Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Nongenetic method for purifying stem cell–derived cardiomyocytes


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: Mitochondrial dyes for cardiomyocyte purification.
Figure 2: Purification of cardiomyocytes from embryonic heart and whole embryo.
Figure 3: Purification of mouse ESC– and mouse iPSC–derived cardiomyocytes using TMRM.
Figure 4: Purification of PSC-derived cardiomyocytes in human and marmoset.
Figure 5: Transplantation of purified mouse ESC–derived cardiomyocytes.


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

    Article  CAS  Google Scholar 

  2. Nemir, M., Croquelois, A., Pedrazzini, T. & Radtke, F. Induction of cardiogenesis in embryonic stem cells via downregulation of Notch1 signaling. Circ. Res. 98, 1471–1478 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Kolossov, E. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Gassanov, N., Er, F., Zagidullin, N. & Hoppe, U.C. Endothelin induces differentiation of ANP-EGFP expressing embryonic stem cells towards a pacemaker phenotype. FASEB J. 18, 1710–1712 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Klug, M.G., Soonpaa, M.H., Koh, G.Y. & Field, L.J. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. J. Clin. Invest. 98, 216–224 (1996).

    Article  CAS  Google Scholar 

  11. Laflamme, M.A. 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).

    Article  CAS  Google Scholar 

  12. Xu, C., Police, S., Hassanipour, M. & Gold, J.D. Cardiac bodies: a novel culture method for enrichment of cardiomyocytes derived from human embryonic stem cells. Stem Cells Dev. 15, 631–639 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Tsukahara, T. 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).

    Article  CAS  Google Scholar 

  16. Recchia, A. 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).

    Article  CAS  Google Scholar 

  17. Woods, N.B. 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).

    Article  CAS  Google Scholar 

  18. van Laake, L.W. 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).

    Article  Google Scholar 

  19. Reinecke, H., Zhang, M., Bartosek, T. & Murry, C.E. Survival, integration, and differentiation of cardiomyocyte grafts: a study in normal and injured rat hearts. Circulation 100, 193–202 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references


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.

Author information

Authors and Affiliations



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.

Corresponding author

Correspondence to Keiichi Fukuda.

Ethics declarations

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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–18, Supplementary Table 1 (PDF 7747 kb)

Supplementary Video 1

Whole embryo (E11.5) treated with TMRM. (MPG 550 kb)

Supplementary Video 2

Purified mouse ESC-cardiomyocyte aggregates. (MPG 270 kb)

Supplementary Video 3

Purified human ESC-cardiomyocyte aggregates. (MPG 376 kb)

Supplementary Video 4

Purified human ESC-cardiomyocyte aggregates cultured for 40 d in non-serum medium supplemented with or without bFGF. (MPG 296 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hattori, F., Chen, H., Yamashita, H. et al. Nongenetic method for purifying stem cell–derived cardiomyocytes. Nat Methods 7, 61–66 (2010).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing