Toxicological evaluation of convulsant and anticonvulsant drugs in human induced pluripotent stem cell-derived cortical neuronal networks using an MEA system

A Odawara,I Suzuki,Y Ishibashi,N Matsuda,R Yokoi

doi:10.1038/s41598-018-28835-7

Abstract

Functional evaluation assays using human induced pluripotent stem cell (hiPSC)-derived neurons can predict the convulsion toxicity of new drugs and the neurological effects of antiepileptic drugs. However, differences in responsiveness depending on convulsant type and antiepileptic drugs, and an evaluation index capable of comparing in vitro responses with in vivo responses are not well known. We observed the difference in synchronized burst patterns in the epileptiform activities induced by pentylentetrazole (PTZ) and 4-aminopryridine (4-AP) with different action mechanisms using multi-electrode arrays (MEAs); we also observed that 100 µM of the antiepileptic drug phenytoin suppressed epileptiform activities induced by PTZ, but increased those induced by 4-AP. To compare in vitro results with in vivo convulsive responses, frequency analysis of below 250 Hz, excluding the spike component, was performed. The in vivo convulsive firing enhancement of the high γ wave and β wave component were observed remarkably in in vitro hiPSC-derived neurons with astrocytes in co-culture. MEA measurement of hiPSC-derived neurons in co-culture with astrocytes and our analysis methods, including frequency analysis, appear effective for predicting convulsion toxicity, side effects, and their mechanism of action as well as the comparison of convulsions induced in vivo.

Highlights

Human iPSC-derived neurons are used to evaluate toxicity to the human nervous system, and are expected to be applied to toxicity evaluations in nonclinical studies[1,2]
Spontaneous firing in human induced pluripotent stem cell (hiPSC)-derived cortical neurons and co-culture with astrocytes
Human iPSC-derived cortical neurons and co-cultures with astrocytes grown on multielectrode arrays (MEAs) chips survived over the long-term without cell aggregation (Fig. 1A–c and b), enabling the measurement of distributed network field activity

Summary

Introduction

Human iPSC-derived neurons are used to evaluate toxicity to the human nervous system, and are expected to be applied to toxicity evaluations in nonclinical studies[1,2]. We have previously reported the effectiveness of MEA measurements for drug responses in human iPSC-derived neurons[6,12,13], which has since been confirmed by several other groups[7,14,15,16]. In the case of human iPSC-derived neurons, it takes time to bring the cells to maturation, so it is necessary to identify a culture period suitable for the functional evaluation of drug responses[6,12,14]. We investigated differences in the responsiveness between Pentylenetetrazol (PTZ, GABAA antagonist) and 4-Aminopyridine (4-AP, K+ channel antagonist), which are typical seizure-inducing drugs with different mechanisms of action We used both neuronal and astrocyte co-cultured samples, and investigated the effect of AEDs by administering Phenytoin upon convulsive-like ignition. Frequency analysis by the administration of PTZ and 4-AP was performed to investigate whether changes observed during in vivo human epilepsy were detected, and whether there was a difference in frequency characteristics between PTZ and 4-AP administration

Methods

Results

Conclusion