Unique hematopoietic growth stimulatory factors have been identified in the conditioned medium of a human bladder carcinoma cell line 5637. One of these, termed Pluripoietin, has been purified to homogeneity and is a protein of Mr18,000 that stimulates human and murine CFU-GM (day 7), BFU-E cfu-Mk., and CFU-GEMM. In addition it induces differentiation of the leukemic cell lines HL-60 and WEH1-3.A related pluripotent activity with alpha CSF-like properties is also identified and is distinguished from the former by ion-exchange chromatography and RP-HPLC. Its action is species restricted. This molecule also will be purifed to homogeneity and both pluripoietins (pCSF's) will be sequenced, cloned and used to raise monoclonal antibodies. Radioiodinated pCSF will be used for RIA development and radioreceptor analysis on normal and leukemic cells. In vitro action of the pCSF's will be investigated under serum-free conditions and in the absence of accessory cells that will be removed by a variety of procedures including monoclonal antibody panning, FACS and micromanipulation of single cells. Synergism between the two pCSF's will be investigated as will the requirement of a second factor such as erythropoietin, thrombopoietin, GM-CSF, CSF-1, G-CSF and Eo.-CSF, for full terminal differentiation of pluripotent stem cell targets. Purification of pluripotent stem cells will be undertaken using monoclonal antibodies in normal or long term human marrow cell suspensions and in murine 5-FU treated marrow and fetal yolk sac and liver. Very early stem cells (pre-CFU-GEMM, HPP-CFC) will be obtained by taking advantage of their lack of certain cell surface antigens defined by monoclonal abs.(anti-la, L4F3), their preferential localization in the adherent marrow, their dormant state and 5-FU resistance. Selective response of these cells to pCSF, Interleukin-3,CSF-2 and other CSF species will be studied and attempts made to develop continuous lines of normal or leukemic pluripotent stem cells. The cell type in the marrow (macrophage, T-cell, preadipocyte, endothelial cell) will be studied for factor production and biological role.
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