- Human placenta–derived mesenchymal stem cell (hPMSC) preparation and isolation of exosomes from hPMSCs
Human placenta stem cells were obtained from CHA General Hospital, Seoul, Republic of Korea. The sample collection and use for research purposes were approved by the Institutional Review Board of the hospital. Preparation and culturing were conducted as previously reported . Human PMSCs were cultured in Minimum Essential Medium (MEM)-alpha GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Thermo Fisher Scientific), 1% penicillin/streptomycin (Thermo Fisher Scientific), 25 ng/mL human fibroblast growth factor 4 (Peprotech Inc., Rocky Hill, NJ, USA), and 1 μg/mL heparin (Sigma-Aldrich, St. Louis, MO, USA). When 80% confluence was reached, the culture medium was replaced with MEM-alpha GlutaMAX containing 10% exosome-free FBS (Thermo Fisher Scientific). The conditioned hPMSCs were harvested from the medium, and residual cells and debris were discarded by centrifuging at 2,000×g for 10 min at 4 °C. After centrifugation, the supernatant was filtered using a 0.2 μm pore filter and transferred to a centrifuge tube (Pall Corporation, Port Washington, NY, USA). The supernatant was centrifuged at 4,000 rpm for 45 min at 4 °C. The collected supernatant again underwent ultracentrifugation at 27,500 rpm for 85 min at 4 °C. Thereafter, the supernatant was removed, and the precipitate was washed with phosphate-buffered saline (PBS) and then ultracentrifuged at 27,500 rpm for 85 min at 4 °C. Finally, the exosome precipitates were dissolved in 100 μL PBS and quantified by the BCA method. The exosomes were stored at -80 °C.
- Cell culture and treatment
Immortalized R28 retinal precursor cells were maintained in low-glucose Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) with 10% FBS (Thermo Fisher Scientific), 1× minimal essential medium nonessential amino acids (Thermo Fisher Scientific), 100 μg/mL gentamicin (Sigma-Aldrich), and 1% penicillin/streptomycin (Thermo Fisher Scientific). A hypoxic condition was induced by exposing the cells to CoCl2 (Sigma-Aldrich). R28 cells (2 × 105) were treated with CoCl2 (200 μM) for 9 h. Then, they were treated with hPMSC exosomes (12 μg/mL). After 24 h, the cells were harvested and prepared for analysis.
- Small-interfering RNA
The target sequence of siRNA (Bioneer Corporation, Daejeon, Republic of Korea) was as follows: siRNA rat UBA2, 5′- GCA CGA AAC CAU GUG AAU AGG A. siRNA Negative Control (Bioneer) was used as the negative control (scramble). R28 cells were transfected using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.
- Cell viability assay
Hypoxic R28 cells were harvested 24 h after exosome (12 μg/mL) treatment, and counted by microscopic examination. The data were expressed as percentage (mean ± SEM) of viable cells in the experimental group compared with those in the control group.
- Reverse transcription–polymerase chain reaction (RT-PCR) analysis
Total RNA was isolated from hOFs using TRIzol reagent (Thermo Fisher Scientific). RT-PCR was performed with nPfu-Forte PCR polymerase (Enzynomics, Daejeon, Republic of Korea). We quantified the gene expression using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and RT-PCR reactions were performed using a CFX-96 machine (Bio-Rad Laboratories, Hercules, CA, USA). The nucleotide sequences of all the primers used were as follows: Rat UBA2 FP: 5′‐ ACG ATT CGG AAC ACA CCT TC, RP: 5′‐ GCT TCA GCC TCT GTT GGT TC; Rat UBE2E3 FP: 5′‐ TCG AGT GCT GTG TTC AAA GG, RP: 5′‐ CTG GTG CTA GGG CTC TCA TC; Rat UBE2I FP: 5′‐ TCT CCC TGC CTG TTA GCT GT, RP: 5′‐ TGG GCT GTA GGG TAA GGT TG.
- Immunoblot analysis
R28 cells were lysed in radioimmunoprecipitation assay (RIPA) buffer. Equal amounts of total protein were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to membranes. The membranes were immunoblotted with anti-Hif-1α (Abcam, Cambridge, UK), Vegf, Thy-1, Gap43 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Ermn (Abcam), Neuroflament (Cell Signaling Technology, Danvers, MA, USA), Wnt3a, β-catenin (GeneTex, Irvine, CA, USA), phospo-GSK3β (Cell Signaling Technology), Lef-1 (GeneTex), UBA2 (Abcam), Skp1, βTrcp (Cell Signaling Technology), and ubiquitin (Abcam). After washing, the membranes were incubated at room temperature for 2 h with horseradish peroxidase–conjugated anti-rabbit/mouse/goat IgG secondary antibodies at a 1:10,000 dilution (GeneTex). Immunoreactive bands were visualized with enhanced chemiluminescence solution (Bio-Rad Laboratories), and analyzed using ImageQuant™ LAS 4000 (GE Healthcare, Chicago, IL, USA).
Proteomic analyses were performed for 4 types of samples: R28 cells with PBS, R28 cells with exosomes, R28 cells treated with CoCl2, and R28 cells treated with CoCl2 and exosomes. Thus, we investigated how exosomes worked in both undamaged and damaged cells.
Tris(2-carboxyethyl)phosphine (TCEP) was supplied by Thermo Fisher Scientific. Formic acid (FA) and iodoacetamide (IAA) were purchased from Sigma-Aldrich. Trypsin was obtained from Promega (Madison, WI, USA). High-performance liquid chromatography (HPLC)-grade water and acetonitrile were purchased from JT Baker (Phillipsburg, NJ, USA).
7.2. Sample preparation
R28 cells (2 × 105) were treated with CoCl2 (200 μM) for 9 h. Then, they were treated with hPMSC exosomes (12 μg/mL). After 24 h, the cells were harvested. Each cell pellet then was mixed with 1 mL of lysis solution (8 M Urea, 0.1 M Tris-HCl buffer, pH 8.5) and 40 µL of protease inhibitor cocktail (25× stock solution) in glass tubes. Cell lysis was performed using a Covaris S2 Focused-Ultrasonicator (Covaris, Woburn, MA, USA) for 8 min. The protein concentrations in the samples were determined using Pierce BCA Protein Assay Kits (Thermo Fisher Scientific). Filter-aided sample preparation (FASP) was performed using Ultracel® YM-30 centrifugal filters (Merck Millipore, Germany), as previously reported . In brief, protein (100 µg) was reduced with TCEP (37 °C, 30 min), alkylated with IAA (25 °C, 30 min, in the dark), and digested with trypsin (37 °C, 18 h, enzyme:protein ratio = 1:50). After digestion, the peptide mixtures were collected. FA was added to inactivate trypsin. The samples were then desalted using C18 Micro spin columns (Harvard Apparatus, MA, USA), vacuum-dried (1,800 rpm, 3 h, ScanSpeed 40 centrifugal evaporator), and reconstituted in 0.1% FA/water (solvent A) prior to analysis.
7.3. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis
An LC-MS/MS setup consisting of a Dionex Ultimate 3000 HPLC system coupled with a Q Exactive™ Hybrid Quadrupole-Orbitrap MS (Thermo Fisher Scientific) system was used for sample analysis. Samples were loaded into an Acclaim™ PepMap™ 100 C18 nano-trap column (75 μm × 2 cm, 3 μm particles, 100 Å pores, Thermo Fisher Scientific) using solvent A at a flow rate of 2.5 μL/min for 5 min. An Acclaim™ PepMap™ C18 100A RSLC nano-column (75 μm × 50 cm, 2 μm particles, 100 Å pores, Thermo Fisher Scientific) was used to separate the peptide mixtures. The solvent consisted of solvent A and solvent B (0.1% FA/80% ACN). The flow rate was fixed at 300 nL/min. A 185 min gradient set up for solvent B was used as follows: 4% (14 min), 4-20% (61 min), 20-50% (81 min), 50-96% (1 min), 96% (10 min), 96-4% B (1 min), and 4% (17 min). The nano-electrospray ionization source was operated in positive mode with a spray voltage of 2.0 kV. The capillary temperature was 320 °C. The isolation width was ± 2 m/z, and the scan range was 400-2,000 m/z. The resolutions in full-MS scans and MS/MS scans at 200 m/z were 70,000 and 17,500, respectively. MS was conducted using a data-dependent acquisition method. The top ten precursor ions with the highest intensity were isolated in the quadrupole and fragmented by higher-energy collisional dissociation with 27% normalized collisional energy. Dynamic exclusion was set at 20 s to minimize repeated analyses of the same abundant precursor ions.
7.4. Data processing and bioinformatics
Database search for proteins and data processing were conducted as previously reported . In brief, raw MS/MS data files were searched against a SwissProt human protein database (https://www.uniprot.org/) using a built-in Andromeda search engine in MaxQuant version 188.8.131.52 (www.coxdocs.org) for label-free quantification (LFQ). The following parameters were used for the search: missed cleavages with trypsin, ≤2; variable modifications, methionine oxidation (+15.995 Da), and carbamylation of protein in N-term (+43.0006 Da); static carbamidomethylation of cysteine (+57.0215 Da); first search peptide tolerance, 20 ppm; and main search peptide tolerance, 4.5 ppm. A false discovery rate (FDR) cutoff of 1% was used. LFQ data from MaxQuant were imported into Perseus software platform version 184.108.40.206 (www.coxdocs.org). Protein LFQ intensities were transformed using log2(x), and samples with missing values for given proteins were assigned random values using the imputation principle (downshift 1.8, width 0.3, total matrix mode). After Z-score normalization, the Student’s T-test was used to compare the protein abundances of the groups. Differentially expressed proteins (DEPs) were filtered with a cutoff p-value ≤ 0.05 and log2FC ≥ 1 (fold-change). Heatmap was generated. Gene ontology (GO) analysis was performed using Panther (http://geneontology.org/). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and protein–protein interactions were analyzed using the String database (https://string-db.org/).
- Statistical analyses
All the results are presented as mean ± standard error of the mean (SEM). Data analyses were conducted using GraphPad Prism (GraphPad, La Jolla, CA, USA). Statistically significant differences were identified using the t-test or nonparametric statistical test, followed by the Mann–Whitney U test at a significance level of 5%.