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

Yasushi Kubota1,2 and Shinya Kimura1

*1Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University 2Department of Transfusion Medicine, Saga University Hospital Japan* 

#### **1. Introduction**

38 Advances in Hematopoietic Stem Cell Research

Zhang, C.C. & Lodish, H.F.(2004). Insulin-like growth factor 2 expressed in a novel fetal

Zhang, J.; Niu, C.; Ye, L.; Huang, H.; He, X.; Tong, W.G.; Ross, J.; Haug, J.; Johnson, T.;

Zhang, P.; Behre, G.; Pan, J.; Iwama, A.; Wara-Aswapati, N.; Radomska, H.S.; Auron, P.E.;

GATA proteins repress PU.1. *Proc Natl Acad Sci U S A*. Jul 20;96(15):8705-10. Zhang, P.; Iwasaki-Arai, J.; Iwasaki, H.; Fenyus, M.L.; Dayaram, T.; Owens, B.M.;

Zon, L.I.(2008). Self-renewal and differentiation at Cell Stem Cell. *Cell Stem Cell*. Jun

1;103(7):2513-21.

23;425(6960):836-41.

Dec;21(6):853-63.

5;2(6):510.

liver cell population is a growth factor for hematopoietic stem cells. *Blood*. Apr

Feng,J.Q.; Harris, S.; Wiedemann, L.M.; Mishina, Y. & Li, L.(2003). Identification of the haematopoietic stem cell niche and control of the niche size. *Nature*. Oct

Tenen, D.G. & Sun, Z.(1999) Negative cross-talk between hematopoietic regulators:

Shigematsu, H.; Levantini, E.; Huettner, C.S.; Lekstrom-Himes, J.A.; Akashi, K. & Tenen, D.G.(2004). Enhancement of hematopoietic stem cell repopulating capacity and self-renewal in the absence of the transcription factor C/EBP alpha. *Immunity*.

> 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).

> 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.

> Understanding the molecular mechanisms underlying HSC regulation is of great importance to basic stem cell biology and for the development of HSCs for use in various clinical applications. Information regarding the regulation of HSC fate has been gained using conventional experimental approaches such as gene deletion, gene overexpression, and the direct stimulation of HSCs with cytokines. Although many studies have elucidated the factors controlling HSC fate using these methods, they can occasionally be misleading

Regulation of Hematopoietic Stem Cell Fate: Self-Renewal, Quiescence and Survival 41

that regulate adult HSCs has largely been achieved through the use of gene-targeted mouse models. Increasing or decreasing HSC cell-cycling results in the accelerated production of more committed progenitors at the expense of self renewal, or the insufficient production of

GATA-2 is highly expressed in immature progenitors within hematopoietic lineages (Tsai & Orkin, 1997; Akashi et al., 2000). The haploinsufficient *GATA-2*+/– mouse model shows mildly increased quiescence of both HSCs and progenitor cells (Rodrigues et al., 2005). However, Tipping et al. recently showed that enforced expression of GATA-2 in a murine

Bmi1 belongs to the polycomb group (PcG) of proteins, which play a role in the transcriptional repression of genes via histone modification (Rajasekhar et al., 2007). Bmi1 is highly expressed in HSCs. The expression of Bmi1 is maintained at high levels in lymphoid lineage cells but is downregulated during myeloid differentiation (Iwama et al., 2004). Although *Bmi1*-/- mice show normal fetal liver hematopoiesis, progressive pancytopenia emerges in postnatal *Bmi1*-/- mice. This hematopoietic defect can be attributed to impaired HSC self-renewal. Transplanted fetal liver and bone marrow cells from *Bmi1*-/- mice cannot contribute to long-term hematopoiesis, although they do maintain the ability to repopulate in the short-term (Park et al., 2003; Iwama et al., 2004). Conversely, enforced expression of Bmi1 promotes HSC self-renewal (Iwama et al., 2004). Thus, Bmi1 is essential for the

The activity of Bmi1 in HSCs largely depends on the silencing of its target, the *Ink4a* locus (Jacobs et al., 1999). The expression of *p16INK4a* and *p19ARF* (both cell-cycle inhibitors encoded by the *Ink4a* locus) is markedly upregulated in hematopoietic cells in *Bmi1*-deficient mice, and the overexpression of *p16INK4a* and *p19ARF* in HSCs induces cell-cycle arrest and p53-dependent apoptosis (Park et al., 2003). On the contrary, the deletion of both *p16INK4a* and *p19ARF* restores the self-renewal ability of *Bmi1*-/- HSCs (Oguro et al., 2006). Thus, Bmi1 prevents the premature loss

Gfi1 is a SNAG-domain–containing zinc-finger transcriptional repressor, which plays a role in T cell proliferation and the development of lymphoid tumors (Gilks et al., 1993). It is suggested that Gfi-1 restricts proliferation and preserves functional integrity of hematopoietic stem cells. Gfi-1-null HSCs show excessive cell cycling and a decreased capacity for self-renewal in

Pbx1 is a TALE class homeodomain transcription factor that critically regulates numerous embryonic processes, including hematopoiesis (DiMartino et al., 2001). Although a potential

of HSCs by repressing the p16INK4a- and p19ARF-dependent senescence pathways.

competitive repopulation assays (Hock et al., 2004; Zeng et al., 2004).

) and progenitors (CD34+CD38+),

progeny cells, which eventually results in BM failure.

cell line (Ba/F3), or human cord blood HSCs (CD34+CD38-

increases quiescence and inhibits proliferation (Tipping, et al, 2009).

**2.1.1 Positive regulation** 

maintenance of HSC self-renewal.

**2.1.1.1 GATA-2** 

**2.1.1.2 Bmi1** 

**2.1.1.3 Gfi-1** 

**2.1.1.4 Pbx1** 

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.


Table 1. Gene expression profiling analyses of adult HSCs

#### **2. Regulators of HSC fate**

#### **2.1 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 that regulate adult HSCs has largely been achieved through the use of gene-targeted mouse models. Increasing or decreasing HSC cell-cycling results in the accelerated production of more committed progenitors at the expense of self renewal, or the insufficient production of progeny cells, which eventually results in BM failure.

#### **2.1.1 Positive regulation**

#### **2.1.1.1 GATA-2**

40 Advances in Hematopoietic Stem Cell Research

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

In this chapter, we briefly review the recent advances in our knowledge of cell-intrinsic

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

regulators of HSC self-renewal, differentiation, quiescence, cycling, and survival.

Table 1. Gene expression profiling analyses of adult HSCs

**2.1 Regulation of HSC self-renewal and quiescence** 

**2. Regulators of HSC fate** 

understood.

GATA-2 is highly expressed in immature progenitors within hematopoietic lineages (Tsai & Orkin, 1997; Akashi et al., 2000). The haploinsufficient *GATA-2*+/– mouse model shows mildly increased quiescence of both HSCs and progenitor cells (Rodrigues et al., 2005). However, Tipping et al. recently showed that enforced expression of GATA-2 in a murine cell line (Ba/F3), or human cord blood HSCs (CD34+CD38- ) and progenitors (CD34+CD38+), increases quiescence and inhibits proliferation (Tipping, et al, 2009).

#### **2.1.1.2 Bmi1**

Bmi1 belongs to the polycomb group (PcG) of proteins, which play a role in the transcriptional repression of genes via histone modification (Rajasekhar et al., 2007). Bmi1 is highly expressed in HSCs. The expression of Bmi1 is maintained at high levels in lymphoid lineage cells but is downregulated during myeloid differentiation (Iwama et al., 2004). Although *Bmi1*-/- mice show normal fetal liver hematopoiesis, progressive pancytopenia emerges in postnatal *Bmi1*-/- mice. This hematopoietic defect can be attributed to impaired HSC self-renewal. Transplanted fetal liver and bone marrow cells from *Bmi1*-/- mice cannot contribute to long-term hematopoiesis, although they do maintain the ability to repopulate in the short-term (Park et al., 2003; Iwama et al., 2004). Conversely, enforced expression of Bmi1 promotes HSC self-renewal (Iwama et al., 2004). Thus, Bmi1 is essential for the maintenance of HSC self-renewal.

The activity of Bmi1 in HSCs largely depends on the silencing of its target, the *Ink4a* locus (Jacobs et al., 1999). The expression of *p16INK4a* and *p19ARF* (both cell-cycle inhibitors encoded by the *Ink4a* locus) is markedly upregulated in hematopoietic cells in *Bmi1*-deficient mice, and the overexpression of *p16INK4a* and *p19ARF* in HSCs induces cell-cycle arrest and p53-dependent apoptosis (Park et al., 2003). On the contrary, the deletion of both *p16INK4a* and *p19ARF* restores the self-renewal ability of *Bmi1*-/- HSCs (Oguro et al., 2006). Thus, Bmi1 prevents the premature loss of HSCs by repressing the p16INK4a- and p19ARF-dependent senescence pathways.

#### **2.1.1.3 Gfi-1**

Gfi1 is a SNAG-domain–containing zinc-finger transcriptional repressor, which plays a role in T cell proliferation and the development of lymphoid tumors (Gilks et al., 1993). It is suggested that Gfi-1 restricts proliferation and preserves functional integrity of hematopoietic stem cells. Gfi-1-null HSCs show excessive cell cycling and a decreased capacity for self-renewal in competitive repopulation assays (Hock et al., 2004; Zeng et al., 2004).

#### **2.1.1.4 Pbx1**

Pbx1 is a TALE class homeodomain transcription factor that critically regulates numerous embryonic processes, including hematopoiesis (DiMartino et al., 2001). Although a potential

Regulation of Hematopoietic Stem Cell Fate: Self-Renewal, Quiescence and Survival 43

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 p53 null 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

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 p18INK4C, suggesting a mechanism by which Nurr1 may regulate HSC quiescence

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

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

(Sirin et al., 2010).

(Sirin et al., 2010).

**2.1.1.9 Reactive oxygen species, FoxOs** 

FoxO-deficient HSC phenotype.

**2.1.1.10 Fbxw7** 

**2.1.1.8 Nurr1** 

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-β 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 TGF-β stimulation *in vitro*. These results suggest that Pbx1 regulates HSC self-renewal and quiescence, at least in part by affecting the response to TGF-β.

#### **2.1.1.5 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).

#### **2.1.1.6 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.

#### **2.1.1.7 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 p53 null 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

#### **2.1.1.8 Nurr1**

(Sirin et al., 2010).

42 Advances in Hematopoietic Stem Cell Research

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-β 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 TGF-β stimulation *in vitro*. These results suggest that Pbx1 regulates HSC self-renewal and

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

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

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

quiescence, at least in part by affecting the response to TGF-β.

**2.1.1.5 Evi-1** 

(Shimabe et al., 2009).

**2.1.1.6 JunB** 

cycle.

**2.1.1.7 p53** 

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 p18INK4C, suggesting a mechanism by which Nurr1 may regulate HSC quiescence (Sirin et al., 2010).

#### **2.1.1.9 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.

#### **2.1.1.10 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

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 TGFβ (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-β. These results suggest that p57 is required for the TGF-β–mediated cell cycle entry of hematopoietic cells and for repressing

Table 2. Genes expressed at higher levels in HSCs than in other subsets.

cells than all other samples are listed.

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

the proliferation of these cells.

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.

### **2.1.1.11 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-1α (HIF-1α) 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-1α 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-1α conditional knockout mice show that HIF-1α– deficient 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-1α levels is required to maintain HSC quiescence.

#### **2.1.1.12 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*; *LKB1fl/fl* or *RosaCreERT2*; *LKB1L/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.

#### **2.1.1.13 Cyclin-dependent kinase inhibitors**

p21cip1/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 p57kip2 (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

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

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-1α (HIF-1α) 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-1α 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-1α conditional knockout mice show that HIF-1α– deficient 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-1α levels is required to maintain HSC

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*; *LKB1fl/fl* or *RosaCreERT2*; *LKB1L/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

p21cip1/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

Although p57kip2 (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

premature exhaustion of HSCs.

**2.1.1.11 HIF-1α**

quiescence. **2.1.1.12 Lkb1** 

mTORC1.

**2.1.1.13 Cyclin-dependent kinase inhibitors** 

hematopoiesis (van Os et al., 2007).

analysis studies of human CD34+ HSC/progenitor cells identified p57 as the only cyclindependent kinase inhibitor induced by TGFβ (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-β. These results suggest that p57 is required for the TGF-β–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.

Regulation of Hematopoietic Stem Cell Fate: Self-Renewal, Quiescence and Survival 47

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 self-

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 (Laurenti et al., 2008). Thus, Myc activity controls important aspects of HSC

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

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

function such as proliferation, survival and differentiation.

renewal. **2.1.2.4 Myc** 

**2.1.2.5 MEF/ELF4** 

stage than p18 and antagonizes p21.

homeostasis or under conditions of stress.

**2.2 Survival of HSCs** 
