The use of murine embryonic stem cells for assessing developmental neurotoxicity in vitro

allemand. Given the significant potential of chemicals and drugs to interfere with development of the nervous system, regulatory test guidelines have been adopted for the prediction andassessment of developmental neurotoxicity (U.S. EPA, OECD). However, current in vivo test methods are laborious, costly and necessitate use of high numbers of laboratory animals.Moreover, the study design is complex and clear recommendations for optimal methodological approaches in DNT studies are lacking. Thus, standardized, predictivescreens for the evaluation of developmental neurotoxicity need to be available with the ultimate goal of increased efficiency in terms of reduced animal use and higher throughputcompared to whole-animal testing using the existing guidelines.Validated alternatives, like the embryonic stem cell test (EST), are already available to assess developmental toxicity in vitro . However, the current experimental procedure is basedon differentiation of murine embryonic stem cells (D3) into contracting cardiomyocytes. Consequently, primarily developmental neurotoxicants may not be fully assessed. This consideration prompted us to expand the EST to other major target tissues including neural cells.Here, we present a new, rapid and reliable differentiation protocol for the development of neural cells, designed with special regard to the testing of chemicals and other compounds.The main advantage is that differentiation can be achieved with adherent monolayer cultures in a defined medium throughout the entire differentiation process; re-plating of cells duringchemical exposure can be completely omitted. The differentiation of D3 cells into neural cells like neuronal precursors, post-mitotic neurons and astrocytes was quantified by flowcytometry of neuron-specific marker gene expression. In addition, to confirm cell-type specificity, the developing neurons were examined by immunofluorescence staining usingneuron-specific antibodies. As a result, we were able to define neuron-specific molecular endpoints for the detection of chemical effects on development. Preliminary chemical testingrevealed differential sensitivity comparing neural cells and cardiomyocytes for a limited number of test compounds.The expansion of the EST to more than one target tissue will considerably improve the accuracy of this predictive screen by reducing false negative results. Moreover, thedifferentiation of murine embryonic stem cells into neural cells might prove to be a useful tool in assessing developmental neurotoxicity in vitro and for use in drug discovery.