Fig 2: Flow cytometry analysis identified the presence of microglia in iPSC-derived MCOs. The MCOs cultured for 35 and 45 days were subjected to flow cytometry analysis with CD11b and P2RY12. Isotype controls of the corresponding antibodies were used to determine the baseline expression of microglia marker. (A) Representative side scatter (SSC) images of one MCO. (B) Diagram graph showing the average percentage of positive cells per MCO. Data represent mean ± SE of three MCOs per group. The **P < 0.01 indicates the significant difference from corresponding marker CD11b at day 35. The ns indicates no significance statistically as compared with corresponding day 35 in each marker.

Fig 3: Effects of papain and miRNA-146a inhibitors on CD11b expression of activated monocytes mediated by MPA formation.(x¯ ± s, n = 3). a) Gating strategy for quantification of Mac-1 in a co-culture system of THP-1 cells and activated platelets by flow cytometry (FCM). Mac-1 was evaluated by expression of CD11b in THP-1 cells mediated by monocyte-platelet attachment following treatment with 100 or 200 U/L of papain with or without miRNA-146a. A: isotype control; B: negative control; C: 100 U/L of papain; D: 200 U/L of papain; E: 100 U/L of papain combined miRNA-146a inhibitors; F: 200 U/L of papain combined miRNA-146a inhbitors. ?, #P < 0.05 compared with negative control and 200 U/L papain group, and ??P < 0.01 compared with negative control, as analyzed by LSD-t test.

Fig 4: Identification of NP-derived G-MDSCs. A) Uniform manifold approximation and projection (UMAP) visualization showing immune cells inside NP tissues. B) Heatmap showing the typically expressed genes in each cell type. C) Dot plot showing scaled expression of selected signature genes for GMP, G-MDSCs, and neutrophils, by average expression of each gene in each cluster scaled across all clusters. Dot size represents the percentage of cells in each cluster with more than one read of the corresponding gene. D) Monocle method reconstructed pseudospace trajectory for GMP, G-MDSC, and neutrophils. E) FACS isolation for CD45+ CD11b+OLR+ cells from mild/severe degenerative NP tisues. F) Safranin O/Fast Green staining of the intervertebral discs sham and experimental rats. Scale bar, 1 mm in left and 100 µm in right. G) Merged immunofluorescence staining of DAPI, CD11b, OLR1 in the intervertebral discs of sham and experimental rat. Scale bar, 1 mm in merge images and 100 µm in others.

Fig 5: Knocking-down STING and STING activation promote both PBMC-DMs and BM-DMs differentiating into pro-inflammatory subtype. (A) Upper panel, flow cytometric analysis of TAMs for pro-inflammatory (CD45+CD11b+CD80+) and anti-inflammatory (CD45+CD11b+CD163+) subtypes; Lower panel, quantification of pro-inflammatory, anti-inflammatory macrophages and the corresponding ratios (n=8); *, p < 0.05; **, p < 0.01. (B, C) Representative flow cytometric analysis of pro-inflammatory (CD11b+/CD80+ in human (B) and mouse(C)) and anti-inflammatory macrophages (CD11b+/CD163+ in human (B) and CD11b+/CD206+ in mouse(C)) in human PBMC-DMs from two healthy donors (B) and mouse BM-DMs (C) treated as indicated. (D, E) Quantifications of pro-inflammatory and anti-inflammatory macrophages in human PBMC-DMs (D) and mouse BM-DMs (E) as in (B) and (C), respectively. ***, p < 0.001; **, p < 0.01; *, p < 0.05. (F, G) RT-PCR analysis of the macrophage subtype markers in human PBMC-DMs and mouse BM-DMs treated as indicated; Data in D, E, F and G are presented as the mean±SD (n=3).***, p < 0.001; **, p < 0.01; *, p < 0.05. Control stands for the average of undistinguishable controls of scrambled sequence (SC; for shSTING), empty vector (EV; for STING overexpression), and PBS treatment (for 2'3'-c-GAMP treatment).