Production of Cardiomyocyte-Like Cells by Fibroblast Reprogramming with Defined Factors.
Production of Cardiomyocyte-Like Cells by Fibroblast Reprogramming with Defined Factors.
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DOI：
10.1007/978-1-0716-1084-8_3
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Fu JD
中科院分区：
文献类型：
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作者：
Bektik E;Fu JD
在过去的十年中，在直接表观遗传重编程领域取得了巨大成就，该领域将一种类型的成体体细胞转化为另一种类型的分化细胞，例如将成纤维细胞直接重编程为心肌细胞，而无需通过未分化的多能阶段。直接心脏重编程的发现提供了预防/减弱患病心脏中的心脏纤维化重构的有前景的治疗策略。此外，成纤维细胞在体外重编程为心肌样细胞提供了新的途径，进行基本的机制研究，测试药物，并在培养皿中模拟心脏疾病。在这里，我们描述了一个详细的一步一步的协议，在体外生产诱导心肌样细胞（iCM）的成纤维细胞。相关程序包括不同来源的高质量成纤维细胞分离（新生儿心脏，尾尖和成人心脏成纤维细胞），重编程因子的逆转录病毒制备，以及通过Gata 4，Mef 2c和Tbx 5的逆转录病毒转导通过成纤维细胞重编程产生iCM。一份详细的书面协议将帮助许多其他实验室，在这一领域缺乏经验，使用和进一步改善这项技术在他们的研究心脏再生医学。
Over the last decade, great achievements have been made in the field of direct epigenetic reprogramming, which converts one type of adult somatic cells into another type of differentiated cells, such as direct reprograming of fibroblasts into cardiomyocytes, without passage through an undifferentiated pluripotent stage. Discovery of direct cardiac reprogramming offers a promising therapeutic strategy to prevent/attenuate cardiac fibrotic remodeling in a diseased heart. Furthermore, in vitro reprogramming of fibroblasts into cardiomyocyte-like cells provides new avenues to conduct basic mechanistic studies, to test drugs, and to model cardiac diseases in a dish. Here, we describe a detailed step-by-step protocol for in vitro production of induced cardiomyocyte-like cells (iCMs) from fibroblasts. The related procedures include high-quality fibroblast isolation of different origins (neonatal cardiac, tail-tip, and adult cardiac fibroblasts), retroviral preparation of reprogramming factors, and iCM generation by fibroblast reprogramming via retroviral transduction of Gata4, Mef2c and Tbx5. A detailed written protocol will help many other laboratories, inexperienced in this area, to use and further improve this technology in their studies of cardiac regenerative medicine.