Title of article :
Cardiomyocytes purified from differentiated embryonic stem cells exhibit characteristics of early chamber myocardium
Author/Authors :
Arnoud C. Fijnvandraat، نويسنده , , Antoni C. G. van Ginneken، نويسنده , , Cees A. Schumacher، نويسنده , , Kenneth R. Boheler، نويسنده , , Ronald H. Lekanne Deprez، نويسنده , , Vincent M. Christoffels، نويسنده , , Antoon F. M. Moorman، نويسنده ,
Issue Information :
روزنامه با شماره پیاپی سال 2003
Pages :
12
From page :
1461
To page :
1472
Abstract :
Mouse embryonic stem (ES) cells easily differentiate towards the cardiac lineage making them suitable as an in vitro model to study cardiogenesis and as a potential source of transplantable cells. In this study, we show by in situ hybridisation that about 30% of the volume of cultures of differentiating ES cells consists of cardiomyocytes. RT-PCR analyses showed that the transcription factors Nkx2.5, Gata4, Mef2c and Irx4 were expressed at levels in the same order of magnitude as the levels observed in embryonic, neonatal and adult hearts. Atrial natriuretic factor and Connexin 40, associated with chamber formation in vivo, are expressed at relatively low levels, similar to those observed at early heart development in vivo. To facilitate the isolation of ES cell-derived cardiomyocytes, a cell line was constructed by stable transfection of the aminoglycoside phosphotransferase cDNA driven by the cardiac-specific distant upstream part of the Na+/Ca2+ exchanger promoter. To accomplish single-copy integration, the construct was inserted into the hypoxanthine phosphoribosyltransferase locus of HM1 ES cells by homologous recombination. Cardiac-specific resistance to G418-sulphate (neomycin) allowed isolation of a pure population of cardiomyocytes. Genetically selected and unselected cell populations were characterised electrophysiologically using patch clamp. To explore whether clusters of cells have a similar differentiation profile, action potentials (APs) were measured in aggregates of differentiating ES cells, using a new method based on the voltage-dependent fluorescent dye di-4-ANEPPS. Both whole-cell recordings using patch-clamp and optical measurements with di-4-ANEPPS of the AP showed that upstroke velocity increases and AP duration decreases with differentiation time, accompanied by a decrease in AP interval, suggesting the initiation of the developmental programme underlying the formation of chamber myocardium.
Keywords :
In vitro differentiation , Embryonic stem cells , Transcription factors , Chamber formation , Genetic selection , NCX1 , ANEPPS
Journal title :
Journal of Molecular and Cellular Cardiology
Serial Year :
2003
Journal title :
Journal of Molecular and Cellular Cardiology
Record number :
528885
Link To Document :
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