Cells and cell culture. AdMSCs and HUVECs were purchased from CEFO (Seoul, Republic of Korea). AdMSCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM)-low glucose (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (P/S, Invitrogen) at 37°C in a 5% CO2 environment. HUVECs were cultured in EGM-2 endothelial cell growth Medium-2 (Lonza, Walkersville, MD, USA) supplemented with 10% FBS and 1% P/S at 37°C in 5% CO2. The culture medium was exchanged every 3 or 4 days until the cells reached approximately 90% confluence. Before reaching 100% confluence, the cells were sub-cultured at a 1:3 or 1:4 ratios for cell expansion and further experiments. To obtain exosomes derived from AdMSCs, AdMSCs were cultured in DMEM containing 5% exosome-depleted FBS (Invitrogen).
Differentiation assay. To assess the differentiation capacity of AdMSCs, the cultured cells were induced in osteogenic, adipogenic, and chondrogenic differentiation medium (Lonza) for 3 weeks. The medium was changed every 3 or 4 days, and 10 ng/mL of TGF-β3 (Lonza) was added to the cells for chondrogenesis. To confirm differentiation capacity, the differentiated cells were stained with von Kossa for osteogenesis, Oil Red O for adipogenesis, and Safranin-O for chondrogenesis. Images of the stained cells were obtained using an inverted phase microscope (Olympus-IX71, Olympus, Tokyo, Japan).
Flow cytometry analysis. For immunophenotyping the AdMSCs, the cells were harvested using 0.05% trypsin/EDTA (Invitrogen) at a single cell level and incubated on ice for 30 min with the following monoclonal antibodies: anti-CD29, anti-CD34, anti-CD44, anti-CD45, anti-CD73, and anti-CD90 (BD Pharmingen, San Diego, CA, USA). After washing with phosphate-buffered saline (PBS, Invitrogen), the cells were analyzed using a Cytomics Flow Cytometer (Beckman Coulter, Fullerton, CA, USA).
Isolation and characterization of exosomes from AdMSCs. Exosomes were isolated from the AdMSC supernatant using an exosome isolation kit (System Biosciences, Palo Alto, CA, USA) according to the manufacturer’s instructions. Briefly, the culture medium was harvested and centrifuged at 1500 × g for 5 min to remove cell debris. Then, the supernatant was transferred to a fresh conical tube, and the ExoQuick-TC reagent was mixed with the supernatant (1:5 ratio). After inverting four times, the mixture was incubated overnight at 5°C. The next day, the mixture was centrifuged at 1500 × g for 30 min, and the supernatant was removed. The isolated exosomes were resuspended in PBS (Invitrogen). The exosomes were quantified using a BCA protein assay kit (Invitrogen), followed by stored at -80 ℃. Transmission electron microscopy (TEM, JEM-1011, JEOL, Tokyo, Japan) was used to observe the morphology of the exosomes. To examine the size distribution, the exosomes were evaluated using a nanoparticle tracking system according to the manufacturer’s protocols (Nanosight NS300, Malvern Panalytical, Malvern, UK).
Western blot. Total proteins were extracted using RIPA lysis buffer (Biosesang, Seongnam, Republic of Korea) containing a protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO, USA). Protein concentration was quantified using a BCA protein assay kit (Invitrogen). Briefly, proteins were fractionated on a 12% sodium dodecyl sulfate polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Redmond, WA, USA). After blocking with 5% skim milk (BD-Pharmingen) for 1 h, samples were incubated with primary antibodies against the following proteins: anti-CD9 (1:500, LSBio #C119436, Seattle, WA, USA), anti-CD63 (1:500, LSBio #B16793), anti-ROCK1 (1:1000, Cell Signaling Technology #4035, Danvers, MA, USA), anti-PTEN (1:1000, Abcam #ab137337, Cambridge, UK), and anti-β-actin (1:1000, Santa Cruz Biotechnology #sc47778, Inc.) for 24 h at 4°C. Subsequently, the samples were incubated with a horseradish peroxidase-conjugated anti-mouse secondary antibody (1:1000, GeneTex #GTX221667-01, CA, USA) and anti-rabbit secondary antibody (1:1000, GeneTex #GTX221666-01) for 1 h at room temperature. After washing three times for 5 min, the membranes were developed using SuperSignal West Femto Maximum Sensitivity Substrate (Invitrogen) according to the manufacturer’s instructions. Signals were detected using the LAS4000 system (GE Healthcare, Uppsala, Sweden).
Real-time quantitative polymerase chain reaction Total RNA was extracted using the RiboEx reagent (GeneAll, Seoul, Republic of Korea). After analyzing the quality and quantity of RNA using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), 500 ng of RNA was used for reverse transcription using HiSenScriptTM RH[-] cDNA synthesis kit (iNtRON, Seongnam, Republic of Korea), according to the manufacturer’s instructions. Real-time qPCR was performed using a Light Cycler 480 II SYBR Green I Master mix (Roche Molecular Systems, Pleasanton, CA, USA) under specific conditions. Experiments were performed in triplicates using the primers listed in Supplementary Table S1. Relative mRNA expression levels were calculated using the comparative CT method, and the values were normalized to glyceraldehyde-6-phosphate dehydrogenase (GAPDH). For miRNA analysis, the U6 primer was used as an internal control.
miRNA inhibitor transfection and inhibition assays. To test the inhibitory effects of ROCK1 and PTEN, 5 × 105 THP-1 cells were plated in 6-well plates (Nunc, Roskilde, Denmark) and cultured with 10 nM ROCK inhibitor (Sigma Chemical Co., Y-27632) and 1 nM PTEN inhibitor (Sigma Chemical Co., SF1670) for 24 h. After washing with PBS, the cells were harvested for real-time qPCR analysis. Lipofectamine 2000 (Invitrogen) was used to transfect miR-132 and miR-146a inhibitor (Sigma Chemical Co.) into HUVECs, according to the manufacturer’s protocol. Briefly, 2.5 ×105 HUVECs were seeded on a 6-well plate (Nunc) and cultured with 50 nM inhibitor for 24 h. Transfected cells were subjected to real-time qPCR and angiogenesis assay using Matrigel.
Cell proliferation assays To examine the effects of exosomes on HUVEC proliferation, 1 × 103 cells were seeded with 5 µg/mL of exosomes in a 96-well plate (BD Falcon, Swedesboro, NJ, USA). After 24 h, the proliferation rate was evaluated using a WST-based assay kit (EZ-Cytox, Daeil Lab, Seoul, Republic of Korea), according to the manufacturer’s instructions. The absorbance of proliferation activity was measured at 450 nm using a microplate reader (Molecular Devices, San Jose, CA, USA). Untreated cells were used as controls, and the absorbance of treated cells was normalized to that of control cells.
Tube formation assay. Capillary network formation was evaluated using a tube formation assay in Matrigel (BD Pharmingen). 2 x 105 of HUVECs were seeded onto Matrigel-coated 24-well plates (BD Falcon) and cultured in EGM-2 supplemented with 10% FBS and 1% P/S for 24 h at 37°C in 5% CO2. The in vitro tube formation was analyzed by microscopy (Olympus).
Data are expressed as the mean ± standard deviation (SD). One-way ANOVA with Tukey’s test and t-test were performed for comparisons (Prism ver. 7, Graph Pad Software, Inc., La Jolla, CA). Statistical significance was defined as p < 0.05, *p < 0.05, and **p < 0.01.