MSCs obtained from human umbilical cord (hUC MSCs) were obtained at the Stem Cell Center of the Children’s Hospital of Chongqing Medical University. To generate exosomes, cells were maintained in DMEM/F12 supplemented with 10% exosome-free fetal bovine serum (FBS). Exosomes in FBS were removed by ultracentrifugation overnight at 100,000 × g and then filtered through a 0.22-µm filter (Millipore). To generate EMVs, cells were maintained in complete DMEM/F12 supplemented with 10% FBS and incubated at 37 °C with 5% CO2. Human umbilical vein endothelial cells (HUVECs) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin streptomycin solution (Gibco, USA) at 37 °C and 5% CO2.
Exosomes were isolated from CM through differential centrifugation. The medium was collected from hUC MSCs when they reached 90% confluence. In summary, the CM was first subjected to serial centrifugation to remove cells (300 × g, 10 min) and cellular debris (2000 × g, 20 min). The CM was centrifuged at 10,000 × g for 30 min to remove large microvesicles. Later, the supernatant was subjected to centrifugation at 100,000 × g for 70 min at 4 °C to pellet the exosomes. The exosome pellets were resuspended in phosphate-buffered saline (PBS, Invitrogen) and centrifuged again at 100,000 × g for 70 min at 4 °C. Finally, the exosome pellets were resuspended in PBS and filtered (through 0.22-μm filters) to remove large particles.
EMVs were extracted following a previously reported protocol . hUC MSCs were harvested, resuspended in PBS at a concentration of 1 × 106/ml and extruded in a mini-extruder with polycarbonate membrane filters with various pore sizes (10, 5, and 1 µm) (Avanti Polar Lipids). The extruded samples were collected and subjected to ultracentrifugation at 100,000 × g for 1 h at 4 °C. After ultracentrifugation, the precipitates were resuspended in PBS and then filtered (with 0.22-µm filters) to ultimately obtain EMVs.
Transmission electron microscopy
Transmission electron microscopy (TEM) was used to confirm the presence of exosomes and EMVs. Approximately, 20 µl of exosomes and EMVs were added separately to copper grids. All excess fluids were removed using filter paper, and the samples were negatively stained with 2% uranyl acetate for 30 s. The grids were rinsed in deionized water and allowed to dry overnight. The samples were then air-dried using an electric incandescent lamp and viewed using an electron microscope (Hitachi, S-3000N).
Nanoparticle tracking analysis
The exosome and EMV particles were resuspended in PBS, and their sizes and concentrations were analyzed by NTA. Then, exosomes and EMVs diluted in solution were injected into the LM10 unit (Malvern Panalytical). NTA software, version 2.3 (Malvern Panalytical, England), was used to collect and analyze the videos.
Western blot analysis
Total proteins were isolated from exosomes and EMVs using RIPA lysis buffer (Beyotime, China) with phenylmethanesulfonyl fluoride (PMSF; Beyotime, China) and centrifuged at 12,000 × g for 20 min at 4 °C. The concentrations were measured using a bicinchoninic acid (BCA) assay. Then, 10 μg of total protein was added to polyacrylamide gels, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk for 1 h and then incubated with different monoclonal primary antibodies overnight at 4℃. Primary antibodies against CD63 (1:500, Abcam, USA), Alix (1:1000, Abcam, USA), and TSG101 (1:1000, Abcam, USA) were used. After washing in Tris-buffered saline/Tween (TBST), the membranes were incubated with goat anti-rabbit or mouse antibodies (1:5000, Zhongshan, China) for 1 h at 37 °C. The immunoblots were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA).
Liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis
Each sample was separated using an Easy nLC system at a nanoliter flow rate. The chromatographic column was equilibrated with 100% solution A (0.1% formic acid in water), and the sample was loaded onto an analytical column (Thermo Fisher Scientific, Acclaim PepMap RSLC 50 µm × 15 cm, nano viper, P/N164943) via an autosampler for separation. The flow rate was 300 nL/min. Solution B was comprised of 0.1% formic acid in acetonitrile aqueous solution; after chromatographic separation, the sample was analyzed with a Q Exactive plus mass spectrometer. The analysis time was 60–90 min, detection was performed in positive ion mode, the scanning range of the precursor ion was 350–1800 m/z, and the primary mass spectrum resolution was 70,000. Then, Proteome Discoverer 2.1 (Thermo Fisher Scientific) software was used to convert the original map file (.raw file) generated with Q Exactive Plus software into a.mgf file, which was submitted to the MASCOT2.6 server for database retrieval. Then, the database search file (.dat file) obtained from the MASCOT server was transmitted back through Proteome Discoverer 2.1 software. The data were filtered on the basis of a false discovery rate (FDR) < 0.01 to obtain highly reliable qualitative results.
Differentially expressed proteins
The exosome-available data from the ExoCarta database (http://www.exocarta.org) was used to compare the identified proteins. Protein differential expression analysis between two different groups was performed with R software (R4.0.1, the EDGER package). Differences in protein expression with a p value < 0.05 and an absolute fold change ≥ 2 were considered to denote differentially expressed proteins.
Gene Ontology (GO) enrichment analysis
Blast2GO was used to perform GO annotations of target protein sets. Proteins were classified into biological process, cellular compartment and molecular function GO categories. For data in each category, a two-tailed Fisher’s exact test was performed to determine the enrichment of differentially expressed proteins compared to all identified proteins. GO terms with a corrected p value < 0.05 were considered to indicate significant enrichment.
Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis
KEGG Orthology (KO) and Links Annotation (KOALA) software was used to analyze the KEGG GENES database to classify target protein sequences identified by KO analysis, and through this process, information on pathways in which the target protein sequence is involved, as indicated by KO classification, was automatically obtained. Fisher’s exact test was performed to compare the KEGG pathways involving target proteins and the overall protein sets to evaluate the significance level of protein enrichment in specific KEGG pathways. The KEGG analysis results with a corrected p value < 0.05 were considered to indicate significant enrichment.
Gene set enrichment analysis
Gene set enrichment analysis (GSEA) was performed using the gene set c2.cp.v7.2.symbols.gmt. Pathway. Significantly enriched protein sets were defined as protein sets with a p value < 0.05, a normalized enrichment score (NES) > 1 or < − 1, and an FDR < 0.25.
Protein–protein interaction network analysis
The STRING database (https://string-db.org/) was used to screen different groups of protein–protein interaction (PPI) networks with an interaction score ≥ 0.4. The Cytoscape cytoHubba plug-in was used to find and identify the 20 most connected hub proteins in each network.
Screening of differentially expressed membrane proteins
First, the proteins located in the cell membrane were selected based on their localization. Second, membrane proteins with high expression in exosomes and EMVs (top 4) were screened according to the expression level detected by sequencing.
Wound healing experiments in vitro and in vivo
Labeling and tracing of exosomes and EMVs in HUVECs
The EMVs and exosomes were labeled with the membrane dye PKH26 in accordance with the manufacturer's instructions. HUVECs were seeded in 24-well plates containing cell sheets, and PKH26-labeled EMVs and exosomes were then added at a concentration of 100 µg/ml. After culturing for 6 h, 12 h and 24 h, the cells were washed with PBS, fixed with 4% paraformaldehyde, stained with DAPI, and photographed under a fluorescence microscope (Nikon, K10587, Japan).
Six-well plates were used for the scratch wound assay. HUVECs were seeded in plates at 200,000 cells/well and incubated until reaching confluence, after which a scratch was made using a pipet tip. The exfoliated cells were washed with PBS and cultured in serum-free medium with or without exosomes or EMVs (100 µg/ml). Images were acquired at 0 h, 12 h and 24 h, and the cell migration distance was measured using NIS-Elements (Nikon, Japan) analysis software. The migration distance (%) was calculated using the following formula: (initial wound width—final wound width)/ initial wound width × 100.
Transwell assays were performed using 24-well transwell plates (Corning, Corning, NY, USA) with 8-µm-pore size filters. HUVECs were routinely digested and resuspended in serum-free medium, and 10,000 cells were seeded into the upper chamber. Medium containing 10% fetal bovine serum was added to the lower chamber, which either contained or did not contain EMVs or exosomes (100 µg/ml). The cells were routinely cultured for 24 h, fixed with 4% paraformaldehyde, stained with crystal violet, photographed under a microscope, and counted by ImageJ software.
Cell Counting Kit-8 analysis was performed to assess cell proliferation. When the HUVECs reached a confluence of approximately 80%, 2000 cells/well were inoculated into 96-well plates. The medium was supplemented with or without EMVs and exosomes (100 µg/ml) derived from MSCs. The cells were cultured routinely, and 10 µl of CCK-8 solution and fresh medium were added to each well at 0 h, 24 h, 48 h, and 72 h. The cells were incubated for another 2 h, and the absorbance was detected at 450 nm using a microplate reader.
Tube formation assay
The matrix gel without growth factors was pre-added to 96-well plates (50 µl/well) and incubated at 37 °C for 30 min to allow solidification. A total of 20,000 cells/well were seeded in plates containing the matrix gel, and the medium was supplemented with or without EMVs or exosomes (100 µg/ml). After culturing for 6 h, tube formation was observed under a microscope. Image-J software was used to measure the number of the branches.
Animals and treatment
Adult male BALB/C mice with an average weight of 20–25 g were purchased from the Experimental Animal Center of Chongqing Medical University (SCXK 2018-0003). All mice were reared at the Animal Center, Children's Hospital of Chongqing Medical University (SYXK 2017-0012) and provided adequate water and food. Under isoflurane (2.5%) anesthesia, the hair on the back of the mouse was shaved, and a full-thickness skin wound (about 10 mm in diameter) was created on the back. Eighteen mice were randomly divided into three groups. The mice were subcutaneously injected with an equal amount of PBS (100 µl), EMVs or exosomes (200 µg dissolved in 100 µl of PBS) at multiple points around the wound for 7 days. Skin incision healing was observed and photographed on days 0, 2, 4 and 7, and the incision area was measured by ImageJ. Wound closure (%) = (the initial wound area – At)/ the initial wound area × 100, where At is the wound area at day 2, 4 or 7 post-operation. The underside of the skin was observed and photographed on day 7 after wounding to examine the formation of new blood vessels. All experiments were approved by the Animal Research Committee of Children's Hospital of Chongqing Medical University.
Principal component analysis (PCA) was performed to evaluate quantitative protein repeatability. Data are presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare the means of multiple groups, and the means between two groups were compared using an independent-sample t test. Graph Pad Prism 8.0 (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses. P values < 0.05 were considered to indicate statistical significance. All experiments were performed three times.