Establishing predictive molecular markers of differentiation as toxicological endpoints in the embryonic stem cell test (EST)

In the field of reproductive toxicity mandatory test guidelines require in vivo experiments for the detection of the embryotoxic potential of chemicals and drugs. A promising alternative method for these purposes has been provided with the Embryonic Stem Cell Test (EST). This assay is based on the capacity of murine embryonic stem cells (ES cells) to differentiate in vitro into a variety of cell types. The EST is able to assess the embryotoxic potential of chemicals by the evaluation of inhibitory effects on differentiation of contracting myocardial cells which can be detected by microscopical analysis. Using a biostatistical prediction model (PM) the assay passed an international validation study and was able to predict the embryotoxic potential of test chemicals with an accuracy of 78%.A joint project was carried out by ZEBET and German pharmaceutical companies to improve the EST by establishing molecular endpoints of differentiation. Cardiac-specific gene expression has been studied at protein and RNA levels by flow cytometry and real-time-PCR under the influence of 10 chemicals with different embryotoxic potentials. The results obtained using cardiac-specific molecular endpoints were comparable to the validated microscopic analysis of beating areas and led to the same predictive outcome. The data clearly demonstrated that the selected molecular markers provide objective endpoints of early embryonic differentiation and are able to predict developmental toxicity in vivo from in vitro data for reference compounds.In conclusion, a modified EST holds promise to be a new predictive screening system for hazard assessment with regard to developmental toxicity.The first two authors contribute equally.