ROLE OF OSTEOBLASTS IN STEM CELL PROLIFERATION

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Description (taken directly from the application): Hematopoietic stem cell survival, self-renewal and differentiation is believed to be highly dependent on the stromal microenvironment, which after birth is largely restricted to the bone marrow. However, investigations of candidate bone marrow stromal cells to date have concentrated on fairly ubiquitous cellular elements, such as fibroblasts and endothelial cells. As important as such cells may be in hematopoiesis, their activities cannot easily explain why hematopoiesis concentrates in the marrow. We therefore have recently begun to focus on a specific cell type, the osteoblast, which is located specifically on the endosteal surface within bone marrow cavities. Based upon our preliminary data, osteoblasts appear to directly communicate with primitive hematopoietic cells and support their proliferation. We propose to explore in depth the cellular and molecular basis for osteoblast-stem interactions. Specifically, we plan to: 1. Compare of the proliferative and self-renewal capacity of primitive hematopoietic cells before and after culture on osteoblast monolayers, utilizing: a. In vitro clonogenic and long term culture initiating cell assays; and b. In vivo transplantation assays using NOD/SCID xenogeneic transplant hosts, via limiting dilution and serial transplantation assays; 2. Determine the effect of bone marrow microenvironmental anatomy (adjacent hematopoietic cells) and physiology (perfusion) on osteoblast hematopoietic function; and 3. Molecularly clone osteoblast derived cDNAs whose protein products stimulate the proliferation of primitive human hematopoietic cells. These experiments should give a comprehensive picture of the hematopoietic role of an important but previously uncharacterized cell in the bone marrow microenvironment, a role which we hypothesize to be central to the maintenance and self-renewal of hematopoietic stem cells. Within the context of this Program Project application, these investigations will focus on the role and mechanisms by which marrow microenvironments govern stem cell self-renewal of the primitive stem cell compartments.