Mobilization of hematopoietic stem cells from the bone marrow niche to the blood compartment

The vast majority of hematopoietic stem cells (HSCs) reside in specialized niches within the bone marrow during steady state, maintaining lifelong blood cell production. A small number of HSCs normally traffic throughout the body; however, exogenous stimuli can enhance their release from the niche and entry into the peripheral circulation. This process, termed mobilization, has become the primary means to acquire a stem cell graft for hematopoietic transplant at most transplant centers. Currently, the preferred method of HSC mobilization for subsequent transplantation is treatment of the donor with granulocyte colony-stimulating factor. The mobilizing effect of granulocyte colony-stimulating factor is not completely understood, but recent studies suggest that its capacity to mobilize HSCs, at least in part, is a consequence of alterations to the hematopoietic niche. The present article reviews some of the key mechanisms mediating HSC mobilization, highlighting recent advances and controversies in the field.


Introduction
Higher organisms have the remarkable capacity to produce and maintain adequate numbers of blood cells throughout their entire lifespan to meet the normal physiological requirements of blood cell turnover, as well as to respond to needs for increased blood cell demand as a consequence of injury or infection. At the center of lifelong blood cell production is the hematopoietic stem cell (HSC), with the capacity to give rise to all mature circulating blood cell types. Regulation of HSC function is a highly complex process involving not only intrinsic cues within the HSC themselves, but signaling from the surrounding microenvironment in which they reside. It was fi rst postulated by Schofi eld that defi ned local microenvironments created specialized stem cell niches that regulated HSCs [1]. Bone marrow is the primary HSC niche in mammals and is composed of stromal cells and an extracellular matrix of collagens, fi bronectin, proteoglycans [2], and endosteal lining osteoblasts [3][4][5][6]. HSCs are thought to be tethered to osteoblasts, other stromal cells, and the extracellular matrix in this stem cell niche through a variety of adhesion molecule interactions, many of which are probably redundant systems.
Disruption of one or more of these niche interactions can result in release of HSCs from the niche and their traffi cking from the bone marrow to the peripheral circulation, a process termed peripheral blood stem cell mobilization. Mobilization can be achieved through adminis tration of chemotherapy [7][8][9], hematopoietic growth factors, chemokines and small-molecule chemokine receptor inhibitors or antibodies against HSC niche interactions [10][11][12].
Th e process of mobilization has been exploited for collection of hematopoietic stem and progenitor cells (HSPCs) and is widely used for hematopoietic transplantation in both the autologous and allogeneic settings. Mobilized peripheral blood hematopoietic stem cell grafts are associated with more rapid engraftment, reduc tion in infectious complications and, in patients with advanced malignancies, lower regimen-related mortality [13][14][15] compared with bone marrow grafts. In many trans plantation centers, mobilized HSC grafts are now the preferred hematopoietic stem cell source used for human leukocyte antigen-identical sibling transplants as well as for matched related and unrelated donor transplants [16,17]. Granulo cyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor and -more recently, for patients who fail to mobilize with a G-CSF or granulocyte-macro phage colony-stimulating factor -plerixafor (AMD3100) are the only US Food and Drug Adminis tration-approved agents for mobilizing HSCs. Despite the clinical prevalence of peripheral blood stem and pro genitor cell mobilization, the mechanisms orchestrating the release of these cells from the hematopoietic niche are still not completely understood. In the following sections, we highlight some of the key mechanistic fi nd ings con cerning HSPC mobilization, with an emphasis on the eff ects of mobilizing agents on bone marrow niche interactions.

CXCR4/SDF-1α: the paradigm of mobilization
Th e most explored HSC niche interaction is between the CXC4 chemokine receptor (CXCR4) and its ligand, stromal cell-derived factor 1α (SDF-1α). SDF-1α is produced by osteoblasts [18], a specialized set of reticular cells found in endosteal and vascular niches [19], endothelial cells and bone itself [20,21], and high levels of SDF-1α were observed recently in nestin-positive mesenchymal stem cells [22]. HSPCs express CXCR4 and are chemoattracted to and retained within the bone marrow by SDF-1α [23][24][25]. Genetic knockout of either CXCR4 [26] or SDF-1α [27] in mice is embryonically lethal, with a failure of HSPCs to traffi c to the bone marrow niche during development. In addition, conditional CXCR4 knock out in mice results in a substantial egress of hemato poietic cells from the bone marrow [28] and impaired ability of CXCR4 knockout HSPCs to be retained within the bone marrow after transplantation [29].
Despite the abundance of evidence supporting a key role for the CXCR4/SDF-1α axis in HSPC retention/ traffi cking/mobilization, it is still not clear which population of cells within the bone marrow niche is the predominate source of SDF-1α. Some studies have demonstrated that SDF-1α production by osteoblasts is reduced after G-CSF treatment [21,40,41], and seminal work by Katayama and colleagues suggests that this reduction in osteoblast SDF-1α is at least partly mediated by the sympathetic nervous system [21]. Notwithstanding the fact that decreased levels of SDF-1α production by osteoblasts are routinely seen following G-CSF administration, however, other studies have questioned the relative importance of osteoblast-derived SDF-1α in HSC mainte nance and mobilization [19,22,42]. A recent study by Christopher and colleagues indicated that reduction in osteoblast production of SDF-1α is a common mechanism of cytokine-induced HSC mobilization and showed a specifi c reduction in SDF-1α production in Col2.3expressing osteoblasts with no reduction in Col2.3negative stromal cells [43]. Mendez-Ferrer and colleagues, however, showed, using a similar approach, a substantial decrease in SDF-1α in a novel population of nestinexpressing mesenchymal stem cells [22], relative to a similar population of stromal cells described by Christopher and colleagues [43], although a direct comparison with defi ned osteoblasts was not made. Future studies are clearly required in order to defi ne the specifi c niche cells responsible for SDF-1α production and HSC retention, and may identify specifi c targets for future HSC therapies.
Osteoblasts also express signifi cant amounts of osteopontin, and HSPCs adhere to osteopontin via β 1 integrins, such as VLA-4 [50]. Osteopontin is a negative regulator of HSC pool size within the bone marrow niche [50,51], and knockout of osteopontin in mice results in endo genous HSPC mobilization and increases the mobilization response to G-CSF [52]. Future therapies that target osteo pontin may not only increase the HSC pool size available for hematopoietic mobilization, but may also act to untether the expanded HSCs from the bone marrow niche, resulting in signifi cantly enhanced HSC mobilization.
Mobilizing regimens of G-CSF are associated with suppression of niche osteoblasts [21,41,53], with increased osteoblast apoptosis [41] and osteoblast fl attening [21], resulting in signifi cant decreases in endosteal niche expres sion of many of the above-mentioned retention molecules. Th is suppression has been reported to be the result of altered sympathetic nervous system signaling to osteoblasts [21]. A recent report by Winkler and colleagues demonstrated that G-CSF treatment results in the reduction of endosteal-lining osteomacs, which results in suppression of osteoblasts [53]. Th is osteomac population of cells is F4/80 + Ly-6G + CD11b + and provides a yet to be deter mined positive supporting role for osteoblasts.
When osteomacs are depleted using Mafi a transgenic mice or by treatment of mice with clodronate-loaded liposomes, signifi cant mobilization of HSPCs was observed. Th ese fi ndings support a mechanistic role for osteoblasts in mediating G-CSF-induced mobilization, independent of the sympathetic nervous system, and highlight that multiple mechanisms may be responsible for the mobilizing eff ects of G-CSF.

What about osteoclasts?
Osteoblasts and osteoclasts regulate/coordinate bone formation and bone resorption, respectively, within the bone marrow niche. A report from Kollet and colleagues suggested that osteoclasts can mediate HSPC mobilization [54], and proposed a model where the balance between osteoblasts and osteoclasts is required for homeo static maintenance of the stem cell niche and HSPC pool size. In their model, increased osteoblastsfor example, after parathyroid hormone administration [3] -increase the stem cell pool size and adherence in the niche, whereas increased osteoclasts degrade the nichefacilitating release and egress of HSPCs.
A role for osteoclasts in mobilization was shown by treating mice with RANK ligand, which increased osteoclast activity that correlated with a moderate increase in hematopoietic progenitor cell (HPC) mobilization [54]. Similarly, bleeding mice or treating them with lipopolysaccharide, two models of physiological stress, resulted in an increase in the number of bone marrow niche osteoclasts as well as HPC mobilization. Inhibition of osteoclasts, either by treat ment with calcitonin or using a genetic knockout model of PTP ε in female mice, resulted in a reduced HPC mobiliza tion response to G-CSF compared with controls, further suggesting that osteoclasts were involved in G-CSF-mediated mobilization. Th e authors proposed that osteoclast-derived proteolytic enzymes, such as cathep sin K, degraded important niche interaction components including SDF-1α and osteo pontin, thereby facilitating mobilization [54]. A more recent study by the same laboratory demonstrated reduced osteoclast maturation and activity in CD45 knockout mice, which correlated with reduced mobilization to RANK ligand and G-CSF [55], providing an additional link between osteoclast activity and HSPC mobilization.
In contrast to studies showing that increased osteoclasts enhance HPC mobilization, an earlier report by Takamatsu and colleagues demonstrated that while G-CSF treatment increases osteoclast number and bone resorption in both BALB/c mice and humans, the increase in osteoclasts did not occur until 10 to 15 days or 6 to 8 days, respectively, after treatment with G-CSF [56] -a fi nding that has also been observed by other groups using similar systems [40,57]. Since HSPC mobiliza tion by G-CSF is typically evaluated after 4 to 5 days, the importance of osteoclasts to HSPC mobilization in response to G-CSF treatment remains unclear. Furthermore, treatment of mice with bisphosphonates, which inhibit osteoclast activity and/or number, prior to G-CSF administration does not result in an impaired HSPC mobilization response [53,56]; in fact, in one case, bisphosphonate treatment increased mobilization by G-CSF [53]. Th ese studies suggest that while osteoclasts elicit mechanisms that can induce hematopoietic stem and progenitor mobilization, their role in clinical HSC mobilization with G-CSF is not suffi ciently defi ned and may not be a primary mechanism of mobilization.
Th e endosteal surface of bone, particularly at the site of resorbing osteoclasts, is a signifi cant source of soluble extracellular calcium within the bone marrow niche. Studies by Adams and colleagues demonstrated that HSCs express calcium-sensing receptors and are chemoattracted to soluble Ca 2+ [58]. When the gene for the calcium-sensing receptor was knocked out, mice had reduced HSC content within the bone marrow niche and increased HSCs in peripheral blood. Moreover, calciumsensing receptor-knockout HSCs failed to engraft in hemato poietic transplantation experiments. Th ese results suggest that Ca 2+ at the endosteal surface is an important retention signal within the hematopoietic niche and that pharmacologic antagonism of the HSC calcium-sensing receptor may represent a possible strategy for HSPC mobilization.

Oxygen regulation of hematopoietic stem cell mobilization
Th e bone marrow hematopoietic niche has been shown to be hypoxic [59,60]. HSCs that reside in hypoxic niches have also been shown to have greater hematopoieticrepopulating ability than those that do not [61]. A known physiolo gical response to hypoxia is stabilization of the trans crip tion factor hypoxia inducible factor 1α (HIF-1α). HIF-1α has been shown to upregulate erythropoietin production [62], numerous cell proliferation and survival genes [63][64][65], the angiogenic vascular endothelial growth factor [66], and other genes. It has also been suggested that the hypoxic bone marrow niche maintains HIF-1α activity, thereby maintaining stem cells [67] -a hypothesis supported by the fact that hypoxic conditions expand human HSCs [68] and HPC populations [69][70][71] in vitro. In response to G-CSF, both the hypoxic environment and HIF-1α expand within the bone marrow compartment [72] and increase production of vascular endothelial growth factor A; however, bone marrow vascular density and permeability are not increased [61]. HIF-1α also increases production of SDF-1α [73] and CXCR4 receptor expression [74], suggesting that hypoxia may be a physiological regulator of this important signaling axis within the hematopoietic niche.
HIF-1α has recently been reported to prevent hematopoietic cell damage caused by overproduction of reactive oxygen species [75], suggesting that the hypoxic niche helps maintain the long lifespan of HSCs. However, some small degree of reactive oxygen species signaling may be necessary for HSC mobilization. A recent report demonstrated that enhanced c-Met activity promotes HSPC mobilization by activating mTOR and increasing reactive oxygen species production in HSPCs [76], while inhibition of mTOR with rapamycin reduced HSC mobilization [76,77]. Genetic knockout of the gene for thioredoxininteracting protein also results in increased HSPC mobili za tion under stress conditions [78], suggesting a role for oxygen tension and reactive oxygen species in regulation of hematopoietic stem and progenitor mobili zation. Th ese fi ndings clearly warrant additional exploration.

Control of the bone marrow niche by the nervous system
It has been known for some time that there is dynamic interaction between the bone marrow niche and the nervous system. Studies by Katayama and colleagues demonstrated that HSPC mobilization by G-CSF requires peripheral β 2 -adrenergic signals [21], showing that G-CSF mobilization was reduced in chemically sym pathecto mized mice treated with 6-hydroxydopamine, in mice treated with the β-blocker propanolol, or in mice genetically defi cient in the gene for dopamine βhydroxylase (Dbh), an enzyme that converts dopamine into norepinephrine. Th ey also showed that treatment with the β 2 -adrenergic agonist clenbuterol reversed the phenotype of Dbh knockout mice [21]. Intriguingly, G-CSF attenuated osteoblast function via the sympathetic nervous system resulting in osteoblasts having a marked fl attened appearance. Th e eff ects of nervous system signaling can also be mediated directly on HSCs, as human CD34 + hemato poietic cells express β 2 -adrenergic and dopamine receptors that are upregulated after G-CSF treatment [79]. Neurotransmitters serve as direct chemoattractants to HSPCs, and treatment with nor epinephrine results in HSC mobilization [79]. Norepi neph rine treatment of mice has also been shown to increase CXCR4 receptor expression [80], perhaps suggesting that adrener gic signal ing could directly aff ect CXCR4/SDF-1α signaling in HSPCs. Additional studies directly assessing eff ects of neurotransmitter signaling in HSPCs will help to further defi ne the role of the nervous system in hematopoietic regulation.
Not only does the sympathetic nervous system aff ect HSC mobilization during stress situations, but it also regu lates HSC traffi cking via a circadian rhythm [81,82]. β 3 -Adrenergic stimulations demonstrate regular oscillations controlling norepinephrine release, CXCR4 expression, and SDF-1α production, leading to rhythmic release of HSPCs from the bone marrow niche. Intriguingly, while optimal mobilization occurs in the morning in mice (Zeitgeber time 5), HSC mobilization circadian control is inverted in humans, with peak mobilization occurring later in the evening [81]. Mobilization by both G-CSF and AMD3100 is aff ected by circadian control of the CXCR4/SDF-1α axis. Recently, it was demonstrated that β 2 -adrenergic signaling upregu lates the vitamin D receptor on osteoblasts; that expres sion of this receptor is necessary for the G-CSF-induced suppression of osteoblast function; and that vitamin D receptor knockout mice have reduced HSC mobilization [83]. Intriguingly, vitamin D recep tor is an important regulator of extracellular calcium and HSPC localization [84] and the receptor is also regulated by circadian rhythms [85], possibly suggesting additional inter con nected mobilization mechanisms. Further assess ment of the role of nervous system signaling and vitamin D recep tor signaling on other niche cells, particularly mesen chymal stem cells, should be performed.

Conclusion
Th ere has been signifi cant progress in understanding the mechanisms of action of G-CSF and other stimuli that increase HSPC traffi cking/mobilization. As described in the present review, however, there is currently an abundance of proposed mechanisms that may be responsible for mobilization. Th is raises the question of whether the proposed mechanisms, be they HSPC intrinsic or manifested through the bone marrow niche, truly represent alternate and independent means to mobilize or enhance egress of HSPCs from bone marrow to the circulation, or whether we have not yet found the unifying mechanism.
Intriguingly, many of the proposed mechanisms of mobilization converge on the CXCR4/SDF-1α pathway ( Figure 1). Alterations of the osteoblast/osteoclast balance result in a reduction of SDF-1α production and/ or degradation of SDF-1α by proteases. Signaling from the sympathetic nervous system, stimulated by G-CSF, can alter the osteoblast/osteoclast balance leading to reduced CXCR4/SDF-1α signaling and HSPC mobilization. Circadian rhythms act to reduce niche SDF-1α production and HSPC CXCR4 expression in an oscillating manner, suggesting that clinical mobilization should be performed at the trough of SDF-1α and CXCR4 expres sion (early night for humans) and perhaps suggesting that clinical transplantation should be performed at the peak of expression (early morning in humans). Th e hypoxic nature of the hematopoietic bone marrow niche may itself regulate the CXCR4/SDF-1α signaling axis, perhaps further identifying this axis as a unifying mobilization mechanism. Th e importance of CXCR4 signaling in HSPC retention and mobilization is certainly supported by the abundance of agents that directly antagonize, or compete with SDF-1α and partially ago nize, the CXCR4 receptor and result in HSPC mobili zation. Even a rapid mobilizing agent such as GROβ (CXCR2 agonist) may function by increasing proteolytic cleavage of SDF-1α [86,87], or altering a homeostatic balance between the CXCR4 and CXCR2 signaling path ways [88].
While perhaps connecting many of the proposed mechanistic pathways for HSPC mobilization, however, the CXCR4/SDF-1α pathway does not appear to be an exclusive target for HSPC mobilization. Continued investigation of the molecular mechanism(s) for action of G-CSF and other HSPC mobilizers is warranted and may defi ne new molecular targets that can be used to enhance the magnitude and/or ease of HSPC collection for hematopoietic transplant.

Competing interests
The authors declare that they have no competing interests.

G-CSF
This article is part of a review series on Stem cell niche. Other articles in the series can be found online at http://stemcellres.com/series/ stemcellniche