The two‐step liquid culture: A novel procedure for studying maturation of human normal and pathological erythroid precursors

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Abstract
We have recently described a novel two-phase liquid culture procedure for growing human erythroid cells in vitro. The two phases are 1) an erythropoietin (EPO)-independent phase, in which the cells are first cultured in the presence of a combination of growth factors excluding EPO; during this phase, early erythroid committed progenitors, burst forming 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-like progenitors continue to proliferate and mature into orthochromatic normoblasts and then enucleated erythrocytes. This procedure yields large (up to 5 × 102) and pure (95-98%) populations of erythroid cells, which allow detailed study of normal and pathologic erythroid maturation, including 1) the effects of growth factors on proliferation and differentiation at various erythroid developmental stages, 2) intracellular iron metabolism in normal and thalassemic erythroid cells and the role of ferritin as an iron donor for heme synthesis, 3) the expression of surface antigens: transferrin receptor, glycophorin, A, B, H, D and I/i antigens, 4) synthesis of erythroid-specific membrane proteins, 5) the kinetics of globin mRNA accumulation during erythroid maturation, 6) the expression of exogenous human β globin gene in β-thalassemic cells as a model for gene therapy, and 7) the enhancement of γ globin chain synthesis by chemical agents.