Directed Differentiation of Human Embryonic Stem Cells to Interrogate the Cardiac Gene Regulatory Network

• Published in: Molecular therapy Vol. 19; no. 9; pp. 1695 - 1703
• Main Authors: Dixon, James E, Dick, Emily, Rajamohan, Divya, Shakesheff, Kevin M, Denning, Chris
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-09-2011; Elsevier Limited; Nature Publishing Group
• Subjects: Actinin - metabolism; Cardiomyocytes; Cell Differentiation; Cell Line; Chromosomal Proteins, Non-Histone; Cloning, Molecular; Cytarabine - pharmacology; Embryonic Stem Cells - cytology; Embryonic Stem Cells - metabolism; Epigenetics; Flow Cytometry; Fluorescent Antibody Technique; GATA4 Transcription Factor - genetics; GATA4 Transcription Factor - metabolism; Gene Expression Regulation; Gene Regulatory Networks; Genes; Genes, Reporter; Genetic Vectors; Heart; Heart - embryology; Homeobox Protein Nkx-2.5; Homeodomain Proteins - genetics; Homeodomain Proteins - metabolism; Humans; Lentivirus - genetics; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Morphology; Myocytes, Cardiac - cytology; Original; Proteins; Red Fluorescent Protein; Stem cells; T-Box Domain Proteins - genetics; T-Box Domain Proteins - metabolism; Tissue engineering; Transcription Factors - genetics; Transcription Factors - metabolism; United Kingdom > UK
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1038/mt.2011.125

The limited ability of the heart to regenerate has prompted development of new systems to produce cardiomyocytes for therapeutics. While differentiation of human embryonic stem cells (hESCs) into cardiomyocytes has been well documented, the process remains inefficient and/or expensive, and progress would be facilitated by better understanding the early genetic events that cause cardiac specification. By maintaining a transgenic cardiac-specific MYH6-monomeric red fluorescent protein (mRFP) reporter hESC line in conditions that promote pluripotency, we tested the ability of combinations of 15 genes to induce cardiac specification. Screening identified GATA4 plus TBX5 as the minimum requirement to activate the cardiac gene regulatory network and produce mRFP+ cells, while a combination of GATA4, TBX5, NKX2.5, and BAF60c (GTNB) was necessary to generate beating cardiomyocytes positive for cTnI and α-actinin. Including the chemotherapeutic agent, Ara-C, from day 10 of induced differentiation enriched for cTnI/α-actinin double positive cells to 45%. Transient expression of GTNB for 5–7 days was necessary to activate the cardiogenesis through progenitor intermediates in a manner consistent with normal heart development. This system provides a route to test the effect of different factors on human cardiac differentiation and will be useful in understanding the network failures that underlie disease phenotypes.