Serum-free culture of enriched hematopoietic progenitors reflects physiologic levels of fetal hemoglobin biosynthesis.
Adult erythroid progenitors produce significantly higher fetal hemoglobin (HbF) levels in cultures containing fetal calf serum (FCS) and erythropoietin (Ep) than in vivo. The precise mechanisms for this increased HbF production in culture have not been elucidated. We examined HbF biosynthesis by enriched human progenitors in serum-free (SF) culture. We measured globin chain biosynthesis by combination of isoelectric focusing and autoradiography and examined percent nucleated erythrocytes containing HbF (%FNRBC) using microscopic immunodiffusion. CD34 (My10)-positive marrow cells from a normal subject yielded an almost negligible amount of gamma-globin in SF culture stimulated by 100 U/mL interleukin-3 (IL-3) and 2 U/mL Ep, while corresponding FCS culture revealed significant gamma-globin biosynthesis. The %FNRBC of the erythroid bursts in SF cultures derived from nine normal adults (2.0% +/- 0.9% F cells) was 3.0% +/- 3.4%, while in FCS culture, it was 25% +/- 12% (mean +/- SD). Dosages of IL-3 between 10 and 10,000 U/mL did not increase %FNRBC in FCS of SF conditions. Mean Hb contents of nucleated erythrocytes (NRBC) assayed by microdensitometry of pericellular immunoprecipitate were similar in FCS and SF cultures. The number of erythroid bursts per 2 x 10(3) CD34-positive marrow cells was 48 +/- 20 in FCS and 36 +/- 12 in SF cultures. In two experiments, progenitors grown for 7 days under SF conditions were isolated and recultured in either SF or FCS conditions for 7 days, and the resulting erythroid bursts were analyzed for FNRBC. The bursts that had been returned to FCS cultures yielded values of %FNRBC intermediate between those obtained from progenitors grown entirely in SF or FCS cultures, indicating that serum effect is not solely due to growth selection for certain subpopulations of erythroid burst-forming units. This experiment also demonstrated that the factors present in serum responsible for HbF augmentation act at both early and late stages during erythroid burst development. SF culture of peripheral blood progenitors of one subject with heterocellular hereditary persistence of fetal hemoglobin (HPFH) yielded elevated levels of FNRBC (19% +/- 5%) that accurately reflected the F cell (18%) of the circulating blood. Similarly, FNRBC in cultures of progenitors from one umbilical cord blood sample (86% F reticulocytes) was 87 +/- 3% FNRBC. The SF culture for enriched human progenitors, which nearly reflects the physiologic HbF programs of the donor, should facilitate studies of the exact mechanisms of postnatal reactivation of HbF production.
Department of Medicine, Medical University of South Carolina, Charleston.
Y
Y
Fujimori
M
M
Ogawa
S C
SC
Clark
G J
GJ
Dover
D001853:Bone Marrow / Q000378:metabolism; D001854:Bone Marrow Cells; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism; D015672:Erythroid Precursor Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D004921:Erythropoietin / Q000494:pharmacology; D005319:Fetal Hemoglobin / Q000096:biosynthesis*; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology
D003470:Culture Media; D007377:Interleukin-3; D004921:Erythropoietin; D005319:Fetal Hemoglobin