Assessment of neurotransmitter release in human iPSC-derived neuronal/glial cells: a missing in vitro assay for regulatory developmental neurotoxicity testing

• Published in: Reproductive toxicology (Elmsford, N.Y.) N.Y.), 2023-04, Vol.117, p.108358-108358, Article 108358
• Main Authors: Cervetto, Chiara, Pistollato, Francesca, Amato, Sarah, Mendoza-de Gyves, Emilio, Bal-Price, Anna, Maura, Guido, Marcoli, Manuela
• Format: Article
• Language: English
• Subjects: Aspartic acid; Cell Differentiation; Developmental neurotoxicity; Glia; Glutamate; Glutamates; Human neural stem cells; Humans; Induced Pluripotent Stem Cells; Multi-electrode array; Neuroglia; Neurons; Neurotoxicity Syndromes - etiology; Neurotransmitters; Toxicity Tests - methods; Vesicular release
• ISSN: 0890-6238; 1873-1708
• DOI: 10.1016/j.reprotox.2023.108358

Human induced pluripotent stem cell (hiPSC)-derived neural stem cells (NSCs) and their differentiated neuronal/glial derivatives have been recently considered suitable to assess in vitro developmental neurotoxicity (DNT) triggered by exposure to environmental chemicals. The use of human-relevant test systems combined with in vitro assays specific for different neurodevelopmental events, enables a mechanistic understanding of the possible impact of environmental chemicals on the developing brain, avoiding extrapolation uncertainties associated with in vivo studies. Currently proposed in vitro battery for regulatory DNT testing accounts for several assays suitable to study key neurodevelopmental processes, including NSC proliferation and apoptosis, differentiation into neurons and glia, neuronal migration, synaptogenesis, and neuronal network formation. However, assays suitable to measure interference of compounds with neurotransmitter release or clearance are at present not included, which represents a clear gap of the biological applicability domain of such a testing battery. Here we applied a HPLC-based methodology to measure the release of neurotransmitters in a previously characterized hiPSC-derived NSC model undergoing differentiation towards neurons and glia. Glutamate release was assessed in control cultures and upon depolarization, as well as in cultures repeatedly exposed to some known neurotoxicants (BDE47 and lead) and chemical mixtures. Obtained data indicate that these cells have the ability to release glutamate in a vesicular manner, and that both glutamate clearance and vesicular release concur in the maintenance of extracellular glutamate levels. In conclusion, analysis of neurotransmitter release is a sensitive readout that should be included in the envisioned battery of in vitro assays for DNT testing. •Human iPSC-derived NSCs and their neuronal/glial derivatives are suitable for DNT testing.•HPLC-based assay can be used to measure neurotransmitter release in human NSCs.•Differentiated NSCs release glutamate through a synaptic vesicle mechanism.•BDE47, lead and chemical mixtures differentially perturb glutamate release and clearance in differentiated NSCs.•Analyses of neurotransmitters should be included in the battery of assays for regulatory DNT.