Production of erythropoietic bursts by progenitor cells from adult human peripheral blood in an improved serum-free medium: role of insulinlike growth factor 1.
Several culture media for the growth of human circulating erythroid burst-forming units (BFU-E) that have been claimed to be "serum-free" ("SF") have actually included albumin preparations known to be contaminated with an undefined burst-promoting activity (BPA); a BPA has also been found in the preparations of other "SF" medium components. This has precluded reliable investigation of the growth factor (GF) requirements of these progenitors. Using a defatted, BPA-free bovine serum albumin (BSA) and the recombinant human growth factors (GFs) erythropoietin (rHu Epo), insulinlike growth factor 1 (rHu IGF-1), and interleukin-3 (rHu IL-3), we have developed an improved serum-free (SF) medium for the production of erythroid bursts from normal adult human peripheral blood mononuclear cells (PBMNC), which requires both hemin and retinyl acetate for its optimal performance. In the presence of BSA without IL-3 or Epo, no burst or colony formation was observed. With IL-3 and Epo alone, only a small number of day 14 erythroid colonies was obtained (12 +/- 1/10(5) PBMNC). Addition of hemin (0.1 mmol/L) allowed the direct scoring of day 14 hemoglobinized colonies and increased their number sevenfold (86 +/- 5). Inclusion of retinyl acetate at physiologic concentrations further augmented the number of colonies threefold to fourfold. Under these apparently optimal conditions, we found that IGF-I could entirely replace Epo. However, IGF-I required a 100-fold higher molar concentration than that of Epo to reach maximal stimulation. The combined effect of Epo and IGF-I was found to be less than the sum of their individual effects, suggesting an overlap in the sensitivities of erythroid progenitors to these GFs. The colony-forming efficiencies of erythroid progenitors in the improved SF medium was very high: 700 single, day 14 erythroid colonies/10(5) PB MNC (at 0.25 mmol/L hemin) distributed as 126 clusters (bursts), with a mean of 5.6 component colonies per burst. These findings show that IGF-I has an Epo-like activity that targets circulating early erythroid progenitors or their progeny, providing strong evidence for the existence of an Epo-independent pathway for normal human adult erythropoiesis, possibly operative when Epo levels are low.
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Department of Anatomy and Cell Biology, Faculty of Medicine, University of Toronto, Ontario, Canada.
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D016895:Culture Media, Serum-Free; D004224:Diterpenes; D015672:Erythroid Precursor Cells / Q000502:physiology*; D004920:Erythropoiesis; D004921:Erythropoietin / Q000494:pharmacology; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007377:Interleukin-3 / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D000084562:Retinyl Esters; D014801:Vitamin A / Q000031:analogs & derivatives / Q000494:pharmacology
D016895:Culture Media, Serum-Free; D004224:Diterpenes; D007377:Interleukin-3; D011994:Recombinant Proteins; D000084562:Retinyl Esters; D004921:Erythropoietin; D014801:Vitamin A; C009166:retinol acetate; D007334:Insulin-Like Growth Factor I; D006427:Hemin