Membrane-bound stem cell factor is a key regulator in the initial lodgment of stem cells within the endosteal marrow region.

OBJECTIVE: The transmembrane isoform of stem cell factor (tm-SCF) has been implicated in the adhesion of hemopoietic stem cells to the extracellular matrix within the bone marrow microenvironment in vitro. In addition, in vivo SCF has been shown to play a role in cell mobilization and migration. The aim of this study was to determine if SCF is an integral component of the hemopoietic "niche" of the bone marrow in situ. MATERIALS AND METHODS: To analyze the role of tm-SCF in cell lodgment, purified populations of primitive progenitors and hemopoietic stem cells (HSC) were transplanted into a hemopoietic microenvironment devoid of tm-SCF, and the spatial distribution of engrafted cells was analyzed. In addition, populations of HSC were isolated using non-neutralizing and neutralizing antibodies to the SCF receptor c-kit, and their spatial distribution was analyzed post-transplant. RESULTS: The data demonstrated a significant impairment in the lodgment of transplanted cells within the endosteal marrow region in mice lacking tm-SCF, with a reduction of almost 30% by 15 hours post-transplant. The role of tm-SCF was confirmed by analyzing the spatial distribution of HSC isolated using a neutralizing antibody to c-kit. CONCLUSION: The data demonstrate that although tm-SCF does not appear to play a role in the homing of transplanted cells to the bone marrow, it is critical in the lodgment and detainment of HSC within their hemopoietic "niche."

Hemopoietic stem cells with higher hemopoietic potential reside at the bone marrow endosteum.

It is now evident that hemopoietic stem cells (HSC) are located in close proximity to bone lining cells within the endosteum. Accordingly, it is unlikely that the traditional method for harvesting bone marrow (BM) from mice by simply flushing long bones would result in optimal recovery of HSC. With this in mind, we have developed improved methodologies based on sequential grinding and enzymatic digestion of murine bone tissue to harvest higher numbers of BM cells and HSC from the endosteal and central marrow regions. This methodology resulted in up to a sixfold greater recovery of primitive hemopoietic cells (lineage(-)Sca(+)Kit(+) [LSK] cells) and HSC as shown by transplant studies. HSC from different anatomical regions of the marrow exhibited important functional differences. Compared with their central marrow counterparts, HSC isolated from the endosteal region (a) had 1.8-fold greater proliferative potential, (b) exhibited almost twofold greater ability to home to the BM following tail vein injection and to lodge in the endosteal region, and (c) demonstrated significantly greater long-term hemopoietic reconstitution potential as shown using limiting dilution competitive transplant assays.

Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells.

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 beta1 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.

Hyaluronan is synthesized by primitive hemopoietic cells, participates in their lodgment at the endosteum following transplantation, and is involved in the regulation of their proliferation and differentiation in vitro.

The localization of adult hemopoiesis to the marrow involves developmentally regulated interactions between hemopoietic stem cells and the stromal cell-mediated hemopoietic microenvironment. Although primitive hemopoietic cells exhibit a broad repertoire of adhesion molecules, little is known about the molecules influencing the site of cell lodgment within the marrow following transplantation. However, our recent studies indicate that hierarchically dependent patterns of migration of transplanted hemopoietic cells result in the retention of primitive cells within the endosteal and lineage-committed cells in the central marrow regions. Herein, we now demonstrate that these 2 subpopulations exhibit a striking difference in the expression of a cell surface adhesion molecule, with populations enriched for murine and human hemopoietic stem cells expressing the carbohydrate hyaluronic acid (HA). Furthermore, the presence of this glycosaminoglycan appears critical for the spatial distribution of transplanted stem cells in vivo. In addition, we also demonstrate that the binding of HA by a surrogate ligand results in marked inhibition of primitive hemopoietic cell proliferation and granulocyte differentiation. Collectively, these data describe an important yet previously unrecognized role for HA in the biology of primitive hemopoietic progenitor cells.