A micro agar culture system for cloning human erythropoietic progenitors in vitro

A simple and reproducible micro agar culture method for cloning erythropoietic progenitor cells from human bone marrow is described. Mononuclear cells (MNC) were immobilized in an agar layer and stimulated by erythropoietin (Ep), which was added to a liquid overlayer. The cultures were routinely incubated, fixed, transferred to microscopic slides, dried, and stained, and erythroid colonies were morphologically examined. The dynamics of growth observed from days 2 to 26 of incubation in the presence of 2.4 U Ep/ml showed basically three kinds of aggregates, which reached maximum growth on different days of incubation. A close Ep dose-response relationship was found for CFUE and BFUE at a concentration of 7.5 x 10(4) seeded cells. By varying the plated cell concentration between 2.5 and 10 x 10(4) cells a linear increase in the aggregates formed was found. On the basis of their growth dynamics and morphologic composition, the existence of three populations of erythropoietic progenitor cells in human bone marrow is tentatively proposed.