Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts.

This study describes a simple approach to generate relatively pure cultures of cardiomyocytes from differentiating murine embryonic stem (ES) cells. A fusion gene consisting of the alpha-cardiac myosin heavy chain promoter and a cDNA encoding aminoglycoside phosphotransferase was stably transfected into pluripotent ES cells. The resulting cell lines were differentiated in vitro and subjected to G418 selection. Immunocytological and ultrastructural analyses demonstrated that the selected cardiomyocyte cultures (> 99% pure) were highly differentiated. G418 selected cardiomyocytes were tested for their ability to form grafts in the hearts of adult dystrophic mice. The fate of the engrafted cells was monitored by antidystrophin immunohistology, as well as by PCR analysis with primers specific for the myosin heavy chain-aminoglycoside phosphotransferase transgene. Both analyses revealed the presence of ES-derived cardiomyocyte grafts for as long as 7 wk after implantation, the latest time point analyzed. These studies indicate that a simple genetic manipulation can be used to select essentially pure cultures of cardiomyocytes from differentiating ES cells. Moreover, the resulting cardiomyocytes are suitable for the formation of intracardiac grafts. This selection approach should be applicable to all ES-derived cell lineages.

[1]  M. Soonpaa,et al.  Potential Approaches For Cell-Mediated Myocardial Repair , 1998 .

[2]  Austin G Smith,et al.  Culture and differentiation of embryonic stem cells , 1991 .

[3]  Ha Won Kim,et al.  Mouse phospholamban gene expression during development in vivo and in vitro. , 1992, Circulation research.

[4]  R. Hruban,et al.  Arterial Delivery of Genetically Labelled Skeletal Myoblasts to the Murine Heart: Long-Term Survival and Phenotypic Modification of Implanted Myoblasts , 1996 .

[5]  P. Howles,et al.  Developmental analysis of tropomyosin gene expression in embryonic stem cells and mouse embryos , 1993, Molecular and cellular biology.

[6]  L. Field.  Transgenic mice in cardiovascular research. , 1993, Annual review of physiology.

[7]  M. Soonpaa,et al.  DNA synthesis and multinucleation in embryonic stem cell-derived cardiomyocytes. , 1995, The American journal of physiology.

[8]  J. Seidman,et al.  Production of homozygous mutant ES cells with a single targeting construct , 1992, Molecular and cellular biology.

[9]  R. C. Chiu,et al.  Cellular cardiomyoplasty: myocardial regeneration with satellite cell implantation. , 1995, The Annals of thoracic surgery.

[10]  Jürgen Hescheler,et al.  Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types , 1993, Mechanisms of Development.

[11]  J. Thomson,et al.  Isolation of a primate embryonic stem cell line. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[12]  R. Arceci,et al.  Localized expression of the atrial natriuretic factor gene during cardiac embryogenesis. , 1987, Genes & development.

[13]  G. Koh,et al.  Differentiation and long-term survival of C2C12 myoblast grafts in heart. , 1993, The Journal of clinical investigation.

[14]  T. Doetschman,et al.  Myosin heavy chain gene expression in mouse embryoid bodies. An in vitro developmental study. , 1991, The Journal of biological chemistry.

[15]  L. Field,et al.  Hypotension in transgenic mice expressing atrial natriuretic factor fusion genes. , 1990, Hypertension.

[16]  D. Zipes,et al.  Stable fetal cardiomyocyte grafts in the hearts of dystrophic mice and dogs. , 1995, The Journal of clinical investigation.

[17]  R Kemler,et al.  The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. , 1985, Journal of embryology and experimental morphology.

[18]  G. Koh,et al.  Long-term survival of AT-1 cardiomyocyte grafts in syngeneic myocardium. , 1993, The American journal of physiology.

[19]  W. Claycomb,et al.  Cardiomyocyte Transfer into the Mammalian Heart , 1995, Annals of the New York Academy of Sciences.

[20]  G. Lyons,et al.  In vitro chamber specification during embryonic stem cell cardiogenesis. Expression of the ventricular myosin light chain-2 gene is independent of heart tube formation. , 1993, The Journal of biological chemistry.

[21]  V. Chapman,et al.  The Frequency of Revertants in mdx Mouse Genetic Models for Duchenne Muscular Dystrophy , 1992, Pediatric Research.

[22]  L. Samuelson,et al.  Transition in cardiac contractile sensitivity to calcium during the in vitro differentiation of mouse embryonic stem cells , 1994, The Journal of cell biology.

[23]  G. Wallukat,et al.  Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. , 1991, Differentiation; research in biological diversity.

[24]  G. Koh,et al.  Formation of nascent intercalated disks between grafted fetal cardiomyocytes and host myocardium. , 1994, Science.

[25]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[26]  P. Boer.  Activation of the gene for type-b natriuretic factor in mouse stem cell cultures induced for cardiac myogenesis. , 1994, Biochemical and biophysical research communications.

[27]  J. Gulick,et al.  Isolation and characterization of the mouse cardiac myosin heavy chain genes. , 1991, The Journal of biological chemistry.

[28]  G. Koh,et al.  Targeted expression of transforming growth factor-beta 1 in intracardiac grafts promotes vascular endothelial cell DNA synthesis. , 1995, The Journal of clinical investigation.

[29]  H. Oberlander.  Tissue Culture Methods , 1980 .