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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
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).
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).
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).
Anderson, D. et al. Transgenic enrichment of cardiomyocytes from human embryonic stem cells. Mol. Ther. 15, 2027–2036 (2007).
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).
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).
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).
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).
Huber, I. et al. Identification and selection of cardiomyocytes during human embryonic stem cell differentiation. FASEB J. 21, 2551–2563 (2007).
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).
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).
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).
Monici, M. Cell and tissue autofluorescence research and diagnostic applications. Biotechnol. Annu. Rev. 11, 227–256 (2005).
Gropp, M. & Reubinoff, B. Lentiviral vector-mediated gene delivery into human embryonic stem cells. Methods Enzymol. 420, 64–81 (2006).
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).
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).
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).
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).
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).
Hattan, N. et al. Purified cardiomyocytes from bone marrow mesenchymal stem cells produce stable intracardiac grafts in mice. Cardiovasc. Res. 65, 334–344 (2005).
Jakobs, S. High resolution imaging of live mitochondria. Biochim. Biophys. Acta. 1763, 561–575 (2006).
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.
Author information
Authors and Affiliations
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.
Corresponding author
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
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). https://doi.org/10.1038/nmeth.1403
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmeth.1403
This article is cited by
-
Telocytes response to cardiac growth induced by resistance exercise training and endurance exercise training in adult male rats
The Journal of Physiological Sciences (2023)
-
Recent advances in pluripotent stem cell-derived cardiac organoids and heart-on-chip applications for studying anti-cancer drug-induced cardiotoxicity
Cell Biology and Toxicology (2023)
-
The Role of Large Animal Models in Cardiac Regeneration Research Using Human Pluripotent Stem Cell-Derived Cardiomyocytes
Current Cardiology Reports (2023)
-
Glucocorticoid receptor antagonization propels endogenous cardiomyocyte proliferation and cardiac regeneration
Nature Cardiovascular Research (2022)
-
Human-induced pluripotent stem cell-derived cardiomyocytes, 3D cardiac structures, and heart-on-a-chip as tools for drug research
Pflügers Archiv - European Journal of Physiology (2021)