Purification of human blood burst-forming units-erythroid and demonstration of the evolution of erythropoietin receptors

Abstract

To facilitate the direct study of the molecular events that control the development of human burst-forming units-erythroid (BFU-E), we have developed a method to purify BFU-E from peripheral blood. Using density centrifugation, rosetting with a mixture of neuraminidase-treated and IgG-coated sheep erythrocytes, positive panning with anti-My10 monoclonal antibody, overnight adherence to plastic dishes, negative panning with monoclonal antibodies, and density centrifugation, human blood BFU-E were purified from 0.04% to 56.6%, a 1,400-fold purification with a 13% yield. More than 90% of purified BFU-E were recombinant interleukin-3 (rIL-3) dependent, which survived for 48 h with rIL-3 in the absence of recombinant erythropoietin (rEP), and 80% gave rise to erythroid bursts of more than 500 hemoglobinized cells. rEP dependency was not evident until after 72 h of incubation in vitro. The purified cells (day 1) were incubated with rIL-3 and rEP in liquid culture for 24 (day 2), 48 (day 3), and 72 (day 4) h and then were transferred into semisolid cultures and incubated until day 15. The size of the erythroid colonies observed in semisolid cultures decreased continuously in association with the incubation time of day 1 purified cells in liquid cultures. The first appearance of colony-forming units-erythroid (CFU-E) that gave rise to colonies of 8 to 49 cells was observed after 72 h of incubation of day 1 cells in the liquid culture. 125I-rEP was incubated for 5 h at 37 degrees C with purified cells (day 1) or with the cells that had been incubated in liquid culture for an additional 24-72 h, and the presence of erythropoietin (EP) receptors was investigated using autoradiography. Specific binding of 125I-rEP was detected in 19 +/- 7% of the initial day 1 BFU-E. The percentage of 125I-rEP-binding to erythroid progenitor cells and the amount of binding continuously increased as day 1 BFU-E matured. 125I-rEP specific binding was observed with all of the erythroid progenitor cells that had been incubated in liquid culture for 72 h. These data demonstrate that primitive BFU-E have a much lower number of EP receptors than CFU-E and develop an increased concentration of EP receptors in association with their maturation and loss of proliferative capacity.