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A human stem cell-derived test system for agents modifying neuronal N-methyl-d-aspartate-type glutamate receptor Ca(2+)-signalling

Methods to assess neuronal receptor functions are needed in toxicology and for drug development. Human-based test systems that allow studies on glutamate signalling are still scarce. To address this issue, we developed and characterized pluripotent stem cell (PSC)-based neural cultures capable of fo...

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Autores principales: Klima, Stefanie, Brüll, Markus, Spreng, Anna-Sophie, Suciu, Ilinca, Falt, Tjalda, Schwamborn, Jens C., Waldmann, Tanja, Karreman, Christiaan, Leist, Marcel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113295/
https://www.ncbi.nlm.nih.gov/pubmed/33713149
http://dx.doi.org/10.1007/s00204-021-03024-0
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author Klima, Stefanie
Brüll, Markus
Spreng, Anna-Sophie
Suciu, Ilinca
Falt, Tjalda
Schwamborn, Jens C.
Waldmann, Tanja
Karreman, Christiaan
Leist, Marcel
author_facet Klima, Stefanie
Brüll, Markus
Spreng, Anna-Sophie
Suciu, Ilinca
Falt, Tjalda
Schwamborn, Jens C.
Waldmann, Tanja
Karreman, Christiaan
Leist, Marcel
author_sort Klima, Stefanie
collection PubMed
description Methods to assess neuronal receptor functions are needed in toxicology and for drug development. Human-based test systems that allow studies on glutamate signalling are still scarce. To address this issue, we developed and characterized pluripotent stem cell (PSC)-based neural cultures capable of forming a functional network. Starting from a stably proliferating neuroepithelial stem cell (NESC) population, we generate “mixed cortical cultures” (MCC) within 24 days. Characterization by immunocytochemistry, gene expression profiling and functional tests (multi-electrode arrays) showed that MCC contain various functional neurotransmitter receptors, and in particular, the N-methyl-d-aspartate subtype of ionotropic glutamate receptors (NMDA-R). As this important receptor is found neither on conventional neural cell lines nor on most stem cell-derived neurons, we focused here on the characterization of rapid glutamate-triggered Ca(2+) signalling. Changes of the intracellular free calcium ion concentration ([Ca(2+)](i)) were measured by fluorescent imaging as the main endpoint, and a method to evaluate and quantify signals in hundreds of cells at the same time was developed. We observed responses to glutamate in the low µM range. MCC responded to kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and a subpopulation of 50% had functional NMDA-R. The receptor was modulated by Mg(2+), Zn(2+) and Pb(2+) in the expected ways, and various toxicologically relevant agonists (quinolinic acid, ibotenic acid, domoic acid) triggered [Ca(2+)](i) responses in MCC. Antagonists, such as phencyclidine, ketamine and dextromethorphan, were also readily identified. Thus, the MCC developed here may fill an important gap in the panel of test systems available to characterize the effects of chemicals on neurotransmitter receptors. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00204-021-03024-0.
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spelling pubmed-81132952021-05-13 A human stem cell-derived test system for agents modifying neuronal N-methyl-d-aspartate-type glutamate receptor Ca(2+)-signalling Klima, Stefanie Brüll, Markus Spreng, Anna-Sophie Suciu, Ilinca Falt, Tjalda Schwamborn, Jens C. Waldmann, Tanja Karreman, Christiaan Leist, Marcel Arch Toxicol In Vitro Systems Methods to assess neuronal receptor functions are needed in toxicology and for drug development. Human-based test systems that allow studies on glutamate signalling are still scarce. To address this issue, we developed and characterized pluripotent stem cell (PSC)-based neural cultures capable of forming a functional network. Starting from a stably proliferating neuroepithelial stem cell (NESC) population, we generate “mixed cortical cultures” (MCC) within 24 days. Characterization by immunocytochemistry, gene expression profiling and functional tests (multi-electrode arrays) showed that MCC contain various functional neurotransmitter receptors, and in particular, the N-methyl-d-aspartate subtype of ionotropic glutamate receptors (NMDA-R). As this important receptor is found neither on conventional neural cell lines nor on most stem cell-derived neurons, we focused here on the characterization of rapid glutamate-triggered Ca(2+) signalling. Changes of the intracellular free calcium ion concentration ([Ca(2+)](i)) were measured by fluorescent imaging as the main endpoint, and a method to evaluate and quantify signals in hundreds of cells at the same time was developed. We observed responses to glutamate in the low µM range. MCC responded to kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and a subpopulation of 50% had functional NMDA-R. The receptor was modulated by Mg(2+), Zn(2+) and Pb(2+) in the expected ways, and various toxicologically relevant agonists (quinolinic acid, ibotenic acid, domoic acid) triggered [Ca(2+)](i) responses in MCC. Antagonists, such as phencyclidine, ketamine and dextromethorphan, were also readily identified. Thus, the MCC developed here may fill an important gap in the panel of test systems available to characterize the effects of chemicals on neurotransmitter receptors. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00204-021-03024-0. Springer Berlin Heidelberg 2021-03-13 2021 /pmc/articles/PMC8113295/ /pubmed/33713149 http://dx.doi.org/10.1007/s00204-021-03024-0 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle In Vitro Systems
Klima, Stefanie
Brüll, Markus
Spreng, Anna-Sophie
Suciu, Ilinca
Falt, Tjalda
Schwamborn, Jens C.
Waldmann, Tanja
Karreman, Christiaan
Leist, Marcel
A human stem cell-derived test system for agents modifying neuronal N-methyl-d-aspartate-type glutamate receptor Ca(2+)-signalling
title A human stem cell-derived test system for agents modifying neuronal N-methyl-d-aspartate-type glutamate receptor Ca(2+)-signalling
title_full A human stem cell-derived test system for agents modifying neuronal N-methyl-d-aspartate-type glutamate receptor Ca(2+)-signalling
title_fullStr A human stem cell-derived test system for agents modifying neuronal N-methyl-d-aspartate-type glutamate receptor Ca(2+)-signalling
title_full_unstemmed A human stem cell-derived test system for agents modifying neuronal N-methyl-d-aspartate-type glutamate receptor Ca(2+)-signalling
title_short A human stem cell-derived test system for agents modifying neuronal N-methyl-d-aspartate-type glutamate receptor Ca(2+)-signalling
title_sort human stem cell-derived test system for agents modifying neuronal n-methyl-d-aspartate-type glutamate receptor ca(2+)-signalling
topic In Vitro Systems
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113295/
https://www.ncbi.nlm.nih.gov/pubmed/33713149
http://dx.doi.org/10.1007/s00204-021-03024-0
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