Msc basal medium

Human DPSCs and BMMSCs were purchased from Lonza, Inc. (Walkersville, MD, USA). DPSCs were cultured in DPSC basal medium (Lonza, Inc.) containing DPSC SingleQuots (Lonza, Inc.) at 37 °C in 5% CO₂ and 95% air. The BMMSCs were cultured in MSC basal medium (Lonza, Inc.), containing MSC-GM SingleQuots (Lonza, Inc.), at 37 °C in 5% CO₂ and 95% air. After primary culture, the cells were subcultured at a density of approximately 1 × 10⁴ cells/cm². Cells from the third to sixth passages were used for the experiments.

U87 glioblastoma and Huh7 hepatocellular carcinoma cells were cultured in Dulbecco's modified eagle medium (DMEM) and human bone marrow-derived MSCs (Lonza) in MSC basal medium (Lonza). Before EV purifications, cells were incubated with EV-depleted medium for 3–4 days (medium was centrifuged overnight at 100,000g to pellet out vesicles). A volume of 360-mL conditioned medium was collected from the culture of approximately 80% confluency and EVs prepared by differential ultracentrifugation (14 ). Briefly, cell debris was pelleted at 500g. Then, MVs were pelleted at 10,000g (30 min), supernatant filtered through a 0.2-µm membrane (Nalgene^® aPES, Thermo Fisher Scientific, Waltham, MA) and exosomes pelleted at 100,000g (90 min). Concentrations and size distribution were measured by Nanoparticle Tracking Analysis (NanoSight NS300, Malvern). Briefly, samples were diluted in phosphate buffered saline (PBS) 1:100–1:1,000, manually injected into the instrument and videos acquired at ambient temperature at camera level 9 for 1 min per sample, and videos processed at threshold level 10. We purified EVs from all 3 source cell types on 6 different days, and used 3 isolates for proteomics and the other 3 isolates for lipidomic analysis. All 6 isolates underwent Nanoparticle Tracking Analysis before being stored at −80°C until further processing for proteomics or lipidomics.

Human mesenchymal stem cells (hMSC) were purchased by Lonza (Walkersville, MD), and all experiments were conducted using hMSCs between passages 3 and 5. To maintain an undifferentiation state, hMSCs were routinely cultured in an MSC basal medium (Lonza) containing 10% of MSC growth supplement (Lonza), 2% l-glutamine, 0.1% GA-1000, and 25 μg of amphotericin B per mL, Sigma-Aldrich Co.) at 37 °C, 5% CO₂ in an incubator. For osteogenic differentiation assay, hMSCs were cultured in α-Minimum Essential Medium (basal medium) supplemented with 10⁻⁸ M dexamethasones (Abcam, Cambridge, UK), 0.2 mM ascorbic acid (Sigma-Aldrich Co), and 10 mM β-glycerophosphate (Sigma-Aldrich Co).