Differentiation and erythropoietin receptor gene expression in human erythroid progenitor cells

Partially purified human burst-forming unit-erythroid (BFU-E) cells from peripheral blood were cultured for 6 to 8 days to obtain colony-forming unit-erythroid (CFU-E) cells. When these BFU-E-derived CFU-E were further purified and recultured in liquid suspension cultures with erythropoietin (EPO), they matured and differentiated into reticulocytes in vitro. A maximum rate of hemoglobin synthesis was observed at day 10 of cumulative culture time by measuring 59Fe incorporation into heme. Withdrawal of EPO from erythroblast cultures at various times during development showed that between day 10 and day 11 (when the majority of the cells are in the polychromatic erythroblast stage), these cells became independent of EPO. The timing of the disappearance of the EPO requirement in these cells coincided with the marked decline in proliferation. Measurement of EPO receptor messenger RNA (mRNA) levels by Northern analysis showed that there is a slight decline during the day 8 to day 10 time period, followed by a rapid decline between days 10 and 14. Binding of 125I-EPO to erythroblasts also showed a steady decline of the cell surface binding during maturation and terminal differentiation. The half-life of the human EPO receptor was 90 minutes in the presence of the transcriptional inhibitor actinomycin D and the half-life measured at two different times during the 8- to 14-day culture period remained constant. These results indicate that human EPO receptor mRNA must be transcribed continuously to maintain the levels seen by Northern analysis. The human cell system described here is well suited for the study of a wide variety of biochemical events during late erythroid differentiation.