Embryonic Stem Cell-Derived Neurons Grown on Multi-Electrode Arrays as a Novel In vitro Bioassay for the Detection of Clostridium botulinum Neurotoxins

Stephen P Jenkinson; Denis Grandgirard; Martina Heidemann; Anne Tscherter; Marc-André Avondet; Stephen L Leib

doi:10.3389/fphar.2017.00073

Embryonic Stem Cell-Derived Neurons Grown on Multi-Electrode Arrays as a Novel In vitro Bioassay for the Detection of Clostridium botulinum Neurotoxins

Front Pharmacol. 2017 Feb 23:8:73. doi: 10.3389/fphar.2017.00073. eCollection 2017.

Authors

Stephen P Jenkinson¹, Denis Grandgirard², Martina Heidemann³, Anne Tscherter³, Marc-André Avondet⁴, Stephen L Leib²

Affiliations

¹ Neuroinfection Laboratory, Institute for Infectious Diseases, University of BernBern, Switzerland; Biology Division, Spiez Laboratory, Swiss Federal Office for Civil ProtectionSpiez, Switzerland; Cluster for Regenerative Neuroscience, Department for Clinical Research, University of BernBern, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of BernBern, Switzerland.
² Neuroinfection Laboratory, Institute for Infectious Diseases, University of BernBern, Switzerland; Cluster for Regenerative Neuroscience, Department for Clinical Research, University of BernBern, Switzerland.
³ Department of Physiology, University of Bern Bern, Switzerland.
⁴ Biology Division, Spiez Laboratory, Swiss Federal Office for Civil Protection Spiez, Switzerland.

Abstract

Clostridium botulinum neurotoxins (BoNTs) are the most poisonous naturally occurring protein toxins known to mankind and are the causative agents of the severe and potentially life-threatening disease botulism. They are also known for their application as cosmetics and as unique bio-pharmaceuticals to treat an increasing number of neurological and non-neurological disorders. Currently, the potency of biologically active BoNT for therapeutic use is mainly monitored by the murine LD₅₀-assay, an ethically disputable test causing suffering and death of a considerable number of mice. The aim of this study was to establish an in vitro assay as an alternative to the widely used in vivo mouse bioassay. We report a novel BoNT detection assay using mouse embryonic stem cell-derived neurons (mESN) cultured on multi-electrode arrays (MEAs). After 21 days in culture, the mESN formed a neuronal network showing spontaneous bursting activity based on functional synapses and express the necessary target proteins for BoNTs. Treating cultures for 6 h with 16.6 pM of BoNT serotype A and incubation with 1.66 pM BoNT/A or 33 Units/ml of Botox^® for 24 h lead to a significant reduction of both spontaneous network bursts and average spike rate. This data suggests that mESN cultured on MEAs pose a novel, biologically relevant model that can be used to detect and quantify functional BoNT effects, thus accelerating BoNT research while decreasing animal use.

Keywords: BoNT; MEA; botulinum neurotoxins; botulism; embryonic stem cell-derived neurons; in vitro bioassay; multi-electrode array; neuronal network.