THE 2-STEP LIQUID CULTURE - A NOVEL PROCEDURE FOR STUDYING MATURATIONOF HUMAN NORMAL AND PATHOLOGICAL ERYTHROID PRECURSORS

We have recently described a novel two-phase liquid culture procedure for growing human erythroid cells in vitro. The two phases are 1) an e rythropoietin (EPO)-independent phase, in which the cells are first cu ltured in the presence of a combination of growth factors excluding EP O; during this phase, early erythroid committed progenitors, burst for ming units (BFU-e), proliferate and differentiate into colony forming unit (CFU-e)-like progenitors; 2) a second phase, in which the latter cells are cultured in an EPO-supplemented medium, in which the CFU-e-l ike progenitors continue to proliferate and mature into orthochromatic normoblasts and then enucleated erythrocytes. This procedure yields l arge (up to 5 x 10(8)) and pure (95-98%) populations of erythroid cell s, which allow detailed study of normal and pathologic erythroid matur ation, including 1) the effects of growth factors on proliferation and differentiation at various erythroid developmental stages, 2) intrace llular iron metabolism in normal and thalassemic erythroid cells and t he role of ferritin as an iron donor iron heme synthesis, 3) the expre ssion of surface antigens: transferrin receptor, glycophorin, A, B, H, D and I/i antigens, 4) synthesis of erythroid-specific membrane prote ins, 5) the kinetics of globin mRNA accumulation during erythroid matu ration, 6) the expression of exogenous human beta globin gene in beta- thalassemic cells as a model for gene therapy, and 7) the enhancement of gamma globin chain synthesis by chemical agents.