Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5.
Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5.
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DOI：
10.3791/53426
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发表时间：
2015-11-13
期刊：
影响因子：
--
通讯作者：
Qian L
中科院分区：
文献类型：
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作者：
Wang L;Liu Z;Yin C;Zhou Y;Liu J;Qian L
将心脏成纤维细胞（CF）直接转化为诱导心肌细胞（iCM），通过提供治疗心脏病的替代策略，为再生医学提供了巨大的潜力。这种转换已经通过强制表达定义的因子如Gata4（G）、Mef2c（M）和Tbx5（T）来实现。传统上，iCM是由表达这些单个因子的病毒混合物产生的。然而，重编程效率相对较低，并且大多数体外G、M、T转导的成纤维细胞没有完全重编程，使得难以研究重编程机制。我们最近已经表明，G，M，T的化学计量对于有效的iCM重编程至关重要。通过使用我们的多顺反子MGT载体（以下称为MGT）实现的具有相对高水平的M和低水平的G和T的G、M、T的最佳化学计量显著增加了体外重编程效率并改善了iCM质量。在这里，我们提供了用于从心脏成纤维细胞生成具有MGT构建体的iCM的方法的详细描述。还包括心脏成纤维细胞的分离、用于重编程的病毒的产生和重编程过程的评价，以提供用于iCM的高效和可再现产生的平台。
Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.