Identification and characterization of embryonic stem cell-derived pacemaker and atrial cardiomyocytes.
The aim of this study was to identify and functionally characterize cardiac subtypes during early stages of development. For this purpose, transgenic embryonic stem cells were generated using the alpha-myosin heavy chain promoter driving the expression of the enhanced green fluorescent protein (EGFP). EGFP-positive clusters of cells were first observed as early as 7 days of development, thus, even before the initiation of the contractile activity. Flow cytometry and single-cell fluorescence measurements evidenced large diversities of EGFP intensity. Patch-clamp experiments showed EGFP expression exclusively in pacemaker and atrial but not ventricular cells. The highest fluorescence intensities were detected in pacemaker-like cardiomyocytes. In accordance, multielectrode-array recordings of whole embryoid bodies confirmed that the pacemaker center coincided with strongly EGFP-positive areas. The cardiac subtypes displayed already at this early stage differential characteristics of electrical activity and ion channel expression. Thus, quantitation of the alpha-myosin heavy chain driven reporter gene expression allows identification and functional characterization of early cardiac subtypes.
Kolossov
E
E
Axiogenesis AG, Cologne, Germany. eugen.kolossov@axiogenesis.com
Lu
Z
Z
Drobinskaya
I
I
Gassanov
N
N
Duan
Y
Y
Sauer
H
H
Manzke
O
O
Bloch
W
W
Bohlen
H
H
Hescheler
J
J
Fleischmann
B K
BK
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002454:Cell Differentiation; D002469:Cell Separation; D002999:Clone Cells; D004594:Electrophysiology; D004622:Embryo, Mammalian / Q000166:cytology*; D005434:Flow Cytometry; D005453:Fluorescence; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006325:Heart Atria / Q000166:cytology* / Q000196:embryology; D006329:Heart Conduction System / Q000166:cytology* / Q000196:embryology; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D018721:Muscarinic Agonists / Q000494:pharmacology; D032383:Myocytes, Cardiac / Q000145:classification / Q000166:cytology* / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D013234:Stem Cells / Q000145:classification / Q000166:cytology* / Q000378:metabolism; D014162:Transfection; D024723:Ventricular Myosins / Q000235:genetics
D018721:Muscarinic Agonists; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002217:Carbachol; D024723:Ventricular Myosins; D018995:Myosin Heavy Chains