Generation of XLSA patient-derived iPSCs and confirmation of X-chromosome inactivation. (A) Schematic representation of the iPSC generation from PBMCs of female patients with XLSA who harbored a heterozygous ALAS2 mutation. The figure was created using BioRender.com. (B) Summary of HUMARA assays of PBMCs from patients with XLSA and the percentage of MT iPSC lines among established iPSCs. (C) Expression levels of OCT3/4, SOX2, and NANOG genes in PBMCs, 1 control iPSC line derived from a healthy donor, 3 WT iPSC lines, and 4 MT iPSC lines. Expression levels of PBMCs were set to 1. Each line was tested in 3 independent experiments. (D) HUMARA assays of WT and MT iPSCs (left). The undigested control is shown at the bottom left. Representative immunofluorescence staining images for Hoechst expression and H3K27me3 expression in iPSCs derived from patients with XLSA (right). Magnification of the objective lens: ×20. Bars represent 50 μm. (E) HUMARA assays of representative iPSC lines (WT1-iPSC3 and MT1-iPSC2) after 5, 20, and 30 passages and HPCs derived from the iPSC lines. Complementary DNA Sanger sequencing data of erythroblasts derived from the iPSC lines are shown on the far right.