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

• Published in: Nature protocols Vol. 12; no. 9; pp. 1890 - 1900
• Main Authors: Bao, Xiaoping, Lian, Xiaojun, Qian, Tongcheng, Bhute, Vijesh J., Han, Tianxiao, Palecek, Sean P.
• Format: Journal Article
• Language: English
• Published: 17.08.2017
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2017.080

The protocol described here efficiently directs human pluripotent stem cells (hPSCs) to 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, then Gsk3 inhibitor is sufficient to produce cells expressing epicardial markers and exhibiting epicardial phenotypes with a high yield and purity from multiple hPSC lines in 16 days. 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 to fibroblasts and smooth muscle cells is also described. In addition, culture in the presence of TGFβ 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.