An Electrophysiological and Pharmacological Study of the Properties of Human iPSC-Derived Neurons for Drug Discovery

Abstract

Human stem cell-derived neurons are increasingly considered powerful models in drug discovery and disease modeling, despite limited characterization of their molecular properties. Here, we have conducted a detailed study of the properties of a commercial human induced Pluripotent Stem Cell (iPSC)-derived neuron line, iCell [GABA] neurons, maintained for up to 3 months in vitro. We confirmed that iCell neurons display neurite outgrowth within 24 h of plating and label for the pan-neuronal marker, βIII tubulin within the first week. Our multi-electrode array (MEA) recordings clearly showed neurons generated spontaneous, spike-like activity within 2 days of plating, which peaked at one week, and rapidly decreased over the second week to remain at low levels up to one month. Extracellularly recorded spikes were reversibly inhibited by tetrodotoxin. Patch-clamp experiments showed that iCell neurons generated spontaneous action potentials and expressed voltage-gated Na and K channels with membrane capacitances, resistances and membrane potentials that are consistent with native neurons. Our single neuron recordings revealed that reduced spiking observed in the MEA after the first week results from development of a dominant inhibitory tone from GABAergic neuron circuit maturation. GABA evoked concentration-dependent currents that were inhibited by the convulsants, bicuculline and picrotoxin, and potentiated by the positive allosteric modulators, diazepam, chlordiazepoxide, phenobarbital, allopregnanolone and mefenamic acid, consistent with native neuronal GABAA receptors. We also show that glycine evoked robust concentration-dependent currents that were inhibited by the neurotoxin, strychnine. Glutamate, AMPA, Kainate and NMDA each evoked concentration-dependent currents in iCell neurons that were blocked by their selective antagonists, consistent with the expression of ionotropic glutamate receptors. The NMDA currents required the presence of the co-agonist glycine and were blocked in a highly voltage-dependent manner by Mg2+ consistent with the properties of native neuronal NMDA receptors. Together, our data suggest that such human iPSC-derived neurons may have significant value in drug discovery and development and may eventually largely replace the need for animal tissues in human biomedical research.

Hyperexcitability and Pharmacological Responsiveness of Cortical Neurons Derived from Human iPSCs Carrying Epilepsy-Associated Sodium Channel Nav1.2-L1342P Genetic Variant

“…We then compared the input resistance and found no significant difference between the control and L1342P neurons (control: 2.0 6 0.2 pF, n = 18; L1342P: 1.6 6 0.2 pF, n = 23; p = 0.16, Student's t test). While a potassium chloride-based solution (we used herein) is widely used to study neurons (Parker et al, 2018;Mis et al, 2019;Halliwell et al, 2021), it may depolarize the cell membrane. To address the potential issue that the potassium chloride in the internal solution may change the firing property, we performed an additional experiment by using a potassium acetate-based solution.…”

The Future of 3D Brain Cultures in Developmental Neurotoxicity Testing

“…Multi-electrode array (MEA) is the most common assay used to assess neuronal functionality by measuring the electrical activity in the test system. Traditional MEA plates were designed for monolayer cultures and are broadly used in neuroscience ( Halliwell et al, 2021 ; Taga et al, 2021 ; Tukker and Westerink, 2021 ). Furthermore, MEA has been applied to assess neurotoxicity and DNT in high throughput manner ( Strickland et al, 2018 ; Shafer et al, 2019 ).…”

Dose-dependent consequences of sub-chronic fentanyl exposure on neuron and glial co-cultures

“…Recently, brain-on-a-chip (BOC) platforms, which often integrate cell cultures with a multi-electrode arrays (MEA), have emerged as a powerful technology to non-invasively record extracellular action potentials from primary rodent or human induced Pluripotent Stem Cell (iPSC)-derived neurons cultured on these systems ( Kamioka et al, 1996 ; Chiappalone et al, 2003 ; Halliwell et al, 2021 ). The ability to monitor the functional dynamics of neural and network activity have been useful in evaluating pharmacological and toxicological effects of compounds on network activity ( Chiappalone et al, 2003 ; Johnstone et al, 2010 ), and better understand how cellular and molecular changes affect the functional properties of neurons and their networks in vitro .…”