Fig 2: Upregulation of the epigenetic regulator Uhrf1 in arthritis tissue.(A) Protocol for analysis of collagen antibody-induced arthritis (CAIA) model. PBS (CtrlP) or LPS (CtrlL) was administered as a control. (B) PCA using microarray data obtained from ankle tissue. (C) Heatmap of differentially expressed gene probes in ankle tissue (log2FC > |1|, P < 0.01). Log10 transformed read counts are scaled from 1.0 to 3.0. (D) Expression of genes related to epigenetic regulation classified by GSEA. Log10 transformed read counts are scaled to minimum to maximum values. (E) Relative probe counts detected in CAIA ankle compared with CtrlP or CtrlL ankles. (F) RT-qPCR of Uhrf1 mRNA expression in CAIA (n = 4) and STA (n = 3) ankles. (G) UHRF1 mRNA expression in synovium biopsies from healthy individuals, patients with OA, and patients with RA by RNA-Seq. Data are registered in the Gene Expression Omnibus (GEO GSE89408). (H) Left, representative images of immunofluorescence staining for Uhrf1 (red); Pdpn, Fap, Thy-1, CD45, F4/80, and CD3 (green); and DAPI (blue) in WT STA ankle tissue. Scale bar: 50 µm. Right, quantification of Uhrf1+ marker cells in hyperplastic synovium. Cell number in 1 field per similar region of independent mice was calculated. (I) Uhrf1 mRNA expression in SFs treated with 20 ng/mL Tnf-a for 24 hours. Mean ± SD is shown. *P < 0.05 and **P < 0.01 by ANOVA followed by Tukey’s test in F (left), G, and H, and unpaired t test in F (right) and I. Data in A–F and H–I were obtained from 3 to 5 independent experiments.

Fig 3: MSC-EVs stimulate the dedifferentiation of myofibroblasts and myofibroblast precursors (FAPa+) in fibrotic pulmonary tissue through the transfer of miR-29c and miR-129 in vivo.a Representative images of pulmonary tissue immunocytochemical analysis for CD90 (top panel), aSMA (green), and fibroblast activation protein A (FAPa, red) (bottom panel) expression. Scale bar = 100 µm. b–d Quantification of CD90- (c), aSMA- (d), and FAPa- (e) positive cells measured by average cell area (c, d) or number of cells (e) in pulmonary tissue sections. The green line indicates the median for the intact group. Ctrl (DMEM 14 days after bleomycin administration), n = 11; EV_t (MSC extracellular vesicles 14 days after bleomycin administration), n = 9; EV_inh (MSC extracellular vesicles transfected by miRNA-29c and miRNA-129 inhibitors 14 days after bleomycin administration), n = 9; inh (miRNA-29c and miRNA-129 inhibitors 14 days after bleomycin administration), n = 5; n = biologically independent animals per group. The number of analyzed fields of view per sample = 5–9. e aSMA dynamics.

Fig 4: MSC-EVs attenuate pulmonary fibrosis and may cause its resolution.a Schematic design of the treatment of bleomycin-induced pulmonary fibrosis in C57BL/6 mice; Ctrl (DMEM 14 days after bleomycin administration), n = 11; EV_t (MSC extracellular vesicles 14 days after bleomycin administration), n = 9; SF_t (MSC soluble factors 14 days after bleomycin administration), n = 6; n = biologically independent animals per group. b Quantification of dynamic changes in the lung tissue density measured via MRI. c–e Representative image of c hematoxylin–eosin (H&E), d Masson trichrome, and e Picrosirius Red (PSR) staining. Scale bar = 100 µm. f Quantification of pulmonary fibrosis severity using the Ashcroft scale. The assessment was performed by two independent blinded experts. g Quantification of ECM deposition on the PSR staining image. h Quantification of immune cell types in BAL fluid smears. The assessment was performed by two independent blinded experts. i Quantification of the major hematopoietic cell lineages in the BAL fluid via flow cytometry. j Quantification of IL-6 in the BAL fluid using ELISAs. k Percentage of animals with a detected rate of MCP-1 in the BAL fluid using ELISAs. l Representative images of pulmonary tissue immunocytochemical analysis for CD163. Scale bar = 100 µm. m Quantification of CD163-positive cells in pulmonary tissue. n Representative images of pulmonary tissue immunocytochemical analysis for aSMA, FAPa, and CD90. Scale bar = 100 µm. o Quantification of FAPa-positive cells or CD90+ and aSMA+ areas in pulmonary tissue. The green line indicates the median for the intact group.

Fig 5: MSC secretome components do not prevent pulmonary fibrosis development when applied during the acute injury period.a Schematic design of the prevention of bleomycin-induced pulmonary fibrosis in C57BL/6 mice; Ctrl (DMEM 1 day after bleomycin administration), n = 11; EV_p (MSC extracellular vesicles 1 day after bleomycin administration), n = 9; SF_p (MSC soluble factors 1 day after bleomycin administration), n = 6, n = biologically independent animals per group. b Quantification of dynamic changes in the lung tissue density measured via MRI. c–e Representative image of c hematoxylin–eosin (H&E), d Masson trichrome, and e Picrosirius Red (PSR) staining. Scale bar = 100 µm. f Quantification of pulmonary fibrosis severity using the Ashcroft scale. The assessment was conducted by two independent blinded experts. g Quantification of ECM deposition on PSR staining images. h Quantification of immune cell types in BAL smears. The assessment was performed by two independent blinded experts. i Quantification of the major hematopoietic cell lineages in the BAL fluid via flow cytometry. j Quantification of IL-6 in the BAL using ELISAs. k Percentage of animals with a detected rate of MCP-1 in the BAL via ELISAs. l Representative images of pulmonary tissue immunocytochemical analysis for CD163. Scale bar = 100 µm. m Quantification of CD163-positive cells in pulmonary tissue. n Representative images of pulmonary tissue immunocytochemical analysis for CD90 (top panel), aSMA (green), and fibroblast activation protein A (FAPa, red) (bottom panel) expression. Scale bar = 100 µm. o–q Quantification of CD90- (o), aSMA- (p), and FAPa- (q) positive cells measured by average cell area (c, d) or number of cells (e) in the pulmonary tissue sections, n = 4, n = biological independent animals per group. The number of analyzed fields of view per sample = 5–9. The green line indicates the median for the intact group.