**3. Hypoxic nature of bone marrow**

136 Advances in Hematopoietic Stem Cell Research

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

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

hypoxic microenvironment.

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, experimental evidence has only been recently provided.

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 studies related to the functional anatomy of bone marrow oxygenation.

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 sinusoids, which are fenestrated and loosely organized.

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 endosteal region is very low.

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 environment.

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 (Shima et al, 2010).

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-

The Hypoxia Regulatory System in Hematopoietic Stem Cells 139

The diagrams represent the regulation of HIF-1 protein and interacting factors under different oxygen conditions. HIF-1 is a substrate for both prolyl and asparaginyl hydroxylases. Under normoxia, proline and asparagine residues are hydroxylated. These modifications regulate the stability and transcriptional activity of HIF-1. bHLH, basic-helix-loop-helix domain; PAS, Per-ARNT-Sim domain; TAD-N, transactivation domain N-terminal; ID, inhibitory domain; TAD-C, C-terminal transactivation domain; PHD, prolyl hydroxylase domain-containing protein; and FIH-1, factor-inhibiting HIF-1.

Fig. 3. Regulation of hypoxia-inducible factor-1 (HIF-1)

lapse and three-dimensional observation with a multi-photon microscope) of single LT-HSCs in the BM has shed light on this paradox. Real-time tracking of murine BM revealed that HSCs gradually move away from bone marrow blood vessels and then detach from them and translocate to the osteoblastic zone of the BM after transplantation (Lo Celso et al, 2009; Xie et al, 2009). Based on these observations, it is possible that subpopulations of HSCs residing in different specific locations have different oxygenation statuses.

In parallel with the hypoxic microenvironment for HSCs *in vivo*, hypoxic culture phenotypically and functionally sustained HSCs more effectively than normoxic culture (20% oxygen). Also, hypoxic culture enhances the colony-forming ability (progenitor ability) and transplantation capacity (HSC capacity) of cultured BM cells or isolated HSCs (Cipolleschi et al, 1993; Danet et al, 2003; Ivanovic et al, 2004). Hypoxic treatment also induces cell cycle quiescence in cultured HSC (Hermitte et al, 2006; Shima et al, 2010). Quiescent HSCs are defined by a high amount of efflux of the DNA-binding dye Hoechst 33342 from the cytosol (Goodell et al, 1996). These cells are called "side population (SP)" cells due to their specific staining pattern by flow cytometric analysis. Hypoxic treatment also sustains the SP phenotype in HSC *in vitro* (Krishnamurthy et al, 2004). Exclusion of Hoechst dye from the HSC cytosol is supported by Bcrp1/ABCG2, an ATP-dependent transporter, at the plasma membrane. When HSCs were cultured under hypoxic conditions, mRNA expression of Bcrp1/ABCG2 was significantly increased and the number of SP cells was also increased as compared to HSCs cultured at normoxia. Interestingly, because Bcrp1- /- mice show no significant defect in hematopoiesis (Zhou et al, 2001), the functional role of Bcrp1 in HSCs is still uncharacterized.
