2.1 Cell culture
A human iPSC line purchased from the Cell Bank of the Chinese Academy of Sciences was cultured in mTESR1 medium (STEMCELL Technologies). The iMSCs were transduced as previously described[16]. Briefly, mTeSR1 medium was replaced with Dulbecco’s Modified Eagle Medium (DMEM, Hyclone) supplemented with 10% foetal bovine serum (FBS; Sigma-Aldrich), 1% penicillin/streptomycin, 2 mM L-glutamine, and 0.1 mM non-essential amino acids (Gibco). These cells were continuously passaged in the MSC medium until they developed a homogeneous fibroblastic morphology. Human dermal fibroblasts (FBs) purchased from the Cell Bank of the Chinese Academy of Sciences were cultured in high-glucose DMEM (Hyclone) with 10% FBS (Sigma-Aldrich) and 1% penicillin/streptomycin. Human NPCs were isolated from normal NP tissue derived from lumbar trauma patients who underwent spinal fusion with no degenerative signal on magnetic resonance imaging (MRI). The method of isolation has been previously described[17]. NPCs were cultured in DMEM/F-12 medium (Hyclone) with 10% FBS (Gibco) and 1% penicillin/streptomycin. These cells were incubated at 37 °C in a humidified atmosphere of 5% CO2.
2.2 Characterization of iMSCs and sEVs
Surface antigens of iMSCs were detected using flow cytometry analysis. Cells were collected and incubated with 3% bovine serum albumin (Sigma-Aldrich) for 30 min to block non-specific antigen binding. The cell suspensions were incubated with antibodies for iMSC-specific surface markers that included CD29, CD34, CD44, CD45, CD73, or HLA-DR (all from BD Biosciences) at 4 °C for 30 min. The Guava easyCyte™ flow cytometer (Millipore) was used to analyze surface antigens.
iMSC-sEVs or FB-sEVs were isolated from iMSC or FB culture supernatants as previously described[18]. Briefly, after reaching 80% confluency, iMSCs and FBs were washed with PBS. The iMSCs were cultured with serum-free MSC medium (StemRD), and FBs were cultured with high-glucose containing extracellular vesicle-depleted FBS (10%) for 48 h at 37 °C in an atmosphere of 5% CO2. The medium were collected and centrifuged at 300 g for 10 min at 4 °C to remove remaining cells, followed by 2000 g for 10 min at 4 °C to remove dead cells, followed by 10000 g for 30 min at 4 °C. The supernatant was ultra-centrifuged at 100000 g for 70 min at 4 °C. This step was repeated once. The sEVs were resuspended in PBS for use in the experiments. The morphology of sEVs was observed by transmission electron microscopy (TEM) by using a model H-7650 device (Hitachi) operating at an accelerating voltage of 80.0 kV. The concentration and size distribution of the sEVs were measured by nanoparticle analysis (NTA) by using a ZetaView PMX 110 (Particle Metrix). Expressions of characteristic markers of sEVs (CD9, CD63, Tsg101, GM130, and Actin) were tested by western blotting.
2.3 Establishment of IVDD model and treatment in rats
Twelve-week-old Sprague–Dawley rats were used. All experimental procedures were approved by the Animal Research Committee of the First Affiliated Hospital of the University of Science and Technology of China. The rat model of IVDD was established as previously described[19]. Briefly, the experimental level rat tail disc (Co5/6) was punctured using a 20-gauge needle (IVDD group). The puncture was made through the center of the disc to the opposite side, and the needle was rotated 180° and held for 10 s. Rats that were not treated were the negative control. One week after the initial surgery, 2 uL of sterile saline containing 1 × 1010 iMSC-sEVs/mL was injected into the punctured discs by using a 33-gauge needle, in the IVDD group. The negative group received an injection of FB-sEVs. The injections were repeated every 2 weeks. At week 8, MRI was performed on all rats, and the rats were euthanized for further analysis.
2.4 MRI
After 8 weeks of the puncture procedure, all rats were examined by MRI examination to evaluate the degenerative changes in sagittal T2-weighted images by using a 3.0 T clinical magnet (Siemens). T2-weighted sections in the median sagittal plane were obtained using the following settings: fast-spin echo sequence with a time to repetition of 5400 ms and time to echo of 920 ms; 320 (h) 256 (v) matrix; field of view of 260; and four excitations. The section was 2 mm thick with a 0-mm gap. The degree of IVDD in the MR images was evaluated by the Pfirrmann grading system[20].
2.5 Histological analysis
Rats were sacrificed and tail samples were fixed in 4% paraformaldehyde, decalcified in 10% EDTA, dehydrated in a gradient alcohol, and embedded in paraffin. The specimens were cut into 5 um-thick sections. Hematoxylin-eosin (H&E) staining was performed to observe IVDD. Briefly, all sections were deparaffinized, rehydrated, and stained in hematoxylin solution for 5 min. After differentiation in 1% acid alcohol for 30 s, the sections were stained in eosin solution for 30 s to 1 min. Sections were observed by optical microscopy. Immunofluorescence (IF) staining was used to detect age-related P16 protein. The sections were deparaffinized, rehydrated, antigen retrieved, and blocked and incubated with primary antibody against P16 (1:200; Invitrogen) overnight at 4 °C. The sections were incubated with Alexa Fluor 594-conjugated secondary antibody (1:400) for 1 h, and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).
2.6 Senescence-associated 𝛽 -galactosidase (SA-𝛽-gal) staining
SA-𝛽-Gal activity of NPCs was detected using a cellular senescence staining kit (Beyotime Biotechnology) according to the manufacturer’s instructions. Senescent cells were identified as blue-stained cells by phase-contrast microscopy. The proportion of positive cells was determined by counting the number of blue cells and dividing by the number of cells observed.
2.7 Proliferation assay
Cell proliferation was measured using a cell counting kit-8 (CCK-8, Dojindo Molecular Technologies). NPCs from different treatment groups were seeded into 96-well plates at a density of 3000 cells per well. Ten microliters of CCK8-solution was added to 100 uL of medium and incubated for 2 h at 37 °C. The absorbance at 450 nm was measured using a model 680 microplate reader (Bio-Rad).
2.8 Western blotting analysis
Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The resolved proteins were transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 for 2 h at room temperature. The membranes were then incubated with primary antibodies against the following proteins: P16 (1:1000; Abcam), collagen II (1:1000; Abcam), aggrecan (1:1000; Abcam), matrix metalloproteinase 3 (MMP3, 1:1000; Abcam), A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4, 1:1000; Abcam), cAMP-specific 3',5'-cyclic phosphodiesterase 4D (PDE4D, 1:1000; Abcam), Sirt6 (1:1000; Abcam), and Actin (1:3000; Abcam) at 4 °C overnight. Subsequently, membranes were incubated with peroxidase-conjugated anti-rabbit IgG (1:3000; Abcam) or anti-mouse IgG (1:3000; Abcam) for 1 h at room temperature. Finally, the proteins were visualized by ECL (Thermo Fisher Scientific).
2.9 qRT-PCR analysis
Extraction of sEV RNA was performed using Exoquick (QIAGEN). qRT-PCR was performed using the QuantiTect® SYBR Green PCR Master Mix. The default PCR setting was 40 cycles of 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 30 s. Specific amplicons were identified by melting curve analysis.
2.10 Uptake of iMSC-sEVs and FB-sEVs
The uptake of sEVs by NPCs was observed using green fluorescent dye (DiO; Life Technologies) to label iMSCs and FBs according to the manufacturer’s instructions. The sEVs released by the labeled iMSCs or FBs were also labeled with DiO. The NPCs were cultured with the conditioned medium containing DiO-labeled sEVs, for 12 h.
2.11 Transport of miRNAs inhibitors into iMSC-sEVs
The miR-105-5p inhibitor and control miRNA inhibitor were purchased from QIAGEN. The miR-105-5p inhibitor and control miRNA inhibitor were transferred to iMSC-sEVs by using the Exo-Fect siRNA/miRNA Transfection Reagent Kit (SBI) according to the manufacturer’s instructions.
2.12 Statistical analysis
The data were presented as means ± standard deviation. Statistical significance (P values) was determined by One-way analysis of variance (ANOVA) or Student’s t-test. Statistical significance was determined to be P < 0.05.