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Abstract

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure1 by providing human cardiomyocytes to support heart regeneration2. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment3,4,5,6,7. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models7, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

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Change history

  • 11 June 2014

    Savannah M. Cook has been added to the author list and an incorrect grant number has been updated.

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Acknowledgements

We thank S. Dupras, B. Brown, D. Rocha, E. Wilson, C. English, J. Randolph-Habecker and T. Goodpaster for assistance with these experiments. This work was supported by National Institutes of Health grants P01HL094374, R01HL084642, U01HL100405 and P01GM081619 and an Institute of Translational Health Sciences/Primate Center Ignition Award. J.J.H.C. was supported by National Health and Medical Research Council of Australia Overseas Training and Australian-American Fulbright Commission Fellowships. X.Y. is supported by an American Heart Association post-doctoral scholarship 12POST11940060. J.J.W. is supported by an American Heart Association post-doctoral scholarship 12POST9330030. H.-P.K. is a Markey Molecular Medicine investigator and the recipient of the Jose Carreras/E.D. Thomas Chair for Cancer Research.

Author information

Author notes

    • James J. H. Chong

    Present address: University of Sydney School of Medicine, Sydney, New South Wales 2006, Australia and Westmead Millennium Institute and Westmead Hospital, Westmead, New South Wales 2145, Australia.

Affiliations

  1. Center for Cardiovascular Biology, University of Washington, Seattle, Washington 98109, USA

    • James J. H. Chong
    • , Xiulan Yang
    • , Elina Minami
    • , Yen-Wen Liu
    • , Jill J. Weyers
    • , William M. Mahoney
    • , Benjamin Van Biber
    • , Nathan J. Palpant
    • , Jay A. Gantz
    • , James A. Fugate
    • , Veronica Muskheli
    • , Lil Pabon
    • , Hans Reinecke
    • , Michael A. Laflamme
    •  & Charles E. Murry
  2. Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington 98109, USA

    • James J. H. Chong
    • , Xiulan Yang
    • , Elina Minami
    • , Yen-Wen Liu
    • , Jill J. Weyers
    • , William M. Mahoney
    • , Benjamin Van Biber
    • , Nathan J. Palpant
    • , Jay A. Gantz
    • , James A. Fugate
    • , Veronica Muskheli
    • , Lil Pabon
    • , Hans Reinecke
    • , Michael A. Laflamme
    •  & Charles E. Murry
  3. Department of Cardiology Westmead Hospital, Westmead, New South Wales 2145, Australia

    • James J. H. Chong
  4. School of Medicine, University of Sydney, Sydney, New South Wales 2006, Australia

    • James J. H. Chong
  5. Department of Pathology, University of Washington, Seattle, Washington 98195, USA

    • James J. H. Chong
    • , Xiulan Yang
    • , Elina Minami
    • , Yen-Wen Liu
    • , Jill J. Weyers
    • , William M. Mahoney
    • , Benjamin Van Biber
    • , Nathan J. Palpant
    • , Jay A. Gantz
    • , James A. Fugate
    • , Veronica Muskheli
    • , Lil Pabon
    • , Hans Reinecke
    • , Hans-Peter Kiem
    • , Michael A. Laflamme
    •  & Charles E. Murry
  6. Department of Medicine/Cardiology, University of Washington, Seattle, Washington 98195, USA

    • Creighton W. Don
    • , Elina Minami
    • , Edward A. Gill
    •  & Charles E. Murry
  7. Department of Comparative Medicine, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington 98109, USA

    • Savannah M. Cook
  8. Department of Bioengineering, University of Washington, Seattle, Washington 98195, USA

    • Jay A. Gantz
    •  & Charles E. Murry
  9. Washington National Primate Research Center, University of Washington, Seattle, Washington 98195, USA

    • G. Michael Gough
    • , Keith W. Vogel
    • , Cliff A. Astley
    • , Charlotte E. Hotchkiss
    •  & Audrey Baldessari
  10. Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA

    • Veronica Nelson
    •  & Hans-Peter Kiem

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Contributions

J.J.H.C., X.Y., C.W.D., E.M., L.P., H.R., H.-P.K., M.A.L. and C.E.M. designed the study. J.J.H.C. and E.M. performed mouse transplantation experiments. J.J.H.C. developed telemetry and analysed recordings. J.J.H.C., C.W.D., C.E.M., G.M.G., K.W.V., C.A.A., E.M. and V.N. performed macaque surgery and procedures. J.J.H.C., E.M., E.A.G. and C.E.H. performed echocardiography and E.M., E.G. and Y.-W.L. performed analysis. A.B. performed necropsies and non-cardiac histopathology. GCaMP3 visualization experiments were carried out and analysed by X.Y. and J.J.H.C. GCaMP3-expressing human ES cells were created by N.J.P., J.A.G. and B.V.B. hESC-CM production was by J.J.H.C., B.V.B., S.M.C., J.A.F. and M.A.L. Microcomputed tomography experiments were performed by J.J.W. and W.M.M. Jr. Immunohistochemistry was performed and analysed by J.J.H.C., V.M. and Y.-W.L. Figures were created by J.J.H.C. with assistance from X.Y., J.J.W., Y.-W.L., N.J.P. and V.M. The manuscript was written principally by J.J.H.C. and C.E.M.

Competing interests

C.E.M. and M.A.L. are equity holders in BEAT BioTherapeutics.

Corresponding author

Correspondence to Charles E. Murry.

Extended data

Supplementary information

Videos

  1. 1.

    GCaMP3-expressing RUES2hESC-CMs exhibit robust fluorescence in vitro

    Representative GCaMP3-expressing hESC-CMs were imaged using an inverted stereo-microscope with halogen and laser light sources. The video file shows the cardiomyocytes first by bright-field microscopy alone and then later with the green fluorescent signal. Note that the cells exhibit robust fluorescent transients with each contractile cycle.

  2. 2.

    GCaMP3 expressing H7hESC-CMs exhibit robust fluorescence in vitro

    Similar to Video S1, representative H7GCAMP3-hESC-CMs are shown first in bright field then with the green fluorescent signal. Note robust fluorescence with each cardiac cycle.

  3. 3.

    Blood vessels extend from the host coronary network into the graft

    3-dimensional rendering of microcomputed tomography (same heart shown in Extended Data Fig. 9) animated with rotation to enable better visualization of the vessels within the graft. Arteries feeding the graft are red, other vessels are gray in the uninjured cardiac tissue, or white within the graft.

  4. 4.

    Modified Langendorff apparatus and GCAMP3 fluorescence imaging equipment

    A harvested macaque heart is seen spontaneously beating after retrograde perfusion of with modified Tyrode’s solution. Electrodes are attached to the heart for electrocardiogram recording and analysis. Placement of the heart under a stereomicroscope with fluorescence imaging capabilities allows 2 visualization and analysis of epicardial fluorescence emitted from GCAMP3 expressing hESC-CM grafts.

  5. 5.

    Intravital imaging of the infarcted macaque heart with GCaMP3-expressing hESC-CM grafts

    A representative macaque heart with numerous GCaMP3-expressing hESC-CM graft regions, harvested at 28 days posttransplantation, mounted ex vivo on a Langendorff apparatus and mechanically arrested with 2,3-butanedione monoxime. This low power video (0.8x) shows several regions of GCaMP3-positive graft (distributed through infarct region and border zones) visible from the epicardial surface. All regions exhibited cyclic fluorescent transients that occurred in synchrony with QRS complexes of the host ECG, indicating 1:1 host-graft coupling. This video corresponds to Figure 2 b in main text.

  6. 6.

    Higher power of intravital imaging of the infarcted macaque heart with GCaMP3-expressing hESC-CM graft

    This video shows the same heart as video S3 at higher (2x) magnification and corresponds to Figure 2c-d in main text.

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DOI

https://doi.org/10.1038/nature13233

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