ToxSci Advance Access originally published online on January 23, 2009
Toxicological Sciences 2009 108(2):389-400; doi:10.1093/toxsci/kfp012
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Embryonic Stem Cell Test Remastered: Comparison between the Validated EST and the New Molecular FACS-EST for Assessing Developmental Toxicity In Vitro
German Federal Institute for Risk Assessment (BfR), Center for Alternative Methods to Animal Experiments - ZEBET, 12277 Berlin, Germany
2 To whom correspondence should be addressed at German Federal Institute for Risk Assessment, Center for Alternative Methods to Animal Experiments – ZEBET, Diedersdorfer Weg 1, 12277 Berlin, Germany. Fax: +49-(0)30-8412 2958. E-mail: Andrea.Seiler{at}bfr.bund.de.
Received December 3, 2008; accepted January 14, 2009
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Abstract |
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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 
-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.
Key Words: in vitro; embryotoxicity; embryonic stem cell test; differentiation; cytotoxicity; developmental toxicity; prediction model; flow cytometry.
1 These authors contributed equally.
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