Department of Chemical and Biological Engineering, University of Wisconsin, Madison, WI, USA.
Nat Protoc. 2013 Jan;8(1):162-75. doi: 10.1038/nprot.2012.150. Epub 2012 Dec 20.
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 β-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.
本文描述的方案通过对经典 Wnt 信号通路调节剂的时间调控，在完全定义的、无生长因子和血清的系统中，有效地将人多能干细胞（hPSC）定向分化为功能性心肌细胞。适当的时间应用糖原合成酶激酶 3（GSK3）抑制剂，加上β-连环蛋白 shRNA 的表达或化学 Wnt 抑制剂，足以在 14 天内从多个 hPSC 系中产生高产量（每平方厘米 0.8-1.3 百万个）、几乎纯（80-98%）的功能性心肌细胞，无需细胞分选或选择。通过免疫染色和流式细胞术对分化细胞进行定性（免疫染色）和定量（流式细胞术）特征分析，以评估心脏转录因子和肌丝蛋白的表达。BrdU 掺入或 Ki67 表达的流式细胞术与心脏肌球蛋白蛋白的表达相结合，可用于确定 hPSC 来源的心肌细胞的增殖能力。通过这些方案分化的功能性人类心肌细胞可能构成心脏病建模、药物筛选和基于细胞的治疗应用的潜在细胞来源。
