Pdgfr

For HSC analysis, femurs were crushed with a mortar and pestle in PBS with 2% fetal bovine serum (FBS) and 2 mM EDTA, then filtered through 30-micron filters. Harvested cells were stained with FcR blocking antibody followed by fluorophore-conjugated antibodies against c-Kit, Sca-1, CD48, CD150, and a hematopoietic lineage marker cocktail (BioLegend^® Inc., San Diego, CA) in PBS containing 0.5% BSA and 2 mM EDTA, and HSC were identified by SLAM-LSK gating (lineage⁻, c-Kit⁺, Sca-1⁺ CD48⁻CD150⁺) by flow cytometry as we have described elsewhere (11 (link)).
For MSC analysis, femurs were crushed as described above and digested for 30 min at 37°C in 0.25% Collagenase type IV (Invitrogen™/Thermo Fisher, Waltham, MA) in PBS supplemented with 10% FBS. Cells were stained with FcR blocking antibody followed by fluorophore-conjugated antibodies against CD45, Ter119, CD31, CD51 and PDGFR (BioLegend). Mesenchymal stromal cells were identified as CD45⁻, Ter119⁻, CD31⁻, CD51⁺ and PDGFR⁺ by flow cytometry, as we have described elsewhere (27 ).
All flow cytometry analyses were performed on an LSR-II flow cytometer (Becton Dickinson, San Jose, CA). Representative gating for HSC and MSC are shown in Supplementary Fig. S1 (https://doi.org/10.1667/RADE-20-00181.1.S1).

Cultured fibroblasts (passages 0-2) were grown in culture to 80% to 90% confluence and trypsinized, as previously described.^{4 (link)} Single-cell suspensions were aliquoted to a 96-well plate. Cells were stained with the following antibody panel: LIVE/DEAD Fixable Aqua, CD31 (PECAM) Brilliant Violet 421 (303124; BioLegend), CD34 PerCP-CY5.5 (343612; BioLegend), FAPa PE (FAB3715P-025; R&D Systems), CD90 Alexa Fluor 647 (328120; BioLegend), and PDGFR (platelet-derived growth factor receptor) PE/Cy7 (323508; BioLegend). After staining, cells were fixed and permeabilized. Single-stained and florescence minus one–stained samples were used as controls for gate selection. Viability Aqua–negative (live) cells were subsequently evaluated by percentage population of fibroblasts (FAP⁺). Fibroblast cells line were grouped according to etiology (n = 3), with rapidly processed autopsy tracheal mucosa–derived nonscar fibroblasts used as controls. All analyses were performed in FlowJo Flow Cytometry Analysis Software (Treestar, Version 10.7.1 for Mac).