Bao Xiaoping, Lian Xiaojun, Qian Tongcheng, Bhute Vijesh J, Han Tianxiao, Palecek Sean P
Department of Chemical &Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Department of Biomedical Engineering, The Pennsylvania State University, University Park, Pennsylvania, USA.
Nat Protoc. 2017 Sep;12(9):1890-1900. doi: 10.1038/nprot.2017.080. Epub 2017 Aug 17.
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.
在此，我们描述了如何通过对经典Wnt信号通路调节因子进行时间调控，在完全确定的无血清体系中高效地将人多能干细胞（hPSC）分化为自我更新的心外膜细胞。适时地分别应用糖原合成酶激酶3（Gsk3）抑制剂、Wnt抑制剂以及Gsk3抑制剂（GiWiGi），足以在16天内从多个hPSC系中高产且高纯度地产生表达心外膜标志物并呈现心外膜表型的细胞。通过流式细胞术和免疫染色对分化细胞进行表征，以评估心外膜细胞特异性蛋白的定量表达和定位。还描述了其在体外分化为成纤维细胞和平滑肌细胞（SMC）的情况。此外，在存在转化生长因子（TGF）-β抑制剂的条件下进行培养，可实现hPSC衍生的心外膜细胞的长期扩增（至少25次群体倍增）。通过该方案分化得到的功能性人心外膜细胞可能构成用于心脏病建模、药物筛选和基于细胞的治疗应用的潜在细胞来源。
