Functional Comparison of Cultures of IPSC-Derived Human Neurons and Rodent Neurons

Abstract

Event Abstract Back to Event Functional Comparison of Cultures of IPSC-Derived Human Neurons and Rodent Neurons Joe Tauskela1*, Slavisa Corluka1, Amy Aylsworth1, Dietmar Hess2, Chris Fleming2 and Greg Luerman2 1 National Research Council Canada (NRC-CNRC), Human Health Therapeutics, Canada 2 Ncardia (Germany), Germany Motivation Human clinical trials in neuroprotection in stroke have been a dismal failure. To help bridge this translational gap, our laboratory develops neuroprotective strategies against ‘stroke in a dish’ insults. It has been recognized for some time that in vitro evaluation of therapeutics based on rodent neurons raises questions regarding suitability for translation to humans. Culturing human neurons derived from iPSCs has recently attracted enormous interest from pharma, academia and clinicians. Regulatory authorities will undoubtedly increasingly require human-derived data. However, the field is young, and the utility of human neurons derived from iPSCs has yet to be meaningfully established, so suitability must first be established. Since the main purpose of employing human neurons will be to evaluate neurotherapeutics, it is necessary to determine which receptors and ion channels are present, particularly at a functional level. A key function of neurons is synaptic activity in a neural network, and electrophysiology is the key technique involved in phenotypic analyses. Multi-electrode arrays (MEAs) have emerged as the most widely adopted technique to characterize human neuron cultures. MEAs allow chronic and non-invasive profiling of development, pharmacology and disease. MEAs will be used to evaluate the presence of functional channels/receptors in Ncardia (formerly Axiogenesis) CNS.4U neuron cultures, based on responses to a panel of neuro-active drugs eliciting exogenous and endogenous responses, with benchmarking provided by responses observed in rodent forebrain neuron cultures. Materials and Methods Cultures of primary rat cortical/hippocampal neurons were prepared from timed-pregnant (embryonic age day 18) Sprague-Dawley rats, based on a previous procedure. Cells were plated at high density as either a drop or covering the entire surface of 60-electrode MEA dishes (MultiChannel Systems, Reutlingen, Germany), pre-treated with either on poly-L-lysine (PLL)/laminin or PEI/laminin. Medium consisted of Eagle’s minimal essential medium (EMEM) supplemented to 25 mM glucose, 10% fetal bovine serum and 10% horse serum. Cultures were treated with mitotic inhibitor to minimize microglia growth, and media was replaced twice per week (50/50%) for the first two weeks, and more frequently as required thereafter to minimize acidity. CNS.4U neurons were provided by Ncardia, and were plated as a drop on MEAs (pre-coated with (PLL)/laminin or PEI/laminin), and maintained according to manufacturer’s instructions. Media contained an antibiotic cyprofloxacin and was replaced 3 times (100/50/50 %) the first two weeks in culture, and twice per week (50/50%) thereafter. CNS.4U cultures contained 10% astrocytes. Following a 20 min incubation period, spontaneous activity was recorded in 20 min sessions in the following manner: 3 pre-drug recordings / 2 drug recordings / 2 wash-out recordings. Results and Discussion Rat neurons plated as drops exhibited a slightly higher tendency to eventually lift, so most data was collected on MEAs with the entire surface plated with neurons, although the trade-off was increased acidity over time, necessitating more frequent media exchanges. Rat neuron cultures displayed robust synchronized bursting synchronized across electrodes at a slightly earlier day in vitro (DIV) for PLL compared to PEI-coated MEAs, so the effect of drugs on spontaneous activity was evaluated from 14-24 DIV. Drugs causing decreased neuronal activity were blockers of Na and Ca channels and glutamate receptor antagonists and GABAAergic agonists. Drugs causing increased neuronal activity were K channel blockers and 0 Mg buffer. Investigations of drugs altering mGluR, nicotinic, serotonin and muscarinic receptors are ongoing. We have also demonstrated the phenomenon of down-regulated synaptic scaling using chronic application of a neuronal stimulant. To maintain viability of CNS.4U neurons, it was crucial to maintain osmolality and to replace medium at the indicated intervals with fresh supplement. CNS.4U neurons took longer than rat cortical neurons to display activity and bursting, at least beyond 30 DIV. The same panel of drugs as used on rat neurons is being investigated on CNS.4U cultures. Completion of the functional neuropharmacological profiles will be an important first step in ascertaining the relevance of IPSC-derived human neurons and rodent neurons in investigation of in vitro paradigms of neurodegenerative and neurological disease. Acknowledgements The authors wish to acknowledge provision of CNS.4U neurons and technical assistance from Ncardia. Keywords: Rat cortical neurons, IPSC-derived neurons, development, Neurotransmitter Agents, Neuropharmacology Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018. Presentation Type: Poster Presentation Topic: Stem cell-derived applications Citation: Tauskela J, Corluka S, Aylsworth A, Hess D, Fleming C and Luerman G (2019). Functional Comparison of Cultures of IPSC-Derived Human Neurons and Rodent Neurons. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00007 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 15 Mar 2018; Published Online: 17 Jan 2019. * Correspondence: Dr. Joe Tauskela, National Research Council Canada (NRC-CNRC), Human Health Therapeutics, Ottawa, Ontario, K1A 0R6, Canada, joetauskela@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Joe Tauskela Slavisa Corluka Amy Aylsworth Dietmar Hess Chris Fleming Greg Luerman Google Joe Tauskela Slavisa Corluka Amy Aylsworth Dietmar Hess Chris Fleming Greg Luerman Google Scholar Joe Tauskela Slavisa Corluka Amy Aylsworth Dietmar Hess Chris Fleming Greg Luerman PubMed Joe Tauskela Slavisa Corluka Amy Aylsworth Dietmar Hess Chris Fleming Greg Luerman Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.