Abstract
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 reprogramming 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.