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Long-term self-renewing human epicardial cells generated from pluripotent stem cells under defined xeno-free conditions

Abstract

The epicardium contributes both multi-lineage descendants and paracrine factors to the heart during cardiogenesis and cardiac repair, underscoring its potential for use in cardiac regenerative medicine. Yet little is known about the cellular and molecular mechanisms that regulate human epicardial development and regeneration. Here, we show that the temporal modulation of canonical Wnt signalling is sufficient for epicardial induction from six different human pluripotent stem cell (hPSC) lines, including a WT1-2A-eGFP knock-in reporter line, under chemically defined, xeno-free conditions. We also show that treatment with transforming growth factor beta (TGF-β)-signalling inhibitors permitted long-term expansion of the hPSC-derived epicardial cells, resulting in more than 25 population doublings of WT1+ cells in homogenous monolayers. The hPSC-derived epicardial cells were similar to primary epicardial cells both in vitro and in vivo, as determined by morphological and functional assays, including RNA sequencing. Our findings have implications for the understanding of self-renewal mechanisms of the epicardium and for epicardial regeneration using cellular or small-molecule therapies.

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Figure 1: Wnt/β-catenin signalling directs the specification of WT1+ epicardial lineages from hPSC-derived cardiac progenitors.
Figure 2: Construction of the WT1-2A-eGFP knock-in ES03 hESC line using Cas9 nuclease.
Figure 3: Molecular analysis of hPSC-derived epicardial cells under chemically defined, albumin-free conditions.
Figure 4: hPSC-derived epicardial cells undergo an EMT in response to bFGF and TGF-β1 treatment, yielding epicardium-derived cells that display characteristics of fibroblasts and vascular smooth muscle cells.
Figure 5: Long-term expansion of hPSC-derived epicardial cells. a,b, H13 hESC-derived day 18 epicardial cells were seeded at a density of 0.05 million cells per cm2 and treated with the indicated small molecules for 3 days (concentrations provided in Supplementary Table 1).
Figure 6: hPSC-derived epicardial cells were similar to primary epicardial cells.
Figure 7

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Acknowledgements

We thank D.A. Roenneburg and X. Wang for their technical support. We also thank members of the Palecek group for critical discussion of the manuscript. This work was supported by NIH grant EB007534 (S.P.P.), NSF grant 1547225 (S.P.P.), and a fellowship from the University of Wisconsin Stem Cell and Regenerative Medicine Center (X.B.).

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Authors

Contributions

X.B. and S.P.P. designed this study and prepared the manuscript. X.B. undertook experimentation and data analysis. X.L. contributed to the study design and assisted in writing the manuscript. T.A.H. and E.G.S. designed and performed the in vivo study. T.H., V.J.B., T.Q. and M.S. assisted in differentiation experiments and data analysis. L.D., A.T.P., Q.-D.W. and M.-J.G. isolated and provided the human primary donor samples for RNA-seq. All authors reviewed and approved the manuscript.

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Correspondence to Sean P. Palecek.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary figures and tables, and movie legends. (PDF 3818 kb)

Supplementary Movie 1

Non-contracting hESC-derived pro-epicardial cells at day 12. (AVI 18384 kb)

Supplementary Movie 2

Spontaneously contracting hESC-derived cardiomyocytes at day 12. (AVI 17886 kb)

Supplementary Movie 3

Spontaneously contracting hESC-derived cardiomyocytes at day 12. (AVI 9065 kb)

Supplementary Movie 4

Non-contracting iPSC-derived pro-epicardial cells at day 12. (AVI 22676 kb)

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Bao, X., Lian, X., Hacker, T. et al. Long-term self-renewing human epicardial cells generated from pluripotent stem cells under defined xeno-free conditions. Nat Biomed Eng 1, 0003 (2017). https://doi.org/10.1038/s41551-016-0003

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