Amelioration of heme synthesis failure and maturation defects of MT erythroblasts with AZA treatment. (A) Schema of a protocol for erythroid differentiation with AZA administration. The image of sorted HPCs was created using BioRender.com. (B) Percentages of CD235a⁺ erythroblasts treated with DMSO or AZA. The generation of CD235a⁺ erythroblasts in MT HPCs was significantly improved after the administration of AZA, whereas the generation of CD235a⁺ erythroblasts in WT HPCs was unchanged. Each line was tested in 3 independent experiments. (C) The pellets of unstained (top left) and o-dianisidine–stained (top right) erythroblasts derived from MT1-iPSC2 treated with DMSO or AZA. o-Dianisidine–stained images of erythroblasts derived from WT1-iPSC1 (left) and MT1-iPSC2 (right) treated with DMSO or AZA. CD235a⁺ cells were sorted on day 34 using FACS. Magnification of the objective lens: ×20. Bars represent 100 μm. (D) Representative Sanger sequencing data of ALAS2 complementary DNA from erythroblasts derived from WT iPSC (WT1-iPSC1) and MT iPSC (MT1-iPSC2) lines treated with DMSO or AZA. (E) Colony formation assay on day 15 of hematopoietic differentiation in WT1-iPSC1– and MT1-iPSC2–derived HPCs treated with DMSO or AZA. Each line was tested in 3 independent experiments. AZA 100, 100 nM AZA; AZA 500, 500 nM AZA. (F) Erythroid and mixed colony counts in panel E. (G) A representative image of mixed colonies derived from MT1-iPSC2 with AZA. Magnification of the objective lens: ×4. Bars represent 200 μm. All data are presented as the mean ± standard error of the mean. P-values were calculated using 1-way analysis of variance with Tukey’s correction and an unpaired, 2-tailed Student t test. *P < .05; **P < .01; N.S., not significant. DMSO, dimethyl sulfoxide.