Fig 2: 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 3: CTGF from preosteoclasts is required to maintain the pool of PSCs and commit PSCs to chondrogenic lineage. After performing fractures on Ccn2Acp5 mice and Ccn2fl/fl controls, the callus tissue was dissected and digested for Flow cytometry analysis (A). Representative plots (B) and quantification (C) of [CD45– Ter-119– Tie2– AlphaV+ Thy– 6C3– CD105–CD200+] PSCs and [CD45– Ter-119– Tie2– AlphaV+ Thy– 6C3– CD105+CD200+] PCPs in the bone callus. (D) Schematic diagram of separating the periosteal graft from Tomato donor mice and transplanting it to the fracture site of Ccn2Acp5 mice and Ccn2fl/fl controls. (E) Representative images of SO staining and DAPI/tdTomato immunofluorescence of fracture tibia after transplantation with tdTomato periosteal graft. In the new periosteum formed 28 days after the fracture, high magnification shows that the scarce periosteum-derived tdTomato+ cells are integrated into the new periosteum (white arrow). After the second fracture, a large number of tdTomato+ cells derived from periosteum were found in day 7 callus and formed cartilage tissue (white arrow). Scale bars, 200 µm. Data are shown as mean ± SD. **P < 0.01.

Fig 4: Remodeled BM microenvironment with age.a, Experimental scheme to investigate endosteal (Endo) and central marrow (Marrow) stromal populations in young (~ 8 week of age) and old (~ 24 months of age) C57BL/6 wild type (WT) mice. Results are from 6 independently analyzed groups of 1 or 2 young or old mice. Cellularity data per mouse are for 1 BM plug for marrow populations and bone chips from 10 bones for endosteal populations. b, Changes in endosteal and marrow endothelial cell (EC) populations with age showing frequency (top) and total cell numbers (bottom). AEC, arteriolar endothelial cell; SEC, sinusoidal endothelial cell. c, Whole mount staining of the BM vasculature in young and old mice. Scale bar, 100 µm. d, Changes in endosteal and marrow mesenchymal populations with age showing frequency (top) and total cell numbers (bottom). MSC, Sca-1+ peri-arteriolar mesenchymal stroma cell; mOPr, PDGFRa+ multipotent osteoprogenitor; OPr, PDGFRa- osteoprogenitor; MSC-L, LepR+ peri-sinusoidal mesenchymal stroma cell; iMSC-L, Sca-1low inflammatory MSC-L. e, Representative image of MSC-L immunofluorescence staining in young and old BM. Scale bar, 38 µm. f, Representative pictures and quantification of fibroblast colony-forming units (CFU-F) from young and old MSC-S, osteoblastic lineage cell (OLC) and MSC-L. Results are from 5 independent cohorts. Data are means ± S.D.; *p = 0.05, **p = 0.01, ***p = 0.001.

Fig 5: Inflammatory nature of the old BM niche.a, Differentially secreted cytokines in young and old BM fluid measured by 200-plex array and clustered based on broad biological functions (n = 5). b, Changes in pro-inflammatory cytokines in young and old BM fluids. Results are from 3 independent cohorts. c, qRT-PCR-based analyses of Il1a, Il1b and Tnf expression in old BM cells and stromal fractions (n = 6). Results are represented as violin plots and are expressed as fold change compared to their respective young counterpart. Neu, Mac-1+/Ly-6G+/Ly-6Cmid neutrophil; Mac, Mac-1+/Ly-6G-/Ly-6C- macrophage; B cell, B220+/CD19+ B cell; T cell, CD4+/TCRß+ T cell; Marrow, Ter119-/CD45- central marrow fraction; Endo, Ter119-/CD45- endosteal fraction. d, Violin plots representation of Il1a, Il1b, and Il1rn expression in the indicated young and old mesenchymal and endothelial like groups. Results are from the plate-based scRNAseq dataset; *padj = 0.05, ** padj = 0.01, *** padj = 0.001. e, Caspase 1 activity (aCASP1) in the indicated young and old BM and stromal populations with representative FACS plots (top) and quantification (bottom). f, Experimental scheme for the indicated co-culture experiments showing the effects of young and old stroma on young HSC expansion as dependent on IL-1ß. Data are means ± S.D.; *p = 0.05, **p = 0.01, ***p = 0.001.