Fig 1: 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.
Fig 2: Glial reactivity in mice on low or adequate α-linolenic acid diet after traumatic brain injury. a Traumatic brain injury (TBI) resulted in distinct increase in Iba-1-positive cells in the peri-contusional cortex at 3 days post-injury. Diets producing different brain docosahexaenoic acid (DHA) contents did not affect the density of Iba-1-positive cells at the injury site. b Cortical GFAP immunofluorescence increased 3 days after TBI was more in mice on low α-linolenic acid (ALA) diet that have less brain DHA than the adequate ALA group. n = 3–4. **p < 0.01 vs control TBI. Data are expressed as mean ± SEM. Scale bar 100 μm
Fig 3: Activation of macro and microglia upon CNF1 injection.(A) Muller cells reactive gliosis 14 days post CNF1 injection. Red: GFAP; green: rhodopsin staining. Arrows point to clusters of residual photoreceptors (green). (B) Retinal whole mount stained with Iba1 antibody against microglia. Cells are in the typical amoeboid, active shape. The focal plane is in the outer retina. The inset shows an example of a quiescent, ramified microglial cell from a control retina.
Fig 4: TYW5 overexpression significantly affected differentiation of mouse neural stem cells. (A and B) Representative immunofluorescence images for GFAP (a marker for astrocyte cells) and MAP2 (a marker for mature neurons) staining. (C) Compared with the controls, the ratio of GFAP-positive cells were significantly decreased in TYW5 overexpression groups, indicating that TYW5 overexpression impaired the differentiation of NSCs into glia cells. (D) In contrast, the ratio of MAP2-positive cells remained unchanged. (C) The quantification data for A. (D) The quantification data for B. (E–H) Western blot results for GFAP and MAP2 expression in TYW5 overexpression and control groups. For MAP2, two bands (MAP2 A/B, molecular weight: 270–300 kDa; and MAP2 C/D, molecular weight: 70–75 kDa) were detected. (F–H) The quantification data of MAP2 A/B, MAP2 C/D and GFAP protein expression for E. Two-tailed Student’s t-test was used to test for a significant difference. Data represent mean ± SD, n = 3 for C, D and F–H).
Fig 5: Localization of GFAP-expressing host cells and rodent nestin-positive cells in glioma xenografts.In mice, murine nestin-expressing host cells (green) at the U251 tumor border and also infiltrating into the tumor (A). These cells did not stain for GFAP, and were shown to be vascular elements. However, a few cells (yellow) expressed both mouse nestin and GFAP (A, inset, arrows). GFAP-expressing cells with characteristic branched morphology (astrocytes) were more numerous in the host brain tissue further away from the tumor border (A, red, GFAP; B, brown, GFAP). We also observed GFAP-positive cells with astrocyte morphology that extended astrocyte foot-like processes onto blood vessels in the vicinity of D566 tumor implant (B inset). Mouse nestin (brown) was strongly expressed at the immediate tumor border (dotted line) (C). GBM biopsy xenograft lesion showing a less demarcated front toward the brain tissue. Numerous double-immunostained cells were seen at the tumor border. GFAP strongly stained host astrocytes as well as glioma cells (D, red, GFAP; green, rat nestin). GFAP and rat nestin co-stained some host cells that showed diverse morphologies. Double-immunostained cells often had elongated cell bodies, with several terminal processes (E, F, red, GFAP; green, rat nestin). An elongated GFAP and rat nestin positive cell within the tumor center, with branched terminal processes (G). U251 (A), D566 (B, C), biopsy spheroid xenografts (D–G). tu tumor. Bars E, F 10 µm, G 15 µm, A insert 25 µm, B insert 75 µm, A, D 100 µm, B, C 500 µm.
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