title: Selection of ventricular-like cardiomyocytes from ES cells in vitro.
abstract: Ischemic disorders of the heart can cause an irreversible loss of cardiomyocytes resulting in a substantial decrease of cardiac output. The therapy of choice is heart transplantation, a technique that is hampered by the low number of donor organs. In the present study, we describe the specific labeling, rapid but gentle purification and characterization of cardiomyocytes derived from mouse pluripotent embryonic stem (ES) cells. To isolate the subpopulation of ventricular-like cardiomyocytes, ES cells were stable transfected with the enhanced green fluorescent protein (EGFP) under transcriptional control of the ventricular-specific 2.1 kb myosin light chain-2v (MLC-2v) promoter and the 0.5 kb enhancer element of the cytomegalovirus (CMV(enh).). First fluorescent cells were detected at day 6 + 8 of differentiation within EBs. Four weeks after initiation of differentiation 25% of the cardiomyocyte population displayed fluorescence. Immunohistochemistry revealed the exclusive cardiomyogenic nature of EGFP-positive cells. This was further corroborated by electrophysiological studies where preferentially ventricular phenotypes, but no pacemaker-like cardiomyocytes, were detected among the EGFP-positive population. The enzymatic digestion of EBs, followed by Percoll gradient centrifugation and fluorescence-activated cell sorting, resulted in a 97% pure population of cardiomyocytes. Based on this study, ventricular-like cardiomyocytes can be generated in vitro from EBs and labeled using CMV(enh)./MLC-2v-driven marker genes facilitating an efficient purification. This method may become an important tool for future cell replacement therapy of ischemic cardiomyopathy especially after the proof of somatic differentiation of human ES cells in vitro.
authors: Internal Medicine II, University of Lübeck, D-23538 Lübeck, Germany.
authors: M
authors: M
authors: Müller
authors: B K
authors: BK
authors: Fleischmann
authors: S
authors: S
authors: Selbert
authors: G J
authors: GJ
authors: Ji
authors: E
authors: E
authors: Endl
authors: G
authors: G
authors: Middeler
authors: O J
authors: OJ
authors: Müller
authors: P
authors: P
authors: Schlenke
authors: S
authors: S
authors: Frese
authors: A M
authors: AM
authors: Wobus
authors: J
authors: J
authors: Hescheler
authors: H A
authors: HA
authors: Katus
authors: W M
authors: WM
authors: Franz
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D004622:Embryo, Mammalian / Q000166:cytology*; D049452:Green Fluorescent Proteins; D006352:Heart Ventricles / Q000166:cytology*; D007545:Isoproterenol / Q000494:pharmacology; D008164:Luminescent Proteins; D008564:Membrane Potentials; D051379:Mice; D018721:Muscarinic Agonists / Q000494:pharmacology; D009206:Myocardium / Q000166:cytology*; D018994:Myosin Light Chains / Q000235:genetics; D018408:Patch-Clamp Techniques; D013234:Stem Cells / Q000166:cytology*; D014162:Transfection
chemical_list: D000318:Adrenergic beta-Agonists; D008164:Luminescent Proteins; D018721:Muscarinic Agonists; D018994:Myosin Light Chains; D049452:Green Fluorescent Proteins; D002217:Carbachol; D007545:Isoproterenol