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To determine the multipotency of MSC overexpressing FasL, cells transduced as described above and were seeded at a 5000 cell/cm² in 24-well plates and left to adhere overnight. Twenty-four hours after transduction, media was changed with adipogenic and osteogenic induction media, respectively, as well as normal MSC media. Media was changed every 2 to 3 days. The adipogenic induction media contained DMEM 1-g/L glucose supplemented with 10% MSC-FBS, 10⁻⁶-M dexamethasone, 100-mM indomethacin and 1% insulin-transferrin-selenium (ITS-G, ThermoFisher, Waltham, MA, USA), and the osteogenic induction media was based on DMEM 1-g/L glucose supplemented with 10% MSC-FBS, 10⁻⁷-M dexamethasone, 10-mM β-glycerophosphate and 0.3-mM ascorbic acid. After 2 weeks, cells were washed with phosphate buffer saline (PBS), fixed with 4% paraformaldehyde and stained with Oil Red O to visualize the lipid droplets accumulated in the differentiated adipocytes or with von Kossa stain represented by incubation in 5% AgNO₃, followed by a short 2-min rinse with 5% sodium thiosulphate to visualize the Ca²⁺ crystals. All the images were taken using an Olympus CKX41 microscope with an Olympus XC30 camera and minimally processed using Adobe Photoshop software.

To determine the multipotency of BALB/c-derived MSC, cells were seeded at a 5000 cell/cm² in 24-well plates and left to adhere overnight. Twenty-four hours after passage, media were changed with adipogenic and osteogenic induction media, respectively, as well as normal MSC media. Media were changed every 2 to 3 days. The adipogenic induction media contained DMEM 1 g/L glucose supplemented with 10% MSC-FBS, 1 µM dexamethasone, 100 mM indomethacin, and 1% insulin–transferrin–selenium (ITS-G, Thermo Fisher Scientific, Waltham, MA, USA), and the osteogenic induction media was based on DMEM 1 g/L glucose supplemented with 10% MSC-FBS, 0.1 µM dexamethasone, 10 mM β-glycerophosphate, and 0.3 mM ascorbic acid. After 2 weeks, cells were washed with phosphate buffer saline (PBS), fixed with 4% paraformaldehyde, and stained with Oil Red O to visualize the lipid droplets accumulated in the differentiated adipocytes or with von Kossa stain represented by incubation in 5% AgNO₃, followed by a short 2 min rinse with 5% sodium thiosulphate to visualize the Ca²⁺ crystals. The images were taken using an Olympus CKX41 inverted microscope with an Olympus XC30 camera and minimally processed using Adobe Photoshop software, version CS3 (Adobe, San Jose, CA, USA).

Approximately 200,000 DCs were placed in a polypropylene conical tube (Corning Inc., USA) with 0.5 ml of chondrogenic media (DMEM-high glucose added with insulin, transferrin, and selenium (ITS-G; Thermo Fisher Scientific, MA, USA)), 5 mg/ml linoleic acid, 10 ng/ml transforming growth factor beta (TGF-β1; R&D Systems, MN, USA), 14 mg/ml ascorbic acid, 10^− 7 M dexamethasone (Sigma-Aldrich, MS, USA), 1.0 mg/ml human serum albumin (Equitech-Bio Inc., KV, USA), and 1% penicillin/streptomycin). The cells were centrifuged (470×g at 4 °C for 5 min) and incubated (37 °C and 5% CO₂) for 3–4 h to allow spheroid formation. For the EV treatment group, the media were replaced with 500 μl of chondrogenic media containing sEVs (5 × 10¹⁰ vesicles/ml). Chondrogenic media without the sEVs served as control. The media were replaced with fresh media with or without sEVs every 48 h, the used media were collected and centrifuged (300×g, 4 °C, 5 min), and the supernatants were stored at − 80 °C for further analysis. The pellets were then harvested at days 7, 14, and 28. Four replicates of pellets were cultured for each group and repeated with different donor cells (n = 5) separately. Two replicates were used for histology and two for biochemical analysis.

For in vitro differentiation, HSAEC_4T53RD cells were co-cultured with TIG3 human lung fibroblasts in Matrigel (BD Bioscience, CA, USA) as described by McQualter et al.[11 (link)] with some modifications. Briefly, HSAEC_4T53RD cells (1 x 10³ cells) and TIG3 (1 x 10⁵ cells) were mixed and resuspended in 100 μL of Matrigel prediluted 1:1 (vol/vol) with the co-culture medium (DMEM/F-12, GlutaMAX (Thermo Fisher Scientific, MA, USA) supplemented with 10% foetal calf serum, ITS-G (Thermo Fisher Scientific, MA, USA) and antibiotic–antimycotic (Thermo Fisher Scientific, MA, USA)). The cells suspended in Matrigel were then added to a 24-well transwell filter insert (Millicell-CM; Merck Millipore, MA, USA) in a 12-well tissue culture plate containing 1 mL of co-culture medium. Cultures were incubated at 37°C in a low-oxygen environment (3% O2 and 5% CO2) in a humidified incubator for 10–14 days and refed three times per week.

The MDA-MB-231 breast cancer cell line was obtained from ATCC and cultured in DMEM with 10% FBS (Hyclone). Cells were passaged regularly when they reached approximately 70% confluency and used between p5 and p15 for all experiments. For patch clamp, 1.5×10⁵ cells were seeded in an uncoated 2 cm plastic dish 24 h prior to the experiment. For voltage sensitive dye experiments, 1.0×10⁴ cells per well were seeded in an uncoated 96-well plastic plate 24 h prior to the experiment. The renal proximal tube epithelial cell (RPTEC/TERT1) line was obtained from ATCC. RPTEC/TERT1 cells were cultured in DMEM/F-12 medium (Thermo Fisher Scientific), containing L-Glutamine and HEPES and supplemented with 25 ng/ml hydrocortisone (Sigma-Aldrich), 5 pM triiodo-L-thyronine (Sigma-Aldrich), 10 ng/ml recombinant human EGF (Thermo Fisher Scientific), 25 ng/ml prostaglandin E1 (Sigma-Aldrich), 3.5 µg/ml L-Ascorbic acid (Sigma-Aldrich), 1× ITS-G (Thermo Fisher Scientific) and 100 µg/ml geneticin (Thermo Fisher Scientific). For patch clamp and voltage sensitive dye measurements, RPTEC/TERT1 cells were seeded at 50% density into 35 mm dish and 12-well plate respectively and were grown for 3 days before starting the experiments. RPTEC/TERT1 cells up to p16 were used for all the experiments. Cells were regularly tested for mycoplasma (every 2 months) by PCR.