(A) Alizarin Red staining for calcification in ACDC iMSCs under osteogenic conditions and treated with etidronate (ETID) for 21 days. Representative images are shown. Data are means ± SEM, n = 3 independent experiments, *P = 0.0356, unpaired two-tailed Student’s t test. (B) Etidronate treatment was administered to hMSCs at day 21 of osteogenic stimulation, and the cells were stained for calcium with Alizarin Red at day 28. Representative images are shown from three independent experiments. (C) iPSC lines generated from two ACDC patients were injected intramuscularly into the hindlimbs of NSG mice. Control animals received an injection of an equal volume of vehicle (Veh). After 6 weeks, teratomas were harvested and subjected to microCT analysis. Calcification percentage was valued in CT Analyzer software. Data are means ± SEM; ACDC etidronate, n = 7 teratomas; ACDC control, n = 6 teratomas; ***P = 0.0004, unpaired two-tailed Student’s t test.