Epicardial cells derived from human embryonic stem cells augment cardiomyocyte-driven heart regeneration

The epicardium and its derivatives provide trophic and structural support for the developing and adult heart. Here we tested the ability of human embryonic stem cell (hESC)-derived

epicardium to augment the structure and function of engineered heart tissue in vitro and to improve efficacy of hESC-cardiomyocyte grafts in infarcted athymic rat hearts. Epicardial cells

markedly enhanced the contractility, myofibril structure and calcium handling of human engineered heart tissues, while reducing passive stiffness compared with mesenchymal stromal cells.

Transplanted epicardial cells formed persistent fibroblast grafts in infarcted hearts. Cotransplantation of hESC-derived epicardial cells and cardiomyocytes doubled graft cardiomyocyte

proliferation rates in vivo, resulting in 2.6-fold greater cardiac graft size and simultaneously augmenting graft and host vascularization. Notably, cotransplantation improved systolic

function compared with hearts receiving either cardiomyocytes alone, epicardial cells alone or vehicle. The ability of epicardial cells to enhance cardiac graft size and function makes them

a promising adjuvant therapeutic for cardiac repair.

The raw data that support the findings of this study are available from the corresponding author upon reasonable request.

This work was supported by the British Heart Foundation (BHF; grants nos. NH/11/1/28922, G1000847, FS/13/29/30024 and FS/18/46/33663), Oxford-Cambridge Centre for Regenerative Medicine

(grant no. RM/13/3/30159), the UK Medical Research Council (MRC) and the Cambridge Hospitals National Institute for Health Research Biomedical Research Centre funding (S.S.), as well as

National Institutes of Health grant nos. P01HL094374, P01GM081619 and R01HL128362 and a grant from the Fondation Leducq Transatlantic Network of Excellence (C.E.M.). J.B. was supported by a

Cambridge National Institute for Health Research Biomedical Research Centre Cardiovascular Clinical Research Fellowship and, subsequently, by a BHF Studentship (grant no. FS/13/65/30441).

D.I. received a University of Cambridge Commonwealth Scholarship. L.G. is supported by BHF Award RM/l3/3/30159 and L.P.O. is funded by a Wellcome Trust Fellowship (grant no. 203568/Z/16/Z).

N.F. was supported by BHF grant no. RG/13/14/30314. N.L.N. was supported by the Biotechnology and Biological Sciences Research Council (Institute Strategic Programmes BBS/E/B/000C0419 and

BBS/E/B/000C0434). S.S. and M.R.B. were supported by the BHF Centre for Cardiovascular Research Excellence. Core support was provided by the Wellcome-MRC Cambridge Stem Cell Institute (grant

no. 203151/Z/16/Z). The authors thank Osiris for providing the primary mesenchymal stem cells47.

These authors contributed equally: Charles E. Murry, Sanjay Sinha.

The Anne McLaren Laboratory, Wellcome Trust – MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK

Johannes Bargehr, Lay Ping Ong, Maria Colzani, Hongorzul Davaapil, Laure Gambardella, Dharini Iyer, Fotios Sampaziotis, William G. Bernard & Sanjay Sinha

Division of Cardiovascular Medicine, University of Cambridge, Cambridge, UK

Johannes Bargehr, Lay Ping Ong, Maria Colzani, Hongorzul Davaapil, Laure Gambardella, Dharini Iyer, William G. Bernard, Nichola Figg, Martin R. Bennett & Sanjay Sinha

Department of Pathology, Center for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA

Peter Hofsteen, Shiv Bhandari, Florian Weinberger, Alessandro Bertero, Andrea Leonard, Amy Martinson & Charles E. Murry

Department of Bioengineering, University of Washington, Seattle, WA, USA

Division of Cardiology, Department of Medicine, University of Washington, Seattle, WA, USA

J.B. was the principal experimentalist and was responsible for study design and conceptualization, data acquisition and interpretation, production of figures and manuscript writing. L.P.O.

performed tissue culture, 3D-EHT generation, force measurement and assisted during surgery. M.C. performed tissue culture and 3D-EHT generation. H.D. performed force measurement and RT–qPCR.

P.H. was responsible for preparation of cell suspensions on the day of transplantation, necropsy, assisting during surgery and postoperative animal care. S.B. performed the casting of

3D-EHT and force measurement. L.G. performed tissue histology, immunofluorescence and sample preparation for RNAseq. N.L.N. performed the bioinformatics analysis. D.I. contributed conceptual

ideas and critically revised the manuscript for important intellectual content. F.S. critically revised the manuscript for important intellectual content. F.W. performed the functional

analysis of echocardiographs. A.B. performed the gene expression analysis. A.L. was responsible for experimental guidance and force measurement data analysis. W.G.B. was responsible for data

interpretation and logistics. A.M. was responsible for animal surgery and logistics. N.F. was responsible for processing of histologic tissue and preparation of slides. M.R. was responsible

for force measurement equipment. M.R.B. critically revised the manuscript for important intellectual content. C.E.M. was responsible for study design and conception, obtaining research

funding, study supervision, and editing and final approval of the manuscript. S.S. was responsible for study design and conception, obtaining research funding, study supervision,

interpretation of data, and editing and final approval of the manuscript.

A patent has been filed on the cardiac application of epicardial cells, on which C.E.M., S.S. and J.B. are coinventors (WO2018170280A1). C.E.M. is a scientific founder and equity holder in

Cytocardia.

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