2 Regulation of Hematopoietic Stem Cell Fate : Self-Renewal , Quiescence and Survival

Hematopoietic stem cells (HSCs) are probably the most extensively characterized somatic stem cells and are the only stem cells that have been clinically used to treat diseases such as leukemia, germ cell tumors, and congenital immunodeficiencies. Because of their capacity for self-renewal and their ability to differentiate into different lineages, HSCs are able to continually replenish the cells that make up the hematopoietic system (Kondo et al., 2003). Decades of intensive study using multicolor cell sorting techniques have allowed investigators to identify these cells within a small population in the mouse bone marrow (BM) (i.e., CD34low/-, Kit+ Sca-1+ lineage marker-negative cells: CD34low/KSL) and thereby allow the prospective isolation of nearly-homogenous HSC populations for further characterization (Osawa et al., 1996).


Introduction
Hematopoietic stem cells (HSCs) are probably the most extensively characterized somatic stem cells and are the only stem cells that have been clinically used to treat diseases such as leukemia, germ cell tumors, and congenital immunodeficiencies. Because of their capacity for self-renewal and their ability to differentiate into different lineages, HSCs are able to continually replenish the cells that make up the hematopoietic system (Kondo et al., 2003). Decades of intensive study using multicolor cell sorting techniques have allowed investigators to identify these cells within a small population in the mouse bone marrow (BM) (i.e., CD34 low/-, Kit + Sca-1 + lineage marker-negative cells: CD34 low/-KSL) and thereby allow the prospective isolation of nearly-homogenous HSC populations for further characterization (Osawa et al., 1996).
Under steady-state conditions, the majority of HSCs are maintained in a quiescent state in which they divide infrequently to produce proliferative progenitors that eventually give rise to the mature hematopoietic cells that sustain blood homeostasis (Cheshier et al., 1999). However, in response to external stresses such as bleeding, myeloablative chemotherapy and total body irradiation, HSCs proliferate extensively to produce very high numbers of primitive progenitor cells, thereby enabling rapid hematological regeneration (Randall et al., 1997). Once recovery from myelosuppression has been achieved, the activated HSCs return to a quiescent state via a number of negative feedback mechanisms (Venezia et al., 2004). The cell fate decisions (including life and death, self-renewal and differentiation) of HSCs are important processes that regulate the number and lifespan of the HSC pool within a host. Defects in these processes may contribute to hematopoietic failures and to the development of hematologic malignancies. 40 because they lack physiological relevance and do not identify phenomena such as genetic redundancy. For example, family genes or alternative pathways can compensate functionally for deleted genes in gene-ablated mouse models in a manner that masks the true physiology. One approach to identifying the individual components involved in the molecular pathways underlying HSC regulation is to define the molecular signature of the HSCs by comparative transcriptional profiling of distinct subsets of hematopoietic cells. Over the past decade, several attempts have been made by independent investigators, including ourselves, to define the molecular signature of HSCs (Park et al., 2002;Ramalho-Santos et al., 2002;Ivanova et al., 2002;Akashi et al., 2003;Venezia et al., 2004;Zhong et al., 2005;Forsberg et al., 2005;Ramos et al., 2006;Chambers et al., 2007;Kubota et al., 2009). A list of gene expression profiling studies using purified mouse HSCs performed to date is shown in Table 1. Although this information has, more or less, clarified the molecular makeup of HSCs and several critical factors have been identified based on the data reported in these studies, it is still extremely time-consuming to elucidate the physiological function of each individual gene involved in HSC regulation. The transcriptional regulation of stem cell fate, particularly by factors that have specific functions in HSCs, is only beginning to be understood.
In this chapter, we briefly review the recent advances in our knowledge of cell-intrinsic regulators of HSC self-renewal, differentiation, quiescence, cycling, and survival.

Regulation of HSC self-renewal and quiescence
The outstanding feature of adult stem cells is their relative quiescence (Orford et al., 2008;Wilson et al., 2008). Quiescence is critical for the maintenance and self-renewal of HSCs. Unscheduled HSC proliferation results in the loss of self-renewal or stem cell exhaustion (Orford et al., 2008;Wilson et al., 2009;Trumpp et al., 2010). Identification of the molecules 42 role was suggested by the observation that Pbx1 is preferentially expressed in long-term repopulating HSCs (LT-HSCs) compared with more mature progenitor cells (Forsberg et al., 2005), its functional analysis in adult HSCs has been hampered because Pbx1 mutant mice are embryonic lethal. Therefore, Pbx1-conditional knockout (KO) mice have been used to study the role of Pbx1 in the adult mouse hematopoietic system (Ficara et al., 2008). Conditional inactivation of Pbx1 in hematopoietic cells results in the loss of HSCs, which is associated with decreased quiescence. This leads to a defect in the maintenance of selfrenewal in serial transplantation assays. Global gene expression profiling analyses show that a significant proportion (~8%) of the downregulated genes in Pbx1-deficient HSCs belong to the TGF-signaling pathway, which has been implicated in maintaining HSC quiescence (Yamazaki et al., 2009). Also, in contrast to WT LT-HSCs, Pbx1-mutant LT-HSCs do not upregulate the expression of several downstream transcripts in response to TGFstimulation in vitro. These results suggest that Pbx1 regulates HSC self-renewal and quiescence, at least in part by affecting the response to TGF-.

Evi-1
The ecotropic viral integration site-1 (Evi-1) was first identified in murine model systems as the integration site for the ecotropic retrovirus that causes myeloid leukemia (Morishita et al., 1988;Mucenski et al., 1988). Several studies using gene-targeting mice show that Evi-1 is required for HSC regulation. Yuasa et al. showed that Evi-1 is preferentially expressed in HSCs in embryos and adult BM. Evi-1-deficient embryonic HSCs are severely decreased in number, and show defective repopulating capacity. In addition, the expression of GATA-2 mRNA is markedly reduced in HSCs from Evi-1-null embryos. GATA-2 promoter analysis revealed that Evi-1 directly binds to the GATA-2 promoter and acts as an enhancer (Yuasa et al., 2005). Another study using conditional Evi-1 knockout mice showed that Evi-1 also regulates adult HSC proliferation in a dose-dependent manner. Evi-1-deficient BM HSCs did not maintain definitive hematopoiesis and lost their ability to reconstitute the cell population. Mutant mice heterozygous for Evi-1 exhibited an intermediate phenotype in terms of HSC activity (Goyama et al., 2008). Furthermore, gene expression profiling of Evi-1-deleted HSCs and leukemic cells identified Pbx1 as a downstream target for Evi-1 in HSCs (Shimabe et al., 2009).

JunB
The AP-1 transcription factor, JunB, is a transcriptional regulator of myelopoiesis and a potential tumor suppressor gene in mice (Passegue et al., 2001). Compared with normal HSCs, JunB-deficient LT-HSCs showed an average 2-fold increase in the percentage of cycling cells, suggesting that JunB functions to limit cell-cycle entry. Gene expression analyses revealed that JunB-deficient LT-HSCs show increased expression of cyclins and decreased expression of cyclin-dependent kinase inhibitors (Santaguida et al., 2009). These results suggest that the absence of JunB induces quiescent cells to enter the cell cycle.

p53
The p53 tumor suppressor protein functions as a transcription factor, regulating the transcription of genes that induce cell-cycle arrest, senescence, and apoptosis. LT-HSCs express high levels of p53 (Dumble et al., 2007). Although p53-deficient mice show almost normal hematopoiesis (Lotem & Suchs., 1993), a number of studies have identified a role for p53 in the proliferation, differentiation, apoptosis, and aging of HSCs (Kastan et al., 1991;Shounan et al., 1996;Park et al., 2003;Dumble et al., 2007). Recent detailed analyses of p53null mice have unraveled other important functions of p53 in HSCs. Liu et al. found that p53 promotes HSC quiescence, and that p53-deficient HSCs enter the cell cycle more easily (Liu et al., 2009). Competitive BM repopulation assays revealed that p53-null cells out-compete wild-type cells (TeKippe et al., 2003;Chen et al., 2008;Liu et al., 2009), indicating that p53 is a negative regulator of HSC self-renewal. In addition, Liu et al. also identified Gfi-1 and necdin as p53 target genes by performing comparative transcriptional profiling of HSCs isolated from wild-type and p53-deficient mice. The results of in vitro overexpression and knockdown experiments identified a role for necdin in the maintenance of HSC quiescence and self-renewal. However, necdin appears to have a modest functional role in HSCs in vivo (Kubota et al., 2009), and necdin overexpression does not result in enhanced HSC quiescence (Sirin et al., 2010).

Nurr1
Gene expression profiling analyses identified Nurr1 (also known as Nr4a2), an orphan nuclear receptor, as a candidate molecule that may play a functional role in HSC quiescence (Venezia et al., 2004;Chambers et al., 2007). Overexpression of Nurr1 resulted in HSC quiescence. On the other hand, loss of one Nurr1 allele resulted in enhanced cycling and sensitivity to the chemotherapeutic agent 5-fluorouracil (5-FU). Molecular analysis showed that Nurr1 overexpression is positively correlated with the upregulation of the cell-cycle inhibitor p18 INK4C , suggesting a mechanism by which Nurr1 may regulate HSC quiescence (Sirin et al., 2010).

Reactive oxygen species, FoxOs
Reactive oxygen species (ROS) play an important role in the regulation of HSC quiescence. The forkhead O (FoxO) family of transcription factors (FoxO1, FoxO3, FoxO4, and FoxO6) participates in various cellular processes, including the induction of cell-cycle arrest, stress resistance, apoptosis, differentiation, and metabolism (Greer & Brunet., 2005). Two groups reported that FoxOs play a regulatory role in a number of physiologic processes that influence HSC numbers and function. Both aged germline FoxO3-deficient mice and conditional triple knockout (FoxO1, 3, 4) mice show a reduction in HSC numbers with a deficient repopulating capacity in competitive reconstitution assays and serial competitive transplantation assays (Tothova et al., 2007;Miyamoto et al., 2007). These phenotypes correlate with increased cell-cycling and apoptosis of HSCs, caused by increased levels of ROS. Furthermore, treatment with the antioxidant, N-acetyl-L-cysteine (NAC), rescues the FoxO-deficient HSC phenotype.

Fbxw7
Fbxw7 is the F-box protein subunit of an SCF-type ubiquitin ligase complex that targets positive regulators of the cell-cycle, including Notch, c-Myc, cyclin E, and c-Jun. Two independent groups investigated the functions of Fbxw7 in HSCs using conditional Fbxw7 knockout mice (Matsuoka et al., 2008;Thompson et al., 2008). Conditional ablation of Fbxw7 rapidly and severely affects hematopoietic progenitor maintenance within the BM. Fbxw7 -/-HSCs show increased cycling and defective long-term repopulation capacity in competitive transplantation assays. As Fbxw7 is able to ubiquitinate several target proteins, studies were conducted to examine the protein expression of Notch1, c-Myc, and cyclin E. The results showed that c-Myc protein was substantially overexpressed in Fbxw7 -/-HSCs, suggesting that the activation of the cell-cycle in Fbxw7-null HSCs induced by excess c-Myc causes the premature exhaustion of HSCs.

HIF-1α
Leukemic stem cells (LSCs) reside in the niches near epiphysis of the bone (Ishikawa et al., 2007) and oxygen concentration of this area is quite low. Thus, it may be very important for leukemic cells, especially for LSCs to survive and adapt to hypoxia (Takeuchi et al., 2010). Cellular responses to hypoxia are mediated by hypoxia-inducible factors (HIFs), which regulate gene expression to facilitate adaptation to hypoxic conditions (Kaelin & Ratcliffe., 2008). Hypoxia inducible factor-1 (HIF-1 ) is stabilized under low-oxygen conditions, such as those present in the BM. Recently, two groups investigated the importance of hypoxia and its related signaling pathways in HSC function using different approaches (Simsek et al., 2010;Takubo et al., 2010). HIF-1 levels are elevated in adult HSCs and its transcription is regulated by the homeodomain protein Meis1, which is essential for hematopoiesis (Hisa et al., 2004;Simsek et al., 2010). HIF-1 conditional knockout mice show that HIF-1deficient HSCs have an increased cell cycling rate and show progressive loss of long-term repopulation ability in serial transplantation assays (Takubo et al., 2010). Taken together, these data indicate that the precise regulation of HIF-1 levels is required to maintain HSC quiescence.

Lkb1
The control of energy metabolism within HSCs is poorly understood, although they are highly sensitive to oxidative stress. Recently, several groups examined the role of the protein, Lkb1, in the metabolic regulation of HSCs (Nakada et al., 2010;Gurumurthy et al., 2010;Gan et al., 2010). Lkb1 is a kinase enzyme that regulates the activity of AMPactivated protein kinase (AMPK). Conditional inactivation of Lkb1 (Mx1-Cre; LKB1 fl/fl or RosaCreERT2; LKB1 L/L ) in adult mice causes the loss of HSC quiescence, rapid HSC depletion, and pancytopenia. Interestingly, Lkb1 seems to regulate HSC homeostasis primarily through pathways that are independent of its downstream effectors, AMPK and mTORC1.

Cyclin-dependent kinase inhibitors
p21 cip1/waf1 (hereafter referred to as p21) is a mammalian member of the CIP/KIP family and was the first cyclin-dependent kinase inhibitor to be identified (Serrano et al., 1993;Harper et al., 1993;Stier et al., 2003). Serial transplantation assays using p21-deficient cells showed premature HSC exhaustion; also, p21-null mice were more sensitive to 5-FU (Cheng et al., 2000). These results suggest that p21 restricts HSC entry into the cell cycle and regulates the size of the HSC pool under conditions of stress. However, a later study demonstrated that p21 plays a minor role in regulating HSC quiescence under conditions of steady-state hematopoiesis (van Os et al., 2007).
Although p57 kip2 (hereafter referred to as p57) is highly expressed in HSCs (Table 2) (Kubota et al., 2009;Umemoto et al., 2005), little is known about its functional role. Microarray www.intechopen.com Regulation of Hematopoietic Stem Cell Fate: Self-Renewal, Quiescence and Survival 45 analysis studies of human CD34 + HSC/progenitor cells identified p57 as the only cyclindependent kinase inhibitor induced by TGF (Scandura et al., 2004). Knockdown of p57 in hematopoietic cell lines using small interfering RNA (siRNA) results in more rapid proliferation of hematopoietic cells in the absence of TGF-. These results suggest that p57 is required for the TGF--mediated cell cycle entry of hematopoietic cells and for repressing the proliferation of these cells. Table 2. Genes expressed at higher levels in HSCs than in other subsets.
Genes showing at least 2-fold higher expression in CD34 -/low KSL cells than in CD34 + KSL cells were selected by microarray analysis. The selected genes were then evaluated by Q-PCR, and genes whose transcripts were expressed at ≥ 2-foltd higher levels in CD34 -/low KSL cells than all other samples are listed.

E3 ubiquitin ligase
The E3 ubiquitin ligase, c-Cbl, is a member of the RING finger-type ubiquitin ligase Cbl (casitas B-cell lymphoma) family. The c-Cbl protein is thought to implement the degradation of various cellular proteins, receptors, and signaling molecules including Notch1, STAT5, and c-Kit (Jehn et al., 2002;Goh et al., 2002;Zeng et al., 2005). c-Cbl-deficient mice were used to study the role of c-Cbl in HSCs (Rathinam et al., 2008). The number of HSCs and progenitors was significantly higher in the BM of c-Cbl-null mice due to increased proliferation. Interestingly, detailed analyses revealed augmented STAT5 phosphorylation in c-Cbl -/-HSCs in response to TPO/c-MPL signaling which is crucial for the proliferation and self-renewal of HSCs (Kimura et al., 1998), and this led to enhanced c-Myc expression. C-Cbl-deficient HSCs also showed an increased repopulating ability in competitive reconstitution assays, including serial transplantation. These results suggest that c-Cbl acts as a negative regulator of both the size of the HSC pool and self-renewal (Rathinam et al., 2008).
Recently, Itch, another E3 ligase belonging to the HECT family (Bernassola et al., 2008), was also identified as a negative regulator of HSC homeostasis and function. The phenotype of Itch -/-HSCs was similar to that of c-Cbl -/-HSCs. However, unlike c-Cbl, Itch-deficient HSCs showed augmented Notch1 signaling. Furthermore, knockdown of Notch1 in Itch-null HSCs resulted in the reversion of the phenotype (Rathinam et al., 2011). Taken together, these studies underscore the pivotal roles of E3 ubiquitin ligases and the importance of posttranslational modification of HSCs in the molecular control of HSC self-renewal.

Egr1
Egr1 is a member of the immediate early response gene family (Gashler et al., 1995). Egr1 is highly expressed in LT-HSCs under steady-state conditions and is downregulated upon proliferative stimulation and migration in response to pharmacological mobilization (Min et al., 2008). Egr1-deficient mice show a significant increase in the frequency of cycling HSCs. This phenomenon results in a slightly higher frequency of HSCs in the BM of Egr1 -/-mice. Interestingly, loss of Egr1 results in a striking increase (up to 10-fold) in the number of circulating HSCs. Importantly, HSCs isolated from both the BM and peripheral blood of Egr1 -/-mice show a greater degree of long-term multi-lineage repopulation after transplantation, although their life span is slightly reduced. Quantitative RT-PCR analysis shows that Bmi1 is upregulated in Egr1 -/-HSCs. In addition, Egr1 -/-HSCs also show the downregulation of p21 CIP1/WAF1 and increased expression of cyclin-dependent kinase 4 (cdk4), which is consistent with their increased cell-cycling status (Min et al., 2008). Taken together, the deletion of Egr1 causes an increase in the number of cycling HSCs but does not lead to stem cell exhaustion. This may be due to Bmi1 upregulation.

Lnk
Lnk is a member of an adaptor protein family that possesses a number of protein-protein interaction domains: a proline-rich amino-terminus, a pleckstrin homology (PH) domain, a Src homology 2 (SH2) domain, and many potential tyrosine phosphorylation motifs (Rudd., 2001). Studies using Lnk-deficient mice show that Lnk-null HSCs are expanded during postnatal development (Ema et al., 2005;Buza-Vidas et al., 2006). The Lnk -/-HSC population contains an increased proportion of quiescent cells and shows decelerated cell cycle kinetics and enhanced resistance to repeat treatment with 5-FU in vivo compared with wild-type HSCs. Genetic evidence demonstrates that Lnk controls HSC self-renewal and quiescence, predominantly through c-Mpl. Furthermore, Lnk-deficient HSCs show higher levels of symmetric proliferation in response to thrombopoietin (TPO) in ex vivo culture than wildtype HSCs (Seita et al., 2007). Biochemical analyses revealed that Lnk directly binds to phosphorylated tyrosine residues in JAK2 after TPO stimulation (Bersenev et al., 2008). Therefore, Lnk is a physiologic negative regulator of JAK2 in HSCs, and TPO/c-Mpl/JAK2/Lnk constitute a major regulatory pathway controlling HSC quiescence and selfrenewal.

Myc
Human c-MYC was the second proto-oncogene to be identified and encodes a basic helixloop-helix leucine zipper transcription factor (c-Myc) (Sheiness et al., 1978). Overexpression of one of the three family members has been detected in numerous human cancers including Burkitt's lymphoma (c-MYC), neuroblastoma (N-MYC), and small cell lung cancer (L-MYC) (Nesbit et al., 1999). Conditional deletion of c-Myc in the BM results in cytopenia and the accumulation of functionally defective HSCs. In the absence of c-Myc, HSC differentiation into more committed progenitors is inhibited because they upregulate a number of adhesion molecules, such as N-cadherin, that anchor them in the niche. Conversely, enforced c-Myc expression in HSCs causes marked repression of N-cadherin and integrin expression leading to the loss of self-renewal ability at the expense of differentiation (Wilson et al., 2004). These results suggest that c-Myc activity controls the first differentiation step of LT-HSCs in vivo. Unexpectedly, conditional ablation of both c-myc and N-myc results in pancytopenia and rapid lethality due to HSC apoptosis via the accumulation of the cytotoxic molecule, Granzyme B . Thus, Myc activity controls important aspects of HSC function such as proliferation, survival and differentiation.

MEF/ELF4
MEF (also known as ELF4), an Ets transcription factor, was identified as a novel component of the transcriptional circuit that dynamically regulates HSC quiescence (Lacorazza et al., 2006). Mef-deficient HSCs grow more slowly than wild-type HSCs in response to cytokine stimulation Pyronin Y staining and BrdU incorporation show increased quiescence. Enhanced HSC quiescence in Mef-null mice also increases HSC resistance to cytotoxic agents that target dividing cells and allows more rapid hematological recovery after chemotherapy or irradiation. These findings suggest that Mef normally functions to induce or facilitate the entry of quiescent HSCs into the cell cycle and imply that Mef expression and/or activity may be dynamically regulated in HSCs. To explain this, Lacorazza et al. proposed a model in which Mef acts at an earlier stage than p18 and antagonizes p21.

Survival of HSCs
HSC self-renewal and apoptosis represent major factors that determine the size of the HSC mass. The number of HSCs is also controlled by their capacity to survive during homeostasis or under conditions of stress.

Bcl-2 family
Accumulating evidence suggests that the suppression of apoptosis is required for HSC survival. Forced expression of Bcl-2 increases the number of HSCs and provides them with enhanced competitive repopulation ability (Domen et al., 1998(Domen et al., , 2000, suggesting that cell death plays a role in regulating HSC homeostasis. Mcl-1, another anti-apoptotic Bcl-2 family member, is also an essential regulator of HSC survival. Mcl-1 is highly expressed in LT-HSCs, and conditional deletion of MCl-1 results in the loss of the early BM progenitor population, including HSCs, leading to fatal hematopoietic failure (Opferman et al., 2005). Recently, it was reported that Mcl-1 is an indispensable regulator of self-renewal in human stem cells and that functional dependence on Mcl-1 defines the human stem cell hierarchy (Campbell et al., 2010).

Scl, Lyl1
Scl/Tal1 is a basic helix-loop-helix (bHLH) transcription factor that is essential for the development of HSCs in the embryo (Robb et al., 1995;Shivdasani et al., 1995). During adult hematopoiesis, Scl/Tal1 is highly expressed in LT-HSCs compared with short-term HSCs and progenitor cells (Lacombe et al., 2010). However, a study using conditional Scl/Tal1 knockout mice revealed that Scl/Tal1 is required for the generation of, but not the maintenance of, adult HSCs (Mikkola et al., 2003). Another group showed that conditional deletion of Scl/Tal1 in adult HSCs has a relatively mild effect: Scl-null HSCs show impaired short-term repopulating ability, but no defect in long-term repopulating capacity (Curtis et al., 2004). Redundant activity caused by the expression of Lyl1, a related bHLH transcription factor, in adult HSCs may provide an explanation for these "mild" phenotypes. While adult HSCs in single-knockout mice show no or only a mild phenotype, Lyl1;Scl-conditional double-knockout mice show a gene dosage defect on HSC survival, as HSCs and progenitor cells are immediately lost due to apoptosis (Souroullas et al., 2009).
Recently, Lacombe et al. demonstrated that Scl/Tal1 is required for the maintenance of the quiescent stem cell pool (Lacombe et al., 2010). Cell-cycle analyses revealed that Scl/Tal1 negatively regulates the G0-G1 transit of LT-HSCs; however, these phenomena were specific to adult HSCs and were not observed in perinatal HSCs. The reconstituting ability of Scl +/-HSCs or HSCs with decreased Scl protein expression induced by RNA interference was impaired in various transplantation assays. Furthermore, gene expression analysis and chromatin immunoprecipitation experiments revealed that the Cdkn1a and Id1 genes are direct SCL targets.

Tel/Etv6
The transcription factor Tel (also known as Etv6), an Ets-related transcriptional repressor, is a frequent target of the diverse chromosomal translocations observed in leukemias (Golub et al., 1994). Tel/ETV6 is also required for HSC survival in adult hematopoiesis. Following conditional inactivation of Tel/Etv6, HSCs are rapidly lost from the adult BM. However, Tel/Etv6 is not required for the maintenance of lineage-committed progenitors. Conditional deletion of Tel/Etv6 after lineage commitment does not affect the differentiation or survival of these progenitors, although it does impair the maturation of megakaryocytes .

Zfx
Zfx is a zinc finger protein belonging to the Zfx/ZFy family. Mammalian Zfx is encoded on the X chromosome and contains an acidic transcriptional activation domain, a nuclear localization sequence, and a DNA binding protein domain consisting of 13 C2H2-type zinc fingers (Schneider-Gadicke et al., 1989). Zfx is highly expressed in both HSCs and undifferentiated embryonic stem cells (ESCs). Using conditional gene targeting, Zfx was identified as an essential transcriptional regulator of HSC function (Galan-Caridad et al., 2007). Constitutive or inducible deletion of Zfx in HSCs (using Tie2-Cre and Mx1-Cre deletion strains, respectively) impairs self-renewal, resulting in increased apoptosis and the upregulation of stress-inducible genes.

ADAR1
ADAR (adenosine deaminase acting on RNA) catalyzes the deamination of adenosine to inosine in double-stranded RNA. Conventional Adar -/-mice die around embryonic day 11.5-12 because of widespread apoptosis and defective hematopoiesis (Hartner et al., 2004;Wang et al., 2004). Conditional deletion of Adar in HSCs shows that ADAR1 is essential for the maintenance of both fetal and adult HSCs, and leads to global upregulation of type I and II interferon-inducible transcripts and rapid apoptosis (Hartner et al., 2009). Interferon regulatory factor-2 (Irf2), a transcriptional suppressor of type I interferon signaling, is a positive regulator of HSC quiescence (Sato et al., 2009). Irf2-deficient HSCs are unable to restore hematopoiesis in irradiated mice, but the reconstituting capacity of Irf2 -/-HSCs can be restored in these cells by disabling type I IFN signaling.

Response to hematopoietic emergency
Various external stresses, such as myelosuppressive chemotherapy, bleeding, infection, and total body irradiation, put HSCs under stress, as they must proliferate to produce large numbers of primitive progenitor cells, thereby enabling rapid hematologic regeneration. Although this property has long been recognized, the molecular basis underlying the reaction of HSCs to hematologic emergency remains enigmatic. However, some key players have been identified.

Heme oxygenase-1
Heme promotes the proliferation and differentiation of hematopoietic progenitor cells (HPCs) (Chertkov et al., 1991) and stimulates hematopoiesis (Porter et al., 1979;Abraham, 1991). The degradation of heme is catalyzed by heme oxygenase (HO). HO-1, encoded by the Hmox1 gene, is the stress-inducible isozyme of HO and is highly expressed in the spleen and BM (Abraham, 1991). Heterozygous HO-1-deficient mice (HO-1 +/-) show accelerated hematologic recovery from myelotoxic injury induced by 5-FU treatment, and mice transplanted with HO-1 +/-BM cells show more rapid hematopoietic repopulation than those transplanted with Ho-1 +/+ BM cells. However, HO-1 +/-HSCs show a reduced capacity to rescue lethally irradiated mice and to serially repopulate irradiated recipients (Cao et al., 2008). These results suggest that HO-1 limits the proliferation and differentiation of HPCs under stressful conditions, and that the failure of this mechanism can lead to the premature exhaustion of the HSC pool.

Necdin
Necdin is a member of the melanoma antigen family of molecules, whose physiological roles have not been well characterized (Xiao et al., 2004). Necdin acts as a cell cycle regulator in post-mitotic neurons (Yoshikawa, 2000). Intriguingly, recent genetic analyses show that aberrant genomic imprinting of NDN on the human 15q11-q13 chromosomal region is, at least in part, responsible for the pathogenesis of Prader-Willi syndrome (MacDonald & Wevrick, 1997;Nakada et al., 1998;Barker et al., 2002), a disorder associated with a mildly increased risk of myeloid leukemia (Davies et al., 2003). Necdin interacts with multiple cell-cycle related proteins, such as SV-40 large T antigen, adenovirus E1A, E2F1, and p53 (Taniura et al., , 1999(Taniura et al., , 2005Hu et al., 2003). As shown in Table 2, necdin is one of 32 genes that show higher expression in HSCs than in differentiated hematopoietic cells (Kubota et al., 2009). Other groups also found that necdin is highly expressed in HSCs (Forsberg et al., 2005;Liu et al., 2009). Necdindeficient mice show accelerated recovery of hematopoietic systems after myelosuppressive stress, such as 5-FU treatment and BM transplantation, whereas no overt abnormality is seen under conditions of steady-state hematopoiesis. Considering necdin as a potential negative cell-cycle regulator, it was reasoned that the enhanced hematologic recovery in necdin-null mice could be the result of an increased number of proliferating HSCs and progenitor cells. As expected, after 5-FU treatment, necdindeficient mice had an increased number of HSCs, but this was only transiently observed during the recovery phase (Kubota et al., 2009). These data suggest that the repression of necdin function in HSCs may present a novel strategy for accelerating hematopoietic recovery, thus providing therapeutic benefits after clinical myelosuppressive treatments (e.g., cytoablative chemotherapy or HSC transplantation).

Slug
Slug belongs to the highly conserved Slug/Snail family of zinc-finger transcriptional repressors found in diverse species ranging from C. elegans to humans. SLUG is a target gene for the E2A-HLF chimeric oncoprotein in pro-B cell acute leukemia (Inukai et al., 1999). Slug-deficient mice show normal peripheral blood counts, but they are very sensitive toirradiation (Inoue et al., 2002). Slug is induced by p53 and protects primitive hematopoietic cells from apoptosis triggered by DNA damage. Slug exerts this function by repressing Puma, a proapoptotic target of p53 (Wu et al., 2005). Sun et al. recently showed that Slug negatively regulates the repopulating ability of HSCs under conditions of stress. Slug deficiency increases HSC proliferation and reconstitution potential in vivo after myelosuppressive treatment, and accelerates HSC expansion during in vitro culture (Sun et al., 2010).

Cancer stem cells
Accumulating evidence strongly suggests that tumors are organized into cellular hierarchies initiated and maintained by a small pool of self-renewing cancer stem cells (CSCs) (Dick, 2008;Reya et al., 2001). CSCs are thought to be resistant to various cancer treatments because of their relative quiescence (Komarova & Wodarz., 2007). Cancer relapses may occur because the dormancy of CSCs protects them from elimination by various cancer therapies (Dick, 2008). In an acute myelogenous leukemia (AML) xenograft model, AML leukemic stem cells (LSCs) localized in the endosteal region of the BM show cellular quiescence and resistance to chemotherapy (Ishikawa et al., 2007;Saito et al., 2010). In patients with chronic myelogenous leukemia (CML), CD34 + progenitor cells contain dormant cells that are resistant to BCR/ABL tyrosine kinase inhibitors (Bhatia et al., 2003).
It is well documented that regulators of HSC maintenance are also involved in the development of leukemias (Rizo et al., 2006). A number of cancer-related proteins, such as Bmi1, c-Myc, p53, Gfi-1, and PTEN, are key participants in HSC regulation, demonstrating the close relationship between normal HSCs and CSCs. Therefore, further understanding the mechanisms regulating HSC fate is needed if we are to develop new strategies for targeting CSCs and successfully treat cancer.

Conclusions
In this review, we have briefly summarized a number of critical regulators involved in the control of HSC self-renewal, quiescence, survival, and responses to external insults. Recent evidence strongly suggests that the BM niche also plays an integral role by providing critical signals that maintain HSCs in a stat of hibernation, thus preventing them from exhausting themselves. However, HSCs are critical for the maintenance and regeneration of an organism after injury/illness. This process must be tightly regulated and coordinated. Intensive studies have uncovered the molecular signatures and key molecules regulating HSC behavior. Moreover, new systems approaches, such as microRNA expression profiling and protein expression profiling, are expected to provide further useful information about HSC biology in the future. However, the overall picture of the molecular mechanisms that govern HSC fate is still unclear. Further understanding of the systems that regulate HSCs will enable the manipulation of stem cells for use in tissue engineering and cell-based therapies.

Acknowledgments
This work was supported by a Grant-in-Aid for Young Scientists to Y.K. (no. 23791083) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.