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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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.
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.
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)
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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