Although recent data suggests that osteoblasts play a key role within the hematopoietic stem cell (HSC) niche, the mechanisms underpinning this remain to be fully defined. The studies described herein examine the role in hematopoiesis of Osteopontin (Opn), a multidomain, phosphorylated glycoprotein, synthesized by osteoblasts, with well-described roles in cell adhesion, inflammatory responses, angiogenesis, and tumor metastasis. We demonstrate a previously unrecognized critical role for Opn in regulation of the physical location and proliferation of HSCs. Within marrow, Opn expression is restricted to the endosteal bone surface and contributes to HSC transmarrow migration toward the endosteal region, as demonstrated by the markedly aberrant distribution of HSCs in Opn^–/– mice after transplantation. Primitive hematopoietic cells demonstrate specific adhesion to Opn in vitro via β₁ integrin. Furthermore, exogenous Opn potently suppresses the proliferation of primitive HPCs in vitro, the physiologic relevance of which is demonstrated by the markedly enhanced cycling of HSC in Opn^–/– mice. These data therefore provide strong evidence that Opn is an important component of the HSC niche which participates in HSC location and as a physiologic-negative regulator of HSC proliferation.

The spatial distribution of subpopulations of hemopoietic progenitor cells following syngeneic transplantation was investigated at the single-cell level. The location of infused hemopoietic progenitor cells within the femoral bone marrow of nonablated recipients was determined by 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester labeling of cells and in situ fixation by perfusion. Analysis performed over 15 hours after infusion demonstrated that the spatial distribution of transplanted marrow cells is not a random process. Although the majority of cells enter the bone marrow from the central marrow vessels, the subsequent localization within the bone marrow varied according to their phenotype. Candidate “stem cells” demonstrated selective redistribution and were significantly enriched within the endosteal region, whereas mature terminally differentiated and lineage-committed cells selectively redistributed away from the endosteal region and were predominantly in the central marrow region. Together, these data strongly support historical evidence of the presence of endosteal hemopoietic stem cell niches.

Mobilized progenitor cells currently represent the most commonly used source of hematopoietic progenitor cells (HPCs) to effect hematopoietic reconstitution following myeloablative chemotherapies. Despite their widespread use, the molecular mechanisms responsible for the enforced egress of HPCs from the bone marrow (BM) into the circulation in response to mobilizing agents such as cytokines remain to be determined. Results of this study indicate that expression of vascular cell adhesion molecule-1 (VCAM-1) is strongly reduced in vivo in the BM during HPC mobilization by granulocyte colony-stimulating factor (G-CSF) and stem cell factor. Two serine proteases, namely, neutrophil elastase and cathepsin G, were identified, which cleave VCAM-1 and are released by neutrophils accumulating in the BM during the course of immobilization induced by G-CSF. The proposal is made that an essential step contributing to the mobilization of HPCs is the proteolytic cleavage of VCAM-1 expressed by BM stromal cells, an event triggered by the degranulation of neutrophils accumulating in the BM in response to the administration of G-CSF.

Osteopontin (OPN), a multifunctional acidic glycoprotein, expressed by osteoblasts within the endosteal region of the bone marrow (BM) suppresses the proliferation of hemopoietic stem and progenitor cells and also regulates their lodgment within the BM after transplantation. Herein we demonstrate that OPN cleavage fragments are the most abundant forms of this protein within the BM. Studies aimed to determine how hemopoietic stem cells (HSCs) interact with OPN revealed for the first time that murine and human HSCs express α₉β₁ integrin. The N-terminal thrombin cleavage fragment of OPN through its binding to the α₉β₁ and α₄β₁ integrins plays a key role in the attraction, retention, regulation, and release of hemopoietic stem and progenitor cells to, in, and from their BM niche. Thrombin-cleaved OPN (trOPN) acts as a chemoattractant for stem and progenitor cells, mediating their migration in a manner that involves interaction with α₉β₁ and α₄β₁ integrins. In addition, in the absence of OPN, there is an increased number of white blood cells and, specifically, stem and progenitor cells in the peripheral circulation.