Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin.
Morphological and biochemical studies of human colony-forming units-erythroid (CFU-E) have been hindered by their extreme rarity. Since burst-forming units-erythroid (BFU-E) develop into CFU-E, we used normal human blood BFU-E to generate large numbers of highly purified CFU-E in vitro. Using density centrifugation, sheep erythrocyte rosetting, surface immunoglobulin-positive cell depletion, adherence to plastic, and negative panning with monoclonal antibodies, human blood BFU-E were purified from 0.017 to 0.368%, a 22-fold purification with a 43% yield. The panned cells were cultured in methylcellulose with recombinant erythropoietin (rEp) and conditioned medium for 9 d. These cells were then collected and CFU-E were further purified using adherence and density centrifugation. This yielded almost 10(7) erythroid colony forming cells with a purity of 70 +/- 18%. Analysis of these cells by light and electron microscopy showed 94% erythroid cells. The prominent cell was a primitive blast with high nuclear/cytoplasmic ratio, dispersed nuclear chromatin and a distinct large nucleolus. The relation between the number of erythroid colonies and the number of day 9 cells plated in plasma clots was a straight line through the origin with a maximum number of erythroid colonies at 1 U/ml of rEp and no erythroid colonies without rEp. Specific binding with 125I-rEp showed that 60% of the binding was inhibited by excess pure erythropoietin (Ep), but not by albumin, fetal calf serum, and a variety of growth factors or glycoproteins. By days 12-13 of cell culture, when the progenitor cells matured to late erythroblasts, specific binding markedly declined. In this study, human CFU-E have been isolated in sufficient purity to characterize the morphology of these rare cells and in sufficient numbers to measure specific binding of Ep.
D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D004900:Erythroblasts / Q000166:cytology* / Q000378:metabolism; D004920:Erythropoiesis; D004921:Erythropoietin / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008747:Methylcellulose; D008854:Microscopy, Electron; D011956:Receptors, Cell Surface / Q000378:metabolism; D017467:Receptors, Erythropoietin; D013997:Time Factors
D000911:Antibodies, Monoclonal; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; D008747:Methylcellulose