Glycogen synthase kinase-3 inhibition and insulin 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 signals for proliferation or maturation remain elusive. To uncover the cues for proliferation, we performed a combinatory cell-cycle screen for insulin and glycogen synthase kinase-3 (GSK3) inhibition in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Our analysis for proliferation, and subsequential downstream sarcomere development, gene expression analysis, and molecular interventions identified a temporal interplay between insulin/Akt/FOXO and CHIR99021/Wnt/GSK3/TCF signaling. Combined pathway activation led to proliferation of immature hiPSC-CMs with low sarcomere and mitochondria content, while, in the absence of pathway activators, cardiomyocytes rapidly exited the cell cycle and fetched higher organization of sarcomeres and mitochondria. Our data demonstrate two important pathways, which enhance proliferation and inhibit maturation, and provide molecular mechanistic understanding of these cell fate decisions in immature hiPSC-CMs. • Insulin and GSK3β inhibition are two important transient cues for proliferation of immature hiPSC-CMs and inhibit maturation • GSK3β inhibition represses sarcomere buildup in immature hiPSC-CMs • Inhibition of FOXO transcription underlies cell-cycle activity in response to insulin Understanding of the molecular cues that drive hiPSC-cardiomyocyte proliferation or maturation would be beneficial for tissue engineering and regenerative approaches for heart failure. Buikema and colleagues demonstrate that insulin and glycogen synthase kinase-3 inhibition concomitantly promote proliferation while inhibiting maturation via FOXO and TCF signaling.