title: Derivation of functional ventricular cardiomyocytes using endogenous promoter sequence from murine embryonic stem cells.
abstract: The purpose of this study is to establish a murine embryonic stem cell (mESC) line for isolation of functional ventricular cardiomyocytes (VCMs) and then to characterize the derived VCMs. By crossing the myosin light chain 2v (Mlc2v)-Cre mouse line with the reporter strain Rosa26-yellow fluorescent protein (YFP), we generated mESC lines from these double transgenic mice, in which Cre-mediated removal of a stop sequence results in the expression of YFP under the control of the ubiquitously active Rosa26 promoter specifically in the VCM. After induction of differentiation via embryoid body (EB) formation, contracting YFP(+) cells were detected within EBs and isolated by fluorescence-activated cell sorting. N-cadherin, the cadherin expressed in cardiomyocytes, and the major cardiac connexin (Cx) isoform, Cx43, were detected in the respective adherens and gap junctions in these VCMs. Using current clamp recordings we demonstrated that mESC-derived VCMs exhibited action potential characteristics comparable to those of neonatal mouse VCMs. Real-time intracellular calcium [Ca(2+)](i) imaging showed rhythmic intracellular calcium transients in these VCMs. The amplitude and frequency of calcium transients were increased by isoproterenol stimulation, suggesting the existence of functional β-adrenergic signaling. Moreover, [Ca(2+)](i) oscillations responded to increasing frequencies of external electrical stimulation, indicating that VCMs have functional excitation-contraction coupling, a key factor for the ultimate cardiac contractile performance. The present study makes possible the production of homogeneous and functional VCMs for basic research as well as for cardiac repair and regeneration.
authors.affiliation: Section of Cardiovascular Medicine, Department of Internal Medicine, Yale School of Medicine, New Haven, CT 06520, USA.
authors.forename: Min Young
authors.initials: MY
authors.lastname: Lee
authors.forename: Baonan
authors.initials: B
authors.lastname: Sun
authors.forename: Simon
authors.initials: S
authors.lastname: Schliffke
authors.forename: Zhichao
authors.initials: Z
authors.lastname: Yue
authors.forename: Mingyu
authors.initials: M
authors.lastname: Ye
authors.forename: Jere
authors.initials: J
authors.lastname: Paavola
authors.forename: Esra Cagavi
authors.initials: EC
authors.lastname: Bozkulak
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Amos
authors.forename: Yongming
authors.initials: Y
authors.lastname: Ren
authors.forename: Rong
authors.initials: R
authors.lastname: Ju
authors.forename: Yong Woo
authors.initials: YW
authors.lastname: Jung
authors.forename: Xin
authors.initials: X
authors.lastname: Ge
authors.forename: Lixia
authors.initials: L
authors.lastname: Yue
authors.forename: Barbara E
authors.initials: BE
authors.lastname: Ehrlich
authors.forename: Yibing
authors.initials: Y
authors.lastname: Qyang
mesh_terms: D000200:Action Potentials / Q000502:physiology; D022005:Adherens Junctions / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D015820:Cadherins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D002469:Cell Separation; D018031:Connexin 43 / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D005434:Flow Cytometry; D017629:Gap Junctions / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology*; D021621:Imaging, Three-Dimensional; D019426:Integrases / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011506:Proteins / Q000378:metabolism; D022661:RNA, Untranslated
chemical_list: D001426:Bacterial Proteins; D015820:Cadherins; D018031:Connexin 43; C110853:Gt(ROSA)26Sor non-coding RNA, mouse; D008164:Luminescent Proteins; D018994:Myosin Light Chains; D011506:Proteins; D022661:RNA, Untranslated; C054562:yellow fluorescent protein, Bacteria; C045073:Cre recombinase; D019426:Integrases; D002118:Calcium
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