Erythropoiesis and hemoglobin regulation will be examined in vitro and in vivo. The hypotheses are: (1) The fetal hemoglobin (Hb F) expression program of adult erythroid progenitor cells can be modified, and (2) The progenitors of fetuses and adults are distinct cell populations. The long term goal is to learn how to increase Hb F in vivo in patients with hemoglobinopathies. The immediate aims are: (1) To modulate erythropoiesis and Hb F in vitro with cellular and soluble factors, (2) To investigate similar modulations in vivo, (3) To characterize the progenitors of fetuses and adults, and (4) To purify erythroid progenitors. Progenitor cells will be obtained from adults, newborn infants, and fetuses, and from normals as well as patients with hemoglobinopathies, and other hematologic diseases. The progenitor cells will be cultured in semi-solid medium with added erythropoietin. Globin synthesis will be assayed by polyacrylamide gel electrophoresis. Cellular components to be evaluated include monocytes, T cells, and T subsets. Soluble factors include conditioned media, burst promoting activity, fetal sheet sera, and hemin. The role of cell cycle active drugs such as hydroxyurea and 5-azacytidine will be investigated. Studies will be performed with agents added in vitro, and patients who receive relevant drugs will be evaluated in vivo and in vitro. The progenitors of fetuses and adults will be characterized according to parameters which affect growth and/or globin gene expression, such as the influence of cellular and soluble factors. Purification of progenitors will employ physical and immunologic methods to remove irrelevant cells (negative selection) as well as select specifically for progenitors (positive selection). The purified cells will be used to prepare monoclonal antibodies to progenitors, to test the clonal model for ontogenic switching, and to examine directly the mechanism of action of factors which influence erythropoiesis and/or globin gene expression.
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