Cd166

Flow cytometry was performed using an LSRFortessa^TM (Becton Dickinson, Franklin Lakes, NJ, USA) cytometer and FlowJo 10.4 was used to analyze the data.
Antibodies used were: LIFRalpha (FAB5990P; R&D Systems, Minneapolis, MN, USA) and pSTAT3 (557815, Becton Dickinson). Cells were sorted on an Aria III^TM (Becton-Dickinson) as described before [82 (link),83 (link)]. Antibodies used for cell sorting were CD45 (25-0451-82; Thermo Fisher), Ter-119 (116222; Biolegend, San Diego, CA, USA), Gr-1 (108416, Biolegend), CD3 (25-0031-82, Thermo Fisher) and CD11b (25-0112-82, Thermo Fisher). CD31 (102422, Biolegend) Sca-1 (108126, Biolegend), CD166 (12166182, eBioscience, San Diego, CA, USA) and CD140 (135906, Biolegend).

This study was approved by the Navy General Hospital Ethical Committee. With the consent of the parents, human umbilical cord Wharton’s jelly was collected from infants delivered by full-term normal labor. The cells from the Wharton's jelly tissue were isolated as described previously [26 (link)]. Briefly, after removal of blood vessels and epithelium, the Wharton’s jelly was cut into pieces about 1.5–2.5 mm³, and digested for 18 hours at 37 °C with 0.2 mg/ml collagenase (Sigma St. Louis, Missouri, USA) solution in serum-free medium containing 100 U/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml amphotericin B. The isolated cells were suspended in Dulbecco's modified Eagle’s medium (DMEM)/F12 nutrient mixture containing 20 % fetal bovine serum (FBS). When WJCs were cultured to reach 80–90 % confluence, they were passaged and seeded at a density of 4 × 10³/cm² in DMEM/F12 with 10 % FBS. WJCs at passage 3 were taken for flow cytometry analysis as described previously [42 (link)]. The antibodies used were phycoerythrin (PE)-conjugated CD105, CD73, CD45, CD29, CD166, human leukocyte antigen (HLA)-DR, HLA-ABC, and fluorescein isothiocyanate (FITC)-conjugated CD34 and CD90 (all eBioscience, Santiago, California, USA).

For analysis of in situ MSCs, ANCs from the hindlimbs of Prx1-Cre;tdTomato mice were collected and stained with a FITC anti-mouse TNFα antibody (BioLegend, USA) at 1:100 or a PerCP anti-mouse p-mTOR antibody (Thermo Fisher Scientific, USA) at 1:100 for 60 min on ice using Intracellular Staining Permeabilization Wash Buffer (BioLegend, USA). For analysis of MSC endocytosis, MSCs were collected after FITC-labeled TNFα and pro-TNFα treatments and fixed in 2% PFA. For analysis of MSC surface markers, cultured MSCs were collected and stained with PE-conjugated antibodies against CD73 (Thermo Fisher Scientific, USA), CD90 (BioLegend, USA), CD105 (Thermo Fisher Scientific, USA), CD146 (Thermo Fisher Scientific, USA), CD166 (Thermo Fisher Scientific, USA), Stem cell antigen 1 (Sca1; BD Biosciences, USA), and CD45 (Thermo Fisher Scientific, USA) and a PerCP-conjugated antibody against CD34 (BioLegend, USA) at 1:100 for 60 min on ice. All samples were analyzed using FACS^Calibur with CellQuest software (BD Bioscience, USA).