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Chemically defined generation of human cardiomyocytes

Abstract

Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. Along with small molecule–based induction of differentiation, this protocol produced contractile sheets of up to 95% TNNT2+ cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell and was effective in 11 hiPSC lines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification.

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Figure 1: Chemically defined cardiac differentiation of hiPSCs.
Figure 2: Characterization and purification of cardiomyocytes produced by chemically defined differentiation.
Figure 3: Characterization of cardiomyocyte subtypes.
Figure 4: Electrophysiological characterization of cardiomyocytes.

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Acknowledgements

We thank J. Odegaard for analysis of teratoma slides, and K.R. Boheler and R.L. Gundry for their insightful comments on this manuscript. This work was supported by the American Heart Association Postdoctoral Fellowship grant 12POST12050254, Beginning Grant-in-Aid 14BGIA20480329 and US National Institutes of Health K99 HL121177 to P.W.B., and American Heart Association Established Investigator Award 14420025, Foundation Leducq, the National Institutes of Health U01 HL099776, R01 HL113006 and R24 HL117756, and the California Institute for Regenerative Medicine TR3-05556 and DR2-05394 to J.C.W.

Author information

Affiliations

Authors

Contributions

P.W.B. conceived, performed and interpreted the experiments and wrote the manuscript; E.M. performed cardiomyocyte immunofluorescence, single-cell RT-PCR and electrophysiology data assessment; P.S., Z.C.L. and O.J.A. performed electrophysiology experiments and assessed data; S.D. provided 'CoMiP' reprogrammed cells; B.H. performed the teratoma assay; J.M.C., A.D.E., F.L., N.M.M. and J.R.P. tested differentiation; B.C. and J.D.G. provided experimental advice; and J.C.W. provided experimental advice, wrote the manuscript and provided funding support.

Corresponding authors

Correspondence to Paul W Burridge or Joseph C Wu.

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Competing interests

J.C.W. is a co-founder of Stem Cell Theranostics.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Tables 1–6 (PDF 12731 kb)

Supplementary Video 1

hiPSCs differentiated using CDM3 at day 9, matches Supplementary Figure 5. (MOV 3610 kb)

Supplementary Video 2

hiPSCs differentiated using CDM3 at day 10, matches Supplementary Figure 5. (MOV 4735 kb)

Supplementary Video 3

hiPSCs differentiated using CDM3 at day 11 matches Supplementary Figure 5. (MOV 4862 kb)

Supplementary Video 4

hiPSCs differentiated using CDM3 on growth factor-reduced Matrigel at day 22, matches Supplementary Figure 6d. (MOV 4477 kb)

Supplementary Video 5

hiPSCs differentiated using CDM3 on recombinant human E-cadherin at day 22, matches Supplementary Figure 6d. (MOV 2295 kb)

Supplementary Video 6

hiPSCs differentiated using CDM3 on recombinant human vitronectin at day 22, matches Supplementary Figure 6d. (MOV 2370 kb)

Supplementary Video 7

hiPSCs differentiated using CDM3 on vitronectin peptide at day 22, matches Supplementary Figure 6d. (MOV 3787 kb)

Supplementary Video 8

hiPSCs differentiated using CDM3 on recombinant human laminin-521 at day 22, matches Supplementary Figure 6d. (MOV 3920 kb)

Supplementary Video 9

hiPSCs differentiated using CDM3 on truncated recombinant human laminin-511 at day 22, matches Supplementary Figure 6d. (MOV 2066 kb)

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Burridge, P., Matsa, E., Shukla, P. et al. Chemically defined generation of human cardiomyocytes. Nat Methods 11, 855–860 (2014). https://doi.org/10.1038/nmeth.2999

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