Bioenergetic Profiling of Human Pluripotent Stem Cells.

Cellular metabolism contributes to cell fate decisions. Bioenergetic profiling can therefore provide considerable insights into cellular identity and specification. Given the current importance of human pluripotent stem cells (hPSCs) for biomedical applications, assessing the bioenergetic properties of hPSCs and derivatives can unveil relevant mechanisms in the context of development biology and molecular disease modeling. Here, we describe a method to facilitate bioenergetic profiling of hPSCs in a reproducible and scalable manner. After simultaneous assessment of mitochondrial respiration and glycolytic capacity using Seahorse XFe96 Analyzer, we measure lactate concentration in the cellular media. Finally, we normalize the values based on DNA amount. We describe the procedures with specific requirements related to hPSCs . However, the same protocol can be easily adapted to other cell types, including differentiated progenies from hPSCs .

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.

HIF1α modulates cell fate reprogramming through early glycolytic shift and upregulation of PDK1-3 and PKM2.

Reprogramming somatic cells to a pluripotent state drastically reconfigures the cellular anabolic requirements, thus potentially inducing cancer-like metabolic transformation. Accordingly, we and others previously showed that somatic mitochondria and bioenergetics are extensively remodeled upon derivation of induced pluripotent stem cells (iPSCs), as the cells transit from oxidative to glycolytic metabolism. In the attempt to identify possible regulatory mechanisms underlying this metabolic restructuring, we investigated the contributing role of hypoxia-inducible factor one alpha (HIF1α), a master regulator of energy metabolism, in the induction and maintenance of pluripotency. We discovered that the ablation of HIF1α function in dermal fibroblasts dramatically hampers reprogramming efficiency, while small molecule-based activation of HIF1α significantly improves cell fate conversion. Transcriptional and bioenergetic analysis during reprogramming initiation indicated that the transduction of the four factors is sufficient to upregulate the HIF1α target pyruvate dehydrogenase kinase (PDK) one and set in motion the glycolytic shift. However, additional HIF1α activation appears critical in the early upregulation of other HIF1α-associated metabolic regulators, including PDK3 and pyruvate kinase (PK) isoform M2 (PKM2), resulting in increased glycolysis and enhanced reprogramming. Accordingly, elevated levels of PDK1, PDK3, and PKM2 and reduced PK activity could be observed in iPSCs and human embryonic stem cells in the undifferentiated state. Overall, the findings suggest that the early induction of HIF1α targets may be instrumental in iPSC derivation via the activation of a glycolytic program. These findings implicate the HIF1α pathway as an enabling regulator of cellular reprogramming.