Abstract

Several applications of pluripotent stem cell (PSC)-derived cardiomyocytes require elimination of undifferentiated cells. A major limitation for cardiomyocyte purification is the lack of easy and specific cell marking techniques. We found that a fluorescent dye that labels mitochondria, tetramethylrhodamine methyl ester perchlorate, could be used to selectively mark embryonic and neonatal rat cardiomyocytes, as well as mouse, marmoset and human PSC-derived cardiomyocytes, and that the cells could subsequently be enriched (>99% purity) by fluorescence-activated cell sorting. Purified cardiomyocytes transplanted into testes did not induce teratoma formation. Moreover, aggregate formation of PSC-derived cardiomyocytes through homophilic cell-cell adhesion improved their survival in the immunodeficient mouse heart. Our approaches will aid in the future success of using PSC-derived cardiomyocytes for basic and clinical applications.

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Acknowledgements

Human ESCs were a gift of N. Nakatsuji at the Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University. Human and mouse iPSCs were a gift of S. Yamanaka at the Center for iPS Cell Research and Application, Institute for Integrated Cell-Material Sciences, Kyoto University. Mouse ESCs were a gift of H. Niwa at the Laboratory of Pluripotent Cell Studies, RIKEN Center for Developmental Biology. This study was supported in part by research grants from the Ministry of Education, Science and Culture, Japan, and by the Program for Promotion of Fundamental Studies in Health Science of the National Institute of Biomedical Innovation.

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Affiliations

  1. Department of Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine, Tokyo, Japan.

    • Fumiyuki Hattori
    • , Hao Chen
    • , Hiromi Yamashita
    • , Shugo Tohyama
    • , Yu-suke Satoh
    • , Shinsuke Yuasa
    • , Weizhen Li
    • , Hiroyuki Yamakawa
    • , Tomofumi Tanaka
    • , Takeshi Onitsuka
    • , Kenichiro Shimoji
    • , Yohei Ohno
    • , Toru Egashira
    • , Ruri Kaneda
    • , Mitsushige Murata
    • , Motoaki Sano
    • , Shinji Makino
    •  & Keiichi Fukuda
  2. Asubio Pharma Co., Ltd., Osaka, Japan.

    • Fumiyuki Hattori
    • , Tomofumi Tanaka
    •  & Shinzo Oikawa
  3. Division of Cardiology, Department of Medicine, Keio University School of Medicine, Tokyo, Japan.

    • Hao Chen
    • , Shugo Tohyama
    • , Hiroyuki Yamakawa
    • , Takeshi Onitsuka
    • , Kenichiro Shimoji
    • , Yohei Ohno
    • , Toru Egashira
    •  & Mitsushige Murata
  4. Division of Basic Biological Sciences, Faculty of Pharmacy, Keio University, Tokyo, Japan.

    • Yu-suke Satoh
    •  & Takeshi Suzuki
  5. Department of Bioscience, National Cardiovascular Center Research Institute, Osaka, Japan.

    • Kyoko Hidaka
    •  & Takayuki Morisaki
  6. Laboratory of Applied Developmental Biology, Marmoset Research Department, Central Institute for Experimental Animals, Kanagawa, Japan.

    • Erika Sasaki

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Contributions

F.H. designed the whole study. F.H. performed most experiments and wrote the manuscript. H.C. participated in cell-sorting experiments and prepared cells. H.Yamashita participated in cell-sorting experiments, PCR experiments, immunofluorescent staining, animal experiments and preparing cells. S.T., Y.S., W.L., T.T., T.O., K.S., Y.O. and T.E. participated in cell preparations. H.Yamakawa and M.M. participated in heart perfusion experiments. K.H. and T.M. provided the Nkx2.5 knock-in ESCs. S.Y., M.M., R.K., M.S., S.M. and S.O. provided advice. E.S. provided cmESCs. T.S. supervised Y.S. K.F. provided advice, obtained the budget and supervised the project.

Competing interests

F.H. and T.T. are employees of Asubio Pharma Co., Ltd. The study was partly supported by grant from Asubio Pharma Co., Ltd.

Corresponding author

Correspondence to Keiichi Fukuda.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–18, Supplementary Table 1

Videos

  1. 1.

    Supplementary Video 1

    Whole embryo (E11.5) treated with TMRM.

  2. 2.

    Supplementary Video 2

    Purified mouse ESC-cardiomyocyte aggregates.

  3. 3.

    Supplementary Video 3

    Purified human ESC-cardiomyocyte aggregates.

  4. 4.

    Supplementary Video 4

    Purified human ESC-cardiomyocyte aggregates cultured for 40 d in non-serum medium supplemented with or without bFGF.

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DOI

https://doi.org/10.1038/nmeth.1403

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