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. 2021 Aug 31;22(17):9473. doi: 10.3390/ijms22179473

Figure 2.

Figure 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 S100β (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.