mtDNA mutations affect the dysfunction of differentiated pancreatic cells. (A) The gene expression of PPC, PDX1, NGN3, and NKX6.1, relative to GAPDH in T2D-2 BiPSCs. All gene expressions in clone 1 were significantly lower compared to other clones (*P < 0.05, 2 biological replicates, 3 technical replicates). (B) PPC-gene expression was inversely proportional to the sum of the heteroplasmy of mtDNA mutations in the positions of change of the amino acid, and RNA. As heteroplasmy increases, the expression of PPC-related genes decreases. (C) Morphology of clones 1 and 2 and pancreatic spheroids derived from them. Clone 1 had 100% mt6678A>G and 13% mt7970G>A mutations in the non-syn protein-coding region and clone 2 had only 5% mt874G>A mutations in the rRNA region. Both clones showed similar spheroid formation. Scale bar = 200 μm. (D) Immunocytochemistry examining the INSULIN (green) expression of pancreatic spheroids. Nuclei were stained with DAPI (blue). Scale bar = 20 μm. (E) Analysis of OCR levels in differentiated pancreatic cells. In clone 1, all OCRs were significantly lower compared with clone 2 (*P < 0.05, 2 biological replicates, and 3 technical replicates). (F) Insulin production in pancreatic spheroids derived from iPSC clones. The insulin in clone 2 was significantly higher than that of clone 1 (*P < 0.05). (G) GSIS analysis of pancreatic spheroids. Clone 2-derived pancreatic spheroids increased the amount of insulin secretion when glucose concentration increased, whereas clone 1 did not (*P < 0.05, 2 biological replicates, 3 technical replicates). PPC: pancreatic progenitor cell; DAPI: 4′,6-diamidino-2-phenylindole; OCR: oxygen consumption rate; GSIS: glucose-stimulated insulin secretion.