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

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          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 LD 50-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.

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          Directed differentiation of embryonic stem cells into motor neurons.

          Hynek Wichterle, Ivo Lieberam, Jeffery Porter … (2002)
          Inductive signals and transcription factors involved in motor neuron generation have been identified, raising the question of whether these developmental insights can be used to direct stem cells to a motor neuron fate. We show that developmentally relevant signaling factors can induce mouse embryonic stem (ES) cells to differentiate into spinal progenitor cells, and subsequently into motor neurons, through a pathway recapitulating that used in vivo. ES cell-derived motor neurons can populate the embryonic spinal cord, extend axons, and form synapses with target muscles. Thus, inductive signals involved in normal pathways of neurogenesis can direct ES cells to form specific classes of CNS neurons.
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            HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells.

            M. Hooper, K. Hardy, A Handyside … (1987)
            Embryonal stem (ES) cell lines, established in culture from peri-implantation mouse blastocysts, can colonize both the somatic and germ-cell lineages of chimaeric mice following injection into host blastocysts. Recently, ES cells with multiple integrations of retroviral sequences have been used to introduce these sequences into the germ-line of chimaeric mice, demonstrating an alternative to the microinjection of fertilized eggs for the production of transgenic mice. However, the properties of ES cells raise a unique possibility: that of using the techniques of somatic cell genetics to select cells with genetic modifications such as recessive mutations, and of introducing these mutations into the mouse germ line. Here we report the realization of this possibility by the selection in vitro of variant ES cells deficient in hypoxanthine guanine phosphoribosyl transferase (HPRT; EC 2.4.2.8), their use to produce germline chimaeras resulting in female offspring heterozygous for HPRT-deficiency, and the generation of HPRT-deficient preimplantation embryos from these females. In human males, HPRT deficiency causes Lesch-Nyhan syndrome, which is characterized by mental retardation and self-mutilation.
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              Neurotoxins affecting neuroexocytosis.

              G Schiavo, M Matteoli, C Montecucco (2000)
              Nerve terminals are specific sites of action of a very large number of toxins produced by many different organisms. The mechanism of action of three groups of presynaptic neurotoxins that interfere directly with the process of neurotransmitter release is reviewed, whereas presynaptic neurotoxins acting on ion channels are not dealt with here. These neurotoxins can be grouped in three large families: 1) the clostridial neurotoxins that act inside nerves and block neurotransmitter release via their metalloproteolytic activity directed specifically on SNARE proteins; 2) the snake presynaptic neurotoxins with phospholipase A(2) activity, whose site of action is still undefined and which induce the release of acethylcholine followed by impairment of synaptic functions; and 3) the excitatory latrotoxin-like neurotoxins that induce a massive release of neurotransmitter at peripheral and central synapses. Their modes of binding, sites of action, and biochemical activities are discussed in relation to the symptoms of the diseases they cause. The use of these toxins in cell biology and neuroscience is considered as well as the therapeutic utilization of the botulinum neurotoxins in human diseases characterized by hyperfunction of cholinergic terminals.
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                Author and article information

                Contributors
                Journal
                Journal ID (nlm-ta): Front Pharmacol
                Journal ID (iso-abbrev): Front Pharmacol
                Journal ID (publisher-id): Front. Pharmacol.
                Title: Frontiers in Pharmacology
                Publisher: Frontiers Media S.A.
                ISSN (Electronic): 1663-9812
                Publication date (Electronic): 23 February 2017
                Publication date Collection: 2017
                Volume: 8
                Electronic Location Identifier: 73
                Affiliations
                [1] 1Neuroinfection Laboratory, Institute for Infectious Diseases, University of Bern Bern, Switzerland
                [2] 2Biology Division, Spiez Laboratory, Swiss Federal Office for Civil Protection Spiez, Switzerland
                [3] 3Cluster for Regenerative Neuroscience, Department for Clinical Research, University of Bern Bern, Switzerland
                [4] 4Graduate School for Cellular and Biomedical Sciences, University of Bern Bern, Switzerland
                [5] 5Department of Physiology, University of Bern Bern, Switzerland
                Author notes

                Edited by: Hani El-Nezami, RMIT University, Australia

                Reviewed by: Dragana D. Četojević-Simin, Oncology Institute of Vojvodina, Serbia; Brigitte G. Dorner, Robert Koch Institute, Germany

                *Correspondence: Stephen L. Leib, stephen.leib@ 123456ifik.unibe.ch

                This article was submitted to Predictive Toxicology, a section of the journal Frontiers in Pharmacology

                Article
                DOI: 10.3389/fphar.2017.00073
                PMC ID: 5322221
                SO-VID: 0a8cf581-c932-41c2-a80f-9171c1c229e6
                Copyright © Copyright © 2017 Jenkinson, Grandgirard, Heidemann, Tscherter, Avondet and Leib.
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                Date received : 11 November 2016
                Date accepted : 06 February 2017
                Page count
                Figures: 5, Tables: 0, Equations: 0, References: 70, Pages: 14, Words: 0
                Categories
                Subject: Pharmacology
                Subject: Original Research

                ScienceOpen disciplines: Pharmacology & Pharmaceutical medicine
                Keywords: botulinum neurotoxins,bont,botulism,embryonic stem cell-derived neurons,neuronal network,in vitro bioassay,multi-electrode array,mea
                Data availability:
                ScienceOpen disciplines: Pharmacology & Pharmaceutical medicine
                Keywords: botulinum neurotoxins, bont, botulism, embryonic stem cell-derived neurons, neuronal network, in vitro bioassay, multi-electrode array, mea

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