Ingrid G. Winkler

Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs.

In the bone marrow, hematopoietic stem cells (HSCs) reside in specific niches near osteoblast-lineage cells at the endosteum. To investigate the regulation of these endosteal niches, we studied the mobilization of HSCs into the bloodstream in response to granulocyte colony-stimulating factor (G-CSF). We report that G-CSF mobilization rapidly depletes endosteal osteoblasts, leading to suppressed endosteal bone formation and decreased expression of factors required for HSC retention and self-renewal. Importantly, G-CSF administration also depleted a population of trophic endosteal macrophages (osteomacs) that support osteoblast function. Osteomac loss, osteoblast suppression, and HSC mobilization occurred concomitantly, suggesting that osteomac loss could disrupt endosteal niches. Indeed, in vivo depletion of macrophages, in either macrophage Fas-induced apoptosis (Mafia) transgenic mice or by administration of clodronate-loaded liposomes to wild-type mice, recapitulated the: (1) loss of endosteal osteoblasts and (2) marked reduction of HSC-trophic cytokines at the endosteum, with (3) HSC mobilization into the blood, as observed during G-CSF administration. Together, these results establish that bone marrow macrophages are pivotal to maintain the endosteal HSC niche and that the loss of such macrophages leads to the egress of HSCs into the blood.

Mobilization by either cyclophosphamide or granulocyte colony-stimulating factor transforms the bone marrow into a highly proteolytic environment

OBJECTIVE Hematopoietic stem and progenitor cells normally reside in the bone marrow but can be mobilized into the peripheral blood following treatment with granulocyte colony-stimulating factor (G-CSF) or myelosuppressive chemotherapy. Although the number of transplants performed with mobilized blood currently exceeds those performed with bone marrow, little is known of the molecular mechanisms responsible for this phenomenon. We sought to determine whether mobilization induced by G-CSF or chemotherapy was triggered by common or distinct mechanisms. METHODS Balb/c mice were mobilized with either G-CSF alone, cyclophosphamide alone, or the combination of both agents. Spleens, peripheral blood, bone marrow extracellular fluids, and cells were taken at different time points and analyzed for the expression of VCAM-1, the number of peripheral blood progenitor cells, concentration of neutrophil proteases, and number of granulocytes. RESULTS Administration of either G-CSF or the myelosuppressive agent cyclophosphamide results in a sharp reduction of VCAM-1/CD106 expression in the bone marrow that coincides with the accumulation of granulocytic precursors and release of active neutrophil proteases neutrophil elastase and cathepsin G that directly cleave VCAM-1/CD106 in vitro. These events follow precisely the kinetics of hematopoietic progenitor cell mobilization into the peripheral blood. CONCLUSION We have identified a commonality of events during mobilization induced by either G-CSF or chemotherapy, which include the accumulation in the bone marrow of active neutrophil proteases that directly cleave VCAM-1 and lead to the sharp reduction of VCAM-1 expression in this tissue.

Vascular niche E-selectin regulates hematopoietic stem cell dormancy, self renewal and chemoresistance

The microenvironment, or niche, surrounding a stem cell largely governs its cellular fate. Two anatomical niches for hematopoietic stem cells (HSCs) have been reported in the bone marrow, but a distinct function for each of these niches remains unclear. Here we report a new role for the adhesion molecule E-selectin expressed exclusively by bone marrow endothelial cells in the vascular HSC niche. HSC quiescence was enhanced and self-renewal potential was increased in E-selectin knockout (Sele−/−) mice or after administration of an E-selectin antagonist, demonstrating that E-selectin promotes HSC proliferation and is a crucial component of the vascular niche. These effects are not mediated by canonical E-selectin ligands. Deletion or blockade of E-selectin enhances HSC survival threefold to sixfold after treatment of mice with chemotherapeutic agents or irradiation and accelerates blood neutrophil recovery. As bone marrow suppression is a severe side effect of high-dose chemotherapy, transient blockade of E-selectin is potentially a promising treatment for the protection of HSCs during chemotherapy or irradiation.

Positioning of bone marrow hematopoietic and stromal cells relative to blood flow in vivo: serially reconstituting hematopoietic stem cells reside in distinct nonperfused niches

Hematopoietic stem cell (HSC) niches have been reported at the endosteum or adjacent to bone marrow (BM) vasculature. To investigate functional attributes of these niches, mice were perfused with Hoechst 33342 (Ho) in vivo before BM cell collection in presence of pump inhibitors and antibody stained. We report that the position of phenotypic HSCs, multipotent and myeloid progenitors relative to blood flow, follows a hierarchy reflecting differentiation stage, whereas mesenchymal stromal cells are perivascular. Furthermore, during granulocyte colony-stimulating factor-induced mobilization, HSCs migrated closer to blood flow, whereas stromal cells did not. Interestingly, phenotypic Lin(-)Sca1(+)KIT(+)CD41(-)CD48(-)CD150(+) HSCs segregated into 2 groups (Ho(neg) or Ho(med)), based on degree of blood/Ho perfusion of their niche. HSCs capable of serial transplantation and long-term bromodeoxyuridine label retention were enriched in Ho(neg) HSCs, whereas Ho(med) HSCs cycled more frequently and only reconstituted a single host. This suggests that the most potent HSC niches are enriched in locally secreted factors and low oxygen tension due to negligible blood flow. Importantly, blood perfusion of niches correlates better with HSC function than absolute distance from vasculature. This technique enables prospective isolation of serially reconstituting HSCs distinct from other less potent HSCs of the same phenotype, based on the in vivo niche in which they reside.

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

The concept of hematopoietic stem cell (HSC) niche was formulated in 1978, but HSC niches remained unidentified 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 most potent HSC – able to reconstitute in serial transplants – 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 ‘osteoblastic’ niches) for HSC, what cell types contribute to these niches with their known physical and biochemical features. In the past decade, a first wave of research uncovered many mechanisms responsible for HSC homing to, and mobilization from, the whole BM tissue. However, the recent discovery of endosteal HSC niches has initiated a second wave of research focusing on the mechanisms by which most primitive HSC lodge into and migrate out of their endosteal niches. The second part of this article reviews the current knowledge of the mechanisms of HSC lodgment into, retention in and mobilization from osteoblastic niches.

Granulocyte colony-stimulating factor induces the release in the bone marrow of proteases that cleave c-KIT receptor (CD117) from the surface of hematopoietic progenitor cells

OBJECTIVE Administration of granulocyte colony-stimulating factor (G-CSF) results in the mobilization of hematopoietic progenitor and stem cells from the bone marrow into the peripheral blood. Although the mechanisms leading to the mobilization of primitive hematopoietic cells is not fully understood, it has been noted that the yield of mobilization in humans is correlated to the down-regulation of c-KIT/CD117 expression on mobilized cells. We sought to determine the mechanisms responsible for the reduced expression of c-KIT on mobilized hematopoietic progenitor cells. MATERIALS AND METHODS Mice were mobilized with G-CSF and primitive hematopoietic cells were collected from bone marrow and blood to analyze c-KIT expression. Using cell lines expressing mouse and human c-KIT and a recombinant protein comprising the entire extracellular domain of human c-KIT, we analyzed by flow cytometry and immunoblotting the proteolytic cleavage of c-KIT by proteases released in bone marrow extracellular fluids extracted from mobilized mice. RESULTS Administration of G-CSF into mice results in the reduction of c-KIT expression on primitive hematopoietic cells in bone marrow and peripheral blood. Bone marrow extracellular fluids isolated from G-CSF-mobilized mice contain serine proteases that cleave c-KIT into discrete fragments. Proteases capable of cleaving c-KIT include neutrophil elastase, cathepsin G, proteinase-3 and matrix metalloproteinase-9. CONCLUSIONS In addition to transcriptional controls, exocytosis, and ligand-induced internalization, the direct proteolytic cleavage of c-KIT by neutrophil and macrophage proteases represents a novel pathway to regulate the levels of c-KIT expression at the surface of hematopoietic cells and may be responsible in part for the down-regulation of c-KIT expression on mobilized hematopoietic progenitors in vivo.

Hematopoietic Progenitor Cell Mobilization Results in Hypoxia with Increased Hypoxia-Inducible Transcription Factor-1α and Vascular Endothelial Growth Factor A in Bone Marrow

Despite the fact that many hypoxia‐inducible genes are important in hematopoiesis, the spatial distribution of oxygen in the bone marrow (BM) has not previously been explored in vivo. Using the hypoxia bioprobe pimonidazole, we showed by confocal laser scanning microscopy that the endosteum at the bone‐BM interface is hypoxic, with constitutive expression of hypoxia‐inducible transcription factor‐1α (HIF‐1α) protein in steady‐state mice. Interestingly, at the peak of hematopoietic stem and progenitor cell (HSPC) mobilization induced by either granulocyte colony‐stimulating factor or cyclophosphamide, hypoxic areas expand through the central BM. Furthermore, we found that HSPC mobilization leads to increased levels of HIF‐1α protein and increased expression of vascular endothelial growth factor A (VEGF‐A) mRNA throughout the BM, with an accumulation of VEGF‐A protein in BM endothelial sinuses. VEGF‐A is a cytokine known to induce stem cell mobilization, vasodilatation, and vascular permeability in vivo. We therefore propose that the expansion in myeloid progenitors that occurs during mobilization depletes the BM hematopoietic microenvironment of O2, leading to local hypoxia, stabilization of HIF‐1α transcription factor in BM cells, increased transcription of VEGF‐A, and accumulation of VEGF‐A protein on BM sinuses that increases vascular permeability.

Hematopoietic stem cell mobilizing agents G-CSF, cyclophosphamide or AMD3100 have distinct mechanisms of action on bone marrow HSC niches and bone formation

The CXCR4 antagonist AMD3100 is progressively replacing cyclophosphamide (CYP) as adjuvant to granulocyte colony-stimulating factor (G-CSF) to mobilize hematopoietic stem cells (HSC) for autologous transplants in patients who failed prior mobilization with G-CSF alone. It has recently emerged that G-CSF mediates HSC mobilization and inhibits bone formation via specific bone marrow (BM) macrophages. We compared the effect of these three mobilizing agents on BM macrophages, bone formation, osteoblasts, HSC niches and HSC reconstitution potential. Both G-CSF and CYP suppressed niche-supportive macrophages and osteoblasts, and inhibited expression of endosteal cytokines resulting in major impairment of HSC reconstitution potential remaining in the mobilized BM. In sharp contrast, although AMD3100 was effective at mobilizing HSC, it did not suppress osteoblasts, endosteal cytokine expression or reconstitution potential of HSC remaining in the mobilized BM. In conclusion, although G-CSF, CYP and AMD3100 efficiently mobilize HSC into the blood, their effects on HSC niches and bone formation are distinct with both G-CSF and CYP targeting HSC niche function and bone formation, whereas AMD3100 directly targets HSC without altering niche function or bone formation.

Serine protease inhibitors serpina1 and serpina3 are down-regulated in bone marrow during hematopoietic progenitor mobilization

Mobilization of hematopoietic progenitor cells into the blood involves a massive release of neutrophil serine proteases in the bone marrow. We hypothesize that the activity of these neutrophil serine proteases is regulated by the expression of naturally occurring inhibitors (serpina1 and serpina3) produced locally within the bone marrow. We found that serpina1 and serpina3 were transcribed in the bone marrow by many different hematopoietic cell populations and that a strong reduction in expression occurred both at the protein and mRNA levels during mobilization induced by granulocyte colony-stimulating factor or chemotherapy. This decreased expression was restricted to the bone marrow as serpina1 expression was maintained in the liver, leading to no change in plasma concentrations during mobilization. The down-regulation of serpina1 and serpina3 during mobilization may contribute to a shift in the balance between serine proteases and their inhibitors, and an accumulation of active neutrophil serine proteases in bone marrow extravascular fluids that cleave and inactivate molecules essential to the retention of hematopoietic progenitor cells within the bone marrow. These data suggest an unexpected role for serpina1 and serpina3 in regulating the bone marrow hematopoietic microenvironment as well as influencing the migratory behavior of hematopoietic precursors.

Mobilization of hematopoietic stem cells: state of the art.

Purpose of reviewHematopoietic stem cells (HSCs) normally reside in the bone marrow but can be forced into the blood, a process termed mobilization used clinically to harvest large numbers of HSCs for transplantation. Currently the mobilizing agent of choice is granulocyte colony-stimulating factor; however, not all patients mobilize well. This article reviews recent advances in understanding the molecular mechanisms responsible for the retention of HSCs in the bone marrow, which are perturbed during HSC mobilization, and the clinical application of these findings. Recent findingsThe interaction between the chemokine SDF-1/CXCL12 and its receptor CXCR4 is critical to retain HSCs within the bone marrow, leading to the discovery that small synthetic CXCR4 antagonists are potent mobilizing agents that synergize with granulocyte colony-stimulating factor. Separate research has shown that HSC numbers in the bone marrow can be boosted by increasing the number of osteoblasts that support HSCs. SummaryHSC mobilization induced by granulocyte colony-stimulating factor may be enhanced by directly targeting the chemotactic interaction between HSCs and bone marrow stroma with CXCR4 antagonists. When the primary problem is reduced, however, HSC numbers in the bone marrow, due to repeated chemotherapy/radiotherapy treatments, an alternative is to enhance HSC content by enhancing bone formation prior to mobilization.