We have isolated Human erythroid progenitor cells from peripheral blood using a two-phase liquid culture system and have studied temporal expression of various genes as the cells matured. In the first phase, peripheral blood mononuclear cells are cultured for one week in the presence of a combination of growth factors, but not of erythropoietin (EPO). During this phase, early erythroid committed progenitors, burst forming units, proliferate and differentiate into colony forming units. In the second phase, the latter cells, following exposure to EPO, continue to proliferate and mature into hemoglobin-containing orthochromatic normoblasts. Cell samples were taken throughout phase II and expression of globins, transcription factors and cytokine receptors was assayed by RT-PCR and quantitated by phosphor-imaging. We have divided Phase II into stages: early (days 0-5), intermediate (days 6- 10) and late (days 11-15) and looked at maximum expression of each gene. During early phase II, gamma-globin, CP2 and SP1 were expressed at their highest level. As the cells matured during the intermediate period, the EPO receptor (EPO-R) and transcription factors EKLF, GATA-1 and NF-E2 reached maximum expression. In late phase II, beta-globin increased and reached its maximum level of expression. GATA-2 was detected in these normal cells early in phase II and declined as the GATA-1 levels increased. We found no expression of the GM-CSF-R in these EPO dependent phase II cells. Expression patterns seen included the reciprocal relationship between the gamma and beta globin genes in which gamma-globin expression is maximum in early phase II and decreases as beta-globin began to increase during the intermediate period. This increase in beta-globin is preceded by a peak in expression of GATA-1, EKLF and EPO-R.
