Extracellular Vesicle Isolation from iMSCs

DMEM (Gibco, Waltham, MA, USA) supplemented with 15% FBS was used for iMSC culture. EV-depleted FBS was prepared as previously described [35 (link)]. Cells from passages 6–7 were collected and seeded at a density of 10,000 cells/cm² in a 150 mm culture dish (SPL, Pocheon-si, Korea). The next day, cells were treated with or without lanifibranor (Cayman, Ann Arbor, M, USA) for 24 h, after which the medium was replaced with DMEM supplemented with EV-depleted FBS and cultured for an additional 24 h. The supernatant was centrifuged for 10 min at 300×g, and the supernatant was transferred to a new tubes, which were then centrifuged for 20 min at 2000×g. This step was followed for another round of centrifugation at 10,000g for 80 min. The supernatant was passed through a 0.2-µm vacuum filter (Merck Millipore, Burlington, MA, USA). Finally, the EVs were isolated by ultracentrifugation at 100,000×g for 80 min, after which the pellet was washed with PBS again (Beckman Coulter, CA, USA). EV pellets were redissolved in EV-free PBS.