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Sawada K, Krantz SB, Kans JS, et al. Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin. J Clin Invest. 1987;80(2):357-66doi: 10.1172/JCI113080.
Sawada, K., Krantz, S. B., Kans, J. S., Dessypris, E. N., Sawyer, S., Glick, A. D., & Civin, C. I. (1987). Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin. The Journal of clinical investigation, 80(2), 357-66. https://doi.org/10.1172/JCI113080
Sawada, K, et al. "Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin." The Journal of clinical investigation vol. 80,2 (1987): 357-66. doi: https://doi.org/10.1172/JCI113080
Sawada K, Krantz SB, Kans JS, Dessypris EN, Sawyer S, Glick AD, Civin CI. Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin. J Clin Invest. 1987 Aug;80(2):357-66. doi: 10.1172/JCI113080. PMID: 3038955; PMCID: PMC442245.
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American Society for Clinical Investigation Free PMC Article

J Clin Invest. 1987 Aug;80(2):357-66. doi: 10.1172/JCI113080.

Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin.

The Journal of clinical investigation

K Sawada, S B Krantz, J S Kans, E N Dessypris, S Sawyer, A D Glick, C I Civin

PMID: 3038955 PMCID: PMC442245 DOI: 10.1172/JCI113080
Free PMC Article

Abstract

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.

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