title: An optimized and simplified system of mouse embryonic stem cell cardiac differentiation for the assessment of differentiation modifiers.
abstract: Generating cardiomyocytes from embryonic stem cells is an important technique for understanding cardiovascular development, the origins of cardiovascular diseases and also for providing potential reagents for cardiac repair. Numerous methods have been published but often are technically challenging, complex, and are not easily adapted to assessment of specific gene contributions to cardiac myocyte differentiation. Here we report the development of an optimized protocol to induce the differentiation of mouse embryonic stem cells to cardiac myocytes that is simplified and easily adapted for genetic studies. Specifically, we made four critical findings that distinguish our protocol: 1) mouse embryonic stem cells cultured in media containing CHIR99021 and PD0325901 to maintain pluripotency will efficiently form embryoid bodies containing precardiac mesoderm when cultured in these factors at a reduced dosage, 2) low serum conditions promote cardiomyocyte differentiation and can be used in place of commercially prepared StemPro nutrient supplement, 3) the Wnt inhibitor Dkk-1 is dispensable for efficient cardiac differentiation and 4) tracking differentiation efficiency may be done with surface expression of PDGFRα alone. In addition, cardiac mesodermal precursors generated by this system can undergo lentiviral infection to manipulate the expression of specific target molecules to assess effects on cardiac myocyte differentiation and maturation. Using this approach, we assessed the effects of CHF1/Hey2 on cardiac myocyte differentiation, using both gain and loss of function. Overexpression of CHF1/Hey2 at the cardiac mesoderm stage had no apparent effect on cardiac differentiation, while knockdown of CHF1/Hey2 resulted in increased expression of atrial natriuretic factor and connexin 43, suggesting an alteration in the phenotype of the cardiomyocytes. In summary we have generated a detailed and simplified protocol for generating cardiomyocytes from mES cells that is optimized for investigating factors that affect cardiac differentiation.
authors.affiliation: Division of Cardiology, Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Hartman
authors.affiliation: Division of Cardiology, Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Librande
authors.affiliation: Division of Cardiology, Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Ivan O
authors.initials: IO
authors.lastname: Medvedev
authors.affiliation: Division of Cardiology, Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Rabiah N
authors.initials: RN
authors.lastname: Ahmad
authors.affiliation: Division of Cardiology, Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Farid
authors.initials: F
authors.lastname: Moussavi-Harami
authors.affiliation: Division of Cardiology, Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Pritha P
authors.initials: PP
authors.lastname: Gupta
authors.affiliation: Division of Cardiology, Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Wei-Ming
authors.initials: WM
authors.lastname: Chien
authors.affiliation: Division of Cardiology, Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Chin
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000172:deficiency / Q000235:genetics; D055415:Bone Morphogenetic Protein 4 / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002470:Cell Survival; D058732:Embryoid Bodies / Q000166:cytology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000502:physiology; D008648:Mesoderm / Q000166:cytology / Q000821:virology; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology*; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000378:metabolism; D012097:Repressor Proteins / Q000172:deficiency / Q000235:genetics; D044967:Serum / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D055415:Bone Morphogenetic Protein 4; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; D020796:Receptor, Platelet-Derived Growth Factor alpha; D012097:Repressor Proteins; D014157:Transcription Factors; C525810:Bmp4 protein, mouse; C485778:Hey2 protein, mouse; C484469:Nkx2-5 protein, mouse