Hematopoiesis occurs within geometric and functional niches In specialized tissues, predominantly yolk sac in the embryo, liver in the fetus and bone marrow In the adult. This dynamic, complex process involves the precise interaction of differentiating stem cells and their progeny with regulatory accessory cells, Including fixed bone marrow stromal cells and circulating mononuclear cells. This application proposes to develop a comprehensive understanding of the role of the critical bone marrow stromal cells in regulating hematopoietic differentiation in the adult bone marrow. Studies will focus on distinct cell types isolated that secrete distinct patterns of hematopoietic cytokines, including multiply passaged normal human bone marrow fibroblasts and individually derived functional stromal clones. The specific aims of the project are to: 1) analyze the spectrum of cytokine gene expression and protein secretion In these cells, In response to controlled variation In serum and nutrient fluxes; 2) measure the functional capacity of these cells, when optimally cultured, as adherent layers In the support of human long-term bone marrow cultures carried out In continuous perfusion bioreactors; and 3) evaluate the effectiveness of optimally stimulated stromal cells to support primitive hematopoietic cell division and proliferation, as judged by retroviral-mediated gene transfer into cells capable of generating mature cells and progenitors over several months in culture. These studies will generate an understanding of the role of adherent stromal cells In the regulation of hematopoiesis that will begin at the molecular level in homogeneous accessory cells and extend to physiologic behavior in reconstructed bone marrow culture systems, utilizing evaluation endpoints that include molecular assays of stromal cell function and cellular assays of at the precursor, progenitor, and pre-progenitor cell levels. The biology described In these studies will contribute directly to our understanding of hematopoietic stem cell biology, and will be directly relevant to many applications In progenitor cell expansion for clinical transplantation and retroviral-mediated gene transfer into stem cells for somatic cell gene therapy.