Journal: Cell Biology and Toxicology

Article Title: Application of the adverse outcome pathway concept for investigating developmental neurotoxicity potential of Chinese herbal medicines by using human neural progenitor cells in vitro

doi: 10.1007/s10565-022-09730-4

Figure Lengend Snippet: Effects of LGT on hNPC migration and differentiation supporting the AOP “Binding to the extracellular matrix protein laminin leading to decreased cognitive function.” Spheres with a defined size of 0.3 mm were plated for hNPC migration analyses onto poly- d -lysine/laminin-coated 96-well plates in presence and absence of LGT for 120 h. Radial glia migration (72 h) was determined by manually measuring the radial migration from the sphere core ( A ). Differentiation into neurons and oligodendrocytes was determined by performing immunocytochemical stainings and using the software Omnisphero (Schmuck et al. ). The number of all β(III)-tubulin-positive (red) and O4-positive (green) cells in the migration area after 120 h of differentiation were counted manually and their percent of Hoechst-positive nuclei (blue) was calculated ( B , D ). In parallel, cytotoxicity ( C ) was assessed by the LDH assay. At least three independent experiments with 5 technical replicates were performed and presented as mean ± SEM. Statistical significance was calculated using one-way ANOVA followed by Bonferroni’s post hoc tests, *significant compared to the solvent control ( p ≤ 0.05 was considered significant). ( E ) The schematic AOP “Binding to the extracellular matrix protein laminin leading to decreased cognitive function” includes the laminin-dependent decreased adhesion of NPCs as a central key event resulting in cortical architecture alterations and adverse outcomes in the developing brain. This AOP is based on published results: *Barenys et al. , and Kühne et al. ; # Graus-Porta et al. ; °Belvindrah et al. ; + Amin and Borrell , Fernández et al. , and Long and Huttner . nq, not quantifiable (relevant for number of neurons and oligodendrocytes)

Article Snippet: Neurospheres (migrated for 5 days) were incubated overnight at 4 °C with a mouse IgM oligodendrocyte O4 antibody solution (1:400 in PBS with 10% GS; R&D System, #MAB1326).

Techniques: Migration, Binding Assay, Software, Lactate Dehydrogenase Assay, Solvent, Control

Journal: Cell Biology and Toxicology

Article Title: Application of the adverse outcome pathway concept for investigating developmental neurotoxicity potential of Chinese herbal medicines by using human neural progenitor cells in vitro

doi: 10.1007/s10565-022-09730-4

Figure Lengend Snippet: Effects of TM on hNPC migration and differentiation. Spheres with a defined size of 0.3 mm were plated onto poly- d -lysine/laminin-coated 96-well plates and exposed to increasing TM concentrations over 120 h. Radial glia migration (72 h) was determined by manually measuring the radial migration from the sphere core ( A ). Differentiation into neurons and oligodendrocytes was determined by performing immunocytochemical stainings and using the software Omnisphero (Schmuck et al. ). The number of all β(III)-tubulin-positive cells ( B ) and O4-positive cells ( B , D ; green) in percent of Hoechst-positive nuclei (blue) in the migration area after 120 h of differentiation was calculated manually. In parallel, viability and cytotoxicity ( A , C ) were assessed by the Alamar Blue and the LDH assay. At least three independent experiments with 5 technical replicates were performed and depicted as mean ± SEM. Statistical significance was calculated using one-way ANOVA followed by Bonferroni’s post hoc tests, *significant compared to the solvent control ( p ≤ 0.05 was considered significant)

Article Snippet: Neurospheres (migrated for 5 days) were incubated overnight at 4 °C with a mouse IgM oligodendrocyte O4 antibody solution (1:400 in PBS with 10% GS; R&D System, #MAB1326).

Techniques: Migration, Software, Lactate Dehydrogenase Assay, Solvent, Control

Journal: Cell Biology and Toxicology

Article Title: Application of the adverse outcome pathway concept for investigating developmental neurotoxicity potential of Chinese herbal medicines by using human neural progenitor cells in vitro

doi: 10.1007/s10565-022-09730-4

Figure Lengend Snippet: Transcriptomic profiling of differentiated hNPCs treated with TM. Differential gene expression between hNPCs exposed to 1 mg/mL TM and untreated hNPCs over 60 h of differentiation was statistically determined using moderated t -test followed by Benjamini–Hochberg tests. Genes (#22) with p ≤ 0.05 and fold change ≥ 2 were termed differentially expressed (DEX). Overrepresented gene ontology (GO) terms for hNPCs differentiated under the influence of 1 mg/mL TM for 60 h ( A ). GO enrichment analysis was performed using the online tool DAVID Bioinformatics Resources 6.8 (DAVID). The significantly regulated GO terms (#12) after 60 h of differentiation were further assigned to 3 superordinate processes based on expert judgment. Numbers in the pie chart represent the percentage of GO terms assigned to each superordinate process. Gene–gene interaction network analysis of the genes regulated by 1 mg/mL TM shows involvement of oxidative stress ( B ). Expression profile (absolute signal intensity) of the 22 differentially regulated genes identified in ( A ) between SC and 1 mg/mL ( C ). Genes are highlighted as reportedly regulated by oxidative stress (red dots), involved in lipid/cholesterol metabolism (green dot) or associated with oligodendrogenesis (blue dots). ROS accumulation ( D ) was measured via DCFDA oxidation over 60 h in hNPCs treated with 0.01 and 1 mg/mL TM. Exposure to 0.01 mM H 2 O 2 was used as a positive control. hNPCs were exposed to 1 mg/mL TM, 100 µM ascorbic acid (Asc) alone and in combination ( E ). Human and rat spheres with a defined size of 0.3 mm were plated onto poly- d -lysine/laminin-coated 96-well plates and exposed to increasing TM concentrations over 120 h ( F ). The differentiation into oligodendrocytes ( E , F ) was determined as number of all O4-positive cells in percent of the total amount of Hoechst-positive nuclei in the migration area. At least three independent experiments with 5 technical replicates were performed and presented as mean ± SEM (in ( D ) TM treatment n = 4; H 2 O 2 n = 3; in ( E ) control, TM and co-treatment n = 4; Asc n = 3). Statistical significance was calculated using one-way ANOVA followed by Bonferroni’s post hoc tests and unpaired t -test for H 2 O 2 (in D , F ), using two-way ANOVA followed by Bonferroni’s post hoc tests (in E ), *significant compared to the solvent control, # significant compared to TM single treatment. ( p ≤ 0.05 was considered significant). SC, solvent control (N2-medium); OSGIN1, oxidative stress induced growth inhibitor 1; HMOX1, heme oxygenase 1; NQO1, NAD(P)H dehydrogenase quinone 1; GCLM, glutamate-cystein ligase modifier subunit; ME1, malic enzyme 1; HDLBP, high-density lipoprotein binding protein; H 2 O 2 , hydrogen peroxide; Asc, ascorbic acid

Article Snippet: Neurospheres (migrated for 5 days) were incubated overnight at 4 °C with a mouse IgM oligodendrocyte O4 antibody solution (1:400 in PBS with 10% GS; R&D System, #MAB1326).

Techniques: Gene Expression, Expressing, Positive Control, Migration, Control, Solvent, Binding Assay

Journal: Cell Biology and Toxicology

Article Title: Application of the adverse outcome pathway concept for investigating developmental neurotoxicity potential of Chinese herbal medicines by using human neural progenitor cells in vitro

doi: 10.1007/s10565-022-09730-4

Figure Lengend Snippet: Putative AOP network for impaired oligodendrocyte development. The previously established oligodendrocyte development AOP network (Klose et al. ) is enlarged by the MIE “increased reactive oxygen species production” and KEs regarding oxidative stress. In addition, the MIE and KE regarding sodium channels and their impact on oligodendrocyte differentiation is included (Hernández-Jerez et al. ). This illustration represents a qualitative network and does not include dashed key event relationships

Article Snippet: Neurospheres (migrated for 5 days) were incubated overnight at 4 °C with a mouse IgM oligodendrocyte O4 antibody solution (1:400 in PBS with 10% GS; R&D System, #MAB1326).

Techniques: