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 interspecies translation. We assessed the suitability of cocultures of human-induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes compared with rodent primary cortical cultures for in vitro seizure liability assessment using microelectrode arrays. hiPSC-derived and rodent primary cortical neuronal cocultures were exposed to 9 known (non)seizurogenic compounds (pentylenetetrazole, amoxapine, enoxacin, amoxicillin, linopirdine, pilocarpine, chlorpromazine, phenytoin, and acetaminophen) to assess effects on neuronal network activity using microelectrode array recordings. All compounds affect activity in hiPSC-derived cocultures. 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 (lowest observed effect concentration [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 cocultures may outperform rodent primary cortical cultures with respect to detecting seizures, thereby paving the way towards animal-free seizure assessment.

Keywords: alternatives to animal testing; human-induced pluripotent stem cell (hiPSC)-derived neuronal models; microelectrode array (MEA); rodent primary cortical cultures; seizure liability assessment.

Figure 1.

Characterization of baseline activity of the 2 models depicting mean spike rate (A), mean burst rate (B), and mean network burst rate (C).

Figure 2.

Effects of modulation of human-induced pluripotent stem cell (hiPSC)-derived cocultures (black) and rat primary cortical cultures (gray) with pentylenetetrazol (A) and amoxapine (B) on mean spike rate (left), mean burst rate (middle), and mean network burst rate (right). Effects are depicted as average % change of control (solvent control set to 100%; dashed line) ± SEM from n = 4–13 wells for hiPSC-derived neuronal cocultures and n = 13–33 wells for rat primary cortical cultures. * indicates a hit.

Figure 3.

Effects of modulation of human-induced pluripotent stem cell (hiPSC)-derived cocultures (black) and rat primary cortical cultures (gray) with enoxacin (A) and amoxicillin (B) on mean spike rate (left), mean burst rate (middle), and mean network burst rate (right). Effects are depicted as average % change of control (solvent control set to 100%; dashed line) ± SEM from n = 10–12 wells for hiPSC-derived neuronal cocultures and n = 26–35 wells for rat primary cortical cultures. * indicates a hit.

Figure 4.

Effects of modulation of human-induced pluripotent stem cell (hiPSC)-derived cocultures (black) and rat primary cortical cultures (gray) with pilocarpine on mean spike rate (A), mean burst rate (B), and mean network burst rate (C). Effects are depicted as average % change of control (solvent control set to 100%; dashed line) ± SEM from n = 9–12 wells for hiPSC-derived neuronal cocultures and n = 15–24 wells for rat primary cortical cultures. * indicates a hit.

Figure 5.

Effects of modulation of human-induced pluripotent stem cell (hiPSC)-derived cocultures (black) and rat primary cortical cultures (gray) with linopirdine on mean spike rate (A), mean burst rate (B), and mean network burst rate (C). Effects are depicted as average % change of control (solvent control set to 100%; dashed line) ± SEM from n = 7–12 wells for hiPSC-derived neuronal cocultures and n = 26–29 wells for rat primary cortical cultures. * indicates a hit.

Figure 6.

Effects of modulation of human-induced pluripotent stem cell (hiPSC)-derived cocultures (black) and rat primary cortical cultures (gray) with chlorpromazine (A) and phenytoin (B) on mean spike rate (left), mean burst rate (middle), and mean network burst rate (right). Effects are depicted as average % change of control (solvent control set to 100%; dashed line) ± SEM from n = 4–16 wells plates for hiPSC-derived neuronal cocultures and n = 21–32 wells for rat primary cortical cultures. * indicates a hit.

Figure 7.

Heatmap of the effects of cys-loop antagonists (green, from top to bottom: pentylenetetrazol, picrotoxin [PTX], amoxapine, and strychnine) on human-induced pluripotent stem cell (hiPSC)-derived neuronal cocultures (black, top) and rat primary cortical cultures (gray, bottom). Color scaling is based on the percentage of change relative to the control with red indicating an increase and blue a decrease. Asterisks indicate a hit. White areas indicate that no average could be calculated. Data are based on n = 1–17 wells for hiPSC-derived neuronal cocultures and n = 9–32 wells for rat primary cortical cultures. PTX and strychnine data have been published previously in Tukker et al. (2020).

Figure 8.

Heatmap of the effects of antibiotics (orange, from top to middle: enoxacin and amoxicillin) and a muscarinic acetylcholine receptor agonist (blue, bottom: pilocarpine) on human-induced pluripotent stem cell (hiPSC)-derived neuronal cocultures (black, top) and rat primary cortical cultures (gray, bottom). Color scaling is based on the percentage of change relative to the control with red indicating an increase and blue a decrease. Asterisks indicate a hit. Data are based on n = 8–12 wells for hiPSC-derived neuronal cocultures and n = 20–35 wells for rat primary cortical cultures.

Figure 9.

Heatmap of the effects of potassium channel blockers (purple, linopirdine and aminopyridine [4-AP]) and inhibitory compounds (red, chlorpromazine and phenytoin) on human-induced pluripotent stem cell (hiPSC)-derived neuronal cocultures (black, top) and rat primary cortical cultures (gray, bottom). Color scaling is based on the percentage of change relative to the control with red indicating an increase and blue a decrease. Asterisks indicate a hit. Data are based on n = 1–16 wells for hiPSC-derived neuronal cocultures and n = 5–31 wells for rat primary cortical cultures. 4-AP data have been published previously in Tukker et al. (2020).

Figure 10.

Scree plot displaying the percentage of variety explained by each component (A) for different GABA_A receptor antagonists. Contribution of each parameter to principal component analysis (PCA) 1 (B) and PCA2 (C). The red line indicates the expected contribution (7%). Bars above the red line indicate importance for the component. The individual compounds are plotted in circles (human-induced pluripotent stem cell-derived neuronal coculture) and triangles (rat primary cortical cultures) visualizing a clear segregation between the 2 models (D). Plot depicting the contribution of parameters to the 2 components in arrows, darker orange arrows indicate higher contribution, whereas arrows in blue depict parameters with a lower contribution (E). Amount of variation explained by a principal component is indicated in parenthesis (D/E).

Figure 11.

Boxplots depicting Lowest observed effect concentration (LOECs) of the different metric parameters per compound on human-induced pluripotent stem cell (hiPSC)-derived neuronal cocultures (A) and rat primary cortical cultures (B). No LOEC could be determined for amoxicillin on the rat primary cortical culture.