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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

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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Figure 1: Experimental protocol for in vivo and in vitro cardiomyocyte maturation.
Figure 2: Isl1+ cardiac progenitor cells.
Figure 3: Surgical transplantation of cardiac progenitor cells for in vivo maturation.
Figure 4: Isolation of mESC-CMs from injected rat hearts.
Figure 5: In vivo-matured mouse and human PSC cardiomyocytes.

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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.

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Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Chulan Kwon.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 FACS gating strategy

A. FACS gating strategy for the sorting of live Isl-1+ mouse CPCs. First plot gating for live cells, then second plot for single and then only for Isl1-1+ RFP cells. B. FACS gating strategy for the analysis of human CPCs. The cells were fixed and stained with a primary mouse anti-Islet-1 antibody and a secondary far red (647 nm emission wavelength) conjugated antibody. First plot gating for fixed cells, then single cells and then Isl-1+ Far Red CPCs.

Supplementary Figure 2 Teratoma formation (asterisk) after injection of unsorted mouse ESC-derived cells

Arrow points to the dissected part of the heart.

Supplementary information

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Supplementary Figures 1 and 2. (PDF 411 kb)

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Cho, GS., Tampakakis, E., Andersen, P. et al. Use of a neonatal rat system as a bioincubator to generate adult-like mature cardiomyocytes from human and mouse pluripotent stem cells. Nat Protoc 12, 2097–2109 (2017). https://doi.org/10.1038/nprot.2017.089

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