Fig 2: Characterization of OM spheres and method to isolate putative OM stem cells.A. SFE of OM samples and fibroblasts in the presence of methylcellulose. Example of a typical OM sphere. B. SFE of OM samples in suspension culture for 3 generations. Data are presented as mean ± SEM (n = 3). C. Immunostaining of OM spheres for Tuj1, Nestin, NSE, S100ß, Vim, FN and CK8. Dapi was used to stain nuclei. D. On the left, example of an OM sphere labeled with PKH26 and Dapi. Representative FACS data of OM cells labeled with PKH26. On the right, sorted populations (positive, intermediate and negative) are shown. Quantification of SFE obtained from each sorted population. Data are presented as mean ± SEM (n = 3). E. Quantitative RTPCR for the expression of CD271 in PKH26-labeled cells. Data were normalized to the reference gene ß-ACTIN (ACT). Bar, 50 µm.

Fig 3: Generation of the iFA Phenotypic Surrogate of Progressive Fibrosis(A) Comparative immunofluorescence (IF) images of primary fibroblasts and mesenchymal-like cell cultures stained for fibroblast (FSP-1) and mesenchymal (VIM) markers cultured on 13-kPa hydrogels. Phase contrast images show monolayer cultures of the primary fibroblasts grown on hydrogels while the mesenchymal-like cells heaped up as scar-like aggregates (also called induced fibroblast activation [iFA] cultures) as shown on day 13. Scale bars, 50 µm.(B) Comparative IF images of primary fibroblasts and mesenchymal-like cell cultures on days 1, 3, 6, and 9 stained with a-SMA, vimentin (VIM), and EdU after a 6-h EdU treatment. Scale bars, 50 µm.(C) Representative IF images of decellularized primary fibroblast (control) and iFA cultures stained for collagen I (top) and a-SMA (bottom). Scale bars, 50 µm.(D) Single-positive cells for SSEA4, CD105, and CD326 (negative for CD45) were sorted from the iFA model and re-cultured on 13-kPa hydrogels (n = 3).(E) The iFA model created from single-positive cells of CD105, SSEA4, and CD326 in (D) were individually reanalyzed using FACS.

Fig 4: AA5 Induces Acute-Phase Response Proteins Promoting Resolution of Fibrosis(A) Representative DMSO-treated, AA5-prevention (AA5p), and AA5-resolution (AA5r) iFA cultures immunostained for VIM and a-SMA (top panels) and collagen I and a-SMA (bottom panels). Scale bar, 50 µm.(B) Quantification of stiffness of the cells in the DMSO-treated, AA5p, and AA5r iFA model.(C) Secreted levels of HMGB1 in the iFA model during AA5p and AA5r treatments in comparison to DMSO-treated controls.(D and E) ClueGO clustering analysis results of up- (red) and downregulated (blue) genes in a pairwise comparison of AA5p (D) or AA5r (E) treatment with DMSO-treated iFA model. p values of = 0.05 are shown.(F) Cytokine profiles of acute-phase proteins in supernatants of the iFA model at day 13 relative to day 4 cytokines in AA5r or AA5p versus DMSO-treated cells(n = 4 in duplicate).(G) Cytokine profiles of proteins secondary to acute-phase response in supernatants from the iFA model at day 13 relative to day 4 cytokines in AA5r or AA5pversus DMSO-treated cells (n = 4 in duplicate).(H) Comparison of gene expression fold change levels of PTX3 and NPTX1 and scavenger receptor CD163L1 in the AA5r- and AA5p-treated iFA model when compared to the DMSO-treated iFA cultures (n = 5).(I) Time-dependent fold increase in secreted PTX3 and NPTX1 on AA5p and AA5r treatment measured in supernatants collected on day 13 (AA5p) and 48 h (AA5r) of treatment when compared to DMSO controls in the iFA model (n = 4).(J) Densitometric analysis depicting fold change of p-SMAD2/3 in the iFA model with DMSO, AA5-prevention, and AA5-resolution treatments analyzed by immunoblotting normalized to total protein.Data are presented as the mean ± SEM. ****p < 0.0001, ***p < 0.001, and **p < 0.01 using one- or two-way ANOVA and Dunnett or Sidaks’s multiple comparison test.

Fig 5: Characterization of the Induced Fibroblast Activation (iFA) Phenotypic Surrogate of Progressive Fibrosis(A) Active TGF-ß secreted during the progression of the iFA phenotype (days 2–14) in a TGF-ß bioassay (n = 3).(B) Representative immunofluorescent image of iFA model stained for total SMAD3 and p-SMAD2/3 (S465/S467).(C) Quantification of cell stiffness of the cells in the iFA model at different time points (days 1–13) (n = 22).(D) Representative image of the iFA model at day 13 of culture stained with p16Ink4 staining and counterstained with VIM.(E and F) Cytokine profiles (E) and HMGB1 (F) levels determined from conditioned media of the iFA model at day 13 relative to day 4 (n = 5 in duplicate).(G) Representative image of the iFA model at day 13 stained for NF-?B p65. HMGB1 was translocated from the nucleus to the cytoplasm.(H) Enrichment analysis of canonical pathways (1,006 MSigDB canonical pathways) in transcriptome data of RNA sequencing in day 13 versus day 4 cultures of the iFA model compared to the fibrotic organ versus healthy tissue data. Fibrotic organs include IPF versus healthy lung (n = 8), advanced (n = 6) versus early (n = 6) liver steatosis, and kidneys from chronic kidney disease (n = 46) versus healthy controls (n = 8). Related to Figure S2E and Table S1.Scale bars, 50 mm. Data in figures represent the mean ± SEM; ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 using two-way ANOVA and Sidak’s multiple comparisons test.