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The location and cellular composition of the hemopoietic stem cell niche
Cytotherapy, 2012; 14: 135–143
REVIEW
The location and cellular composition of the hemopoietic stem cell niche
SARAH L. ELLIS1 & SUSAN K. NILSSON2,3 1Peter
MacCallum Cancer Centre, Melbourne, Australia, 2CMSE, CSIRO, Clayton, Australia, and 3Department of Pathology, Melbourne University, Melbourne, Australia
Abstract While it is accepted that hemopoietic stem cells (HSC) are located in a three-dimensional microenvironment, termed a niche, the cellular and extracellular composition, as well as the multifaceted effects the components of the niche have on HSC regulation, remains undefined. Over the past four decades numerous advances in the field have led to the identification of roles for some cell types and propositions of potentially a number of HSC niches. We present evidence supporting the roles of multiple cell types and extracellular matrix molecules in the HSC niche, as well as discuss the potential significant overlap and intertwining of previously proposed distinct HSC niches. Key Words: blood vessels, bone, hemopoietic stem cells, niche
Background The concept that non-hemopoietic stromal cells surrounding hemopoietic stem cells (HSC) can direct the differentiation of these cells was shown elegantly by Trentin’s laboratory more than 40 years ago (1). He revealed that both the bone marrow (BM) and spleen stroma are ‘geographically segmented into microenvironments’, each of which directs differentiation along a specific lineage. Building on this work, Schofield (2) introduced the ‘niche’ concept to describe the specialized domains in the BM where HSC reside. In this context, the stem cell represents a ‘fixed tissue cell’ whose maturation is prevented and proliferation controlled by its association with other cells. The hypothesis is that when HSC leave the niche, they differentiate. Over the last 30 years, the niche concept has been adopted to describe the three-dimensional (3-D) location of HSC within the BM but, despite a considerable body of research, the precise anatomical location of the HSC BM niche and the cells within it remains unresolved. The niche is close to bone Almost 40 years ago, examination of the distribution of primitive hemopoietic cells [spleen colony-forming
units (CFUs)] across the longitudinal axis of the femur resulted in HSC being described as residing close to the inner surface of the bone: the endosteum (3). Subsequently, using a differential wash-out technique to fractionate the BM into central and endosteal cells, Lord et al. (4) revealed that differentiated cells were located more centrally. The observation that CFUs were more enriched near the bone was confirmed independently by a number of laboratories (5–7), although there were conflicting reports of a random CFUs distribution (8,9). Lambertsen & Weiss (10) conducted a detailed stereologic spatial assessment of marrow colonies formed post-irradiation, and found preferential seeding of undifferentiated (primitive) colonies to the endosteal regions. Collectively, this body of work provides provocative evidence that the HSC niche is located close to bone. With subsequent improvements in the methods designed to enrich for HSC, a number of laboratories, including our own CSIRO laboratory, developed new assays to examine the location of transplanted HSC. For example, our laboratory examined the location of transplanted HSC (identified using Rhodamine123/ Hoechst3342) in situ in non-ablated mice 6 weeks and 6 months post-transplant using fluorescence in situ hybridization (11,12). All donor-derived cells
Correspondence: Dr Susie K. Nilsson, CSIRO, Niche Laboratory, Bag 10, Clayton South MDC, Melbourne, 3168 Australia. E-mail: [email protected] (Received 15 September; accepted 5 October 2011) ISSN 1465-3249 print/ISSN 1477-2566 online © 2012 Informa Healthcare DOI: 10.3109/14653249.2011.630729
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were within 12 cell diameters of bone, leading to this region being classified as the endosteal niche. Although this was in line with earlier studies, it is possible that the proliferating cells migrated to the endosteum post-transplant. Consequently we developed a novel methodology for visualizing the location of transplanted HSC (Lineage– Sca-1 c-Kit ⫹ ; LSK) in the long bones at 15 h post-transplant, by labeling isolated HSC with the fluorescent tracking dye 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) prior to injecting (13). The 15-h time-point was chosen to ensure that the fate of transplanted cells and not that of their daughter progeny was analyzed (14). Using this technique, the spatial distribution of these cells could be examined more accurately and it demonstrated that primitive HSC preferentially lodged in the endosteal region whereas committed hemopoietic cells were distributed randomly throughout the BM (13). The development of assays to visualize HSC homing was an important step in advancing studies on the anatomical location of the HSC niche. Transgenic mice constitutively expressing green fluorescent protein (GFP) made it possible to follow the route of transplanted GFP ⫹ HSC over time. Yoshimoto et al. (15) injected GFP ⫹ HSC (LSK) into wild-type mice to examine the temporal and spatial behavior of these cells. However, because of the inability to visualize single cells through the thickness of cortical bone, the earliest time-point GFP ⫹ cells could be viewed with stereo microscopy was 3 days. At this time, clusters of GFP ⫹ cells were observed in the trabecular regions of femurs, ribs and vertebrae, suggesting an endosteal HSC niche. However, as with earlier work, it was possible that the progeny of transplanted cells had migrated to these regions. Most recently, advancements in optical microscopy have permitted the visualization of transplanted fluorescently tagged HSC (lineage–, LSK, LSKCD34–, LSKCD48– fetal liver kinase-2–, LSKFlk2–, Gata2-expressing 5-Fluorouracil-resistant BM cells) at very short time-points post-transplant, eliminating the complication of cells dividing (16–21). However, only the calvarium, because of the thin layer of bone covering the marrow, permits optical imaging without damage to the bone. The use of the calvarium to determine the anatomical location of the HSC niche is restrictive because it is largely composed of trabecular bone, and HSC homing kinetics and spatial distribution may not be representative of long bones, where ⬎ 75% of the marrow is considered ‘central’ marrow (13). Imaging HSC homing to long bones is difficult as these bones are optically dense. Various innovative techniques have been utilized to circumvent this, including grinding away the outer surface of the bone (16), cutting the bone to
create a window (22), slicing the bone open ex vivo (20,23) and inserting a probe into the bone (17). As blood flow is continuous between bone and cellular marrow (24), damaging this structure would cause an immediate stress response. Additionally, ablative conditioning regimes are often used to ‘free’ up niche sites prior to studying the spatial and temporal behavior of transplanted HSC in animals (17–20,25). Ablation severely alters the BM architecture, such as dilating and destroying vasculature and decreasing BM cellularity (26–32). Furthermore, ablative conditioning such as irradiation elicits the up-regulation of a number of molecules involved in the process of HSC homing (17,20,33–35) and produces changes in the microenvironment that enhance proliferation and differentiation. Collectively, these factors would alter cell homing and consequently the spatial distribution of transplanted HSC within the BM. One, two, three or more HSC niches within the BM? Within the literature, controversy remains regarding the cellular components of the HSC niche and their importance in HSC regulation. Three cell types, bone-lining osteoblasts (36,37), sinusoidal endothelial cells (38) and reticular stromal cells (39–43), have been promoted as pivotal. In addition, direct contact with each of these cell types has been suggested as a requirement for HSC regulation. The terms osteoblastic niche, vascular niche and reticular stromal niche have been used to describe these reportedly separate HSC niches (44,45). How these niches relate to each other, whether they are truly separate, overlapping or entwined (Figure 1), has generated considerable debate within the stem cell field. An overview of the evidence for the existence of each of these niches is provided below. Osteoblasts The earlier evidence proposing that the HSC niche was close to bone prompted the concept that boneforming osteoblasts directly regulate hemopoiesis. In vitro differentiated osteoblasts expand human hemopoietic progenitors (long-term culture initiating cells) when co-cultured ex vivo (46,47), as well as enhance the lodgment of HSC when co-transplanted in vivo (46,47). Genetic evidence for a role of osteoblasts within the HSC niche was provided simultaneously by two laboratories in 2003, both of which reported a concomitant increase in HSC (LSK) numbers when osteoblasts were stimulated to proliferate (36,37). The first group genetically altered mice specifically to activate the osteoblast receptor for the parathyroid hormone (PTH), leading to an increase in
HSC niche
Figure 1. Within the literature three different HSC niches have been proposed: the osteoblastic niche, the vascular niche and the reticular niche. Osteoblasts (Ob) are the main cellular component of the osteoblastic niche, blood vessels (BV) form the vascular niche, while the reticular niche proposes reticular cells (R) are pivotal components of the HSC niche.
both osteoblast numbers and HSC (LSK) (36). The overall production of the osteoblast-expressed Notch ligand, Jagged 1 (48), was also increased. As Jagged 1 bound to Notch expressed by HSC enhanced proliferation in vitro (49,50), it was suggested that the Jagged 1–Notch interaction comprised a functional component of the HSC endosteal niche. In the second study, Zhang et al. (37) used mutant mice with a conditional inactivation of the bone morphogenic protein (BMP) receptor type 1A (BMPR1A) to examine the role of BMP in regulating HSC development in vivo. Mutants increased bone formation with a simultaneous increase in HSC (LSK), suggesting that BMP signaling through BMPR1A indirectly affects HSC number by increasing the size of the endosteal niche. The authors further described a spindle-shaped N-cadherin ⫹ CD45– subset of osteoblasts (SNO cells) that were predominantly found around metaphyseal trabeculae and the cancellous bone of the epiphysis adjacent to HSC (defined in situ by expression of Stem Cell Antigen (Sca-1) and c-Kit or by the retention of 5-bromodeoxyuridine). Homophilic interactions between N-cadherin expressed on HSC and osteoblasts were proposed to maintain HSC within the niche (37,51,52). As increasing the numbers of osteoblasts results in increased numbers of HSC, does the reverse hold true? Two laboratories have investigated this premise through the use of transgenic mice with a lineage-specific expression of thymidine kinase in
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developing osteoblasts (53,54). Treatment of mice with ganciclovir results in the conditional ablation of osteoblasts followed by progressive bone loss, reduced BM cellularity (53–55) and a subsequent decrease in HSC (LSK) (53,54). In contrast, Kiel et al. (56) reported normal hemopoiesis and numbers of HSC (LSK CD150 ⫹ CD48– CD41–) in biglycan-deficient mice, despite significant reductions in trabecular bone and osteoblasts. It was suggested that osteoblasts may not even be needed for HSC regulation. However, as osteoblasts are not entirely eliminated from these mice and decline gradually over the first 24 weeks of age (57), it is possible that those remaining provided the necessary maintenance for HSC. In addition to the genetic evidence supporting the integral involvement of osteoblasts within the HSC niche, osteoblasts secrete a range of cytokines that regulate quiescence and hence maintain HSC, including thrombopoietin (58–60), osteopontin (61–63) and angiopoietin (64), as well as chemokines such as the CXC-chemokine ligand 12 (CXCL12) (35,65) that attract HSC to the niche. While there is a large body of evidence supporting a role of osteoblasts in HSC regulation, a requirement for cell–cell contact remains controversial. As described above, it is proposed that contact of HSC with osteoblasts though the Jagged 1–Notch interaction and homophilic N-cadherin adhesion is important for HSC proliferation (36,37,51,52,64,66). In addition, it has also been suggested that HSC (CD34 ⫹ ) make direct contact with osteoblasts to elicit the increased release of stromal derived factor-1 (SDF-1), leading to increased survival and proliferation (67). However, recent studies seriously question the need for intimate contact between HSC and osteoblasts for HSC regulation. Inducible gene-targeted mice for Jagged 1 revealed that its inactivation in primary BM stromal cells did not impair HSC maintenance or differentiation. In addition, Notchdeficient HSC could competitively reconstitute mice in which Jagged 1 had been inactivated (68). HSC (LSK CD150 ⫹ CD48– CD41–) do not appear to express N-cadherin in vitro and N-cadherin is not required for HSC maintenance or function in vivo (56,69,70). And as mentioned above, biglycandeficient mice have reduced numbers of osteoblasts but normal numbers of HSC and exhibit no defects in hemopoiesis (56). Furthermore, human HSC (Lin–CD34 ⫹ ) cultured in vitro with, but not touching, osteoblasts still proliferate in response to factors secreted by these cells (71). Finally, in vivo imaging of homing using fluorescently tagged transplanted HSC into either irradiated or non-irradiated mice revealed few if any HSC actually attached to osteoblasts (16,18). Osteoblasts exist within the BM in
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various stages of differentiation, with recent evidence suggesting only certain differentiation stages, which tend to be at least one cell diameter away from the bone, are important to HSC regulation (20). These cells have been suggested to be a more primitive osteoblast precursor (72,73). The lack of osteoblasts in sites of extramedullary hemopoiesis suggests that their contribution to HSC regulation is dispensable. Collectively, these data suggest that osteoblasts regulate HSC by the secretion of various factors but that cell–cell contact may not be necessary. Sinusoidal endothelial cells Under normal physiologic conditions, HSC periodically move out of and then return to the BM (74–81), migrating through BM sinusoids (82,83). The sinusoids are critical for HSC homing to the BM niche and interact with HSC through adhesion molecules and chemokines expressed on the surface of the sinusoidal endothelium. Recently, sinusoidal endothelial cells have been suggested to play an even larger role in HSC physiology. Sipkins et al. (82) imaged primitive hemopoietic cells (LSK mixed with Lin– Sca ⫹ and Lin– Kit ⫹ cells) homing to the trabecular-rich calvarium. Seventy days post-transplant, these cells were detected next to blood vessels and it was proposed that the sinusoidal endothelium was important in the regulation of HSC. In addition, Kiel et al. (84) demonstrated that 60% of HSC identified in sections of long bone by the Signaling lymphocyte activation molecule (SLAM) repertoire of markers (Lin– CD150 ⫹ CD48– CD41–) were located next to sinusoids, with almost all within five cells of sinusoids (56). Furthermore, the majority of these cells (57%) were also amongst trabecular bone (84). The positioning of HSC within regions of trabecular bone raises the possibility that these cells are touching or close to osteoblasts lying outside the plane of section and emphasizes the difficulty of gauging the proximity of one cell type to another in two-dimensional (2-D) sections. These studies led to the term ‘vascular niche’. One caveat regarding the existence of a separate vascular niche is the location of the blood vessels themselves. As described above, the BM is richly vascularized, with blood vessels permeating throughout the cellular marrow. If the vascular niche is a truly independent HSC niche, HSC would be expected to be uniformly distributed throughout the marrow instead of localized to specific areas. It is possible that the sinusoidal vessels in long bones differentially express certain molecules that influence the location of HSC, as has been reported in the calvarium (82), but this is yet to be examined extensively. Very recently, we demonstrated that hyaluronic acid
is highly expressed on the blood vessel endothelium in the metaphyseal region of long bones, and this plays a previously unrecognized role in directing the homing of HSC and progenitors (LSK) to the metaphysis (85). Another consideration for HSC adjacent to BM vasculature is that they may be in transit either in or out of the BM. However, the high percentage of HSC (Lin–CD150 ⫹ CD48– CD41–) observed adjacent to vasculature (56,86) coupled with the very low numbers of HSC circulating during homeostasis (74–81) argues against this. Given the heterogeneity of isolated HSC populations, which is made up of variable numbers of progenitor cells, it has also been proposed that different HSC subsets localize to either the sinusoidal endothelium or osteoblasts, with more differentiated progenitors localizing to the endothelium and more primitive subsets localizing to osteoblasts (87–92). However, this has yet to be proven. We have now utilized a multifaceted approach to investigate the micro-anatomical location of HSC (LSK) with respect to blood vessels and bone in long bones without damage to either the bone or BM. A combination of micro-computed tomography, histomorphometry, homing and spatial distribution assays has demonstrated that HSC preferentially home to the metaphyseal region, or an endosteal niche, in which blood vessels are integral but do not form a separate niche (85). Reticular stromal cells More than 40 years ago, ultrastructural studies of the BM revealed a close association of adventitial reticular cells with both the sinusoidal endothelium and hemopoietic cells (42,93). The adventitial reticular cells extended cytoplasmic processes into the cellular marrow, forming a reticular network upon which hemopoietic cells rested (24,42). This observation led to the suggestion that adventitial reticular cells participated in the regulation of hemopoiesis (93). More recently, using GFP– CXCL12 knock-in mice, reticular cells were shown to express high concentrations of the HSC chemoattractant CXCL12. In addition, 97% of HSC (CD150 ⫹ CD48– CD41–) were in direct contact with these cells (41). As the CXCL12-abundant reticular (CAR) cells occurred in both the vascular and osteoblastic niches, it was further suggested that CAR cells may be a key element linking the vascular niche with the osteoblastic niche. What is unclear is the region where HSC are located, with these cells being described as ‘scattered throughout the BM’. Previously, phenotypically similar ‘barrier’ cells (identified ultrastructurally as dark reticular
HSC niche cells) were described as restricted to the distal medial metaphysis (43). These cells were proposed to envelope putative HSC and provide a regulatory barrier to the premature release of primitive cells into the blood. As reticular cells extend long thin processes throughout the cellular marrow, the probability of an HSC residing next to a CAR cell by chance needs to be determined. Definitive proof of a regulatory role for reticular cells in HSC maintenance remains to be shown. Indirect evidence for the role of reticular cells in the regulation of HSC was provided 20 years ago by Yamazaki & Allen (94), who performed a rigorous ultrastructural examination of the BM and established that peri-arterial reticular cells, which surrounded both arteries and associated nerves, were connected with stromal reticular cells and per-sinusoidal reticular cells by gap junctions. This reticular cell network was proposed to form a ‘neuroreticular complex’, which was hypothesized to regulate cell egress and the release of growth factors. This hypothesis lay dormant until recently, when the role of the sympathetic nervous system (SNS) in controlling cell egress gained momentum. Katayama et al. (95) demonstrated that granulocyte–colony-stimulating factor (G-CSF)-induced mobilization of HSC was absent in mice deficient in an enzyme required for myelination of nerves and normal nerve conduction. Furthermore, the release of HSC into the blood was shown to follow a circadian rhythm in anti-phase, with the expression of CXCL12 under the control of the SNS (79). In the latter study, transgenic mice expressing GFP under the regulatory elements of the Nestin promoter revealed that the BM cells targeted by the SNS were CD45– Nestin ⫹ perivascular stromal cells expressing high levels of CXCL12. In addition, immunohistochemistry demonstrated that 60% of HSC (Lin– CD150 ⫹ CD48–) were adjacent to CXCL12-expressing Nestin ⫹ cells, with 90% of HSC located within five cells. Interestingly, these percentages of HSC adjacent to, or in close proximity to, the Nestin ⫹ cells closely corresponds with the figures presented by Kiel et al. (84) for HSC adjacent to blood vessels. A functional relationship between Nestin ⫹ cells and HSC was suggested based on the expression of a number of HSC-retention signals (vascular cell adhesion molecule 1 (VCAM-1), stem cell factor SCF (KitL), CXCL12, angiopoietin 1 and interleukin (IL)-7) by these cells (79). While it is tantalizing to speculate that the Nestin ⫹ cells and CAR cells are one and the same and that both communicate with other reticular cells in the cellular marrow through gap junctions, this has yet to be demonstrated. Also yet to be determined is the importance of CXCL12-expressing reticular cells compared with other stromal cells that express
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CXCL12, such as osteoblasts, in HSC physiology and the direct involvement of reticular cells in HSC regulation. The HSC regulatory capabilities of cells other than osteoblasts, sinusoidal endothelial cells and reticular cells The HSC niche is a 3-D structure and logically must incorporate cells other than osteoblasts, sinusoidal endothelial cells and reticular cells. One such possible cell is the osteoclast. Osteoclasts exist in close association with osteoblasts, forming a functional bone remodeling unit (reviewed in 96). Osteoclasts are derived from fused monocytes and are involved in the mobilization of HSC during both homeostasis and stress-induced conditions (97). Stimulation of osteoclasts through mild bleeding, lipopolysaccharide (LPS), which mimics bacterial infection, or via the specific osteoclast-differentiating cytokine, receptor activator of nuclear factor-κB ligand (RANKL), results in an increase in osteoclasts and concomitant increase in HSC mobilization (97). Stimulated osteoclasts secrete enzymes that cleave osteoblast-expressed niche molecules, including CXCL12 and membranebound stem cell factor (SCF; c-Kit ligand), and also lead to a decrease in the expression of osteopontin, culminating in a release of HSC from the niche (97). This suggests a delicate balance between osteoblasts, the production of HSC-regulating cytokines and HSC niche function (97). The release of calcium is also associated with bone remodeling by osteoclasts (98). Mononuclear cells from the fetal liver of knockout mice deficient in the calcium receptor were capable of homing to the BM post-transplant but, unlike wild-type controls, were unable to lodge within the endosteal niche. This suggests calcium concentrations are important in retaining HSC within the niche (99). Together, these results suggest a dual role for osteoclasts in the HSC niche, influencing both release and maintenance of HSC. In addition to osteoclasts, it has been revealed that adipocytes secrete a number of proteins, or adipokines, that can influence hemopoiesis (100) and appear in the femoral BM 7 days post-irradiation, or the time when hemopoiesis is initiated (94). These observations suggest a role of adipocytes in hemopoiesis. This hypothesis was recently strengthened with a study revealing adipocytes as negative regulators of HSC (LSK Flk2–) through a combination of in vitro and in vivo assays (101). HSC from fatty areas of the marrow are reduced in number compared with non-fatty marrow, and HSC transplanted into irradiated ‘fatless’ mice or mice treated with an inhibitor of adipogenesis exhibit accelerated engraftment compared with wild-type
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or untreated recipients. Additional evidence for a role of adipocytes in regulating hemopoiesis was derived from a study on ovariectomized mice (102). The ovariectomized mice exhibited a decrease in the volume of hemopoietic tissue and osteoblasts and an increase in adipose tissue and osteoclasts, suggesting a three-way co-regulation of hemopoiesis, osteogenesis and adipogenesis. One further cell that has gained momentum in the literature as a putative HSC regulator is the megakaryocyte. Megakaryocytes are usually associated with a vascular niche, yet this cell has been implicated in the regulation of osteoblasts and osteocytes through in vitro cultures. Co-cultures of megakaryocytes with osteoclasts inhibited osteoclast numbers and activity (103,104). Conversely, co-culture of megakaryocytes with osteoblasts increased proliferation of these cells (105) and modulated their expression of osteoblast-secreted factors that influence bone remodeling (106). Most recently, using immunohistochemistry, megakaryocytes were implicated in restoring the osteoblastic niche postirradiation (107). By impacting on bone remodeling, megakaryocytes have the potential to affect HSC lodgment and regulation within the niche, suggesting an indirect influence of megakaryocytes on HSC. Conclusion Overall, the HSC niche is the best understood in mammals, with multiple cell types and extracellular molecules being identified as playing key roles in the attraction, retention and regulation of stem cells to specific regions of the marrow. However, the concept of multiple unique and distinct ‘niches’, comprising a dominant cell type (e.g. osteoblastic, vascular and reticular niches) versus multifaceted intertwined niches, where a combination of these key cell types plays synergistic regulatory roles, still causes significant debate within the field. Given the importance of the hemopoietic system, it is highly unlikely that one cell type is solely responsible for the regulation of these rare pluripotent cells, offering a plausible explanation for the overlap in marrow cells expressing the same stem cell-regulatory molecules. It is only through the development of new animal models, unique stem cell-identifying reagents and improved techniques for stem cell visualization within dense structures, that all the interactions of the microenvironment and the stem cells that live within it will ultimately be defined. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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REVIEW
The location and cellular composition of the hemopoietic stem cell niche
SARAH L. ELLIS1 & SUSAN K. NILSSON2,3 1Peter
MacCallum Cancer Centre, Melbourne, Australia, 2CMSE, CSIRO, Clayton, Australia, and 3Department of Pathology, Melbourne University, Melbourne, Australia
Abstract While it is accepted that hemopoietic stem cells (HSC) are located in a three-dimensional microenvironment, termed a niche, the cellular and extracellular composition, as well as the multifaceted effects the components of the niche have on HSC regulation, remains undefined. Over the past four decades numerous advances in the field have led to the identification of roles for some cell types and propositions of potentially a number of HSC niches. We present evidence supporting the roles of multiple cell types and extracellular matrix molecules in the HSC niche, as well as discuss the potential significant overlap and intertwining of previously proposed distinct HSC niches. Key Words: blood vessels, bone, hemopoietic stem cells, niche
Background The concept that non-hemopoietic stromal cells surrounding hemopoietic stem cells (HSC) can direct the differentiation of these cells was shown elegantly by Trentin’s laboratory more than 40 years ago (1). He revealed that both the bone marrow (BM) and spleen stroma are ‘geographically segmented into microenvironments’, each of which directs differentiation along a specific lineage. Building on this work, Schofield (2) introduced the ‘niche’ concept to describe the specialized domains in the BM where HSC reside. In this context, the stem cell represents a ‘fixed tissue cell’ whose maturation is prevented and proliferation controlled by its association with other cells. The hypothesis is that when HSC leave the niche, they differentiate. Over the last 30 years, the niche concept has been adopted to describe the three-dimensional (3-D) location of HSC within the BM but, despite a considerable body of research, the precise anatomical location of the HSC BM niche and the cells within it remains unresolved. The niche is close to bone Almost 40 years ago, examination of the distribution of primitive hemopoietic cells [spleen colony-forming
units (CFUs)] across the longitudinal axis of the femur resulted in HSC being described as residing close to the inner surface of the bone: the endosteum (3). Subsequently, using a differential wash-out technique to fractionate the BM into central and endosteal cells, Lord et al. (4) revealed that differentiated cells were located more centrally. The observation that CFUs were more enriched near the bone was confirmed independently by a number of laboratories (5–7), although there were conflicting reports of a random CFUs distribution (8,9). Lambertsen & Weiss (10) conducted a detailed stereologic spatial assessment of marrow colonies formed post-irradiation, and found preferential seeding of undifferentiated (primitive) colonies to the endosteal regions. Collectively, this body of work provides provocative evidence that the HSC niche is located close to bone. With subsequent improvements in the methods designed to enrich for HSC, a number of laboratories, including our own CSIRO laboratory, developed new assays to examine the location of transplanted HSC. For example, our laboratory examined the location of transplanted HSC (identified using Rhodamine123/ Hoechst3342) in situ in non-ablated mice 6 weeks and 6 months post-transplant using fluorescence in situ hybridization (11,12). All donor-derived cells
Correspondence: Dr Susie K. Nilsson, CSIRO, Niche Laboratory, Bag 10, Clayton South MDC, Melbourne, 3168 Australia. E-mail: [email protected] (Received 15 September; accepted 5 October 2011) ISSN 1465-3249 print/ISSN 1477-2566 online © 2012 Informa Healthcare DOI: 10.3109/14653249.2011.630729
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were within 12 cell diameters of bone, leading to this region being classified as the endosteal niche. Although this was in line with earlier studies, it is possible that the proliferating cells migrated to the endosteum post-transplant. Consequently we developed a novel methodology for visualizing the location of transplanted HSC (Lineage– Sca-1 c-Kit ⫹ ; LSK) in the long bones at 15 h post-transplant, by labeling isolated HSC with the fluorescent tracking dye 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) prior to injecting (13). The 15-h time-point was chosen to ensure that the fate of transplanted cells and not that of their daughter progeny was analyzed (14). Using this technique, the spatial distribution of these cells could be examined more accurately and it demonstrated that primitive HSC preferentially lodged in the endosteal region whereas committed hemopoietic cells were distributed randomly throughout the BM (13). The development of assays to visualize HSC homing was an important step in advancing studies on the anatomical location of the HSC niche. Transgenic mice constitutively expressing green fluorescent protein (GFP) made it possible to follow the route of transplanted GFP ⫹ HSC over time. Yoshimoto et al. (15) injected GFP ⫹ HSC (LSK) into wild-type mice to examine the temporal and spatial behavior of these cells. However, because of the inability to visualize single cells through the thickness of cortical bone, the earliest time-point GFP ⫹ cells could be viewed with stereo microscopy was 3 days. At this time, clusters of GFP ⫹ cells were observed in the trabecular regions of femurs, ribs and vertebrae, suggesting an endosteal HSC niche. However, as with earlier work, it was possible that the progeny of transplanted cells had migrated to these regions. Most recently, advancements in optical microscopy have permitted the visualization of transplanted fluorescently tagged HSC (lineage–, LSK, LSKCD34–, LSKCD48– fetal liver kinase-2–, LSKFlk2–, Gata2-expressing 5-Fluorouracil-resistant BM cells) at very short time-points post-transplant, eliminating the complication of cells dividing (16–21). However, only the calvarium, because of the thin layer of bone covering the marrow, permits optical imaging without damage to the bone. The use of the calvarium to determine the anatomical location of the HSC niche is restrictive because it is largely composed of trabecular bone, and HSC homing kinetics and spatial distribution may not be representative of long bones, where ⬎ 75% of the marrow is considered ‘central’ marrow (13). Imaging HSC homing to long bones is difficult as these bones are optically dense. Various innovative techniques have been utilized to circumvent this, including grinding away the outer surface of the bone (16), cutting the bone to
create a window (22), slicing the bone open ex vivo (20,23) and inserting a probe into the bone (17). As blood flow is continuous between bone and cellular marrow (24), damaging this structure would cause an immediate stress response. Additionally, ablative conditioning regimes are often used to ‘free’ up niche sites prior to studying the spatial and temporal behavior of transplanted HSC in animals (17–20,25). Ablation severely alters the BM architecture, such as dilating and destroying vasculature and decreasing BM cellularity (26–32). Furthermore, ablative conditioning such as irradiation elicits the up-regulation of a number of molecules involved in the process of HSC homing (17,20,33–35) and produces changes in the microenvironment that enhance proliferation and differentiation. Collectively, these factors would alter cell homing and consequently the spatial distribution of transplanted HSC within the BM. One, two, three or more HSC niches within the BM? Within the literature, controversy remains regarding the cellular components of the HSC niche and their importance in HSC regulation. Three cell types, bone-lining osteoblasts (36,37), sinusoidal endothelial cells (38) and reticular stromal cells (39–43), have been promoted as pivotal. In addition, direct contact with each of these cell types has been suggested as a requirement for HSC regulation. The terms osteoblastic niche, vascular niche and reticular stromal niche have been used to describe these reportedly separate HSC niches (44,45). How these niches relate to each other, whether they are truly separate, overlapping or entwined (Figure 1), has generated considerable debate within the stem cell field. An overview of the evidence for the existence of each of these niches is provided below. Osteoblasts The earlier evidence proposing that the HSC niche was close to bone prompted the concept that boneforming osteoblasts directly regulate hemopoiesis. In vitro differentiated osteoblasts expand human hemopoietic progenitors (long-term culture initiating cells) when co-cultured ex vivo (46,47), as well as enhance the lodgment of HSC when co-transplanted in vivo (46,47). Genetic evidence for a role of osteoblasts within the HSC niche was provided simultaneously by two laboratories in 2003, both of which reported a concomitant increase in HSC (LSK) numbers when osteoblasts were stimulated to proliferate (36,37). The first group genetically altered mice specifically to activate the osteoblast receptor for the parathyroid hormone (PTH), leading to an increase in
HSC niche
Figure 1. Within the literature three different HSC niches have been proposed: the osteoblastic niche, the vascular niche and the reticular niche. Osteoblasts (Ob) are the main cellular component of the osteoblastic niche, blood vessels (BV) form the vascular niche, while the reticular niche proposes reticular cells (R) are pivotal components of the HSC niche.
both osteoblast numbers and HSC (LSK) (36). The overall production of the osteoblast-expressed Notch ligand, Jagged 1 (48), was also increased. As Jagged 1 bound to Notch expressed by HSC enhanced proliferation in vitro (49,50), it was suggested that the Jagged 1–Notch interaction comprised a functional component of the HSC endosteal niche. In the second study, Zhang et al. (37) used mutant mice with a conditional inactivation of the bone morphogenic protein (BMP) receptor type 1A (BMPR1A) to examine the role of BMP in regulating HSC development in vivo. Mutants increased bone formation with a simultaneous increase in HSC (LSK), suggesting that BMP signaling through BMPR1A indirectly affects HSC number by increasing the size of the endosteal niche. The authors further described a spindle-shaped N-cadherin ⫹ CD45– subset of osteoblasts (SNO cells) that were predominantly found around metaphyseal trabeculae and the cancellous bone of the epiphysis adjacent to HSC (defined in situ by expression of Stem Cell Antigen (Sca-1) and c-Kit or by the retention of 5-bromodeoxyuridine). Homophilic interactions between N-cadherin expressed on HSC and osteoblasts were proposed to maintain HSC within the niche (37,51,52). As increasing the numbers of osteoblasts results in increased numbers of HSC, does the reverse hold true? Two laboratories have investigated this premise through the use of transgenic mice with a lineage-specific expression of thymidine kinase in
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developing osteoblasts (53,54). Treatment of mice with ganciclovir results in the conditional ablation of osteoblasts followed by progressive bone loss, reduced BM cellularity (53–55) and a subsequent decrease in HSC (LSK) (53,54). In contrast, Kiel et al. (56) reported normal hemopoiesis and numbers of HSC (LSK CD150 ⫹ CD48– CD41–) in biglycan-deficient mice, despite significant reductions in trabecular bone and osteoblasts. It was suggested that osteoblasts may not even be needed for HSC regulation. However, as osteoblasts are not entirely eliminated from these mice and decline gradually over the first 24 weeks of age (57), it is possible that those remaining provided the necessary maintenance for HSC. In addition to the genetic evidence supporting the integral involvement of osteoblasts within the HSC niche, osteoblasts secrete a range of cytokines that regulate quiescence and hence maintain HSC, including thrombopoietin (58–60), osteopontin (61–63) and angiopoietin (64), as well as chemokines such as the CXC-chemokine ligand 12 (CXCL12) (35,65) that attract HSC to the niche. While there is a large body of evidence supporting a role of osteoblasts in HSC regulation, a requirement for cell–cell contact remains controversial. As described above, it is proposed that contact of HSC with osteoblasts though the Jagged 1–Notch interaction and homophilic N-cadherin adhesion is important for HSC proliferation (36,37,51,52,64,66). In addition, it has also been suggested that HSC (CD34 ⫹ ) make direct contact with osteoblasts to elicit the increased release of stromal derived factor-1 (SDF-1), leading to increased survival and proliferation (67). However, recent studies seriously question the need for intimate contact between HSC and osteoblasts for HSC regulation. Inducible gene-targeted mice for Jagged 1 revealed that its inactivation in primary BM stromal cells did not impair HSC maintenance or differentiation. In addition, Notchdeficient HSC could competitively reconstitute mice in which Jagged 1 had been inactivated (68). HSC (LSK CD150 ⫹ CD48– CD41–) do not appear to express N-cadherin in vitro and N-cadherin is not required for HSC maintenance or function in vivo (56,69,70). And as mentioned above, biglycandeficient mice have reduced numbers of osteoblasts but normal numbers of HSC and exhibit no defects in hemopoiesis (56). Furthermore, human HSC (Lin–CD34 ⫹ ) cultured in vitro with, but not touching, osteoblasts still proliferate in response to factors secreted by these cells (71). Finally, in vivo imaging of homing using fluorescently tagged transplanted HSC into either irradiated or non-irradiated mice revealed few if any HSC actually attached to osteoblasts (16,18). Osteoblasts exist within the BM in
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various stages of differentiation, with recent evidence suggesting only certain differentiation stages, which tend to be at least one cell diameter away from the bone, are important to HSC regulation (20). These cells have been suggested to be a more primitive osteoblast precursor (72,73). The lack of osteoblasts in sites of extramedullary hemopoiesis suggests that their contribution to HSC regulation is dispensable. Collectively, these data suggest that osteoblasts regulate HSC by the secretion of various factors but that cell–cell contact may not be necessary. Sinusoidal endothelial cells Under normal physiologic conditions, HSC periodically move out of and then return to the BM (74–81), migrating through BM sinusoids (82,83). The sinusoids are critical for HSC homing to the BM niche and interact with HSC through adhesion molecules and chemokines expressed on the surface of the sinusoidal endothelium. Recently, sinusoidal endothelial cells have been suggested to play an even larger role in HSC physiology. Sipkins et al. (82) imaged primitive hemopoietic cells (LSK mixed with Lin– Sca ⫹ and Lin– Kit ⫹ cells) homing to the trabecular-rich calvarium. Seventy days post-transplant, these cells were detected next to blood vessels and it was proposed that the sinusoidal endothelium was important in the regulation of HSC. In addition, Kiel et al. (84) demonstrated that 60% of HSC identified in sections of long bone by the Signaling lymphocyte activation molecule (SLAM) repertoire of markers (Lin– CD150 ⫹ CD48– CD41–) were located next to sinusoids, with almost all within five cells of sinusoids (56). Furthermore, the majority of these cells (57%) were also amongst trabecular bone (84). The positioning of HSC within regions of trabecular bone raises the possibility that these cells are touching or close to osteoblasts lying outside the plane of section and emphasizes the difficulty of gauging the proximity of one cell type to another in two-dimensional (2-D) sections. These studies led to the term ‘vascular niche’. One caveat regarding the existence of a separate vascular niche is the location of the blood vessels themselves. As described above, the BM is richly vascularized, with blood vessels permeating throughout the cellular marrow. If the vascular niche is a truly independent HSC niche, HSC would be expected to be uniformly distributed throughout the marrow instead of localized to specific areas. It is possible that the sinusoidal vessels in long bones differentially express certain molecules that influence the location of HSC, as has been reported in the calvarium (82), but this is yet to be examined extensively. Very recently, we demonstrated that hyaluronic acid
is highly expressed on the blood vessel endothelium in the metaphyseal region of long bones, and this plays a previously unrecognized role in directing the homing of HSC and progenitors (LSK) to the metaphysis (85). Another consideration for HSC adjacent to BM vasculature is that they may be in transit either in or out of the BM. However, the high percentage of HSC (Lin–CD150 ⫹ CD48– CD41–) observed adjacent to vasculature (56,86) coupled with the very low numbers of HSC circulating during homeostasis (74–81) argues against this. Given the heterogeneity of isolated HSC populations, which is made up of variable numbers of progenitor cells, it has also been proposed that different HSC subsets localize to either the sinusoidal endothelium or osteoblasts, with more differentiated progenitors localizing to the endothelium and more primitive subsets localizing to osteoblasts (87–92). However, this has yet to be proven. We have now utilized a multifaceted approach to investigate the micro-anatomical location of HSC (LSK) with respect to blood vessels and bone in long bones without damage to either the bone or BM. A combination of micro-computed tomography, histomorphometry, homing and spatial distribution assays has demonstrated that HSC preferentially home to the metaphyseal region, or an endosteal niche, in which blood vessels are integral but do not form a separate niche (85). Reticular stromal cells More than 40 years ago, ultrastructural studies of the BM revealed a close association of adventitial reticular cells with both the sinusoidal endothelium and hemopoietic cells (42,93). The adventitial reticular cells extended cytoplasmic processes into the cellular marrow, forming a reticular network upon which hemopoietic cells rested (24,42). This observation led to the suggestion that adventitial reticular cells participated in the regulation of hemopoiesis (93). More recently, using GFP– CXCL12 knock-in mice, reticular cells were shown to express high concentrations of the HSC chemoattractant CXCL12. In addition, 97% of HSC (CD150 ⫹ CD48– CD41–) were in direct contact with these cells (41). As the CXCL12-abundant reticular (CAR) cells occurred in both the vascular and osteoblastic niches, it was further suggested that CAR cells may be a key element linking the vascular niche with the osteoblastic niche. What is unclear is the region where HSC are located, with these cells being described as ‘scattered throughout the BM’. Previously, phenotypically similar ‘barrier’ cells (identified ultrastructurally as dark reticular
HSC niche cells) were described as restricted to the distal medial metaphysis (43). These cells were proposed to envelope putative HSC and provide a regulatory barrier to the premature release of primitive cells into the blood. As reticular cells extend long thin processes throughout the cellular marrow, the probability of an HSC residing next to a CAR cell by chance needs to be determined. Definitive proof of a regulatory role for reticular cells in HSC maintenance remains to be shown. Indirect evidence for the role of reticular cells in the regulation of HSC was provided 20 years ago by Yamazaki & Allen (94), who performed a rigorous ultrastructural examination of the BM and established that peri-arterial reticular cells, which surrounded both arteries and associated nerves, were connected with stromal reticular cells and per-sinusoidal reticular cells by gap junctions. This reticular cell network was proposed to form a ‘neuroreticular complex’, which was hypothesized to regulate cell egress and the release of growth factors. This hypothesis lay dormant until recently, when the role of the sympathetic nervous system (SNS) in controlling cell egress gained momentum. Katayama et al. (95) demonstrated that granulocyte–colony-stimulating factor (G-CSF)-induced mobilization of HSC was absent in mice deficient in an enzyme required for myelination of nerves and normal nerve conduction. Furthermore, the release of HSC into the blood was shown to follow a circadian rhythm in anti-phase, with the expression of CXCL12 under the control of the SNS (79). In the latter study, transgenic mice expressing GFP under the regulatory elements of the Nestin promoter revealed that the BM cells targeted by the SNS were CD45– Nestin ⫹ perivascular stromal cells expressing high levels of CXCL12. In addition, immunohistochemistry demonstrated that 60% of HSC (Lin– CD150 ⫹ CD48–) were adjacent to CXCL12-expressing Nestin ⫹ cells, with 90% of HSC located within five cells. Interestingly, these percentages of HSC adjacent to, or in close proximity to, the Nestin ⫹ cells closely corresponds with the figures presented by Kiel et al. (84) for HSC adjacent to blood vessels. A functional relationship between Nestin ⫹ cells and HSC was suggested based on the expression of a number of HSC-retention signals (vascular cell adhesion molecule 1 (VCAM-1), stem cell factor SCF (KitL), CXCL12, angiopoietin 1 and interleukin (IL)-7) by these cells (79). While it is tantalizing to speculate that the Nestin ⫹ cells and CAR cells are one and the same and that both communicate with other reticular cells in the cellular marrow through gap junctions, this has yet to be demonstrated. Also yet to be determined is the importance of CXCL12-expressing reticular cells compared with other stromal cells that express
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CXCL12, such as osteoblasts, in HSC physiology and the direct involvement of reticular cells in HSC regulation. The HSC regulatory capabilities of cells other than osteoblasts, sinusoidal endothelial cells and reticular cells The HSC niche is a 3-D structure and logically must incorporate cells other than osteoblasts, sinusoidal endothelial cells and reticular cells. One such possible cell is the osteoclast. Osteoclasts exist in close association with osteoblasts, forming a functional bone remodeling unit (reviewed in 96). Osteoclasts are derived from fused monocytes and are involved in the mobilization of HSC during both homeostasis and stress-induced conditions (97). Stimulation of osteoclasts through mild bleeding, lipopolysaccharide (LPS), which mimics bacterial infection, or via the specific osteoclast-differentiating cytokine, receptor activator of nuclear factor-κB ligand (RANKL), results in an increase in osteoclasts and concomitant increase in HSC mobilization (97). Stimulated osteoclasts secrete enzymes that cleave osteoblast-expressed niche molecules, including CXCL12 and membranebound stem cell factor (SCF; c-Kit ligand), and also lead to a decrease in the expression of osteopontin, culminating in a release of HSC from the niche (97). This suggests a delicate balance between osteoblasts, the production of HSC-regulating cytokines and HSC niche function (97). The release of calcium is also associated with bone remodeling by osteoclasts (98). Mononuclear cells from the fetal liver of knockout mice deficient in the calcium receptor were capable of homing to the BM post-transplant but, unlike wild-type controls, were unable to lodge within the endosteal niche. This suggests calcium concentrations are important in retaining HSC within the niche (99). Together, these results suggest a dual role for osteoclasts in the HSC niche, influencing both release and maintenance of HSC. In addition to osteoclasts, it has been revealed that adipocytes secrete a number of proteins, or adipokines, that can influence hemopoiesis (100) and appear in the femoral BM 7 days post-irradiation, or the time when hemopoiesis is initiated (94). These observations suggest a role of adipocytes in hemopoiesis. This hypothesis was recently strengthened with a study revealing adipocytes as negative regulators of HSC (LSK Flk2–) through a combination of in vitro and in vivo assays (101). HSC from fatty areas of the marrow are reduced in number compared with non-fatty marrow, and HSC transplanted into irradiated ‘fatless’ mice or mice treated with an inhibitor of adipogenesis exhibit accelerated engraftment compared with wild-type
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or untreated recipients. Additional evidence for a role of adipocytes in regulating hemopoiesis was derived from a study on ovariectomized mice (102). The ovariectomized mice exhibited a decrease in the volume of hemopoietic tissue and osteoblasts and an increase in adipose tissue and osteoclasts, suggesting a three-way co-regulation of hemopoiesis, osteogenesis and adipogenesis. One further cell that has gained momentum in the literature as a putative HSC regulator is the megakaryocyte. Megakaryocytes are usually associated with a vascular niche, yet this cell has been implicated in the regulation of osteoblasts and osteocytes through in vitro cultures. Co-cultures of megakaryocytes with osteoclasts inhibited osteoclast numbers and activity (103,104). Conversely, co-culture of megakaryocytes with osteoblasts increased proliferation of these cells (105) and modulated their expression of osteoblast-secreted factors that influence bone remodeling (106). Most recently, using immunohistochemistry, megakaryocytes were implicated in restoring the osteoblastic niche postirradiation (107). By impacting on bone remodeling, megakaryocytes have the potential to affect HSC lodgment and regulation within the niche, suggesting an indirect influence of megakaryocytes on HSC. Conclusion Overall, the HSC niche is the best understood in mammals, with multiple cell types and extracellular molecules being identified as playing key roles in the attraction, retention and regulation of stem cells to specific regions of the marrow. However, the concept of multiple unique and distinct ‘niches’, comprising a dominant cell type (e.g. osteoblastic, vascular and reticular niches) versus multifaceted intertwined niches, where a combination of these key cell types plays synergistic regulatory roles, still causes significant debate within the field. Given the importance of the hemopoietic system, it is highly unlikely that one cell type is solely responsible for the regulation of these rare pluripotent cells, offering a plausible explanation for the overlap in marrow cells expressing the same stem cell-regulatory molecules. It is only through the development of new animal models, unique stem cell-identifying reagents and improved techniques for stem cell visualization within dense structures, that all the interactions of the microenvironment and the stem cells that live within it will ultimately be defined. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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