Applicability of hiPSC-Derived Neuronal Cocultures and Rodent Primary Cortical Cultures for In Vitro Seizure Liability Assessment

Abstract

Seizures are life-threatening adverse drug reactions which are investigated late in drug development using rodent models. Consequently, if seizures are detected, a lot of time, money and animals have been used. Thus, there is a need for in vitro screening models using human cells to circumvent inter-species translation. We assessed the suitability of co-cultures of human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes compared to rodent primary cortical cultures for in vitro seizure liability assessment using micro-electrode arrays (MEA). HiPSC-derived and rodent primary cortical neuronal co-cultures were exposed to nine known (non-)seizurogenic compounds (pentylenetetrazole, amoxapine, enoxacin, amoxicillin, linopirdine, pilocarpine, chlorpromazine, phenytoin and acetaminophen) to assess effects on neuronal network activity using MEA recordings. All compounds affect activity in hiPSC-derived co-cultures. In rodent primary cultures all compounds, except amoxicillin changed activity. Changes in activity patterns for both cell models differ for different classes of compounds. Both models had a comparable sensitivity for exposure to amoxapine (LOEC 0.03 μM), linopirdine (LOEC 1 μM) and pilocarpine (LOEC 0.3 μM). However, hiPSC-derived cultures were about 3 times more sensitive for exposure to pentylenetetrazole (LOEC 30 μM) than rodent primary cortical cultures (LOEC 100 μM). Sensitivity of hiPSC-derived cultures for chlorpromazine, phenytoin and enoxacin was 10-30 times higher (LOECs 0.1, 0.3 and 0.1 μM respectively) than in rodent cultures (LOECs 10, 3 and 3 μM respectively). Our data indicate that hiPSC-derived neuronal co-cultures may outperform rodent primary cortical cultures with respect to detecting seizures, thereby paving the way towards animal-free seizure assessment.

A human stem cell-derived test system for agents modifying neuronal N-methyl-d-aspartate-type glutamate receptor Ca2+-signalling

“…Moreover, the first data from such systems did not provide strong evidence for a robust NMDA-R response. However, compounds acting on other glutamate receptors like domoic acid, have shown clear responses (Nimtz et al 2020 ; Tukker et al 2020 ). As MCCs can be generated relatively fast, and Ca 2+ imaging is performed in 96-well format, our system allows a sufficient throughput for small screens and relatively extensive follow-up characterizations.…”

“…The hitherto most robust MEA data have been generated with rat primary neurons. More recently, murine and human-stem cell-derived neurons have been demonstrated to be principally suitable for the analysis of network signalling on MEA (Pagan-Diaz et al 2020 ; Tukker et al 2020 ). However, the production of cultures at a reasonable price, time effort and robustness has proven challenging.…”

Sodium channel Nav1.2-L1342P variant displaying complex biophysical properties renders hyperexcitability of cortical neurons derived from human iPSCs

“…Neurons derived from hiPSCs are amenable for long-term studies and highthroughput parallel screening for targeted drug discovery. Pairing with MEA, a highthroughput, extracellular recording assay of neuronal activity (Odawara et al, 2016) (Odawara et al, 2018Tukker et al, 2020b), study has shown a successful application of patient-specific disease modeling to discover epilepsy therapies (Tidball et al, 2020). Using the hiPSC-derived neurons, it was found that riluzole and phenytoin can target specific Nav1.6 variants to attenuate bursting phenotypes in vitro, and suppress patient seizure events in the clinical study (Tidball et al, 2020).…”

“…Neurons derived from hiPSCs are amenable for high-throughput parallel screening for targeted drug discovery. Pairing with MEA, a high-throughput, extracellular recording assay of neuronal activity, 57,74,75 such studies have been attempted with neurons carrying Nav1.6 variants. 64 Using the hiPSC-derived neurons, it was recently found that riluzole and phenytoin can target specific Nav1.6 variants to attenuate bursting phenotypes in vitro.…”

Culture of Rat Primary Cortical Cells for Microelectrode Array (MEA) Recordings to Screen for Acute and Developmental Neurotoxicity

“…Consequently, the availability of MEA data derived from human cells is still scarce. The sensitivity between cells from different species can differ, with rat cortical cultures being more sensitive to methoxetamine (Hondebrink et al., 2017), and, on the other hand, hiPSC‐derived neurons being more sensitive to several seizureogenic compounds (Tukker, Wijnolts, de Groot, & Westerink, 2020b). Notably, however, differences in sensitivity between cells from different species are generally small, i.e., less than a factor of 10 (Tukker et al., 2020b), or negligible (Kasteel & Westerink, 2017).…”