Towards an in vitro test system for developmental neurotoxicity: a new test method using mouse embryonic stem cells

Today, on a large number of chemicals we lack information on their adverse effects on the developing nervous system. Existing in vivo test methods are time consuming, laborious, expensive and require high numbers of laboratory animals. Thus, standardized, predictive in vitro screens for the evaluation of developmental neurotoxicity need to be available. The long-term goal is to increase efficiency in terms of reduced animal use and higher throughput compared to whole-animal testing using the existing regulatory guidelines. In this study we established a method for differentiation of mouse embryonic stem cells into neural cells, designed with special regard to the testing of chemicals. It is based on a modified protocol for adherent monolayer cultures in a defined medium and offers the advantage of a reproducible development of neural cells in a comparatively short time. The differentiation of D3 cells into neural cells was determined by analysis of neuron-specific marker protein expression using flow cytometry. In addition, the developing neurons were characterized by immunofluorescence staining using neuron- and glia-specific antibodies. In this way, we also identified neurotransmitter subtypes. As a result, we were able to define neural-specific molecular endpoints for the detection of chemical effects on neural development. To investigate the assay performance, we assessed concentration-dependent effects on neurons and glial cells using a limited number of model substances. This in vitro method might prove to be a useful component of a more complex modular in vitro test system assessing the impact of neurotoxicants on the developing nervous system.