**2. Quiescence of hematopoietic stem cells**

Somatic stem cells contribute to tissue homeostasis throughout life (Suda et al., 2011). Because proliferation will induce senescence, the proper maintenance of stem cells without senescence is mandatory. There are two states for tissue stem cells in terms of the cell cycle. One is the quiescent state. Stem cells in the quiescent state are out of the active cell cycle (S/G2/M phase) and in the G0 phase. The other is the cycling state. Cycling stem cells actively reproduce themselves (self-renewal) to generate progeny. Cycling cells are in the non-G0 phase of the cell cycle. Quiescence is thought to be an effective strategy for stem cells to avoid various forms of cytotoxic damage. If stem cells lost quiescence, they would become susceptible to intrinsic and extrinsic stresses.

Mammalian HSCs are included in the heterogeneous population of lineage marker-, Sca-1+, and c-Kit+ (LSK) cells. LSK cells are a mixture of progenitors and HSCs. Within the LSK population, CD34- CD150hiCD48- CD41-Flt3 cells, as well as side population (SP) cells (Osawa et al, 1996; Kiel et al, 2005; Goodell et al, 1996), are quiescent.

Measurement of the cell cycle in HSCs by the staining of DNA with Hoechst 33342, DAPI, and/or anti-Ki67 indicates that more than 70% of highly purified HSC (CD34–CD48–CD150hi LSK) are in the G0 phase, whereas less than 10% of CD34+ LSK cells (differentiated progenitors) are in the quiescent phase (Wilson et al, 2008).

Slow-cycling HSCs have long-term (LT) reconstitution activity when they are transplanted into lethally irradiated recipient mice. In contrast, actively cycling HSCs and progenitors exhibit only short-term (ST) reconstitution activity and only maintain hematopoiesis for 3–4 months. Thus, the former are termed "LT-HSC(s)" and the latter are "ST-HSC(s)". LT-HSCs produce ST-HSCs, multipotent progenitors (MPPs), lineage-restricted progenitors, and terminally differentiated hematopoietic cells including erythrocytes, platelets, lymphocytes, granulocytes, and macrophages (Figure 1).

Because slow-cycling stem cells are not in the S or M phase of the cell cycle, they are more resistant to cytotoxic agents such as ultraviolet (UV) light, ionizing radiation and chemicals, compared with actively cycling cells. Recent reports indicate that quiescent stem cell fractions are present in several tissues. For example, in the hair follicle, cell cycle progression of stem cells in bulge regions is suppressed by Wnt inhibitors (Fuchs and Horsley, 2011). In contrast, stem cells in the murine intestinal and gastric epithelia divide every 24 hours

generation during ontogeny. However, a detailed analysis of the contribution of HIF-1to

We analyzed HSCs in HIF-1– and VHL-deficient mice and found that the cellular pool and cell cycle status of HSCs were regulated by the HIF-1 level (Takubo et al., 2010). Our analysis revealed that the regulation of the HIF-1 dose is critical for HSC maintenance in the hypoxic niche microenvironment of the BM. The critical role for HIF-1 in HSC cell cycle regulation broadens the involvement of oxygen status in the stem cell niche. It also implies a novel strategy for maintaining and expanding HSC resources based on cellular oxygen metabolism reprogramming, including the modulation of HSC quiescence through the

Somatic stem cells contribute to tissue homeostasis throughout life (Suda et al., 2011). Because proliferation will induce senescence, the proper maintenance of stem cells without senescence is mandatory. There are two states for tissue stem cells in terms of the cell cycle. One is the quiescent state. Stem cells in the quiescent state are out of the active cell cycle (S/G2/M phase) and in the G0 phase. The other is the cycling state. Cycling stem cells actively reproduce themselves (self-renewal) to generate progeny. Cycling cells are in the non-G0 phase of the cell cycle. Quiescence is thought to be an effective strategy for stem cells to avoid various forms of cytotoxic damage. If stem cells lost quiescence, they would

Mammalian HSCs are included in the heterogeneous population of lineage marker-, Sca-1+, and c-Kit+ (LSK) cells. LSK cells are a mixture of progenitors and HSCs. Within the LSK population, CD34-CD150hiCD48-CD41-Flt3- cells, as well as side population (SP) cells (Osawa

Measurement of the cell cycle in HSCs by the staining of DNA with Hoechst 33342, DAPI, and/or anti-Ki67 indicates that more than 70% of highly purified HSC (CD34–CD48–CD150hi LSK) are in the G0 phase, whereas less than 10% of CD34+ LSK cells (differentiated

Slow-cycling HSCs have long-term (LT) reconstitution activity when they are transplanted into lethally irradiated recipient mice. In contrast, actively cycling HSCs and progenitors exhibit only short-term (ST) reconstitution activity and only maintain hematopoiesis for 3–4 months. Thus, the former are termed "LT-HSC(s)" and the latter are "ST-HSC(s)". LT-HSCs produce ST-HSCs, multipotent progenitors (MPPs), lineage-restricted progenitors, and terminally differentiated hematopoietic cells including erythrocytes, platelets, lymphocytes,

Because slow-cycling stem cells are not in the S or M phase of the cell cycle, they are more resistant to cytotoxic agents such as ultraviolet (UV) light, ionizing radiation and chemicals, compared with actively cycling cells. Recent reports indicate that quiescent stem cell fractions are present in several tissues. For example, in the hair follicle, cell cycle progression of stem cells in bulge regions is suppressed by Wnt inhibitors (Fuchs and Horsley, 2011). In contrast, stem cells in the murine intestinal and gastric epithelia divide every 24 hours

the maintenance of adult HSCs has not yet been reported.

**2. Quiescence of hematopoietic stem cells** 

become susceptible to intrinsic and extrinsic stresses.

et al, 1996; Kiel et al, 2005; Goodell et al, 1996), are quiescent.

progenitors) are in the quiescent phase (Wilson et al, 2008).

granulocytes, and macrophages (Figure 1).

oxygenation status of HIF-1.

(Snippert and Clevers, 2011). Therefore, quiescence itself is not the only strategy for the long-term maintenance of stem cells.

Life-long hematopoiesis is maintained by long-term (LT)-HSCs and their progeny. LT-HSCs have a two cell cycle states: a quiescent state (G0 phase) and a cycling state (non-G0; i.e., G1/S/G2/M phase). LT-HSCs in the former state are resistant to various cytotoxic stresses. Reactive oxygen species (ROS) change the cell cycle state of LT-HSCs from quiescent to cycling. Cycling LT-HSCs are also promoted to differentiate into short-term (ST)-HSCs and multipotent progenitors (MPPs). These differentiated progenitors actively produce various terminally differentiated hematopoietic cells.

Fig. 1. Quiescent and cycling hematopoietic stem cells (HSCs)

The Hypoxia Regulatory System in Hematopoietic Stem Cells 137

Although molecular oxygen is critically important for living organisms, HSCs are susceptible to reactive oxygen species or oxidative stresses that are derived from molecular oxygen. To maintain life-long hematopoiesis, it is reasonable for HSCs to avoid high-oxygen conditions. Although classical observations and theoretical studies supported these views,

Classically, bone marrow has been thought to be hypoxic. Recently, its exact nature and dynamic regulation were studied. This section will summarize the classical and recent

Genetic studies have postulated that LT-HSCs reside primarily in the endosteal zone of the bone marrow (BM) (Calvi et al, 2003; Zhang J, 2003; Arai et al, 2004). Vascular organization around the endosteal zone is unique (Draenert and Draenert, 1980). Nutrient arteries penetrate the cortical bone, enter the medullary canal, and then proceed in a spiral pattern into the metaphyseal region of the bone marrow. The blood in arterial capillaries drains into

As a result, hematopoietic cells can easily move across the sinusoidal endothelium. Accordingly, the perfusion of the BM is limited and the partial oxygen pressure (PO2) in the

In addition to hypoperfusion, the BM is tightly packed with blood cells. Oxygen consumption by hematopoietic cells is relatively high, and a simulation of O2 diffusion in the bone marrow suggested that the PO2 is decreased 10-fold at a distance of several cells from the nearest capillary (Chow et al, 2001). The average PO2 in the BM is approximately 55 mmHg and the mean O2 saturation is 87.5% (Harrison et al, 2002). Thus, based on this simulation study, HSCs may well reside in a severely hypoxic

In support of this idea, it has also been reported that murine HSCs live in a hypoxic BM niche. By administering a perfusion tracer into mice, one group found that HSCs accumulated in a hypoperfusion cellular fraction in the BM (Parmar et al, 2007). These hypoperfused cells retained pimonidazole, a probe that selectively binds and forms adducts with protein thiol groups in a hypoxic environment. Administration of a toxin selective for hypoxic cells (tirapazamine) resulted in the depletion of HSCs *in vivo*. It was also shown that LT-HSCs are positive for pimonidazole in mice (Takubo et al, 2010). Moreover, human cord blood stem cells transplanted into super-immunodeficient NOD/scid/IL-2R (NOG) mice homed to the BM niche and became both hypoxic and quiescent after BM transplantation

Collectively, these findings suggest the hypoxic nature of HSCs. The hypoxic character of LT-HSCs is potentially determined by their position within the BM. However, in contrast to the simple O2 gradient model for the BM hypoxic niche, immunohistochemical observation of a two-dimensional segment of the murine BM suggests that 60% of LT-HSCs localize closely to BM endothelial cells (Kiel et al, 2005; Sugiyama et al, 2006). These findings do not fit the simple O2 gradient model for the hypoxic status of HSCs in the niche. However, as noted above, the vasculature in the niche near the endosteal zone of the BM may perfuse the bone marrow very poorly. Four-dimensional tracking (time-

**3. Hypoxic nature of bone marrow** 

experimental evidence has only been recently provided.

sinusoids, which are fenestrated and loosely organized.

endosteal region is very low.

environment.

(Shima et al, 2010).

studies related to the functional anatomy of bone marrow oxygenation.

One important regulator for the quiescence of HSCs is reactive oxygen species (ROS). ROS are an intrinsic and extrinsic stress for HSCs (Figure 2). Intrinsically, ROS are mainly produced by mitochondria, the energy factory of the cell, as a by-product of the electron transport chain. Because anaerobic energy metabolism in mitochondria utilizes oxygen to generate ATP, oxygen-rich conditions produce intracellular ROS in HSCs. In addition, various immune cells utilize oxygen to generate ROS as an anti-microbial agent. ROS have favourable and unfavourable effects on HSCs. ROS are a signal transducer for essential cytokine signalling in HSCs (Sattler et al., 1999). However, excessive or prolonged ROS exposure is detrimental to HSCs (Naka et al., 2008). Aberrant exposure to ROS induces senescence, apoptosis, or the accumulation of DNA damage in HSCs. These damaged cells are dysfunctional and a potential source for leukemic transformation. Therefore, it is reasonable to hypothesize that HSCs reside in a hypoxic microenvironment.

HSCs are exposed to ROS from various sources, including endogenous mitochondria and adjacent immune cells. High O2 pressure in the microenvironment also promotes ROS generation. This ROS burden results in the oxidation of DNA, protein, and lipid in HSCs. Also, the appropriate dose of ROS mediates cytokine signalling in HSCs. These balances determine the fate of HSCs: survival, premature senescence, apoptosis, differentiation, or malignant transformation.

Fig. 2. Intrinsic and extrinsic oxidative stresses and HSCs
