Mouse embryonic stem cells as a model for in vitro developmental neurotoxicity

Немецкий. Given the significant potential of chemicals and drugs to interfere with the development of the nervous system, regulatory test guidelines have been adopted for the prediction and assessment of developmental neurotoxicity (DNT; U.S.EPA OPPTS 870.6300 and OECD TG 426). However, current in vivo test methods are laborious, costly and necessitate the use of high numbers of laboratory animals. Due to a complex study design, clear and straight-forward recommendations for optimized methodological approaches in DNT testing are not available to date. In addition, under the REACH program of the European Commission it is planned to evaluate approximately 30,000 existing chemicals for their toxicological properties. Prediction of developmental neurotoxic effects is a key feature in the toxicological profile of a compound and triggered DNT studies are recommended under REACH (ECHA, May, 2008). This situation certainly will dramatically increase the number of laboratory animals used for toxicity testing in Europe. Conversely, validated alternative methods for developmental neurotoxicity testing are still not available to date. Thus, standardized, predictive screens for the evaluation of developmental neurotoxicity soon need to be made available with the ultimate goal of increased efficiency in terms of reduced animal use and higher throughput compared to whole-animal testing according to existing guidelines.Using the mouse embryonic stem cell line D3, we established a new, rapid and reliable protocol of efficient neurogenesis in vitro (e.g. neuronal precursors, post-mitotic neurons astrocytes and oligodendrocytes) specifically adapted for testing of chemicals and other compounds. Applying this protocol, differentiation can be achieved in a defined medium throughout the entire differentiation process and re-plating of cells during chemical exposure can be completely omitted.The formation of neural cells during differentiation of D3 cells was quantified by analysis of both neuron- and glial-specific marker protein expression using flow cytometry. To confirm cell-type specificity, developing neural cells were examined by immunofluorescence staining. In terms of a higher throughput, the protocol was also adapted to a 96-well plate format. As a result we were able to establish predictive toxicological endpoints for proliferation and neural cell differentiation suited to detect adverse effects of chemicals on the developing nervous system. Preliminary chemical testing revealed differences in the sensitivity of stem cells, 3T3 fibroblasts, and stem cells differentiating into neurons when compared to each other. Furthermore, we could show that the mouse embryonic stem cell model provides trustful and reliable results in the detection of developmental neurotoxicants.Overall our results suggest that differentiation of murine embryonic stem cells into neural cells might prove useful as a model in the assessment of developmental neurotoxicity in vitro.