Wnt Activation and Reduced Cell-Cell Contact Synergistically Induce Massive Expansion of Functional Human iPSC-Derived Cardiomyocytes
Authors: Renee G Maas
Larissa M Dorsch
William R Goodyer
Srilatha Swami
Guang Li
Jan W Buikema
Haodi Wu
James Hu
Paul Wijnker
Caroline Geisen
Siyeon Rhee
Aimee Beck
Alain van Mil
Maike Schuldt
K Christopher Garcia
Vahid Serpooshan
Soah Lee
Lei Tian
Sharon L Paige
Kristy Red-Horse
Jolanda van der Velden
Joy Y Wu
Pieter A Doevendans
Joost P G Sluijter
Stefan Jovinge
David T Paik
Joseph C Wu
Bernd K Fleischmann
Orlando Chirikian
Sneha Venkamatran
Sean M Wu
Francisco X Galdos
Benjamin Beyersdorf
Daniel Lee
Michael Hesse
Yi Miao
Nazan Puluca
Journal: Cell stem cell (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: GSK3; Wnt signaling; cardiomyocytes; expansion; induced pluripotent stem cells
Affiliations: Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; Utrecht Regenerative Medicine Center, Circulatory Health Laboratory, University Utrecht, Department of Cardiology, University Medical Center Utrecht, 3508 GA Utrecht, the Netherlands., Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA., Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; Division of Pediatric Cardiology, Department of Pediatrics, Stanford School of Medicine, Stanford, CA 94305, USA., Utrecht Regenerative Medicine Center, Circulatory Health Laboratory, University Utrecht, Department of Cardiology, University Medical Center Utrecht, 3508 GA Utrecht, the Netherlands., Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, and Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA., Department of Biology, Stanford University, Stanford, CA 94305, USA., Division of Endocrinology and Metabolism, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA., Amsterdam Cardiovascular Sciences, Department of Physiology, Amsterdam University Medical Centers, Amsterdam, the Netherlands., Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Biology, Stanford University, Stanford, CA 94305, USA., Institute of Physiology I, Life and Brain Center, Medical Faculty, University of Bonn, 53105 Bonn, Germany., Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, Atlanta, GA 30322, USA., DeVos Cardiovascular Research Program of Spectrum Health and Van Andel Research Institute, 100 Michigan Street NE, Grand Rapids, MI 49503, USA; Michigan State University, College of Human Medicine, 15 Michigan Street NE, Grand Rapids, MI, USA., Utrecht Regenerative Medicine Center, Circulatory Health Laboratory, University Utrecht, Department of Cardiology, University Medical Center Utrecht, 3508 GA Utrecht, the Netherlands; Netherlands Heart Institute, Holland Heart House, 3511 EP Utrecht, the Netherlands., Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA., Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address: .

Abstract:  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 (&lt;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. 

Retracted: No

PubPeer comments:
 Figure 3A appears to have a repeated image (cyan boxes). Last author now appears to be at University of Maryland. *** ![file] , 
 #1 Note, the images in panel A are not photographs. They are diagrams, which plot quantitative expression data (from another study) in a color-coded way onto a cartoon map of different root tissues. In all four parts of panel A the cartoon map is identical. They differ only in the pattern of expression (the coloring). As the authors note, the expression patterns for PGP1 and PIN1 are strongly similar. It does not seem to me that there is a genuine issue or mistake here, but obviously the authors are in a better position to clarify. , 
 Yes, the cartoons in question were the somewhat crude expression maps that were available online previous to the beautiful expression study was published by Brady et al. later that year. They are not duplicates, but do indicate the poor resolution that existed before Brady et al. 2007 , 
 Thank you both. I apologize for my confusion. ,