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

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 immunglobulin positive cell depletion, adherence to plastic, and negative pn with monoclonal antibodies, human blood BFU-E were purified from 0.017 to 0368%, 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 107 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 hih 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 ', SI-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.

Thrombopoietin, a Direct Stimulator of Viability and Multilineage Growth of Primitive Bone Marrow Progenitor Cells

“…The reason for this is most likely that whereas c-mpl appears to be expressed on primitive/multipotent progenitors [18][19][20], the EPO receptor might not be. In fact, previous studies have suggested that even the most primitive stages of erythroid progenitor cells might not yet express the EPO receptor [37,38].Based on these and other studies [5, 30,[33][34][35],39-431, we can conclude that TPO along with some other early-acting cytokines can promote viability of primitive HPC in the presence of little or no proliferation (Table 2). At least on Lin-Sca-1' bone marrow progenitor cells, TPO has a potent ability to promote viability of in vitro clonogenic and multipotent progenitor cells.…”

“…However, unlike TPO, EPO had no effect on the viability of Lin-Sca-1' progenitors. The reason for this is most likely that whereas c-mpl appears to be expressed on primitive/multipotent progenitors [18][19][20] [37,38].…”

“…The reason for this is most likely that whereas c-mpl appears to be expressed on primitive/multipotent progenitors [18][19][20], the EPO receptor might not be. In fact, previous studies have suggested that even the most primitive stages of erythroid progenitor cells might not yet express the EPO receptor [37,38].…”

Development of Erythroid Progenitors under Erythropoietin Stimulation inXenopus laevisLarval Liver

“…Epo supports the survival of late erythroid progenitors and enhances their proliferation and differentiation by binding to Epo receptor (Epor) (Krantz, 1991;Jelkmann, 2010;D'Andrea and Zon, 1990). Epo stimulates burst-forming unit-erythroid cells (BFU-e) expressing Epor on the surface, and successively colony-forming unit-erythroid cells (CFU-e), at sites of erythropoiesis (Broudy et al, 1991;Gregory and Eaves, 1978;Sawada et al, 1987). Further, mature erythrocytes are produced through proerythroblasts, erythroblasts, and reticulocytes.…”

Stem cell factor increases the expression of FLIP that inhibits IFNγ-induced apoptosis in human erythroid progenitor cells

“…A further negative selection and removal of contaminant cells with CD2, CD11b, CD16, and CD45 monoclonal antibodies was performed as previously described. 18,27 The day Ϫ1 BFU-Es were suspended in Iscove modified Dulbecco medium (IMDM) containing 20% heat-inactivated fetal calf serum (FCS), 5% heat-inactivated, pooled, human AB serum, 1% deionized bovine serum albumin (BSA), 5 ϫ 10 Ϫ5 M 2-mercaptoethanol, 10 g/mL insulin (Sigma, St Louis, MO), 2 U/mL erythropoietin (Amgen, Thousand Oaks, CA), 50 U/mL IL-3 (R&D Systems, Minneapolis, MN), 100 ng/mL SCF (Amgen), and streptomycin plus penicillin to generate ECFCs. After 5 days of culture the average purity of day Ϫ6 ECFCs was 60% or higher as measured by the plasma clot assay.…”