Abstract Body (Do not enter title and authors here): Introduction: With the growing understanding of cell fate decisions, metabolism is increasingly recognized as a potential signaling axis in developmental regulation. Human pluripotent stem cells (hPSCs) offer significant potential to advance our understanding of heart development. However, variability in differentiation efficiency and the limited reproducibility of hPSC-derived cardiomyocyte (CM) production remain major challenges.
Objectives: We aimed to investigate the metabolic regulation in CM differentiation and develop the metabolic method to promote CM differentiation.
Methods and Results: We first investigated metabolic changes during CM differentiation using real-time assessment of mitochondrial function. We found that CM differentiation involved marked suppression of oxidative phosphorylation from the mesendoderm to the cardiac mesoderm, which was regulated by PHGDH, a rate-limiting enzyme in the serine synthesis pathway (SSP). To evaluate the importance of the SSP during CM differentiation, we applied a small molecule inhibitor targeting PHGDH to the CM differentiation protocol. Addition of a PHGDH inhibitor from the mesendoderm to the cardiac mesoderm increased generation of CMs by 2 folds than control conditions. Next, we performed scRNA-seq to identify cell populations with altered differentiation fates upon PHGDH inhibition. scRNA-seq analysis revealed that PHGDH inhibition increased CM populations, while decreasing cardiopharyngeal mesoderm (CPM) populations, which gave rise to skeletal system and head development. Moreover, trajectory inference successfully identified a bifurcation, with distinct branches leading to CM and CPM. Metabolically, we found that PHGDH inhibition decreased the glutathione synthesis and rescue experiments demonstrated that antioxidants counteracted the effects of PHGDH inhibition, promoting CPM differentiation while inhibiting CM differentiation. Finally, we evaluated the effects of PHGDH inhibition on differentiation signals. We identified that SSP regulated FGF/ERK signaling pathway, one of the major regulators of cardiopharyngeal development. The inhibition of FGF/ERK pathway recapitulated PHGDH inhibition-induced CM differentiation.
Conclusions: Collectively, our findings indicate that metabolism can regulate CM differentiation by modulating differentiation signals. Our study has implications in elucidating the potential mechanisms of heart development as well as providing a cellular source for transplantation.