Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions

被引：1237

作者：

Lian, Xiaojun ^{[1
]}

Zhang, Jianhua ^{[2
]}

Azarin, Samira M. ^{[1
]}

Zhu, Kexian ^{[1
]}

Hazeltine, Laurie B. ^{[1
]}

Bao, Xiaoping ^{[1
]}

Hsiao, Cheston ^{[1
]}

Kamp, Timothy J. ^{[2
]}

Palecek, Sean P. ^{[1
]}

机构：

[1] Univ Wisconsin, Dept Chem & Biol Engn, Madison, WI 53706 USA

[2] Univ Wisconsin, Dept Med, Madison, WI USA

来源：

NATURE PROTOCOLS | 2013年 / 8卷 / 01期

基金：

美国国家科学基金会; 美国国家卫生研究院;

关键词：

CARDIAC DIFFERENTIATION; CARDIOMYOGENESIS; SPECIFICATION; PROGENITORS; RENEWAL;

D O I：

10.1038/nprot.2012.150

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The protocol described here efficiently directs human pluripotent stem cells (hPSCs) to functional cardiomyocytes in a completely defined, growth factor- and serum-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate temporal application of a glycogen synthase kinase 3 (GSK3) inhibitor combined with the expression of beta-catenin shRNA or a chemical Wnt inhibitor is sufficient to produce a high yield (0.8-1.3 million cardiomyocytes per cm(2)) of virtually pure (80-98%) functional cardiomyocytes in 14 d from multiple hPSC lines without cell sorting or selection. Qualitative (immunostaining) and quantitative (flow cytometry) characterization of differentiated cells is described to assess the expression of cardiac transcription factors and myofilament proteins. Flow cytometry of BrdU incorporation or Ki67 expression in conjunction with cardiac sarcomere myosin protein expression can be used to determine the proliferative capacity of hPSC-derived cardiomyocytes. Functional human cardiomyocytes differentiated via these protocols may constitute a potential cell source for heart disease modeling, drug screening and cell-based therapeutic applications.

引用

页码：162 / 175

页数：14

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