Assessing the bioenergetic profile of human pluripotent stem cells

Assessing the bioenergetics of human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provides considerable insight into their mitochondrial functions and cellular properties. This might allow exposing potential energetic defects caused by mitochondrial diseases. However, certain challenges have to be met due to unique growth conditions in highly specialized and costly culture media. Here, we describe a method that facilitates the assessment of the bioenergetic profiles of hPSCs in a noninvasive fashion, while requiring only small sample sizes and allowing for several replicates. Basal respiratory and glycolytic capacities are assessed using a XF24 Extracellular Flux Analyzer by simultaneous measurements of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively. In addition, bioenergetic parameters are estimated by monitoring OCR and ECAR values upon metabolic perturbations via the consecutive introduction of mitochondria-specific inhibitors.