Compensated variability in the expression of globin-related genes in erythroblasts generated ex vivo from different donors

BACKGROUND: Ex vivo generated erythroblasts are being evaluated for transfusion. Expression of balanced levels of globin mRNA is essential for normal red blood cell function and survival but it is unknown whether the expression of the globin genes in ex vivo expanded cells is balanced. STUDY DESIGN AND METHODS: Immature erythroblasts (IEs) were expanded in human erythroid massive amplification cultures from blood mononuclear cells of 19 normal donors and four β⁰-thalassemia patients (for comparison) and induced to mature for 4 days in the presence of erythropoietin. mRNA was prepared from IEs and mature erythroblasts to evaluate the expression of α-, β-, and γ-globin genes and of adult hemoglobin-stabilizing protein (AHSP) and BCL11A, two proteins directly controlling globin function and/or production. Results were analyzed using Pearson's correlation coefficient, the Wilcoxon signed rank, and the Mann-Whitney rank sum tests. RESULTS: The absolute levels of globin, AHSP, and BCL11A mRNA expressed by erythroblasts generated ex vivo from normal donors were distributed along a 2-log range. With maturation, the levels of γ-globin and BCL11A mRNA did not decrease while those of α-globin, γ + β-globins, and AHSP mRNA greatly increased. In normal cells, the modest imbalance (two- to fourfold) observed between α- and γ + β-globin mRNA was fully compensated by AHSP expression. Thus, the levels of α-globin mRNA were correlated with those of γ + β-globin (R² = 0.93, p < 0.0001) and AHSP (R² = 0.86, p < 0.0001). CONCLUSIONS: Ex vivo expanded erythroblasts from normal donors express modestly imbalanced levels of α-globin and γ + β-globin fully compensated by AHSP expression, likely ensuring normal function and survival.