Fig 2: The effect of EV treatment on astrocyte and oligodendrocyte populations in trisomic CS. (A) Single and merged channels of confocal images showing DAPI staining (blue) with IHC labeling of GFAP (green) and TUJ1 (magenta) in euploid and trisomic untreated and EV-treated CS generated from isogenic cell lines. (B) Single and merged channels of confocal images showing DAPI staining (blue) with IHC labeling of CC1 (green) and MAP2 (magenta) in euploid and trisomic untreated and EV-treated CS generated from isogenic cell lines. Top panels in A and B show low magnification images, scale bar = 100 µm. White boxes represent high magnification insets in bottom panels, scale bar = 20 µm. (C) Bar graphs showing the quantification of the percentage of GFAP expression and (D) the percentage of CC1 expression in euploid CS and trisomic untreated and EV-treated trisomic CS. The percentage of each marker was quantified as the ratio of the area occupied by the label (GFAP or CC1) to the area occupied by DAPI in each image and normalized to the euploid control that was calculated as 100% for each independent experiment. The quantification was performed through particle analysis via ImageJ; euploid, n = 18; trisomic, n = 22; trisomic EV+ n = 22 CS per group. These graphs display results from three independent experiments. Statistical analysis was performed via unpaired t-test with Welch’s correction comparing all three groups to each other. Results are shown as mean ± SE. * p < 0.05, *** p < 0.001, **** p < 0.0001, ns = non-significant.

Fig 3: Measurements of proliferative markers in CS are unaffected by EV treatment. (A) Single and merged channels of confocal images showing DAPI staining (blue) with IHC labeling of MAP2 (magenta), SOX2 (green), and Ki67 (red) in euploid and trisomic untreated and EV-treated CS generated from isogenic cell lines showing VZ-like structures. The top panels show low magnification images, scale bar = 100 µm. White boxes represent high magnification insets seen in the bottom panels, scale bar = 20 µm. (B,C) Bar graphs showing quantification of the percentages of SOX2+ and Ki67+ cells in euploid CS and trisomic untreated and EV-treated CS, quantified as the ratio of the numbers of SOX2+ and Ki67+ to the number of all cells marked with DAPI and normalized to the euploid control that was calculated as 100% for each independent experiment. Cells were counted using the ACEq counting app; euploid n = 20; trisomic n = 23; trisomic EV+ n = 25 CS per group. These graphs reflect the results obtained from data collected from three independent experiments. Statistical analysis was performed via unpaired t-test with Welch’s correction comparing all three groups to each other. Results are shown as mean ± SE, ns = non-significant.