Differentiation of human erythroid cells in culture

A culture procedure for growing erythroid progenitors in liquid medium is described. The procedure is divided into two phases. The first is an erythropoietin (EPO)--independent phase in which peripheral blood mononuclear cells are isolated and cultured in the presence of a combination of growth factors, but in the absence of EPO. During this phase, early erythroid-committed progenitors proliferate and differentiate into more mature progenitors. In the second phase, the latter cells, cultured in an EPO-supplemented medium, continue to proliferate and mature into hemoglobin-containing nucleated erythroid cells. The culture procedure yields populations that are large, relatively pure, and synchronized (in terms of differentiation), and which recapitulate in vivo erythropoiesis. Since the cells are grown in suspension, samples of cells can be withdrawn at any time, without disturbing the cultures, and assayed for various parameters, e.g., morphology, size, number, viability, apoptosis, cell cycle, surface antigens, or gene expression. Several procedures for analyzing the cultured cells with respect to their hemoglobin content during their differentiation are included.