EARLY HEMATOPOIETIC CELL REGULATORS OF HUMAN ORIGIN

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The objective of this project is to study the roles early hematopoietic cell regulatory factors such as erythroid burst promoting activities (BPA's) megakaryocyte colony stimulating factor (MEF-CSF) and colony forming unit spleen (CFU-S) survival enhancing factor (CFU-S-F) play with respect to the replication, commitment and differentiation of hematopoietic progenitor cells. It is intended to pursue the following aims: Isolate, progressively purify and characterize relevant factors derived from human urine and from human mononuclear cell conditioned media by techniques involving size selective ultrafiltration, fractional precipitation, chromatography and ultracentrifugation. Study the mechanism of action of these factors, e.g. with respect to reversibility, time course, dose dependence by procedures involving preincubation of the target cells in suspension culture with a factor followed by erythropoietin (EPO) challenge of the cells in semi-solid medium culture or assay of the cells for their content of CFU-S. Determine whether the increase in numbers of erythroid burst forming units (BFU-E) or CFU-S brought about by these factors is based on cell replication or on cell activation (sensitization). Investigate the nature of the target cells for the factors (direct vs. indirect action, unipotent vs. multipotent progenitor cells). Study the specificity of the factors of the factors, at various stages of purification. Study the mechanisms governing the elaboration of the factors and ascertain their physiological and clinical significance. To this end cell populations will be fractionated and the isolates subjected to appropriate stimuli in vitro. The BPA, MEG-CSF and CFU-S-F content of urine from patients with anemias, polycythemias and platelet disorders due to different causes and from normal individuals will be measured to detect possible correlations with other hematological parameters. The blood mononuclear cells of patients will be tested for the ability to produce BPA after PHA treatment and also for the ability to respond to BPA in the direct and the preincubation BFU-E assays in order to find out whether these measurements provide clinically useful information. Based on information gained in the course of this project assays for the factors will be further developed.