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Generation of the epicardial lineage from human pluripotent stem cells

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.

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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.

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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.

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Authors

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.

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Correspondence to Gordon Keller.

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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)

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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

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