Lower mitochondrial respiration in EpiSC, compared with ESC, is not due to mitochondrial immaturity, low mitochondria number or lack of pyruvate accessibility to mitochondria. (A) The differences in OCR between ESC, EpiSC and hESC were abolished when oligomycin was present, confirming that the difference in the aerobic respiration was caused by difference in the oxidative phosphorylation. FCCP treatment following oligomycin resulted in higher OCR increase in ESC than in EpiSC and hESC, confirming the higher level of mitochondrial electron transport chain (ETC) activity in ESC. (B) Another mitochondrial uncoupler 2,4-DNP confirms that EpiSC and hESC have lower mitochondrial activity. Results were summarized from two independent experiments, and the error bars represented the standard error of the mean. (C–F) Electron microscopy shows that EpiSC and hESC contain more elongated mitochondria than ESC; 20 EM images containing 194 and 296 mitochondria were used, respectively, for ESC and EpiSC mitochondrial quantification, and 30 images containing 212 mitochondria were used for hESC mitochondrial quantification. (G) EpiSC and hESC have higher mitochondrial DNA copy number than ESC. (H) ESC but not EpiSC increase OCR in response to DCA. SeaHorse results were summarized from two independent experiments, each consisting of independent cell plating on five SeaHorse microplate wells. The error bars represented the standard error of the mean. *Indicates P<0.05.