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Derivation of functional ventricular cardiomyocytes using endogenous promoter sequence from murine embryonic stem cells

Authors
Lee, Min YoungSun, BaonanSchliffke, SimonYue, ZhichaoYe, MingyuPaavola, JereBozkulak, Esra CagaviAmos, Peter J.Ren, YongmingJu, RongJung, Yong WooGe, XinYue, LixiaEhrlich, Barbara E.Qyang, Yibing
Issue Date
1월-2012
Publisher
ELSEVIER SCIENCE BV
Citation
STEM CELL RESEARCH, v.8, no.1, pp.49 - 57
Indexed
SCIE
SCOPUS
Journal Title
STEM CELL RESEARCH
Volume
8
Number
1
Start Page
49
End Page
57
URI
https://scholar.korea.ac.kr/handle/2021.sw.korea/106247
DOI
10.1016/j.scr.2011.08.004
ISSN
1873-5061
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 [Ca2+](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 beta-adrenergic signaling. Moreover, [Ca2+](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. (C) 2011 Elsevier B.V. All rights reserved.
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