The embryonic stem cell test (EST) represents a reliable, scientifically validated in vitro system for the detection and classification of compounds according to their teratogenic potency. However, some serious issues were frequently raised against the widespread implementation and practicability of the EST in its original version. Most importantly, the evaluation of the morphological endpoint of beating cell agglomerates requires extensive experimental experience and is prone to misjudgment. Also, the testing period of 10 days is too long and costly to be attractive for industries interested in high-throughput screening of potential drug candidates. These drawbacks prompted us to work out a new molecular approach based on analysis of the expression of certain marker proteins specific for developing heart tissue. We have previously reported that quantitative flow cytometry of marker proteins (i.e., sarcomeric myosin heavy chain and alpha-actinin) can be performed at day 7 in embryonic stem cells from mice and combined with concurrent cell viability analysis. In the present study, extensive investigations were performed in order to explore the predictive power and validity of the newly established EST, subsequently referred to as molecular fluorescence activated cell sorting (FACS)-EST, by applying and comparing a set of 10 well-known embryotoxicants that encompasses the full range of chemical inherent embryotoxic potencies possible. While the molecular FACS-EST offered the same sensitivity compared to the validated EST protocol, the test duration could be significantly reduced. Due to significant improvements, this new molecular method holds promise as a sensitive, more rapid and reproducible screen highly suited to predict developmental toxicity in vivo from in vitro data.
The embryonic stem cell test (EST) takes advantage of the potential of murine embryonic stem (ES) cells to differentiate in culture to test embryotoxicity in vitro. The EST represents a scientifically validated in vitro system for the classification of compounds according to their teratogenic potential based on the morphological analysis of beating cardiomyocytes in embryoid body outgrowths compared to cytotoxic effects on murine ES cells and differentiated 3T3 fibroblasts. Through a number of prevalidation and validation studies, the EST has been demonstrated to be a reliable alternative method for embryotoxicity testing based on the most important mechanisms in embryotoxicity-cytotoxicity and differentiation--as well as on differences in sensitivity between differentiated and embryonic tissues. Improvements of the EST protocol using flow cytometry analysis showed that differential expression of sarcomeric myosin heavy chain and alpha-actinin proteins quantified under the influence of a test compound is a useful marker for detecting potential teratogenicity. The in vitro embryotoxicity test described in this chapter is rapid, simple, and sensitive and can be usefully employed as a component of the risk/hazard assessment process.
Teratogenicity can be predicted in vitro using the embryonic stem cell test (EST). The EST, which is based on the morphometric measurement of cardiomyocyte differentiation and cytotoxicity parameters, represents a scientifically validated method for the detection and classification of chemicals according to their teratogenic potency. Furthermore, an abbreviated protocol applying flow cytometry of intracellular marker proteins to determine differentiation into the cardiomyocyte lineage is available. Although valproic acid (VPA) is in worldwide clinical use as antiepileptic drug, it exhibits two severe side effects, i.e., teratogenicity and hepatotoxicity. These limitations have led to extensive research into derivatives of VPA. Here we chose VPA as model compound to test the applicability domain and to further evaluate the reliability of the EST. To this end, we study six closely related congeners of VPA and demonstrate that both the standard and the molecular flow cytometry-based EST are well suited to indicate differences in the teratogenic potency among VPA analogs that differ only in chirality or side chain length. Our data show that identical results can be obtained by using the standard EST or a shortened protocol based on flow cytometry of intracellular marker proteins. Both in vitro protocols enable to reliably determine differentiation of murine stem cells toward the cardiomyocyte lineage and to assess its chemical-mediated inhibition.
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