Establishment of an embryotoxicity assay with green fluorescence protein-expressing embryonic cell-derived cardiomyocytes

Transgenic embryonic stem cells were used to determine the embryotoxic effe cts of chemicals on the development of embryonic tissues. This investigatio n supports an ongoing validation study, aimed at reducing the time-consumin g procedure currently in use, and at providing more-objective and more-deta iled information on the embryotoxic potentials of chemicals. Green fluoresc ence protein (GFP) was used as a reporter gene and was linked to a human al pha-cardiac-specific promoter. The expression of GFP was switched on after specific activation of the human alpha-actin promoter. This permitted the e asy quantification of cardiac cells by using a fluorescence-activated cell sorter (FACS). The percentage of cardiac precursor cells was calculated fro m the FACS-distribution pattern of cells which fluoresced versus the total number of cells. The percentage of cardiac precursor cells increased from 2 5% in embryoid bodies on day 3, to 86% on day 7. However, in 11-day-old emb ryoid bodies, the percentage decreased to 35%. Five chemicals with known em bryotoxic potentials were compared with respect to the IC50 (concentration causing 50% inhibition of measured effect) values obtained by various in vi tro 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 e mbryotoxicology.