SUMMARY A general mechanism for how intracellular signaling pathways in human pluripotent cells are co-ordinated and how they maintain self-renewal remain to be elucidated. In this report, we describe a novel signaling mechanism where PI3K/Akt activity maintains self-renewal by restraining pro-differentiation signaling through suppression of the Raf/Mek/Erk and canonical Wnt signaling pathways. When active, PI3K/Akt establishes conditions where Activin A/Smad2,3 performs a pro-self renewal function by activating target genes, including Nanog. When PI3K/Akt signaling is low, Wnt effectors are activated and function in conjunction with Smad2,3 to promote differentiation. The switch in Smad2,3 activity following inactivation of PI3K/Akt requires the activation of canonical Wnt signaling by Erk, which targets Gsk3β. In sum, we define a signaling framework that converges on Smad2,3 and determines its ability to regulate the balance between alternative cell states. This signaling paradigm has far-reaching implications for cell fate decisions during early embryonic development.

SUMMARY As human pluripotent stem cells (hPSCs) exit pluripotency they are thought to switch from a glycolytic mode of energy generation to one more dependent on oxidative phosphorylation. Here we show that although metabolic switching occurs during early mesoderm and endoderm differentiation, high glycolytic flux is maintained and in fact essential during early ectoderm specification. The elevated glycolysis observed in hPSCs requires elevated MYC/MYCN activity. Metabolic switching during endodermal and mesodermal differentiations coincides with a reduction in MYC/MYCN, and can be reversed by ectopically restoring MYC activity. During early ectodermal differentiation, sustained MYCN activity maintains the transcription of 'switch' genes that are rate-limiting for metabolic activity and lineage commitment. Our work, therefore, shows that metabolic switching is lineage-specific and not a required step for exit of pluripotency in hPSCs, and identifies MYC and MYCN as developmental regulators that couple metabolism to pluripotency and cell fate determination.

Metabolism has been shown to alter cell fate in human pluripotent stem cells (hPSC). However, current understanding is almost exclusively based on work performed at 20% oxygen (air), with very few studies reporting on hPSC at physiological oxygen (5%). In this study, we integrated metabolic, transcriptomic, and epigenetic data to elucidate the impact of oxygen on hPSC. Using 13C-glucose labeling, we show that 5% oxygen increased the intracellular levels of glycolytic intermediates, glycogen, and the antioxidant response in hPSC. In contrast, 20% oxygen increased metabolite flux through the TCA cycle, activity of mitochondria, and ATP production. Acetylation of H3K9 and H3K27 was elevated at 5% oxygen while H3K27 trimethylation was decreased, conforming to a more open chromatin structure. RNA-seq analysis of 5% oxygen hPSC also indicated increases in glycolysis, lysine demethylases, and glucose-derived carbon metabolism, while increased methyltransferase and cell cycle activity was indicated at 20% oxygen. Our findings show that oxygen drives metabolite flux and specifies carbon fate in hPSC and, although the mechanism remains to be elucidated, oxygen was shown to alter methyltransferase and demethylase activity and the global epigenetic landscape.

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