Glycogen Synthase Kinase-3 Inhibition and Insulin Synergistically Enhance Proliferation and Inhibit Maturation of Human iPSC-derived Cardiomyocytes via TCF and FOXO Signaling

Embryonic signaling pathways exert stage-specific effects during cardiac development, yet the precise cues for proliferation or maturation remain elusive. To address this, we utilized spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) at day 12 of differentiation and performed a combinatory screen for various dosages of the glycogen synthase kinase-3 (GSK3) inhibitor CHIR99021 and Insulin for the analysis of cell cycle in hiPSC-CMs. Our combinatory screen for proliferation, subsequential downstream sarcomere development and RNA-seq analyses for Insulin/Akt and CHIR99021/Wnt demonstrate synergistic effects on proliferation of immature hiPSC-CMs. Conversely, removal of the Wnt and Insulin stimuli leads to rapid cell cycle exit and facilitates the terminal differentiation of immature hiPSC-CMs. Detailed characterization reveals that Wnt/CHIR99021, but not Insulin, regulates sarcomere homeostasis and architecture of immature hiPSC-CMs. Moreover, we further identify a temporal interplay between CHIR99021/Wnt via TCF and Insulin via FoxO signaling as regulators between proliferation and maturation in immature hiPSC-CMs. This work describes the cues that control proliferation versus terminal differentiation in functional immature hiPSC-CMs, and provides molecular mechanistic understanding between proliferation and maturation development of hiPSC-CMs.