title: Is the time right for in vitro neurotoxicity testing using human iPSC-derived neurons?
abstract: Current neurotoxicity testing heavily relies on expensive, time consuming and ethically debated in vivo animal experiments that are unsuitable for screening large number of chemicals. Consequently, there is a clear need for (high-throughput) in vitro test strategies, preferably using human cells as this increases relevance and eliminates the need for interspecies translation. However, human stem cell-derived neurons used to date are not well characterised, require prolonged differentiation and are potentially subject to batch-to-batch variation, ethical concerns and country-specific legislations. Recently, a number of human induced pluripotent stem cell (iPSC)-derived neurons became commercially available that may circumvent these concerns. We therefore used immunofluorescent stainings to demonstrate that human iPSC-derived neurons from various suppliers form mixed neuronal cultures, consisting of different types of (excitatory and inhibitory) neurons. Using multi-well microelectrode array (mwMEA) recordings, we demonstrate that these human iPSC-derived cultures develop spontaneous neuronal activity over time, which can be modulated by different physiological, toxicological and pharmacological compounds. Additional single cell calcium imaging illustrates the presence of functional GABA, glutamate, and acetylcholine receptors as well as voltage-gated calcium channels. While human iPSC-derived neuronal cultures appear not yet suitable to fully replace the rat primary cortical model, our data indicate that these rapidly differentiating, commercially available human iPSC-derived neuronal cultures are already suitable for in vitro prioritisation and effect screening studies. Further characterisation and toxicological validation is now required to facilitate acceptance and large-scale implementation of these animal-free, physiologically-relevant human iPSC-based modelsfor future neurotoxicity testing.
authors.affiliation: Neurotoxicology Research Group, Toxicology Division, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Anke M
authors.initials: AM
authors.lastname: Tukker
authors.affiliation: Neurotoxicology Research Group, Toxicology Division, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Martje W G D M
authors.initials: MW
authors.lastname: de Groot
authors.affiliation: Neurotoxicology Research Group, Toxicology Division, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Fiona M J
authors.initials: FM
authors.lastname: Wijnolts
authors.affiliation: Neurotoxicology Research Group, Toxicology Division, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Emma E J
authors.initials: EE
authors.lastname: Kasteel
authors.affiliation: National Poisons Information Center (NVIC), University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Laura
authors.initials: L
authors.lastname: Hondebrink
authors.affiliation: Neurotoxicology Research Group, Toxicology Division, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Remco H S
authors.initials: RH
authors.lastname: Westerink
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D055724:Electrophysiological Phenomena; D015200:Fluoroimmunoassay; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology*; D007473:Ion Channels / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D051381:Rats; D017981:Receptors, Neurotransmitter / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D007473:Ion Channels; D017981:Receptors, Neurotransmitter
keywords: alternatives to animal testing; human induced pluripotent stem cell-derived neurons; in vitro neurotoxicity screening; mixed neuronal cultures; multi-electrode array (MEA)