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Alternatives to Laboratory Animals - ATLA

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Establishment of an Embryotoxicity Assay with Green Fluorescence Protein-expressing Embryonic Cell-derived Cardiomyocytes

Susanne Bremer, Maaike van Dooren, Martin Paparella, Eugen Kossolov, Bernd Fleischmann and Juergen Hescheler

Transgenic embryonic stem cells were used to determine the embryotoxic effects of chemicals on the development of embryonic tissues. This investigation supports an ongoing validation study, aimed at reducing the time-consuming procedure currently in use, and at providing more-objective and more-detailed information on the embryotoxic potentials of chemicals. Green fluorescence protein (GFP) was used as a reporter gene and was linked to a human α-cardiac-specific promoter. The expression of GFP was switched on after specific activation of the human α-actin promoter. This permitted the easy quantification of cardiac cells by using a fluorescence-activated cell sorter (FACS). The percentage of cardiac precursor cells was calculated from the FACS-distribution pattern of cells which fluoresced versus the total number of cells. The percentage of cardiac precursor cells increased from 25% in embryoid bodies on day 3, to 86% on day 7. However, in 11-day-old embryoid bodies, the percentage decreased to 35%. Five chemicals with known embryotoxic potentials were compared with respect to the IC50 (concentration causing 50% inhibition of measured effect) values obtained by various in vitro endpoints (for example, cytotoxicity, morphology). The results showed a higher sensitivity of endpoints used for the analysis of specific effects on the selected target tissue. The data also showed the need to develop in vitro methods with specific endpoints which account for the complexity of embryotoxicology.