**5.2 How some extrinsic factors act in the niche**

Stem cell factor receptor, also known as *c-kit* and its ligand *SCF* play a central role in hematopoiesis, melanogenesis and gametogenesis (Edling & Hallberg, 2007; Kent et al., 2008). *C-kit* is a member of the type-III subfamily of receptor tyrosine kinases that also includes the receptor for *M-CSF*, *Flt-3* and *PDGF*. It is expressed in HSCs (LT-HSCs, ST-HSCs and MPPs) (Zayas et al., 2008), normal B- and T-cell progenitors, mast cells, germ cells, melanocytes, neurons, glial cells, placenta, kidney, lung and gut cells. Deficiency and/or deregulation in *SCF* or *c-kit* produce defects in hematopoiesis leading to Acute Myeloid Leukemia (AML) (Scholl et al., 2008). Sporadic mutations of *c-kit* and autocrine/paracrine activation pathways of the *SCF/c-kit* pathway have been implicated in a variety of malignancies. Gain of function mutations of *c-kit* are associated with malignancies such AML, gastrointestinal tumors and mastocytomas. Moreover, expression of a defective *c-kit* leads to a decrease in repopulating HSCs (Ikuta & Weissman, 1992).

Binding of SCF to c-kit promotes dimerization and activation of protein kinase that autophosphorylates the receptor. Although SCF may not be essential for the generation of HSCs, numerous studies have shown that it prevents HSC apoptosis. Almost all cytokine combinations used to date for culturing HSCs include SCF. SCF potentiates the greater ability of fetal liver HSCs than adult HSCs to undergo symmetric self-renewal in culture this activity likely needs the cooperation of other factors. The membrane-bound form of SCF is also an adhesive molecule for HSCs to the bone marrow environment (Heissig et al., 2002) as interruption of the interaction between the membrane-bound stem cell factor on osteoblasts with the c-kit receptor on HSCs by blocking antibodies has demonstrated that *ckit* signaling is essential to maintain HSC dormancy and function (Suzuki et al., 2006), and an increased number of osteoclasts was associated with HSC mobilization. Receptor activator of nuclear factor (NF)-κB (RANK) ligand and cathepsin K mediate the cleavage of membrane-bound SCF; this decreases the abundance of SCF and, therefore, increases HSC mobilization (Kollet et al., 2006). The involvement of SCF in survival, mobility and possibly self-renewal of HSCs in culture and in the HSC niche likely reflects the complex relationship of different cell fates of HSCs.

Transforming growth factor (TGF)-β potently inhibits HSC activity *in vitro* (Blank et al., 2008). However, a *TGF-β* signaling deficiency *in vivo* does not affect proliferation of HSCs. TGF-β and BMP are secreted ligands that are recognized by different receptors that dimerizes and activates downstream cytosolic targets, culminating with the translocation of these activated transcription factors to the nucleus. BMPs, members of the TGF-β

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

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.

Stem cell factor receptor, also known as *c-kit* and its ligand *SCF* play a central role in hematopoiesis, melanogenesis and gametogenesis (Edling & Hallberg, 2007; Kent et al., 2008). *C-kit* is a member of the type-III subfamily of receptor tyrosine kinases that also includes the receptor for *M-CSF*, *Flt-3* and *PDGF*. It is expressed in HSCs (LT-HSCs, ST-HSCs and MPPs) (Zayas et al., 2008), normal B- and T-cell progenitors, mast cells, germ cells, melanocytes, neurons, glial cells, placenta, kidney, lung and gut cells. Deficiency and/or deregulation in *SCF* or *c-kit* produce defects in hematopoiesis leading to Acute Myeloid Leukemia (AML) (Scholl et al., 2008). Sporadic mutations of *c-kit* and autocrine/paracrine activation pathways of the *SCF/c-kit* pathway have been implicated in a variety of malignancies. Gain of function mutations of *c-kit* are associated with malignancies such AML, gastrointestinal tumors and mastocytomas. Moreover, expression of a defective

Binding of SCF to c-kit promotes dimerization and activation of protein kinase that autophosphorylates the receptor. Although SCF may not be essential for the generation of HSCs, numerous studies have shown that it prevents HSC apoptosis. Almost all cytokine combinations used to date for culturing HSCs include SCF. SCF potentiates the greater ability of fetal liver HSCs than adult HSCs to undergo symmetric self-renewal in culture this activity likely needs the cooperation of other factors. The membrane-bound form of SCF is also an adhesive molecule for HSCs to the bone marrow environment (Heissig et al., 2002) as interruption of the interaction between the membrane-bound stem cell factor on osteoblasts with the c-kit receptor on HSCs by blocking antibodies has demonstrated that *ckit* signaling is essential to maintain HSC dormancy and function (Suzuki et al., 2006), and an increased number of osteoclasts was associated with HSC mobilization. Receptor activator of nuclear factor (NF)-κB (RANK) ligand and cathepsin K mediate the cleavage of membrane-bound SCF; this decreases the abundance of SCF and, therefore, increases HSC mobilization (Kollet et al., 2006). The involvement of SCF in survival, mobility and possibly self-renewal of HSCs in culture and in the HSC niche likely reflects the complex relationship

Transforming growth factor (TGF)-β potently inhibits HSC activity *in vitro* (Blank et al., 2008). However, a *TGF-β* signaling deficiency *in vivo* does not affect proliferation of HSCs. TGF-β and BMP are secreted ligands that are recognized by different receptors that dimerizes and activates downstream cytosolic targets, culminating with the translocation of these activated transcription factors to the nucleus. BMPs, members of the TGF-β

*c-kit* leads to a decrease in repopulating HSCs (Ikuta & Weissman, 1992).

certain circumstances.

of different cell fates of HSCs.

**5.2 How some extrinsic factors act in the niche** 

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

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 crosstalk of FGF signaling with other pathways is complex.

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 manner.

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

Networks Establishing Hematopoietic Stem Cell Multipotency and Self-Renewal 11

differentiation, or homing of HSCs is unclear. Interestingly, IGF-2 was found to bind and stimulate self-renewal of human embryonic stem cells (Bendall et al., 2007). Angiopoietinlike proteins (Angptls) were also implicated in HSC expansion. Angptls are a family of seven secreted glycoproteins that share sequence homology with the angiopoietins (Morisada et al., 2006). Similar to the angiopoietins, each Angptl contains an N-terminal coiledcoil domain and a C-terminal fibrinogen-like domain. However, unlike angiopoietins, Angptls do not bind to Tie-2 or Tie-1 and their receptors are unknown. This suggests that Angptls may have different functions from the angiopoietins. Angptl7 was suggested to be a target of the *Wnt/β-catenin* signaling pathway. However, most of the physiological activities of the Angptls remain unknown. Recently Angptl2 and Angptl3 were identified as growth factors that stimulate *ex vivo* expansion of bone marrow HSCs. Other analogues, including

In addition to signaling pathways as described above, extracellular matrix components of the niche have also been shown to play role in regulating the HSC dynamics. A matrix glycoprotein, osteopontin (OPN), as a constraining factor on HSCs within the bone marrow microenvironment is produced by osteoblasts in response to stimulation (Stier et al., 2005). Using studies that combine OPN-deficient mice and exogenous OPN, Stier et al. (2005) demonstrated that OPN modifies primitive hematopoietic cell number and function in a stem cell non-autonomous manner. The 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. The activation of the stem cell microenvironment with PTH was shown to induce a super-physiologic increase in stem cells in the absence of OPN. Therefore, OPN seems to be a negative regulatory element of the stem cell niche that limits the size of the stem cell pool and may provide a mechanism for

The production of OPN by osteoblasts is likely to be an essential requirement as shown by Karahuseyinoglu et al. (2007). Osteogenically induced umbilical cord stromal cells express OPN during the first week of induction followed by a third week expression of another matricellular protein, bone sialoprotein-2 (BSP-2). In the following weeks, in conditioned media differentiating osteoblasts express osteonectin and osteocalcin that led us to suggest that all those proteins have roles in autocrine regulation of osteoblast maturation and thus might serve to determine the conditional status of the partner cell(s) in hematopoietic niche

Previous studies showed that cell adhesion molecules, such as cadherins and integrins, are crucial for the interactions between HSCs and the osteoblastic niche. N-cadherin−mediated adhesion mediates slowing cell cycling of HSCs and may keep HSCs quiescent. Some studies showed that specialized spindle-shaped N-cadherin+ osteoblasts are a key component of the bone marrow stem cell niche. HSCs are thought to be anchored to spindleshaped N-cadherin+ osteoblast cells via a homotypic N-cadherin interaction. Also, Ncadherin and β1-integrin are identified as the downstream targets in *Tie-2/Ang-1* signaling and *TPO/MPL* signaling (Yoshihara et al., 2007) in HSCs, respectively, suggesting a link between adhesion molecules and cell-cycle regulators in modulating the HSC−niche interaction. These data suggest cell-adhesion molecules not only contribute to the anchoring

Angptl5, Angptl7, and Mfap4, also support *ex vivo* expansion of HSCs.

restricting excess stem cell expansion under conditions of niche stimulation.

**5.3 Signaling through cell adhesion molecules** 

microenvironment.

self-renewal activity. These findings show that *Wnt/β-catenin* activity is crucial for the maintenance of HSC quiescence in the bone marrow niche.

The angiopoietin (*Ang*) family of growth factors is composed of four members that bind to the Tie-2 tyrosine kinase receptor; Ang growth factors are important modulators of angiogenesis. Members of the angiopoietin family of proteins contain an N-terminal coiled– coil domain that mediates homo-oligomerization and a C-terminal fibrinogen-like domain that binds Tie-2. To identify the HSCs in situ, Arai et al. (2004) analyzed the receptor tyrosine kinase Tie-2 expression in bone marrow and found that 5-FU-resistant Tie-2 expressing HSCs adhere to osteoblasts at the endosteal surface, in agreement with previous findings of Calvi et al. (2003) and Zhang et al (2003). They also demonstrated that angiopoietin-1 (*Ang-1*), a Tie-2 receptor ligand, is produced primarily by osteoblasts, indicating that Tie-2 and Ang-1 are expressed complementarily in the niche. Tie-2 together with Tie-1 was also found required for homing of HSCs to bone marrow. Taken together, Tie receptors seem one group of the likely candidates for localizing stem cells to the stem cell niche.

Mineral content of bone contributes to compose a unique extracellular matrix in bone marrow and distinguishes it from other mesenchymal tissues. The extracellular calcium concentrations are recognized by the seven-transmembrane calcium-sensing receptors and therefore can initiate an intracellular G protein–coupled response. Those receptors are found on hematopoietic cells and have also been identified on the surface of HSCs (Adams et al., 2006). Local calcium gradient is involved in retaining HSCs in close physical proximity to the endosteal surface of bone. Extracellular calcium ion concentrations in the endosteum are likely higher than in the central marrow region (Silver et al., 1988). In receptor deficient mice models, HSCs were found not to engraft in the bone marrow (Adams et al., 2006) suggesting that the ability of stem cells to sense and respond to the increased calcium concentrations at the endosteal surface participates in creating the unique stem cell-niche interaction that enables bone marrow hematopoiesis.

Most slow-cycling hematopoietic cells are found in the hypoxic zones close to bone surface and distant from capillaries (Kubota et al., 2008), raising the possibility that these hypoxic niches are important for diminished HSC proliferation. Evidence for quiescent HSCs situated in a hypoxic environment has lately been confirmed by analyzing bone marrow cells from mice injected with a Hoechst dye. Transplantation results showed that the bone marrow fraction with the lowest Hoechst-dye uptake, inferred to be hypoxic, had the highest amount of long-term repopulating cells (Parmar et al., 2007). Consistently, HSCs were found to be the most positive for binding of the hypoxic probe pimonidazole. The molecular mechanisms involve the hypoxia-inducible factor-1a regulated gene expressions in stromal cells, such as c-Kit, stromal cell derived factor−1, and others (Ceradini et al., 2004).

Other molecules were recently identified to have role in signaling pathways inside the niche. DNA array experiments showed that, among other proteins, IGF-2 is specifically expressed in cells that do support HSC expansion in culture. Moreover, it was showed that all fetal liver and bone marrow HSCs express receptors for IGF-2. The inclusion of IGF-2 with SCF, TPO, and FGF-1 supports an eight-fold increase of highly enriched HSCs in culture (Zhang & Lodish, 2004). Whether IGF-2 acts on self-renewal, apoptosis, differentiation, or homing of HSCs is unclear. Interestingly, IGF-2 was found to bind and stimulate self-renewal of human embryonic stem cells (Bendall et al., 2007). Angiopoietinlike proteins (Angptls) were also implicated in HSC expansion. Angptls are a family of seven secreted glycoproteins that share sequence homology with the angiopoietins (Morisada et al., 2006). Similar to the angiopoietins, each Angptl contains an N-terminal coiledcoil domain and a C-terminal fibrinogen-like domain. However, unlike angiopoietins, Angptls do not bind to Tie-2 or Tie-1 and their receptors are unknown. This suggests that Angptls may have different functions from the angiopoietins. Angptl7 was suggested to be a target of the *Wnt/β-catenin* signaling pathway. However, most of the physiological activities of the Angptls remain unknown. Recently Angptl2 and Angptl3 were identified as growth factors that stimulate *ex vivo* expansion of bone marrow HSCs. Other analogues, including Angptl5, Angptl7, and Mfap4, also support *ex vivo* expansion of HSCs.

#### **5.3 Signaling through cell adhesion molecules**

10 Advances in Hematopoietic Stem Cell Research

self-renewal activity. These findings show that *Wnt/β-catenin* activity is crucial for the

The angiopoietin (*Ang*) family of growth factors is composed of four members that bind to the Tie-2 tyrosine kinase receptor; Ang growth factors are important modulators of angiogenesis. Members of the angiopoietin family of proteins contain an N-terminal coiled– coil domain that mediates homo-oligomerization and a C-terminal fibrinogen-like domain that binds Tie-2. To identify the HSCs in situ, Arai et al. (2004) analyzed the receptor tyrosine kinase Tie-2 expression in bone marrow and found that 5-FU-resistant Tie-2 expressing HSCs adhere to osteoblasts at the endosteal surface, in agreement with previous findings of Calvi et al. (2003) and Zhang et al (2003). They also demonstrated that angiopoietin-1 (*Ang-1*), a Tie-2 receptor ligand, is produced primarily by osteoblasts, indicating that Tie-2 and Ang-1 are expressed complementarily in the niche. Tie-2 together with Tie-1 was also found required for homing of HSCs to bone marrow. Taken together, Tie receptors seem one group of the likely candidates for localizing stem cells to the stem

Mineral content of bone contributes to compose a unique extracellular matrix in bone marrow and distinguishes it from other mesenchymal tissues. The extracellular calcium concentrations are recognized by the seven-transmembrane calcium-sensing receptors and therefore can initiate an intracellular G protein–coupled response. Those receptors are found on hematopoietic cells and have also been identified on the surface of HSCs (Adams et al., 2006). Local calcium gradient is involved in retaining HSCs in close physical proximity to the endosteal surface of bone. Extracellular calcium ion concentrations in the endosteum are likely higher than in the central marrow region (Silver et al., 1988). In receptor deficient mice models, HSCs were found not to engraft in the bone marrow (Adams et al., 2006) suggesting that the ability of stem cells to sense and respond to the increased calcium concentrations at the endosteal surface participates in creating the unique stem cell-niche interaction that

Most slow-cycling hematopoietic cells are found in the hypoxic zones close to bone surface and distant from capillaries (Kubota et al., 2008), raising the possibility that these hypoxic niches are important for diminished HSC proliferation. Evidence for quiescent HSCs situated in a hypoxic environment has lately been confirmed by analyzing bone marrow cells from mice injected with a Hoechst dye. Transplantation results showed that the bone marrow fraction with the lowest Hoechst-dye uptake, inferred to be hypoxic, had the highest amount of long-term repopulating cells (Parmar et al., 2007). Consistently, HSCs were found to be the most positive for binding of the hypoxic probe pimonidazole. The molecular mechanisms involve the hypoxia-inducible factor-1a regulated gene expressions in stromal cells, such as c-Kit, stromal cell derived factor−1, and others (Ceradini et al.,

Other molecules were recently identified to have role in signaling pathways inside the niche. DNA array experiments showed that, among other proteins, IGF-2 is specifically expressed in cells that do support HSC expansion in culture. Moreover, it was showed that all fetal liver and bone marrow HSCs express receptors for IGF-2. The inclusion of IGF-2 with SCF, TPO, and FGF-1 supports an eight-fold increase of highly enriched HSCs in culture (Zhang & Lodish, 2004). Whether IGF-2 acts on self-renewal, apoptosis,

maintenance of HSC quiescence in the bone marrow niche.

cell niche.

2004).

enables bone marrow hematopoiesis.

In addition to signaling pathways as described above, extracellular matrix components of the niche have also been shown to play role in regulating the HSC dynamics. A matrix glycoprotein, osteopontin (OPN), as a constraining factor on HSCs within the bone marrow microenvironment is produced by osteoblasts in response to stimulation (Stier et al., 2005). Using studies that combine OPN-deficient mice and exogenous OPN, Stier et al. (2005) demonstrated that OPN modifies primitive hematopoietic cell number and function in a stem cell non-autonomous manner. The 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. The activation of the stem cell microenvironment with PTH was shown to induce a super-physiologic increase in stem cells in the absence of OPN. Therefore, OPN seems to be a negative regulatory element of the stem cell niche that limits the size of the stem cell pool and may provide a mechanism for restricting excess stem cell expansion under conditions of niche stimulation.

The production of OPN by osteoblasts is likely to be an essential requirement as shown by Karahuseyinoglu et al. (2007). Osteogenically induced umbilical cord stromal cells express OPN during the first week of induction followed by a third week expression of another matricellular protein, bone sialoprotein-2 (BSP-2). In the following weeks, in conditioned media differentiating osteoblasts express osteonectin and osteocalcin that led us to suggest that all those proteins have roles in autocrine regulation of osteoblast maturation and thus might serve to determine the conditional status of the partner cell(s) in hematopoietic niche microenvironment.

Previous studies showed that cell adhesion molecules, such as cadherins and integrins, are crucial for the interactions between HSCs and the osteoblastic niche. N-cadherin−mediated adhesion mediates slowing cell cycling of HSCs and may keep HSCs quiescent. Some studies showed that specialized spindle-shaped N-cadherin+ osteoblasts are a key component of the bone marrow stem cell niche. HSCs are thought to be anchored to spindleshaped N-cadherin+ osteoblast cells via a homotypic N-cadherin interaction. Also, Ncadherin and β1-integrin are identified as the downstream targets in *Tie-2/Ang-1* signaling and *TPO/MPL* signaling (Yoshihara et al., 2007) in HSCs, respectively, suggesting a link between adhesion molecules and cell-cycle regulators in modulating the HSC−niche interaction. These data suggest cell-adhesion molecules not only contribute to the anchoring

Networks Establishing Hematopoietic Stem Cell Multipotency and Self-Renewal 13

niche offers an alternative niche for mobilized stem cells and promotes proliferation and further differentiation or maturation into the circulatory system. It would be interesting to further define the respective contributions of endothelial and endosteal niches to HSC

Recent studies have shown that Polycomb group (PcG) proteins and their interaction are important in the regulation of HSC self-renewal and lineage restriction. In particular, members of the PRC1 (Polycomb repression complex 1), such as *Bmi1*, *Mel18* and *Rae28*, have been implicated. *Bmi1* plays an important role in regulating the proliferative activity of stem and progenitor cells. It is required for the self-renewal of both adult HSCs and neural stem cells (Molofsky et al., 2005; Park et al, 2003). *Bmi1* enhances symmetrical expansion of the stem cell pool through self-renewal, induces a marked *ex vivo* expansion of multipotent progenitors, and increases the ability of HSCs to repopulate bone marrow *in vivo* (Iwama et al., 2004). Leukemic cells lacking *Bmi1* undergo proliferation arrest, differentiation and apoptosis, leading to failure of leukemia in a mouse transplant model (Lessard & Sauvageau, 2003). In *Bmi1*-deficient bone marrow there is an up-regulation of cell cycle inhibitors *p16* and *p19*, and the *p53*-induced gene *Wig1*, and a down-regulation of the apoptosis inhibitor *AI-6*. This suggests that a mechanism exists whereby *Bmi1* functions by modulating proliferation and preventing apoptosis (Park et al., 2004). *Bmi1* has also been shown to regulate the expression of *Hox* genes that are required for differentiation during hematopoiesis (van der Lugt et al., 1996).Loss or knockdown of another Polycomb gene, *Mel18*, leads to increased expression of *Hoxb4* (Kajiume et al., 2004), and transplanted *Mel18*-deficient bone marrow showed an increase in overall HSC numbers but a decrease in their activity owing to arrest in G0 phase of the cell cycle. *Rae28*-deficient HSCs were defective in their long-term repopulating ability in serial transplantation experiments (Kim et al., 2004; Ohta et al., 2002). Taken together, these studies show the importance of the Polycomb proteins in HSC self-renewal and

Transcriptional repression by PcG proteins is essential for maintenance of HSC identity. Part of the mechanism by which it functions is by repression of genes that promote lineage specification, cell death and cell cycle arrest. More recently, PcG complexes have been shown to be essential for maintenance of the undifferentiated state in murine embryonic stem (ES) cells and human ES cells by directly repressing a large number of developmental regulators (Boyer et al., 2006; Lee et al., 2006). PcG complexes bind to and presumably repress the expression of a subset of these genes linked to differentiation. This represents a dynamic repression of genes required for differentiation, and a scenario in which PcG proteins act as transcription repressors by cooperating with a specific set of transcription factors in stem cells. Some target genes include *Hox* family members important for induction of differentiation. Expression of *Hox* genes that are involved in differentiation is repressed in the ES cells by PcG proteins. Thus, PcG complex repression is also necessary for ES cell identity. Taken together, these studies suggest that differentiation is the default state during stem cell replication, and self-renewal requires active repression of transcription factors that

behaviour.

**5.4 Cell intrinsic responses** 

maintenance of the blood system.

prevent self-renewal

of HSCs to the niche, but also regulate cell-cycle quiescence of HSCs in the niche. However, the studies by conditional deletion of N-cadherin fail to support the effects of N-cadherin on hematopoiesis (Kiel et al., 2007).

The members of the *Notch* family are developmental morphogens shown to be expressed in self-renewing tissues, enhance the self-renewal capacity of HSCs and promote T-cell differentiation. *Notch* signaling is initiated by the involvement of the extracellular portion of Notch with its ligands Jagged/Delta. Activation of the *Notch* signaling pathway has been shown to potentiate self-renewal of HSCs. It is initiated by the binding of Jagged ligand to Notch protein followed by metalloproteinase (-secretase) cleavage in the extracellular receptor portion leading to the intracellular release of Notch (NICD). Then Notch translocate into the nucleus, where it forms a multimeric transcriptional complex with other transcription factors (Huntly & Gilliland, 2005). Inhibitors of -secretase abrogate the Notch signaling activation (Rizzo et al., 2008; Shih & Wang, 2007).

Calvi et al*.* (2003) and Duncan et al*.* (2005) demonstrated that the *Notch* signaling pathway plays a role in the osteoblast bone marrow HSCs niche. Notch ligands have positive effects on *ex vivo* expansion of HSCs: activated *Notch* is able to immortalize primitive mouse hematopoietic progenitors and Notch ligands support HSC expansion in culture (Chiba, 2006). Recently, by culturing human cord blood cells in serum-free medium supplemented with SCF, TPO, Flt3L, IL-3, IL-6/sIL-6R, and Delta 1, Suzuki et al*.* (Suzuki et al., 2006) reported an approximate six-fold increase in SCID-repopulating cell (SRC) number. It is noteworthy that there exists a dose effect for Notch ligands in HSC culture. Whereas a low amount of Delta 1 supports human cord blood SRC expansion, high amounts of the cytokine induce apoptosis (Chiba, 2006).

This emphasizes the complicated relationship among the different fates of HSCs. As conditional knockouts of *Notch1* and *Jagged1* have normal *in vivo* HSC activities (Mancini et al., 2005), there likely is functional redundancy of different *Notch* isoforms and their ligands.

Endothelial cells in the vascular niche environment contacting HSCs also provide maintenance signals on the HSC behaviour (Coultas et al., 2005 ; Li & Li, 2006). The main components of vascular niche – hematopoietic cells and endothelial cells – are closely related during development since they are both derived from haemangioblasts (Kopp et al., 2005). Previous studies have suggested that the vascular niche is the place for HSC differentiation and mobilization (Avencilla et al., 2004). Endothelial cells expressing vascular cell-adhesion molecule-1 (VCAM-1) associate closely with megakaryocytes and their progenitors through VLA-4 in response to chemotactic factors, stromal cell-derived factor- 1 (SDF1) and fibroblast growth factor-4 (FGF4), and thus provide a niche for megakaryocyte maturation and platelet production. The immediate juxtaposition of HSCs to endothelial cells also facilitates their rapid mobilization and entry into circulation in response to stress and, in the case of megakaryocytes, release of platelets directly into the blood. Endothelial cells promote survival of HSCs in culture, but this seems to be limited to certain populations of endothelial cells (Li et al., 2004). Fractions of HSCs in both adult bone marrow and spleen were found in close association with endothelial sinusoids (Kiel et al., 2005), suggesting that endothelial cells provide support to HSCs *in vivo*. Depending on these data, it is now plausible to note that while the osteoblastic niche provides a quiescent environment for HSC maintenance, the vascular niche offers an alternative niche for mobilized stem cells and promotes proliferation and further differentiation or maturation into the circulatory system. It would be interesting to further define the respective contributions of endothelial and endosteal niches to HSC behaviour.

#### **5.4 Cell intrinsic responses**

12 Advances in Hematopoietic Stem Cell Research

of HSCs to the niche, but also regulate cell-cycle quiescence of HSCs in the niche. However, the studies by conditional deletion of N-cadherin fail to support the effects of N-cadherin on

The members of the *Notch* family are developmental morphogens shown to be expressed in self-renewing tissues, enhance the self-renewal capacity of HSCs and promote T-cell differentiation. *Notch* signaling is initiated by the involvement of the extracellular portion of Notch with its ligands Jagged/Delta. Activation of the *Notch* signaling pathway has been shown to potentiate self-renewal of HSCs. It is initiated by the binding of Jagged ligand to Notch protein followed by metalloproteinase (-secretase) cleavage in the extracellular receptor portion leading to the intracellular release of Notch (NICD). Then Notch translocate into the nucleus, where it forms a multimeric transcriptional complex with other transcription factors (Huntly & Gilliland, 2005). Inhibitors of -secretase abrogate the Notch

Calvi et al*.* (2003) and Duncan et al*.* (2005) demonstrated that the *Notch* signaling pathway plays a role in the osteoblast bone marrow HSCs niche. Notch ligands have positive effects on *ex vivo* expansion of HSCs: activated *Notch* is able to immortalize primitive mouse hematopoietic progenitors and Notch ligands support HSC expansion in culture (Chiba, 2006). Recently, by culturing human cord blood cells in serum-free medium supplemented with SCF, TPO, Flt3L, IL-3, IL-6/sIL-6R, and Delta 1, Suzuki et al*.* (Suzuki et al., 2006) reported an approximate six-fold increase in SCID-repopulating cell (SRC) number. It is noteworthy that there exists a dose effect for Notch ligands in HSC culture. Whereas a low amount of Delta 1 supports human cord blood SRC expansion, high amounts of the cytokine

This emphasizes the complicated relationship among the different fates of HSCs. As conditional knockouts of *Notch1* and *Jagged1* have normal *in vivo* HSC activities (Mancini et al., 2005), there likely is functional redundancy of different *Notch* isoforms and their ligands. Endothelial cells in the vascular niche environment contacting HSCs also provide maintenance signals on the HSC behaviour (Coultas et al., 2005 ; Li & Li, 2006). The main components of vascular niche – hematopoietic cells and endothelial cells – are closely related during development since they are both derived from haemangioblasts (Kopp et al., 2005). Previous studies have suggested that the vascular niche is the place for HSC differentiation and mobilization (Avencilla et al., 2004). Endothelial cells expressing vascular cell-adhesion molecule-1 (VCAM-1) associate closely with megakaryocytes and their progenitors through VLA-4 in response to chemotactic factors, stromal cell-derived factor- 1 (SDF1) and fibroblast growth factor-4 (FGF4), and thus provide a niche for megakaryocyte maturation and platelet production. The immediate juxtaposition of HSCs to endothelial cells also facilitates their rapid mobilization and entry into circulation in response to stress and, in the case of megakaryocytes, release of platelets directly into the blood. Endothelial cells promote survival of HSCs in culture, but this seems to be limited to certain populations of endothelial cells (Li et al., 2004). Fractions of HSCs in both adult bone marrow and spleen were found in close association with endothelial sinusoids (Kiel et al., 2005), suggesting that endothelial cells provide support to HSCs *in vivo*. Depending on these data, it is now plausible to note that while the osteoblastic niche provides a quiescent environment for HSC maintenance, the vascular

hematopoiesis (Kiel et al., 2007).

induce apoptosis (Chiba, 2006).

signaling activation (Rizzo et al., 2008; Shih & Wang, 2007).

Recent studies have shown that Polycomb group (PcG) proteins and their interaction are important in the regulation of HSC self-renewal and lineage restriction. In particular, members of the PRC1 (Polycomb repression complex 1), such as *Bmi1*, *Mel18* and *Rae28*, have been implicated. *Bmi1* plays an important role in regulating the proliferative activity of stem and progenitor cells. It is required for the self-renewal of both adult HSCs and neural stem cells (Molofsky et al., 2005; Park et al, 2003). *Bmi1* enhances symmetrical expansion of the stem cell pool through self-renewal, induces a marked *ex vivo* expansion of multipotent progenitors, and increases the ability of HSCs to repopulate bone marrow *in vivo* (Iwama et al., 2004). Leukemic cells lacking *Bmi1* undergo proliferation arrest, differentiation and apoptosis, leading to failure of leukemia in a mouse transplant model (Lessard & Sauvageau, 2003). In *Bmi1*-deficient bone marrow there is an up-regulation of cell cycle inhibitors *p16* and *p19*, and the *p53*-induced gene *Wig1*, and a down-regulation of the apoptosis inhibitor *AI-6*. This suggests that a mechanism exists whereby *Bmi1* functions by modulating proliferation and preventing apoptosis (Park et al., 2004). *Bmi1* has also been shown to regulate the expression of *Hox* genes that are required for differentiation during hematopoiesis (van der Lugt et al., 1996).Loss or knockdown of another Polycomb gene, *Mel18*, leads to increased expression of *Hoxb4* (Kajiume et al., 2004), and transplanted *Mel18*-deficient bone marrow showed an increase in overall HSC numbers but a decrease in their activity owing to arrest in G0 phase of the cell cycle. *Rae28*-deficient HSCs were defective in their long-term repopulating ability in serial transplantation experiments (Kim et al., 2004; Ohta et al., 2002). Taken together, these studies show the importance of the Polycomb proteins in HSC self-renewal and maintenance of the blood system.

Transcriptional repression by PcG proteins is essential for maintenance of HSC identity. Part of the mechanism by which it functions is by repression of genes that promote lineage specification, cell death and cell cycle arrest. More recently, PcG complexes have been shown to be essential for maintenance of the undifferentiated state in murine embryonic stem (ES) cells and human ES cells by directly repressing a large number of developmental regulators (Boyer et al., 2006; Lee et al., 2006). PcG complexes bind to and presumably repress the expression of a subset of these genes linked to differentiation. This represents a dynamic repression of genes required for differentiation, and a scenario in which PcG proteins act as transcription repressors by cooperating with a specific set of transcription factors in stem cells. Some target genes include *Hox* family members important for induction of differentiation. Expression of *Hox* genes that are involved in differentiation is repressed in the ES cells by PcG proteins. Thus, PcG complex repression is also necessary for ES cell identity. Taken together, these studies suggest that differentiation is the default state during stem cell replication, and self-renewal requires active repression of transcription factors that prevent self-renewal

Networks Establishing Hematopoietic Stem Cell Multipotency and Self-Renewal 15

has been identified as a positive regulator of HSC quiescence through analysis of *p53*−/<sup>−</sup> mice (Liu et al., 2009). Furthermore, in the same study, it was demonstrated that the increased quiescence of HSCs from MEF null mice, in which both *p53* and *p21* are upregulated, is dependent on *p53*, but not *p21*, further confirming the positive role of *p53* in maintaining HSC in quiescence. *Gfi1* was identified as *p53* target gene, which is both shown important in regulating HSC quiescence by up-regulation or knockdown experiments.

Stem cell leukemia/T-cell acute lymphoblastic leukemia 1 (*SCL/TAL1*) plays a key role in controlling development of primitive and definitive hematopoiesis during mouse development. In adult HSCs, it is highly expressed in LT-HSCs compared with short-term HSCs and progenitors (Lacombe et al., 2010). SCL impedes G0-G1 transition in HSCs. The function of HSCs from *Scl* +/<sup>−</sup> mice or with decreased dosage of SCL protein by *in vitro* interference was shown decreased in various transplantation assays. At the molecular level,

Recently, many other transcriptional factors, such as interferon regulatory factor−2, a transcriptional suppressor of interferon signaling (Sato et al., 2009); *Nurr1*, a nuclear receptor transcription factor (Sirin et al., 2010); and thioredoxin-interacting protein, a transcriptional repressor (Shao et al., 2010), have been identified as positive regulators of HSC quiescence. Loss of HSC quiescence was observed in mice with deletion of each of

Individual member of Retinoblastoma (*Rb*) tumor suppressor gene family serves critical roles in the control of cellular proliferation and differentiation with functional redundancy for each other. The mice with conditional triple knockout of *Rb* family genes including *Rb*, *p107*, and *p130* display a cell-intrinsic myeloproliferation that originates from hyperproliferative early hematopoietic progenitors due to the loss of quiescence, and the mutant HSCs show strong short-term repopulation capacity but impaired long-term repopulation ability on transplantation. Thus, *Rb* family members collectively maintain HSC

It has been shown that the conditional inactivation of *c-Myc* induces excessive expression of *integrins* and *N-cadherin* in HSCs, leading to the enhanced HSC interaction with the niche, which subsequently enable *Myc*-deficient HSCs stay in quiescence. Conversely, enforced *c-Myc* expression in HSCs downregulates *N-cadherin* and *integrins*, leading to a loss of HSC

*p21* mRNA expression levels are dramatically lower in the *Gfi1*-deficient HSCs. *p21* itself has been implicated in the regulation of HSCs (Cheng et al., 2000). In its absence, HSCs have an impaired serial transplantation capacity. Another cell cycle inhibitor, *p18* , has also been shown to affect HSC self-renewal. The absence of *p18* leads to increased HSC self-renewal (Yuan et al., 2004; Yu et al., 2006). Therefore, intricate control of the cell cycle and

In contrast to *p21*, little is known about the role of *p57* in adult stem cell populations. Using primary human hematopoietic cells and microarray analysis, Scandura et al. (2004) identified *p57* as the only cyclin-dependent kinase inhibitor induced by TGF-β. Upregulation of *p57* is essential for TGF-β−induced cell-cycle arrest in these cells, which may represent the mechanisms by which TGF-β affects cell-cycle arrest and stem cell quiescence.

proliferation machinery is required for self-renewal regulation.

SCL maintains HSC quiescence by regulating gene expression of *Cdkn1a* and *Id1*.

these factors.

quiescence (Viatour et al., 2008).

function (Wilson et al., 2004).

The transcription factor *Tel* (Translocation Ets leukemia; also known as Etv6 [Ets variant gene 6]), an *Ets* (E-26 transforming-specific)-related transcriptional repressor, is also required for HSC maintenance. Conditional inactivation of *Tel/Etv6* in HSCs rapidly leads to the depletion of *Tel/Etv6*-deficient bone marrow. However, *Tel/Etv6* is not required for the maintenance of committed precursors. When it is conditionally inactivated in most hematopoietic lineages, it does not affect their differentiation or survival (Hock et al., 2004). At the moment, the mechanism by which *Tel/Etv6* modulates adult HSCs renewal is not known. Study of the downstream targets it represses should shed light on other players essential for HSC maintenance.

*Pbx1* (pre–B-cell acute lymphoblastic leukemia) is a TALE class homeodomain transcription factor that critically regulates numerous embryonic processes, including hematopoiesis. *Pbx1* is preferentially expressed in LT-HSCs compared to more mature short-term HSCs and multipotent progenitor cells (Ficara et al., 2008). By using *Pbx1*-conditional knockout mice, it was revealed that *Pbx1* positively regulates HSC quiescence. Transcriptional profiling showed that a significant proportion of *Pbx1*-dependent genes are associated with the *TGF-β* pathway.

The homeobox (*Hox*) genes encode transcription factors that regulate embryonic body patterning and organogenesis. They play a role in the regulation of hematopoiesis. Overexpression of *HoxB4* in bone marrow leads to expansion of HSCs *in vivo* and *in vitro*, therefore appearing to be a positive regulator of HSC self-renewal (Antonchuck et al., 2002; Krosl et al., 2003; Miyake et al., 2006; Sauvageau et al., 1995). It therefore came as a surprise when *HoxB4*-deficient mice had normal hematopoietic development but exhibited only mild proliferative HSC defects (Brun et al., 2004). In an attempt to determine if this was due to compensatory mechanisms, the entire *HoxB* cluster was deleted. However, this did not lead to major defects in hematopoiesis (Bijl et al., 2006), possibly owing to compensation by *HoxA4* and/or *HoxC4*.

*Gfi1* (Growth factor independence 1), a zinc-finger repressor, has been recently implicated as a regulator of HSC self-renewal. Two groups working independently determined that *Gfi1* controls self-renewal of HSCs by restraining their proliferative potential (Hock et al 2004; Zeng et al., 2004). They showed that *Gfi1*-deficient HSCs display increased proliferation rates and are also functionally compromised in competitive repopulation and serial transplantation assays. *Gfi1* might exert its effects on HSC proliferation by regulating the cell cycle inhibitor p21.

*Gfi1* is originally recognized for its role in T-cell differentiation and lymphoma. *Gfi1* gene knockout is one of the first targeted mutants to exhibit the combination of an increase in cycling HSCs at the expense of HSC function. Both *Gfi1* knockout models displayed an increase in cycling cells within the HSC pool, a large decrease in HSC function in transplantation experiments. Profoundly reduced expression of *p21*, the cyclin-dependent kinase inhibitor, in *Gfi1* null HSCs may account for the mechanism. Thus, under normal homeostasis, *Gfi1* is thought to suppress the proliferation of HSCs, thereby keeping HSCs in quiescence.

Numerous studies have identified roles for *p53* in the proliferation, differentiation, apoptosis, and aging of hematopoietic cells. LT-HSCs express high levels of *p53* transcripts, which is an indication of roles of *p53* in HSC physiology (Dumble et al., 2007). Recently, *p53*

The transcription factor *Tel* (Translocation Ets leukemia; also known as Etv6 [Ets variant gene 6]), an *Ets* (E-26 transforming-specific)-related transcriptional repressor, is also required for HSC maintenance. Conditional inactivation of *Tel/Etv6* in HSCs rapidly leads to the depletion of *Tel/Etv6*-deficient bone marrow. However, *Tel/Etv6* is not required for the maintenance of committed precursors. When it is conditionally inactivated in most hematopoietic lineages, it does not affect their differentiation or survival (Hock et al., 2004). At the moment, the mechanism by which *Tel/Etv6* modulates adult HSCs renewal is not known. Study of the downstream targets it represses should shed light on other players

*Pbx1* (pre–B-cell acute lymphoblastic leukemia) is a TALE class homeodomain transcription factor that critically regulates numerous embryonic processes, including hematopoiesis. *Pbx1* is preferentially expressed in LT-HSCs compared to more mature short-term HSCs and multipotent progenitor cells (Ficara et al., 2008). By using *Pbx1*-conditional knockout mice, it was revealed that *Pbx1* positively regulates HSC quiescence. Transcriptional profiling showed that a significant proportion of *Pbx1*-dependent genes are associated with the *TGF-β*

The homeobox (*Hox*) genes encode transcription factors that regulate embryonic body patterning and organogenesis. They play a role in the regulation of hematopoiesis. Overexpression of *HoxB4* in bone marrow leads to expansion of HSCs *in vivo* and *in vitro*, therefore appearing to be a positive regulator of HSC self-renewal (Antonchuck et al., 2002; Krosl et al., 2003; Miyake et al., 2006; Sauvageau et al., 1995). It therefore came as a surprise when *HoxB4*-deficient mice had normal hematopoietic development but exhibited only mild proliferative HSC defects (Brun et al., 2004). In an attempt to determine if this was due to compensatory mechanisms, the entire *HoxB* cluster was deleted. However, this did not lead to major defects in hematopoiesis (Bijl et al., 2006), possibly owing to compensation by

*Gfi1* (Growth factor independence 1), a zinc-finger repressor, has been recently implicated as a regulator of HSC self-renewal. Two groups working independently determined that *Gfi1* controls self-renewal of HSCs by restraining their proliferative potential (Hock et al 2004; Zeng et al., 2004). They showed that *Gfi1*-deficient HSCs display increased proliferation rates and are also functionally compromised in competitive repopulation and serial transplantation assays. *Gfi1* might exert its effects on HSC proliferation by regulating the

*Gfi1* is originally recognized for its role in T-cell differentiation and lymphoma. *Gfi1* gene knockout is one of the first targeted mutants to exhibit the combination of an increase in cycling HSCs at the expense of HSC function. Both *Gfi1* knockout models displayed an increase in cycling cells within the HSC pool, a large decrease in HSC function in transplantation experiments. Profoundly reduced expression of *p21*, the cyclin-dependent kinase inhibitor, in *Gfi1* null HSCs may account for the mechanism. Thus, under normal homeostasis, *Gfi1* is thought to suppress the proliferation of HSCs, thereby keeping HSCs in

Numerous studies have identified roles for *p53* in the proliferation, differentiation, apoptosis, and aging of hematopoietic cells. LT-HSCs express high levels of *p53* transcripts, which is an indication of roles of *p53* in HSC physiology (Dumble et al., 2007). Recently, *p53*

essential for HSC maintenance.

pathway.

*HoxA4* and/or *HoxC4*.

cell cycle inhibitor p21.

quiescence.

has been identified as a positive regulator of HSC quiescence through analysis of *p53*−/<sup>−</sup> mice (Liu et al., 2009). Furthermore, in the same study, it was demonstrated that the increased quiescence of HSCs from MEF null mice, in which both *p53* and *p21* are upregulated, is dependent on *p53*, but not *p21*, further confirming the positive role of *p53* in maintaining HSC in quiescence. *Gfi1* was identified as *p53* target gene, which is both shown important in regulating HSC quiescence by up-regulation or knockdown experiments.

Stem cell leukemia/T-cell acute lymphoblastic leukemia 1 (*SCL/TAL1*) plays a key role in controlling development of primitive and definitive hematopoiesis during mouse development. In adult HSCs, it is highly expressed in LT-HSCs compared with short-term HSCs and progenitors (Lacombe et al., 2010). SCL impedes G0-G1 transition in HSCs. The function of HSCs from *Scl* +/<sup>−</sup> mice or with decreased dosage of SCL protein by *in vitro* interference was shown decreased in various transplantation assays. At the molecular level, SCL maintains HSC quiescence by regulating gene expression of *Cdkn1a* and *Id1*.

Recently, many other transcriptional factors, such as interferon regulatory factor−2, a transcriptional suppressor of interferon signaling (Sato et al., 2009); *Nurr1*, a nuclear receptor transcription factor (Sirin et al., 2010); and thioredoxin-interacting protein, a transcriptional repressor (Shao et al., 2010), have been identified as positive regulators of HSC quiescence. Loss of HSC quiescence was observed in mice with deletion of each of these factors.

Individual member of Retinoblastoma (*Rb*) tumor suppressor gene family serves critical roles in the control of cellular proliferation and differentiation with functional redundancy for each other. The mice with conditional triple knockout of *Rb* family genes including *Rb*, *p107*, and *p130* display a cell-intrinsic myeloproliferation that originates from hyperproliferative early hematopoietic progenitors due to the loss of quiescence, and the mutant HSCs show strong short-term repopulation capacity but impaired long-term repopulation ability on transplantation. Thus, *Rb* family members collectively maintain HSC quiescence (Viatour et al., 2008).

It has been shown that the conditional inactivation of *c-Myc* induces excessive expression of *integrins* and *N-cadherin* in HSCs, leading to the enhanced HSC interaction with the niche, which subsequently enable *Myc*-deficient HSCs stay in quiescence. Conversely, enforced *c-Myc* expression in HSCs downregulates *N-cadherin* and *integrins*, leading to a loss of HSC function (Wilson et al., 2004).

*p21* mRNA expression levels are dramatically lower in the *Gfi1*-deficient HSCs. *p21* itself has been implicated in the regulation of HSCs (Cheng et al., 2000). In its absence, HSCs have an impaired serial transplantation capacity. Another cell cycle inhibitor, *p18* , has also been shown to affect HSC self-renewal. The absence of *p18* leads to increased HSC self-renewal (Yuan et al., 2004; Yu et al., 2006). Therefore, intricate control of the cell cycle and proliferation machinery is required for self-renewal regulation.

In contrast to *p21*, little is known about the role of *p57* in adult stem cell populations. Using primary human hematopoietic cells and microarray analysis, Scandura et al. (2004) identified *p57* as the only cyclin-dependent kinase inhibitor induced by TGF-β. Upregulation of *p57* is essential for TGF-β−induced cell-cycle arrest in these cells, which may represent the mechanisms by which TGF-β affects cell-cycle arrest and stem cell quiescence.

Networks Establishing Hematopoietic Stem Cell Multipotency and Self-Renewal 17

expression of the oncogene *Bcl2* resulted in increased numbers of transgenic HSCs *in vivo* and gave these cells a competitive edge over wild type HSCs in competitive reconstitution experiments (Domen et al., 1998; Domen et al., 2000) suggesting that cell death plays a role in regulating the homeostasis of HSCs. Recently, *Mcl1* (Myeloid cell leukemia 1), another anti-apoptotic *Bcl2* family member, has been shown to be required for HSC survival

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

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

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

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

competitive repopulating capacity, eventually causing hematological failure.

the pathophysiological origins of many related disorders.

it depends on a complex network of signals.

(Opferman et al., 2005).

**6. Quiescence or self-renewal** 

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 *ATM*−/− HSCs.

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

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' quiescent status.

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 malignancies that harbor constitutively activated Stats.

Studies using transgenic mice constitutively expressing *BCL2* (*B-cell lymphoma 2*) in all hematopoietic tissues provide evidence directly supporting this theory. The forced expression of the oncogene *Bcl2* resulted in increased numbers of transgenic HSCs *in vivo* and gave these cells a competitive edge over wild type HSCs in competitive reconstitution experiments (Domen et al., 1998; Domen et al., 2000) suggesting that cell death plays a role in regulating the homeostasis of HSCs. Recently, *Mcl1* (Myeloid cell leukemia 1), another anti-apoptotic *Bcl2* family member, has been shown to be required for HSC survival (Opferman et al., 2005).
