Fig 1: OECs inhibited microglial activation in vitro and changed the expression of pro- and anti-inflammatory factors. (A1) Optical microscopy image showing density of normal cultured BV2 cells (BV2-only). Lower panel (A1’) shows an enlargement of the area marked, which shows morphology of microglia. (A2) The same for BV2 cells treated with LPS for 4 h (BV2-LPS). (A3) The same for BV2 cells treated with LPS for 4 h and co-cultured with OECs (BV2-LPS-OEC). (A4) The same for BV2 cells treated with LPS for 4 h and co-cultured with ONFs (BV2-LPS-ONF). Scale bars: 100 μm. in upper panels, 20 μm in lower panels (B1–B4) Immunohistochemistry images showing staining with Iba1 (red) and DAPI (blue) for the same groups as in A1–A4. Scale bars: 50 μm. (B1’–B4’) Lower panel shows an enlargement of the cells in B1–B4. Scale bars: 20 μm. (C1–C4) The same for staining with TMEM119. Scale bars: 50 μm. (C1’–C4’) Lower panel shows an enlargement of the cells in C1–C4. Scale bars: 20 μm. (D) mRNA expression level of Iba1 after 24 h co-culture. (E) TMEM119 mRNA expression. (F,G) Expression of pro-inflammatory cytokines: TNF-α and IL-6, respectively. (H,I) Expression of anti-inflammatory cytokines: Arg1 and IL-4, respectively. (J) Example WB of JAK2 and STAT3, with β-actin as loading control. (K) Quantified JAK2 protein expression by WB (n = 3 per group). (L) Same for STAT3 (n = 3 per group). *p < 0.05, ∗∗p < 0.01.
Fig 2: Developmental dynamics of microglia in the cortex(A) Representative laminar structure of the developing cortex with its transient zones observed from CS23 (9th pcw) in humans and representative cortical columns from 10 to 12 pcw showing (1) the development of the pre-subplate to the subplate at 12.5 pcw and the alignment of microglial cells at the CP-PSP/SP boundary and (2) the distribution of microglial cells across transient zones. Scale bars: 2 mm (left); 100 μm (right).(B) Corrected microglial densities (against fold change in frontal telencephalic wall thickness with age) and proliferative dynamics in the telencephalon during development (CS10 [late 3rd/early 4th pcw]) until term (38 pcw) (n = 50). ∗n/k, not known. Embryonic and early fetal temporal windows are most significant for proliferation against other temporal windows, while the early fetal window is the most significant against other temporal windows.(C) Equally spaced temporal windows for proliferation levels (top) and densities (bottom) around the most significant first wave. Data are represented as mean ± SEM.(D) Mean apoptotic index around the first peak of densities (n = 15, 8 cases between 7 and 11 pcw and 7 cases between 12 and 16 pcw) (top panel). Data are represented as mean ± SEM. Representative microglial cell death photomicrographs observed in wave 1 following the decrease in densities (bottom panel, black arrows in B). Scale bars: 20 μm.(E) Migratory profile of microglia and type of migration in representative cases from the telencephalon (n = 12).(F) Representative profile (top panel) of TMEM119+ and IBA1+ cell densities around the first significant density wave (10–16 pcw) (n = 6). Mean TMEM119+ and IBA1+ cell densities across the prenatal period (10–28 pcw, n = 10) (bottom panel). Data are shown as mean ± SEM.(G) Ratio of labeled TMEM119+/IBA1+ cells during the prenatal period (10–28 pcw, n = 10). Data are presented as mean ±SEM.(H) Representative confocal images of TMEM119+ cells in the MZ and the VZ of a 13-pcw case (left) and double labeling of TMEM119/IBA1 in bright field in a neocortical column at 13 pcw observed in the MZ (right). Scale bars (left): 50 μm; scale bars (right): 100 μm.(I) Wave 2 microglial cell death at 18 and 23 pcw. Scale bars: 20 μm.(J) Non-microglial death observed in the GE and in cortical transient zones at CS23 (9th pcw) (top, scale bars: 100 μm) and at 24 pcw (bottom, scale bars: 50 μm).(K) Proliferative dynamics and densities by sex across human development shown as relative cumulative frequency distribution plots (top panel) and mean values between the sexes (27M:23F) (bottom panel). Data are shown as mean ± SEM. R, right; L, left; CP, cortical plate; GE, ganglionic eminence; IZ, intermediate zone; M, meninges; MZ, marginal zone; PSP, pre-subplate; SP, subplate; SVZ, subventricular zone; VZ, ventricular zone. For all panels, asterisks represent adjusted p value significance as follows: ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p < 0.0001, and ns p > 0.05.
Fig 3: The expression level of pro- and anti-inflammatory factors shift in activated microglia induced by OECs. (A1) Optical microscopy image showing density of BV2 cells treated with LPS for 4 h (BV2-LPS). Lower panel (A1’) shows an enlargement of the area marked which shows the morphology of microglia. (A2) The same for BV2 cells treated with LPS for 4 h and co-cultured with OECs (BV2-LPS-OEC). (A3) The same for BV2 cells treated with LPS for 4 h and co-cultured with OECs pre-treated with AG490 (BV2-LPS-OEC-AG490). Scale bars: 100 μm in upper panels, 20 μm in lower panels (B1–B3) Immunohistochemistry images showing staining with Iba1 (red) and DAPI (blue) for the same groups as in A1–A3. Scale bars: 50 μm. (B1’–B3’) Lower panel shows an enlargement of the cells in B1–B3. Scale bars: 20 μm. (C) mRNA expression level of Iba1 after 24 h co-culture. (D) TMEM119 mRNA expression. (E,F) Expression of pro-inflammatory factors: TNF-α and IL-6, respectively. (G,H) Expression of anti-inflammatory factors: Arg1 and IL-4, respectively. (I) Example WB of JAK2 and STAT3, with β-actin as loading control. (J) Quantified JAK2 protein expression by WB (n = 3 per group). (K) Same for STAT3 (n = 3 per group). *p < 0.05, ∗∗p < 0.01.
Fig 4: Distribution and activation marker change of resident microglia and infiltrated macrophage in RCS rats. (A) Immunohistological images, labeled with Iba1 (red), and DAPI (blue) in retinal slices from control (rdy) rats (A1,A3,A5), and RCS rats of different post-natal ages (A2,A4,A6). (B) Quantitative group analysis of relative Iba1 fluorescence intensity at different ages, normalized to each control (n = 3 per bar). (C,D) Same as (A,B), but for TMEM119 (red) (E) mRNA expression level of Iba1 in RCS retinas, relative to expression in control retinas of the same age (n = 3 per bar). (F) Example western blot of Iba1 protein levels. (G) Quantitative group data of Iba1 protein levels in RCS retinas, normalized to β-actin expression levels, and compared to each control retinas (n = 3 per bar). (H–J) Same as (E–G) but for TMEM119. *p < 0.05, ∗∗p < 0.01; scale bars: 50 μm.
Fig 5: Single-cell transcriptomic analysis of vessel organoids (VOrs).(A) UMAP plot showing the nine major cell types isolated from day (D) 40 VOrs. (B) Violin plots showing the expression value of the typical markers in each cluster. (C) Expression pattern of cell-type-specific markers in VOrs. Relative expression level is plotted from gray (low) to blue (high) colors. (D) Immunostaining of aSMA for representing the smooth muscle cells in VOrs. Scale bar, 10 µm. (E) Immunostaining of PDGFRß for representing the pericytes in VOrs. Scale bar, 10 µm. (F) Immunostaining of microglia markers (IBA1, TREM2, TMEM119) and endothelial marker CD31 in VOrs at D40. Scale bar, 20 µm. (G) Violin plots showing the expression value of the venous marker EPHB4 and arterial marker DLL4 in endothelial cell (EC) clusters. (H) Expression pattern of arterial and venous markers in EC clusters. Relative expression level is plotted from gray to green (EPHB4) or red (DLL4) colors.
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