Leukemia (2010) 24, 1979–1992 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 www.nature.com/leu SPOTLIGHT REVIEW

The endosteal ‘osteoblastic’ niche and its role in hematopoietic stem cell homing and mobilization

J-P Le´vesque1,2, FM Helwani1 and IG Winkler1

1Biotherapies Program, Haematopoietic Stem Cell Laboratory, Mater Medical Research Institute, South Brisbane, Queensland, Australia and 2School of Medicine, University of Queensland, Brisbane, Queensland, Australia

The concept of hematopoietic stem cell (HSC) niche was granulocyte colony-stimulating factor (G-CSF), cytotoxic drugs formulated in 1978, but HSC niches remained unidentified or chemokine analogs.8–10 for the following two decades largely owing to technical limitations. Sophisticated live microscopy techniques and genetic manipulations have identified the endosteal region of the bone marrow (BM) as a preferential site of residence for the The first HSC hitch-hiker’s guide to getting in and out most potent HSC – able to reconstitute in serial transplants – of the BM with osteoblasts and their progenitors as critical cellular elements of these endosteal niches. This article reviews the path to the discovery of these endosteal niches (often called It is now well established that homing to the BM tissue involves ‘osteoblastic’ niches) for HSC, what cell types contribute to a capture step in which circulating HSPC directly interact with these niches with their known physical and biochemical the BM endothelium via cell–cell adhesive interactions. This is features. In the past decade, a first wave of research uncovered made possible by the unique feature of BM sinusoidal endo- many mechanisms responsible for HSC homing to, and thelial cells, to constitutively express the adhesion molecules mobilization from, the whole BM tissue. However, the recent (1) P-selectin (CD62P), (2) E-selectin (CD62E) and (3) vascular discovery of endosteal HSC niches has initiated a second wave 11,12 of research focusing on the mechanisms by which most cell adhesion molecule-1 (VCAM-1 or CD106). HSPC primitive HSC lodge into and migrate out of their endosteal express receptors for both endothelial selectins, most impor- niches. The second part of this article reviews the current tantly P-selectin glycoprotein ligand-1 (or CD162), CD44,13 and knowledge of the mechanisms of HSC lodgment into, retention other less well-defined E-selectin receptors,14 as well as in and mobilization from osteoblastic niches. receptors for VCAM-1, such as integrins a4b1,15,16 a4b717 and Leukemia (2010) 24, 1979–1992; doi:10.1038/leu.2010.214; a9b1.18,19 Experiments in mice using function-blocking anti- published online 23 September 2010 Keywords: hematopoietic stem cell; niche; osteoblast; bone bodies or targeted deletions of these genes have clearly shown marrow; homing; mobilization their essential role in HSPC homing to the BM. P- and E-selectin mediate the initial rolling of fast flowing HSPC onto BM endothelium. Once slowed, HSPC can firmly adhere to the vasculature via integrins and transmigrate through the endothe- lium into the BM stroma15,20,21 to migrate and lodge into their respective niches. Although these same three cell adhesion Introduction molecules are utilized to recruit immune cells to sites of inflammation/injury in other tissues, they only become Following the discovery that virtually all long-term reconstitut- expressed at the surface of endothelial cells after pro-inflam- ing hematopoietic activity is contained within the bone marrow 1,2 matory stimuli. A unique feature of BM endothelium is the (BM) tissue in adult mammals, many aspects of hematopoietic constitutive expression of endothelial selectins and VCAM-1 cell stem cell (HSC) regulation within the BM have now been adhesion molecules enabling homing of HSPC into the BM in elucidated. Some of the most significant discoveries have been steady-state conditions. that (1) HSC must reside in specialized niches in the BM to exert SPOTLIGHT 3 The chemokine CXCL12 (or stromal cell-derived factor SDF-1) their function properly, and (2) a small proportion of HSC and also plays a critical role in recruiting HSPC into the BM and more committed hematopoietic progenitors cells (HPC) physio- guiding them to their niches by interacting with its receptor logically traffic continually from the BM into the circulation4 5 CXCR4 expressed by HSPC. This CXCL12:CXCR4 interaction and concomitantly re-home to the BM. Although little is known triggers chemotaxis via intracellular GTPase proteins. Indeed, about the mechanisms leading to the spontaneous release pre-treatment of human HSPC with a blocking anti-CXCR4 of a small proportion of hematopoietic stem and progenitor 6,7 antibody impairs their homing into the BM of non-obese cells (HSPC) from their niches into the blood, much more diabetes/severe-combined immunodeficiency (NOD/SCID) research has been devoted to HSPC homing into the BM and mice.22 Similarly in the mouse, homing of HSPC isolated from extraphysiological mobilization as observed following the the fetal liver of CXCR4À/À mice (CXCR4 gene deletion is administration of therapeutic doses of cytokines, such as perinatally lethal) have a severe homing defect in host BM. However, this homing defect can be rescued by increasing the Correspondence: Associate Professor J-P Le´vesque, Biotherapies cell dose,23 possibly because other cytokines such as KIT ligand Program, Haematopoietic Stem Cell Laboratory, Mater Medical (KL) can also promote HSPC adhesion and migration via integrin Research Institute, Aubigny Place, Raymond Terrace, South Brisbane, activation.16,24 Integration of the adhesive and chemotactic Queensland 4101, Australia. E-mail: [email protected] signaling from CXCR4, c-KIT and many other receptors that Received 28 May 2010; revised 5 August 2010; accepted 18 August contribute to homing (such as adrenergic and prostaglandin E2 2010; published online 23 September 2010 receptors) depends on several small GTPases such as Rac1, In and out of hematopoietic stem cell niches J-P Le´vesque et al 1980 Rac2,25 cdc42,26 and guanine nucleotide binding protein to HSPC mobilization has been questioned by the fact that complexes Gi27 and Gs,28 which could be upstream of Rac targeted deletion of these genes in the C57BL/6 inbred mouse and cdc42. Indeed, pharmacological inhibition or conditional strain does not decrease mobilization in response to G-CSF.45 deletion of genes encoding these proteins impairs HSPC This was highly unexpected as in humans, shed soluble VCAM-1 homing. In addition to recruiting HSC to the BM, CXCL12 is fragments resulting from proteolytic cleavage have increased also critical to the retention of HSPC within the BM and concentrations in the blood of mobilized donors, and this consequently in mobilization. This will be reviewed further increase is correlated with the mobilization response.34,46 In below. addition, soluble VCAM-1, neutrophil proteases and circulating HSPC also increase in idiopathic myelofibrosis.47 Interestingly, inbred mouse strains that mobilize poorly in response to G-CSF HSC mobilization is not the mirror image of homing (typically C57BL/6, the strain in which most protease knockout During the past decade, it has also emerged that HSPC have been made) have much lower release of neutrophil mobilization is not the reverse of homing, but rather a disruption proteases in the BM in response to G-CSF than strains that of the mechanisms that retain HSPC within the BM and their mobilize well (such as BALB/c and 129Sv, strains in which niches as the mechanisms of homing do not perfectly overlap initial in vivo studies on proteases and protein cleavages were or mirror those of retention and mobilization. For instance, carried out). Therefore, before totally excluding the possible role conditional deletion of both Rac1 and Rac2 genes in HSPC of proteases in HSPC mobilization, experiments should be causes a mild homing defect (40% reduction in homing of repeated in knockout mice backcrossed onto BALB/c, DBA/2 or colony-forming cells 16 h post transplant), whereas it causes 129Sv strains, which are known to mobilize well and release SPOTLIGHT massive HSPC mobilization in vivo.25 Similarly, homing of large amounts of proteases in the BM in response to G-CSF. CXCR4À/À HSPC is severely impaired but can be rescued by the Furthermore, although protease knockout C57BL/6 mice still administration of large cell doses. However, once engrafted long mobilize in response to G-CSF, mobilization can be dramati- term into recipients, CXCR4À/À HSPC are poorly retained in the cally reduced by transient inhibition of these same proteases BM and large numbers circulate in the blood.23 From these following injection of protease inhibitors.36,39,42 In summary, experiments, it was concluded that mobilization is not caused although proteases are likely to be an important mechanism of by a homing defect, but rather by a breakdown of retention HSC mobilization in normal circumstance, other mechanisms mechanisms. The mechanistic difference between homing and are available. Taken together, experiments in C57BL/6 mice retention in the BM is further illustrated by the conditional show that there are alternative mechanisms that can lead to deletion of the Gsa gene. Although inhibition of the hetero- HSPC mobilization in response to cytokines and chemotherapy. trimeric guanyl binding protein Gi, which inhibits adenyl Indeed, the C57BL/6 strain has been very helpful in identifying cyclase and the formation of cAMP, impairs both homing such alternative mechanisms of HSPC mobilization involving, 27,29 to and retention within the BM, conditional deletion of Gsa for instance, the transmembrane dipeptidyl peptidase in hematopoietic cells, which is necessary to activate adenyl CD26,48,49 complement cascade activation and complement cyclase and the formation of cAMP, prevents HSPC homing to fragments C3 and C5,50,51 or the plasminolytic pathway.52,53 the BM during fetal development, as well as in adults following Although these studies have provided invaluable insights on transplantation without affecting HSPC retention within the how HSPC home into and mobilize from the whole BM tissue, BM.28 This suggests that cAMP, whose synthesis is controlled by they did not investigate the mechanisms of migration and the dual action of Gs and Gi proteins, may be rheostat regulating lodgment into their specific niches, and whether mobilization homing and retention differentially.30 involves perturbation within the niche itself. The recent identification of cellular and molecular components of HSC niches in the BM has initiated a second wave of research to Are proteases important? dissect the mechanisms of homing into and mobilization from Conditional deletion experiments have delineated some of the these niches. most critical pathways in HSPC retention and mobilization. In particular, the VCAM-1/a4 integrin interaction, the CXCL12/ CXCR4 interactions and the signaling mediated by Gi protein, Evidence supporting osteoblastic (or endosteal) niches Rac1 and 2, and cdc42. Conditional deletion of any of these genes23,25,26,31–33 or treatment with pertussis toxin that blocks The nature and composition of HSC niches remained unknown Gi protein29 causes massive HSPC mobilization into the blood. until the second half of this decade. Preceding the formulation of Nonetheless, how these mechanisms are disrupted during the HSC niche concept by Schofield, Brian Lord and co-workers clinical mobilization, for instance, following the administration provided the first evidence that the distribution of primitive of G-CSF or cytotoxic therapy, is still not completely elucidated HSPC was not isotropic throughout the mouse BM, but enriched and under intense investigation. We and others have found that in proximity to the bone – the endosteal region. By applying in humans and inbred mouse strains that mobilize well in serial BM flushes with increasing strength, BM leukocyte response to G-CSF or chemotherapy (such as BALB/c or 129Sv), fractions were eluted radially according to their distance from neutrophil proteases such as neutrophil elastase, cathepsin G, the central longitudinal axis of the BM. Short-term reconstituting matrix metalloproteinase-9 or membrane type-1 matrix metal- spleen colony-forming units were three times more frequent in loproteinase accumulate in the BM space with concomitant the endosteal region within 50 mm of the bone surface than in downregulation of their naturally occurring inhibitors serpins the more central BM.54 This anisotropic distribution of most and tissue inhibitors of metalloproteinases.34–40 As these primitive HSPC led Russell Taichman to hypothesize that bone- proteases cleave and inactivate a range of proteins involved in forming osteoblasts may be the critical cells that support HSC HSPC retention within the BM, such as VCAM-1,34,35 CXCL12, in vivo, and showed that in culture, human osteoblasts purified its receptor CXCR4,41,42 KIT43 and KL,44 they are tempting from trabecular bones could support the expansion of long-term candidates to mediate some aspects of mobilization. However, culture-initiating cells (LT-CIC) derived from CD34 þ human BM the whole concept that secreted neutrophil proteases contribute cells55; an effect mediated in part by the secretion of G-CSF and

Leukemia In and out of hematopoietic stem cell niches J-P Le´vesque et al 1981 hepatocyte growth factor by osteoblasts.56 Further evidence of endosteum and 10 mm (or two-cell diameters) from osteoblasts the privileged properties of the endosteum to support primitive in irradiated recipients, whereas these HSC homed more HSPC was provided by Susie Nilsson in mice. Lineage-negative distant from the endosteum and osteoblasts in non-irradiated (LinÀ) wheat germ agglutinin (WGA)dim rhodamine123 (Rho)dull recipients. On the other hand, more mature HPC such as cells, which represent a primitive population of less metaboli- LinÀSca1 þ KIT þ CD34 þ Flt3 þ progenitors and linage com- cally active HSPC with high dye efflux activity, and mitted LinÀSca1ÀKIT þ HPC homed more distant from osteo- LinÀ WGAdim Rhobright, which represent more proliferative blasts and endosteum.60 Similar three-dimensional microscopy HPC, were loaded with the fluorescent tracker CFSE (5-(and-6)- studies on whole femurs explanted 4 h post transplant showed a carboxyfluorescein diacetate, succinimidyl ester) and trans- preferential homing of 60% transplanted LinÀSca1 þ KIT þ HSPC planted into non-ablated mice. Strikingly, within 5 h post within two-cell diameters from osteoblasts at the trabecular transplant, 60% of primitive LinÀ WGAdim Rhodull HSPC endosteum in irradiated recipients but not in non-irradiated distributed preferentially in the endosteal region within 12-cell recipients. Interestingly, transplanted HSPC proliferated exten- diameters from the bone surface, whereas Lin þ and LinÀ sively in irradiated recipients in the trabecular metaphysis but WGAdim Rhobright HPC distributed randomly within the BM.57 not in the diaphysis 1 week post transplant. In non-irradiated Altogether, these studies showed that most primitive HSPC recipients, transplanted HSPC did not proliferate and were lost preferentially reside near and migrate close to the endosteum, from recipient BM within 2 weeks.61 These two congruent and that osteoblast lineage cells could support these HSPC studies and others62,63 confirmed that transplanted HSC rapidly in vitro. This suggested that the endosteal region and osteoblast homed to specific niches located within two-cell diameters from lineage cells provide a specific microenvironment favorable osteoblasts at endosteal surfaces in both flat and long bones and to HSC in vivo. that these niches need first to be vacated (for example, following In 2003, two papers published in the same issue of Nature lethal irradiation) in order for HSC to successfully home in these propelled osteoblasts into the spotlight. Two strains of geneti- endosteal niches. Furthermore, a recent study has shown in cally modified mice with increased osteoblast function and adult mice that LT-CRC with the capacity to serially transplant bone formation constructed in David Scadden’s and Liheng Li’s are deeply quiescent in steady state, dividing on average laboratories had increased the number of HSC and hematopoi- once every 145 days, or five times in a mouse lifetime.64 These esis in the BM. In the first paper, mice overexpressed a deeply quiescent, slow-cycling HSC (able to retain bromodeoxy- constitutively active mutant of parathyroid hormone 1 receptor uridine over a 70-day chase) also reside close to the endosteal (PTHR1), the common receptor for parathyroid hormone (PTH) surfaces64 and distal to rapid blood flow.65 and paracrine PTH-related peptide specifically under the control The notion that the bone provides a privileged environment of the 2.3-kb fragment of the rat collagen 1a promoter, which is supporting HSC self-renewal is also supported by experiments in specific of maturing and mature osteoblasts (col2.3PTHR1 which bone ossicles are grown ectopically under the mouse skin mice).58 As expected, constitutive activation of PTHR1 in or kidney capsule. In these experiments, ossicles are formed by osteoblasts dramatically increased trabecular bone formation transplanting cultured plastic adherent mesenchymal stromal in tibial metaphysis at the expense of the BM space. Less cells in a gelatin sponge under the skin.66 At 21 days post expected was the doubling in the frequency of LinÀSca1 þ KIT þ transplant, bone ossicles develop containing BM with HSC, HSPC, LT-CIC and long-term competitive repopulating cells leukocytes and endothelium derived from the host. This ossicle (LT-CRC), whereas the frequency of more mature colony- BM is very similar in composition to endogenous femoral BM forming cells was unaltered. In these mice, expression of and contains HSC defined as LT-CRC. Interestingly, administra- interleukin-6, KL and CXCL12 by osteoblasts was increased and tion of bone anabolic PTH during the growth of the ossicles a direct role of the transgenic BM stroma in supporting wild-type doubles bone density and trebles LT-CRC frequency in the (WT) LT-CIC was shown in vitro. The role of PTH and paracrine ossicles.67 When BM cells are injected directly into ectopic PTH-related peptide in supporting HSC niche function was ossicles or in the circulation after lethal irradiation to eliminate further supported by the observation that daily administration of host HSC, donor HSC colonize and self-renew within the PTH to WT mice also increased LinÀSca1 þ KIT þ HSPC, LT-CIC ossicles, and reconstitute hematopoiesis.67 Taken together, these and LT-CRC frequencies in the BM, and increased the data show that small ectopic ossicles can form bona fide HSC engraftment of suboptimal doses of BM donor cells into lethally niches supporting HSC self-renewal and hematopoietic reco- irradiated recipients treated with PTH.58 In the second paper, nstitution. the bone morphogenic protein receptor BMPR1a gene, which is The proto-oncogene c-myc has been proposed to play a SPOTLIGHT expressed by osteoblasts and most hematopoietic lineages, critical role in the regulation of HSC self-renewal in the except LinÀSca1 þ KIT þ HSPC, was floxed and deleted in adults endosteal niche.68 This conclusion was based on the findings mice following induction of the Mx1Cre transgene with that: (1) phenotypic LinÀSca1 þ KIT þ Flt3À HSC express much polyI:polyC. The somatic deletion of the BMPR1a gene resulted lower levels of c-myc than their more differentiated in a phenotype similar to that observed in col2.3PTHR1 and committed progenies; (2) conditional deletion of c-myc in transgenic mice, including increased bone formation and hematopoietic cells enhances HSC self-renewal with the reduced BM cavity associated with increased absolute number accumulation of HSC in the BM tissue, but inhibits their of LinÀSca1 þ KIT þ HSPC, LT-CIC and LT-CRC per femur, differentiation; (3) once transplanted c-myc-deficient HSC home as well as lower HSPC proliferation in vivo as measured by and lodge into endosteal niches, self-renew and accumulate in bromodeoxyuridine incorporation.59 host BM, but fail to differentiate into mature leukocytes; and The suggestion that osteoblasts and endosteum support HSC (4) overexpression of c-myc compromises HSC reconstitution in vivo was further supported by refined in vivo and ex vivo potential of lethally irradiated hosts.68 Although c-myc is an three-dimensional imaging of HSC in whole mouse bones. In the important rheostat that regulates HSC self-renewal versus calvaria, a flat bone which unlike long bones is not formed by differentiation, localization analyses of HSC in the BM and endochondral ossification but by intramembranous ossification, endosteum are necessary to confirm whether c-myc also LinÀSca1 þ KIT þ CD48ÀCD150 þ or LinÀSca1 þ KIT þ CD34À regulates HSC location. For instance, do self-renewing c-myc- Flt3À phenotypic HSC rapidly homed within 20 mm of the deficient HSC accumulate specifically in endosteal niches, and

Leukemia In and out of hematopoietic stem cell niches J-P Le´vesque et al 1982 do c-myc-overexpressing HSC fail to lodge at the endosteum HSC were closer to the endosteal surface than normal and following transplant? proliferated more; B lymphopoiesis was reduced and myelopoi- esis increased. Transplantation of WT HSC into lethally irradiated osxCre þ Dicer1f/f recapitulated the phenotype. Are mature osteoblasts important? Here comes the Finally, secondary leukemia emerged in these mice showing osteoprogenitors that a defective HSC niche can also support the expansion of malignant clones that would be repressed in a normal niche. Most studies show that following transplantation, a proportion of Interestingly, deletion of the Shwachman–Diamond–Bodian phenotypic HSC rapidly homed to endosteal niches within syndrome (Sbds) gene from osteoprogenitors, whose expression two-cell diameters (or 10 mm) of osteoblasts lining the endo- is reduced in osxCre þ Dicer1f/f osteoprogenitors, resulted in a steum.33,59,61,69 However, in non-manipulated naı¨ve mice, an similar phenotype.78 Todate, these results provide the most important proportion of these phenotypic HSC also reside in compelling evidence that osteoprogenitors are crucial to HSC perivascular locations in direct contact with the abluminal side function and essential components of HSC niches. of endothelial cells away from the endosteum.33,70,71 From these data emerged the concept of vascular niches. Importantly, these two niches cannot be easily distinguished in flat bones What do HSC particularly like in endosteal niches? such as the calvaria, in which the BM is so thin that most osteoblasts are perivascular,60 or in femurs where 30% of Although the precise cellular and molecular composition of osteoblasts in trabecular areas are in the vicinity of the endosteal HSC niches is far from fully understood, it clearly SPOTLIGHT vasculature.61 Therefore, the vascular and endosteal niche need involves cells from the osteoblastic lineage, particularly not be mutually exclusive and are likely to partially overlap in osteoprogenitors. In addition to this, CXCL12-abundant reticular location and function.71 (CaR) cells have been identified in mouse BM in direct contact In addition, the initially described direct association of with phenotypic HSC regardless of the location of the niche spindle-shaped N-cadherin þ osteoblasts with N-cadherin þ (endosteal or vascular).33 Further characterization of these cells HSC59 has been challenged by a competing group that could will enable to better understand how niches control HSC fate. not detect N-cadherin in functional HSC.72,73 Further doubts on Toshio Suda’s group has recently reported that CD45À Ter119À the role of mature osteoblasts in directly supporting HSC was CD31À endosteal cells, which are neither hematopoietic nor provided by col2.3HTK transgenic mice, in which the suicide endothelial, can be sorted into three populations according to Herpes virus thymidine kinase (HTK) gene is specifically their expression of Sca1 and activated leukocyte cell adhesion expressed in maturing osteoblasts under the control of col2.3 molecule (ALCAM or CD166), with Sca1ÀALCAM þ osteoblast promoter. When these mice are treated with ganciclovir, this lineage-restricted osteoprogenitors, Sca1ÀALCAMÀ osteochon- guanosine analog is phosphorylated and incorporated into DNA droprogenitors and Sca1 þ ALCAMÀ immature mesenchymal specifically killing osteoblasts expressing HTK. Osteoblast cells with osteogenic and adipogenic potential.79 Interestingly, depletion in vivo resulted in ablation of the BM and relocation each of these three sorted cell types were able to support of hematopoiesis and HSC in the spleen and the liver.74 expansion of LT-CRC in co-cultures with sorted LinÀSca1 þ KIT þ However, the surprise was that HSC ablation in the BM was HSPC, despite expression of very different arrays of genes.79 slow, 2–3 weeks after osteoblast depletion, whereas medullar B Paul Frenette’s group has just reported that a rare population of lymphopoiesis, which requires functional osteoblasts in vivo BM reticular cells expressing the intermediate filament protein and in vitro, was ablated within few days.75 Similarly, in a nestin is perivascular throughout the BM, including the mouse model of chronic inflammatory arthritis, bone formation endosteum, with clonal self-renewal in vitro and osteogenic, was reduced with reduced number of B-cell progenitors in the chondrogenic and adipogenic potential in vitro and in vivo, the BM. However, the content in phenotypic and functional LT-CRC hallmark of a true MSC. LinÀCD48ÀCD150 þ are spatially was unaltered.76 This disconnection between mature osteoblasts associated with these nestin þ cells in the BM, and upon and B lymphopoiesis on the one hand, and slow mobilization of transplantation, HSC rapidly lodge near these nestin þ cells. medullar HSC on the other, questioned the role of mature Selective depletion of nestin þ cells in the BM resulted in a 50% osteoblasts in supporting HSC niches.77 Furthermore, deletion of reduction in phenotypic HSC numbers in the BM, and a 90% the biglycan gene, an extracellular protein produced by mature reduction in homing of transplanted colony-forming cells. These osteoblasts and chondrocytes, results in reduced trabecular nestin þ MSC express high levels of KL, CXCL12 and angio- bone and osteoblasts, but not in reduced frequency of poietin-1 (ang-1), cytokines well known to promote HSC self- phenotypic HSC and LT-CRC in the BM.72 In fact, it has very renewal.80,81 Therefore, several cell types from the mesenchy- recently emerged that osteoprogenitors are more important than mal and osteogenic lineages may act in concert to form HSC mature osteoblasts in supporting HSC.78 Global gene regulation niches. was targeted specifically in either mature osteoblasts or A number of proteins with effects on HSC proliferation and osteoprogenitors in two distinct mouse strains by breeding mice self-renewal in vivo or in vitro have been found to be expressed with a floxed Dicer-1 gene (necessary to the processing and by osteoblasts and osteoblast lineage cells (Table 1). CXCL1233 maturation of all regulatory microRNA), with mice containing a and VCAM-1,32 which are critical to the retention of HSPC Cre recombinase driven by either the osterix promoter – active within the whole BM tissue, are also expressed by osteoblast specifically in osteoprogenitors (osxCre þ Dicer1f/f), or by the lineages cells and osteoblasts at the endosteum.75,82–84 In osteocalcin promoter – only active in mature osteoblasts addition, many proteins that affect HSC proliferation and self- (osxCre þ Dicer1f/f). Both strains had similar bone defects; renewal capacity are also expressed by osteoblasts such as however, only osxCre þ Dicer1f/f mice had a severe hemato- ang-1, KL, thrombopoietin, BMP-4, secreted frizzled related poietic defect, whereas in sharp contrast, oscalCre þ Dicer1f/f protein-1 (sfrp-1) and connexin-43 (Table 1). Mutations, had normal hematopoiesis.78 The specific ablation of microRNA deletions or overexpression of these genes, which are expressed in osteoprogenitors resulted in a myelodysplastic syndrome with by osteoblast lineage cells, all alter HSC function in vivo peripheral pancytopenia. Furthermore, in the BM, phenotypic (Table 1). Osteoblasts also express a number of Notch ligands

Leukemia In and out of hematopoietic stem cell niches J-P Le´vesque et al 1983 Table 1 Components of the endosteal niche involved in HSC function

Components Effects in vivo or in vitro References

Proteins Angiopoietin-1 Overexpression in HSC increases HSC quiescence and recovery from cytotoxic insult 69 and irradiation Transmembrane KIT-ligand Lack of expression in host causes severe anemia and reduced HSC lodgment into 133,134 endosteal niches Thrombopoietin Anti-Mpl antibody increases HSC proliferation in vivo. TPO administration increases 135 the number of quiescent HSC in vivo BMP-4 Deletion in host increases HSC proliferation, but decreases serial reconstitution (HSC 136 self-renewal) Sfrp1 Deletion in host decreases HSC proliferation and serial reconstitution (self-renewal) 88 Connexin-43 Deletion compromises recovery from cytotoxic insult 137 CXCL12 Conditional deletion of the receptor CXCR4 causes HSC to enter cell cycle, compromise 33 recovery from cytotoxic insult and mobilize into the blood Osteopontin Gene deletion causes increased HSC proliferation, accumulation in BM and reduced 99,100 lodgment to the endosteum following transplant VCAM-1 Deletion of VCAM-1 gene induces HSPC mobilization. Deletion of its receptor on HSC 32,63 reduced HSC lodgment into endosteal niches N-cadherin N-cadherin knockdown or overexpression of dominant-negative mutant in HSC increases 103,104 HSC proliferation, reduces lodgment to the endosteum and compromises long-term reconstitution Annexin II Anti-annexin II antibody treatment of HSC reduces HSPC homing to the BM, and long-term 106 reconstitution following transplant. HSPC adhere less to annexin II-deficient osteoblasts

Pathways Notch signaling Inhibition compromises HSC self-renewal and increases differentiation 58,86 Wnt signaling Overexpression of Dikkopf-1 in osteoblasts to inhibit Wnt signaling increases HSC 89 proliferation and decreases serial reconstitution (HSC self-renewal)

Physicochemical properties Soluble calcium ion HSC lacking the calcium receptor have hypocellular BM with reduced number of HSC. 98 They fail to lodge near endosteum upon transplantation Low blood perfusion, hypoxia Dormant serially transplantable HSC reside in poorly perfused hypoxic niches and the 65,91,93 endosteal niches are hypoxic Abbreviations: BM, bone marrow; BMP-4, bone morphogenetic protein 4; HSC, hematopoietic stem cell; HSPC, hematopoietic stem and progenitor cells; Sfrp1, secreted frizzled related protein-1; TPO, thrombopoietin; VCAM-1, vascular cell adhesion molecule-1. such as Jagged-1 and -2, Delta-1 and -4, and Hes-1 and -5.85 with Ca2 þ receptors sensing this gradient and enabling them to Furthermore, experiments using reporter mice in which cells home to endosteal niches. The second characteristic of HSC become fluorescent when Notch signaling is activated have niches are their very low blood perfusion and oxygen tension. shown that Notch signaling is activated in phenotypic HSC in In steady state, the endosteal region of the BM is hypoxic with 58 col2.3PTHR1 transgenic mice. Inhibition of Notch signaling less than 2% O2, with high cellular content in the hypoxia- 91 by retroviral expression of a dominant-negative mutant of inducible factor-1a, an O2-labile transcription factor rapidly xenopus suppressor of hairless, a pan inhibitor of Notch degraded by the proteasome when O2 concentration is in excess signaling, also resulted in compromised reconstitution and of 2%.92 Furthermore, long-term reconstituting HSC bind to increased HSC differentiation in vivo.86 The fact that osteoblasts chemical probes such as pimonidazole that detect hypoxic express several Notch ligands, which may trigger several Notch environment in vivo,93 suggesting that most potent and primitive receptors on HSC, may explain why deletion of Jagged-1 and HSC reside in a hypoxic environment in the BM. We have Notch-1 does not compromise hematopoiesis in vivo.87 Notch recently found that the most potent and deeply quiescent HSC SPOTLIGHT and Wnt signaling pathways are coactivated in vivo,86 suggest- (retaining bromodeoxyuridine label for over 70 days with ing an important role for the latter, a conclusion strengthened by unique ability to serially transplant in secondary recipients) the finding that HSC function is altered in the BM of mice after are located in niches with the poorest blood perfusion. Indeed, deletion of sfrp-1 gene in the stroma (sfrp1 is a canonical Wnt following intravenous injection of Hoechst33342, a cell-perme- signaling inhibitor), or overexpression of the Dickkopf-1 able fluorescent dye was administered for the last 10 min before transgene (another Wnt signaling inhibitor) in osteoblast lineage being killed; only phenotypic HSC not exposed to this dye in the cells under the control of the col2.3 promoter88,89 (Table 1). BM in vivo (furthest from rapid blood flow) were able to serially In addition to proteins, endosteal niches have a peculiar transplant, whereas HSC of identical phenotype, which had physical–chemical environment compared with the central BM. incorporated some dye in vivo (located in more perfused The endosteum, particularly in the metaphyseal spongiosa niches), were less potent with faster cycling and able to where most HSC reside, is the site of active bone remodeling reconstitute a single host only.65 The poor perfusion of niches, with coupled bone formation and degradation. Consequently, which contain the most potent HSC, may be due to very low soluble Ca2 þ ions are released by osteoclasts degrading the blood velocity in BM endothelial sinusoids. Indeed, sinusoidal bone and re-deposited in the neo-formed bone matrix or blood flow at the endosteum of the rabbit fibula is only one- osteoid. Ca2 þ concentrations around active osteoclast-degrad- tenth of that in capillaries.94 In addition, mathematic modeling 90 ing bone fragments can reach up to 40 mM, thus creating a predicts that a layer of two myeloid progenitors is sufficient 2 þ 95 Ca gradient from the endosteal surfaces. HSC are equipped to deplete most O2 provided by a nearby BM sinusoid.

Leukemia In and out of hematopoietic stem cell niches J-P Le´vesque et al 1984 It is therefore possible that the blood velocity in these sinusoids number of circulating LinÀSca1 þ KIT þ HSPC in steady state.18 is so low that phenotypic HSC need not be located very far away Integrin a4b1, which is critical to HSPC homing and retention from these sinusoids to be in hypoxia. Thus, exposure to blood within the BM tissue, is also critical to HSC lodgment into the perfusion may determine HSC function and niche characteristics endosteal region as integrin a4-deficient LinÀKIT þ cells failed to more than absolute distance from the vasculature. Low blood preferentially lodge at the endosteum of WT mice.63 Consider- velocity in BM sinusoids may therefore reconcile the observa- ing that two of the integrin a4 ligands are expressed by tions that (1) most long-term reconstituting HSC reside in osteoblast lineage cells, namely VCAM-1 and osteopontin, this hypoxic93 and poorly perfused areas of the BM, and is not so surprising. (2) phenotypic HSC are also observed in proximity to blood sinusoids.33,70 Other consequences of poor perfusion of HSC niches containing the most dormant and primitive HSC may also Transmembrane KL be (i) greater concentration of locally secreted factors/mediators Similarly, transmembrane KL, which is also expressed by produced by support cells of the niche (factors/mediators diluted osteoblasts, is necessary for HSC lodgment near the endosteum. by the blood flow in more perfused/less dormant niches), and Transplanted WT HSPC have significantly reduced lodgment at 65 d d (ii) very low concentrations of blood-borne nutrients and O2. the endosteum of Steel Dickie (Sl /Sl ) mice, which express only a soluble form of KL due to a deletion of the region encoding the transmembrane and intracellular domains.101 What drives HSC lodgment into endosteal niches? SPOTLIGHT Once circulating HSC have crossed the endothelium into the Cadherins BM (homing phase), they rapidly migrate through the BM stroma In the initial work describing enhanced HSC number in the BM to lodge in their niches near the endosteum (lodgment) within a cavity of mice with somatic deletion of the BMPR1a gene,59 few hours.96,97 Many molecular determinants of HSC lodgment HSC directly attached to spindle-shaped osteoblasts. Both cell into their endosteal niches have recently been identified. Most types were described to express the cell adhesion molecule of these signals are either produced by osteoblasts and N-cadherin. However, the expression of N-cadherin by HSC osteoblast lineage cells, deposited in the extracellular matrix was later contested by Sean Morrison’s group who could not at or near the endosteum, or released from the endosteum detect N-cadherin by quantitative reverse transcriptase-poly- during its remodeling. merase chain reaction or flow cytometry in sorted LinÀSca1 þ KIT þ CD48ÀCD150 þ HSC or in HSC from trans- genic mice expressing the lacZ reporter under the N-cadherin Calcium promoter.72 Furthermore, polyI:polyC treatment of Mx1Cre þ Soluble Ca2 þ ions released from the remodeling endosteum N-cadherinfl/À mice, which deletes the N-cadherin gene from together with the calcium ion receptor CaR expressed by HSPC both osteoblasts and hematopoietic cells did not alter the are essential to HSC lodgment at the endosteum. CaR-deficient frequency or the number of phenotypic HSC in the BM, long- mice have hypocellular BM, reduced frequency of phenotypic term reconstitution potential in competitive repopulations LinÀSca1 þ KIT þ HSPC and LT-CIC in the BM, and consequently assays, nor their serial reconstitution potential.73 Following a increased the number of LinÀSca1 þ KIT þ HSPC in the blood mediation by Len Zon, it was concluded that HSC either do not and spleen. Furthermore, on histological sections, fluorescently express N-cadherin or the level is so low or expressed by a very labeled LinÀSca1 þ KIT þ HSPC from CaR-deficient mice failed limited HSC subpopulation that N-cadherin is not reliably to properly lodge within two-cell diameters of the endosteum of detected. Furthermore, serial dilution analysis in competitive WT recipients,98 showing that soluble Ca2 þ and CaR are critical repopulations assays of HSC that underwent N-cadherin gene to HSC lodgment into endosteal niches. deletion using the Mx1Cre þ N-cadherinfl/À mice model showed that N-cadherin deletion in HSC did not alter long-term engraftment and reconstitution.102 In a more recent twist, Osteopontin however, Toshio Suda’s laboratory has recently reported that Osteopontin, a bone matrix protein particularly abundant at the lentiviral transduction of a dominant-negative mutant of endosteum, is produced by both osteoblasts and osteoclasts and N-cadherin that inhibits both homotypic and heterotypic exerts many functions on HSC. Lodgment of transplanted WT interactions of N-cadherin in donor HSC significantly reduced LinÀSca1 þ KIT þ HSPC at the endosteum is reduced in non- lodgment to the endosteum and compromised long-term ablated osteopontin-deficient recipients compared with WT engraftment and chimerism, whereas overexpression of WT recipients.99 In one report, osteopontin negatively regulated N-cadherin caused increased lodgment at the endosteum and HSC proliferation in vivo. Indeed, phenotypic HSC were more increased ability to self-renew and serially reconstitute lethally numerous and proliferative, as measured by bromodeoxyuridine irradiated hosts.103 Finally, overexpression of two distinct short incorporation, in the BM of osteopontin-deficient mice.99 In hairpin RNA specific for N-cadherin in HSC, accelerated HSC another report, osteopontin deletion from the mouse BM stroma proliferation, severely reduced long-term engraftment in a was found to decrease apoptosis of LinÀSca1 þ KIT þ HSPC in competitive repopulation assay and reduced HSC lodgment to the BM and increase expression of Jagged-1 and ang-1 by endosteal surfaces,104 a result at odd with the conditional osteoblasts, suggesting that osteopontin is also a negative deletion of the N-cadherin gene.102 Therefore, the expression of regulator of niche function.100 These effects of osteopontin are N-cadherin on HSC and its role on HSC retention and lodgment not mediated by the intact protein, but via a cryptic site exposed are still controversial and need further clarification. upon proteolytic cleavage by thrombin. Once cleaved by thrombin, these osteopontin fragments were chemoattractive to HSPC via integrins a4b1 and a9b1 expressed at the surface of Annexin II both mouse and human HSPC.18 Consequently, mice with Annexin II, which forms a heterotetramer with the Ca2 þ -binding deleted osteopontin gene have a 2- to 3-fold increase in the protein S100A10 and is involved in plasminogen binding and

Leukemia In and out of hematopoietic stem cell niches J-P Le´vesque et al 1985 activation,105 is expressed on both BM endothelial cells and move from very poorly perfused niches in the BM to more osteoblasts.106 Annexin II may play an important role in the perfused and perivascular areas of the BM.65 lodgment of HSC in osteoblastic niches as adhesion of WT An interesting parallel can be drawn between the observa- HSPC to annexin II-deficient osteoblasts is reduced by 50%. tions that upon G-CSF treatment, (1) phenotypic HSC migrate Furthermore, treatment of transplant recipients with blocking from poorly perfused niches to more perfused niches,65 and antibodies or annexin II peptide reduced WT HSPC homing to (2) phenotypic HSC proliferate more rapidly in the BM of the BM and compromised long-term reconstitution.106 How- G-CSF-treated animals64 (IGW and JPL, unpublished data). This ever, as annexin II is expressed by both endothelial cells and opens the question as to whether the exit of HSC from their osteoblasts, it is not clear from these experiments whether endosteal niche causes entry into cell cycle before their egress annexin II is necessary for the initial adhesive interaction into the circulation. between BM endothelial cells and circulating HSPC or for the final lodgment in endosteal niches. Further experiments are clearly needed. The sympathetic nervous system The precise mechanisms by which endosteal niches are inhibited in response to mobilizing cytokines or chemotherapy are still poorly understood and clearly do not involve neutrophil What drives HSC out of endosteal niches? serine proteases as mice deficient for dipeptidylprotease I (also called cathepsin C), which cannot mature active neutrophil Cytokines such as G-CSF mobilize long-term reconstituting elastase or cathepsin G, have similar osteoblast inhibition in HSC. One would anticipate HSC in endothelial niches to be response to G-CSF as WT mice.111 A first type of mechanism rapidly mobilized owing to their proximity to the circulation. was proposed by Paul Frenette’s group and involves adrenergic However, as most serially reconstituting dormant HSC are found sympathetic nerves.30,84,112 Neonate mice knocked out for the in poorly perfused hypoxic niches65,91,93 and are enriched at the uridine diphosphate-galactose ceramide galactosyltransferase endosteum,64 one would expect a proportion of HSC to be also (an enzyme necessary for myelin synthesis) or for dopamine mobilized from endosteal niches. Recent research has clearly b-hydroxylase (enzyme converting dopamine to norepinephr- shown that mobilizing cytokines and cytotoxic drugs do indeed ine) genes have abnormal osteoblast morphology at the severely alter the endosteal landscape. endosteum with poor HSPC mobilization in response to G-CSF. Conversely, serial injections of 6-hydroxydopamine, which kill dopaminergic and adrenergic neurons, reduced Inhibition of osteoblasts and bone formation osteoblast inhibition and HSPC mobilization in response to As early as the mid-1990s, before the recognition of endosteal G-CSF.84 A 3-week treatment with the b-adrenergic antagonist and osteoblastic HSC niches, bone pain was readily identified proranolol in WT adult mice reduced HSPC mobilization in as a common side effect of G-CSF treatment in humans.107,108 response to G-CSF by 30%, whereas the b-adrenergic agonist In 1998, we found that short-term G-CSF regimens to mobilize clenbuterol partly rescued mobilization in dopamine b-hydro- HSPC transiently inhibited bone formation and osteoblast xylase knockout neonates and enhanced mobilization in control function in humans with decreased osteocalcin concentration heterozygous littermates.84 This suggests that noradrenergic in blood plasma.109 The identification of endosteal niches nerves radiating through the BM and bone play an important renewed interest in the effect of mobilizing agents on these role in mediating the response of osteoblasts, niches and HSC to niches. G-CSF. In a separate work, these authors found that mouse In mice, G-CSF causes a rapid and transient loss in HSPC are spontaneously released in the BM with a circadian morphologically recognizable mature osteoblasts, with marked periodicity, with oscillations in CXCL12 mRNA and protein in reduction of the osteoid deposited by active osteoblasts at the the BM following an opposite circadian pattern. Furthermore, endosteum, due in part to increased apoptosis.110 Dynamic denervation of the tibia by sectioning the sciatic and femoral histomorphometry studies on endosteal bone surfaces in the nerves, or killing of adrenergic and dopaminergic nerves by secondary spongiosa of tibial metaphysis showed that bone injection of 6-hydroxydopamine abrogated oscillations in formation was reduced by 90% during a 6-day treatment with CXCL12 concentration and HSPC release into the blood. This G-CSF, whereas periosteal bone formation was not affected.111 effect was found to be mediated by b3-adrenergic receptors as a As a result, mRNA encoding cytokines essential to HSC specific antagonist could prevent these oscillations, whereas a SPOTLIGHT maintenance in osteoblastic niches such as KL and ang-1 were specific agonist could alone reduce CXCL12 concentration in significantly reduced at the endosteum.111 In particular, CXCL12 the BM and elicit HSPC mobilization.7 An interesting twist to expression, which is critical to HSPC retention within the BM, these findings is that if blood from G-CSF-treated mice or was reduced both at the endosteum and in the central humans is harvested in synchrony with the circadian time when BM,83,84,111 suggesting that G-CSF not only downregulates HSPC spontaneously mobilize in steady-state, circulating HSPC endosteal niche function, but also CXCL12 expression by other harvest is significantly increased.112 Although it is clear that cells such as CaR cells, osteoprogenitors and endothelial cells. A b2- and b3-adrenergic receptors are important for HSPC functional consequence of the decrease in CXCL12-, KL- and mobilization in response to G-CSF, they are clearly not the ang-1-mediated signaling could be the weakening of adhesive only players. Indeed, in mice knocked out for both b2- and b3- interactions between HSC and niche cells, and initiation of a adrenergic receptors, HSPC mobilization in response to G-CSF motion away from the niche by HSC autonomous mechanisms is reduced by 50% only.113 Furthermore, although adrenergic requiring Gi proteins and small GTPases, such as Rac1, Rac2 nerves radiate through the bone, only endosteal osteoblasts are and cdc42. This is supported by the fact that (1) loss of inhibited by G-CSF, and periosteal osteoblasts function nor- osteoblasts on endosteal surfaces starts as early as 24 h of G-CSF mally. This observation clearly indicates that other cells from the administration and precedes HSC mobilization, which only BM compartment are also necessary for osteoblast inhibition in starts 48 h after the initiation of G-CSF treatment,111 and response to G-CSF. This was proven using reciprocal transplan- (2) LinÀSca1 þ KIT þ CD41ÀCD48ÀCD150 þ phenotypic HSC tation chimeras between G-CSF receptor (GCSFR) knockout and

Leukemia In and out of hematopoietic stem cell niches J-P Le´vesque et al 1986 WT mice. Indeed, WT recipients reconstituted with GCSFRÀ/À of endosteal osteoblasts and CXC12 expression may explain BM (all hematopoietic cells are GCSFRÀ/À, while non-hemato- why cyclophosphamide mobilizes better than G-CSF in mice. poietic cells express GCSFR) have no osteoblast inhibition in However, it must be noted that some mobilizing agents do not response to G-CSF, whereas GCSFRÀ/À recipients reconstituted perturb endosteal niches. This is typically the case of the CXCR4 with WT BM had reduced osteoblast function in response to inhibitor AMD3100. Continuous administration of AMD3100 G-CSF.110 Thus, BM leukocytes expressing GCSFR are required via osmotic pump mobilizes HSPC, but does not reduce osteoid to inhibit osteoblasts. formation, osteoblast number or expression of CXCL12, KL or ang-1 at the endosteum (IGW, JPL, NA Sims, LJ Bendall, unpublished data). Back to BM myeloid cells Some reports suggest that osteoclasts could play an important Several experiments indicate that myeloid cells and particularly part in destabilizing endosteal niches and mobilizing HSPC in BM phagocytes play an important role in the G-CSF response response to G-CSF.122 These conclusions were based on the and maintaining osteoblastic niche integrity. In the mouse, a observation that (i) excessive bleeding, (ii) bacterial lipopoly- population of F4/80 þ macrophages called osteomacs, which saccharide and (iii) the osteoclast-stimulating cytokine RANKL form a canopy over active osteoblasts, were found to be (receptor activator for nuclear factor-kB ligand) all increase necessary for osteoblast differentiation in vitro and in vivo.114 (a) the number of tartrate-resistant alkaline phosphatase-positive We have found that this layer of osteomacs vanishes from the osteoclasts at the endosteum, (b) mobilize HSPC in the endosteum, but not from the periosteum in response to G-CSF, circulation and (c) decrease CXCL12 concentration in the BM suggesting that the loss of osteomacs may play a part in the fluid. In addition, cathepsin K, a cysteine protease produced SPOTLIGHT inhibition of endosteal niches in response to G-CSF.111 specifically by osteoclasts, degrades CXCL12 and may therefore Furthermore, depletion of BM phagocytes with liposomes cause the drop in CXCL12 concentration observed in mobilized loaded with clodronate, a non-cell-permeable drug that has no BM.122 From these data, the authors concluded that osteoclasts cell toxicity in aqueous solution and requires active phagocy- play a unique role in the mobilization of HSPC in response to tosis via liposomes to penetrate and kill cells, ablated osteomacs bleeding, bacterial infections, RANKL as well as G-CSF. and osteoblasts, elicited mobilization of HSPC and LT-CRC Although osteoclasts could induce HSPC mobilization via within 48 h of administration, with marked downregulation of cathepsin K in response to RANKL, bleeding or lipopolysacchar- CXCL12, KL and ang-1 expression at the endosteum.111 ide, it is doubtful that osteoclasts play a major role in the Similarly, Dan Link’s group has found that activation of G-CSF clinically relevant mobilization of HSPC in response to G-CSF or signaling in monocytes/macrophages alone is sufficient to chemotherapy. In 1998, we indeed showed that (1) bone inhibit osteoblast function and elicit a robust HSPC mobiliza- degradation (as measured by urine concentration of the collagen tion. This was shown by introducing a GCSFR transgene under degradation product deoxypyridinoline) and (2) the number of the control of the CD68 promoter to express the transgene osteoclasts lining the endosteum both increased in humans and specifically in monocytes and macrophages. These transgenic BALB/c mice following G-CSF administration. Importantly, mice were then backcrossed into GCSFRÀ/À mice. The resulting however, this increase occurs very late, between days 6 and 8 CD68GCSFR Tg þ GCSFRÀ/À mice express GCSFR exclusively in humans and 10–15 in BALB/c mice following G-CSF in CD68 þ monocytes/macrophages and were consequently administration, at a stage when mobilization is over and HSPC neutropenic with normal numbers of monocytes. Once injected are not circulating in the blood anymore.109 It has been with G-CSF, these CD68GCSFR Tg þ GCSFRÀ/À mice mobilized independently confirmed in C57BL/6 mice that osteoclast HSPC as much as WT with inhibition of osteoblasts.115 numbers do not increase during G-CSF administration when Therefore, activation of monocytes and macrophages may be HSPC mobilize, but later when G-CSF is ceased and HSPC sufficient to elicit both HSPC mobilization and inhibition of mobilization stops.110 Furthermore, we find that pre-treatment endosteal niches. The mechanisms by which these BM of BALB/c mice with pamidronate109 or C57BL/6 mice with monocytes and macrophages maintain HSC niches and osteo- zoledronate, two potent therapeutic inhibitors of osteoclasts, blasts are unknown and remain to be investigated. However, the does not reduce mobilization of colony-forming cells and facts that the hypoxic state of the BM cavity raises dramatically LT-CRC in response to G-CSF.111 Interestingly, however, it has during HSC mobilization with G-CSF or cyclophosphamide,91 recently been published that osteoclasts mediate the accumula- and that expression of hypoxia-inducible factor-1a is absolutely tion of colony-forming units fibroblast, which correspond to required for macrophage activation in vivo116 suggests that multipotent mesenchymal progenitor cells, in the BM in increased hypoxia and the hypoxia-sensing pathway may play response to G-CSF by releasing these colony-forming units an important part in this mechanism. Similarly, the complement fibroblast from the compact bone.123 Again this accumulation of cascade is activated during mobilization,50,51,117,118 and com- colony-forming units fibroblast in the BM, which was prevented plement proteins activate macrophages.119,120 by co-administration of osteoprotegerin, was not detectable Very importantly, impairment of osteoblastic niche function is before day 7 of G-CSF administration,123 probably because not restricted to G-CSF. It is also observed during mobilization osteoclasts do not accumulate in the BM before this time point. elicited by KIT Ligand and Flt3 ligand with similar down- Therefore, while osteoclasts may promote the accumulation of regulation of CXCL12 expression.121 Nor is this phenomenon colony-forming units fibroblast in the BM at late stages of G-CSF limited to mobilizing cytokines. We have found that a single administration, they are unlikely to play a major role in the injection of cyclophosphamide causes a rapid increase in mobilization of HSPC and inhibition of osteoblastic niches, osteoblast number and osteoid formation during the myeloa- which occur within the first 4 days of G-CSF administration. blative phase of the treatment when HSPC do not mobilize, but this is followed by a complete ablation of osteoblasts, an arrest in bone formation, and reduced CXCL12 mRNA expression at Are humans the same as mice? the endosteum from day 6 to day 10, when HSPC are Most data in this review on the existence of endosteal abundantly mobilized into the blood (IGW, NA Sims, JPL, osteoblastic niches and how mobilization alters their function unpublished data). This rapid swing from up- to downregulation have been derived from mouse models. For obvious ethical

Leukemia In and out of hematopoietic stem cell niches J-P Le´vesque et al 1987 reasons, many of these experiments cannot be repeated in with enhanced human HSC content. In this mouse model with humans. So are human HSC niches similar? Reassuringly, human bone and human HSC, effects of Wnt antagonists, Notch several lines of evidence do suggest that human BM niches signaling activation on bone formation and HSC content could are organized similar to the mouse. further show the functional relationship between human For a start, hematopoietic development in human and mouse osteoblasts and HSC. Similarly, HSC content in trabecular bone embryos follows a very similar sequence, with emergence of versus central BM could be compared using non-human definitive HSC from hemogenic endothelium in the placenta primates or cadaveric human bones to confirm whether and dorsal aorta, migration of these fetal HSC to liver and spleen transplantable HSC (in a non-obese diabetes/severe-combined until ossification of bones and BM formation cavity occurs, and immunodeficiency xenotransplant model, for instance) are more then finally HSC migration to the nascent BM in the bone- frequent in trabecular bone than central BM. forming cavity.124,125 Pioneering experiments show that osteo- In respect to mobilization, both human and mouse species are blasts isolated from trabecular bone that could support LT-CIC known to elicit similar responses to G-CSF and cyclophos- in vitro were performed using human osteoblasts and CD34 þ phamide, such as release of neutrophil proteases in the BM, cells.55,56,126,127 Similar to mice, the density of CD34 þ KIT þ cleavage of VCAM-1, CXCL12 and inactivation of CXCR4.34,41 HSPC diminishes with increased distance from the endosteum in In regards to the response of the endosteal microenvironment to human cancellous bones.128 In addition, similar to mouse MSC, mobilizing agents, we have found that similar to mice, a rare population of CD45À CD146 þ adventitial reticular cells mobilization with cytokines such as G-CSF and KL results in a from the human BM, when transplanted subcutaneously in a arrest in bone formation in humans, as suggested by a significant hydroxyapatite–fibrin gel in immunocompromised mice, can reduction in blood osteocalcin, which was correlated with the spontaneously form ectopic bone ossicles containing a BM concentration of mobilized CFU-GM into the blood.109 How- microenvironment and mouse leukocytes.129 This suggests that ever, additional experiments must be conducted in non-human similar to mice, the endosteum and osteoblast lineage cells in primates in order to confirm whether endosteal niches are humans support HSC in the BM. Additional experiments are shutdown during mobilization, such as bone formation arrest now required to confirm the existence of endosteal ‘osteoblastic’ and disappearance of osteoblasts by histomorphometry, and niches in humans or primates. For instance, immunocompro- downregulation of cytokines supporting HSC at the endosteum. mised mice with ectopic human ossicles formed from Clinical trials could be designed to determine whether PTH CD45ÀCD146 þ adventitial reticular cells could be irradiated or bisphosphonate treatment enhances HSPC mobilization and then transplanted with human CD34 þ CD38À HSC to show in humans as observed in mice. Pre-treatment with PTH direct interaction between human HSC and human osteoblast or bisphosphonates could be advantageous to mobilize patients lineage cells. PTH treatment could be included to extend the with perturbed bone turn-over, such as menopausal women, observation that enhanced human bone formation is coupled elderly or multiple myeloma patients. Finally, the central role of SPOTLIGHT

Figure 1 Schematic representation of some of the interactions between HSC and their endosteal and vascular niches. Quiescent HSC are lodged in endosteal niche at two-cell diameters from osteoblasts, which are covered by a layer of osteomacs. These dormant HSC are in equilibrium with active, more proliferative HSC, which may reside in perivascular and more oxygenating niches. Osteoclasts provide soluble calcium ions that bind to CaR expressed by HSC. Proteins expressed by osteoblasts, osteoprogenitors, HSC and endothelial cells are in boxes. Details of these interactions are explained in the text.

Leukemia In and out of hematopoietic stem cell niches J-P Le´vesque et al 1988 BM macrophages and osteomacs in maintaining niches and stromal cells) can favor the development of hematopoietic initiating HSC mobilization could be easily assessed in primates malignancies78,130,131 introducing the novel concept of using clodronate-loaded liposomes, which kill phagocytic ‘malignant niches’. A number of myelodysplasia cannot be macrophages regardless of the species. easily cured by HSC transplantation, probably because the BM niches are dysfunctional. Detailed studies of the molecular interactions between HSC and their niches will therefore Conclusion provide additional clues and potential therapeutic targets to restore the functionality of these niches, to improve HSC It is now clear that the bone and hematopoietic tissue are engraftment particularly in situations where the number of functionally intertwined, and that this relationship extends well HSC in the graft is a limitation (for example, cord blood beyond bone chewing osteoclasts formed from myeloid transplants), or to further boost HSC mobilization yields 132 precursors. Deregulation of osteoblasts and osteoprogenitors particularly in poor mobilizers. can profoundly perturb hematopoiesis, and reciprocally, dereg- ulation or stress of the hematopoietic system can profoundly Conflict of interest perturb bone metabolism. The complex relationship between the bone, osteoblastic and mesenchymal lineages, innate The authors declare no conflict of interest. immunity and HSPC function, lodgment and mobilization are summarized in Figures 1 and 2.

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