Derivation of functional ventricular cardiomyocytes using endogenous promoter sequence from murine embryonic stem cells.
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Derivation of functional ventricular cardiomyocytes using endogenous promoter sequence from murine embryonic stem cells. / Lee, Min Young; Sun, Baonan; Schliffke, Simon; Yue, Zhichao; Ye, Mingyu; Paavola, Jere; Bozkulak, Esra Cagavi; Amos, Peter J; Ren, Yongming; Ju, Rong; Jung, Yong Woo; Ge, Xin; Yue, Lixia; Ehrlich, Barbara E; Qyang, Yibing.
in: STEM CELL RES, Jahrgang 8, Nr. 1, 1, 2012, S. 49-57.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Derivation of functional ventricular cardiomyocytes using endogenous promoter sequence from murine embryonic stem cells.
AU - Lee, Min Young
AU - Sun, Baonan
AU - Schliffke, Simon
AU - Yue, Zhichao
AU - Ye, Mingyu
AU - Paavola, Jere
AU - Bozkulak, Esra Cagavi
AU - Amos, Peter J
AU - Ren, Yongming
AU - Ju, Rong
AU - Jung, Yong Woo
AU - Ge, Xin
AU - Yue, Lixia
AU - Ehrlich, Barbara E
AU - Qyang, Yibing
PY - 2012
Y1 - 2012
N2 - 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 ?-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.
AB - 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 ?-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.
KW - Animals
KW - Mice
KW - Base Sequence
KW - Flow Cytometry
KW - Imaging, Three-Dimensional
KW - Cell Line
KW - Cell Separation
KW - Action Potentials/physiology
KW - Calcium/metabolism
KW - Adherens Junctions/metabolism
KW - Bacterial Proteins/metabolism
KW - Cadherins/metabolism
KW - Cell Culture Techniques/methods
KW - Connexin 43/metabolism
KW - Embryonic Stem Cells/cytology/metabolism
KW - Gap Junctions/metabolism
KW - Heart Ventricles/cytology
KW - Integrases/metabolism
KW - Luminescent Proteins/metabolism
KW - Myocytes, Cardiac/cytology/metabolism
KW - Myosin Light Chains/metabolism
KW - Promoter Regions, Genetic/genetics
KW - Proteins/metabolism
KW - Animals
KW - Mice
KW - Base Sequence
KW - Flow Cytometry
KW - Imaging, Three-Dimensional
KW - Cell Line
KW - Cell Separation
KW - Action Potentials/physiology
KW - Calcium/metabolism
KW - Adherens Junctions/metabolism
KW - Bacterial Proteins/metabolism
KW - Cadherins/metabolism
KW - Cell Culture Techniques/methods
KW - Connexin 43/metabolism
KW - Embryonic Stem Cells/cytology/metabolism
KW - Gap Junctions/metabolism
KW - Heart Ventricles/cytology
KW - Integrases/metabolism
KW - Luminescent Proteins/metabolism
KW - Myocytes, Cardiac/cytology/metabolism
KW - Myosin Light Chains/metabolism
KW - Promoter Regions, Genetic/genetics
KW - Proteins/metabolism
M3 - SCORING: Journal article
VL - 8
SP - 49
EP - 57
JO - STEM CELL RES
JF - STEM CELL RES
SN - 1873-5061
IS - 1
M1 - 1
ER -
