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Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro

Nature Protocols volume 8, pages 1204–1215 (2013)Cite this article

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Abstract

Cardiac fibroblasts can be reprogrammed to cardiomyocyte-like cells by the introduction of three transcription factors: Gata4, Mef2c and Tbx5 (collectively referred to here as GMT). Resident cardiac fibroblasts can be converted in vivo into induced cardiomyocyte-like cells (iCMs) that closely resemble endogenous cardiomyocytes and electrically integrate with the host myocardium. In contrast, in vitro reprogramming yields many partially reprogrammed iCMs, with a few that reprogram fully into contracting myocytes (∼3 out of 10,000 GMT-transduced cells). iCMs can be observed as early as 3 d after viral infection, and they continue to mature over 2 months before beating is observed. Despite the success of multiple groups, the inefficiency of in vitro reprogramming has made it challenging for others. However, given the advantages of in vitro iCMs for performing mechanistic studies and, if refined, for testing drugs or small molecules for personalized medicine and modeling cardiac disease in a dish, it is important to standardize the protocol to improve reproducibility and enhance the technology further. Here we describe a detailed step-by-step protocol for in vitro cardiac reprogramming using retroviruses encoding GMT.

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Figure 1: Timeline of the cardiac reprogramming process.
Figure 2: The range of sarcomere structures observed in in vitro iCMs.

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Acknowledgements

We thank B. Taylor for editorial assistance. D.S. was supported by grants from the US National Institutes of Health (NIH), National Heart, Lung and Blood Institute (NHLBI) (U01 HL100406), the California Institute for Regenerative Medicine, the William Younger Family Foundation, the L.K. Whittier Foundation and the Eugene Roddenberry Foundation. This work was supported by a NIH National Center for Research Resources (NCRR) grant (C06 RR018928) to the Gladstone Institute.

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Author notes

  1. Li Qian

    Present address: Present address: Department of Pathology and Laboratory Medicine, McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.,

Authors and Affiliations

  1. Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA

    Li Qian, Emily C Berry, Ji-dong Fu & Deepak Srivastava

  2. Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, California, USA

    Li Qian, Emily C Berry, Ji-dong Fu & Deepak Srivastava

  3. Department of Pediatrics, University of California, San Francisco, California, USA

    Li Qian, Emily C Berry, Ji-dong Fu & Deepak Srivastava

  4. Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA

    Li Qian, Emily C Berry, Ji-dong Fu & Deepak Srivastava

  5. Department of Clinical and Molecular Cardiovascular Research, School of Medicine, Keio University, Tokyo, Japan

    Masaki Ieda

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Contributions

All authors contributed to the writing and editing of this paper, and to the refinement of the protocol.

Corresponding author

Correspondence to Deepak Srivastava.

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Competing interests

D.S. is a cofounder and scientific advisory board member of iPierian, Inc., and also a scientific advisory board member of RegeneRx Pharmaceuticals.

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Qian, L., Berry, E., Fu, Jd. et al. Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro. Nat Protoc 8, 1204–1215 (2013). https://doi.org/10.1038/nprot.2013.067

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