Techniques for studying stimulation of fetal hemoglobin production in human erythroid cultures

This report describes in detail the procedures for growing human erythroid cells in liquid culture for evaluating the potential of pharmacological age nts to affect hemoglobin production. The procedure consists of two phases: an erythropoietin-independent phase in which peripheral blood mononuclear c ells are first cultured in the presence of a combination of hemopoietic gro wth factors, but in the absence of erythropoietin, where early erythroid co mmitted progenitors proliferate and differentiate into more mature progenit ors. In the second phase, the latter cells, cultured in an erythropoietin-s upplemented medium, continue to proliferate and mature into orthochromatic normoblasts and enucleated erythrocytes. This procedure produces large cult ures of relatively pure and synchronized erythroid cell populations derived from normal donors or patients with beta hemoglobinopathies. The cultured cells recapitulate many aspects of erythropoiesis in vivo, including the do nor's pattern of hemoglobin production (types and proportions). Tested comp ounds, at different concentrations, are added at different stages of the cu lture. The various types of hemoglobins and globin chains produced can be m easured by high performance liquid chromatographic techniques and their cel lular distribution analyzed by flow cytometry using fluorescently labeled a ntibodies against specific hemoglobins. This approach provides a screening system for compounds with potential therapeutic efficacy in patients with b eta hemoglobinopathies.