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Directed differentiation and long-term maintenance of epicardial cells derived from human pluripotent stem cells under fully defined conditions

Abstract

Here, we describe how to efficiently direct human pluripotent stem cells (hPSCs) differentiation into self-renewing epicardial cells in a completely defined, xeno-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate differentiation-stage-specific application of Gsk3 inhibitor, Wnt inhibitor, and Gsk3 inhibitor (GiWiGi) is sufficient to produce cells expressing epicardial markers and exhibiting epicardial phenotypes with a high yield and purity from multiple hPSC lines in 16 d. Characterization of differentiated cells is performed via flow cytometry and immunostaining to assess quantitative expression and localization of epicardial cell–specific proteins. In vitro differentiation into fibroblasts and smooth muscle cells (SMCs) is also described. In addition, culture in the presence of transforming growth factor (TGF)-β inhibitors allows long-term expansion of hPSC-derived epicardial cells (for at least 25 population doublings). Functional human epicardial cells differentiated via this protocol may constitute a potential cell source for heart disease modeling, drug screening, and cell-based therapeutic applications.

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Figure 1: Schematic of protocol to differentiate cardiac progenitor cells from hPSCs with small-molecule modulators of canonical Wnt signaling (Steps 1–8).
Figure 2: Schematic of protocol to differentiate epicardial cells by treatment of hPSC-derived cardiac progenitor cells with the Gsk3 inhibitor CHIR99021 (Steps 9–14).
Figure 3: Schematic of protocol to maintain self-renewing hPSC-derived epicardial cells (Step 15A).
Figure 4: Immunostaining analysis of epicardial cells differentiated from hPSCs via small-molecule modulation of Wnt signaling (Step 15B).
Figure 5: Quantitative analysis of marker expression in epicardial cells differentiated from hPSCs by small-molecule modulation of Wnt signaling (Step 15C).
Figure 6: Differentiation of hPSC-derived epicardial cells to fibroblasts and smooth muscle cells (Step 15D).

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Acknowledgements

This study was supported by NIH grant EB007534 (S.P.P.), NSF grant 1547225 (S.P.P.), and a fellowship from the University of Wisconsin–Madison Stem Cell and Regenerative Medicine Center (X.B.).

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Contributions

X.B. and S.P.P. designed the study and prepared the manuscript. X.B. undertook the experimentation and the data analysis. T.Q., X.L., V.J.B., and T.H. contributed to the development of this protocol.

Corresponding author

Correspondence to Sean P Palecek.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Flow cytometry data analysis of WT1+ cells.

(A) The intact cell population is gated using forward (FWD) and side scatter to exclude cell debris. The numbers in the pink polygon show the percentage of total events that are intact cells in the pink gated region. (B) The WT1+ gated region is identified using both isotype and WT1 antibody stained samples (red) with a ranged tool. The numbers above black line show the percentage of WT1+ cells in the gated population from (A). (C) Overlay histogram showing percentage of WT1+ cells in the gated population from (A).

Supplementary information

Supplementary Figure 1

Supplementary Figure 1. Flow cytometry data analysis of WT1+ cells. (a) The intact cell population is gated using forward (FWD) and side scatter to exclude cell debris. The numbers in the pink polygon show the percentage of total events that are intact cells in the pink gated region. (b) The WT1+ gated region is identified using both isotype control (blue) and WT1-antibody-stained (red) samples with a ranged tool. The numbers above the black line show the percentage of WT1+ cells in the gated population from a. (c) Overlay histogram showing percentage of WT1+ cells in the gated population from a. (PDF 89 kb)

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Bao, X., Lian, X., Qian, T. et al. Directed differentiation and long-term maintenance of epicardial cells derived from human pluripotent stem cells under fully defined conditions. Nat Protoc 12, 1890–1900 (2017). https://doi.org/10.1038/nprot.2017.080

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