Fig 1: Glucocorticoid impairs neurogenesis through activating A1-like astrocytes. (a, b) After exposure to prenatal vehicle or corticosterone (10 mg/kg) at E14, the hippocampus from mice at P1 was collected. a For BrdU labeling, mice were injected intraperitoneally with BrdU (100 mg/kg) 18 h before sacrifice. Then, dissociated hippocampal cells were immunostained with BrdU (green) and c-Fos (red). The ratio of c-Fos-positive cells to BrdU-positive cells was analyzed by flow cytometry. The ratio of c-Fos- to BrdU-positive cells was decreased by prenatal corticosterone. n = 5. b The cells dissociated from the hippocampus were immunostained with DCX (green) and NeuN (red). The ratio of DCX-positive cells to NeuN-positive cells was analyzed by flow cytometry. The ratio of DCX- to NeuN-positive cells was increased by prenatal corticosterone. n = 5. (c, d) Human NSCs were incubated with astrocytic differentiation media with cortisol (1 µM) for 5 days. c The percentages of apoptotic cells (annexin V-positive cells) were analyzed by annexin V/PI analysis, measured by flow cytometry. Apoptotic cells were increased by cortisol. n = 5. d The differentiated cells were immunostained with GFAP (green) and PI (red). The ratio of GFAP-positive cells to PI-positive cells was analyzed by flow cytometry, which was increased by cortisol. n = 5. (e, f) Human NSCs were incubated with astrocytic differentiation media for 5 days and then cortisol (1 µM) was treated for 48 h. e Astrocytes were immunostained with GFAP (green) and DAPI (blue). Astrocytic surface area was analyzed by Image J, which was increased by cortisol. Scale bars, 20 µm (magnification × 1000). Three technical replicates for one dish were analyzed. Six biological replicates were performed. f The expressions of GFAP, ALDH1L1, STAT3, C3, and ß-actin were detected by western blot. The levels of GFAP/ALDH1L1 and C3 were increased but STAT3 expression was reduced by cortisol. Five biological replicates were performed. (g, h) Human NSCs were incubated with astrocytic differentiation media for 5 days, and were then treated with cortisol (1 µM) for 48 h. Then, the ACM were collected and administered to the human NSCs incubated with neuronal differentiation media for 5 days. After 5 days of treatment, the neurons were immunostained as appropriate. g The cells were incubated with 10 µM BrdU (green) for 45 min at 37 °C and then immunostained with c-Fos (red). The ratio of c-Fos-positive cells to BrdU-positive cells was analyzed by flow cytometry, which was reduced by cortisol. n = 5. h The cells were immunostained with DCX (green) and NeuN (red). The ratio of DCX-positive cells to NeuN-positive cells was analyzed by flow cytometry, which was increased by cortisol. n = 5. All immunofluorescence images are representative. n = 5 from independent experiments with two technical replicates each. Quantitative data are presented as a mean ± S.E.M. The representative images were acquired by SRRF imaging system. Two-sided unpaired student’s t test was conducted. *,**indicates p < 0.05 and p < 0.01 versus control, respectively
Fig 2: Characterization of WT- and 3Tg-iAstro lines.a Phase contrast images of four WT-iAstro and four 3Tg-iAstro lines at passage 15. Bar, 100 µm. b Immunofluorescence images of WT-iAstro#2 and 3Tg-iAstro#2, stained with anti-AQP4, anti-GS, anti-Aldh1l1 and anti-GFAP antibodies. Bar, 50 µm. The images shown in (a, b) are representative of n = 3 independent experiments. c Quantification of GFAP-positive cells in WT-iAstro#2 and 3Tg-iAstro#2 lines. Data expressed as mean ± SD % of 15 fields of GFAP-positive cells evaluating a total of 359 WT- and 514 3Tg-iAstro cells. In (b, c), other characterized independently generated iAstro lines show similar results in immunostaining of astroglial markers and in quantification of GFAP-positive cells (data not shown)
Fig 3: Neuron-induced astrocytic gene expression is reversible and disrupted in neurodegenerative disease.(a) Mouse astrocytes were isolated by immunopanning (using an anti-GLAST antibody) at P7. Expression of the indicated neuron-induced astrocytic genes was measured both immediately post-isolation and after 4 days of ex vivo maintenance (neuron-free). *P<0.05 unpaired t-test, (n=5). (b) Mouse astrocytes isolated as in a, and maintained for 22 days in culture, with (black) or without (grey) the last 11 days being in the presence of rat neurons. *P<0.05 unpaired t-test, (n=7). (c) Example micrographs showing phospho-tau immunoreactivity in spinal cord sections of the Thy1-P301S transgenic mouse (at week 20). Scale bar, 500 µm. (d) (Left) Example micrographs showing loss of ChAT-positive motor neurons in spinal cord sections of the Thy1-P301S transgenic mouse at week 20. Scale bar, 50 µm. (Right) Quantitation of loss of ChAT-positive motor neurons in spinal cord sections of the Thy1-P301S transgenic mouse. *P<0.05 unpaired t-test, (n=5 animals of both genotypes). (e) Expression of the indicated astrocyte-specific, neuron-induced genes in the spinal cord of week 20 wild type (WT) and P301S mice, normalized to astrocyte-specific gene Aldh1l1. Rik: 2900052N01Rik. P<0.0001, two-way analysis of variance, P value indicates main effect of genotype (n=6 animals of both genotypes). All error bars represent s.e.m.
Fig 4: Generation of hiPSC-astrocytes. (a) Schematic of hiPSC differentiation and RFP viral transduction. (b) Expression of RFP (left), ALDH1L1 (middle), GFAP and S100B (right) in astrocytes before engraftment (here shown for D20–D22). Scale = 200 µm [Color figure can be viewed at wileyonlinelibrary.com]
Fig 5: MT5-MMP-deficient astrocyte-like cells display altered expression of several astrocytic markers. (A) Representative images of hiPSC-derived astrocyte-like cells from wild-type and MT5-MMP KO hiPSCs, immunostained at DIV 45 for different astrocytic markers as indicated (scale bar: 20 µm). (B–E) Bar charts showing the quantification of the mean fluorescence intensity for different astrocytic markers within each morphological category. MT5-MMP-deficient astrocytes were characterized by no difference for GFAP (nested t-test, N = 3, n = 245; arborized: n = 67, F = 3.92, p = 0.12; arrowhead: n = 64, F = 0.381, p = 0.57; fibroblast-like: n = 114, F = 0.433, p = 0.55, in (B), ALDH1L1 (nested t-test, N = 3, n = 121; arborized: n = 36, F = 0.04, p = 0.85; arrowhead: n = 30, F = 1.86, p = 0.24; fibroblast-like: n = 55, F = 0.746, p = 0.44, in (C). Meanwhile, the significant difference was observed for GLAST (nested t-test, N = 3, n = 181; arborized: n = 57, F = 31.2, ** p = 0.005; arrowhead: n = 32, F = 5.80, * p = 0.02; fibroblast-like: n = 92, F = 14.0, * p = 0.019, in (D). Analysis of S100ß fluorescence intensity (nested t-test, N = 3, n = 139; arborized: n = 49, F = 9.67, p = 0.04; arrowhead: n = 42, F = 1.48, p = 0.07; fibroblast-like: n = 48, F = 0.05, p = 0.83, in (E) reveals a significant difference between the arborized morphotype of WT and MT5-MMP KO astrocytes. Data are presented as mean ± SEM.
Supplier Page from Abcam for Anti-ALDH1L1 antibody - Astrocyte Marker