The proposed studies will examine the effects of two novel hematopoietic growth factors - stem cell factor (SCF) and Rauscher factor - on the self- renewal and differentiation of highly enriched populations of murine and human hematopoietic stem cells (HSC). Recombinant purified rat SCF will be studied for its effect alone and with other growth factors such as IL-1, IL-3, IL-6, GM-CSF and erythropoietin (Epo) on the self renewal and differentiation of HSC isolated by physical means. Suspensions of HSC will be used in """"""""read out"""""""" assays for the generation of colony-forming cells (CFC). In vivo, murine HSC function will be assayed by day 12 spleen colony-formation and long-term marrow reconstitution. The effects of recombinant human HSC will be studied using highly enriched populations of human progenitor cells (from marrow and umbilical cord blood) separated by flow cytometry or derived from blast cell colonies. SCF will be tested alone and in combination with other hematopoietic growth factors for generation of CFU-Mix and unilineage CFC in read-out assays. Rauscher factor, a potentially novel growth factor which is not species- specific, is most easily detectable when hematopoietic CFC come into contact with irradiated Rauscher cells. Thus, Rauscher cells may form a matrix containing Rauscher factor which can be studied by adding enriched murine or human SCF in low numbers to an established feeder. All cultures will be conducted under serum-deprived conditions to eliminate factors or co-factors provided in fetal bovine serum. These studies should provide insight into the mechanisms of action of these factors and the range of progenitor cells acted upon. The results also may allow a basis for the use of combinations of these and other factors to amplify HSC numbers appropriate for transplantation or quantities of differentiated cells appropriate for transfusion.
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