Mitochondrial metabolism and glutamine are essential for mesoderm differentiation of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) generate energy mainly by aerobic glycolysis, with glutamine oxidation in the tricarboxylic acid (TCA) cycle providing additional ATP required for survival.1–3 During the exit from pluripotency and initial differentiation into multiple germ lineage precursors, energy production shifts from mainly aerobic glycolysis to mitochondrial oxidative phosphorylation (OXPHOS).1 Until recently, consensus in the field was that as PSCs exit pluripotency, a metabolic switch from aerobic glycolysis to OXPHOS is required. However, a more detailed examination of nascent ectoderm (EC) metabolism showed unexpected maintenance of a high, MYC-dependent glycolytic flux, resembling sustained hPSC metabolism, in contrast to mesoderm (ME) and endoderm (EN),4 generating questions for the role(s) of mitochondrial metabolism in early hPSC tri-lineage differentiation. To examine this issue, we differentiated hPSCs into early EN, ME, and EC lineages using a non-limiting, nutrient-balanced culture media that differed only by established lineage-driving cytokines,5,6 so that intrinsic metabolic preferences were not derived from a variance in nutrient composition (Supplementary information, Data S1). Principal component analysis (PCA) of these early lineages using RNA-Seq was equivalent to a previous study using nutrient-balanced and chemically defined growth media.4 Furthermore, transcriptomic and protein biomarker expression matched established profiles for hPSCs and hPSC-derived EN, ME, and EC (Supplementary information, Fig. S1a–c, Table S1),7 confirming the validity of our model system.