Rapamycin efficiently promotes cardiac differentiation of mouse embryonic stem cells
Rapamycin efficiently promotes cardiac differentiation of mouse embryonic stem cells
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雷帕霉素有效促进小鼠胚胎干细胞心脏分化
DOI：
10.1042/bsr20160552
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发表时间：
2017-06-30
影响因子：
4
通讯作者：
Zhou, Chunyan
中科院分区：
文献类型：
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作者：
Lu, Qin;Liu, Yinan;Zhou, Chunyan
为了研究雷帕霉素对心肌分化的影响，用10（-4）M抗坏血酸（AA）、20 nM雷帕霉素单独或0.01%溶剂DMSO诱导小鼠胚胎干细胞（ESC）向心肌细胞分化。我们发现单独的雷帕霉素不足以启动心肌形成。然后，用AA和雷帕霉素（20 nM）或AA和DMSO（0.01%）处理ESC作为对照。与对照组相比，用雷帕霉素（20 nM）和AA处理的小鼠ESC（mESC）产生了显著更高的心肌细胞百分比，如通过搏动胚胎体（EBs）的百分比、免疫荧光和FACS分析所证实的。雷帕霉素显著增加了一组心脏标志物的表达，包括Gata 4、α-Mhc、β-Mhc和Tnnt 2。此外，雷帕霉素增强了中胚层和心脏转录因子的表达，如Mesp 1、Brachyury T、Eomes、Isl 1、Gata 4、Nkx2.5、Tbx 5和Mef 2c。机制研究表明，雷帕霉素抑制Wnt/β-catenin和Notch信号传导，但在早期促进成纤维细胞生长因子（Fgf 8）、Fgf 10和Nodal的表达，在后期促进骨形态发生蛋白2（Bmp 2）的表达。雷帕霉素的顺序处理表明，雷帕霉素促进心脏分化的早期和晚期阶段。有趣的是，另一种哺乳动物雷帕霉素靶蛋白（mTOR）抑制剂Ku 0063794（1 μ M）对心肌发生有类似的作用。总之，我们的研究结果突出了一个实用的方法来产生心肌细胞从mESC雷帕霉素。
To investigate the effects of rapamycin on cardiac differentiation, murine embryonic stem cells (ESCs) were induced into cardiomyocytes by 10(-4) M ascorbic acid (AA), 20 nM rapamycin alone or 0.01% solvent DMSO. We found that rapamycin alone was insufficient to initiate cardiomyogenesis. Then, the ESCs were treated with AA and rapamycin (20 nM) or AA and DMSO (0.01%) as a control. Compared with control, mouse ESCs (mESCs) treated with rapamycin (20 nM) and AA yielded a significantly higher percentage of cardiomyocytes, as confirmed by the percentage of beating embryonic bodies (EBs), the immunofluorescence and FACS analysis. Rapamycin significantly increased the expression of a panel of cardiac markers including Gata4, alpha-Mhc, beta-Mhc, and Tnnt2. Additionally, rapamycin enhanced the expression of mesodermal and cardiac transcription factors such as Mesp1, Brachyury T, Eomes, Isl1, Gata4, Nkx2.5, Tbx5, and Mef2c. Mechanistic studies showed that rapamycin inhibits Wnt/beta-catenin and Notch signaling but promotes the expression of fibroblast growth factor (Fgf8), Fgf10, and Nodal at early stage, and bone morphogenetic protein 2 (Bmp 2) at later stages. Sequential treatment of rapamycin showed that rapamycin promotes cardiac differentiation at the early and later stages. Interestingly, another mammalian target of rapamycin (mTOR) inhibitor Ku0063794 (1 mu M) had similar effects on cardiomyogenesis. In conclusion, our results highlight a practical approach to generate cardiomyocytes from mESCs by rapamycin.