TGF-beta activated PDHB promotes mitochondrial pyruvate metabolism and contributes to human endoderm differentiation via ATP-dependent BRG1

Glucose metabolism is a major cellular energy pathway, encompassing early glycolysis, which catalyzes glucose into pyruvate and lactate, as well as downstream tricarboxylic acid (TCA) cycle and oxidative phosphorylation, which convert pyruvate-derived metabolites into acetyl-CoA within mitochondria. Despite its central role in cellular energetics, the importance of glucose metabolism during the differentiation of embryonic stem cells (ESCs) toward definitive endoderm (DE) remains incompletely understood. To systematically investigate the metabolic regulators of DE differentiation, we performed a CRISPR-based functional genetic screen in our DE differentiation model using a metabolism-focused sgRNA library. In this screening, CXCR4 expression was used as a quantitative readout to assess DE differentiation efficiency. Cells were sorted into the top 30% CXCR4-positive and bottom 30% CXCR4-negative populations, and sgRNA representation was compared between these two groups to identify differentially enriched genes that promote or inhibit DE differentiation.