Mouse embryonic stem cells in developmental neurotoxicitiy testing: aiming at higher throughput

Alemán. Testing for developmental neurotoxicity (DNT) is a key element in the toxicological portfolio of a compound and triggered DNT studies are recommended under REACH. This situation will considerably increase the number of laboratory animals used inchemical safety testing. Validated alternative methods for DNT testing are currently not available. Thus, standardised, predictive screens for the evaluation of DNT need to be established with the ultimate goal of increased efficiency in terms of reducedanimal use and higher throughput compared to whole-animal testing using existing guidelines. Mouse embryonic stem cells (mESCs) are pluripotent cells with an unlimited capacity for differentiation and thus provide an attractive cellular system for in vitro studies in developmental biology as well as toxicology.In a previous study we demonstrated that these cells differentiate efficiently into neural cells, including neural progenitors, neurons, astrocytes, oligodendrocytes and radial glial cells, and might have the potential to develop into functionally active networks.Therefore, mESCs offer the opportunity to study adverse effects on neural cell development after exposure to toxic compounds in an in vitro setting. Murine ESCs (line D3) were differentiated into neural cell types. Proliferation in differentiating and undifferentiated stem cell cultures was determined using a BrdU-ELISA system with a chemiluminescence read-out. Cell viability was analysed as the ability of the cells to reduce resazurin into the fluorescent product resorufin. For quantification of neuron differentiation the expression of the neuron-specific marker protein beta-III tubulin was assessed in a new cell-based ELISA method, which we developed specifically for this purpose.Recently, we developed an in vitro test method suitable to assess adverse effects of chemicals and other compounds on neural development. Molecular and mechanistic endpoints for differentiation and proliferation were successfully established.Furthermore, our model seemed applicable to detect a diverse group of positive and negative developmental neurotoxicants. However, it also turned out that the detection of specific neurodevelopmental effects depends on the exposure regimes used. Therefore different exposure regimes, along with the selected set of positive and negative developmental neurotoxicants, were evaluated. In this way we were able to establish the most specific and predictive endpoints for DNT testing in our model, clearlyseparating specific DNT effects from general cytotoxicity. The mESC model represents a useful component in an in vitro testing strategy for developmental neurotoxicity. The tools described here provide the possibility for a higher throughputand for the detection of developmental neurotoxic effects with increased specificity, thus facilitating application in extensive chemical testing