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

Abstract

Summary Pyruvate is a key metabolite in glycolysis and the 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 toward 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 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.

AMPKβ1 and AMPKβ2 define an isoform-specific gene signature in human pluripotent stem cells, differentially mediating cardiac lineage specification

“…Wnt and activin signaling are modulated by YAP (61,62), that can directly be phosphorylated by AMPK (63) and is under certain circumstances a target of O-GlcNAcylation (64). In addition, metabolite availability in hESCs can affect lineage decision via activation of AMPK (65). Taken together, the mechanism underlying the phenotype observed in our stem cell model is most likely multifaceted and might include direct phosphorylation and posttranslational modifications of effector proteins at different levels of regulation.…”

Human iPSC-derived cerebral organoids model features of Leigh Syndrome and reveal abnormal corticogenesis

“…Clinical data available from the patient with the PDH mutation showed elevated blood pyruvate levels. High concentrations of pyruvate have been shown to potentiate the differentiation of human embryonic stem cells (hESCs) into endodermal and mesodermal lineages while suppressing the expression of ectodermal markers in a lineage-specific fashion (Song et al, 2019). Finally, mesodermal lineage was confirmed by the staining of the protein markers Brachyury and CXCR4 (Figure 2E) and the expression of the genes TBXT and NCAM (Figure 2F).…”

“…Clinical data available from the patient with the PDH mutation reports elevated blood pyruvate levels (Schubert and Vilarinho, 2020; Sofou et al, 2014). High concentrations of pyruvate in the media have been shown to potentiate the differentiation of human embryonic stem cells (hESCs) into endodermal and mesodermal lineages in a lineage-specific fashion (Song et al, 2019), suggesting a differential capacity of these cells to preferentially commit into certain cell fates due to metabolic dysregulation.…”

Optimization of the nutritional environment for differentiation of human‐induced pluripotent stem cells using design of experiments—A proof of concept

“…This effect is more pronounced than when hiPSCs are exposed to the same conditions with higher pyruvate concentration (0.50 mM), since the presence of exogenous pyruvate potentiates the differentiation of stem cells toward mesodermal lineages. 43 Depleting glucose from media while keeping the physiological environmental levels of oxygen and pyruvate (2%, 0.14 mM, respectively) enhances all endodermal markers from Day 2 onward (Figures 1 and 2). The deprivation of all three factors induced endodermal expression with a maximum of 500-fold increase of SOX17 and 40-fold increase of CXCR4 at Day 3 without inducing the definitive endodermal marker (FOXA2) (Figure 2).…”