Wnt Activation and Reduced Cell-Cell Contact Synergistically Induce Massive Expansion of Functional Human iPSC-Derived Cardiomyocytes
Jan W Buikema, Soah Lee, William R Goodyer, Renee G Maas, Orlando Chirikian, Guang Li, Yi Miao, Sharon L Paige, Daniel Lee, Haodi Wu, David T Paik, Siyeon Rhee, Lei Tian, Francisco X Galdos, Nazan Puluca, Benjamin Beyersdorf, James Hu, Aimee Beck, Sneha Venkamatran, Srilatha Swami, Paul Wijnker, Maike Schuldt, Larissa M Dorsch, Alain van Mil, Kristy Red-Horse, Joy Y Wu, Caroline Geisen, Michael Hesse, Vahid Serpooshan, Stefan Jovinge, Bernd K Fleischmann, Pieter A Doevendans, Jolanda van der Velden, K Christopher Garcia, Joseph C Wu, Joost P G Sluijter, Sean M Wu
Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (<5-fold). Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e., 100- to 250-fold) by glycogen synthase kinase-3β (GSK-3β) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show that GSK-3β inhibition suppresses CM maturation, while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10 to 25 times greater expansion beyond GSK-3β inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from yes-associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. In summary, we uncovered a molecular interplay that enables massive hiPSC-CM expansion for large-scale drug screening and tissue engineering applications.