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.

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The prevalence and use of new psychoactive substances (NPS) is increasing and currently over 600 NPS exist. Many illicit drugs and NPS increase brain monoamine levels by inhibition and/or reversal of monoamine reuptake transporters (DAT, NET and SERT). This is often investigated using labor-intensive, radiometric endpoint measurements. We investigated the applicability of a novel and innovative assay that is based on a fluorescent monoamine mimicking substrate. DAT, NET or SERT-expressing human embryonic kidney (HEK293) cells were exposed to common drugs (cocaine, dl-amphetamine or MDMA), NPS (4-fluoroamphetamine, PMMA, α-PVP, 5-APB, 2C-B, 25B-NBOMe, 25I-NBOMe or methoxetamine) or the antidepressant fluoxetine. We demonstrate that this fluorescent microplate reader-based assay detects inhibition of different transporters by various drugs and discriminates between drugs. Most IC values were in line with previous results from radiometric assays and within estimated human brain concentrations. However, phenethylamines showed higher IC values on hSERT, possibly due to experimental differences. Compared to radiometric assays, this high-throughput fluorescent assay is uncomplicated, can measure at physiological conditions, requires no specific facilities and allows for kinetic measurements, enabling detection of transient effects. This assay is therefore a good alternative for radiometric assays to investigate effects of illicit drugs and NPS on monoamine reuptake transporters.

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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 b s t r a c tThe use of new psychoactive substances (NPS) is steadily increasing. One commonly used NPS is methoxetamine (MXE), a ketamine analogue. Several adverse effects have been reported following MXE exposure, while only limited data are available on its neuropharmacological modes of action.We investigated the effects of MXE and ketamine on several endpoints using multiple in vitro models. These included rat primary cortical cells, human SH-SY5Y cells, human induced pluripotent stem cell (hiPSC)-derived iCell ® Neurons, DopaNeurons and astrocyte co-cultures, and human embryonic kidney (HEK293) cells. We investigated effects on several neurotransmitter receptors using single cell intracellular calcium [Ca 2þ ] i imaging, effects on neuronal activity using micro-electrode array (MEA) recordings and effects on human monoamine transporters using a fluorescence-based plate reader assay.In rat primary cortical cells, 10 mM MXE increased the glutamate-evoked increase in [Ca 2þ ] i , whereas 10 mM ketamine was without effect. MXE and ketamine did not affect voltage-gated calcium channels (VGCCs), but inhibited spontaneous neuronal activity (IC 50 0.5 mM and 1.2 mM respectively). In human SH-SY5Y cells, 10 mM MXE slightly inhibited the K Our combined in vitro data provide an urgently needed first insight into the multiple modes of action of MXE. The use of different models and different (neuronal) endpoints can be complementary in pharmacological profiling. Rapid in vitro screening methods as those presented here, could be of utmost importance for gaining a first mechanistic insight to aid the risk assessment of emerging NPS.

PurposeThe combination of systemic cisplatin with local and regional radiotherapy as primary treatment of head and neck squamous cell carcinoma (HNSCC) leads to cure in approximately half of the patients. The addition of cisplatin has significant effects on outcome, but despite extensive research the mechanism underlying cisplatin response is still not well understood.MethodsWe examined 19 HNSCC cell lines with variable cisplatin sensitivity. We determined the TP53 mutational status of each cell line and investigated the expression levels of 11 potentially relevant genes by quantitative real-time PCR. In addition, we measured cisplatin accumulation and retention, as well as the level of platinum-DNA adducts.ResultsWe found that the IC50 value was significantly correlated with the platinum-DNA adduct levels that accumulated during four hours of cisplatin incubation (p = 0.002). We could not find a significant correlation between cisplatin sensitivity and any of the other parameters tested, including the expression levels of established cisplatin influx and efflux transporters. Furthermore, adduct accumulation did not correlate with mRNA expression of the investigated influx pumps (CTR1 and OCT3) nor with that of the examined DNA repair genes (ATR, ATM, BRCA1, BRCA2 and ERCC1).ConclusionOur findings suggest that the cisplatin-DNA adduct level is the most important determinant of cisplatin sensitivity in HNSCC cells. Imaging with radio-labeled cisplatin might have major associations with outcome.