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.

journal_name

Stem Cell Res

journal_title

Stem cell research

authors

Lee MY,Sun B,Schliffke S,Yue Z,Ye M,Paavola J,Bozkulak EC,Amos PJ,Ren Y,Ju R,Jung YW,Ge X,Yue L,Ehrlich BE,Qyang Y

doi

10.1016/j.scr.2011.08.004

subject

Has Abstract

pub_date

2012-01-01 00:00:00

pages

49-57

issue

1

eissn

1873-5061

issn

1876-7753

pii

S1873-5061(11)00110-3

journal_volume

8

pub_type

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