Distinct Metabolic States Can Support Self-Renewal and Lipogenesis in Human Pluripotent Stem Cells under Different Culture Conditions

• Medium choice influences the metabolic state of hPSCs
• Chemically defined medium reprograms hPSC lipogenic and redox metabolic pathways
• hPSCs cultured in lipid-replete medium exhibit active oxidative mitochondrial flux
• Glutamine fuels mitochondrial metabolism in self-renewing hPSCs

SummaryRecent studies have suggested that human pluripotent stem cells (hPSCs) depend primarily on glycolysis and only increase oxidative metabolism during differentiation. Here, we demonstrate that both glycolytic and oxidative metabolism can support hPSC growth and that the metabolic phenotype of hPSCs is largely driven by nutrient availability. We comprehensively characterized hPSC metabolism by using 13C/2H stable isotope tracing and flux analysis to define the metabolic pathways supporting hPSC bioenergetics and biosynthesis. Although glycolytic flux consistently supported hPSC growth, chemically defined media strongly influenced the state of mitochondrial respiration and fatty acid metabolism. Lipid deficiency dramatically reprogramed pathways associated with fatty acid biosynthesis and NADPH regeneration, altering the mitochondrial function of cells and driving flux through the oxidative pentose phosphate pathway. Lipid supplementation mitigates this metabolic reprogramming and increases oxidative metabolism. These results demonstrate that self-renewing hPSCs can present distinct metabolic states and highlight the importance of medium nutrients on mitochondrial function and development.

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