The Neurosphere Assay as an In Vitro Method for Developmental Neurotoxicity (DNT) Evaluation

Nimtz, Laura; Klose, Jördis; Masjosthusmann, Stefan; Barenys, Marta; Fritsche, Ellen

doi:10.1007/978-1-4939-9228-7_8

Cited by 10 publications

(17 citation statements)

References 35 publications

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“…NPC proliferation is a fundamental neurodevelopmental KE that, when disturbed, like in Zika virus infected primary NPC, leads to microcephaly in children (Tang et al, 2016;Devakumar et al, 2018). Proliferation of primary hNPC of fetal origin (Lonza, Verviers, Belgium) grown as neurospheres in 3D is studied by measuring increase in sphere size over time using phase contrast microscopy (Baumann et al, 2015(Baumann et al, , 2014Gassmann et al, 2010Gassmann et al, , 2012Moors et al, 2009;Schreiber et al, 2010;Tofighi et al, 2011;Nimtz et al 2019) and/or by measuring BrdU incorporation after 3 days in vitro (DIV) using a luminescence-based BrdU Assay (Roche) and a luminometer (Baumann et al, 2014(Baumann et al, , 2015Nimtz et al, 2019). This assay is part of a 'high content DNT test', the 'Neurosphere Assay' (NPC1-5) and uses withdrawal of growth factors as an endpoint-specific control Nimtz et al, 2019).…”

Section: Primary Hnpc Proliferation Assay (Npc1 Test)mentioning

confidence: 99%

“…the src kinase inhibitor PP2 is used to reduces migration distance (Baumann et al, 2015;Nimtz et al, 2019). Secondarily, after radial glia differentiation, neurons and oligodendrocytes arise (Schmuck et al, 2017).…”

Section: Primary Hnpc Migration Assay (Npc2 Test)mentioning

confidence: 99%

“…With this the a) radial glia cell, b) early neuronal and c) oligodendrocyte migration can be assessed (Schmuck et al, 2017). The NPC2 assay can be multiplexed with early fetal neuronal and oligodendrocyte differentiation (NPC3 and 5) as well as neuronal morphology (NPC4), yet these are described as separate assays as they can also be studied without migration measures (Baumann et al, 2015;Nimtz et al, 2019).…”

Section: Primary Hnpc Migration Assay (Npc2 Test)mentioning

confidence: 99%

“…In addition, multiplexing of NPC3 with NPC2 and NPC5 (oligodendrocyte differentiation and positioning, see below) after five days in vitro reveals information on neuronal and oligodendrocyte differentiation and migration within one assay (Schmuck, unpublished data). EGF inhibits neuronal differentiation and is used as an endpoint-specific control (Nimtz et al, 2019;Baumann et al, 2015).…”

Section: Primary Hnpc Neuronal Differentiation Assay (Npc3)mentioning

confidence: 99%

“…Oligodendrocytes differentiate in co-culture with astrocytes and neurons in secondary 3D structures from hNPC within 5 DIV (Dach et al, 2017;Alépée et al, 2014;Moors et al, 2009;Schreiber et al, 2010;Schmuck et al, 2017) into a mixed culture of pre-oligodendrocytes, immature, and non-myelinating, mature oligodendrocytes ( Figure 5). The hNPC oligodendrocyte differentiation and maturation was recently put into an assay format (NPC5; (Dach et al, 2017;Nimtz et al, 2019). DAPI-stained nuclei that co-localize for the epitope O4 after 5 DIV are identified.…”

Section: Oligodendrocyte Differentiation (Npc5)mentioning

confidence: 99%

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Establishment of an a priori protocol for the implementation and interpretation of an in‐vitro testing battery for the assessment of developmental neurotoxicity

Masjosthusmann

Blum

Bartmann

et al. 2020

EFSA Supporting Publications

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In this project we set up a human cell-based DNT in vitro testing strategy that is based on test methods with high readiness and data generated therefrom. The methods underwent a fit-for-purpose evaluation that considered four key elements: 1. The test system, 2. the exposure scheme, 3. the assay and analytical endpoint(s) and 4. the classification model. This testing battery was challenged with 119 chemicals for which rich toxicological information was available (for some of them also on their DNT hazard). Testing was performed in 5 test systems measuring 10 DNT-specific endpoints and additional 9 viability/ cytotoxicity-related parameters. For approximately half of the compounds, additional and complementary data from DNT in vitro tests was added by the US-EPA. This extended battery was also evaluated. Testing results revealed that the test methods of this current DNT in vitro battery are reliable and reproducible. The endpoints had to a large extent low redundancy. Battery performance, as assessed with compounds well-characterized for DNT hazard had a sensitivity of 82.7 % and a specificity of 88.2 %. Gap analyses suggested that radial, astro-and microglia as well as myelination endpoints may be added to the battery. Two case studies, one for screening and prioritization of 14 flame retardants, and one on hazard characterization of 2 pesticides, were presented. Hypothetical AOPs were developed based on the latter case study. In conclusion, the DNT testing strategy explored here is a very promising first approach for DNT hazard identification and characterization. The performance is encouraging and may be improved by inclusion of further tests. Some uncertainties in DNT in vitro battery testing outcomes could be reduced by incorporating test data and modelling approaches related to in vitro and in vivo toxicokinetics of test compounds.

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Section: Primary Hnpc Proliferation Assay (Npc1 Test)mentioning

confidence: 99%

Section: Primary Hnpc Migration Assay (Npc2 Test)mentioning

confidence: 99%

Section: Primary Hnpc Migration Assay (Npc2 Test)mentioning

confidence: 99%

Section: Primary Hnpc Neuronal Differentiation Assay (Npc3)mentioning

confidence: 99%

Section: Oligodendrocyte Differentiation (Npc5)mentioning

confidence: 99%

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Establishment of an a priori protocol for the implementation and interpretation of an in‐vitro testing battery for the assessment of developmental neurotoxicity

Masjosthusmann

Blum

Bartmann

et al. 2020

EFSA Supporting Publications

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150

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show abstract

Reliable identification and quantification of neural cells in microscopic images of neurospheres

et al. 2021

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Neurosphere cultures consisting of primary human neural stem/progenitor cells (hNPC) are used for studying the effects of substances on early neurodevelopmental processes in vitro. Differentiating hNPCs migrate and differentiate into radial glia, neurons, astrocytes, and oligodendrocytes upon plating on a suitable extracellular matrix and thus model processes of early neural development. In order to characterize alterations in hNPC development, it is thus an essential task to reliably identify the cell type of each migrated cell in the migration area of a neurosphere. To this end, we introduce and validate a deep learning approach for identifying and quantifying cell types in microscopic images of differentiated hNPC. As we demonstrate, our approach performs with high accuracy and is robust against typical potential confounders. We demonstrate that our deep learning approach reproduces the dose responses of well‐established developmental neurotoxic compounds and controls, indicating its potential in medium or high throughput in vitro screening studies. Hence, our approach can be used for studying compound effects on neural differentiation processes in an automated and unbiased process.

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Neurodevelopmental toxicity assessment of flame retardants using a human DNT in vitro testing battery

et al. 2021

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Due to their neurodevelopmental toxicity, flame retardants (FRs) like polybrominated diphenyl ethers are banned from the market and replaced by alternative FRs, like organophosphorus FRs, that have mostly unknown toxicological profiles. To study their neurodevelopmental toxicity, we evaluated the hazard of several FRs including phased-out polybrominated FRs and organophosphorus FRs: 2,2′,4,4′-tetrabromodiphenylether (BDE-47), 2,2′,4,4′,5-pentabromodiphenylether (BDE-99), tetrabromobisphenol A, triphenyl phosphate, tris(2-butoxyethyl) phosphate and its metabolite bis-(2-butoxyethyl) phosphate, isodecyl diphenyl phosphate, triphenyl isopropylated phosphate, tricresyl phosphate, tris(1,3-dichloro-2-propyl) phosphate, tert-butylphenyl diphenyl phosphate, 2-ethylhexyl diphenyl phosphate, tris(1-chloroisopropyl) phosphate, and tris(2-chloroethyl) phosphate. Therefore, we used a human cell–based developmental neurotoxicity (DNT) in vitro battery covering a large variety of neurodevelopmental endpoints. Potency according to the respective most sensitive benchmark concentration (BMC) across the battery ranked from <1 μM (5 FRs), 1<10 μM (7 FRs) to the >10 μM range (3 FRs). Evaluation of the data with the ToxPi tool revealed a distinct ranking (a) than with the BMC and (b) compared to the ToxCast data, suggesting that DNT hazard of these FRs is not well predicted by ToxCast assays. Extrapolating the DNT in vitro battery BMCs to human FR exposure via breast milk suggests low risk for individual compounds. However, it raises a potential concern for real-life mixture exposure, especially when different compounds converge through diverse modes-of-action on common endpoints, like oligodendrocyte differentiation in this study. This case study using FRs suggests that human cell–based DNT in vitro battery is a promising approach for neurodevelopmental hazard assessment and compound prioritization in risk assessment. Graphical abstract

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The Neurosphere Assay as an In Vitro Method for Developmental Neurotoxicity (DNT) Evaluation

Cited by 10 publications

References 35 publications

Establishment of an a priori protocol for the implementation and interpretation of an in‐vitro testing battery for the assessment of developmental neurotoxicity

Establishment of an a priori protocol for the implementation and interpretation of an in‐vitro testing battery for the assessment of developmental neurotoxicity

Reliable identification and quantification of neural cells in microscopic images of neurospheres

Neurodevelopmental toxicity assessment of flame retardants using a human DNT in vitro testing battery

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