Transgenic enrichment of cardiomyocytes from human embryonic stem cells.

To realize the full scientific and clinical potential of human embryonic stem cell (hESC)-cardiomyocytes, strategies to overcome the high degree of heterogeneity of differentiated populations are required. Here we demonstrate the utility of two transgenic approaches in enrichment of cardiomyocytes derived from HUES-7 cells: (i) negative selection of proliferating cells with the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system; and (ii) positive selection of cardiomyocytes expressing a bicistronic reporter [green fluorescent protein (GFP)-internal ribosome entry site (IRES)-puromycin-N-acetyltransferase (PAC)] from the human alphamyosin heavy chain promoter. Parental and transgenic HUES-7 cells were similar with regard to morphology, pluripotency marker expression, differentiation, and cardiomyocyte electrophysiology. Whereas immunostaining of dissociated cardiomyocyte preparations expressing HSVtk or PAC contained <7% cardiomyocytes, parallel cultures treated with GCV or puromycin, respectively, contained 33.4 +/- 2.1% or 91.5 +/- 4.3% cardiomyocytes corresponding to an enrichment factor of 6.7- or 14.5-fold. Drug-selected cardiomyocytes responded to chronotropic stimulation and displayed cardiac-specific action potentials, demonstrating that functionality was retained. Both transgenic strategies will be generically applicable and should readily translate to the enrichment of many other differentiated lineages derived from hESCs.

[1]  A M Wobus,et al.  Selection of ventricular‐like cardiomyocytes from ES cells in vitro , 2000, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[2]  J. R. Smith,et al.  Intraclonal variation in proliferative potential of human diploid fibroblasts: stochastic mechanism for cellular aging. , 1980, Science.

[3]  Austin G Smith,et al.  Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture , 2003, Nature Biotechnology.

[4]  Johannes Gerdes,et al.  The Ki‐67 protein: From the known and the unknown , 2000, Journal of cellular physiology.

[5]  B. Lawrenz,et al.  Highly sensitive biosafety model for stem-cell-derived grafts. , 2004, Cytotherapy.

[6]  C. Denning,et al.  Bystander effects of different enzyme-prodrug systems for cancer gene therapy depend on different pathways for intercellular transfer of toxic metabolites, a factor that will govern clinical choice of appropriate regimes. , 1997, Human gene therapy.

[7]  J. Itskovitz‐Eldor,et al.  Molecular analysis of cardiomyocytes derived from human embryonic stem cells , 2005, Development, growth & differentiation.

[8]  S. Harding,et al.  The human embryonic stem cell-derived cardiomyocyte as a pharmacological model. , 2007, Pharmacology & therapeutics.

[9]  A. Moorman,et al.  Chamber formation and morphogenesis in the developing mammalian heart. , 2000, Developmental biology.

[10]  R. Bronson,et al.  Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene , 1991, Cell.

[11]  Takumi Miura,et al.  Properties of four human embryonic stem cell lines maintained in a feeder‐free culture system , 2004, Developmental dynamics : an official publication of the American Association of Anatomists.

[12]  M. Schuldiner,et al.  Selective Ablation of Human Embryonic Stem Cells Expressing a “Suicide” Gene , 2003, Stem cells.

[13]  J. Hescheler,et al.  Beta-adrenergic and Muscarinic Modulation of Human Embryonic Stem Cell-derived Cardio-myocytes , 2004, Cellular Physiology and Biochemistry.

[14]  J. Thomson,et al.  Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells , 2004, Nature Biotechnology.

[15]  James A Thomson,et al.  Human Embryonic Stem Cells Develop Into Multiple Types of Cardiac Myocytes: Action Potential Characterization , 2003, Circulation research.

[16]  F. Nishimura,et al.  Potential Use of Embryonic Stem Cells for the Treatment of Mouse Parkinsonian Models: Improved Behavior by Transplantation of In Vitro Differentiated Dopaminergic Neurons from Embryonic Stem Cells , 2003, Stem cells.

[17]  Robert Passier,et al.  Genome‐Wide Transcriptional Profiling of Human Embryonic Stem Cells Differentiating to Cardiomyocytes , 2006 .

[18]  L Gepstein,et al.  Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. , 2001, The Journal of clinical investigation.

[19]  J. Fletcher,et al.  Deletion of the α(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep , 2001, Nature Biotechnology.

[20]  P. Burridge,et al.  Improved Human Embryonic Stem Cell Embryoid Body Homogeneity and Cardiomyocyte Differentiation from a Novel V‐96 Plate Aggregation System Highlights Interline Variability , 2007, Stem cells.

[21]  Roberto Bolli,et al.  Life and Death of Cardiac Stem Cells: A Paradigm Shift in Cardiac Biology , 2006, Circulation.

[22]  E. Huang,et al.  Unique spectrum of activity of 9-[(1,3-dihydroxy-2-propoxy)methyl]-guanine against herpesviruses in vitro and its mode of action against herpes simplex virus type 1. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[23]  L. V. Van Laake,et al.  Human embryonic stem cells: genetic manipulation on the way to cardiac cell therapies. , 2005, Reproductive toxicology.

[24]  G. Koh,et al.  Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. , 1996, The Journal of clinical investigation.

[25]  Robert Passier,et al.  Increased Cardiomyocyte Differentiation from Human Embryonic Stem Cells in Serum‐Free Cultures , 2005, Stem cells.

[26]  W. Freed,et al.  Karyotypic stability, genotyping, differentiation, feeder-free maintenance, and gene expression sampling in three human embryonic stem cell lines derived prior to August 9, 2001. , 2004, Stem cells and development.

[27]  Stefan Przyborski,et al.  An Autogeneic Feeder Cell System That Efficiently Supports Growth of Undifferentiated Human Embryonic Stem Cells , 2005, Stem cells.

[28]  Seung Jun Yoo,et al.  Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment. , 2006, Differentiation; research in biological diversity.

[29]  I. Germano,et al.  Adenovirus/Herpes Simplex-Thymidine Kinase/Ganciclovir Complex: Preliminary Results of a Phase I trial in Patients with Recurrent Malignant Gliomas , 2003, Journal of Neuro-Oncology.

[30]  S. Grant,et al.  Expression of Transgenes Targeted to the Gt(ROSA)26Sor Locus Is Orientation Dependent , 2006, PloS one.

[31]  C. Férec,et al.  Efficient gene transfer into human epithelial cell lines using glycosylated cationic carriers and neutral glycosylated co-lipids. , 2004 .

[32]  T. Self,et al.  Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and HUES-7. , 2006, The International journal of developmental biology.

[33]  M. Schuldiner,et al.  Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells , 2001, Current Biology.

[34]  J. Itskovitz‐Eldor,et al.  Assessment of the ultrastructural and proliferative properties of human embryonic stem cell-derived cardiomyocytes. , 2003, American journal of physiology. Heart and circulatory physiology.

[35]  Arnold Munnich,et al.  Mutation in myosin heavy chain 6 causes atrial septal defect , 2005, Nature Genetics.

[36]  Chunhui Xu,et al.  Characterization and Enrichment of Cardiomyocytes Derived From Human Embryonic Stem Cells , 2002, Circulation research.

[37]  Yue-zhong Wu,et al.  Restriction landmark genome scanning identifies culture-induced DNA methylation instability in the human embryonic stem cell epigenome. , 2007, Human molecular genetics.

[38]  B. Fleischmann,et al.  Identification and characterization of embryonic stem cell‐derived pacemaker and atrial cardiomyocytes , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[39]  Chad A. Cowan,et al.  Derivation of embryonic stem-cell lines from human blastocysts. , 2004, The New England journal of medicine.