Cell tradition, identification, and remedy
MSCs have been remoted from the recent human umbilical cords as described beforehand [16]. After the permission of moms, human umbilical cords have been positioned in phosphate buffer answer (PBS) containing penicillin-streptomycin for 30 min, adopted by the removing of arteries and veins. Subsequently, the umbilical cords have been minimize into 2-cm items, pasted on the underside of tradition dishes, positioned upside-down within the tradition incubator for 60 min, and maintained in serum-free DMEM (Life Applied sciences, USA) at 37 °C with 5% CO2. The tradition medium was modified each 3 days. Major cells have been trypsinized and passaged for additional enlargement. MSCs at passage 3 have been cultured in normoxic (21% O2) or hypoxic (1% O2) situations at 37 °C with 5% CO2 respectively. Normoxic conditioned MSCs (Nor-MSCs) and hypoxic preconditioned MSCs (Hyp-MSCs) have been then seeded in osteogenic medium (Cyagen Biosciences, USA) and adipogenic medium (Cyagen Biosciences, USA) for two weeks, and stained with Alizarin Pink S and Oil Pink O to detect their differentiation potential. Movement cytometry assay was used to detect the markers of Nor-MSCs and Hyp-MSCs together with CD29, CD44, CD73, CD11b, CD34, and CD45.
The 661 W cells have been bought from the Chinese language Academy of Sciences (Shanghai, China) and have been characterised beforehand to be of cone photoreceptor cell lineage [17]. The 661 W cells have been maintained in high-glucose DMEM (Bioind, Israel) with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 g/ml streptomycin at 37 °C with 5% CO2. In vitro remedy of 661 W cells with MNU (Sigma-Aldrich, USA) was carried out on the focus of 300 µg/ml for six h, adopted by the administration of sEVs (20 µg/mL) for twenty-four h.
Isolation and identification of sEVs
sEVs have been remoted from the cell supernatant of MSCs cultured with serum-free medium by ultracentrifugation. Briefly, the conditioned medium was centrifuged at 300 × g for 15 min, 2,000 × g for 20 min and 10,000 × g for 30 min to take away cells and cell particles. The clarified supernatant was then ultracentrifuged at 100,000 × g for 90 min to acquire sEVs precipitation. After washing with PBS, the sEVs have been ultracentrifuged at 100,000 × g for 90 min, dissolved with PBS, handed by a 0.22-mm filter, and saved at -80 °C. The morphology of sEVs was noticed by the transmission electron microscopy (TEM). The scale distribution and particle variety of sEVs have been analyzed by NanoSight monitoring evaluation (NTA). The expressions of protein markers together with CD9, CD63, Alix, tumor susceptibility gene 101 (TSG101), and endoplasmic reticulum protein calnexin have been detected by Western blot.
Animal mannequin and remedy
All animal experiments have been accredited by the Medical Ethics Committee and Ethics Committee for Experimental Animals of Jiangsu College (2,020,161). The 12-week-old C57BL/6 male mice have been bought from the Animal Middle of Jiangsu College, and housed in a pathogen-free surroundings with a 12 h gentle/darkish cycle on the temperature of 25 °C with free entry to meals and water. For the institution of MNU-induced retinal degeneration mannequin, mice have been intraperitoneally injected with MNU (50 mg/kg in sterile saline). The collected sEVs have been adjusted to the protein focus of 1 µg/µl and injected at 1 µl into every vitreous chamber at 6 h after MNU remedy with a 33-G Hamilton syringe. All mice at 7 d after MNU remedy have been sacrificed with 4% isoflurane and the eyeballs have been collected for additional evaluation. The variety of mice utilized in every experiment is indicated within the determine legend.
Tracing of sEVs
The membrane fluorescent dye PKH26 (5 µM) was incubated with Nor-sEVs and Hyp-sEVs for 30 min at 37 °C. Subsequently, the stained Nor-sEVs and Hyp-sEVs have been washed with PBS and centrifuged at 100,000 g for 90 min. For the retinal tracing of sEVs, the retinal tissues have been remoted at 24 h after the intravitreal injection of PKH26-labeled Nor-sEVs and Hyp-sEVs, fastened in 4% paraformaldehyde, cryoprotected with 30% sucrose, embedded within the OCT compound, and minimize into 15-µm retinal sections. After the counterstaining with Hoechst 33,342 (Sigma-Aldrich, USA), retinal sections have been noticed underneath a confocal microscope (DeltaVision Elite, GE, USA). For in vitro tracing, 661 W cells have been handled with PKH26-labeled Nor-sEVs and Hyp-sEVs for twenty-four h, fastened in 4% paraformaldehyde, counterstained with Hoechst 33,342 (Sigma-Aldrich, USA), and noticed underneath a confocal microscope (DeltaVision Elite, GE, USA).
Electroretinogram (ERG) evaluation
After in a single day darkish adaptation, mice have been anesthetized by the intraperitoneal injection with ketamine (87.5 mg/kg) and xylazine (12.5 mg/kg), and the cornea was anesthetized with 0.5% proxymetacaine. The bottom electrodes, reference electrodes and document electrodes have been then positioned on the tail, mouth, and cornea respectively. The flash depth was 3.0 cd.s/m2. The UTAS Visible Diagnostic System (LKC Applied sciences, USA) was used to document the scotopic and photopic responses for analyzing the a-wave and b-wave.
Hematoxylin and eosin (H&E) staining
After enucleation, retinal tissues have been quickly remoted, fastened in 4% paraformaldehyde in a single day, embedded in paraffin, and minimize into 4-µm sections. Retinal sections have been stained with H&E for the statement of retinal morphology in line with the usual protocols.
Immunofluorescence staining
After deparaffinization, retinal sections have been handled with boiled citrate buffer (pH 6.0, 10 mM) to restore antigens, blocked with 5% bovine serum albumin (BSA) for 1 h and incubated with the first antibody at 4 °C in a single day. The 661 W cells have been fastened with 4% paraformaldehyde on the slide for 30 min, permeabilized with 0.1% Triton X-100 for 15 min, blocked with 5% BSA for 1 h and incubated with the first antibody at 4 °C in a single day. Subsequently, the sections have been stained with fluorescent-conjugated secondary antibody for 1 h at 37 °C, counterstained with Hoechst 33,342 (Sigma-Aldrich, USA), and noticed underneath a confocal microscope (DeltaVision Elite, GE, USA). The first antibodies included S-opsin (1:100, ABN1660, Millipore, USA), Ki-67 (1:100, 34,330, CST, USA), Cleaved Caspase-3 (1:100, 9661, CST, USA), and GAP43 (1:100, 16971-1-AP, Proteintech, China).
Terminal deoxynucleotidyl transferase dUTP nick finish labelling (TUNEL) staining
TUNEL staining was carried out in line with the producer’s directions of TUNEL BrightRed Apoptosis Detection Package (Vazyme, China). Briefly, every part was incubated with 100 µL of proteinase Okay answer for 20 min, balanced in 100 µL of 1 × equilibration buffer for 30 min, handled with 100 µL of TDT buffer for 1 h at 37 °C to label apoptotic cells, and counterstained with Hoechst 33,342 (Sigma-Aldrich, USA). After washing with PBS, the sections have been photographed utilizing a confocal microscope (DeltaVision Elite, GE, USA).
Liquid chromatography-tandem mass spectrometry (LC-MS/MS)
50 µg of every protein pattern was subjected to digestion with the sequencing-grade trypsin, adopted by the peptide labeling. The peptides have been analyzed by LC-MS/MS. A number of databases have been used for the practical annotation evaluation of recognized proteins. Correlation evaluation on differentially expressed proteins was additionally carried out.
Transfection of siRNA and plasmid
Particular focusing on siRNA and overexpressing plasmid have been designed and synthesized by Genepharma (Suzhou, China). The total-length cDNA of the human HIF-1α gene was inserted into the pCDH-CMV-MCS-GFP + Puro plasmid vector to assemble the overexpression plasmid. Cells have been resuspended and seeded in 6-well plates (2 × 105 cells/effectively). When the cell density reached 50–70%, cells have been transfected with siRNA and plasmid utilizing Lipofectamine 2000 (Life Applied sciences, USA) in serum-free medium in line with the producer’s directions. At 6 h after transfection, cells have been uncovered to the recent full medium and cultured for an additional 24 h. The sequences of siRNA have been as follows:
siRNA NC: sense: UUCUCCGAACGUGUCACGUTT.
siRNA NC: antisense: ACGUGACACGUUCGGAGAATT.
si-HIF-1α: sense: CUCCCUAUAUCCCAAUGGATT.
si-HIF-1α: antisense: UCCAUUGGGAUAUAGGGAGTT.
Lentiviral knockdown of GAP43 in Hyp-MSCs
The lentiviral vectors containing GAP43 quick hairpin RNA (shRNA) sequence and destructive management vector have been designed and synthesized by Genepharma (Suzhou, China). Recombinant lentiviruses have been produced by the transfection in HEK293T cells utilizing Lipofectamine 2000 (Life Applied sciences, USA). Hyp-MSCs have been then transfected with recombinant lentivirus. The secure cell strains have been cultured in serum-free medium for 48 h to acquire supernatants, adopted by the isolation of sEVs by ultracentrifugation. Western blot was used to guage the effectivity of GAP43 knockdown in Hyp-MSCs and Hyp-sEVs. The sequences of shRNA have been as follows:
Management shRNA: CCTAAGGTTAAGTCGCCCTCG.
GAP43 shRNA: GCTCATAAGGCCGCAACCAAA.
Western blot
The whole protein of retinas, cells and sEVs was remoted utilizing radio-immunoprecipitation assay lysis buffer. The protein focus was decided utilizing the BCA assay equipment. Equal quantities of protein samples have been separated by 12% SDS-PAGE gel and transferred onto PVDF membranes (Millipore, USA). After blocking in 5% skim milk for 1 h, the membranes have been incubated with main antibodies at 4 °C in a single day, handled with HRP-conjugated secondary antibodies at 37 °C for 1 h, and detected by enhanced chemiluminescence. The first antibodies included Oct4 (1:1000, 2750, CST, USA), Sall4 (1:1000, 8459, CST, USA), Lin28a (1:1000, 11724-1-AP, Proteintech, China), Sox2 (1:1000, 3579, CST, USA), TSG101 (1:1000, 72,312, CST, USA), Alix (1:1000, 92,880, CST, USA), CD9 (1:1000, 98,327, CST, USA), CD63 (1:2000, 52,090, CST, USA), calnexin (1:1000, 2433, CST, USA), proliferating cell nuclear antigen (PCNA; 1:1000, 10205-2-AP, Proteintech, China), Bcl-2 (1:500, 12789-1-AP, Proteintech, China), Bax (1:500, 50599-2-Ig, Proteintech, China), GAP43 (1:1000, 16971-1-AP, Proteintech, China), HIF-1α (1:1000, 36,169, CST, USA), Ubiquitin (1:2000, 58,395, CST, USA), TRIM25 (1:1000, 13,773, CST, USA), and β-actin (1:5000, 4970, CST, USA).
Co-immunoprecipitation (Co-IP) assay
Cells have been lysed in Co-IP buffer and incubated with the GAP43 antibody (1:100, 16971-1-AP, Proteintech, China) at 4 °C in a single day, adopted by the remedy with magnetic beads for 4 h. After washing with Co-IP buffer, protein complexes have been detected by western blot.
qRT-PCR
The whole RNA of cells was remoted utilizing Trizol reagent (Gibco, USA) and reverse-transcribed to cDNA in line with the producer’s protocol of HiScript II 1st Strand cDNA Synthesis Package (Vazyme, China). Primarily based on the ABI Prism 2720 PCR system, qRT-PCR was carried out with SYBR Inexperienced PCR equipment (CWBIO, China). The relative mRNA expression was evaluated utilizing the two−ΔΔCt methodology. The sequences of primers have been as follows:
β-actin: ahead: GACCTGTACGCCAACACAGT.
β-actin: reverse: CTCAGGAGGAGCAATGATCT.
GAP43: ahead: GGCCGCAACCAAAATTCAGG.
GAP43: reverse: CGGCAGTAGTGGTGCCTTC.
CCK8 assay
After remedy, cells have been resuspended and seeded in 96-well plates (3,000 cells/effectively). After the incubation for twenty-four, 48, 72 and 96 h, cells have been handled with 100 µL of recent medium containing 10 µL of CCK8 reagent (Vazyme, China) for 3 h. The absorbance of every effectively was decided at 450 nm utilizing the spectrophotometer (FLX800, BioTek, USA).
Cell apoptosis assay
Annexin V–FITC/PI Apoptosis Detection Package (Vazyme, China) was used to evaluate the impact of sEVs on cell apoptosis. After the remedy with MNU and sEVs, 661 W cells have been washed with PBS, centrifuged at 800 rpm for five min, resuspended in 1× binding buffer, and stained with 5 µl of Annexin V-FITC and 5 µl of PI at room temperature. The stained cells have been then detected by move cytometry (FACSCalibur, BD, USA). The odds of early apoptotic (Annexin V+PI−) plus late apoptotic (Annexin V+PI+) cells have been thought-about as apoptotic percentages.
Statistical evaluation
All statistical evaluation was carried out utilizing GraphPad Prism software program (GraphPad, San Diego, USA). All knowledge have been introduced because the means ± SEM. Unpaired Scholar’s t-test was used for comparability between two teams. The numerous variations amongst a number of teams have been assessed by evaluation of variance with Newman-Keuls take a look at. P worth < 0.05 was thought-about statistically vital.
