Wang Li, Liu Ziqing, Yin Chaoying, Zhou Yang, Liu Jiandong, Qian Li
Department of Pathology and Laboratory Medicine, McAllister Heart Institute, University of North Carolina, Chapel Hill.
Department of Pathology and Laboratory Medicine, McAllister Heart Institute, University of North Carolina, Chapel Hill;
J Vis Exp. 2015 Nov 13(105):53426. doi: 10.3791/53426.
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.
将心脏成纤维细胞（CFs）直接转化为诱导性心肌细胞（iCMs），为再生医学提供了治疗心脏病的替代策略，具有巨大潜力。这种转化已通过强制表达特定因子如Gata4（G）、Mef2c（M）和Tbx5（T）得以实现。传统上，iCMs是通过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，大多数体外经G、M、T转导的成纤维细胞并未完全重编程，这使得研究重编程机制变得困难。我们最近表明，G、M、T的化学计量对于高效的iCM重编程至关重要。通过使用我们的多顺反子MGT载体（以下简称MGT）实现的G、M、T的最佳化学计量，即相对高水平的M以及低水平的G和T，显著提高了重编程效率并改善了体外iCM的质量。在此，我们详细描述了使用MGT构建体从心脏成纤维细胞生成iCMs的方法。还包括心脏成纤维细胞的分离、用于重编程的病毒生成以及重编程过程的评估等内容，以提供一个高效且可重复生成iCMs的平台。
