Fig 2: Erythrocyte differentiation of BH1 and BH2 iPSCs. (A): Diagrammatic sketch of differentiation procedure. To generate red blood cells, the iPSCs underwent three stages of spin-EB differentiation, erythroid differentiation, and erythrocyte terminal maturation (TM) in feeder-free and xeno-free culture conditions. (B): Flow cytometric analyses of hematopoietic stem/progenitor cells (CD34+CD45+) cells at EB day 14. (C): Flow cytometric analyses of erythrocytes (CD235a+CD45-) at TM day 8. The differentiation derivatives from BC1 iPSCs were used as controls. (D): Western blot to detect HBB proteins of erythrocytes from various iPSCs after terminal differentiation. GAPDH was used as loading control. Lane 1: BC1; Lane 2: BH1; Lane 3: BH2; Lanes 4, 5: two other control iPSC lines. Abbreviations: EB, embryoid bodies; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iPSCs, induced pluripotent stem cells.

Fig 3: The GFP expression under the control of the HBB locus from induced pluripotent stem cell (iPSC)-derived erythrocytes after genome editing. (A): Histogram of flow cytometric analyses at terminally differentiated erythrocytes from genome edited BC1 iPSCs. BC1 cells with a heterozygous integration of the HBB-GFP allele (#6C2) or homozygous integration (#8C8) were used, together with parental BC1 iPSCs. (B): Real-time quantitative PCR analyses of HBB and HBG gene expression. GFP positive and negative cells were isolated by fluorescence-activated cell sorting at day 8 after terminal differentiation. The relative gene expression levels were normalized by housekeeping gene GAPDH. (C): Flow cytometric analyses of other marker expression in the GFP positive versus negative cells at day 8 of terminal differentiation of #6C2 iPSCs. CD235a and CD45 were used to characterize erythrocytes. A DNA-staining fluorescent dye DRAQ5 was used to evaluate enucleation of erythrocytes lacking nuclear DNA. Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig 4: Expression of HBB protein in erythrocytes from the HBB-corrected BH1 and BH2 iPSCs. (A): Histogram of flow cytometric analyses of erythrocytes from HBB gene targeted BH1, BH2, and BC1 iPSCs at TM day 8. Efficient erythrocyte generation was demonstrated by a high percentage of cells expressing CD235a and a low percentage of cells expressing CD45. (B): Western blot of HBB expression in erythrocytes at TM day 8. The defective beta hemoglobin expression in erythrocytes from beta thalassemia patient iPSCs was rescued by mono allelic HBB gene targeting (heterozygous). The undifferentiated iPSCs of each line were used as negative controls. (C): Quantification (mean ± SEM) of HBB protein expression based on Western blot data of multiple experiments (n = 3). Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iPSC, induced pluripotent stem cells; TM, terminal maturation.

Fig 5: Overexpression of PALI1 triggers K562 cell differentiation along the erythroid lineage, with the effect altered by a separation-of-function allosteric-defective PALI1 mutant (K1241A).a Schematic illustration of the competitive cell proliferation assay used to directly compare the proliferation of cells transduced with different constructs. Cells overexpressing different proteins and fluorophores are co-cultured, with the higher-proliferating cells outcompete the lower-proliferating cells. At the end of the experiment, the number of cells carries each vector is quantified using flow cytometry. b Competitive cell proliferation assay of human chronic myeloid leukaemia (K562) cells overexpressing either PALI1 wild-type (WT), the separation-of-function PALI1 mutant (K1241A) or the LacZ negative control, as indicated. The bar plot represents the mean percentage of each cell population after 7 days of competition, as quantified using flow cytometry. Error bars represent standard deviations derived from three independent experiments carried out on different days with the observed values indicated by dots. Evidence for protein expression and nuclear localisation are in Supplementary Fig. 5a. Source data are provided as a Source Data file. c Colour photographs of pelleted K562 cells overexpressing different proteins: LacZ (left), PALI1 WT (middle) and PALI1-K1241A mutant (right). d Representative histograms generated from flow cytometric analysis of the differentiation markers CD235a (top) and CD44 (bottom) exhibited by K562 cells overexpressing PALI1 WT (red), PALI1 K1241A mutant (orange) or LacZ (blue). The other two replicates shown in Supplementary Fig. 9a, b and evidence for protein expression are in Supplementary Fig 8a. e Illustration of the expected expression level of CD235a and CD44 during erythropoiesis (grey) and the actual observed values represented in dashed lines coloured in red and blue for PALI1 and the negative control LacZ, respectively.