Abstract Body: Introduction: With the growing understanding of cell fate decisions, metabolism is now regarded as a potential signaling axis in developmental regulation. Human pluripotent stem cells (hPSCs) hold significant potential for advancing our understanding of heart development. However, variations in differentiation efficiency and poor reproducibility of hPSC-derived cardiomyocyte (CM) production remain a challenge.
Objectives: We aimed to investigate the metabolic regulation in CM differentiation and develop the metabolic method to promote CM differentiation.
Methods: We first investigated metabolic changes during CM differentiation using RNA sequencing (RNA-seq) and real-time assessment of mitochondrial function. We focused on serine synthesis pathway (SSP) and evaluated the effects of SSP inhibition on CM differentiation. We then performed single-cell RNA-seq (scRNA-seq) to identify the mechanisms and cell populations with altered differentiation fates upon SSP inhibition. Metabolome and electron transport chain (ETC) activities were assessed to identify the biological process, and rescue experiments were conducted following SSP inhibition to confirm its function. Finally, we evaluated the effects of SSP inhibition on signaling pathways.
Results: 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 SSP. hPSCs could be differentiated into CMs stably and efficiently with PHGDH inhibition. scRNA-seq analysis revealed that PHGDH inhibition increased CM populations, while decreasing cardiopharyngeal mesoderm (CPM) populations. Trajectory inference successfully identified a bifurcation, with distinct branches leading to CM and CPM. PHGDH inhibition induced reactive oxygen species via impairing ETC complex I function. Rescue experiments demonstrated that antioxidants counteracted the effects of PHGDH inhibition, promoting CPM differentiation while inhibiting CM differentiation, and cell fate divergence was regulated by FGF/MAPK signaling pathway.
Conclusions: Our findings show that SSP can regulate CM differentiation and have implications in elucidating the potential mechanisms of heart development.