Abstract: Metabolism is well known as an active driver of physiological changes rather than merely a housekeeping process during development. With the expanding understanding of metabolic control over cell fate specification, metabolic regulation is now recognized as a potential signaling axis in the regulation of development. Human pluripotent stem cells (hPSCs) hold significant potential for advancing our understanding of heart development. Differentiating hPSCs into cardiac cells allows for modeling early cardiac developmental processes and exploring key signaling pathways. However, variations in differentiation efficiency and poor reproducibility of hPSC-derived cardiomyocytes (hPSC-CMs) production have remained a challenge. Here, we report a unique metabolic method to promote hPSC-CM differentiation that involves marked suppression of the mitochondrial oxidative phosphorylation from the mesendoderm to the cardiac mesoderm, which is regulated by PHGDH, a rate-limiting enzyme in the serine synthesis pathway (SSP). Mechanistically, the analysis of metabolomics and single-cell RNA-sequencing revealed that SSP inhibition impaired mitochondrial respiration by blocking the electron transport chain, resulting in elevated reactive oxygen species (ROS) levels and promoting the cardiomyocyte lineage specification by disrupting the cardiopharyngeal mesoderm lineage differentiation. Additionally, antioxidant supplementation can scavenge ROS and eliminate the effects of SSP inhibition. Collectively, our findings show that SSP can regulate cardiac lineage specification and have implications in providing a cellular source for transplantation and elucidating the potential mechanisms of heart development and pathogenesis of heart disease.
