BMI1 represses HbF in primary adult erythroblasts. (A) Schematic of the validation experiment in primary adult erythroid cells. CD34⁺ HSPCs isolated from peripheral blood of healthy donors were cultured with indicated cytokines and electroporated with Cas9 ribonucleoprotein at day 4 or 5 of the differentiation. (B-D) HBG:(HBB+HBG) mRNA levels (B) (n = 3 independent donors), HbF⁺ cell fraction (C) (n = 2), and hemoglobin HPLC profile (D) (n = 2) in control and BMI1-depleted primary adult erythroid cells. Values are presented as mean ± SEM. P values were calculated by unpaired 2-tailed Student t test. HPLC, high-performance liquid chromatography; SEM, standard error of the mean.