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Use of a neonatal rat system as a bioincubator to generate adult-like mature cardiomyocytes from human and mouse pluripotent stem cells

Nature Protocols volume 12, pages 20972109 (2017) | Download Citation

Abstract

Pluripotent stem cells (PSCs), including induced PSCs, hold great potential for personalized disease modeling, drug testing and cell-based therapeutics. However, cells differentiated from PSCs remain immature in a dish, and thus there are serious caveats to their use in modeling adult-onset diseases such as cardiomyopathies and Alzheimer's disease. By taking advantage of knowledge gained about mammalian development and from bioinformatics analyses, we recently developed a neonatal rat system that enables maturation of PSC-derived cardiomyocytes into cardiomyocytes analogous to those seen in adult animals. Here we describe a detailed protocol that describes how to initiate the in vitro differentiation of mouse and human PSCs into cardiac progenitor cells, followed by intramyocardial delivery of the progenitor cells into neonatal rat hearts, in vivo incubation and analysis. The entire process takes 6 weeks, and the resulting cardiomyocytes can be analyzed for morphology, function and gene expression. The neonatal system provides a valuable tool for understanding the maturation and pathogenesis of adult human heart muscle cells, and this concept may be expanded to maturing other PSC-derived cell types, including those containing mutations that lead to the development of diseases in the adult.

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References

  1. 1.

    et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

  2. 2.

    , , & Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell 10, 16–28 (2012).

  3. 3.

    , , & Induced pluripotent stem cells: the new patient? Nat. Rev. Mol. Cell Biol. 13, 713–726 (2012).

  4. 4.

    , , , & Pluripotent stem cell models of human heart disease. Cold Spring Harb. Perspect. Med. 3 (2013).

  5. 5.

    , & Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes. Circ. Res. 114, 511–523 (2014).

  6. 6.

    & Pluripotent stem cells in regenerative medicine: challenges and recent progress. Nat. Rev. Genet. 15, 82–92 (2014).

  7. 7.

    et al. Transcriptional landscape of cardiomyocyte maturation. Cell Rep. 13, 1705–1716 (2015).

  8. 8.

    , & Regenerative medicine for the heart: perspectives on stem-cell therapy. Antioxid. Redox Signal. 21, 2018–2031 (2014).

  9. 9.

    , & Mortality from ischaemic heart disease by country, region, and age: statistics from World Health Organisation and United Nations. Int. J. Cardiol. 168, 934–945 (2013).

  10. 10.

    & iPS cells: a source of cardiac regeneration. J. Mol. Cell. Cardiol. 50, 327–332 (2011).

  11. 11.

    , & Concise review: maturation phases of human pluripotent stem cell-derived cardiomyocytes. Stem Cells 31, 829–837 (2013).

  12. 12.

    Ultrastructural and functional features of the developing mammalian heart: a brief overview. Reprod. Fertil. Dev. 7, 451–461 (1995).

  13. 13.

    , & Excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes. Front. Cell Dev. Biol. 3, 59 (2015).

  14. 14.

    Induced pluripotent stem cell-derived cardiomyocytes: boutique science or valuable arrhythmia model? Circ. Res. 112, 969–976 (2013).

  15. 15.

    et al. Biowire: a platform for maturation of human pluripotent stem cell-derived cardiomyocytes. Nat. Methods 10, 781–787 (2013).

  16. 16.

    et al. Combinatorial polymer matrices enhance in vitro maturation of human induced pluripotent stem cell-derived cardiomyocytes. Biomaterials 67, 52–64 (2015).

  17. 17.

    et al. Mechanical stress promotes maturation of human myocardium from pluripotent stem cell-derived progenitors. Stem Cells 33, 2148–2157 (2015).

  18. 18.

    et al. Glucocorticoids promote structural and functional maturation of foetal cardiomyocytes: a role for PGC-1alpha. Cell Death Differ. 22, 1106–1116 (2015).

  19. 19.

    et al. Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells. J. Mol. Cell. Cardiol. 72, 296–304 (2014).

  20. 20.

    , , & In vivo maturation of human induced pluripotent stem cell-derived cardiomyocytes in neonatal and adult rat hearts. Stem Cell Rep. 8, 278–289 (2017).

  21. 21.

    et al. Neonatal transplantation confers maturation of PSC-derived cardiomyocytes conducive to modeling cardiomyopathy. Cell Rep. 18, 571–582 (2017).

  22. 22.

    et al. Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature 433, 647–653 (2005).

  23. 23.

    et al. Islet-1 cells are cardiac progenitors present during the entire lifespan: from the embryonic stage to adulthood. Stem Cells Dev. 19, 1601–1615 (2010).

  24. 24.

    & Comparative gene expression analysis of mouse and human cardiac maturation. Genomics Proteomics Bioinformatics 14, 207–215 (2016).

  25. 25.

    et al. Hallmarks of Alzheimer's disease in stem-cell-derived human neurons transplanted into mouse brain. Neuron 93, 1066–1081 (2017).

  26. 26.

    et al. Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors. ELife 3, e02164 (2014).

  27. 27.

    et al. PDE5A suppression of acute beta-adrenergic activation requires modulation of myocyte beta-3 signaling coupled to PKG-mediated troponin I phosphorylation. Basic Res. Cardiol. 105, 337–347 (2010).

  28. 28.

    et al. Electrical stimulation systems for cardiac tissue engineering. Nat. Protoc. 4, 155–173 (2009).

  29. 29.

    et al. Single-cell RNA-Seq with waterfall reveals molecular cascades underlying adult neurogenesis. Cell Stem Cell 17, 360–372 (2015).

  30. 30.

    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).

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Acknowledgements

We thank Kwon laboratory members for critical reading and discussions. E.T. was supported by a Johns Hopkins School of Medicine Clinician Scientist Award. This work was supported by the Magic that Matters Fund and grants from the MSCRF (2015-MSCRFI-1622), NHLBI/NIH (R01HL111198) and NICHD/NIH (R01HD086026) to C.K.

Author information

Author notes

    • Gun-Sik Cho
    •  & Emmanouil Tampakakis

    These authors contributed equally to this work.

Affiliations

  1. Division of Cardiology, Department of Medicine, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Gun-Sik Cho
    • , Emmanouil Tampakakis
    • , Peter Andersen
    •  & Chulan Kwon
  2. Laboratory of Stem Cells, NEXEL, Seoul, Republic of Korea.

    • Gun-Sik Cho

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Contributions

G.-S.C. and C.K. designed the experiments. G.-S.C., E.T. and P.A. performed the experiments. G.-S.C., E.T. and P.A. analyzed the data. E.T. and P.A. created the figures. E.T., G.-S.C., P.A. and C.K. wrote the manuscript. All authors approved the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Chulan Kwon.

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https://doi.org/10.1038/nprot.2017.089

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