title: Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro.
abstract: Cardiac fibroblasts can be reprogrammed to cardiomyocyte-like cells by the introduction of three transcription factors: Gata4, Mef2c and Tbx5 (collectively referred to here as GMT). Resident cardiac fibroblasts can be converted in vivo into induced cardiomyocyte-like cells (iCMs) that closely resemble endogenous cardiomyocytes and electrically integrate with the host myocardium. In contrast, in vitro reprogramming yields many partially reprogrammed iCMs, with a few that reprogram fully into contracting myocytes (~3 out of 10,000 GMT-transduced cells). iCMs can be observed as early as 3 d after viral infection, and they continue to mature over 2 months before beating is observed. Despite the success of multiple groups, the inefficiency of in vitro reprogramming has made it challenging for others. However, given the advantages of in vitro iCMs for performing mechanistic studies and, if refined, for testing drugs or small molecules for personalized medicine and modeling cardiac disease in a dish, it is important to standardize the protocol to improve reproducibility and enhance the technology further. Here we describe a detailed step-by-step protocol for in vitro cardiac reprogramming using retroviruses encoding GMT.
authors.affiliation: Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Qian
authors.forename: Emily C
authors.initials: EC
authors.lastname: Berry
authors.forename: Ji-dong
authors.initials: JD
authors.lastname: Fu
authors.forename: Masaki
authors.initials: M
authors.lastname: Ieda
authors.forename: Deepak
authors.initials: D
authors.lastname: Srivastava
mesh_terms: D000818:Animals; D054338:Cell Transdifferentiation / Q000235:genetics / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology*; D050991:GATA4 Transcription Factor / Q000378:metabolism; D005822:Genetic Vectors; D064326:MEF2 Transcription Factors / Q000378:metabolism; D051379:Mice; D009206:Myocardium / Q000166:cytology*; D032383:Myocytes, Cardiac / Q000166:cytology*; D012190:Retroviridae; D020825:T-Box Domain Proteins / Q000378:metabolism
chemical_list: D050991:GATA4 Transcription Factor; C484171:Gata4 protein, mouse; D064326:MEF2 Transcription Factors; C578041:Mef2c protein, mouse; D020825:T-Box Domain Proteins; C103172:T-box transcription factor 5