The overall goal of these studies is to understand how marrow stroma or adherent cells regulate lymphohemopoiesis. The understanding of the basic mechanisms regulating production of lymphocytes and myeloid cells is critical to our understanding of disordered marrow cell production and to our understanding of the primary lymphoid and hematologic malignancies. A characterization of growth factors regulating these systems could lead to therapeutic approaches to correct deficient cell production or to alter growth of malignant cell populations. We plan to continue the study of a lymphohemopoietic growth factor (LHGF) produced by the murine TC-1 adherent marrow cell line. This growth factor appears to synergize with hemopoietic growth factors Interleukin-3, granulocyte-macrophage colony stimulating activity, colony stimulating factor-I, and erythropoietin, to induce pre-B cell differentiation in both human and murine cells and to stimulate B cell and factor dependent cell line proliferation. We plan to purify the growth factor(s) to homogeneity utilizing DEAE cellulose, Sephadex and Sephacryl sizing, and Conconavalin affinity chromatography, and then either HPLC or FPLC chromatographic applications. We also plan to clone the gene for this growth factor using expression vector cloning. We will continue studies on the characterization of the target cells for this growth factor separating marrow cells with Thy-I antigen utilizing separative techniques including adherence, density, centrifugation, and separation by fluorescent activated cell sorting or panning with a variety of monoclonal antibodies directed to cell surface determinants. We will assess the capacity of the LHGF(s) to stimulate progressively purified stem cell populations and the potential role of accessory cells. Lastly, with purification or cloning of the gene for this growth factor we will assess its effects on stimulating in vivo lymphohemopoiesis or in generating marrow renewing cells in in-vitro liquid culture. We will also continue to support direct growth of hemopoie-parent cell line which have differential capacity to support direct growth of hemopoietic cells and one of which produces a retrovirus. During the course of this work, we will use clonal soft agar and methyl cellulose marrow cultures, liquid cultures of cell lines and pre-B cells, fluorescent microscopy, analysis of DNA and RNA by southern and northern blot techniques and a variety of cell kinetic techniques.
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