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Acknowledgements
This work was supported by US National Institutes of Health (NIH) grant R01EB007534. K.K.D. was funded in part by an NIH Chemistry Biology Interface Training Grant (NIGMS T32 GM008505).
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T.J.K. is a founder and consultant for Cellular Dynamics International, a company that uses human stem cells for drug testing.
Integrated supplementary information
Supplementary Figure 1 Optimization of chemically defined, albumin-free generation of cardiomyocytes from hPSCs.
(A-B) Purity of cardiomyocytes, determined by flow cytometric analysis of cTnT expression, differentiated from ES03 hESCs in albumin-free media with the indicated concentrations of CH (A) or IWP2 (B). (C) Purity of cardiomyocytes differentiated from ES03 hESCs in RPMI medium supplemented with indicated components.3F: sodium selenite, progesterone, putrescine; 3-Se: progesterone, putrescine; 3-Pu: sodium selenite, progesterone; 3-Pr: sodium selenite, putrescine; Se: sodium selenite; Pu: putrescine; Pr: progesterone (D) Purity of cardiomyocytes differentiated from ES03 hESCs in indicated basal media, B27-ins as a control. #P<0.005, indicated basal medium versus RPMI; Student's t test. Error bars indicate s.e.m. of three biological replicates.
Supplementary Figure 3 Purity of cardiomyocytes differentiated from hESCs in RPMI.
Purity of cardiomyocytes, determined by flow cytometric analysis of cTnT and sarcomeric myosin heavy chain (MF20 antibody) expression on day 15, differentiated from H1 hESCs in RPMI. This plot is representative of three independent experiments.
Supplementary Figure 4 Purity of cardiomyocytes differentiated from hESCs expanded in mTeSR1 or E8.
Purity of cardiomyocytes, determined by flow cytometric analysis of cTnT expression on day 15, differentiated from ES03 hESCs expanded in mTeSR1 or E8. These plots are representative of three independent experiments.
Supplementary Figure 5 Flow cytometry analysis of cardiac-specific markers.
(A-B) Purity of cardiomyocytes, determined by flow cytometric analysis of cTnT/MF20 (A) or α-actinin (B) expression, differentiated from ES03 hESCs in RPMI. (C) Purity of cardiomyocytes, determined by flow cytometric analysis of GFP expression, differentiated from ES03 Nkx2.5-GFP hESCs in RPMI. These plots are representative of three independent experiments.
Supplementary Figure 6 Calcium-handling properties of hPSC-derived cardiomyocytes.
(A-B) Cardiomyocytes differentiated from 19-9-11iPSCs in RPMI were treated with 10 μM Fluo-4 AM for 15 min and then Ca2+ transients were recorded with a temporal resolution at 10 frames per second. Box (white arrow head) in panel (A) denotes the site of analysis of absolute fluorescence normalized to initial fluorescence (F/F0) shown in panel (B).
Supplementary Figure 7 Cardiomyocytes differentiated from 19-9-11 iPSCs in RPMI were cultured in CMM for 60 d.
(A) Coimmunolabeling of α-actinin and connexin-43 in 19-9-11 iPSC-derived cardiomyocytes. (B) Immunolabeling of cTnT in 19-9-11iPSC-derived cardiomyocytes. These images are representative of three independent experiments. Scale bars, 50 μm.
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Supplementary Text and Figures
Supplementary Figures 1–7, Supplementary Tables 1–4 and Supplementary Methods (PDF 1118 kb)
Supplementary Video 1
Movie of day 240 H9 hESC-derived cardiomyocytes (MOV 5146 kb)
Supplementary Video 2
Movie of day 10 H9 hESC-derived cardiomyocytes (MOV 3963 kb)
Supplementary Video 3
Movie of day 10 H1 hESC-derived cardiomyocytes (MOV 10133 kb)
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Lian, X., Bao, X., Zilberter, M. et al. Chemically defined, albumin-free human cardiomyocyte generation. Nat Methods 12, 595–596 (2015). https://doi.org/10.1038/nmeth.3448
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DOI: https://doi.org/10.1038/nmeth.3448
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