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Production of erythropoietic bursts by progenitor cells from adult human
peripheral blood in an improved serum-free medium: role of insulinlike
growth factor 1
PN Correa and AA Axelrad
Department of Anatomy and Cell Biology, Faculty of Medicine, University of
Toronto, Ontario, Canada.
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.
Volume 78,
Issue 11,
pp. 2823-2833,
12/01/1991
Copyright © 1991 by The American Society of Hematology
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