Augmentation of fetal hemoglobin (HbF) synthesis in culture by human erythropoietic precursors in the marrow and peripheral blood: studies in sickle cell anemia and nonhemoglobinopathic adults

Abstract

We cultured marrow and peripheral blood erythropoietic precrusors in methylcellulose clonal assay and measured the synthetic rates of HbA, A2, F, and S in patients with and without sickle cell anemia. Hb was labeled with 14C-amino acid in culture, separated by slab gel isoelectric focusing techniques, and quantitated by autoradiographic methods. Comparison of marrow late (CFU-E) and early (BFU-E) precursors from patients without hemoglobinopathies showed that preferential synthesis of HbF is limited to early precursors. Simultaneous examinations of Hb synthesis by blood and marrow early erythropoietic precursors confirmed the similarity of the biosynthetic capabilities of the precursors from the two sources. Increasing concentrations of erythropoietin (Ep) in culture corresponded with increases in the percentages of HbF synthesized by blood BFU-E of normal individuals. HbF biosynthesis by blood BFU-E from sickle cell anemia patients was significantly higher than that synthesized by nonanemic individuals and showed significant individual variations. HbF synthesis in patients with sickle cell anemia was partially dependent on Ep concentrations in culture. Cell culture of circulating erythropoietic precursors in man appears to provide a unique tool for studying the control mechanisms of Hb synthesis in man.

Hb Switching in neonatal cultures. Increase of Hb A synthesis in presence of an erythroid potentiating activity (EPA)

“…This action was impressive since Mo medium elicited a 36% increase in Hb A synthesis in comparison to standard cultures. In some cultures, the Hb A synthesis reached up to 78% and was nearly comparable to the ratio obtained in culture of adult BFU-E (Kidoguchi et al, 1978a;Papayannopoulou et al, 1978b).…”

“…Using this approach, it has been showed by several investigators that the expression of Hb in culture is reflective of each ontogenic state, but with some peculiarities. The progeny of early erythroid precursors (BFU-E) in adults produce a moderate amount of Hb F (usually less than 10% in mature erythroblasts; Housman et al, 1979;Kidoguchi et al, 1978a;Papayannopoulou et al, 1978a,b;Peschle et al, 1980;Testa et al, 1979;Vainchenker et al, 1980a,b. ) In erythroid cultures from neonates Hb A synthesis is slightly higher than in reticulocytes (Beuzard et al, 1979;Comi et al, 1980;Hassan et al, 1979;Kidoguchi et al, 1978b;Stamatoyannopoulos et al, 1979); in contrast, in the fetus similar levels of Hb A and Hb F synthesis are found in vivo and in culture (Beuzard et al, 1979;Rowley et al, 1979;Stamatoyannopoulos et al, 1979).…”

Human hemoglobin switching in culture

“…The results of Kidoguchi et a1 [26], obtained in methylcellulose culture, fundamentally agreed with those of the Seattle group. They reported that the marrow cultures labeled between days 3 and 5 of incubation showed no detectable HbF synthesis, whereas those labeled between days 12 and 14 revealed high levels of HbF production [26]. These results were interpreted to indicate that late CFU-E are unable to reactivate the synthesis of HbF, whereas BFU-E are capable of marked augmentation of HbF.…”

“…Comparison of the HbF synthetic capabilities between marrow and peripheral blood BFU-E yielded conflicting results between laboratories. While Kidoguchi et a1 [26] observed similarity between marrow and blood BFU-E in their HbF biosynthetic capabilities, C1ark.e and his co-workers [27] proposed that only peripheral blood BFU-E but not normal marrow BFU-E synthesize significant amounts of HbF in their more differentiated progenies. Recently, Dubart et al [28] reported results similar to those of Kidoguchi et al [26] and supported the hypothesis proposed by Papayannopoulou et al [23] that HbF synthetic capabilities inversely relate to the maturational stages of adult precursors.…”

“…Kinetics of Hb biosynthesis by adult erythroid precursors in culture have also been investigated in several laboratories. While Clarke and Housman [29] and Kidoguchi et a1 [26] reported constant ratios of the biosynthesis in peripheral blood cultures incubated for 10 and 14 days, Papayannopoulou et al [30] observed a gradual decline in the HbF biosynthetic rates by adult precursors in culture. The latter observation appears to be in agreement with that of Dover and Boyer [31] that cellular accumulation of HbF is completed earlier than that of HbA during terminal erythroid maturation in vivo.…”