, Hoxb4 overexpression induces an expansion of Bmi1-/- STR-HSCs leading to a rescue of their repopulation defect. Together, these results support the emerging concept that fate and sustainability of this fate are two critical components of self-renewal in adult stem cells such as HSCs.

 Moreover Polycomb group (PcG) proteins play a role in the transcriptional repression of genes through histone modifications. Recent studies have clearly demonstrated that PcG proteins are required for the maintenance of embryonic as well as a broad range of adult stem cells, including hematopoietic stem cells (HSCs). PcG proteins maintain the self-renewal capacity of HSCs by repressing tumor suppressor genes and keep differentiation programs poised for activation in HSCs by repressing a cohort of hematopoietic developmental regulator genes via bivalent chromatin domains. Enforced expression of one of the PcG genes, Bmi1, augments the self-renewal capacity of HSCs. PcG proteins also maintain redox homeostasis to prevent premature loss of HSCs. These findings established PcG proteins as essential regulators of HSCs and underscored epigenetics as a new field of HSC research (Li et al., 2010; Komuna, 2010).

Networks Establishing Hematopoietic Stem Cell Multipotency and Self-Renewal 23

a self-renewal division are identical to the parental cell. Strictly speaking, this is not possible because DNA is continuously damaged and repaired by DNA-repair mechanisms that are

It is important to note that the efficiency of DNA repair varies greatly among different stem cell types. For example, embryonic stem cells are quite resistant to DNA damage and maintain the length of telomere repeats on serial passage, whereas HSCs are quite sensitive to DNA damage and less able to maintain telomere length. This idea has given rise to the notion that many aspects of normal aging could primarily reflect limitations in DNA repair and telomere-maintenance pathways in the (stem) cells of the soma (Lansdorp et al., 2005). The loss of telomere repeats in adult hematopoietic cells (including purified "candidate" HSCs) relative to fetal hematopoietic cells also fits a model that postulates a finite and limited replicative potential of HSCs (Vaziri et al., 1994; Lansdorp et al., 1995; Lansdorp et al., 1997). How this collaborates with the model of LT-HSCs given rise to ST-HSCs has not

Eukaryotic chromosomes are capped by special structures called telomeres, which are guanine-rich, simple repeat sequences. Telomeres act to guarantee chromosome integrity by preventing illegitimate recombination, degradation, and end fusions (Blackburn et al., 1991;

Synthesis and maintenance of telomeric repeats are accomplished by a specialized ribonucleoprotein complex known as telomerase. Telomerase consists of an essential RNA template and protein components, one of which appears to resemble reverse transcriptase. In the absence of telomerase, the failure of DNA polymerase to fully synthesize DNA

In contrast to mice were short telomeres maintain cell survival for some generations, a modest two fold reduction in telomerase levels in humans (resulting from haploinsufficiency for the telomerase RNA template gene) typically results in premature death from complications of aplastic anemia or immune deficiency. Recent studies indicate that short telomeres and eventual marrow failure may also result from haploinsufficiency

Moreover the large number of HSCs typically used in clinical transplant settings may effectively prevent their replicative exhaustion. Variations in telomere length between cells and individuals have even made it difficult to reproducibly document a decline in telomere length following transplantation. Nevertheless, a significant shortening of telomeres was observed in the first year after allogeneic bone marrow transplantation (Landsdorp et al., 2005). Furthermore, marrow failure with pronounced telomere shortening has been described in a few long-term survivors of HSC transplants. Although there is little evidence to suggest that telomere shortening will result in an epidemic of marrow failure in HSC transplant recipients, caution remains warranted when the cell number available for transplantation is limited or when the telomere length in HSCs for transplantation is short, as in cells from old donors or patients with telomerase deficiencies. It is tempting to speculate that some of the advantages of cord blood HSC transplants are related to the longer telomeres in individual cord blood HSCs (Awaya et

termini leads to chromosome shortening (Stain et al., 2004; Lee et al., 1998).

for the telomerase reverse transcriptase(hTERT) gene (Yamaguchi et al., 2005).

not 100% efficient (Lansdorp et al., 2005).

still been addressed.

Stain et al ., 2004).

al ., 2002).

Recently we demonstrated that another polycomb group member, Suz12 gene, is activated by the non canonical Wnt pathway and may epigenetically inhibit genes involved in hematopoietic differentiation. These data pointed to cell cycle changes, deregulation of early differentiation genes and regulation of PRC2 polycomb complex genes, due to Suz12 role in CML blast crisis. This observation indicates that the cross talk between Wnt and Polycomb pathways may promotes hematopoietic differentiation. (Pizzatti et al., 2010).

Taken together all these data fits in a model were HSCs fated to self renew are in contact in the endosteal niches with osteoblasts expressing *Notch* legands (*Jagged*) so the pathway that will be induced is *Notch* pathway.

The *Hedgehog* (*Hh*) is a ligand that binds and represses the *Patched* receptor and thereby releases the latent activity of the multipass membrane protein Smoothened, which is essential for transducing the *Hh* signal. Using *Patched*+/− mouse with increased *Hh* signaling activity, it was demonstrated that constitutive activation of the *Hh* signaling pathway results in the steady-state accumulation of phenotypically defined HSCs and an increase in the proportion of cycling cells within this population (Kuhn et al., 1995). However, HSC activity on secondary transplantation is reduced 3-fold, indicating the functional exhaustion of the HSC pool in this mutant. In vivo treatment with an inhibitor of the *Hh* pathway rescues these transcriptional and functional defects in HSCs. This study establishes *Hh* signaling as a negative regulator of the HSC quiescence. In contrast to the germline *Patched*+/− mode, the mode of conditional deletion of Smoothened in the adult hematopoietic compartment was used in other two studies. However, the negative effects of the Hh pathway on HSC quiescence were shown in one study (Walkley & Orkin, 2006) and not in another (Stead et al., 2002). The discrepancy is possibly due to a distinct mode of deletion. How this pathway collaborate with the two others is not clear although interaction through GSK3 have been already proposed.

The outcome of *Hh* signaling varies according to the receiving cell type. GLI, the cytoplasmic effector of *Hh* signaling activates the transcription of several target genes as *CyclinD1 and D2,N-Myc,Wnts, FoxM1,Hes 1,Bcl2,Osteopontin* and others. If these genes are activated in HSCs has not yet been defined but if they are, a clear interconnection between several important signaling pathways is visualized.

One important point when talking about self-renewal is how to prevent exhaustion of the HSC pool.
