Abstract
Neurotoxicity testing still relies on ethically debated, expensive and time consuming in vivo experiments, which are unsuitable for high-throughput toxicity screening. There is thus a clear need for a rapid in vitro screening strategy that is preferably based on human-derived neurons to circumvent interspecies translation. Recent availability of commercially obtainable human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes holds great promise in assisting the transition from the current standard of rat primary cortical cultures to an animal-free alternative.We therefore composed several hiPSC-derived neuronal models with different ratios of excitatory and inhibitory neurons in the presence or absence of astrocytes. Using immunofluorescent stainings and multi-well micro-electrode array (mwMEA) recordings we demonstrate that these models form functional neuronal networks that become spontaneously active. The differences in development of spontaneous neuronal activity and bursting behavior as well as spiking patterns between our models confirm the importance of the presence of astrocytes. Preliminary neurotoxicity assessment demonstrates that these cultures can be modulated with known seizurogenic compounds, such as picrotoxin (PTX) and endosulfan, and the neurotoxicant methylmercury (MeHg). However, the chemical-induced effects on different parameters for neuronal activity, such as mean spike rate (MSR) and mean burst rate (MBR), may depend on the ratio of inhibitory and excitatory neurons. Our results thus indicate that hiPSC-derived neuronal models must be carefully designed and characterized prior to large-scale use in neurotoxicity screening.
Highlights
Human induced pluripotent stem cell-derived neuronal cultures are becoming increasingly important for in vitro neurotoxicity testing
We have recently shown that these commercially available models have the potential to develop spontaneously active neuronal networks that can be used for screening and prioritization of chemically induced effects on neuronal activity (Tukker et al, 2016), whereas others have already shown that hiPSCderived neurons exhibit the behavior and function of mature neurons (Hyysalo et al, 2017; Odawara et al, 2016; Paavilainen et al, 2018)
While costly in comparison to primary cultures and neural progenitor cells, an important benefit of Human induced pluripotent stem cell (hiPSC)-derived neurons is the rapid development of a functional neuronal network, in contrast to the time consuming differentiation of hiPSCs into neural progenitor cells that subsequently need to be cultured into functional neurons, a process that can take several weeks (Görtz et al, 2004; Hyysalo et al, 2017; Kuijlaars et al, 2016) till months (Odawara et al, 2016; Paavilainen et al, 2018)
Summary
Human induced pluripotent stem cell (hiPSC)-derived neuronal cultures are becoming increasingly important for in vitro neurotoxicity testing. While costly in comparison to (rodent) primary cultures and (human) neural progenitor cells, an important benefit of hiPSC-derived neurons is the rapid development of a functional neuronal network, in contrast to the time consuming differentiation of hiPSCs into neural progenitor cells that subsequently need to be cultured into functional neurons, a process that can take several weeks (Görtz et al, 2004; Hyysalo et al, 2017; Kuijlaars et al, 2016) till months (Odawara et al, 2016; Paavilainen et al, 2018) Opting for these commercially obtainable cells comes with the additional benefit that they are available in high quantity, allowing for efficient screening (Anson et al, 2011). We composed different (co-)cultures of iCell Glutaneurons® (∼90% excitatory glutamatergic neurons/ 10% inhibitory GABAergic neurons) and iCell neurons® (∼30% excitatory glutamatergic neurons/70% inhibitory GABAergic neurons) in the absence or presence of astrocytes to explore the role of different cell types in development of neuronal activity and suitability for in vitro neurotoxicity testing, including seizure liability testing
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