Isolation and Transnasal Administration of CD73+ Cells

CD73-positive cells were isolated from the subcutaneous fat tissue of 8-week-old C57BL/6-Tg CAG-EGFP mice. Similar to the isolation from human tissues, for collagenase treatments, the subcutaneous fat was chopped and minced in DMEM containing 2% FBS and 2 mg/mL collagenase (FUJIFILM Wako Pure Chemical) for 1 h at 37 °C with shaking. The cells were then diluted in HBSS containing 2% FBS, 10 mM HEPES, and 1% penicillin/streptomycin (Gibco). PI fluorescence was used to remove dead cells, and APC-conjugated anti-CD73 (Biolegend) was used for single-colour staining, followed by sorting with flow cytometry analysis on the FACS Aria II system (BD). The cells were cultured in MSCs medium as previously described^{14 (link)}. After culture for 14 days, non-adherent cells were removed, and the cells (7.0 × 10⁴ in 25 μL of PBS) were administered to the mice through transnasal injection 14 and 21 days after the first BLM administration. Control animals received an equal volume of PBS.