TISSUE-SPECIFIC STEM CELLS

Transcription

1 TISSUE-SPECIFIC STEM CELLS Concise Review: Multiple Niches for Hematopoietic Stem Cell Regulations IL-HOAN OH, a,b KYUNG-RIM KWON a,b a The Catholic High-Performance Cell Therapy Center, Division of Regenerative Medicine, College of Medicine, Seoul; b The Catholic University of Korea, Korea Key Words. Stem cell niche Microenvironment Osteoblast Vascular niche ABSTRACT Two types of stem cell niches in bone marrow (BM), endosteal osteoblastic, and vascular niches are involved in the microenvironmental regulation of hematopoietic stem cells (HSCs). Recently, redundant features of the two niches were identified, based on their common cellular origins or chemical mediators being produced in each niche. In contrast, studies have also revealed that HSCs are localized differentially in the niches with respect to their distinct functional status, and that the biological activity of each Disclosure of potential conflicts of interest is found at the end of this article. niche is differentially influenced by extrinsic conditions. An important question is, therefore, whether these two niches play distinct roles in regulating HSCs and whether they respond differentially to environmental stimuli/stress for compartmentalized niche organization in BM. In this review, recent discoveries related to the characteristics of each type of niche and their common or unique features are discussed, along with the possibility of multiniche regulation of HSCs in BM. STEM CELLS 2010;28: INTRODUCTION Organisms have adopted a reservoir system for replenishing effector cells in each tissue, as such cells have short lifetimes and are vulnerable to damage and aging. The hematopoietic system is a representative model for tissue regeneration, displaying a life-long regeneration of all lineages of hematopoietic cells through a balance between the self-renewal and differentiation of hematopoietic stem cells (HSCs). The regenerative activities of HSCs in reconstituting lympho-myeloid systems have been documented, following their transplantation into irradiated recipients [1, 2] and quantitatively measured in a murine model through competitive repopulating analysis [3]. HSCs can be initially identified in the developing embryo in aorta-gonad-mesonephros regions, after which they migrate into the fetal liver and subsequently move into the bone marrow (BM) after birth [4]. During development, HSCs undergo active self-renewal to expand the pool size, but then become largely quiescent and stay in a steady state in adult BM, where their maintenance is tightly regulated [5]. Multiple intrinsic factors have been shown to regulate the behavior and maintenance of HSCs in a complex network of signaling molecules, but more recent studies point to microenvironmental factors as key regulators of HSCs. Such microenvironmental regulation of HSCs largely occurs in a special architecture of the BM, referred to as a niche, that is, HSCs are selectively localized in endosteal regions or in close proximity to the sinusoidal endothelium, where they interact with secreted or membrane-bound factors, and these HSC-niche interactions influence the self-renewal [6, 7], quiescence [8 10], or mobilization of HSCs [11]. Although a growing body of evidence indicates that both types of niches participate in hematopoiesis, the difference between the two is not currently fully understood. One possibility is that the two compartments of niches in the BM function in a redundant manner, even though they are spatially separated in BM. Another possibility is that each niche has a distinct function during hematopoiesis and therefore each represents a part of multiple niches in the hematopoietic system. In this review, therefore, recent studies on the characterization of these two types of BM niches are discussed, as well as how similarly or distinctively they are organized in BM for stem cell regulation, in an attempt to gain further insights into the microenvironmental regulation of HSCs. OSTEOBLASTIC NICHE Localization of HSCs in the Endosteum Studies in the 1970s indicated that undifferentiated hematopoietic cells are localized close to the endosteal bone surface, but that differentiated cells move toward the central axis of the marrow. While the cells identified in these earlier studies were progenitors that form colonies in the spleen of irradiated animals, known as a colony-forming unit-spleen (CFU-S) [12, 13], subsequent investigations also revealed a similar spatial distribution of undifferentiated cells near the endosteal region, over a time course of 15 hours after transplantation [14]. Author contributions: IHO: conception and design, manuscript writing, financial support, KRK: conception and design, collection and assembly of material. Correspondence: Dr. Il-Hoan Oh, M.D., Ph.D., Catholic High-Performance Cell Therapy Center, The Catholic University of Korea, 505, Banpo-Dong, Seocho-Ku, Seoul, Korea Telephone: , Fax: ; iho@catholic.ac. kr Received April 13, 2010; accepted for publication May 10, 2010; first published online in STEM CELLS EXPRESS June 1, 2010; available online without subscription through the open access option. VC AlphaMed Press /2009/$30.00/0 doi: /stem.453 STEM CELLS 2010;28:

2 1244 Distinct Niches Regulate Hematopoietic Stem Cells Although those studies showed the endosteal localization of phenotypically defined HSCs, a recent study demonstrated that the HSC populations functionally defined by the longterm retention of bromo-deoxy uridine (LRC-BrdU) were similarly localized in the surface of the trabecular endosteum adhering to the surface through N-cadherin-mediated cell cell interactions [7]. Thus, the specific localization of primitive cell populations near the endosteum support the view that the endosteal region of the BM might play a role as a specific niche for primitive hematopoietic cell populations. Production of Factors Regulating HSCs by Osteoblasts The concept of the endosteal osteoblastic niche is also supported by findings that osteoblasts produce factors that have the ability to regulate quiescence and the maintenance of HSCs, such as angiopoietin-1 [15], osteopontin [8], or chemokine (C- X-C motif)ligand 12 (CXCL-12) [16]. Similarly, osteoblasts support the maintenance of long-term culture-initiating cells (LTC-IC) during the ex vivo culture of primitive hematopoietic cells [17]. On the contrary, receptor activator of NF-kappa B ligand (RANKL)-activated osteoclasts, which resorb bone, were found to decrease the expression of stem cell factor (SCF), CXCL-12, or osteopontin in the endosteum and caused the egress of HSCs from the niche, suggesting that the balance in the endosteal osteoblast is linked to the production of active factors that influence HSC niche function [18]. Genetic Model Supporting the Endosteal Niche In support of the concept of the osteoblastic niche, several genetic studies have demonstrated a correlation between osteoblastic mass and the size of the HSC pool. For example, in transgenic mice expressing thymidine kinase under the control of the 2.3 kb collagen type 1 promoter, the conditional ablation of osteoblast lineages by the administration of ganciclovir resulted in a severe loss of lymphoid, erythroid, and myeloid progenitors in the BM, followed by a decrease in the number of HSCs, while extramedullary hematopoiesis was activated in the spleen or liver. Conversely, the recovery of osteoblasts by the withdrawal of ganciclovir led to the recovery of BM cells [19]. On the other hand, two genetic models demonstrated an association between increasing osteoblastic mass and an increase in HSC pool size. For example, the transgenic expression of constitutively active PTH/parathyroid hormonerelated protein (PTHrP) receptor under the control of the Col1a 2.3kB promoter resulted in an increase in trabecular osteoblast numbers, which caused an expansion of primitive hematopoietic cells, characterized as LTC-ICs. In this study, the expansion of HSCs occurred in a manner dependent on the induction of jagged one in osteoblasts, indicating that the concomitant functional activation of osteoblasts is necessary, in addition to the increased mass of osteoblasts [6]. Similarly, the conditional disruption of bone morphogenic protein receptor 1a (Bmpr1a), caused an increase in the numbers of ectopically formed trabecular osteoblasts in mutant mice, which was associated with a concomitant increase in HSCs characterized by the LRC-BrdU [7]. Cellular Composition of Osteoblastic Niche With the identification of niches in the endosteal region, cells in the endothelial lining were proposed as a cellular component of the niche. However, heterogeneous cell populations were found in endosteal regions that included mature bone lining cells, osteoblasts, and preosteoblasts [20]. Although genetic models targeting selective stages of osteoblasts have been utilized to identify the exact cell types in the osteoblastic niche, controversies remain and await further characterization [19, 20]. Moreover, endosteal regions are highly vascularized [21], raising the possibility that vascular structures, in addition to osteoblasts, could also participate in the endosteal niche (discussed in more detail below). PERIVASCULAR NICHE Identification of the Vascular Niche The observation that HSCs also reside at a distance from the endosteum triggered the concept of the vascular niche as alternative niche in BM, that is, histochemical analyses of BM sections using SLAM (stimulated lymphocyte activating molecule) family markers (CD150 þ CD244 CD48 CD41 ) showed that about 60% of SLAM family HSCs were localized in the vicinity of sinusoidal endothelial cells (SECs) suggesting the presence of an additional niche in the vascular area [22, 23]. Interestingly, 95% of these perisinusoidal HSCs were located within a five cell layer of the sinusoidal endothelium, whereas only 8% 20% of SLAM family HSCs were located within a five cell layer of the endosteum [23]. Additional evidence for the vascular niche was also provided from a genetic study using the green fluorescent protein (GFP) reporter in the CXCL-12 locus [24]. The study identified specific reticular cell populations that secrete high levels of CXCL-12, referred to as CAR cells (CXCL-12 abundant reticular cells) which interacted with over 90% of the HSCs (either recognized by SLAM markers or lin ckit þ Sca1 þ ) [24]. Complex Cellular Composition of the Vascular Niche As in the case of the osteoblastic niche, the cellular components of the vascular niche also display heterogeneities in their nature and origin. First, reticular cells around the sinusoid appear to constitute one component of the vascular niche, as evidenced by a study of CAR cells [24]. In human BM, such reticular cells constitute the subendothelial (adventitial) layer of sinusoidal walls projecting a reticular process that is in close contact with HSCs [25]. Interestingly, these reticular cells were derived from a specific subset of mesenchymal cells (CD146 þ ) that had been shown to produce either reticular or endosteum of the ectopic hematopoietic microenvironment (HME), referred to as skeletal stem cells. Similarly, another study in mice also identified CD105 þ Thy-1 mesenchymal cells that were capable of generating heterotrophic HME when injected into the renal capsule [26]. These studies indicate that reticular cells derived from mesenchymal (skeletal) stem cells function as active building blocks in vascular niche formation. On the other hand, other studies suggest that endothelial cells are also involved in the vascular niche. For example, earlier studies showed that endothelial cells are capable of supporting the maintenance of HSCs in culture [27, 28]. In addition, the disruption of gp130 in Tie-2 expressing cells resulted in hypocellularity in most blood lineages with dilatation of the vascular sinusoids, which were reproduced when wild-type hematopoietic cells were transplanted into gp130 / mice, indicating their microenvironmental origin in endothelial components [29]. Moreover, recent studies demonstrated the role of endothelial cells during hematopoietic regeneration, that is, an infusion of endothelial progenitor cells accelerated the recovery of BM sinusoidal vessels after irradiation, which was associated with higher recoveries of BM cellularity and HSCs [30]. Similarly, a recent study demonstrated that the inhibition of vascular endothelial growth factor receptor 2

3 Oh and Kwon 1245 (VEGFR2) signaling during the recovery of BM after lethal irradiation prevented not only the regeneration of sinus endothelial cell (SEC), but also the hematopoietic reconstitution of transplanted HSCs, pointing to the specific role of SECs in hematopoietic regeneration [31]. In addition, a study using adenovirus E4 ORF-immortalized endothelial cells showed that co-culturing HSC with immortalized endothelial cells under cytokine-free conditions supported the expansion of the longterm repopulating HSCs during the serial subculture of HSCs, which was dependent on notch signal activation in HSCs [32]. Collectively, these studies show that multiple cellular populations, including reticular and endothelial cell populations participate in HSC regulation in the vascular niche. However, the interplay between the two cell populations is not currently clear, hence, CD146 þ mesenchymal cells neither express endothelial markers nor did they differentiate into endothelial cells [25]. Similarly, pericytes, perivascular cells in the vasculature of multiple organs also did not differentiate into endothelial cells [33]. Therefore, a more likely possibility is that the mesenchymal cells exert a tropic effect rather than playing a role in direct differentiation. Further studies will be necessary to develop a complete understanding of the functional interplays in the vascular niche. Genetic Model Supporting the Vascular Niche In contrast to the models for the osteoblastic niche, relatively fewer genetic models are available for studies of the vascular niche. Mice, in which VEGFR2 is deleted [31], represent one such model, as described above. Of note, we recently identified another model, where a targeted disruption of Bis (Bcl-2- interacting cell death suppressor), a molecule implicated in the antiapoptotic or antistress response, caused the deterioration of the vascular niche [34, 35]. Mutant mice (bis / ) exhibited early neonatal death and a series of hematological derangements that included the loss of B-cell lineages along with HSCs [34]. However, such defects were not observed when hematopoietic cells of mutant mice were transplanted into wild-type littermates, but the reciprocal transplantation of wild-type hematopoietic cells into bis / mice reproduced the phenotype, indicating microenvironmental origin of the phenotypes. The BM of mutant mice exhibited a marked loss of CAR cells and a decreased proliferation in vitro [34]. Moreover, the BM of bis / mice exhibited markedly dilated sinusoids, a decrease in capillary density and the loss of endothelial cells in the BM, thus demonstrating the association between the mesenchymal (reticular) and endothelial systems [34]. However, in contrast to the vascular niche, the osteoblastic component of the bis / BM was not affected in the mutant mice. Collectively, bis / mice may be a model where cell populations of the vascular niche undergo selective deterioration without the osteoblastic compartment of the niche being affected. FACTORS THAT CAN REGULATE HEMATOPOIETIC ACTIVITY IN THE NICHE As the HSC niche plays a key role in regulating hematopoietic activity, various physiological conditions that modulate and integrate hematopoietic needs may be reflected in the niche to change their activities. Although diverse repertoires of factors are now being identified, many of the factors appear to share common mechanisms for altering HSC activities such as the activation of notch signals or CXCL-12 signaling pathways (Schematically shown in Fig. 1) PTH/PTHrP Signaling As described above, PTH/PTHrP activation factor that can regulate niche activity through increasing trabecular osteoblastic mass and inducing the production of jagged-1 in osteoblasts [6]. However, it should be pointed out that the quantity of the bone mass itself could not be a factor responsible for the increase in the HSC pool because HSC have been shown to be not affected under conditions where trabecular bone and osteoblasts were reduced by a Biglycan deficiency [22]. In fact, PTH signaling is also involved in the maintenance of calcium levels and HSCs express the calcium sensing receptor (CaR), a defect of which leads to reduced cellularity as well as the defective localization of HSCs in endosteal osteoblasts [36]. In addition, stromal cells from PTH transgenic mice expressed higher levels of stem cell factor (SCF), CXCL-12, or interleukin-6 (IL-6), implicating PTH as a pleiotrophic regulator of the osteoblastic niche [6]. Wnt/ß-Catenin Signaling Despite the widespread interest in the effects of Wnt signals in hematopoiesis, the effects of canonical Wnt/ß-catenin signals on HSC self-renewal remains controversial with conflicting observations being reported [37]. The reasons for these conflicting observations remain unclear at present. However, we recently showed that distinct biological outcomes can be caused by wnt/b-catenin signals depending on the target site of their activation, that is, while the direct stabilization of b- catenin in HSCs resulted in the loss of their repopulating activity, stabilization in the stroma led to the enhanced selfrenewal of HSCs through cross-talk between the stroma and HSCs in a manner that was dependent on notch signal activation in HSCs [38]. Importantly, endosteal N-cadherin þ osteoblasts exhibited the selective activation of b-catenin under physiological conditions for stimulating hematopoiesis, indicating the physiological relevance of Wnt/b-catenin signals in the stroma [38]. A similar insight into the stromal effects of b-catenin arose from studies involving the inducible disruption of b-catenin in the stroma, where co-culturing HSCs with b-catenin null stroma caused a significant decrease in the maintenance of hematopoietic progenitors or spleen colonies (CFU-S) [39]. In support of the in vivo function of b-catenin-activated mesenchymal stromal cells (MSCs), we recently demonstrated that the direct intrafemoral injection of b-catenin-activated MSCs stimulated self-renewal of transplanted HSCs several fold higher than HSCs that had been injected with naïve MSCs [40]. On the contrary, a transgenic mice model expressing Dickkopf homolog 1 (DKK-1), an inhibitor of canonical wnt signals, under the osteoblastspecific promoter, resulted in a decrease in the frequency of HSCs and the loss of HSC pools over serial transplantation [41]. Thus, Wnt/b-catenin signals should be a signal that can regulate niche activity and, thereby, HSC self-renewal in response to physiological stimuli for hematopoietic regeneration. Osteopontin Osteopontin (OPN) is an acidic glycoprotein secreted by osteoblasts. It binds to cells through integrins or CD44, both of which are expressed by HSCs. An OPN-null microenvironment increased the number of HSCs associated with elevated stromal jagged1 and Angiopoietin-1 expression [8]. In contrast, exogenous OPN significantly lowered the number of LTC-ICs. Collectively, these findings suggest that OPN serves as a negative regulator of the HSC pool and restricts stem cell expansion.

4 1246 Distinct Niches Regulate Hematopoietic Stem Cells Figure 1. Factors regulating hematopoietic stem cells (HSC) niche activity in a shared mechanism. In the osteoblastic niche, several extrinsic factors such as PTH signaling or canonical wnt/b-catenin induce Jagged-1 in the niche to activate notch in HSCs, whereas OPN suppresses the induction of jagged- 1. Thus, jagged-1/notch axis represents one of shared elements to modulate niche activity (yellow area). In contrast, other groups of signals such as PTH or 5-FU induce the expression of CXCL-12, and G-CSF or sympathetic nerve system down regulates CXCL-12 to release HSCs. Thus, CXCL-12/CXCR4 axis represents another shared signaling mechanism (blue area). Notably, these conserved elements of Jagged-1 or CXCL-12 axis are also similarly shared by cells in the vascular niche (reticular cells or skeletal stem cells). Intrinsic molecules such as Nf2, Rb, FANCB, RAR-c, or Bis regulate niche activities in a poorly defined manner. (þ) represent upregulation, ( ), downregulation, curved arrows represent self-renewal of HSCs. Abbreviations: 5-FU, 5-fluorouracil; CXCL-12, chemokine (C-X-C motif)ligand 12; CXCR4, chemokine (C-X-C motif) receptor 4; FANCB, fanconi anemia, complementation group B; G-CSF, granulocyte-colony stimulating factor; OPN, osteopontin; PTH, parathyroid hormone; Rb, retinoblastoma; RARc, retinoic acid receptor c. Cyclophosphamide/Granulocyte-Colony Stimulating Factor A recent study showed that granulocyte-colony stimulating factor (G-CSF) induced mobilization with cytotoxic treatment, which alters hematopoietic activity, may also activate osteoblasts [11]. The study showed that osteoblasts can be prospectively identified by markers (OPN þ CD45 TER119 ), and that such osteoblasts are expanded in vivo after exposure to cyclophosphamide/g-csf treatments in an ataxia telangiectasia mutated-dependent manner, exhibiting a higher ability to support HSCs than unstimulated osteoblasts [11]. Sympathetic Nervous System Recent study on mice with defective nerve conduction showed that HSC mobilization by G-CSF is dependent on the intact adrenergic nerve system and that norepinephrine downregulate osteoblast and its expression of CXCL-12 [42]. Moreover, it was shown that egress of HSCs from BM is influenced by circadian oscillations in CXCL-12 expression and the cyclical release of HSCs was regulated by molecular clock through circadian noradrenaline secretion, which stimulated the b 3 adrenergic receptor in BM stromal cells [43]. These findings indicate that sympathetic nerve systems is another regulator of BM niche as reviewed by Spiegel et al. [44]. Intrinsic Molecules That Control the HSC Niche In addition to the extrinsic factors that regulate the HSC niche, recent studies began to identify intrinsic molecules that can influence the integrity or function of the niche. For example, mice deficient in retinoic acid receptor c developed a myeloproliferative syndrome in a microenvironment-dependent manner [45]. Similarly, the disruption of Rb caused a defective interaction in hematopoietic cells with the microenvironment leading to the myeloproliferative disease of BMs and mobilization of primitive cells into extramedullary organs [46]. In the case of mice with a disruption in Nf2/merlin, HSC frequencies were increased and shifted into the circulation, which was associated with an increase in trabecular bone mass and stromal cell numbers, as well as increased vascularity and VEGF levels [47]. On the contrary, molecules whose disruption can cause a defect in the vascular niche have been identified, as seen in the targeted disruption of bis [34]. Similarly, loss of the murine homologue of FANCB led to microenvironmental defects mimicking the hematological signs of fanconi anemia, which was rescued by the adoptive transfer of wild-type MSCs [48]. Thus, studies have begun to unveil the regulation of niche at the molecular levels although the mechanisms are not clear at present. OSTEOBLASTIC VS. PERIVASCULAR NICHE As described so far, both the osteoblastic and perivascular niche seem to participate in the regulation of HSCs. An emerging question should be, therefore, whether the two niches represent a common, but anatomically separate niche, or whether they represent parts of multiple niches for distinct functions. Likewise, the question arises as to whether these niches differ in their response to stress, or in the way they respond to extrinsic stimuli. These possibilities are discussed below and are schematically summarized in Figure 2. Evidence Supporting a Common Niche Common Cellular Structure in Both Niche. As described above, CAR cells that interact with 90% of HSCs were found

5 Oh and Kwon 1247 Figure 2. Schematic illustration of the common, but distinct features of HSC niches in bone marrow. (A): Common features of niches in the endosteal regions and central marrow region. Both regions of the niches are populated by SEC and reticular cells around the sinusoids. Moreover, common cellular elements such as SSC or pericytes can regenerate both osteoblasts and reticular cells thus sharing their origins. In addition, cells in both types of niches produce common signaling mediators such as CXCL-12, angiopoietin-1, or jagged-1. (B): Distinction between the endosteal niche (contributed by both osteoblastic and vascular component) and central marrow regions (exclusively contributed by the vascular component). Transplanted hematopoietic cells are differentially localized in the bone marrow (BM) in such a way that more immature cells migrate closer to endosteum particularly when BM was stimulated, and, under stimulated conditions, cell proliferation predominantly occurs in the endosteal area. In addition, osteoblastic and vascular niche respond differently to environmental stress in a manner that vascular compartments undergo selective deteriorations while osteoblastic niches are preserved, or activated in response to the same stress. Red-colored cells represent proliferating, blue represents quiescent HSCs. Abbreviations: CXCL-12, chemokine (C-X-C motif) ligand 12; SEC, sinusoidal endothelial cell; SSC, skeletal stem cell. scattered throughout the intertrabecular region in the vicinity of the perisinusoidal area as well as near the endosteal area raising the possibility that the endosteal and vascular compartments of the niche could share common cellular elements [24]. In addition, the three-dimensional reconstitution of live animal BM demonstrated that over 90% of the osteoblasts were localized within a 20-lm layer of the vasculature [21, 49]. These findings suggest that niches in the endosteal and vascular areas may, to a certain extent, share common structural elements. Contribution of Both Niches from the Same Type of Stem Cells. As inferred from studies of heterotrophic HMEs, an infusion of the same type of mesenchymal cells (CD146 þ for human and CD105 þ for murine) could give rise to both perisinusoidal reticular cells and osteoblasts in the newly formed microenvironment for both humans [25] and a murine model [26]. Moreover, pericytes share common features with mesenchymal cells (CD146 þ ) and differentiate into multiple tissues including osteoblasts [33], suggesting that those mesenchymal cells (or skeletal stem cells) constitute a part of pericytes sharing the common origins [50]. In addition, a recent study demonstrated that, when cultured MSCs were directly injected into the femur, the cells underwent differentiation into pericytes, reticular cells, osteocytes and bone-lining osteoblasts and interacted with transplanted human CD34 þ cells [51]. Collectively, these findings show that the common origin of stem cells can contribute to both the endosteal and vascular compartments of the niche. Common Chemical Mediators of Niche. Another insight into the common niche function is supported by the findings that both types of niches produce common chemical mediators that regulate HSCs. For example, the notch ligand, jagged-1 was shown to be expressed in CaR cells that are producing high levels of CXCL-12 [24] as well as in osteoblasts [6]. On the other hand, osteoblasts also express CXCL12 at a steady state [24], and its production is increased in the presence of DNA damaging agents (irradiation, 5-FU, cyclophosphamide) or by PTH activation [52]. Similarly, angiopoietin- 1, which regulates the quiescence of HSCs in the osteoblastic niche was also shown to have the characteristics of reticular cells in the vascular niche of BM [25]. Collectively, these findings point to the possibility that the two compartments of the niche share common chemical factors and exert similar influences on HSCs. Evidence for a Distinctive Niche Function Distinct Spatial Distribution of HSCs in the BMs with Physiological Conditions. Despite the commonality of the two niches, the findings that HSCs are localized differentially, depending on the intrinsic or extrinsic conditions, suggest that each niche might be unique in the milieu it provides for HSCs. As described above, earlier studies on the distribution of transplanted hematopoietic cells demonstrated the selective redistribution of transplanted cells with respect to their differentiation status [14]. Recent studies, using real-time in vivo imaging of transplanted HSCs, further revealed the selective, nonrandom distribution of HSCs, where transplanted HSCs (Lin SCa-1 þ c-kit þ CD34 Flk2 ) were localized closest to the endosteum and osteoblasts, with more mature subsets (multipotent-progenitors or commited progenitors) residing progressively farther away from the endosteum [21, 49]. Moreover, HSCs were localized closer to the endosteum in irradiated mice or kit W /kitv W-v (WW v ) mice (a model for a c-kit mutation that permits engraftment under non-irradiated conditions), indicating that the conditions for engraftment of transplanted HSCs are correlated with the localization of HSCs closer to the endosteum [21, 49]. Of note, the distinct pattern of HSC

6 1248 Distinct Niches Regulate Hematopoietic Stem Cells localization was also found to be dependent on cell cycling status as well as physiological conditions, that is, during homeostatic conditions, quiescent cells were localized near the endosteum with proliferating cells distributed further from the endosteum, whereas, in irradiated BM, more proliferating cells were localized near the endosteum than in the central marrow [21, 49]. Similarly, environmental stress such as the treatment of BM with 5-FU also influenced the localization of HSCs in a such a way that Tie2 þ HSC is redistributed from the vascular-enriched central area to the bone surface adhering to osteoblasts [15]. Collectively, these distinct localizations of HSCs with respect to their functional status suggest that a certain extent of compartmentalization may exist in the BM for the distinct organization of multiple niches. Alternatively, it is also possible that each different population of hematopoietic cells are preferably home to specific types of niches, which might provide a distinct milieu for HSCs during physiological hematopoiesis. Independent Regulation of the Two Components of the Niche. Although both niches are influenced by physiological conditions, the same conditions could cause a differential response of each niche compartment. For example, treatment of BM with 5 FU suppressed the vascular niche by inducing the regression of SECs [31], but caused an elevated expression of CXCL12 in osteoblasts [52] suggesting that the pattern of HSC-niche interactions might change with physiological conditions. Similarly, our model for bis / mice, shows that a lack of functional Bis, a protein implicated in the anti-apoptosis or anti-stress response, led to the selective loss of the vascular niche, but not the osteoblastic niche [34]. Interestingly, in either case of the models, the vascular niche was found to be vulnerable to environmental stress, whereas the osteoblastic component was preserved or activated under the same conditions. The issue of whether these differences represent a distinct response to environmental stress should be further pursued. Taken together, the findings that reveal differential response of each niche to environmental stress raises the possibility that each niche is endowed with a distinct role during the physiological or environmental stress for compartmentalized regulation of HSCs. However, further studies will be necessary to confirm this hypothesis and to elucidate the interplay between the two compartments during the orchestration of hematopoietic activities. CONCLUSION Although the paradigms for interplay between stem cells and the niche are shared among many vertebrates [53], the regulation of HSC in the BM niche is becoming more complex because of the possible existence of different types of niches. Although both compartments of the niche appear to employ shared structural and functional mediators, distinctions between the two niches are emerging for the possible multiniche regulation of HSCs. One simplistic scenario is that the endosteal niche is preserved or activated during stress or stimulation, whereas central marrow regions undergo deterioration. During such stress, primitive hematopoietic cells tend to move into the endosteum and begin to proliferate. In this light, it is possible that the endosteum plays a more stimulatory role under conditions of stress, whereas the central vascular regions play a homeostatic role during steady state conditions. However, further studies will be clearly needed to confirm this scenario. First, the issue of whether the two compartments of the niche indeed provide a distinct milieu for HSCs in signaling molecules, oxygen tension, or intercellular interaction needs to be clarified. In addition, the responses of osteoblastic and vascular niches under specific stress or injury signals should be further characterized. With additional elucidation of these issues, new horizons for the functional distinction of the niche for the compartmentalized regulation of HSCs could be envisioned. ACKNOWLEDGMENTS This study was supported by a grant for high-performance cell therapy R&D project (0405-DB ) from the Ministry of Health, Welfare and Family and a grant from Korea Science and Engineering Foundation (KOSEF) ( ). DISCLOSURE OF POTENTIAL CONFLICT OF INTEREST The authors indicate no potential conflict of interest. REFERENCES 1 Dick JE, Magli MC, Huszar D et al. Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice. Cell 1985;42: Lemischka IR, Raulet DH, Mulligan RC. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell 1986;45: Szilvassy SJ, Humphries RK, Lansdorp PM et al. Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy. Proc Natl Acad Sci USA 1990;87: Dzierzak E. Embryonic beginnings of definitive hematopoietic stem cells. Ann N Y Acad Sci 1999;872: ; discussion Dzierzak E. Ontogenic emergence of definitive hematopoietic stem cells. Curr Opin Hematol 2003;10: Calvi LM, Adams GB, Weibrecht KW et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 2003;425: Zhang J, Niu C, Ye L et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 2003;425: Stier S, Ko Y, Forkert R et al. Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size. J Exp Med 2005;201: Nilsson SK, Johnston HM, Whitty GA et al. Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells. Blood 2005;106: Arai F, Suda T. Maintenance of quiescent hematopoietic stem cells in the osteoblastic niche. Ann N Y Acad Sci 2007;1106: Mayack SR, Wagers AJ. Osteolineage niche cells initiate hematopoietic stem cell mobilization. Blood 2008;112: Lord BI, Testa NG, Hendry JH. The relative spatial distributions of CFUs and CFUc in the normal mouse femur. Blood 1975;46: Gong JK. Endosteal marrow: A rich source of hematopoietic stem cells. Science 1978;199: Nilsson SK, Johnston HM, Coverdale JA. Spatial localization of transplanted hemopoietic stem cells: inferences for the localization of stem cell niches. Blood 2001;97: Arai F, Hirao A, Ohmura M et al. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004;118: Petit I, Szyper-Kravitz M, Nagler A et al. G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol 2002;3: Taichman RS, Reilly MJ, Emerson SG. Human osteoblasts support human hematopoietic progenitor cells in vitro bone marrow cultures. Blood 1996;87: Kollet O, Dar A, Shivtiel S et al. Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells. Nat Med 2006;12: Visnjic D, Kalajzic Z, Rowe DW et al. Hematopoiesis is severely altered in mice with an induced osteoblast deficiency. Blood 2004; 103:

7 Oh and Kwon Askmyr M, Sims NA, Martin TJ et al. What is the true nature of the osteoblastic hematopoietic stem cell niche? Trends Endocrinol Metab 2009;20: Xie Y, Yin T, Wiegraebe W et al. Detection of functional haematopoietic stem cell niche using real-time imaging. Nature 2009;457: Kiel MJ, Radice GL, Morrison SJ. Lack of evidence that hematopoietic stem cells depend on N-cadherin-mediated adhesion to osteoblasts for their maintenance. Cell Stem Cell 2007;1: Kiel MJ, Yilmaz OH, Iwashita T et al. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 2005;121: Sugiyama T, Kohara H, Noda M et al. Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches. Immunity 2006;25: Sacchetti B, Funari A, Michienzi S et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 2007;131: Chan CK, Chen CC, Luppen CA et al. Endochondral ossification is required for haematopoietic stem-cell niche formation. Nature 2009; 457: Ohneda O, Fennie C, Zheng Z et al. Hematopoietic stem cell maintenance and differentiation are supported by embryonic aorta-gonadmesonephros region-derived endothelium. Blood 1998;92: Li W, Johnson SA, Shelley WC et al. Hematopoietic stem cell repopulating ability can be maintained in vitro by some primary endothelial cells. Exp Hematol 2004;32: Yao L, Yokota T, Xia L et al. Bone marrow dysfunction in mice lacking the cytokine receptor gp130 in endothelial cells. Blood 2005;106: Salter AB, Meadows SK, Muramoto GG et al. Endothelial progenitor cell infusion induces hematopoietic stem cell reconstitution in vivo. Blood 2009;113: Hooper AT, Butler JM, Nolan DJ et al. Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2-mediated regeneration of sinusoidal endothelial cells. Cell Stem Cell 2009;4: Butler JM, Nolan DJ, Vertes EL et al. Endothelial cells are essential for the self-renewal and repopulation of notch-dependent hematopoietic stem cells. Cell Stem Cell 2010;6: Crisan M, Yap S, Casteilla L et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 2008;3: Kwon KR, Ahn JY, Kim MS et al. Disruption of bis leads to the deterioration of the vascular niche for hematopoietic stem cells. Stem Cells 2010;28: Youn DY, Lee DH, Lim MH et al. Bis deficiency results in early lethality with metabolic deterioration and involution of spleen and thymus. Am J Physiol Endocrinol Metab 2008;295:E1349 E Adams GB, Chabner KT, Alley IR et al. Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor. Nature 2006;439: Trowbridge JJ, Moon RT, Bhatia M. Hematopoietic stem cell biology: Too much of a Wnt thing. Nat Immunol 2006;7: Kim JA, Kang YJ, Park G et al. Identification of a stroma-mediated Wnt/beta-catenin signal promoting self-renewal of hematopoietic stem cells in the stem cell niche. Stem Cells 2009;27: Nemeth MJ, Mak KK, Yang Y et al. beta-catenin expression in the bone marrow microenvironment is required for long-term maintenance of primitive hematopoietic cells. Stem Cells 2009;27: Ahn JY, Park G, Shim JS et al. Intramarrow injection of beta-cateninactivated, but not naive mesenchymal stromal cells stimulates selfrenewal of hematopoietic stem cells in bone marrow. Exp Mol Med 2010;42: Fleming HE, Janzen V, Lo Celso C et al. Wnt signaling in the niche enforces hematopoietic stem cell quiescence and is necessary to preserve self-renewal in vivo. Cell Stem Cell 2008;2: Katayama Y, Battista M, Kao WM et al. Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow. Cell 2006;124: Mendez-Ferrer S, Lucas D, Battista M et al. Haematopoietic stem cell release is regulated by circadian oscillations. Nature 2008;452: Spiegel A, Kalinkovich A, Shivtiel S et al. Stem cell regulation via dynamic interactions of the nervous and immune systems with the microenvironment. Cell Stem Cell 2008;3: Walkley CR, Olsen GH, Dworkin S et al. A microenvironmentinduced myeloproliferative syndrome caused by retinoic acid receptor gamma deficiency. Cell 2007;129: Walkley CR, Shea JM, Sims NA et al. Rb regulates interactions between hematopoietic stem cells and their bone marrow microenvironment. Cell 2007;129: Larsson J, Ohishi M, Garrison B et al. Nf2/merlin regulates hematopoietic stem cell behavior by altering microenvironmental architecture. Cell Stem Cell 2008;3: Li Y, Chen S, Yuan J et al. Mesenchymal stem/progenitor cells promote the reconstitution of exogenous hematopoietic stem cells in Fancg-/- mice in vivo. Blood 2009;113: Lo Celso C, Fleming HE, Wu JW et al. Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature 2009;457: Caplan AI. Why are MSCs therapeutic? New data: New Insight. J Pathol 2009;217: Muguruma Y, Yahata T, Miyatake H et al. Reconstitution of the functional human hematopoietic microenvironment derived from human mesenchymal stem cells in the murine bone marrow compartment. Blood 2006;107: Ponomaryov T, Peled A, Petit I et al. Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function. J Clin Invest 2000;106: Kiel MJ, Morrison SJ. Uncertainty in the niches that maintain haematopoietic stem cells. Nat Rev Immunol 2008;8: