Trends in improving the embryonic stem cell test (EST): an overview

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Roland Buesen, Anke Visan, Elke Genschow, Birgitta Slawik, Horst Spielmann, Andrea Seiler
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The embryonic stem cell test (EST) is an in vitro assay that has been developed to assess the teratogenic and embryotoxic potential of drugs and chemicals. It is based on the capacity of murine ES cells (cell line D3) to differentiate into contracting myocardial cells under specific cell culture conditions. To assess the embryotoxic potential of a test substance the appearance of beating cardiomyocytes in embryoid body (EB) outgrowths is used as an toxicological endpoint. Applying linear analysis of discriminance, a biostatistical prediction model (PM) was developed to assign test chemicals to three classes of embryotoxicity. In an international validation study the EST predicted the embryotoxic potential of chemicals and drugs in the same manner as two other in vitro embryotoxicity tests employing embryonic cells and tissues from pregnant animals.
In a joint research project with German drug companies we have successfully improved the EST by establishing molecular endpoints of differentiation in cultured ES cells. The quantification of cardiac-specific protein expression by intracellular flow cytometry has been studied in the presence of chemicals with different embryotoxic potentials. The results obtained using molecular endpoints specific for differentiated cardiomyocytes employing FACS (fluorescence activated cell sorting) analysis will be presented in comparison to the validated endpoint - the microscopic analysis of beating areas. FACS analysis provides a more objective endpoint for predicting the embryotoxic potential of chemicals than the validated method. Furthermore, flow cytometry holds promise to be suitable for high-throughput screening systems (HTS).
In addition, our partners from the joint project have improved the EST by developing protocols that stimulate differentiation of ES cells into neural and endothelial cells, chondrocytes and osteoblasts because some substances might have embryotoxic effects on specific cell-types other than cardiomyocytes. These protocols have been successfully established at ZEBET and in the participating laboratories. Additionally, molecular endpoints have been established for the detection of specific differentiation pathways.
Furthermore, new prediction models (PMs) have been developed using single endpoints of the EST.

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How to Cite
Buesen, R. (2004) “Trends in improving the embryonic stem cell test (EST): an overview”, ALTEX - Alternatives to animal experimentation, 21(1), pp. 15–22. Available at: (Accessed: 19 June 2024).

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