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An Improved Serum-free Medium for the Growth of Normal Human Circulating Erythroid Progenitor Cells and its Application to the Study of Erythropoiesis in Polycythemia vera

Paulo N. Correa, Department of Anatomy and Cell Biology, University of Toronto, 1991, Ph.D. Thesis


We have developed a novel serum-free (SF) medium for the growth of normal human adult circulating burst-forming units erythroid (BFU-E). This medium, which utilizes a defatted albumin devoid of contaminants having erythropoietic activity, allowed us to reliably investigate the growth factor requirements of circulating BFU-E. In the presence of a defined BPA composed of recombinant human interleukin-3 (rHu IL-3) and hemin, optimal growth of BFU-E required the addition of erythropoietin (rHu Epo), insulin-like growth factor-I (rHu IGF-I) and retinyl acetate or all-trans-retinoic acid. The retinoids synergised with either Epo or IGF-I to increase 3 to 4-fold the number of burst-component colonies (BCC). IGF-I could entirely replace Epo for erythroid burst-formation, but required a 100-fold higher concentration than the latter. The effect of IGF-I was shown to occur in the presence of an anti-Epo antibody which inhibited the effect of Epo.  These findings show that IGF-I has an Epo-like activity and strongly indicate that an Epo-independent pathway exists for normal human erythropoiesis in vitro.  Removal of adherent cells prevented  burst-formation; this was partially restored by the addition of stem cell factor (rHu SCF).

The sensitivities of circulating BFU-E from patients with Polycythemia vera (PV) to Epo, IGF-I and IL-3 were examined in the SF medium.  We found that the Epo and IL-3 sensitivities of PV and normal BFU-E were identical; in contrast, the sensitivity of BFU-E to IGF-I in PV was >100-fold greater than normal. Thus, the erythrocytosis characteristic of PV cannot be explained by hypersensitivity to either Epo or IL-3.  Instead, the apparent Epo-independence of erythroid progenitors in PV can be explained by their pronounced hypersensitivity to IGF-I, given that we show that IGF-I can replace Epo in SF medium. Moreover, burst-formation in PV was unaffected by adherent cell removal. This suggests that the PV phenotype is due to a combination of an IGF-I-hypersensitivity with a relative independence from the influence of adherent cells.


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