Techniques for Studying Stimulation of Fetal Hemoglobin Production in Human Erythroid Cultures

Abstract

This report describes in detail the procedures for growing human erythroid cells in liquid culture for evaluating the potential of pharmacological agents to affect hemoglobin production. The procedure consists of two phases: an erythropoietin-independent phase in which peripheral blood mononuclear cells are first cultured in the presence of a combination of hemopoietic growth factors, but in the absence of erythropoietin, where early erythroid committed progenitors proliferate and differentiate into more mature progenitors. In the second phase, the latter cells, cultured in an erythropoietin-supplemented medium, continue to proliferate and mature into orthochromatic normoblasts and enucleated erythrocytes. This procedure produces large cultures of relatively pure and synchronized erythroid cell populations derived from normal donors or patients with β hemoglobinopathies. The cultured cells recapitulate many aspects of erythropoiesis in vivo, including the donor's pattern of hemoglobin production (types and proportions). Tested compounds, at different concentrations, are added at different stages of the culture. The various types of hemoglobins and globin chains produced can be measured by high performance liquid chromatographic techniques and their cellular distribution analyzed by flow cytometry using fluorescently labeled antibodies against specific hemoglobins. This approach provides a screening system for compounds with potential therapeutic efficacy in patients with β hemoglobinopathies.