Wnt Activation and Reduced Cell-Cell Contact Synergistically Induce Massive Expansion of Functional Human iPSC-Derived Cardiomyocytes

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...

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Published in:	Cell stem cell Vol. 27; no. 1; pp. 50 - 63.e5
Main Authors:	Buikema, Jan W., Lee, Soah, Goodyer, William R., Maas, Renee G., Chirikian, Orlando, Li, Guang, Miao, Yi, Paige, Sharon L., Lee, Daniel, Wu, Haodi, Paik, David T., Rhee, Siyeon, Tian, Lei, Galdos, Francisco X., Puluca, Nazan, Beyersdorf, Benjamin, Hu, James, Beck, Aimee, Venkamatran, Sneha, Swami, Srilatha, Wijnker, Paul, Schuldt, Maike, Dorsch, Larissa M., van Mil, Alain, Red-Horse, Kristy, Wu, Joy Y., Geisen, Caroline, Hesse, Michael, Serpooshan, Vahid, Jovinge, Stefan, Fleischmann, Bernd K., Doevendans, Pieter A., van der Velden, Jolanda, Garcia, K. Christopher, Wu, Joseph C., Sluijter, Joost P.G., Wu, Sean M.
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 02-07-2020
Subjects:	cardiomyocytes Cell Differentiation Cells, Cultured expansion Glycogen Synthase Kinase 3 beta GSK3 Humans Induced Pluripotent Stem Cells maturation Myocytes, Cardiac proliferation Wnt signaling cardiomyocytes maturation Wnt signaling proliferation GSK3 induced pluripotent stem cells expansion
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Summary:	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. [Display omitted] •GSK-3β inhibition-mediated hiPSC-cardiomyocyte proliferation is cell density dependent•GSK-3β inhibition with reduced cell-cell contact massively expands hiPSC-cardiomyocytes•LEF/TCF activity inhibits hiPSC-cardiomyocyte maturation without promoting cell cycling•Long-term expansion does not alter cardiomyocyte contractile function Deriving a large number of hiPSC-cardiomyocytes would be beneficial for large-scale tissue engineering and drug screening applications. Buikema et al. show that GSK-3β inhibition combined with removal of cell-cell contact enables massive expansion of hiPSC-cardiomyocytes with comparable function to non-expanded cells.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 JB, SL and SW conceived the study and wrote the manuscript. WG, RG and JS provided significant input on the experimental design. JB, SL, SL, WG, OC, GL, YM, SP, DL, HW, DP, SR, LT, FG, NP, BB, JH, AB, SV, SW, PW, MS, LD, AM and CG performed experiments. MR, JW, CG, MH, VS, SJ, BF, PD, JV, JS, CG and JW made critical revisions to the manuscript. These authors contributed equally to this work accordingly to their positions. Author Contributions
ISSN:	1934-5909 1875-9777
DOI:	10.1016/j.stem.2020.06.001