Fig. 2. Differentiation of ALDH2*1/*1 hiPSCs and ALDH2*1/*2 hiPSCs-derived OBs. (A) Schematic representation of the osteogenic differentiation process of ALDH2*1/*1-hiPSCs and ALDH2*1/*2-hiPSCs. (B) Bright-field microscopy images showing the mineralized cell morphology. Scale bar=40 μm. (C) Calcium deposition and hydroxyapatite quantification in ALDH2*1/*1-OBs and ALDH2*1/*2-OBs on day 7, visualized using Alizarin Red S and OsteoImage staining. Scale bar=40 μm. (D) Quantitative assessment of Alizarin Red S staining by absorbance measurement at 405 nm. (E) Verification of OsteoImage staining using the ImageJ software. (F) Quantified expression levels of pluripotent marker OCT4 and osteogenic marker genes, including RUNX2, COL1A1, and OCN, in ALDH2*1/*1-hiPSCs (n=3) and ALDH2*1/*2-hiPSCs (n=3) after 7 days of osteogenic differentiation. Data are presented as the mean±SEM. Statistical significance: ^#p<0.05, ^##p<0.01, ^###p<0.001 (t-test), **p<0.01 (two-way ANOVA). E8: Essential 8, ALDH2: acetaldehyde dehydrogenase 2, hiPSCs: human induced pluripotent stem cells, OBs: osteoblasts.