Fig 2: Characterization of glial population in BrainSpheres. (A) Representative bright field images of NPCs (upper panel) and BrainSpheres (lower panel). (B) Development-dependent oligodendrocyte gene expression in BrainSphere (two, four, eight weeks of differentiation compared to baseline expression in NPCs). Gene expression was normalized to GAPDH. (C) Representative confocal images of MBP (myelin marker, green) and NF (axonal marker, red) expression over BrainSpheres differentiation process. (D) Number of pixels on confocal images of axonal marker (NF) and myelin marker (MBP) at two, four and eight weeks of BrainSpheres differentiation (mean ± SD, n = 10). (E) Myelination in BrainSpheres (% of myelinated axons) calculated using CEM plugin at two, four and eight weeks of differentiation. (F) Developmental-dependent GFAP (astrocyte marker, red) and O4 (oligodendrocyte marker, green) protein expression. (G) Development-dependent S100ß (astrocytes marker, red) and PLP1 (myelin marker, green) protein expression. (H) Cell viability measured after a seven-day chemical exposure in the eighth week of differentiation (8/8) by resazurin test. Data are expressed as percentage of vehicle (DMSO) control and displayed as mean ± SD. n = 6–8 samples coming from two independent experiments. Non-parametric Kruskal–Wallis test was used to determine significant differences between NPCs and BrainSpheres (* p = 0.05; ** p = 0.01; *** p = 0.001; **** p = 0.0001). Scale bars = 100 µm.

Fig 3: Immunofluorescent labeling of differentiated neural stem cellsTertiary neurospheres were dissociated and plated on GFR-Matrigel coated coverslips in a 24-well plate for differentiation. Cells were immunolabeled after 14 days to detect co-expression of RFP in oligodendrocyte progenitor cells (OPCs) with NG2 antibody, oligodendrocytes with MBP antibody, astrocytes with GFAP antibody and proliferation with EdU labeling. BF = Brightfield, RFP = Red Fluorescent Protein, MBP = Myelin Basic Protein, GFAP = Glial Fibrillary Acidic Protein, Scale=10 µm.

Fig 4: Chemical exposure altered myelin in BrainSpheres. (A) Example of binary conversion of confocal images using CEM plugin. (B) Confocal images of representative BrainSpheres exposed to the highest non-cytotoxic concentration of each chemical. Treatment, concentration, and antibody used are indicated in each picture. White bars correspond to 50 µm. (C) Quantification of BrainSpheres myelination after chemical exposure. Percentage of myelination was quantified as co-localization of MBP and NF immunocytochemical staining in confocal images of BrainSpheres taken at the end of the eighth week of differentiation following a seven-day exposure to test chemicals during eighth (8/8) week. The percentage of myelination was normalized to the mean percentage of myelination in the vehicle (DMSO) control samples. Each bar in the 8/8 experiment represents pooled results from three experiments (first experiment n = 5, second experiment n = 10, third experiment n = 5 total n = 20 BrainSpheres). Kruskal–Wallis no-parametric test was used to determine significant differences between control and exposed samples (* p = 0.05; ** p = 0.01; *** p = 0.001; **** p = 0.0001). We also performed total quantification of PLP1, another myelin marker.

Fig 5: Representative images of oligodendrocyte-microglia co-culture following different schedules of LPS treatment. Representative images of the oligodendrocyte-microglia co-culture subjected to immunostaining after in vitro treatment with vehicle (control) (A) or 0.01 µg/mL LPS at D1 (early LPS group) (B), D3 (late LPS group) (C), or D1 and D3 (double LPS group) stages. Scale bar = 100 µm (200 × magnification).LPS = lipopolysaccharide, MBP = myelin basic protein.