Combination of multiple neural crest migration assays to identify environmental toxicants from a proof-of-concept chemical library.

Many in vitro tests have been developed to screen for potential neurotoxicity. However, only few cell function-based tests have been used for comparative screening, and thus experience is scarce on how to confirm and evaluate screening hits. We addressed these questions for the neural crest cell migration test (cMINC). After an initial screen, a hit follow-up strategy was devised. A library of 75 compounds plus internal controls (NTP80-list), assembled by the National Toxicology Program of the USA (NTP) was used. It contained some known classes of (developmental) neurotoxic compounds. The primary screen yielded 23 confirmed hits, which comprised ten flame retardants, seven pesticides and six drug-like compounds. Comparison of concentration-response curves for migration and viability showed that all hits were specific. The extent to which migration was inhibited was 25-90%, and two organochlorine pesticides (DDT, heptachlor) were most efficient. In the second part of this study, (1) the cMINC assay was repeated under conditions that prevent proliferation; (2) a transwell migration assay was used as a different type of migration assay; (3) cells were traced to assess cell speed. Some toxicants had largely varying effects between assays, but each hit was confirmed in at least one additional test. This comparative study allows an estimate on how confidently the primary hits from a cell function-based screen can be considered as toxicants disturbing a key neurodevelopmental process. Testing of the NTP80-list in more assays will be highly interesting to assemble a test battery and to build prediction models for developmental toxicity.

Design of a high-throughput human neural crest cell migration assay to indicate potential developmental toxicants.

Migration of neural crest cells (NCCs) is one of the pivotal processes of human fetal development. Malformations arise if NCC migration and differentiation are impaired genetically or by toxicants. In the currently available test systems for migration inhibition of NCC (MINC), the manual generation of a cell-free space results in extreme operator dependencies, and limits throughput. Here a new test format was established. The assay avoids scratching by plating cells around a commercially available circular stopper. Removal of the stopper barrier after cell attachment initiates migration. This microwell-based circular migration zone NCC function assay (cMINC) was further optimized for toxicological testing of human pluripotent stem cell (hPSC)-derived NCCs. The challenge of obtaining data on viability and migration by automated image processing was addressed by developing a freeware. Data on cell proliferation were obtained by labelling replicating cells, and by careful assessment of cell viability for each experimental sample. The role of cell proliferation as an experimental confounder was tested experimentally by performing the cMINC in the presence of the proliferation-inhibiting drug cytosine arabinoside (AraC), and by a careful evaluation of mitotic events over time. Data from these studies led to an adaptation of the test protocol, so that toxicant exposure was limited to 24 h. Under these conditions, a prediction model was developed that allows classification of toxicants as either inactive, leading to unspecific cytotoxicity, or specifically inhibiting NC migration at non-cytotoxic concentrations.