Elevated Exogenous Pyruvate Potentiates Mesodermal Differentiation through Metabolic Modulation and AMPK/mTOR Pathway in Human Embryonic Stem Cells

Pyruvate is a key metabolite in glycolysis and tricarboxylic acid (TCA) cycle. Exogenous pyruvate modulates metabolism, provides cellular protection, and is essential for the maintenance of human preimplantation embryos and human embryonic stem cells (hESCs). However, little is known about how pyruvate contributes to cell fate determination during epiblast stage. In this study, we used hESCs as a model to demonstrate that elevated exogenous pyruvate shifts metabolic balance towards oxidative phosphorylation in both maintenance and differentiation conditions. During differentiation, pyruvate potentiates mesoderm and endoderm lineage specification. Pyruvate production and its mitochondrial metabolism are required in BMP4-induced mesoderm differentiation. However, the TCA cycle metabolites do not have the same effect as pyruvate on differentiation. Further study shows that pyruvate increases AMP/ATP ratio, activates AMPK, and then modulates the mTOR pathway to enhance mesoderm differentiation. This study reveals that exogenous pyruvate not only controls metabolism, but also modulates signaling pathways in hESC differentiation. Total RNA obtained from H1 human embryonic stem cell (hESC) culture in E8 medium (E8_2 Days), E8 medium with Pyruvate 16 mM treatment (Pyr 16mM_2 Days); HESCs were treated with BMP4 for 2 or 6 days for mesoderm differentiation in E8 medium, BMP4 treated for 2 days (BMP4_2 Days), BMP4 treatment with Pyruvate 16mM for 2 days (BMP4+Pyr 16mM_2 Days) or for 6 days (BMP4+Pyr 16mM_6 Days).