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


Type
Article
Change log
Authors
Bao, Xiaoping 
Lian, Xiaojun 
Qian, Tongcheng 
Bhute, Vijesh J 
Han, Tianxiao 
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.

Description
Keywords
Cell Culture Techniques, Cell Differentiation, Cell Engineering, Humans, Pericardium, Pluripotent Stem Cells, Wnt Signaling Pathway
Journal Title
Nat Protoc
Conference Name
Journal ISSN
1754-2189
1750-2799
Volume Title
12
Publisher
Springer Science and Business Media LLC