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.

调节Wnt和Hippo等信号通路可以在体内诱导心肌细胞的增殖。在体外将这些信号调节剂应用于人类诱导的多能干细胞衍生的心肌细胞（hiPSC-CM）可使CM适度扩增（<5倍）。在这里，我们展示了hiPSC-CMs在体外的大规模扩展使用CHIR99021抑制糖原合酶激酶-3β（GSK-3β）（100-250倍），并同时去除细胞间接触。我们表明，GSK-3β抑制抑制了CM的成熟，而去除接触阻止了CM从细胞周期退出。值得注意的是，去除接触物后，其扩展作用比单独的GSK-3β抑制作用大10至25倍。从机制上讲，持续的CM增殖既需要LEF / TCF活性也需要AKT磷酸化，但独立于yes相关蛋白（YAP）信号传导。来自扩增的hiPSC-CM的工程心脏组织显示出与未扩增的hiPSC-CM相似的收缩力，证明了扩增后细胞功能未受损。总而言之，我们发现了一种分子相互作用，可实现大规模hiPSC-CM扩展，以用于大规模药物筛选和组织工程应用。