Kristina Bartmann, IUF-Leibniz Research Institute
Conference: 2020 Society of Toxicology Annual Meeting, Anaheim, CA
Abstract Title: A human iPSC-based in vitro neuronal network formation assay for regulatory developmental neurotoxicity testing
Abstract: The spatiotemporal orchestration of key neurodevelopmental processes (KNDP) is essential for brain development. If at least one KNDP is impaired due to exposure towards a compound during a critical period of neurodevelopment, an adverse outcome is expected. To study developmental neurotoxicity (DNT) hazards, animal-free new approach methods (NAM) have been developed, which model certain KNDP in vitro and in silico. One of these KNDP is the formation and function of neuronal networks (NNF), the ultimate, functional readout for nervous system function. For studying NNF, the NNF assay based on hiPSC-derived human glutamatergic excitatory and GABAergic inhibitory neurons, as well as primary human astroglia (NeuCyte, USA) was developed. These pre-differentiated cells were seeded in standardized ratio on 48-well microelectrode array (MEA) plates. We let the cells differentiate for one week until we received first recording as an internal control for a ‘positive’ (firing) well. After one week of differentiation in absence of test compounds, cell and network activity was measured via the assessment of spike-related (e.g. mean firing rate), burst-related (e.g bursting ratio) and network-related parameters (e.g. network synchronicity) using the Axion Maestro Pro System. These measures served as the baseline for subsequent neurodevelopmental testing. Treatments started on active wells by exposing from day 7 to day 35. With this protocol, the positive compounds BIS-I and Mevastatin were established as endpoint-specific controls for the assay. Both compounds reduced the values of the above described parameters in a time- and concentration-dependent manner. Data from control wells demonstrate low plate-to-plate and well-to-well variability of the assay. Currently, a set of 35 pesticides, that are known to either affect or not affect brain development based on the rodent DNT guideline study (OECD TG426), is tested in the NNF assay. These results are presented. The human NNF assay will be a valuable addition to the current DNT in vitro testing battery as neuronal network formation converges on multiple neurodevelopmental KE like neurite outgrowth, dendritic spine formation and synaptogenesis.