Erythroid cell differentiation of corrected α⁰-thal-iPSCs. (A) Chromatograms illustrating the results of HPLC analyses of globin chain expression performed after 14 days (14 FHB 14LC) or 24 days (14FHB 24 LC) of liquid culture of corrected iPSCs differentiated into erythroid cells. The 3 chromatograms on the left illustrate the globin expression pattern in orthochromatic erythroblasts obtained from culture of CD34⁺ cells from cord blood, fetal liver, and peripheral blood from a transfusion dependent adult with α⁰-thalassemia. β1-Het indicates heterozygous transgene integrated in same orientation as PPP1R12C, driven by β-globin promoter; β1-Hom, homozygous transgene in same orientation as PPP1R12C, driven by β-globin promoter; α1-Hom, homozygous transgene in same orientation as PPP1R12C, driven by α-globin promoter. Almost complete correction of chain imbalance in mature erythroid cells was obtained when homozygous transgenes driven by the α-globin promoter were inserted at AAVS1. The β-globin promoter was less effective than the α-globin promoter. Orientation of the transgene had no major effect on expression. (B) Q-RT-PCR analysis of globin expression in hESCs and iPSCs after differentiation into basophilic erythroblasts (14 days of liquid culture). The y-axis indicates the fold-difference compared with GAPDH. (C) IEF electrophoresis on lysates of orthochromatic erythroblasts illustrating the hemoglobin tetramers expressed in controls, in H1 and in corrected iPSCs. Globin content of each tetramer was determined by HPLC after cutting-out each major band from the gel. Corrected iPSCs express Hb F and Hb Gower II in addition to embryonic globins. A vertical line has been inserted to indicate a repositioned gel lane.