A human iPSC-based in vitro neural network formation assay to investigate neurodevelopmental toxicity of pesticides.

Proper brain development is based on the orchestration of key neurodevelopmental processes (KNDP), including the for­mation and function of neural networks. If at least one KNDP is affected by a chemical, an adverse outcome is expected. To enable a higher testing throughput than the guideline animal experiments, a developmental neurotoxicity (DNT) in vitro testing battery (DNT IVB) comprising a variety of assays that model several KNDPs was set up. Gap analysis revealed the need for a human-based assay to assess neural network formation and function (NNF). Therefore, we established the human NNF (hNNF) assay. A co-culture comprised of human induced pluripotent stem cell (hiPSC)-derived excitatory and inhibitory neurons as well as primary human astroglia was differentiated for 35 days on microelectrode arrays (MEA), and spontaneous electrical activity, together with cytotoxicity, was assessed on a weekly basis after washout of the compounds 24 h prior to measurements. In addition to the characterization of the test system, the assay was challenged with 28 com­pounds, mainly pesticides, identifying their DNT potential by evaluating specific spike-, burst-, and network parameters. This approach confirmed the suitability of the assay for screening environmental chemicals. Comparison of benchmark con­centrations (BMC) with an NNF in vitro assay (rNNF) based on primary rat cortical cells revealed differences in sensitivity. Together with the successful implementation of hNNF data into a postulated stressor-specific adverse outcome pathway (AOP) network associated with a plausible molecular initiating event for deltamethrin, this study suggests the hNNF assay as a useful complement to the DNT IVB.