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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  1. 1.

    et al. Transient inhibition of BMP signaling by Noggin induces cardiomyocyte differentiation of mouse embryonic stem cells. Nat. Biotechnol. 23, 607–611 (2005).

  2. 2.

    , , & Induction of cardiogenesis in embryonic stem cells via downregulation of Notch1 signaling. Circ. Res. 98, 1471–1478 (2006).

  3. 3.

    et al. Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 107, 2733–2740 (2003).

  4. 4.

    et al. Transgenic enrichment of cardiomyocytes from human embryonic stem cells. Mol. Ther. 15, 2027–2036 (2007).

  5. 5.

    et al. Engraftment of engineered ES cell-derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium. J. Exp. Med. 203, 2315–2327 (2006).

  6. 6.

    et al. Chamber-specific differentiation of Nkx2.5-positive cardiac precursor cells from murine embryonic stem cells. FASEB J. 17, 740–742 (2003).

  7. 7.

    et al. Cardiomyocytes purified from differentiated embryonic stem cells exhibit characteristics of early chamber myocardium. J. Mol. Cell. Cardiol. 35, 1461–1472 (2003).

  8. 8.

    , , & Endothelin induces differentiation of ANP-EGFP expressing embryonic stem cells towards a pacemaker phenotype. FASEB J. 18, 1710–1712 (2004).

  9. 9.

    et al. Identification and selection of cardiomyocytes during human embryonic stem cell differentiation. FASEB J. 21, 2551–2563 (2007).

  10. 10.

    , , & Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. J. Clin. Invest. 98, 216–224 (1996).

  11. 11.

    et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat. Biotechnol. 25, 1015–1024 (2007).

  12. 12.

    , , & Cardiac bodies: a novel culture method for enrichment of cardiomyocytes derived from human embryonic stem cells. Stem Cells Dev. 15, 631–639 (2006).

  13. 13.

    Cell and tissue autofluorescence research and diagnostic applications. Biotechnol. Annu. Rev. 11, 227–256 (2005).

  14. 14.

    & Lentiviral vector-mediated gene delivery into human embryonic stem cells. Methods Enzymol. 420, 64–81 (2006).

  15. 15.

    et al. Murine leukemia virus vector integration favors promoter regions and regional hot spots in a human T-cell line. Biochem. Biophys. Res. Commun. 345, 1099–1107 (2006).

  16. 16.

    et al. Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells. Proc. Natl. Acad. Sci. USA 103, 1457–1462 (2006).

  17. 17.

    et al. Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis. Blood 101, 1284–1289 (2003).

  18. 18.

    et al. Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction. Stem Cell Rev. 1, 9–24 (2007).

  19. 19.

    , , & Survival, integration, and differentiation of cardiomyocyte grafts: a study in normal and injured rat hearts. Circulation 100, 193–202 (1999).

  20. 20.

    et al. Purified cardiomyocytes from bone marrow mesenchymal stem cells produce stable intracardiac grafts in mice. Cardiovasc. Res. 65, 334–344 (2005).

  21. 21.

    High resolution imaging of live mitochondria. Biochim. Biophys. Acta. 1763, 561–575 (2006).

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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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  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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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

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–18, Supplementary Table 1


  1. 1.

    Supplementary Video 1

    Whole embryo (E11.5) treated with TMRM.

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    Supplementary Video 2

    Purified mouse ESC-cardiomyocyte aggregates.

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    Supplementary Video 3

    Purified human ESC-cardiomyocyte aggregates.

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    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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