The haematopoietic stem cell niche at a glance

Haematopoietic stem cells (HSCs) regulate the balanced turnover of erythrocytes, platelets and all immune cells by switching between self-renewal, differentiation, quiescence and dormancy (Trumpp et al., 2010) and, thereby, maintain homeostasis both in the steady state as well as in response to injury. HSCs are among the longest studied and utilised somatic stem cells. However, they cannot yet be maintained and expanded in vitro because a complex and dynamic molecular crosstalk between HSC and their endogenous microenvironment (or ‘niche’) directs their fate. The importance of the stem cell niche in regulating HSC function was first postulated in 1978 by Ray Schofield, when observing that the spleen is unable to support HSCs in the same way that bone marrow can (Schofield, 1978). Since then, it has become clear that not only HSCs but all somatic stem cells maintain homeostasis because they sense and respond to the need of an organism for their differentiated progeny as well as to stem cells themselves. The stem cell niche is the functional and anatomical ‘node’ that allows integration of signals from the periphery into the appropriate stem cell behaviour. The constantly evolving technological and experimental approaches have provided insights into the nature of the HSC niche, the specific function of different niche components and how perturbations in this highly integrated system are involved in the development of haematopoietic disease.In this Cell Science at a Glance article, we provide an update of the cellular and molecular components of the HSC niche, describe how they adapt in response to specific stimuli, and discuss their potential role in the development of haematopoietic disease. As several controversial observations have led to the most recently proposed niche models and because future studies are likely to further change our views again, we do not present a definitive, detailed portrait of the HSC niche but, instead, integrate currently accepted niche elements and dynamic models to provide an up-to-date overview. Box 1 contains details on the specific nomenclature used in this article. Box 2 contains a brief summary of recent experimental models and technological advances that were used to study the HSC niche.HSCs reside within the bone marrow, which presents a complex microenvironment that is made up of different cell types and extracellular elements. An increasing number of bone marrow lineages, structures and molecular components have been demonstrated to affect HSC fate and function.The endosteal surface of the bone and cells of the osteoblastic lineage were shown first to be components of the HSC niche (Calvi et al., 2003; Grassinger et al., 2010; Lord et al., 1975; Visnjic et al., 2004; Zhang et al., 2003). Whether a specific subpopulation of osteoblastic cells is interacting with HSCs is currently under investigation. Recently, secreted phosphoprotein 1 [SPP1; also known as osteopontin (OPN) and one of the main extracellular proteins secreted by osteoblasts] and ALCAM (an adhesion molecule widely expressed across different lineages) were proposed as markers of a subpopulation of osteoblastic cells that affect HSC function. (Mayack and Wagers, 2008; Nakamura et al., 2010). Interestingly, haematopoiesis is dramatically affected by the conditional deletion of the ribonuclease Dicer in osteoprogenitors. However, the same deletion in fully mature, osteocalcin-positive osteoblasts does not lead to haematopoietic defects (Raaijmakers et al., 2010), and the selective ablation of terminally differentiated osteoblasts does not affect haematopoiesis either (Corral et al., 1998; Raaijmakers et al., 2010). The association between HSCs and osteoprogenitors might be rooted even earlier within the osteoblastic lineage, as was suggested by the fact that nestin-positive MSC-like cells are marrow stroma cells that can interact with HSCs (Mendez-Ferrer et al., 2010). Furthermore, human CD146+ osteoprogenitor cells are able to direct ectopic bone formation accompanied by haematopoietic seeding (Sacchetti et al., 2007). PDGFRα+, Sca1+ mesenchymal progenitors have been successfully transplanted and have been shown to localise to areas of the bone marrow that are generally recognised to contain HSC niches (Morikawa et al., 2009). Taken together, these studies show that osteoblastic cells at various stages of differentiation can support different HSC functions and states.The endosteal HSC niche model was challenged, however, by immunofluorescence studies, which showed that the only bone marrow structure that is consistently located adjacent to HSCs is the sinusoidal vasculature (Kiel et al., 2005). Integrity and regeneration of bone marrow vasculature are, indeed, fundamental for HSC recovery from myeloablative injuries and following bone marrow transplantation (Hooper et al., 2009; Kobayashi et al., 2010). Several reports agree on the location of functional, engrafting haematopoietic stem and progenitor cells (HSPCs, Box 1) being near the endosteal surface but not exclusively adjacent to osteoblastic cells (Jiang et al., 2009; Lo Celso et al., 2009; Xie et al., 2009). Whereas endosteal surfaces are highly vascularised, the question remains whether HSCs that are located at varying distances from osteoblastic cells are functionally distinct from those located near osteoblastic cells.Other HSC regulators include perivascular, non-endothelial supportive cells (Sugiyama et al., 2006), adipocytes, which have been shown to inhibit HSC engraftment (Naveiras et al., 2009), and the autonomous nervous system, which influences HSC mobilisation (Katayama et al., 2006). In addition, several cells of haematopoietic origin have a role in the HSC niche. For example, the activation of osteoclasts, a specialised subpopulation of endosteal macrophages that are responsible for bone resorption, leads to HSC egress from the bone marrow (Kollet et al., 2006), whereas their pharmacological inhibition leads to a reduction of HSPC numbers (Lymperi et al., 2011). A different subpopulation of bone marrow macrophages, the ‘osteomacs’, form a canopy of cells near active osteoblasts and carry out the opposite role: their depletion leads to the loss of osteoblast activity and increased HSC mobilisation (Winkler et al., 2010). Very recently regulatory T cells have been demonstrated to make the HSC niche a site of immune privilege (Fujisaki et al., 2011).The question remains whether we should think about one or several HSC niches. The observation that different HSPC localisations exist within the marrow and that a growing number of cell types are involved in HSC regulation (as illustrated above), together with the increasing number of reports that describe the heterogeneity of even highly purified HSPC populations (Dykstra et al., 2007; Lo Celso et al., 2009; Wilson et al., 2008), could be an indication of the complex microenvironments through which HSCs navigate. One proposed model suggests that osteoblastic cells provide a context for HSC dormancy, whereas a perivascular, quiescent niche provides an intermediate niche for activated HSCs that are ready to either generate differentiating progeny or revert to dormancy, depending on the needs of the organism (Malhotra and Kincade, 2009a; Trumpp et al., 2010). It is possible that the number of functional niches increases further if additional HSC subpopulations and states are identified. Real-time long-term cell tracking coupled with a greater number of reporter strategies to simultaneously highlight multiple stroma components and HSPC subpopulations will be instrumental to fully understand the interactions between HSCs and their niche.Independent of the identity of the niche cell that generates a signal for the HSC, a multitude of molecular regulators of HSC fate have been described. Some are known products of osteoblastic cells; however, it is possible that an increasing number of these factors are produced by multiple cell types.The stroma-derived factor 1 (SDF1; also known as CXCL12)–C-X-C chemokine receptor type 4 (CXCR4) axis is the best-defined regulator of HSC localisation in the bone marrow. Osteoblastic cells produce SDF1 and upregulate its expression in response to irradiation (Xie et al., 2009). However, analysis of SDF1-reporter mice has highlighted numerous perivascular cells throughout the marrow that are SDF1-positive (Sugiyama et al., 2006), which indicates that SDF1 directs HSC localisation not only near osteoblastic cells but probably also throughout perivascular areas.If SDF1 and CXCR4 are responsible for HSC localisation, other cytokines, signalling pathways and adhesion molecules known to have a role in the HSC niche might, instead, regulate HSC fate. Indeed, the ligand–receptor pairs stem cell factor (SCF) and KIT, thrombopoietin (TPO) and myeloproliferative leukemia virus oncogene (MPL), as well as angiopoietin 1 (ANGPT1) and TIE2 tyrosine kinase (officially known as TEK) have pivotal roles in regulating the interaction between osteoblasts and HSCs and, specifically, HSC maintenance and in vivo quiescence (Arai et al., 2004; Barker, 1997; Yoshihara et al., 2007). Recently, angiopoietin-like 3 (ANGPTL3), which is primarily expressed by endothelial cells, has been shown to control HSC quiescence as well as the number of HSCs both in the steady state and following transplantation (Zheng et al., 2011).The WNT signalling cascade has been implicated with a role in HSC regulation; but this is still highly controversial, mostly because of the complexity of the signalling pathway itself (Malhotra and Kincade, 2009b). Following initial reports that the obliteration of the canonical WNT pathway does not affect HSCs, later studies that focussed on interactions between HSCs and their niche provided further detailed information on the involvement of this pathway (Cobas et al., 2004; Koch et al., 2008). It was shown that osteoblast-specific overexpression of the WNT inhibitor dickkopf homolog 1 (DKK1) leads to impairment of HSC self-renewal (Fleming et al., 2008). Similarly, the knockout of secreted frizzled-related protein 1 (SFRP1) – another negative modulator of WNT signalling – leads to an initial increase in long-term reconstituting HSPCs (LT-HSPCs), followed by their premature exhaustion (Renstrom et al., 2009). Interestingly, early B-cell factor 2 (EBF2, a transcription factor known to synergise with WNT signalling in certain cells and under certain conditions) knockout mice are affected by environment-dependent loss of HSCs, and their osteoblastic cells have altered expression of – among others – SFRP1 and SFRP2 (Kieslinger et al., 2010). Another modulator of WNT signalling is the secreted eicosanoid prostaglandin E2 (PGE2), which stabilises WNT signalling in HSCs and is, therefore, a new player in the complexity of WNT-mediated crosstalk between HSCs and the niche (Goessling et al., 2009; North et al., 2007). Further interactions between WNT and other signalling pathways affecting the HSC–osteoblastic-cell crosstalk were uncovered very recently by studying transgenic mice that express WNT inhibitor factor 1 (WIF) in osteoblasts, which display a similar phenotype to that observed following overexpression of Dkk1 (Shaniel et al., 2011).A controversy similar to that surrounding WNT signalling accompanies the role of Notch signalling in the HSC niche. Initial gain-of-function studies indicated its ability to expand HSCs (Varnum-Finney et al., 1998). Furthermore, studies in which transgenic reporters have been used revealed Notch activity in transplanted HSPCs (Duncan et al., 2005), but in vivo knockout studies could not confirm these results (Maillard et al., 2008; Mancini et al., 2005). However, the Notch ligand JAG1 (jagged1), was first found to be upregulated at the endosteal surface of parathyroid hormone receptor transgenic mice – which are characterised by increased HSC number and activity (Calvi et al., 2003) – and is present on human CD146+ HSC supportive bone marrow stroma cells (Sacchetti et al 2007). Moreover, expression of Notch1 and Notch2 in HSCs was recently shown to be important for their response to endothelial-derived maintenance factors (Butler et al., 2010).Studies that investigate the role of hedgehog (HH) signalling in the HSC niche seem to follow a similar paradigm compared with those focusing Notch signalling (Bhardwaj et al., 2001; Hofmann et al., 2009). The initial indication that HH signalling can cause HSC expansion through activation of bone morphogenetic protein (BMP) signalling pathways (Bhardwaj et al., 2001) was followed by the finding that HH signalling is dispensable for adult haematopoiesis (Bhardwaj et al., 2001; Hofmann et al., 2009). However, stromal BMP4 was shown to contribute to HSC maintenance (Goldman et al., 2009).As a result of their ability to influence stem cell fate, components of the extracellular matrix (ECM) have gained increasing attention with regards to the HSC niche (Connelly et al., 2010). Although it is still impossible to test how contact area, shape and matrix stiffness impact HSC fate in vivo, it could be shown that HSCs seeded on microwells actively produce their own ECM and undergo quiescence or proliferation depending on the size of the well (Kurth et al., 2009). In addition, SPP1 was the first osteoblast-derived ECM protein that was shown to influence HSC number and function (Nilsson et al., 2005; Stier et al., 2005). Lack of SPP1 leads to a stroma-dependent increase of LT-HSPCs and increased JAG1 and ANGPT1 expression in stroma cells, which perhaps explains how, in Spp1-deficient mice, HSC expansion is not accompanied by their exhaustion (Nilsson et al., 2005; Staal and Clevers, 2005).Glycans are non-protein components of the bone marrow stroma that have a role in the HSC niche. They are likely to mediate the formation of chemokine and growth factor gradients (Haylock and Nilsson, 2006). Moreover, eicosanoids (including PGE2, as mentioned above) affect the strength of signalling cascades, and neurotransmitters regulate the response to HSC mobilizing agents (see below).For a number of years, the role of β1 integrins in the crosstalk between HSCs and their niche has been of interest. Integrin α4β1 (ITGA4; also known as VLA4) mediates HSC retention within the bone marrow microenvironment (Priestley et al., 2006), whereas integrins α1β1 and α5β1 (ITGA3 and ITGA5; also known as VLA1 and VLA5, respectively) mediate adhesion of HSCs to – among others – SPP1 (Nilsson et al., 2005). Furthermore, these three integrins mediate SDF1 function (Peled et al., 2000). Interestingly, there is a link between WNT signalling and integrin expression in HSCs: the expression of constitutively active β-catenin leads to the loss of HSCs and rapid exhaustion of their progeny, but also to higher expression of integrins α2, β1 and β7 in HSPCs (Kirstetter et al., 2006).Integrins interact with ECM proteins, but integrin α4β1 is also the main binding partner of vascular cell adhesion molecule 1 (VCAM1), which is expressed on the surface of endothelial cells (Ulyanova et al., 2007) as well as cells of the osteoblastic lineage (Jiang et al., 2009). Interestingly, VCAM1 expression correlates with HSC homing (Lewandowski et al., 2010) and is upregulated in response to WNT signalling (Malhotra and Kincade, 2009a). Cadherins, which make up another class of adhesion molecules, might also contribute to HSC fate but their role in HSC homeostasis remains controversial (Levesque et al., 2010). Future studies will indicate whether the HSC niche is similar to other niches in that the direct interaction of HSCs with osteoblastic, endothelial and other cell types is fundamental to ensure their correct function.Calcium and oxygen are the main chemical elements that have been studied in relation to the HSC niche. Ca2+ is released by osteoclasts during bone resorption, which leads to the formation of a concentration gradient that spreads out from the endosteal surface (Levesque et al., 2010). HSCs express the G-protein coupled Ca2+-sensing receptor (CASR) and depend on it for their peri-endosteal localisation and function (Adams et al., 2006).In contrast to the acknowledged role of Ca2+ in the HSC nice, the relevance of oxygen tension is a controversial topic. Adaptations of the protocol for the isolation of side populations 2005), which in vivo to cells, suggested that HSCs reside in areas of the bone marrow et al., 2007; et al., 2010). However, both endosteal and LT-HSPCs are near vasculature (Kiel et al., 2005; Lo Celso et al., 2009). in vivo of bone marrow concentration are to whether gradients are present within the marrow and their and function question has recently gained further as were shown to affect transcription and WNT signalling through the activity of the transcription factor that is also expressed in HSPCs (Levesque et al., HSC niche is a of the complex molecular interactions that in and the of haematopoietic on the of its molecular detailed analysis of the molecular of the HSC niche will to and new into a functional HSC niche somatic stem cell niches are characterised by to the of stem cells between self-renewal and The HSC niche, however, has to a HSCs the niche and to later the bone marrow et al., 2009; et al., is during bone marrow bone marrow engraftment of the by of irradiation or bone marrow transplantation provides a of the potential of the HSC niche. The perturbations by pharmacological HSC mobilisation and transplantation have been studied for a in to and recent technological advances have to the that in the HSC niche during and throughout the development of factor known as factor 3 is the most widely used in It has a highly of results in the reduction of SDF1 the activation of that VCAM1 and as well as the of from the nervous system, which the of osteoblasts and their ability to HSCs within the niche et al., 2010; et al., 2006). and oxygen have recently been as non-protein of the of (Jiang et al., et al., 2010; et al., 2011). Interestingly, whereas HSC mobilisation is the of the SDF1 can this et al., homing is the first of a transplantation and on the signalling axis and β1 the in response to a Ca2+ mediates the of HSC near the (see above) (Adams et al., et al., 2006). Although HSCs and the niche molecules are dispensable during haematopoiesis but are for transplanted HSC For example, the class of the of is for transplanted HSPCs to and the bone marrow (Adams et al., 2009). Furthermore, might have a role in HSC and to the niche but, defects have only been observed in very specific transplantation and marrow with agents or irradiation to transplantation several molecular signals and is for HSC homing in the endosteal (Jiang et al., 2009; Lo Celso et al., 2009). The of on cells of the osteoblastic and other stroma are only but of of VCAM1 and vascular endothelial growth factor endothelial recovery have been highlighted as of the by irradiation (Hooper et al., 2009; et al., 2010; Xie et al., studies uncovered the ability of the HSC niche to dynamic to injuries through the of An analysis of the response to activation of the immune was the first study that highlighted a clear function for cell 1) et al., 2009), which has been recognised as a of egress of HSCs and their the bone marrow niche is probably by that are similar to those to engraftment and recovery from on a affect niche and the number of HSCs (Mendez-Ferrer et al., 2008), and bone itself might have a role in HSC homeostasis (Levesque et al., 2010). is another that is likely to affect Although long-term follow up of endogenous HSCs is currently in cell and to the endosteal surface were observed in and transplanted HSPCs et al., is for the HSC niche both the development of and being affected by haematopoietic disease. bone marrow stroma can lead not only to loss of HSC function but also to and et al., 2009; Raaijmakers et al., 2010; et al., For example, mice with in the osteoblastic lineage bone defects as well as which can into (Raaijmakers et al., 2010). Whereas it is clear that on the of in cells of the haematopoietic lineage, this model indicates that the bone marrow stroma has a role in by different that support cells or a to haematopoietic cells. might, therefore, the development of disease. In addition, cells might the ability to respond to niche signals For example, growth factor is to contribute to the dormancy of HSCs et al., and, by to the and proliferation of cells in a model of et al., HSCs and stem cells for the same niche is still an but it to be that haematopoiesis is in the of Indeed, the homing of human HSPCs is dramatically altered when they are transplanted into mice et al., 2008). Whether HSC niche function is et al., 2009), but human stem (or cells have been shown to reside near the endosteal surface when into mice et al., 2010). HSCs, whether or might have a role in their niche and it remains an whether that interaction can be to affect the support of or cells et al., number of known cellular components and molecular that regulate interactions has dramatically increased during the An in the HSC niche has been the from several to the that HSC niches present a that is complex Whereas initial studies highlighted the role of osteoblastic cells in regulating HSC number and it is widely accepted that several other stromal cell from mesenchymal stem cells to various differentiated lineages, interact with HSC and influence their fate through an of secreted and adhesion molecules as well as by affecting the protein and chemical of bone marrow extracellular The of the components involved in the HSC niche is likely to in and the same is for the of signalling pathways and of on and localisation of technological advances and experimental models will to new of the HSC niche itself and to several that are currently under For example, an question is how functionally distinct HSC niches exist and whether they are dynamic in the bone marrow on niches have indicated their ability to regulate during stem cell and to the of differentiating cells to a stem cell Further development of and functional will the of whether this is also the for the HSC niche. the between and interactions will to these for through vivo expansion of HSCs and the development of new for haematopoietic disease.