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NKX2-5eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes

Abstract

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5eGFP/w hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP+ cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.

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Figure 1: Characterization of cardiomyocytes generated from NKX2-5eGFP/w hESCs.
Figure 2: NKX2-5eGFP/w hESCs facilitate real-time monitoring of cardiac differentiation.
Figure 3: Expression profiling of NKX2-5 eGFP+ cells identifies cardiac cell-surface markers.

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Acknowledgements

This work was funded by the Australian Stem Cell Centre (to E.G.S., A.G.E., D.A.E., D.M.K., J.M.H. and C.W.P.), the National Health and Medical Research Council of Australia (D.A.E., grant 606586; O.W.J.P., grant 573707), National Heart Foundation (Australia) (E.G.S. and A.G.E., grant G 08M 3711; O.W.J.P., Career Development Award grant CR 08S 3958), the Netherlands Organization for Scientific Research and the Netherlands Institute of Regenerative Medicine (S.R.B. and C.L.M.), the Victorian State Government Operational Infrastructure Support and the National Health and Medical Research Council of Australia's Independent Research Institutes Infrastructure Support Scheme (O.W.J.P. and C.B.). R.P.D. is funded by a Rubicon fellowship from the Netherlands Organization for Scientific Research and the Marie Curie Co-fund Action. E.G.S. and A.G.E. receive Senior Research Fellowships and D.M.K. receives a Principal Research Fellowship from the National Health and Medical Research Council of Australia.

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Authors and Affiliations

Authors

Contributions

D.A.E., A.G.E., E.G.S. and C.L.M. designed the study. D.A.E., S.R.B., K.K., E.S.N., R.J., E.L.L., C.B., T.H., R.J.P.S., O.K., D.W.O., X.L., S.M.H., S.M.L., R.P., R.P.D., A.L.G., O.W.J.P., A.G.E., X.L., S.M.H., J.M.H., C.W.P. and D.M.K. performed and analyzed experiments. C.E.H. and Q.C.Y. performed bioinformatics analyses. D.A.E., C.L.M., A.G.E. and E.G.S. wrote the manuscript.

Corresponding authors

Correspondence to Andrew G Elefanty or Edouard G Stanley.

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Competing interests

D.A.E., A.G.E. and E.G.S. have applied for a patent (US provisional USSN 61/492,099) in relation to results described in this paper.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 1–3 (PDF 4994 kb)

Supplementary Video 1

NKX2-5eGFP/w derived embryoid bodies express eGFP in contractile areas. Brightfield and green fluorescence of a day-14 NKX2-5eGFP/w embryoid body. This video demonstrates that contractile areas of the embryoid body express eGFP. (MOV 774 kb)

Supplementary Video 2

NKX2-5eGFP/w hESC cultured with the END2 endodermal cell line express eGFP in contractile areas. Movie shows a Z-dimension stack through the beating area demonstrating that eGFP is expressed with contracting clusters. (MOV 2962 kb)

Supplementary Video 3

Calcium flux across a contraction cycle. Green fluorescence (top left). Red fluorescence (top right). Brightfield (bottom left). Pseudo-colored video showing calcium flux (bottom right). (MOV 1931 kb)

Supplementary Video 4

Monolayer differentiation with NKX2-5eGFP/w hESCs. Brightfield and green fluorescence shows that beating foci are eGFP+. (MOV 936 kb)

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Elliott, D., Braam, S., Koutsis, K. et al. NKX2-5eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nat Methods 8, 1037–1040 (2011). https://doi.org/10.1038/nmeth.1740

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