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Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions

Abstract

The protocol described here efficiently directs human pluripotent stem cells (hPSCs) to functional cardiomyocytes in a completely defined, growth factor– and serum-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate temporal application of a glycogen synthase kinase 3 (GSK3) inhibitor combined with the expression of β-catenin shRNA or a chemical Wnt inhibitor is sufficient to produce a high yield (0.8–1.3 million cardiomyocytes per cm2) of virtually pure (80–98%) functional cardiomyocytes in 14 d from multiple hPSC lines without cell sorting or selection. Qualitative (immunostaining) and quantitative (flow cytometry) characterization of differentiated cells is described to assess the expression of cardiac transcription factors and myofilament proteins. Flow cytometry of BrdU incorporation or Ki67 expression in conjunction with cardiac sarcomere myosin protein expression can be used to determine the proliferative capacity of hPSC-derived cardiomyocytes. Functional human cardiomyocytes differentiated via these protocols may constitute a potential cell source for heart disease modeling, drug screening and cell-based therapeutic applications.

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Figure 1: Analysis of undifferentiated hPSCs.
Figure 2: Analysis of cardiomyocyte progenitor marker expression.
Figure 3: Schematic of the protocol for the differentiation of cardiomyocytes from hPSCs with small-molecule modulators of canonical Wnt signaling (Step 12B).
Figure 4: Structural characterization of cardiomyocytes generated from hPSCs via small-molecule modulation of Wnt signaling.
Figure 5: Quantitative analysis of cardiomyocytes differentiated from hPSCs via small-molecule modulation of Wnt signaling.

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Acknowledgements

This study was supported by US National Institutes of Health grant nos. R01 EB007534 and U01 HL099773 and National Science Foundation grant no. EFRI 0735903.

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Authors

Contributions

X.L. designed and performed experiments, analyzed the data and wrote the paper. J.Z., S.M.A., K.Z., L.B.H., X.B. and C.H. contributed to the development of this protocol. T.J.K. and S.P.P. supervised the project, and wrote and approved the final paper.

Corresponding author

Correspondence to Sean P Palecek.

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Competing interests

T.J.K. is a founder and consultant for Cellular Dynamics International, a company that uses human stem cells for drug testing. All the other authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Spontaneous contraction of cardiomyocytes differentiated using Step 12A (GiSB method). 19-9-11 inducible β-catenin shRNA cells were treated with 12 μM CHIR99021 at day 0 and 2 μg/ml dox at 36 hr. Movie S1 shows day 15 cardiomyocytes. (WMV 4866 kb)

Supplementary Video 2

Spontaneous contraction of cardiomyocytes differentiated using Step 12A (GiSB method). 19-9-11 inducible β-catenin shRNA cells were differentiated as described in Movie S1. Movie S2 shows day 180 cardiomyocytes. (WMV 4943 kb)

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Lian, X., Zhang, J., Azarin, S. et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nat Protoc 8, 162–175 (2013). https://doi.org/10.1038/nprot.2012.150

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