Cell culture, identification, and treatment

MSCs were isolated from the fresh human umbilical cords as described previously [16]. After the permission of mothers, human umbilical cords were placed in phosphate buffer solution (PBS) containing penicillin-streptomycin for 30 min, followed by the removal of arteries and veins. Subsequently, the umbilical cords were cut into 2-cm pieces, pasted on the bottom of culture dishes, placed upside-down in the culture incubator for 60 min, and maintained in serum-free DMEM (Life Technologies, USA) at 37 °C with 5% CO2. The culture medium was changed every 3 days. Primary cells were trypsinized and passaged for further expansion. MSCs at passage 3 were cultured in normoxic (21% O2) or hypoxic (1% O2) conditions at 37 °C with 5% CO2 respectively. Normoxic conditioned MSCs (Nor-MSCs) and hypoxic preconditioned MSCs (Hyp-MSCs) were then seeded in osteogenic medium (Cyagen Biosciences, USA) and adipogenic medium (Cyagen Biosciences, USA) for 2 weeks, and stained with Alizarin Red S and Oil Red O to detect their differentiation potential. Flow cytometry assay was used to detect the markers of Nor-MSCs and Hyp-MSCs including CD29, CD44, CD73, CD11b, CD34, and CD45.

The 661 W cells were purchased from the Chinese Academy of Sciences (Shanghai, China) and have been characterized previously to be of cone photoreceptor cell lineage [17]. The 661 W cells were 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 treatment of 661 W cells with MNU (Sigma-Aldrich, USA) was conducted at the concentration of 300 µg/ml for 6 h, followed by the administration of sEVs (20 µg/mL) for 24 h.

Isolation and identification of sEVs

sEVs were isolated 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 remove cells and cell debris. The clarified supernatant was then ultracentrifuged at 100,000 × g for 90 min to obtain sEVs precipitation. After washing with PBS, the sEVs were ultracentrifuged at 100,000 × g for 90 min, dissolved with PBS, passed through a 0.22-mm filter, and stored at -80 °C. The morphology of sEVs was observed by the transmission electron microscopy (TEM). The size distribution and particle number of sEVs were analyzed by NanoSight tracking analysis (NTA). The expressions of protein markers including CD9, CD63, Alix, tumor susceptibility gene 101 (TSG101), and endoplasmic reticulum protein calnexin were detected by Western blot.

Animal model and treatment

All animal experiments were approved by the Medical Ethics Committee and Ethics Committee for Experimental Animals of Jiangsu University (2,020,161). The 12-week-old C57BL/6 male mice were purchased from the Animal Center of Jiangsu University, and housed in a pathogen-free environment with a 12 h light/dark cycle at the temperature of 25 °C with free access to food and water. For the establishment of MNU-induced retinal degeneration model, mice were intraperitoneally injected with MNU (50 mg/kg in sterile saline). The collected sEVs were adjusted to the protein concentration of 1 µg/µl and injected at 1 µl into each vitreous chamber at 6 h after MNU treatment with a 33-G Hamilton syringe. All mice at 7 d after MNU treatment were sacrificed with 4% isoflurane and the eyeballs were collected for further analysis. The number of mice used in each experiment is indicated in the figure 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 were washed with PBS and centrifuged at 100,000 g for 90 min. For the retinal tracing of sEVs, the retinal tissues were isolated at 24 h after the intravitreal injection of PKH26-labeled Nor-sEVs and Hyp-sEVs, fixed in 4% paraformaldehyde, cryoprotected with 30% sucrose, embedded in the OCT compound, and cut into 15-µm retinal sections. After the counterstaining with Hoechst 33,342 (Sigma-Aldrich, USA), retinal sections were observed under a confocal microscope (DeltaVision Elite, GE, USA). For in vitro tracing, 661 W cells were treated with PKH26-labeled Nor-sEVs and Hyp-sEVs for 24 h, fixed in 4% paraformaldehyde, counterstained with Hoechst 33,342 (Sigma-Aldrich, USA), and observed under a confocal microscope (DeltaVision Elite, GE, USA).

Electroretinogram (ERG) analysis

After overnight dark adaptation, mice were 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 ground electrodes, reference electrodes and record electrodes were then placed on the tail, mouth, and cornea respectively. The flash intensity was 3.0 cd.s/m2. The UTAS Visual Diagnostic System (LKC Technologies, USA) was used to record the scotopic and photopic responses for analyzing the a-wave and b-wave.

Hematoxylin and eosin (H&E) staining

After enucleation, retinal tissues were rapidly isolated, fixed in 4% paraformaldehyde overnight, embedded in paraffin, and cut into 4-µm sections. Retinal sections were stained with H&E for the observation of retinal morphology according to the standard protocols.

Immunofluorescence staining

After deparaffinization, retinal sections were treated with boiled citrate buffer (pH 6.0, 10 mM) to repair antigens, blocked with 5% bovine serum albumin (BSA) for 1 h and incubated with the primary antibody at 4 °C overnight. The 661 W cells were fixed 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 primary antibody at 4 °C overnight. Subsequently, the sections were stained with fluorescent-conjugated secondary antibody for 1 h at 37 °C, counterstained with Hoechst 33,342 (Sigma-Aldrich, USA), and observed under a confocal microscope (DeltaVision Elite, GE, USA). The primary 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 end labelling (TUNEL) staining

TUNEL staining was performed according to the manufacturer’s instructions of TUNEL BrightRed Apoptosis Detection Kit (Vazyme, China). Briefly, each section was incubated with 100 µL of proteinase K solution for 20 min, balanced in 100 µL of 1 × equilibration buffer for 30 min, treated 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 were photographed using a confocal microscope (DeltaVision Elite, GE, USA).

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

50 µg of each protein sample was subjected to digestion with the sequencing-grade trypsin, followed by the peptide labeling. The peptides were analyzed by LC-MS/MS. Multiple databases were used for the functional annotation analysis of identified proteins. Correlation analysis on differentially expressed proteins was also performed.

Transfection of siRNA and plasmid

Specific targeting siRNA and overexpressing plasmid were designed and synthesized by Genepharma (Suzhou, China). The full-length cDNA of the human HIF-1α gene was inserted into the pCDH-CMV-MCS-GFP + Puro plasmid vector to construct the overexpression plasmid. Cells were resuspended and seeded in 6-well plates (2 × 105 cells/well). When the cell density reached 50–70%, cells were transfected with siRNA and plasmid using Lipofectamine 2000 (Life Technologies, USA) in serum-free medium according to the manufacturer’s instructions. At 6 h after transfection, cells were exposed to the fresh complete medium and cultured for another 24 h. The sequences of siRNA were 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 short hairpin RNA (shRNA) sequence and negative control vector were designed and synthesized by Genepharma (Suzhou, China). Recombinant lentiviruses were produced by the transfection in HEK293T cells using Lipofectamine 2000 (Life Technologies, USA). Hyp-MSCs were then transfected with recombinant lentivirus. The stable cell lines were cultured in serum-free medium for 48 h to obtain supernatants, followed by the isolation of sEVs by ultracentrifugation. Western blot was used to evaluate the efficiency of GAP43 knockdown in Hyp-MSCs and Hyp-sEVs. The sequences of shRNA were as follows:

Control shRNA: CCTAAGGTTAAGTCGCCCTCG.

GAP43 shRNA: GCTCATAAGGCCGCAACCAAA.

Western blot

The total protein of retinas, cells and sEVs was isolated using radio-immunoprecipitation assay lysis buffer. The protein concentration was determined using the BCA assay kit. Equal amounts of protein samples were separated through 12% SDS-PAGE gel and transferred onto PVDF membranes (Millipore, USA). After blocking in 5% skim milk for 1 h, the membranes were incubated with primary antibodies at 4 °C overnight, treated with HRP-conjugated secondary antibodies at 37 °C for 1 h, and detected by enhanced chemiluminescence. The primary 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 were lysed in Co-IP buffer and incubated with the GAP43 antibody (1:100, 16971-1-AP, Proteintech, China) at 4 °C overnight, followed by the treatment with magnetic beads for 4 h. After washing with Co-IP buffer, protein complexes were detected by western blot.

qRT-PCR

The total RNA of cells was isolated using Trizol reagent (Gibco, USA) and reverse-transcribed to cDNA according to the manufacturer’s protocol of HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China). Based on the ABI Prism 2720 PCR system, qRT-PCR was performed with SYBR Green PCR kit (CWBIO, China). The relative mRNA expression was evaluated using the 2−ΔΔCt method. The sequences of primers were as follows:

β-actin: forward: GACCTGTACGCCAACACAGT.

β-actin: reverse: CTCAGGAGGAGCAATGATCT.

GAP43: forward: GGCCGCAACCAAAATTCAGG.

GAP43: reverse: CGGCAGTAGTGGTGCCTTC.

CCK8 assay

After treatment, cells were resuspended and seeded in 96-well plates (3,000 cells/well). After the incubation for 24, 48, 72 and 96 h, cells were treated with 100 µL of fresh medium containing 10 µL of CCK8 reagent (Vazyme, China) for 3 h. The absorbance of each well was determined at 450 nm using the spectrophotometer (FLX800, BioTek, USA).

Cell apoptosis assay

Annexin V–FITC/PI Apoptosis Detection Kit (Vazyme, China) was used to assess the effect of sEVs on cell apoptosis. After the treatment with MNU and sEVs, 661 W cells were washed with PBS, centrifuged at 800 rpm for 5 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 were then detected by flow cytometry (FACSCalibur, BD, USA). The percentages of early apoptotic (Annexin V+PI−) plus late apoptotic (Annexin V+PI+) cells were considered as apoptotic percentages.

Statistical analysis

All statistical analysis was performed using GraphPad Prism software (GraphPad, San Diego, USA). All data were presented as the means ± SEM. Unpaired Student’s t-test was used for comparison between two groups. The significant differences among multiple groups were assessed by analysis of variance with Newman-Keuls test. P value < 0.05 was considered statistically significant.