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What is the true nature of the osteoblastic hematopoietic stem cell niche?
Review
What is the true nature of the osteoblastic hematopoietic stem cell niche? Maria Askmyr1, Natalie A. Sims1,2, T. John Martin1,2 and Louise E. Purton1,2 1 2
St Vincent’s Institute, Fitzroy, Victoria, 3065, Australia Department of Medicine, The University of Melbourne, Fitzroy, Victoria, 3065, Australia
The recently revitalized interest in the regulation of hematopoietic stem cells (HSCs) by the bone marrow microenvironment has resulted in the identification of some important cell types that potentially form the HSC niche. The term ‘osteoblast’ has commonly been used to describe the endosteal elements of the HSC niche, but these cells are part of a larger family that functions in bone at different stages of differentiation. Given that there is much controversy as to what cell types have important roles in the HSC niche, this review offers an overview of the diverse osteoblastic cell types and discusses the current evidence regarding what roles they have in the HSC niche. The identification of the adult hematopoietic stem cell niche The bone marrow (BM) cavity serves as the main site of hematopoiesis in the human adult. This lifelong process of constant replenishment of mature hematopoietic cells is sustained by the hematopoietic stem cells (HSCs). To accomplish this, it is thought that HSCs have the potential to self-renew to maintain the HSC pool. In 1978, Schofield proposed that HSCs needed to be localized in a particular location (termed the HSC niche) within the BM to retain their multipotency [1] and that if the HSCs were located elsewhere, they would probably commit to differentiation rather than self-renew [1]. However, until recently, the nature of the HSC niche was largely unknown. It should be noted here that sites of hematopoiesis differ between rodents and humans; whereas mice sustain blood formation in all bones and spleen throughout life, human hematopoiesis is restricted to the proximal regions of long bones, cranium, sternum, ribs, vertebra and ilium [2]. The studies discussed below were predominantly performed in the mouse model, and it is not clear whether all the properties and components of the HSC niche described in these models are translatable to humans. However, the mouse model, to date, is the best small animal model of bone and hematopoiesis and enables extensive in vivo analyses that cannot be performed in humans. A series of studies in 2003 and 2005 described two different types of HSC niches: one at the bone surfaces in contact with BM and the other in proximity to the BM vasculature [3–6]. These are commonly referred to as the Corresponding author: Purton, L.E. ([email protected]).
osteoblastic (or endosteal) and perivascular niches, respectively. Furthermore, cells of the sympathetic nervous system were recently identified as having important roles in retaining HSCs in the BM [7–10]. Given the complexity of the three-dimensional structure of BM, it is unclear whether these niches are distinct or overlap in both location and function [11]. The contributions of each niche to HSC regulation are currently the subject of ongoing investigations in various laboratories and have been recently reviewed in detail elsewhere [11–16]. This review focuses on recent findings regarding the nature of cells of the osteoblast lineage that probably participate in the HSC niche. Roles of cells of the osteoblast lineage in the HSC niche The development of long-term BM cultures in the 1970s described methods whereby hematopoiesis could be sustained in culture for many months [17]. The success of this culture system relied on the development of adherent cells, termed ‘stromal cells’. The identity of the different types of stromal cells remained elusive; however, these stromal cells expressed alkaline phosphatase (an enzyme present in, but not exclusive to, osteoblast lineage cells) [18]. Almost 20 years later, a series of reports demonstrated that human osteoblasts could support hematopoiesis in ex vivo culture systems [19–21]. These human osteoblasts were defined as alkaline phosphatase-expressing cells and, more importantly, were shown to be capable of mineralization in vitro, clearly demonstrating their osteoblastic nature [19–21]. These in vitro findings were the first indication that cells of the osteoblastic lineage had important roles in the regulation of HSCs. Three studies utilizing genetically modified mice further supported roles for cells of the osteoblast lineage in the retention and regulation of HSCs in the BM [3,4,22]. However, these findings also resulted in some confusion in the field regarding which cell type of the osteoblast lineage was important. For example, the interchangeable terms of ‘osteoblast niche’ and ‘endosteal niche’ became common terminology when referring to the HSC niche. As we will discuss in detail below, the nature of the participating cell types of the HSC niche is still to be clearly determined. Misleading terminology in describing the HSC niche As has been raised recently by Bianco [23], the term ‘osteoblastic niche’ is oversimplified and potentially incorrect in regard to which cells actively regulate HSC maintenance
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Figure 1. Differentiation of skeletal stem cells along the osteoblast lineage in bone marrow. Black arrows indicate the direction of differentiation, starting with (i) the immature skeletal stem cells. Dashed red arrows indicate potential differentiation pathways. Genes expressed in each cell type are listed below each cell type, with key proteins in bold and transcription factors in italics. The symbol ‘?’ indicates that mRNA expression has been demonstrated but protein studies have not yet been performed or that the cell type expressing the protein – (ii) pre-osteoblasts, (iii) osteoblasts, (iv) bone-lining cells or (v) osteocytes – has not been conclusively identified. Abbreviations: ALP, alkaline phosphatase; Ang-1, angiopoietin-1; DMP1, dental matrix protein 1; Jag-1, Jagged-1; N-cad, N-cadherin; PTH1R, receptor for PTH/PTHrP; SOST, sclerostin.
and self-renewal. Researchers have used the generic term ‘osteoblasts’ to refer to a spectrum of cell types of the osteoblastic lineage. However, osteoblasts are a well-defined population of differentiated cells actively involved in bone formation. The terminology ‘endosteal niche’ is also misrepresentative because the endosteal surface of bone includes all cells that line the interface between bone and marrow, including both endocortical (i.e. inner cortical) and trabecular surfaces. In contrast to this, most studies support the trabecular region of bone as the most important anatomical location for the HSC niche [3,4], whereas there is little evidence that cortical bone plays an important part with respect to the HSC niche [11]. Note that although hematopoietic cells have been well defined by fluorescence-activated cell sorting (FACS) techniques and assays of hematopoietic potential [24], there is much less definition of the full spectrum of cells of the osteoblast lineage, from the most immature cells (skeletal stem cells) to the most mature cells (osteocytes) (Figure 1). This is, in part, due to a lack of cell surface markers or antibodies that can be used to isolate these cells by FACS and the difficulty of performing in vivo assays of the bone lineage cells. Yet there are some distinguishing properties of cells of the osteoblast lineage, discussed below. Skeletal stem cells, adventitial reticular cells and preosteoblasts In postnatal stages, the osteoblast lineage is derived from mesenchymal stem cells (MSCs). A population of MSCs can 304
be obtained from bone marrow stromal cells (BMSCs), nonhematopoietic cells that adhere to cell culture dishes. The cells that give rise to these plastic-adherent colonies were originally identified as colony-forming fibroblasts (CFU-F) [25], and progeny of single bone-marrow-derived CFU-F differentiate into osteoblast-lineage cells, cartilage cells (chondrocytes) or adipocytes both in vivo and in vitro [26]. It remains controversial whether CFU-F also differentiate into cell types such as neurons, endothelial cells and muscle cells; hence, the terminology utilized to describe these cells has been suggested to be skeletal stem cells (SSCs) [26]. The identity of the SSCs has been predominantly restricted to functional properties, such as the ability to adhere to plastic (CFU-F-derived populations), that have identified them as a heterogeneous population of cells [27]. A recent study isolated pure populations of SSCs from human BM using a panel of antibodies; however, the phenotype of mouse SSCs is yet to be defined [26,27]. Human SSCs are found in perivascular sites in the BM [28] and express high levels of alkaline phosphatase and runt-related transcription factor 2, markers of early osteoblast lineage cells [27]. Human SSCs share similar properties to pericytes, including expression of a-smooth muscle cell actin (a-SMA) and platelet-derived growth factor receptor beta [27,29]. Like SSCs, in vitro cultured pericytes can differentiate into osteocytes, chondrocytes and adipocytes, which implies that SSCs might be pericytes or belong to a subset of pericytes; however, this is yet to be proven [29].
Review SSCs express high levels of CXCL12 [27], a chemokine with major roles in chemotaxis, homing, survival and retention of HSCs in the BM niche [30]. CXCL12 binds to CXCR4, which is expressed on HSCs. Blocking the interaction of CXCL12 and CXCR4 by an antagonist against CXCR4 results in rapid HSC mobilization from the BM into the peripheral blood, revealing the profound effects CXCL12 has on retaining HSCs in the BM microenvironment [30]. In the mouse, CXCL12 has also been shown to be highly expressed by reticular cells in the BM [31], termed ‘CXCL12-abundant reticular cells’ (CAR) and found in both perivascular and endosteal locations [31]. Although not proven, it is likely that murine CAR cells are analogous to human SSCs, sharing the same perivascular location and functional properties [23,32]. Recent studies using green fluorescent protein (GFP) transgenic mice have given more insight into the potential nature of mouse SSCs. In two complementary reports, an a-SMA–GFP reporter mouse was used to isolate a population of mesenchymal progenitor cells from murine BMSCs [33,34]. The a-SMA–GFP+ cells comprised approximately 0.02–0.03% of mononuclear cells [33] and were located perivascularly in the BM [34]. Furthermore, aSMA–GFP+ cells sorted from cultures of BMSCs contained all CFU-F potential and differentiated into osteoblasts and adipocytes [34]. These studies, therefore, provided further evidence that cells expressing a-SMA (which is a classical marker of pericytes) were potentially SSCs. In a more recent report, transgenic mice in which GFP is driven by regulatory elements of the Nestin promoter were used to isolate Nestin+CD45 and Nestin CD45 cells from BM [35]. As reported for a-SMA GFP+ cells, Nestin+CD45 cells were located in perivascular spaces, all CFU-F potential was contained in the Nestin+CD45 cells and these cells robustly differentiated into osteoblasts and adipocytes in culture [35]. Although it is likely that
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both a-SMA GFP+ cells and Nestin+CD45 cells are akin to SSCs (and CAR cells), their chondrogenic potential has not yet been tested; hence, they are currently best termed ‘mesenchymal progenitor cells’. Osteoblasts and bone lining cells The committed osteoblast population is heterogeneous, and there are at least two structurally and functionally distinct subpopulations that cover the endosteal bone surface: osteoblasts and bone lining cells [36]. Bone lining cells are frequently overlooked in studies of the HSC niche because mononuclear cells surrounding bone are commonly referred to as osteoblasts. Although both cell types are always found in close contact with the bone surface, both at the endosteum (marrow surface of bone) and the periosteum (outer bone surface) [37], actively synthesizing osteoblasts are by far outnumbered by quiescent bone lining cells [38,39]. Bone lining cells are inactive, at least in regard to the production of bone (osteoid). These flat cells stretch over the bone surface to form a protective layer, and although they are largely considered terminally differentiated, it has not yet been determined whether osteoblasts turn into lining cells [38]. Nor has it been clearly established that lining cells can become active bone-producing cells, although they have been proposed as one of the sources of osteoblasts formed in response to anabolic parathyroid hormone (PTH) [40]. Studies in transgenic mice, in which GFP is under the control of promoters of osteoblast-lineage-specific genes, have distinguished mature cells of the osteoblast lineage. Two different rat type 1 collagen (Col1a1) promoter constructs have been used to direct GFP expression to pre-osteoblasts (pOBCol3.6GFP) versus mature osteoblasts and osteocytes (pOBCol2.3GFP) (Figure 1) [41]. Because a recent report has demonstrated that bone lining cells express pOBCol2.3GFP, it can be assumed that they are mature osteoblast cells [42].
Figure 2. Histology of mouse tibia. Toluidine blue staining of undecalcified plastic-embedded tibia [(i) and (ii)] showing trabecular bone (i) and cortical bone (ii). Mineralized bone is stained dark blue, and the areas of the bone with lighter blue staining are osteoid (unmineralized bone). (iii) Hematoxylin and eosin (H&E) staining of decalcified tibia showing trabecular bone. The specific cell types are highlighted as indicated. Scale bar = 10 mm.
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Review Although specific markers that distinguish osteoblasts from bone lining cells have not been identified, these cells do have distinct morphologies. Bone lining cells have a flattened, spindle shape, whereas active osteoblasts are plump and cuboidal (Figure 2). Bone lining cells have been reported to be negative for alkaline phosphatase and osteocalcin expression, with only occasional expression of osteonectin [38], but systematic studies have not yet been carried out. In contrast to lining cells, active cuboidal osteoblasts have a high density of Golgi and endoplasmic reticulum required for their intense protein production. These cells actively lay down osteoid and regulate its mineralization to form bone, a process that requires the production of numerous proteins including type 1 collagen, osteocalcin, osteonectin, sialoprotein and osteopontin. Bone formation is tightly coupled with bone resorption by hematopoieticderived osteoclasts to regulate skeletal mass [43]. Studies linking HSCs and the osteoblast lineage: which cells of the osteoblast lineage comprise the HSC niche? The two pioneering studies describing the osteoblastic HSC niche reported that an increase in the amount of trabecular bone and/or trabecular osteoblasts correlated with increased HSC numbers [3,4]. A subsequent study demonstrated that ablating osteoblast-lineage cells in vivo resulted in relocation of most HSCs from the BM into the extramedullary sites such as spleen and liver [22]. These studies have, importantly, directed attention to the previously understudied roles of the microenvironment in regulating hematopoiesis. However, widespread misinterpretation of the term ‘osteoblastic’ as meaning mature osteoblasts has resulted in a common belief that the mature osteoblast is the cell type mediating the changes in HSC numbers. Furthermore, even though subsequent studies have revealed HSC-supportive roles for osteoblast-associated proteins, it remains to be shown that these proteins are exclusively derived from osteoblasts [15].
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Indeed, mature osteoblasts are unlikely to have a direct role as part of the HSC-supportive niche. Osteopontin, a protein expressed abundantly by osteoblasts, osteoclasts and macrophages, has been shown to be a negative regulator of HSCs [44,45]. Additional studies using Col2.3D thymidine kinase (TK) transgenic mice have also been used to explore the effects of mature osteoblasts in hematopoiesis. TK converts gancyclovir to a toxic substrate by phosphorylation; hence, cells expressing TK (in this case, mature osteoblasts, bone lining cells and pre-osteoblasts that express Col2.3) are killed after gancyclovir treatment. This results in a reduction in BM hematopoiesis and cells having an immunophenotype representative of HSCs and progenitors (HSC-like cells) accompanied by extramedullary hematopoiesis [22,46]. Although ablation of osteoblasts did eventually result in loss of HSCs from the BM at day 25 post-gancyclovir treatment, this loss did not occur concurrent with death of osteoblasts; hence, osteoblasts were not likely to be the cell type that directly altered HSC numbers in the BM. If mature osteoblasts are not the true HSC niche, which cells of the osteoblast lineage are likely to regulate HSCs? We have listed some cell-extrinsic factors in the BM microenvironment that are important for HSC regulation in Box 1. We summarize some of the key discoveries of two pioneering studies below. These studies, using in vivo mouse models, primarily relied on anatomical locations and immunohistochemistry to identify HSCs and the cells comprising their niche. Furthermore, although the in vivo model is the most precise in which to assess hematopoiesis, it is also the most complex because the contributions of direct effects of distinct cell types on hematopoiesis versus those mediated by other cell types in the body cannot be distinguished. PTH/PTHrP receptor in cells of the microenvironment The mouse Col1a1 2.3kB promoter was used to generate transgenic mice in which osteoblasts express the constitu-
Box 1. Cell-extrinsic factors in the BM microenvironment important for HSC regulation Osteopontin (Opn) Glycoprotein produced by cells of the osteoblast and monocyte lineages in the BM. It binds to integrin a4 and CD44 present on HSCs. The increase of the HSC pool in Opn / mice showed that Opn acts as a negative regulator of HSCs [44].
CXCL12 CXCL12 binds CXCR4 on HSCs, and this is important for seeding of HSCs in BM during ontogeny [64]. Blocking interaction of CXCL12 and CXCR4 results in mobilization of HSCs from the BM to the periphery, indicating the importance of CXCL12 for retention of HSCs in the BM [30].
Stem cell factor (SCF) Transmembrane SCF is important for lodgment and detainment in the BM microenvironment [60]. Function of the SCF receptor c-kit has been shown to be important for maintaining quiescent HSCs in the niche [61].
Thrombopoietin (TPO) Deletion of TPO or its receptor, Mpl, in mice results in reduction of HSCs in the BM [65,66]. Interaction between TPO and Mpl maintains a quiescent HSC population in the HSC niche [67].
Angiopoietin 1 (Ang-1) Ang-1 interacts with its receptor Tie2 on HSCs and has a role in maintaining HSC quiescence in the HSC niche [62].
Ca2+ ions The cell-surface receptor calcium-sensing receptor plays a role in the lodgment of HSCs in the HSC niche [68].
Jagged1 Jagged1-dependent activation of Notch1 was crucial for the increase of HSCs in the PTH/PTHrP model [3]. However, deletion of Jagged1 in hematopoietic cells of BM stromal cells did not affect HSC maintenance [63]. In addition, transplantation of Notch1-negative HSCs did not affect reconstitution [63]. The essential role of Jagged1 in the HSC niche, therefore, is uncertain.
N-cadherin Bmpr1a mutant mice have an increased number of HSCs in the BM that correlates with an increase of N-cadherin+ osteoblasts. HSCs were also shown to express N-cadherin [4]. However, the expression and function of N-cadherin on HSCs is controversial because later studies failed to identify N-cadherin on HSCs and N-cadherin deletion from HSCs does not alter stem cell function [69].
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Review tively active PTH/parathyroid hormone-related protein (PTHrP) receptor [47]. Bone histomorphometry revealed increased trabecular bone volume, with increased numbers of osteoblasts, osteoclasts and fibrotic stroma-like cells [47]. In situ hybridization showed that the fibrotic cells expressed alkaline phosphatase and other markers of the osteoblast lineage, including osteopontin [47]. The numbers of osteoblasts, fibrotic cells and osteoclasts were highest in mice that were two weeks old but remained increased compared with wild-type mice at 12 weeks of age. In contrast to the increased trabecular bone observed in Col2.3PTH/PTHrP transgenic mice, cortical bone thickness was decreased, and the cortical bone was porous, a result of inhibitory effects on periosteal osteoblast proliferation and mineral apposition rate [47]. A subsequent study revealed that the marrow cavity in Col2.3PTH/PTHrP transgenic mice was not well established in mice that were two weeks old but was relatively normal by four to five months of age [48]. In these older mice, the numbers of CFU-F derived from BM were reduced compared with wild-type mice; however, the proliferative capacity of the transgenic CFU-F was higher than that of wild-type [48]. In vivo transplantation studies of CFU-F-derived cells revealed that Col2.3PTH/PTHrP transgenic cells formed bone in preexisting mineralized scaffold to a similar extent to wild-type CFU-F [48]. However, unlike wild-type CFU-F-derived cells, transgenic CFU-F-derived cells were unable to form hematopoiesissupporting ossicles in a non-osteoconductive collagen carrier, indicating that the cells were pre-osteoblastic rather than multipotent skeletal stem cells [48]. Therefore, these studies demonstrated that Col2.3PTH/PTHrP transgenic mice had increased numbers of pre-osteoblasts, osteoblasts, fibrotic cells and trabecular bone in their BM. In a separate study using the same source of Col2.3PTH/ PTHrP transgenic mice on the same genetic background [3], significantly increased numbers of immunophenotypical HSCs and primitive long-term-culture-initiating cells (LTC-ICs) were observed in BM obtained from 12-week-old transgenic mice. To determine whether HSCs were increased in these mice, competitive transplant assays were performed in which BM cells obtained from male wild-type or transgenic mice were mixed with female BM cells and transplanted into female recipient mice. At eight weeks post-transplant, a twofold increase in male donor cells was detected in BM of female recipients of Col2.3PTH/PTHrP transgenic cells [3]. Note, however, that this assay was performed at a time point that has since been recognized as being too early to measure long-term repopulating HSCs, and LTC-ICs and cells having HSC immunophenotypes are not necessarily HSCs [24]. To further determine how constitutively active PTH/ PTHrP impacts HSCs, stromal cell cultures derived from Col2.3PTH/PTHrP transgenic or wild-type mice were established. Cultures established from transgenic mice were shown to have augmented support of LTC-ICs, and this required cell–cell contact between hematopoietic cells and stromal cells [3]. Immunohistochemistry studies of proximal tibiae revealed elevated expression of the HSCsupportive proteins CXCL12, stem cell factor (SCF) and Jagged-1 in osteoblastic cells that also expressed osteo-
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pontin [3]. However, the majority of these ‘osteoblastic’ cells were not morphologically mature osteoblasts but, instead, appeared similar to fibrotic stromal cells that surrounded the trabeculae [3,47]. In the same report, PTH treatment of wild-type stroma resulted in a significant increase in support of LTC-ICs, accompanied by increases in alkaline phosphatase-positive cells [3]. Furthermore, hematopoietic cells obtained from PTH-treated mice had superior engraftment capacity in the BM of transplanted recipients, and PTH treatment of irradiated recipients improved survival of the mice posttransplant [3]. The potential clinical benefits of PTH in improving recovery after HSC transplant, therefore, are extremely promising. However, the nature of the cell type mediating these effects is less clear but is more likely to be a pre-osteoblast than a mature osteoblast. Impact of Bmpr1a loss in cells of the microenvironment The bone morphogenetic proteins (BMPs) have been shown to be important in regulating HSC specification during embryonic development and, also, in regulating the proliferation of adult HSCs [49]. To investigate its potential role in regulating adult HSCs, Bmpr1a was conditionally deleted in murine hematopoietic and BM stromal cells using myxovirus resistance 1-Cre (Mx1-Cre) [4]. There was an increase in ectopic bone formation after the deletion, accompanied by increased numbers of osteoblasts [4]. The dramatic effects on bone suggest the osteoblast as the target cell responsible for the bone phenotype. However, postnatal osteoblast-specific deletion of Bmpr1a using an osteocalcin2 (Og2)-Cre reporter (which deletes in mature osteoblasts; Figure 1) resulted in loss of total bone volume, including trabecular bone, attributed to impaired function of osteoblasts and osteoclasts [50]. Given the contrasting bone phenotypes between the Mx1-Cre and Og2-Cre conditional Bmpr1a knockout mice, it is unlikely that the mature osteoblast was the target cell responsible for the bone phenotype observed in the Mx1-Cre conditional Bmpr1a knockout. Mx1-Cre can target deletion in osteoblasts, as well as in a wide variety of cells including hematopoietic cells, heart, liver, lung [51] and endothelial cells [52]. The increased numbers of HSCs observed in the Mx1-Cre Bmpr1a conditional knockout were not intrinsic to the HSC; HSCs do not express Bmpr1a and increased numbers of HSCs were also observed when wild-type cells were transplanted into Mx1-Cre Bmpr1a knockout mice (which resulted in chimeric mice having wild-type hematopoietic cells and Bmpr1a deleted in BM microenvironment cells) [4]. The target cell responsible for the increased bone phenotype and HSC numbers in Mx1-Cre Bmpr1a conditional knockout mice remains unclear. Nevertheless, there was an increase in N-cadherin-expressing spindle-shaped cells located at the bone surface (referred to as SNO cells), and these SNO cells were reported to be the supportive cells for HSCs in the Mx1-Cre Bmpr1a conditional knockout mouse [4]. However, N-cadherin is expressed by other cell types implicated in the HSC niche, including endothelial cells [53] and nerve cells [54]. It must also be noted that the antibodies used to detect N-cadherin in HSC niche studies were recently stated to potentially recognize other cadher307
Review ins (see the ‘online methods’ section of Ref. [55]). Indeed, the importance of N-cadherin in regulating HSCs has been a topic of contention [56,57], although studies have not yet addressed the crucial question as to whether loss of Ncadherin in microenvironment cells alters HSC numbers. A recent report further defining the nature of the Ncadherin+ osteoblastic cells showed that the majority of these cells were located perivascularly but did not express the endothelial cell marker CD31 [55]. The N-cadherin+ cells were also not of the mature osteoblast lineage because immunohistochemical staining for N-cadherin did not colocalize with GFP+ osteoblast cells in pOBCol2.3-GFP transgenic mice [55]. Some (but not all) N-cadherin+ SNO cells co-expressed osterix [55], a transcription factor primarily associated with maturing pre-osteoblasts but also shown to be expressed in other tissues including brain [58]. Hence, although it has not yet been conclusively proven, it is highly possible that the N-cadherin+ cells closely associated with HSCs are immature pre-osteoblast cells located primarily in perivascular regions of the trabecular bone. However, their relationship to the ‘osteoblastic’ fibrotic stromal cells detected in Col2.3PTH/PTHrP mice remains to be determined. Other candidate HSC niches of the osteoblast lineage The relationship and function of Nestin+CD45 cells [35], a-SMA GFP+ cells [33,34] and SSCs is also of interest. Although extensive studies have not yet examined these candidate HSC niche cells, preliminary studies have shown that Nestin+CD45 cells express mRNA for proteins shown to regulate HSCs, including CXCL12, SCF and angiopoietin-1 (Ang-1) [15,35]. Furthermore, human SSCs have been shown to express high levels of mRNA for Ncadherin, Jagged-1, CXCL12, SCF and Ang-1 [27]. These cells could potentially be the true HSC niche, and their anatomical location might also resolve the previous controversy regarding whether endothelial cells or cells of the osteoblast lineage are the true HSC niche. Functional studies showing the importance of pre-osteoblasts and SSCs as participants in the HSC niche, therefore, are of future importance. Concluding remarks Precursors of the osteoblast lineage, such as pre-osteoblasts and SSCs, express molecules shown to be important for HSC regulation (such as CXCL12, Ang-1 and SCF) and might play an active part in the HSC niche. Indeed, stromal cells that support hematopoiesis are reminiscent of immature osteoblasts and are grown in culture conditions in which spontaneous mature osteoblast cell differentiation does not normally occur [17–21]. In contrast, mature cells of the osteoblast lineage (including osteoblasts, bone lining cells and osteocytes) are unlikely to be active participants in the HSC-supportive niche. The increasing use of osteoblastic GFP transgenic mice (many of which have been reviewed in detail recently [59]), together with conditional mutants specific to cells of the osteoblast lineage, should be useful in future studies to resolve the controversy surrounding the roles of cells of the osteoblastic lineage in regulating HSCs and reveal the true nature of the HSC niche. 308
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Acknowledgements We thank C.R. Walkley and D.T. Scadden for critically reviewing the manuscript. M.A. is a Swedish Research Council Post-doctoral Fellow, N.A.S is a Senior Research Fellow of the National Health and Medical Research Council (NHMRC), T.J.M. is a John Holt Fellow and L.E.P. is an NHMRC RD Wright Fellow.
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receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow. J. Cell Biol. 167, 1113–1122 Larsson, J. and Karlsson, S. (2005) The role of Smad signaling in hematopoiesis. Oncogene 24, 5676–5692 Mishina, Y. et al. (2004) Bone morphogenetic protein type IA receptor signaling regulates postnatal osteoblast function and bone remodeling. J. Biol. Chem. 279, 27560–27566 Kuhn, R. et al. (1995) Inducible gene targeting in mice. Science 269, 1427–1429 Hayashi, M. et al. (2004) Targeted deletion of BMK1/ERK5 in adult mice perturbs vascular integrity and leads to endothelial failure. J. Clin. Invest. 113, 1138–1148 Ferreri, D.M. et al. (2008) N-cadherin levels in endothelial cells are regulated by monolayer maturity and p120 availability. Cell Commun. Adhes. 15, 333–349 Wanner, I.B. et al. (2006) Role of N-cadherin in Schwann cell precursors of growing nerves. Glia 54, 439–459 Xie, Y. et al. (2009) Detection of functional haematopoietic stem cell niche using real-time imaging. Nature 457, 97–101 Kiel, M.J. et al. (2007) Lack of evidence that hematopoietic stem cells depend on N-cadherin-mediated adhesion to osteoblasts for their maintenance. Cell Stem Cell 1, 204–217 Kiel, M.J. et al. (2008) CD150- cells are transiently reconstituting multipotent progenitors with little or no stem cell activity. Blood 111, 4413–4414 Milona, M.A. et al. (2003) Expression of alternatively spliced isoforms of human Sp7 in osteoblast-like cells. BMC Genomics 4, 43 van Koevering, K. and Williams, B. (2008) Transgenic mouse strains for conditional gene deletion during skeletal development. IBMS BoneKEy 5, 151–170 Driessen, R.L. et al. (2003) Membrane-bound stem cell factor is a key regulator in the initial lodgment of stem cells within the endosteal marrow region. Exp. Hematol. 31, 1284–1291 Thoren, L.A. et al. (2008) Kit regulates maintenance of quiescent hematopoietic stem cells. J. Immunol. 180, 2045–2053 Arai, F. et al. (2004) Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 118, 149–161 Mancini, S.J. et al. (2005) Jagged1-dependent Notch signaling is dispensable for hematopoietic stem cell self-renewal and differentiation. Blood 105, 2340–2342 Ara, T. et al. (2003) Long-term hematopoietic stem cells require stromal cell-derived factor-1 for colonizing bone marrow during ontogeny. Immunity 19, 257–267 Kimura, S. et al. (1998) Hematopoietic stem cell deficiencies in mice lacking c-Mpl, the receptor for thrombopoietin. Proc. Natl. Acad. Sci. U. S. A. 95, 1195–1200 Qian, H. et al. (2007) Critical role of thrombopoietin in maintaining adult quiescent hematopoietic stem cells. Cell Stem Cell 1, 671–684 Yoshihara, H. et al. (2007) Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche. Cell Stem Cell 1, 685–697 Adams, G.B. et al. (2006) Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor. Nature 439, 599–603 Kiel, M.J. et al. (2009) Hematopoietic stem cells do not depend on Ncadherin to regulate their maintenance. Cell Stem Cell 4, 170–179
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What is the true nature of the osteoblastic hematopoietic stem cell niche? Maria Askmyr1, Natalie A. Sims1,2, T. John Martin1,2 and Louise E. Purton1,2 1 2
St Vincent’s Institute, Fitzroy, Victoria, 3065, Australia Department of Medicine, The University of Melbourne, Fitzroy, Victoria, 3065, Australia
The recently revitalized interest in the regulation of hematopoietic stem cells (HSCs) by the bone marrow microenvironment has resulted in the identification of some important cell types that potentially form the HSC niche. The term ‘osteoblast’ has commonly been used to describe the endosteal elements of the HSC niche, but these cells are part of a larger family that functions in bone at different stages of differentiation. Given that there is much controversy as to what cell types have important roles in the HSC niche, this review offers an overview of the diverse osteoblastic cell types and discusses the current evidence regarding what roles they have in the HSC niche. The identification of the adult hematopoietic stem cell niche The bone marrow (BM) cavity serves as the main site of hematopoiesis in the human adult. This lifelong process of constant replenishment of mature hematopoietic cells is sustained by the hematopoietic stem cells (HSCs). To accomplish this, it is thought that HSCs have the potential to self-renew to maintain the HSC pool. In 1978, Schofield proposed that HSCs needed to be localized in a particular location (termed the HSC niche) within the BM to retain their multipotency [1] and that if the HSCs were located elsewhere, they would probably commit to differentiation rather than self-renew [1]. However, until recently, the nature of the HSC niche was largely unknown. It should be noted here that sites of hematopoiesis differ between rodents and humans; whereas mice sustain blood formation in all bones and spleen throughout life, human hematopoiesis is restricted to the proximal regions of long bones, cranium, sternum, ribs, vertebra and ilium [2]. The studies discussed below were predominantly performed in the mouse model, and it is not clear whether all the properties and components of the HSC niche described in these models are translatable to humans. However, the mouse model, to date, is the best small animal model of bone and hematopoiesis and enables extensive in vivo analyses that cannot be performed in humans. A series of studies in 2003 and 2005 described two different types of HSC niches: one at the bone surfaces in contact with BM and the other in proximity to the BM vasculature [3–6]. These are commonly referred to as the Corresponding author: Purton, L.E. ([email protected]).
osteoblastic (or endosteal) and perivascular niches, respectively. Furthermore, cells of the sympathetic nervous system were recently identified as having important roles in retaining HSCs in the BM [7–10]. Given the complexity of the three-dimensional structure of BM, it is unclear whether these niches are distinct or overlap in both location and function [11]. The contributions of each niche to HSC regulation are currently the subject of ongoing investigations in various laboratories and have been recently reviewed in detail elsewhere [11–16]. This review focuses on recent findings regarding the nature of cells of the osteoblast lineage that probably participate in the HSC niche. Roles of cells of the osteoblast lineage in the HSC niche The development of long-term BM cultures in the 1970s described methods whereby hematopoiesis could be sustained in culture for many months [17]. The success of this culture system relied on the development of adherent cells, termed ‘stromal cells’. The identity of the different types of stromal cells remained elusive; however, these stromal cells expressed alkaline phosphatase (an enzyme present in, but not exclusive to, osteoblast lineage cells) [18]. Almost 20 years later, a series of reports demonstrated that human osteoblasts could support hematopoiesis in ex vivo culture systems [19–21]. These human osteoblasts were defined as alkaline phosphatase-expressing cells and, more importantly, were shown to be capable of mineralization in vitro, clearly demonstrating their osteoblastic nature [19–21]. These in vitro findings were the first indication that cells of the osteoblastic lineage had important roles in the regulation of HSCs. Three studies utilizing genetically modified mice further supported roles for cells of the osteoblast lineage in the retention and regulation of HSCs in the BM [3,4,22]. However, these findings also resulted in some confusion in the field regarding which cell type of the osteoblast lineage was important. For example, the interchangeable terms of ‘osteoblast niche’ and ‘endosteal niche’ became common terminology when referring to the HSC niche. As we will discuss in detail below, the nature of the participating cell types of the HSC niche is still to be clearly determined. Misleading terminology in describing the HSC niche As has been raised recently by Bianco [23], the term ‘osteoblastic niche’ is oversimplified and potentially incorrect in regard to which cells actively regulate HSC maintenance
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Figure 1. Differentiation of skeletal stem cells along the osteoblast lineage in bone marrow. Black arrows indicate the direction of differentiation, starting with (i) the immature skeletal stem cells. Dashed red arrows indicate potential differentiation pathways. Genes expressed in each cell type are listed below each cell type, with key proteins in bold and transcription factors in italics. The symbol ‘?’ indicates that mRNA expression has been demonstrated but protein studies have not yet been performed or that the cell type expressing the protein – (ii) pre-osteoblasts, (iii) osteoblasts, (iv) bone-lining cells or (v) osteocytes – has not been conclusively identified. Abbreviations: ALP, alkaline phosphatase; Ang-1, angiopoietin-1; DMP1, dental matrix protein 1; Jag-1, Jagged-1; N-cad, N-cadherin; PTH1R, receptor for PTH/PTHrP; SOST, sclerostin.
and self-renewal. Researchers have used the generic term ‘osteoblasts’ to refer to a spectrum of cell types of the osteoblastic lineage. However, osteoblasts are a well-defined population of differentiated cells actively involved in bone formation. The terminology ‘endosteal niche’ is also misrepresentative because the endosteal surface of bone includes all cells that line the interface between bone and marrow, including both endocortical (i.e. inner cortical) and trabecular surfaces. In contrast to this, most studies support the trabecular region of bone as the most important anatomical location for the HSC niche [3,4], whereas there is little evidence that cortical bone plays an important part with respect to the HSC niche [11]. Note that although hematopoietic cells have been well defined by fluorescence-activated cell sorting (FACS) techniques and assays of hematopoietic potential [24], there is much less definition of the full spectrum of cells of the osteoblast lineage, from the most immature cells (skeletal stem cells) to the most mature cells (osteocytes) (Figure 1). This is, in part, due to a lack of cell surface markers or antibodies that can be used to isolate these cells by FACS and the difficulty of performing in vivo assays of the bone lineage cells. Yet there are some distinguishing properties of cells of the osteoblast lineage, discussed below. Skeletal stem cells, adventitial reticular cells and preosteoblasts In postnatal stages, the osteoblast lineage is derived from mesenchymal stem cells (MSCs). A population of MSCs can 304
be obtained from bone marrow stromal cells (BMSCs), nonhematopoietic cells that adhere to cell culture dishes. The cells that give rise to these plastic-adherent colonies were originally identified as colony-forming fibroblasts (CFU-F) [25], and progeny of single bone-marrow-derived CFU-F differentiate into osteoblast-lineage cells, cartilage cells (chondrocytes) or adipocytes both in vivo and in vitro [26]. It remains controversial whether CFU-F also differentiate into cell types such as neurons, endothelial cells and muscle cells; hence, the terminology utilized to describe these cells has been suggested to be skeletal stem cells (SSCs) [26]. The identity of the SSCs has been predominantly restricted to functional properties, such as the ability to adhere to plastic (CFU-F-derived populations), that have identified them as a heterogeneous population of cells [27]. A recent study isolated pure populations of SSCs from human BM using a panel of antibodies; however, the phenotype of mouse SSCs is yet to be defined [26,27]. Human SSCs are found in perivascular sites in the BM [28] and express high levels of alkaline phosphatase and runt-related transcription factor 2, markers of early osteoblast lineage cells [27]. Human SSCs share similar properties to pericytes, including expression of a-smooth muscle cell actin (a-SMA) and platelet-derived growth factor receptor beta [27,29]. Like SSCs, in vitro cultured pericytes can differentiate into osteocytes, chondrocytes and adipocytes, which implies that SSCs might be pericytes or belong to a subset of pericytes; however, this is yet to be proven [29].
Review SSCs express high levels of CXCL12 [27], a chemokine with major roles in chemotaxis, homing, survival and retention of HSCs in the BM niche [30]. CXCL12 binds to CXCR4, which is expressed on HSCs. Blocking the interaction of CXCL12 and CXCR4 by an antagonist against CXCR4 results in rapid HSC mobilization from the BM into the peripheral blood, revealing the profound effects CXCL12 has on retaining HSCs in the BM microenvironment [30]. In the mouse, CXCL12 has also been shown to be highly expressed by reticular cells in the BM [31], termed ‘CXCL12-abundant reticular cells’ (CAR) and found in both perivascular and endosteal locations [31]. Although not proven, it is likely that murine CAR cells are analogous to human SSCs, sharing the same perivascular location and functional properties [23,32]. Recent studies using green fluorescent protein (GFP) transgenic mice have given more insight into the potential nature of mouse SSCs. In two complementary reports, an a-SMA–GFP reporter mouse was used to isolate a population of mesenchymal progenitor cells from murine BMSCs [33,34]. The a-SMA–GFP+ cells comprised approximately 0.02–0.03% of mononuclear cells [33] and were located perivascularly in the BM [34]. Furthermore, aSMA–GFP+ cells sorted from cultures of BMSCs contained all CFU-F potential and differentiated into osteoblasts and adipocytes [34]. These studies, therefore, provided further evidence that cells expressing a-SMA (which is a classical marker of pericytes) were potentially SSCs. In a more recent report, transgenic mice in which GFP is driven by regulatory elements of the Nestin promoter were used to isolate Nestin+CD45 and Nestin CD45 cells from BM [35]. As reported for a-SMA GFP+ cells, Nestin+CD45 cells were located in perivascular spaces, all CFU-F potential was contained in the Nestin+CD45 cells and these cells robustly differentiated into osteoblasts and adipocytes in culture [35]. Although it is likely that
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both a-SMA GFP+ cells and Nestin+CD45 cells are akin to SSCs (and CAR cells), their chondrogenic potential has not yet been tested; hence, they are currently best termed ‘mesenchymal progenitor cells’. Osteoblasts and bone lining cells The committed osteoblast population is heterogeneous, and there are at least two structurally and functionally distinct subpopulations that cover the endosteal bone surface: osteoblasts and bone lining cells [36]. Bone lining cells are frequently overlooked in studies of the HSC niche because mononuclear cells surrounding bone are commonly referred to as osteoblasts. Although both cell types are always found in close contact with the bone surface, both at the endosteum (marrow surface of bone) and the periosteum (outer bone surface) [37], actively synthesizing osteoblasts are by far outnumbered by quiescent bone lining cells [38,39]. Bone lining cells are inactive, at least in regard to the production of bone (osteoid). These flat cells stretch over the bone surface to form a protective layer, and although they are largely considered terminally differentiated, it has not yet been determined whether osteoblasts turn into lining cells [38]. Nor has it been clearly established that lining cells can become active bone-producing cells, although they have been proposed as one of the sources of osteoblasts formed in response to anabolic parathyroid hormone (PTH) [40]. Studies in transgenic mice, in which GFP is under the control of promoters of osteoblast-lineage-specific genes, have distinguished mature cells of the osteoblast lineage. Two different rat type 1 collagen (Col1a1) promoter constructs have been used to direct GFP expression to pre-osteoblasts (pOBCol3.6GFP) versus mature osteoblasts and osteocytes (pOBCol2.3GFP) (Figure 1) [41]. Because a recent report has demonstrated that bone lining cells express pOBCol2.3GFP, it can be assumed that they are mature osteoblast cells [42].
Figure 2. Histology of mouse tibia. Toluidine blue staining of undecalcified plastic-embedded tibia [(i) and (ii)] showing trabecular bone (i) and cortical bone (ii). Mineralized bone is stained dark blue, and the areas of the bone with lighter blue staining are osteoid (unmineralized bone). (iii) Hematoxylin and eosin (H&E) staining of decalcified tibia showing trabecular bone. The specific cell types are highlighted as indicated. Scale bar = 10 mm.
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Review Although specific markers that distinguish osteoblasts from bone lining cells have not been identified, these cells do have distinct morphologies. Bone lining cells have a flattened, spindle shape, whereas active osteoblasts are plump and cuboidal (Figure 2). Bone lining cells have been reported to be negative for alkaline phosphatase and osteocalcin expression, with only occasional expression of osteonectin [38], but systematic studies have not yet been carried out. In contrast to lining cells, active cuboidal osteoblasts have a high density of Golgi and endoplasmic reticulum required for their intense protein production. These cells actively lay down osteoid and regulate its mineralization to form bone, a process that requires the production of numerous proteins including type 1 collagen, osteocalcin, osteonectin, sialoprotein and osteopontin. Bone formation is tightly coupled with bone resorption by hematopoieticderived osteoclasts to regulate skeletal mass [43]. Studies linking HSCs and the osteoblast lineage: which cells of the osteoblast lineage comprise the HSC niche? The two pioneering studies describing the osteoblastic HSC niche reported that an increase in the amount of trabecular bone and/or trabecular osteoblasts correlated with increased HSC numbers [3,4]. A subsequent study demonstrated that ablating osteoblast-lineage cells in vivo resulted in relocation of most HSCs from the BM into the extramedullary sites such as spleen and liver [22]. These studies have, importantly, directed attention to the previously understudied roles of the microenvironment in regulating hematopoiesis. However, widespread misinterpretation of the term ‘osteoblastic’ as meaning mature osteoblasts has resulted in a common belief that the mature osteoblast is the cell type mediating the changes in HSC numbers. Furthermore, even though subsequent studies have revealed HSC-supportive roles for osteoblast-associated proteins, it remains to be shown that these proteins are exclusively derived from osteoblasts [15].
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Indeed, mature osteoblasts are unlikely to have a direct role as part of the HSC-supportive niche. Osteopontin, a protein expressed abundantly by osteoblasts, osteoclasts and macrophages, has been shown to be a negative regulator of HSCs [44,45]. Additional studies using Col2.3D thymidine kinase (TK) transgenic mice have also been used to explore the effects of mature osteoblasts in hematopoiesis. TK converts gancyclovir to a toxic substrate by phosphorylation; hence, cells expressing TK (in this case, mature osteoblasts, bone lining cells and pre-osteoblasts that express Col2.3) are killed after gancyclovir treatment. This results in a reduction in BM hematopoiesis and cells having an immunophenotype representative of HSCs and progenitors (HSC-like cells) accompanied by extramedullary hematopoiesis [22,46]. Although ablation of osteoblasts did eventually result in loss of HSCs from the BM at day 25 post-gancyclovir treatment, this loss did not occur concurrent with death of osteoblasts; hence, osteoblasts were not likely to be the cell type that directly altered HSC numbers in the BM. If mature osteoblasts are not the true HSC niche, which cells of the osteoblast lineage are likely to regulate HSCs? We have listed some cell-extrinsic factors in the BM microenvironment that are important for HSC regulation in Box 1. We summarize some of the key discoveries of two pioneering studies below. These studies, using in vivo mouse models, primarily relied on anatomical locations and immunohistochemistry to identify HSCs and the cells comprising their niche. Furthermore, although the in vivo model is the most precise in which to assess hematopoiesis, it is also the most complex because the contributions of direct effects of distinct cell types on hematopoiesis versus those mediated by other cell types in the body cannot be distinguished. PTH/PTHrP receptor in cells of the microenvironment The mouse Col1a1 2.3kB promoter was used to generate transgenic mice in which osteoblasts express the constitu-
Box 1. Cell-extrinsic factors in the BM microenvironment important for HSC regulation Osteopontin (Opn) Glycoprotein produced by cells of the osteoblast and monocyte lineages in the BM. It binds to integrin a4 and CD44 present on HSCs. The increase of the HSC pool in Opn / mice showed that Opn acts as a negative regulator of HSCs [44].
CXCL12 CXCL12 binds CXCR4 on HSCs, and this is important for seeding of HSCs in BM during ontogeny [64]. Blocking interaction of CXCL12 and CXCR4 results in mobilization of HSCs from the BM to the periphery, indicating the importance of CXCL12 for retention of HSCs in the BM [30].
Stem cell factor (SCF) Transmembrane SCF is important for lodgment and detainment in the BM microenvironment [60]. Function of the SCF receptor c-kit has been shown to be important for maintaining quiescent HSCs in the niche [61].
Thrombopoietin (TPO) Deletion of TPO or its receptor, Mpl, in mice results in reduction of HSCs in the BM [65,66]. Interaction between TPO and Mpl maintains a quiescent HSC population in the HSC niche [67].
Angiopoietin 1 (Ang-1) Ang-1 interacts with its receptor Tie2 on HSCs and has a role in maintaining HSC quiescence in the HSC niche [62].
Ca2+ ions The cell-surface receptor calcium-sensing receptor plays a role in the lodgment of HSCs in the HSC niche [68].
Jagged1 Jagged1-dependent activation of Notch1 was crucial for the increase of HSCs in the PTH/PTHrP model [3]. However, deletion of Jagged1 in hematopoietic cells of BM stromal cells did not affect HSC maintenance [63]. In addition, transplantation of Notch1-negative HSCs did not affect reconstitution [63]. The essential role of Jagged1 in the HSC niche, therefore, is uncertain.
N-cadherin Bmpr1a mutant mice have an increased number of HSCs in the BM that correlates with an increase of N-cadherin+ osteoblasts. HSCs were also shown to express N-cadherin [4]. However, the expression and function of N-cadherin on HSCs is controversial because later studies failed to identify N-cadherin on HSCs and N-cadherin deletion from HSCs does not alter stem cell function [69].
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Review tively active PTH/parathyroid hormone-related protein (PTHrP) receptor [47]. Bone histomorphometry revealed increased trabecular bone volume, with increased numbers of osteoblasts, osteoclasts and fibrotic stroma-like cells [47]. In situ hybridization showed that the fibrotic cells expressed alkaline phosphatase and other markers of the osteoblast lineage, including osteopontin [47]. The numbers of osteoblasts, fibrotic cells and osteoclasts were highest in mice that were two weeks old but remained increased compared with wild-type mice at 12 weeks of age. In contrast to the increased trabecular bone observed in Col2.3PTH/PTHrP transgenic mice, cortical bone thickness was decreased, and the cortical bone was porous, a result of inhibitory effects on periosteal osteoblast proliferation and mineral apposition rate [47]. A subsequent study revealed that the marrow cavity in Col2.3PTH/PTHrP transgenic mice was not well established in mice that were two weeks old but was relatively normal by four to five months of age [48]. In these older mice, the numbers of CFU-F derived from BM were reduced compared with wild-type mice; however, the proliferative capacity of the transgenic CFU-F was higher than that of wild-type [48]. In vivo transplantation studies of CFU-F-derived cells revealed that Col2.3PTH/PTHrP transgenic cells formed bone in preexisting mineralized scaffold to a similar extent to wild-type CFU-F [48]. However, unlike wild-type CFU-F-derived cells, transgenic CFU-F-derived cells were unable to form hematopoiesissupporting ossicles in a non-osteoconductive collagen carrier, indicating that the cells were pre-osteoblastic rather than multipotent skeletal stem cells [48]. Therefore, these studies demonstrated that Col2.3PTH/PTHrP transgenic mice had increased numbers of pre-osteoblasts, osteoblasts, fibrotic cells and trabecular bone in their BM. In a separate study using the same source of Col2.3PTH/ PTHrP transgenic mice on the same genetic background [3], significantly increased numbers of immunophenotypical HSCs and primitive long-term-culture-initiating cells (LTC-ICs) were observed in BM obtained from 12-week-old transgenic mice. To determine whether HSCs were increased in these mice, competitive transplant assays were performed in which BM cells obtained from male wild-type or transgenic mice were mixed with female BM cells and transplanted into female recipient mice. At eight weeks post-transplant, a twofold increase in male donor cells was detected in BM of female recipients of Col2.3PTH/PTHrP transgenic cells [3]. Note, however, that this assay was performed at a time point that has since been recognized as being too early to measure long-term repopulating HSCs, and LTC-ICs and cells having HSC immunophenotypes are not necessarily HSCs [24]. To further determine how constitutively active PTH/ PTHrP impacts HSCs, stromal cell cultures derived from Col2.3PTH/PTHrP transgenic or wild-type mice were established. Cultures established from transgenic mice were shown to have augmented support of LTC-ICs, and this required cell–cell contact between hematopoietic cells and stromal cells [3]. Immunohistochemistry studies of proximal tibiae revealed elevated expression of the HSCsupportive proteins CXCL12, stem cell factor (SCF) and Jagged-1 in osteoblastic cells that also expressed osteo-
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pontin [3]. However, the majority of these ‘osteoblastic’ cells were not morphologically mature osteoblasts but, instead, appeared similar to fibrotic stromal cells that surrounded the trabeculae [3,47]. In the same report, PTH treatment of wild-type stroma resulted in a significant increase in support of LTC-ICs, accompanied by increases in alkaline phosphatase-positive cells [3]. Furthermore, hematopoietic cells obtained from PTH-treated mice had superior engraftment capacity in the BM of transplanted recipients, and PTH treatment of irradiated recipients improved survival of the mice posttransplant [3]. The potential clinical benefits of PTH in improving recovery after HSC transplant, therefore, are extremely promising. However, the nature of the cell type mediating these effects is less clear but is more likely to be a pre-osteoblast than a mature osteoblast. Impact of Bmpr1a loss in cells of the microenvironment The bone morphogenetic proteins (BMPs) have been shown to be important in regulating HSC specification during embryonic development and, also, in regulating the proliferation of adult HSCs [49]. To investigate its potential role in regulating adult HSCs, Bmpr1a was conditionally deleted in murine hematopoietic and BM stromal cells using myxovirus resistance 1-Cre (Mx1-Cre) [4]. There was an increase in ectopic bone formation after the deletion, accompanied by increased numbers of osteoblasts [4]. The dramatic effects on bone suggest the osteoblast as the target cell responsible for the bone phenotype. However, postnatal osteoblast-specific deletion of Bmpr1a using an osteocalcin2 (Og2)-Cre reporter (which deletes in mature osteoblasts; Figure 1) resulted in loss of total bone volume, including trabecular bone, attributed to impaired function of osteoblasts and osteoclasts [50]. Given the contrasting bone phenotypes between the Mx1-Cre and Og2-Cre conditional Bmpr1a knockout mice, it is unlikely that the mature osteoblast was the target cell responsible for the bone phenotype observed in the Mx1-Cre conditional Bmpr1a knockout. Mx1-Cre can target deletion in osteoblasts, as well as in a wide variety of cells including hematopoietic cells, heart, liver, lung [51] and endothelial cells [52]. The increased numbers of HSCs observed in the Mx1-Cre Bmpr1a conditional knockout were not intrinsic to the HSC; HSCs do not express Bmpr1a and increased numbers of HSCs were also observed when wild-type cells were transplanted into Mx1-Cre Bmpr1a knockout mice (which resulted in chimeric mice having wild-type hematopoietic cells and Bmpr1a deleted in BM microenvironment cells) [4]. The target cell responsible for the increased bone phenotype and HSC numbers in Mx1-Cre Bmpr1a conditional knockout mice remains unclear. Nevertheless, there was an increase in N-cadherin-expressing spindle-shaped cells located at the bone surface (referred to as SNO cells), and these SNO cells were reported to be the supportive cells for HSCs in the Mx1-Cre Bmpr1a conditional knockout mouse [4]. However, N-cadherin is expressed by other cell types implicated in the HSC niche, including endothelial cells [53] and nerve cells [54]. It must also be noted that the antibodies used to detect N-cadherin in HSC niche studies were recently stated to potentially recognize other cadher307
Review ins (see the ‘online methods’ section of Ref. [55]). Indeed, the importance of N-cadherin in regulating HSCs has been a topic of contention [56,57], although studies have not yet addressed the crucial question as to whether loss of Ncadherin in microenvironment cells alters HSC numbers. A recent report further defining the nature of the Ncadherin+ osteoblastic cells showed that the majority of these cells were located perivascularly but did not express the endothelial cell marker CD31 [55]. The N-cadherin+ cells were also not of the mature osteoblast lineage because immunohistochemical staining for N-cadherin did not colocalize with GFP+ osteoblast cells in pOBCol2.3-GFP transgenic mice [55]. Some (but not all) N-cadherin+ SNO cells co-expressed osterix [55], a transcription factor primarily associated with maturing pre-osteoblasts but also shown to be expressed in other tissues including brain [58]. Hence, although it has not yet been conclusively proven, it is highly possible that the N-cadherin+ cells closely associated with HSCs are immature pre-osteoblast cells located primarily in perivascular regions of the trabecular bone. However, their relationship to the ‘osteoblastic’ fibrotic stromal cells detected in Col2.3PTH/PTHrP mice remains to be determined. Other candidate HSC niches of the osteoblast lineage The relationship and function of Nestin+CD45 cells [35], a-SMA GFP+ cells [33,34] and SSCs is also of interest. Although extensive studies have not yet examined these candidate HSC niche cells, preliminary studies have shown that Nestin+CD45 cells express mRNA for proteins shown to regulate HSCs, including CXCL12, SCF and angiopoietin-1 (Ang-1) [15,35]. Furthermore, human SSCs have been shown to express high levels of mRNA for Ncadherin, Jagged-1, CXCL12, SCF and Ang-1 [27]. These cells could potentially be the true HSC niche, and their anatomical location might also resolve the previous controversy regarding whether endothelial cells or cells of the osteoblast lineage are the true HSC niche. Functional studies showing the importance of pre-osteoblasts and SSCs as participants in the HSC niche, therefore, are of future importance. Concluding remarks Precursors of the osteoblast lineage, such as pre-osteoblasts and SSCs, express molecules shown to be important for HSC regulation (such as CXCL12, Ang-1 and SCF) and might play an active part in the HSC niche. Indeed, stromal cells that support hematopoiesis are reminiscent of immature osteoblasts and are grown in culture conditions in which spontaneous mature osteoblast cell differentiation does not normally occur [17–21]. In contrast, mature cells of the osteoblast lineage (including osteoblasts, bone lining cells and osteocytes) are unlikely to be active participants in the HSC-supportive niche. The increasing use of osteoblastic GFP transgenic mice (many of which have been reviewed in detail recently [59]), together with conditional mutants specific to cells of the osteoblast lineage, should be useful in future studies to resolve the controversy surrounding the roles of cells of the osteoblastic lineage in regulating HSCs and reveal the true nature of the HSC niche. 308
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Acknowledgements We thank C.R. Walkley and D.T. Scadden for critically reviewing the manuscript. M.A. is a Swedish Research Council Post-doctoral Fellow, N.A.S is a Senior Research Fellow of the National Health and Medical Research Council (NHMRC), T.J.M. is a John Holt Fellow and L.E.P. is an NHMRC RD Wright Fellow.
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