title: Transgenic enrichment of cardiomyocytes from human embryonic stem cells.
abstract: 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.
authors: Wolfson Centre for Stem Cells, Tissue Engineering and Modelling, University of Nottingham, Nottingham, UK.
authors: David
authors: D
authors: Anderson
authors: Tim
authors: T
authors: Self
authors: Ian R
authors: IR
authors: Mellor
authors: Gareth
authors: G
authors: Goh
authors: Stephen J
authors: SJ
authors: Hill
authors: Chris
authors: C
authors: Denning
mesh_terms: D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D019076:Transgenes / Q000235:genetics*