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Scientific Validation of Human Neurosphere Assays for Developmental Neurotoxicity Evaluation - PubMed

  • ️Sat Jan 01 2022

Scientific Validation of Human Neurosphere Assays for Developmental Neurotoxicity Evaluation

Katharina Koch et al. Front Toxicol. 2022.

Abstract

There is a call for a paradigm shift in developmental neurotoxicity (DNT) evaluation, which demands the implementation of faster, more cost-efficient, and human-relevant test systems than current in vivo guideline studies. Under the umbrella of the Organisation for Economic Co-operation and Development (OECD), a guidance document is currently being prepared that instructs on the regulatory use of a DNT in vitro battery (DNT IVB) for fit-for-purpose applications. One crucial issue for OECD application of methods is validation, which for new approach methods (NAMs) requires novel approaches. Here, mechanistic information previously identified in vivo, as well as reported neurodevelopmental adversities in response to disturbances on the cellular and tissue level, are of central importance. In this study, we scientifically validate the Neurosphere Assay, which is based on human primary neural progenitor cells (hNPCs) and an integral part of the DNT IVB. It assesses neurodevelopmental key events (KEs) like NPC proliferation (NPC1ab), radial glia cell migration (NPC2a), neuronal differentiation (NPC3), neurite outgrowth (NPC4), oligodendrocyte differentiation (NPC5), and thyroid hormone-dependent oligodendrocyte maturation (NPC6). In addition, we extend our work from the hNPCs to human induced pluripotent stem cell-derived NPCs (hiNPCs) for the NPC proliferation (iNPC1ab) and radial glia assays (iNPC2a). The validation process we report for the endpoints studied with the Neurosphere Assays is based on 1) describing the relevance of the respective endpoints for brain development, 2) the confirmation of the cell type-specific morphologies observed in vitro, 3) expressions of cell type-specific markers consistent with those morphologies, 4) appropriate anticipated responses to physiological pertinent signaling stimuli and 5) alterations in specific in vitro endpoints upon challenges with confirmed DNT compounds. With these strong mechanistic underpinnings, we posit that the Neurosphere Assay as an integral part of the DNT in vitro screening battery is well poised for DNT evaluation for regulatory purposes.

Keywords: 3D in vitro models; developmental neurotoxicity; human induced pluripotent stem cells; neural progenitor cells; neurons; new approach methodologies; oligodendrocytes; radial glia cells.

Copyright © 2022 Koch, Bartmann, Hartmann, Kapr, Klose, Kuchovská, Pahl, Schlüppmann, Zühr and Fritsche.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1

Schematic description of the human Neurosphere Assay test methods and the identification of their scientific bases. The outer ring highlights the five questions which were the basis of the scientific validation of all six assays. The inner ring contains the neurodevelopmental key events modelled in the individual NPC assays. Each color represents one NPC assay. The color scheme is kept throughout the manuscript. Abbreviation: OL, oligodendrocytes. Created with

biorender.com

.

FIGURE 2
FIGURE 2

The NPC1ab assay identifies chemicals disturbing NPC proliferation. (A+B) Primary hNPC neurospheres (Lonza, Verviers, Belgium) were cultivated for 3 days in proliferation medium containing 20 ng/ml of the growth factors EGF and FGF (control) or in medium without growth factors (w/o growth factors). Representative pictures (A) and quantifications of the sphere size (B), as assessed within the NPC1a assay, showed that growth factors are necessary for hNPC proliferation. (D) Proliferating hNPC neurospheres issued from three different individuals (062, 263, and 806) were analyzed using flow cytometry analysis, confirming high expression of the neural stem/progenitor markers nestin and SOX2. The percentage of double-positive cells is indicated in the upper right quartile. (C, E). Exposure of proliferating hNPCs for 3 days to increasing concentrations of (C) the EGFR inhibitor PD153035 (0.01–0.64 µM) or (E) cadmium chloride (0.027–20 µM) concentration-dependently decreased hNPC proliferation compared to the respective solvent controls (adapted from Masjosthusmann et al., 2020). Proliferation was assessed by sphere size increase (NPC1a) and BrdU incorporation into the DNA (NPC1b). The values of the chemical-treated conditions are expressed as % of the respective solvent controls. Cytotoxicity (LDH release) was assessed in parallel and is depicted as % of a lysis control (spheres treated with 0.2% Triton-X100). Data are presented as mean ± SEM. Statistical significance was calculated using one-way ANOVA (C, E) and two-tailed Student’s t-tests (B). A p-value below 0.05 was termed significant. * and # significantly changed compared to the solvent control of the respective endpoint if not marked otherwise.

FIGURE 3
FIGURE 3

The NPC2a assay identifies chemicals disturbing radial glia migration. Primary hNPCs were differentiated on PDL-laminin-coated 96-well plates in the absence of growth factors. (A) After 24 h, immunocytochemical stainings for nestin, GFAP and Ki-67 were performed confirming radial glia-typic marker expression and morphology. Nuclei were counterstained with Hoechst33258. (B) Over the 5 days of differentiation, cells radially migrate out of the sphere core and form a circular migration area. The migration distance of hNPCs increased gradually over time, as assessed by determining the distance from the sphere core to the furthest migrated cells at four opposite positions in brightfield images every 24 h (C) hNPC migration over 5 days was assessed in presence of EGF (0.5–1 ng/ml) alone, in combination with the EGFR inhibitor PD153035 (1–2 µM), or the respective solvent. While EGF increased hNPC migration compared to the solvent control, PD153035 inhibited the EGF-induced effect. (D) A negative effect of the SRC-family kinase inhibitor PP2 on hNPC migration was confirmed by differentiating hNPCs for 3 and 5 days in presence of 10 µM PP2 or the respective solvent (SC). (E) hNPCs differentiation in presence of increasing concentrations of methyl-mercury (MeHg, 0.005–1 µM) for 3 days concentration-dependently reduced the migration distance (adapted from Fritsche et al., 2018a). For (D,E), cytotoxicity (LDH release) was assessed in parallel and is depicted as % of a lysis control (differentiated hNPCs treated with 0.2% Triton-X100). Data are presented as mean ± SEM. Statistical significance was calculated using one-way ANOVA (B, E) and two-tailed Student’s t-tests (C, D). A p-value below 0.05 was termed significant. *significantly changed compared to the respective solvent control. #significantly changed compared to the respective EGF concentration.

FIGURE 4
FIGURE 4

The NPC3 and NPC4 assays identify disruption of neuronal differentiation and morphology. Primary hNPC neurospheres were differentiated on PDL-laminin-coated matrices for 5 days without growth factors. (A) Immunocytochemical stainings for β(III)tubulin (neurons, red) and Hoechst33258 (nuclei, blue) confirmed neuronal marker expression and morphology. Scale bar: 100 µm. (B) Neuronal differentiation, assessed as the percentage of β(III)tubulin-positive neurons compared to the total nuclei count within the migration area, increased gradually over the 5 days of differentiation. (C) hNPCs differentiation for 5 days in presence of increasing concentrations of the Notch inhibitor DAPT (0.01 µM–10 µM) concentration-dependently decreased neuronal differentiation compared to the solvent control. (D,E) Treatment with the RhoA activator narciclasine (0.00014 µM–0.1 µM) for 5 days concentration-dependently decreased neuronal differentiation compared to the solvent control (SC). Representative pictures of β(III)tubulin- and Hoechst33258-stained cells (D) and concentration-response curves (E) are shown (adapted from Masjosthusmann et al. (2020). Scale bar: 100 µm. Cytotoxicity (LDH release) was assessed in parallel and is depicted as % of a lysis control (differentiated hNPCs treated with 0.2% Triton-X100). (F) The NPC4 assay detects the neuronal morphology of hNPC-derived β(III)tubulin-positive neurons. After the neurons were annotated by the convolutional neural network of the AI, neurite length and area were calculated by the Omnisphero software (Schmuck et al., 2017). (G+H) Neurite area and total subneurite length gradually increased over the 5 days of differentiation. (I+J) Both the RhoA activator narciclasine and the PKC inhibitor bisindolylmaleimide 1 (Bis-I) concentration-dependently decreased the neurite area and subneurite length compared to the respective solvent control in hNPCs differentiating over 5 days (adapted from Masjosthusmann et al., 2020). Cytotoxicity (LDH release) was assessed in parallel and is depicted as % of a lysis control (differentiated hNPCs treated with 0.2% Triton-X100). Data are presented as mean ± SEM. Statistical significance was calculated using one-way ANOVA. A p-value below 0.05 was termed significant. * significantly changed compared to the respective solvent control.

FIGURE 5
FIGURE 5

Oligodendrocyte differentiation is assessed with the NPC5 assay. (A) Primary hNPCs were differentiated for 5 days in differentiation medium without growth factors on PDL-laminin-coated plates. Immunocytochemical stainings were performed to identify cells of the OL lineage (O4) and cell nuclei (Hoechst33258). (B) mRNA expression of OL lineage markers PDGFRA, CNP, GALC, PLP1 and MBP was assessed in proliferating hNPCs and hNPC differentiated for 60 h using quantitative real-time PCR. Expression was calculated as copy numbers (CN) per CN of the reference gene ACTB multiplied by 10.000. The expression in differentiated hNPCs is displayed as fold of expression in proliferating hNPCs. Expression of all markers increased during hNPC differentiation (adapted from Klose et al. (2021a)). (C) Oligodendrocyte differentiation, assessed as the percentage of O4-positive OLs compared to the total nuclei count within the migration area, increased gradually over the 5 days of differentiation. (D) hNPCs differentiation for 5 days in presence of increasing concentrations of the Notch inhibitor DAPT (0.01 µM–10 µM) concentration-dependently decreased OL differentiation compared to the solvent control. (E) hNPCs differentiation in presence of 100 µM ascorbic acid (Asc) increased the percentage of OLs in the differentiated culture. (F+G) Both exposure to the insecticide deltamethrin (DM, 0.027–20 µM, adapted from Masjosthusmann et al., 2020) and the flame retardant tetrabromobisphenol A (TBBPA, 0.027–20 µM, adapted from Klose et al., 2021a) during the 5 days of differentiation concentration-dependently decreased OL differentiation compared to the respective solvent controls. For deltamethrin and TBBPA the mitochondrial activity was assessed in parallel and is depicted as % of solvent control. For (E–G), representative pictures of O4+ OLs exposed to solvent control or the respective treatments are shown. Data are presented as mean ± SEM. Statistical significance was calculated using one-way ANOVA (C,D,F,G) and two-tailed Student’s t-tests (B,E). A p-value below 0.05 was termed significant. *significant compared to the respective solvent control.

FIGURE 6
FIGURE 6

The NPC6 assay identifies disruptors of TH-dependent oligodendrocyte maturation. (A) Primary hNPCs were differentiated for 5 days in differentiation medium without growth factors on PDL-laminin coated plates either in presence of 3 nM T3 or solvent control. Immunocytochemical stainings were performed to identify cells of the OL lineage (O4) and cell nuclei (Hoechst33258). (B) mRNA expression of the OL lineage marker MBP was assessed in hNPCs differentiated in the presence of 3 nM T3 or solvent for 24, 72 or 120 h using quantitative real-time PCR. Expression is displayed as MBP mRNA copy numbers (CN) per CN of the reference gene ACTB multiplied by 10.000. The expression is displayed as fold of expression after 24 h (adapted from Dach et al., 2017). (C) OL maturation was quantified using the maturation quotient (QM), which is calculated by dividing the MPB mRNA CN ((copy number MBP/ copy number ACTB) *10.000) by the percentage of O4+ cells. Exposure to 3 nM T3 significantly increased the QM compared to the solvent control (SC). (D+E) The QM was calculated for hNPCs differentiating for 5 days in presence of solvent (SC), 3 nM T3 alone or T3 in combination with increasing concentrations of the TH receptor antagonist NH-3 (4–400 nM) or the flame retardant TBBPA (0.01–1 µM). Both NH-3 and TBBPA concentration-dependently decreased the QM compared to 3 nM T3 and thus impaired T3-depedent OL maturation (adapted from Klose et al. (2021b)). Data are presented as mean ± SEM. Statistical significance was calculated using one-way ANOVA (B, D, E) and two-tailed Student’s t-tests (C). A p-value below 0.05 was termed significant. *significant compared to the respective solvent control. #significant compared to 3 nM T3.

FIGURE 7
FIGURE 7

The proliferation of hiNPCs is assessed with the iNPC1ab assay. (A,B) Human iPSC-derived hiNPCs were cultivated for 3 days in proliferation medium containing 20 ng/ml of the growth factors EGF and FGF (control) or in medium without growth factors (w/o growth factors). Representative pictures (A) and quantifications of the sphere size (B), as assessed within the iNPC1a assay, showed that growth factors are necessary for hiNPC proliferation. (D) Proliferating hiNPC neurospheres issued from an iPS11 neural induction were analyzed using flow cytometry analysis, confirming high expression of the neural stem/progenitor markers nestin and SOX2. The percentage of double-positive cells is indicated in the upper right quartile. (C, E, F) Exposure of proliferating hiNPCs for 3 days to increasing concentrations of the EGFR inhibitor PD153035 (C) or the mitochondrial complex I inhibitor rotenone (E,F) concentration-dependently decreased hiNPC proliferation compared to the respective solvent controls. Proliferation was assessed by sphere size increase (iNPC1a) and BrdU incorporation into the DNA (iNPC1b). The values of the chemical-treated conditions are expressed as % of the respective solvent controls. Cytotoxicity (LDH release) was assessed in parallel and is depicted as % of a lysis control (spheres treated with 0.2% Triton-X100). For rotenone-treatment, mitochondrial activity (F) was assessed in parallel and is depicted as % of solvent control. Data are presented as mean ± SEM. Statistical significance was calculated using one-way ANOVA (C, E, F) and two-tailed Student’s t-tests (B). A p-value below 0.05 was termed significant. Symbols * and # show statistical differences in comparison to the solvent control of the respective endpoint if not marked otherwise.

FIGURE 8
FIGURE 8

The hiNPC2 assay identifies disturbances of neural migration. Human hiNPCs were differentiated on PDL-laminin-coated 96-well plates in the absence of growth factors. (A) After 3 days of differentiation, immunocytochemical stainings of hiNPCs in early (p8) and later (p25) passages confirmed expression of the neuronal marker β(III)tubulin and the glial marker S100β. Nuclei were counterstained with Hoechst33258. Stars indicate RG-like cells and triangles indicate astrocyte-like morphology. (B) The migration distance of hiNPCs increased gradually over time, as assessed by determining the distance from the sphere core to the furthest migrated cells at four opposite positions in brightfield images every 24 h (C) hiNPC migration over 3 days was assessed in presence of EGF (0.5–1 ng/ml) alone, in combination with the EGFR inhibitor PD153035 (1–2 µM), or the respective solvent. While EGF increased hiNPC migration, PD153035 inhibited the EGF-induced effect. (D) A negative effect of the SRC-family kinase inhibitor PP2 on hiNPC migration was confirmed by differentiating hiNPCs for 3 days in presence of 10 µM PP2 or the respective solvent (SC). (E) hiNPCs differentiation in presence of increasing concentrations of narciclasine (MeHg, 0.0001–0.1 µM) for 3 days concentration-dependently reduced the migration distance. For (D,E), cytotoxicity (LDH release) was assessed in parallel and is depicted as % of a lysis control (differentiated hiNPCs treated with 0.2% Triton-X100). Data are presented as mean ± SEM. Statistical significance was calculated using one-way ANOVA (B, E) and two-tailed Student’s t-tests (C, D). A p-value below 0.05 was termed significant. *significant compared to the respective solvent control; #significant compared to the respective EGF concentration.

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