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

Abstract

BackgroundOur 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.MethodsThe 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.ResultsDuring 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).ConclusionsCumulative 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 materialThe online version of this article (doi:10.1186/s40360-016-0107-4) contains supplementary material, which is available to authorized users.

Bisphenol A and rotenone induce S-nitrosylation of protein disulfide isomerase (PDI) and inhibit neurite outgrowth of primary cultured cells of the rat hippocampus and PC12 cells

“…3A). These results were consistent with previous findings showing that BPA (10 μM) inhibited neurite outgrowth (Wang et al, 2019;Wu et al, 2016). BPA could change neurite outgrowth and S-nitrosylation of PDI at low concentrations (5 or 10 μM).…”

Strain-Dependent Consequences of Zika Virus Infection and Differential Impact on Neural Development

“…The authors, using their well-characterized hNP to neuron differentiation process, could characterize the pathogenic effects of ZIKV infection on populations of both immature and mature neurons. After 14 DIV in the absence of FGF2, hNP cell cultures presented a neuronal phenotype with reduced SOX1 expression and a portion of HuC/HuD+ and βIII-tubulin+ cells, indicative of maturing neurons ( Figure 1 A) [ 28 ]. The full 28 DIV of differentiation in vitro resulted in a highly homogeneous population of post-mitotic and mature neurons characterized by microtubule-associated protein 2 (MAP2) expression ( Figure 1 A) [ 34 , 36 ].…”

“…hNP cells and hNP-derived neurons are advantageous when assessing developmental malformations and provide a platform to examine the effects toxins and pathogens exert in a controlled, homogenous, and reproducible setting [ 39 ]. In vitro assays designed to evaluate critical functions of hNP and hNP-derived neurons provide critical insight into how environmental stresses and pathogens can alter normal functions including cell migration and neurite outgrowth [ 28 , 39 , 40 ]. In order to evaluate the impact of Asian and African isolates of ZIKV on human neural cell development, both undifferentiated hNP cells and hNP-derived neurons were infected to observe ZIKV-induced changes in differentiation, proliferation, migration, and neurite outgrowth.…”

“…To further address the lineage-specific effects of ZIKV infection, the authors employed a representative in vitro model of CNS development to directly evaluate the effects of ZIKV infection using both African-lineage and Asian-lineage isolates of ZIKV. Previously, the authors demonstrated that hNP cells could yield mature neurons in a predictable and well-defined manner representative of in vivo human development [ 26 , 27 , 28 ]. In vitro culture of hNP cells and hNP-derived neurons enabled the evaluation of viral replication and cytopathic effect of both Asian and African lineages of ZIKV.…”

Time and space-resolved quantification of plasma membrane sialylation for measurements of cell function and neurotoxicity

“…Although they offer the possibility of high-throughput screening, viability measurements do not reveal alterations caused by compounds at sub-cytotoxic concentrations. In this case, neuron-specific morphologic endpoints such as neurite outgrowth (for developing neurons) (Ceresa et al 2014;Delp et al 2018b;Flaskos et al 2011;Frimat et al 2010;Krug et al 2013;Stiegler et al 2011;Wu et al 2016) or neurite integrity (for mature neurons) (Axelsson et al 2006;Krug et al 2013;Saxena and Caroni 2007) can be measured and quantified. If compounds display neurotoxicity based on these endpoints, at concentrations lower than those which produce cytotoxic effects, they can be considered as potentially neurotoxic.…”