Massive Expansion of Functional Human iPSC-derived Cardiomyocytes by Concomitant Glycogen Synthase Kinase-3 Beta Inhibition and Removal of Cell-Cell Contact. Massive Expansion of Functional Human iPSC-derived Cardiomyocytes by Concomitant Glycogen Synthase Kinase-3 Beta Inhibition and Removal of Cell-Cell Contact

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 only to modest extent (< 5-fold). Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e. 100-250-fold) by glycogen synthase kinase-3β (GSK-3β) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show 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, cell cycle re-activation required both LEF/TCF activity and AKT phosphorylation, but it was independent from Yes associated protein (YAP) activity. Engineered heart tissues from expanded hiPSC-CMs showed the comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. In sum, we uncovered a molecular interplay that enables massive expansion hiPSC-CMs for large-scale drug screening and tissue engineering. Overall design: 24 hours treatment with various Wnt signaling pathway modulators

调控包括Wnt信号通路（Wnt signaling pathway）与Hippo信号通路（Hippo signaling pathway）在内的多条信号通路，可在活体中诱导心肌细胞（cardiomyocyte）增殖。此前研究显示，在体外将此类信号通路调节剂应用于人类诱导多能干细胞衍生心肌细胞（human induced pluripotent stem cell-derived cardiomyocytes，简称hiPSC-CMs）时，仅能实现有限程度的扩增（扩增倍数<5倍）。本研究证实，通过使用CHIR99021抑制糖原合酶激酶3β（glycogen synthase kinase-3β，简称GSK-3β），并同时去除细胞间接触，可在体外实现hiPSC-CMs的大规模扩增（扩增倍数达100~250倍）。研究发现，抑制GSK-3β会抑制心肌细胞成熟，而去除细胞间接触则可阻止心肌细胞退出细胞周期。值得注意的是，相较于仅抑制GSK-3β的方案，去除细胞间接触可使扩增效率提升10~25倍。机制研究显示，细胞周期再激活同时依赖LEF/TCF活性与AKT磷酸化，但不依赖Yes相关蛋白（Yes associated protein，简称YAP）的活性。由扩增后的hiPSC-CMs构建的工程化心脏组织，其收缩能力与未扩增hiPSC-CMs构建的工程化心脏组织相当，证实扩增后的心肌细胞功能未受损害。综上，本研究揭示了一种可实现hiPSC-CMs大规模扩增的分子互作机制，可用于大规模药物筛选与组织工程研究。实验整体设计：使用多种Wnt信号通路调节剂处理24小时。