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

doi:10.1016/j.stem.2020.06.001

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

Cell Stem Cell. 2020 Jul 2;27(1):50-63.e5. doi: 10.1016/j.stem.2020.06.001.

Authors

Jan W Buikema¹, Soah Lee², William R Goodyer³, Renee G Maas⁴, Orlando Chirikian², Guang Li², Yi Miao⁵, Sharon L Paige³, Daniel Lee², Haodi Wu², David T Paik², Siyeon Rhee⁶, Lei Tian², Francisco X Galdos², Nazan Puluca², Benjamin Beyersdorf², James Hu², Aimee Beck², Sneha Venkamatran², Srilatha Swami⁷, Paul Wijnker⁸, Maike Schuldt⁸, Larissa M Dorsch⁸, Alain van Mil⁴, Kristy Red-Horse⁹, Joy Y Wu⁷, Caroline Geisen¹⁰, Michael Hesse¹⁰, Vahid Serpooshan¹¹, Stefan Jovinge¹², Bernd K Fleischmann¹⁰, Pieter A Doevendans¹³, Jolanda van der Velden⁸, K Christopher Garcia⁵, Joseph C Wu¹⁴, Joost P G Sluijter⁴, Sean M Wu¹⁵

Affiliations

¹ 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.
² Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
³ 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.
⁴ Utrecht Regenerative Medicine Center, Circulatory Health Laboratory, University Utrecht, Department of Cardiology, University Medical Center Utrecht, 3508 GA Utrecht, the Netherlands.
⁵ Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, and Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
⁶ Department of Biology, Stanford University, Stanford, CA 94305, USA.
⁷ Division of Endocrinology and Metabolism, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
⁸ Amsterdam Cardiovascular Sciences, Department of Physiology, Amsterdam University Medical Centers, Amsterdam, the Netherlands.
⁹ Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Biology, Stanford University, Stanford, CA 94305, USA.
¹⁰ Institute of Physiology I, Life and Brain Center, Medical Faculty, University of Bonn, 53105 Bonn, Germany.
¹¹ 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.
¹² 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.
¹³ 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.
¹⁴ 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.
¹⁵ 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: smwu@stanford.edu.

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 (<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.

Keywords: GSK3; Wnt signaling; cardiomyocytes; expansion; induced pluripotent stem cells; maturation; proliferation.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Cell Differentiation
Cells, Cultured
Glycogen Synthase Kinase 3 beta
Humans
Induced Pluripotent Stem Cells*
Myocytes, Cardiac

Substances

Glycogen Synthase Kinase 3 beta

Abstract

Publication types

MeSH terms

Substances

Grants and funding