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

Robert F Halliwell,William S Cao,Hamed Salmanzadeh,Leanne Coyne

doi:10.3390/cells10081953

Robert F Halliwell, William S Cao + Show 2 more

Open Access

https://doi.org/10.3390/cells10081953

Copy DOI

Journal: Cells	Publication Date: Jul 31, 2021
Citations: 8	License type: CC BY 4.0

Affiliation: University of the Pacific

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.

Highlights

Alternative approaches have used recombinant human receptors and ion channels expressed in human cell lines or Xenopus oocytes to investigate receptor pharmacology or drug interactions on these systems (e.g., [4]) but the complex neural networks and diverse neuronal cell types are absent in this approach
We have previously reported on the electrophysiological characteristics acterized [5]
We have previously reported on the electrophysiological characteristics ofof neuronsderived derivedfrom fromaahuman humanstem stemcell cellline linethat thatisissimple simpleand andinexpensive inexpensivetotoculture cultureand and neurons differentiate[24,25]

Summary

Introduction

The instruments used to investigate animal electricity have since evolved radically from the differential rheotome built by Bernstein (1868) to the patch-clamp technique invented by Nobel Laureates, Neher and Sakmann (1976). Electrophysiological and neuropharmacological studies of human neurons have, until recently, been technically, legally and ethically difficult and often conducted on small sections (slices) of neocortex resected from patients undergoing neurosurgery to treat epilepsy, or to remove arterio-vascular malformations or neoplasms (e.g., [3]). This model retains some of the complexity of neural networks and cell diversity but may or may not represent the ‘normal’ properties of human neurons. Alternative approaches have used recombinant human receptors and ion channels expressed in human cell lines or Xenopus oocytes to investigate receptor pharmacology or drug interactions on these systems (e.g., [4]) but the complex neural networks and diverse neuronal cell types are absent in this approach

Methods

Results

Conclusion