Abstract

Induced pluripotent stem cells (iPSCs) constitute a potential source of autologous patient-specific cardiomyocytes for cardiac repair, providing a major benefit over other sources of cells in terms of immune rejection. However, autologous transplantation has substantial challenges related to manufacturing and regulation. Although major histocompatibility complex (MHC)-matched allogeneic transplantation is a promising alternative strategy1, few immunological studies have been carried out with iPSCs. Here we describe an allogeneic transplantation model established using the cynomolgus monkey (Macaca fascicularis), the MHC structure of which is identical to that of humans. Fibroblast-derived iPSCs were generated from a MHC haplotype (HT4) homozygous animal and subsequently differentiated into cardiomyocytes (iPSC-CMs). Five HT4 heterozygous monkeys were subjected to myocardial infarction followed by direct intra-myocardial injection of iPSC-CMs. The grafted cardiomyocytes survived for 12 weeks with no evidence of immune rejection in monkeys treated with clinically relevant doses of methylprednisolone and tacrolimus, and showed electrical coupling with host cardiomyocytes as assessed by use of the fluorescent calcium indicator G-CaMP7.09. Additionally, transplantation of the iPSC-CMs improved cardiac contractile function at 4 and 12 weeks after transplantation; however, the incidence of ventricular tachycardia was transiently, but significantly, increased when compared to vehicle-treated controls. Collectively, our data demonstrate that allogeneic iPSC-CM transplantation is sufficient to regenerate the infarcted non-human primate heart; however, further research to control post-transplant arrhythmias is necessary.

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Acknowledgements

We thank Y. Ichihara, N. Ishimine, Y. Karatsu, the Rigaku Corporation, Brainvision Inc. and the Keyence Corporation for assistance with the experiments and Astellas Pharma Inc. for the gift of tacrolimus. We also appreciate the scientific advice of M. A. Laflamme, J. Chong and N. Saito. This work was supported by research grants (to Y.S.) from the Japan Society for the Promotion of Science KAKENHI (grant no. 26293182), Japan Agency for Medical Research and Development, Takeda Science Foundation, Astellas Foundation for Research on Metabolic Disorders, Mochida Memorial Foundation for Medical and Pharmaceutical Research, and Japan Heart Association. The experiments to generate the G-CaMP7.09 plasmid were supported by grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to J.N. (grant no. 26115504) and M.O. (grant no. 25116504) and a grant from the Regional Innovation Cluster Program (City Area Type, Central Saitama Area) to J.N.

Author information

Author notes

    • Yuji Shiba
    •  & Toshihito Gomibuchi

    These authors contributed equally to this work.

Affiliations

  1. Institute for Biomedical Sciences, Shinshu University, Matsumoto 390-8621, Japan

    • Yuji Shiba
  2. Department of Cardiovascular Medicine, Shinshu University School of Medicine, Matsumoto 390-8621, Japan

    • Yuji Shiba
    •  & Uichi Ikeda
  3. Department of Cardiovascular Surgery, Shinshu University School of Medicine, Matsumoto 390-8621, Japan

    • Toshihito Gomibuchi
    • , Tatsuichiro Seto
    • , Yuko Wada
    • , Hajime Ichimura
    • , Yuki Tanaka
    • , Tatsuki Ogasawara
    •  & Kenji Okada
  4. Department of Pediatrics, Shinshu University School of Medicine, Matsumoto 390-8621, Japan

    • Naoko Shiba
  5. Ina Research Inc., Ina 399-4501, Japan

    • Kengo Sakamoto
    •  & Daisuke Ido
  6. Department of Molecular Life Science, Tokai University School of Medicine, Isehara 259-1193, Japan

    • Takashi Shiina
  7. Brain Science Institute, Saitama University, Saitama 338-8570, Japan

    • Masamichi Ohkura
    •  & Junichi Nakai
  8. Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan

    • Masamichi Ohkura
    •  & Junichi Nakai
  9. Chromosome Engineering Research Center, Tottori University, Yonago 683-8503, Japan

    • Narumi Uno
    • , Yasuhiro Kazuki
    •  & Mitsuo Oshimura
  10. Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8501, Japan

    • Itsunari Minami

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Contributions

Y.S. designed the study. Y.S., T.G., T.Se., Y.W., H.I., Y.T., K.S. and D.I. performed all animal procedures. T.O., N.S. and Y.S. performed histological analysis. K.S. and D.I. analysed Holter ECGs. N.U., Y.K., and M.Os. performed karyotype analysis of iPSCs. T.Sh. analysed RNA sequences of cynomolgus MHC. K.O. and U.I. analysed all other data and provided administrative assistance. M.Oh. and J.N. generated the G-CaMP7.09 plasmid. In vitro fluorescent imaging studies were performed by I.M. The manuscript was written by Y.S., T.Sh., M.Oh., I.M. and N.U.

Competing interests

K.S. and D.I. are employees of Ina Research, where all animal procedures in this study were performed. The remaining authors have no competing interests to declare.

Corresponding author

Correspondence to Yuji Shiba.

Reviewer Information Nature thanks T. Braun, K. Fukuda, T. Kamp and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains gel source data for Extended Data Figures 2 and 4.

Videos

  1. 1.

    In vitro fluorescent imaging of G-CaMP7.09-expressing cynomolgus iPSC-CMs

    Monolayer-cultured cardiomyocytes exhibit robust fluorescent flashes in synchrony with their contraction.

  2. 2.

    In vitro experiments with G-CaMP7.09-expressing iPSC-CMs by using nifedipine, ryanodine, and caffeine

    Representative video of G-CaMP7.09 fluorescent transients and contraction of cardiomyocytes before and after treatment with the L-type calcium-channel blocker nifedipine, the ryanodine receptor blocker ryanodine, and the ryanodine receptor activator caffeine.

  3. 3.

    In vitro G-CaMP7.09 transients sustained after cessation of spontaneous contraction of iPSC-CMs by BDM.

    Treatment with 40 mM BDM resulted in cessation of spontaneous contraction of iPSC-CMs, but G-CaMP7.09 fluorescent transients were sustained for a few minutes.

  4. 4.

    Myocardial ischemia/reperfusion model in cynomolgus monkey

    The myocardial infarction model was produced by 3 h of ischemia followed by reperfusion using polyethylene tubing 2 weeks before transplantation.

  5. 5.

    Intravital imaging of G-CaMP7.09-expressing iPSC-CMs in cynomolgus heart

    G-CaMP7.09⁺ iPSC-CMs were transplanted into infarcted cynomolgus hearts. The hearts were excised and mechanically arrested ex vivo by perfusion with BDM on a Langendorff apparatus at 12 weeks post-transplantation. All graft regions exhibited cyclic changes in fluorescent intensity that occurred synchronously in a 1:1 relationship with the host ECG when the heart beat spontaneously or was electrically paced at rates from 3 to 5 Hz. Note that some, but not all hearts could be paced up to 5 Hz.

  6. 6.

    Cardiac contractile function assessed by mCT at 4 weeks post-transplantation

    Cardiac function was assessed by mCT pre- and post-transplantation. The first and second segments of the video show left ventricular contractions of the short axis at the base and apex, respectively. Note that while contraction at the base is similar, that at the apex in iPSC-CM recipients looks better than that in PSC-vehicle recipients. The last segment represents a long-axis view of the left ventricle.

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https://doi.org/10.1038/nature19815

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