Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Generation of the epicardial lineage from human pluripotent stem cells

Nature Biotechnology volume 32, pages 1026–1035 (2014)Cite this article

Subjects

Abstract

The epicardium supports cardiomyocyte proliferation early in development and provides fibroblasts and vascular smooth muscle cells to the developing heart. The epicardium has been shown to play an important role during tissue remodeling after cardiac injury, making access to this cell lineage necessary for the study of regenerative medicine. Here we describe the generation of epicardial lineage cells from human pluripotent stem cells by stage-specific activation of the BMP and WNT signaling pathways. These cells display morphological characteristics and express markers of the epicardial lineage, including the transcription factors WT1 and TBX18 and the retinoic acid–producing enzyme ALDH1A2. When induced to undergo epithelial-to-mesenchymal transition, the cells give rise to populations that display characteristics of the fibroblast and vascular smooth muscle lineages. These findings identify BMP and WNT as key regulators of the epicardial lineage in vitro and provide a model for investigating epicardial function in human development and disease.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: BMP specifies the differentiation of cardiomyocytes from hESC-derived mesoderm.
Figure 2: BMP4 treatment specifies WT1+ cells.
Figure 3: WT1+ cells generate epithelial sheets following passage.
Figure 4: BMP and WNT signaling modulate cardiomyocyte and pre-epicardium specification.
Figure 5: Epicardial cells undergo EMT in response to TGFB1 and bFGF treatment.
Figure 6: Epicardium-derived cells display characteristics of fibroblasts and vascular smooth muscle cells.
Figure 7: Functional assessment of EPDCs.

Similar content being viewed by others

References

  1. DeRuiter, M.C., Poelmann, R.E., VanderPlas-de Vries, I., Mentink, M.M. & Gittenberger-de Groot, A.C. The development of the myocardium and endocardium in mouse embryos. Fusion of two heart tubes? Anat. Embryol. (Berl.) 185, 461–473 (1992).

    Article  CAS  Google Scholar 

  2. Limana, F., Capogrossi, M.C. & Germani, A. The epicardium in cardiac repair: from the stem cell view. Pharmacol. Ther. 129, 82–96 (2011).

    Article  CAS  Google Scholar 

  3. Komiyama, M., Ito, K. & Shimada, Y. Origin and development of the epicardium in the mouse embryo. Anat. Embryol. (Berl.) 176, 183–189 (1987).

    Article  CAS  Google Scholar 

  4. Austin, A.F., Compton, L.A., Love, J.D., Brown, C.B. & Barnett, J.V. Primary and immortalized mouse epicardial cells undergo differentiation in response to TGFbeta. Dev. Dyn. 237, 366–376 (2008).

    Article  CAS  Google Scholar 

  5. Bax, N.A. et al. In vitro epithelial-to-mesenchymal transformation in human adult epicardial cells is regulated by TGFbeta-signaling and WT1. Basic Res. Cardiol. 106, 829–847 (2011).

    Article  CAS  Google Scholar 

  6. von Gise, A. et al. WT1 regulates epicardial epithelial to mesenchymal transition through beta-catenin and retinoic acid signaling pathways. Dev. Biol. 356, 421–431 (2011).

    Article  CAS  Google Scholar 

  7. Morabito, C.J., Dettman, R.W., Kattan, J., Collier, J.M. & Bristow, J. Positive and negative regulation of epicardial-mesenchymal transformation during avian heart development. Dev. Biol. 234, 204–215 (2001).

    Article  CAS  Google Scholar 

  8. Smith, C.L., Baek, S.T., Sung, C.Y. & Tallquist, M.D. Epicardial-derived cell epithelial-to-mesenchymal transition and fate specification require PDGF receptor signaling. Circ. Res. 108, e15–e26 (2011).

    Article  CAS  Google Scholar 

  9. Lie-Venema, H. et al. Origin, fate, and function of epicardium-derived cells (EPDCs) in normal and abnormal cardiac development. ScientificWorldJournal 7, 1777–1798 (2007).

    Article  CAS  Google Scholar 

  10. Christoffels, V. Regenerative medicine: muscle for a damaged heart. Nature 474, 585–586 (2011).

    Article  CAS  Google Scholar 

  11. Moore, A.W., McInnes, L., Kreidberg, J., Hastie, N.D. & Schedl, A. YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland and throughout nephrogenesis. Development 126, 1845–1857 (1999).

    CAS  PubMed  Google Scholar 

  12. Haenig, B. & Kispert, A. Analysis of TBX18 expression in chick embryos. Dev. Genes Evol. 214, 407–411 (2004).

    Article  CAS  Google Scholar 

  13. Moss, J.B. et al. Dynamic patterns of retinoic acid synthesis and response in the developing mammalian heart. Dev. Biol. 199, 55–71 (1998).

    Article  CAS  Google Scholar 

  14. Xavier-Neto, J., Shapiro, M.D., Houghton, L. & Rosenthal, N. Sequential programs of retinoic acid synthesis in the myocardial and epicardial layers of the developing avian heart. Dev. Biol. 219, 129–141 (2000).

    Article  CAS  Google Scholar 

  15. Huang, G.N. et al. C/EBP transcription factors mediate epicardial activation during heart development and injury. Science 338, 1599–1603 (2012).

    Article  CAS  Google Scholar 

  16. Lepilina, A. et al. A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell 127, 607–619 (2006).

    Article  CAS  Google Scholar 

  17. Cai, C.L. et al. A myocardial lineage derives from Tbx18 epicardial cells. Nature 454, 104–108 (2008).

    Article  CAS  Google Scholar 

  18. Zhou, B. et al. Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature 454, 109–113 (2008).

    Article  CAS  Google Scholar 

  19. Smart, N. et al. De novo cardiomyocytes from within the activated adult heart after injury. Nature 474, 640–644 (2011).

    Article  CAS  Google Scholar 

  20. van Tuyn, J. et al. Epicardial cells of human adults can undergo an epithelial-to-mesenchymal transition and obtain characteristics of smooth muscle cells in vitro. Stem Cells 25, 271–278 (2007).

    Article  CAS  Google Scholar 

  21. Kattman, S.J. et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8, 228–240 (2011).

    Article  CAS  Google Scholar 

  22. Klaus, A., Saga, Y., Taketo, M.M., Tzahor, E. & Birchmeier, W. Distinct roles of Wnt/beta-catenin and Bmp signaling during early cardiogenesis. Proc. Natl. Acad. Sci. USA 104, 18531–18536 (2007).

    Article  CAS  Google Scholar 

  23. Watt, A.J., Battle, M.A., Li, J. & Duncan, S.A. GATA4 is essential for formation of the proepicardium and regulates cardiogenesis. Proc. Natl. Acad. Sci. USA 101, 12573–12578 (2004).

    Article  CAS  Google Scholar 

  24. MacNeill, C., French, R., Evans, T., Wessels, A. & Burch, J.B. Modular regulation of cGATA-5 gene expression in the developing heart and gut. Dev. Biol. 217, 62–76 (2000).

    Article  CAS  Google Scholar 

  25. Ma, Q., Zhou, B. & Pu, W.T. Reassessment of Isl1 and Nkx2–5 cardiac fate maps using a Gata4-based reporter of Cre activity. Dev. Biol. 323, 98–104 (2008).

    Article  CAS  Google Scholar 

  26. Liu, J. & Stainier, D.Y. Tbx5 and Bmp signaling are essential for proepicardium specification in zebrafish. Circ. Res. 106, 1818–1828 (2010).

    Article  CAS  Google Scholar 

  27. Bochmann, L. et al. Revealing new mouse epicardial cell markers through transcriptomics. PLoS ONE 5, e11429 (2010).

    Article  Google Scholar 

  28. Mahtab, E.A. et al. Cardiac malformations and myocardial abnormalities in podoplanin knockout mouse embryos: correlation with abnormal epicardial development. Dev. Dyn. 237, 847–857 (2008).

    Article  CAS  Google Scholar 

  29. Dubois, N.C. et al. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nat. Biotechnol. 29, 1011–1018 (2011).

    Article  CAS  Google Scholar 

  30. Mellgren, A.M. et al. Platelet-derived growth factor receptor beta signaling is required for efficient epicardial cell migration and development of two distinct coronary vascular smooth muscle cell populations. Circ. Res. 103, 1393–1401 (2008).

    Article  CAS  Google Scholar 

  31. Phillips, M.D., Mukhopadhyay, M., Poscablo, C. & Westphal, H. Dkk1 and Dkk2 regulate epicardial specification during mouse heart development. Int. J. Cardiol. 150, 186–192 (2011).

    Article  Google Scholar 

  32. Yu, P.B. et al. Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. Nat. Chem. Biol. 4, 33–41 (2008).

    Article  CAS  Google Scholar 

  33. Kruithof, B.P. et al. BMP and FGF regulate the differentiation of multipotential pericardial mesoderm into the myocardial or epicardial lineage. Dev. Biol. 295, 507–522 (2006).

    Article  CAS  Google Scholar 

  34. Casanova, J.C., Travisano, S. & de la Pompa, J.L. Epithelial-to-mesenchymal transition in epicardium is independent of Snail1. Genesis 51, 32–40 (2013).

    Article  CAS  Google Scholar 

  35. Cheung, C., Bernardo, A.S., Trotter, M.W., Pedersen, R.A. & Sinha, S. Generation of human vascular smooth muscle subtypes provides insight into embryological origin-dependent disease susceptibility. Nat. Biotechnol. 30, 165–173 (2012).

    Article  CAS  Google Scholar 

  36. Acharya, A. et al. The bHLH transcription factor Tcf21 is required for lineage-specific EMT of cardiac fibroblast progenitors. Development 139, 2139–2149 (2012).

    Article  CAS  Google Scholar 

  37. El-Mounayri, O. et al. Serum-free differentiation of functional human coronary-like vascular smooth muscle cells from embryonic stem cells. Cardiovasc. Res. 98, 125–135 (2013).

    Article  CAS  Google Scholar 

  38. Yang, L. et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453, 524–528 (2008).

    Article  CAS  Google Scholar 

  39. Lian, X. et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc. Natl. Acad. Sci. USA 109, E1848–E1857 (2012).

    Article  CAS  Google Scholar 

  40. Ueno, S. et al. Biphasic role for Wnt/beta-catenin signaling in cardiac specification in zebrafish and embryonic stem cells. Proc. Natl. Acad. Sci. USA 104, 9685–9690 (2007).

    Article  CAS  Google Scholar 

  41. David, R. et al. MesP1 drives vertebrate cardiovascular differentiation through Dkk-1-mediated blockade of Wnt-signalling. Nat. Cell Biol. 10, 338–345 (2008).

    Article  CAS  Google Scholar 

  42. Weeke-Klimp, A. et al. Epicardium-derived cells enhance proliferation, cellular maturation and alignment of cardiomyocytes. J. Mol. Cell. Cardiol. 49, 606–616 (2010).

    Article  CAS  Google Scholar 

  43. Gaudesius, G., Miragoli, M., Thomas, S.P. & Rohr, S. Coupling of cardiac electrical activity over extended distances by fibroblasts of cardiac origin. Circ. Res. 93, 421–428 (2003).

    Article  CAS  Google Scholar 

  44. de la Cuesta, F. et al. Deregulation of smooth muscle cell cytoskeleton within the human atherosclerotic coronary media layer. J. Proteomics 82, 155–165 (2013).

    Article  CAS  Google Scholar 

  45. Jonasson, L., Holm, J., Skalli, O., Bondjers, G. & Hansson, G.K. Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis 6, 131–138 (1986).

    Article  CAS  Google Scholar 

  46. Winter, E.M. et al. Preservation of left ventricular function and attenuation of remodeling after transplantation of human epicardium-derived cells into the infarcted mouse heart. Circulation 116, 917–927 (2007).

    Article  CAS  Google Scholar 

  47. Winter, E.M. et al. A new direction for cardiac regeneration therapy: application of synergistically acting epicardium-derived cells and cardiomyocyte progenitor cells. Circ Heart Fail 2, 643–653 (2009).

    Article  Google Scholar 

  48. Kennedy, M., D'Souza, S.L., Lynch-Kattman, M., Schwantz, S. & Keller, G. Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood 109, 2679–2687 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Clarke, R.L. et al. The expression of Sox17 identifies and regulates haemogenic endothelium. Nat. Cell Biol. 15, 502–510 (2013).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank T. Araki and B. Neel (Ontario Cancer Institute, Toronto) for providing the Sendai hiPSC line, O. El-Mounayri and M. Husain for their advice on epithelial-to-mesenchymal transition, Mark Gagliardi for his assistance in the culture of hESC-derived epicardial cells and the Sick Kids/UHN Flow Cytometry Facility for their assistance with cell sorting. We thank members of the Keller laboratory for their advice on the studies and comments on the manuscript. This work was supported by the Canadian Institute of Health Research (MOP-84524; MOP-119507; MOP-106538; CPG-127793), the Natural Sciences and Engineering Research Council of Canada (CHRPJ 446379-13) and the US National Institutes of Health (5U01 HL100405). This work was funded in part by VistaGen Therapeutics, Inc.

Author information

Author notes

  1. Steven J Kattman

    Present address: Present address: Cellular Dynamics International, Madison, Wisconsin, USA.,

Authors and Affiliations

  1. McEwen Centre for Regenerative Medicine, University Health Network, Toronto, Ontario, Canada

    Alec D Witty, Anton Mihic, Alexander Mikryukov, Molly S Shoichet, Ren-Ke Li, Steven J Kattman & Gordon Keller

  2. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

    Alec D Witty & Gordon Keller

  3. Heart and Stroke Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto, Toronto, Ontario, Canada

    Anton Mihic & Ren-Ke Li

  4. Toronto General Research Institute, Toronto, Ontario, Canada

    Anton Mihic & Ren-Ke Li

  5. Department of Surgery, University of Toronto, Toronto, Ontario, Canada

    Anton Mihic & Ren-Ke Li

  6. The Donnelly Centre for Cellular and Biomolecular Research, Toronto, Ontario, Canada

    Roger Y Tam, Stephanie A Fisher & Molly S Shoichet

  7. Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada

    Roger Y Tam, Stephanie A Fisher & Molly S Shoichet

  8. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada

    Roger Y Tam, Stephanie A Fisher & Molly S Shoichet

  9. Department of Chemistry, University of Toronto, Toronto, Ontario, Canada

    Molly S Shoichet

  10. Princess Margaret Cancer Centre, Toronto, Ontario, Canada

    Gordon Keller

Authors
  1. Alec D Witty
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anton Mihic
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Roger Y Tam
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephanie A Fisher
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alexander Mikryukov
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Molly S Shoichet
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ren-Ke Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Steven J Kattman
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Gordon Keller
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.D.W. contributed to designing the study, designing and performing experiments, analyzing the data, and writing and editing the manuscript. A.M. contributed to designing, performing and analyzing experiments concerning calcium imaging, and editing the manuscript. R.Y.T. contributed to designing, performing and analyzing experiments concerning Matrigel invasion. S.A.F. contributed to designing, performing and analyzing experiments concerning Matrigel invasion. A.M. contributed data acquisition concerning cell quantification and editing the manuscript. M.S.S. contributed to designing experiments concerning Matrigel invasion. R.-K.L. contributed to designing experiments concerning calcium imaging. S.J.K. contributed to designing the study and editing the manuscript. G.K. contributed to designing the study, writing and editing the manuscript.

Corresponding author

Correspondence to Gordon Keller.

Ethics declarations

Competing interests

G.K. is on the scientific advisory board and a shareholder of VistaGen Therapeutics, which partially funded this work. A.D.W., S.J.K. and G.K. are co-inventors on a patent application covering the generation of human pluripotent stem cell--derived epicardial cells described here.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1 (PDF 39006 kb)

Supplementary Video 1

Representative video depicts Fluo 4-AM-generated fluorescence signal on D8 after EMT initiation during the 12-min recording at 30x speed. (MOV 6280 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Witty, A., Mihic, A., Tam, R. et al. Generation of the epicardial lineage from human pluripotent stem cells. Nat Biotechnol 32, 1026–1035 (2014). https://doi.org/10.1038/nbt.3002

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.3002

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing