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High content imaging quantification of multiple in vitro human neurogenesis events after neurotoxin exposure

BACKGROUND: Our objective was to test neural active compounds in a human developmental neurotoxicity (DNT) model that represents neural tube stages of vulnerability. Previously we showed that 14 days in vitro (DIV 14) was sufficient to generate cryopreserved neuronal cells for post thaw neurite reco...

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Autores principales: Wu, Xian, Majumder, Anirban, Webb, Robin, Stice, Steven L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131404/
https://www.ncbi.nlm.nih.gov/pubmed/27903287
http://dx.doi.org/10.1186/s40360-016-0107-4
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author Wu, Xian
Majumder, Anirban
Webb, Robin
Stice, Steven L.
author_facet Wu, Xian
Majumder, Anirban
Webb, Robin
Stice, Steven L.
author_sort Wu, Xian
collection PubMed
description BACKGROUND: Our objective was to test neural active compounds in a human developmental neurotoxicity (DNT) model that represents neural tube stages of vulnerability. Previously we showed that 14 days in vitro (DIV 14) was sufficient to generate cryopreserved neuronal cells for post thaw neurite recovery assays. However, short exposure and assessment may not detect toxicants that affect an early neurogenesis continuum, from a mitotic human neural progenitor (hNP) cell population through the course of neurite outgrowth in differentiating neurons. Therefore, we continuously exposed differentiating hNP cells from DIV 0 through DIV 14 to known toxicants and endocrine active compounds in order to assess at DIV 14 effects of these compounds in a human DNT maturation model for neurogenesis. METHODS: The Human DNT continuum (DIV 0 to DIV 14) was determined using immunocytochemistry for SOX1+ (proliferating hNP) and HuC/D+ (post mitotic neurons). The cumulative effects of five compounds was observed on neurite outgrowth in (βIII-tubulin+) and (HuC/D+) cells using high content imaging. All data were analyzed using a one-way ANOVA with a significance threshold of p < 0.05. RESULTS: During maturation in vitro, the neural cultures transitioned from uniform hNP cells (DIV 0) to predominantly mature post mitotic neuronal neurons (HuC/D+, 65%; DIV14) but also maintained a smaller population of hNP cells (SOX1+). Using this DNT maturation model system, Bis-1, testosterone, and β-estradiol inhibited neuronal maturation at micromolar levels but were unaffected by acetaminophen. β-estradiol also disrupted neurite extension at 10 μM. Treating cells in this window with Bisphenol A (BPA) significantly inhibited neurite outgrowth and branching in these continuum cultures but only at the highest concentrations tested (10 μM). CONCLUSIONS: Cumulative effects of neurotoxicant exposure during a maturation continuum altered human neurogenesis at lower exposure levels than observed in acute exposure of static cryopreserved neurite recovery neurons cultures. Unlike prior acute studies, β-estradiol was highly toxic when present throughout the continuum and cytotoxicity was manifested starting early in the continuum via a non-estrogen receptor α (ER α) mechanism. Therefore, the effect of neural developmental neurotoxins can and should be determined during the dynamic process of human neural maturation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40360-016-0107-4) contains supplementary material, which is available to authorized users.
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spelling pubmed-51314042016-12-12 High content imaging quantification of multiple in vitro human neurogenesis events after neurotoxin exposure Wu, Xian Majumder, Anirban Webb, Robin Stice, Steven L. BMC Pharmacol Toxicol Research Article BACKGROUND: Our objective was to test neural active compounds in a human developmental neurotoxicity (DNT) model that represents neural tube stages of vulnerability. Previously we showed that 14 days in vitro (DIV 14) was sufficient to generate cryopreserved neuronal cells for post thaw neurite recovery assays. However, short exposure and assessment may not detect toxicants that affect an early neurogenesis continuum, from a mitotic human neural progenitor (hNP) cell population through the course of neurite outgrowth in differentiating neurons. Therefore, we continuously exposed differentiating hNP cells from DIV 0 through DIV 14 to known toxicants and endocrine active compounds in order to assess at DIV 14 effects of these compounds in a human DNT maturation model for neurogenesis. METHODS: The Human DNT continuum (DIV 0 to DIV 14) was determined using immunocytochemistry for SOX1+ (proliferating hNP) and HuC/D+ (post mitotic neurons). The cumulative effects of five compounds was observed on neurite outgrowth in (βIII-tubulin+) and (HuC/D+) cells using high content imaging. All data were analyzed using a one-way ANOVA with a significance threshold of p < 0.05. RESULTS: During maturation in vitro, the neural cultures transitioned from uniform hNP cells (DIV 0) to predominantly mature post mitotic neuronal neurons (HuC/D+, 65%; DIV14) but also maintained a smaller population of hNP cells (SOX1+). Using this DNT maturation model system, Bis-1, testosterone, and β-estradiol inhibited neuronal maturation at micromolar levels but were unaffected by acetaminophen. β-estradiol also disrupted neurite extension at 10 μM. Treating cells in this window with Bisphenol A (BPA) significantly inhibited neurite outgrowth and branching in these continuum cultures but only at the highest concentrations tested (10 μM). CONCLUSIONS: Cumulative effects of neurotoxicant exposure during a maturation continuum altered human neurogenesis at lower exposure levels than observed in acute exposure of static cryopreserved neurite recovery neurons cultures. Unlike prior acute studies, β-estradiol was highly toxic when present throughout the continuum and cytotoxicity was manifested starting early in the continuum via a non-estrogen receptor α (ER α) mechanism. Therefore, the effect of neural developmental neurotoxins can and should be determined during the dynamic process of human neural maturation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40360-016-0107-4) contains supplementary material, which is available to authorized users. BioMed Central 2016-12-01 /pmc/articles/PMC5131404/ /pubmed/27903287 http://dx.doi.org/10.1186/s40360-016-0107-4 Text en © The Author(s). 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Wu, Xian
Majumder, Anirban
Webb, Robin
Stice, Steven L.
High content imaging quantification of multiple in vitro human neurogenesis events after neurotoxin exposure
title High content imaging quantification of multiple in vitro human neurogenesis events after neurotoxin exposure
title_full High content imaging quantification of multiple in vitro human neurogenesis events after neurotoxin exposure
title_fullStr High content imaging quantification of multiple in vitro human neurogenesis events after neurotoxin exposure
title_full_unstemmed High content imaging quantification of multiple in vitro human neurogenesis events after neurotoxin exposure
title_short High content imaging quantification of multiple in vitro human neurogenesis events after neurotoxin exposure
title_sort high content imaging quantification of multiple in vitro human neurogenesis events after neurotoxin exposure
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131404/
https://www.ncbi.nlm.nih.gov/pubmed/27903287
http://dx.doi.org/10.1186/s40360-016-0107-4
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