Experimental animal source. The study has been approved by the Ethics Committee of Chongqing Medical University. Experimental animals were provided by the Experimental Animal Center of Chongqing Medical University.
Collection of B104 supernatant. The frozen B104 cell strain was rapidly dissolved in a 37 °C water bath, and the dissolved cell suspension was added to a medium containing 12% fetal bovine serum (FBS, Zhejiang Tianhang Biotechnology, 11011 − 8511). Centrifuged at 1000 rpm for 5 min. The cells were then planted in a petri dish containing 12% FBS medium, which was changed every other day until the cells were 70%-80% confluent. The medium was replaced with a medium containing 1% N-2 supplement (Gibco, A1370701), and cells were incubated for 4 days in the incubator at 37 °C. Then, the supernatant was centrifuged, filtered, collected and stored at -80 °C.
Primary culture OPCs. The cerebral cells of 1-3-day-old Sprague-Dawley (SD) rats were cultured, and 12% FBS (Gibco, 10099–141) medium was added. After 4–5 days, the medium was replaced with selective medium which containing 20% B104 supernatant and 1% N-2 supplement. Cells were cultivated for 4–6 days. OPCs were separated by 0.01% EDTA digest which cannot digest ASTs because of its weak digestion, and OPCs were seeded in a new medium.
Primary culture ASTs. The cerebral tissue of newborn SD rats was trypsinized (Gibco, 40127ES60) for 5 min, and the cells were seeded in poly-D-lysine (PDL)-coated dishes and cultured for 5–6 days in a medium containing 12% FBS.
Experimental groups (Fig. 1).
OPCs mono-culture (group O). Primary cultured OPCs were digested with 0.01% EDTA and seeded in petri dishes coated with PDL. A medium containing 1% N-2 and 20% B104 supernatant was added, and cells were incubated for 4–6 days.
OPCs cultured with ASTs secretion (group C). ASTs and OPCs were digested with 0.25% trypsin and 0.01% EDTA, respectively, and ASTs were seed in the upper chamber of the OPCs. A medium containing 1% N-2 and 20% B104 supernatant was added. Cells were incubated for 4–6 days.
ASTs and OPCs co-culture (group A). After ASTs were seed in PDL-coated dishes for 1 day, OPCs were then seed on the cell surface of ASTs. A medium containing 1% N-2 and 20% B104 supernatant was added. Cells were incubated for 4–6 days. Digest with 0.01% EDTA when collecting cells.
siRNA interference Cx47 (group Cx47si and group NCsi1). Interfering agents were added to co-cultured ASTs and OPCs according to the instructions of the siRNA Interference Kit (RiboBio). NC siRNA was a disordered RNA used as a control, and the Cx47 interference sequence was CCGAGAAGACTGTCTTCTT. Digest with 0.01% EDTA when collecting cells.
siRNA interference Cx47 + exosomes ( group Cx47si+)
Purified OPCs in direct contact with ASTs were cultured in OPC proliferation medium for 24 h. Then, the Cx47 siRNA was transfected and about 9 × 1010 particles/ml of exosomes were added for 24 h at the same time.
Added exogenous Chi3l1 (group 5Ch and group 10Ch). In these groups, 5 ng/ml and 10 ng/ml exogenous Chi3l1 (CLOUD-CLONE CORP, APB463Ra01) was added to the OPCs mono-culture.
siRNA interference Myh9 (groups M1, M2 and M3, group NCsi2). Interfering agents were added to OPCs mono-culture according to the instructions of the siRNA Interference Kit (RiboBio). The interference sequence of Myh9 siRNA1 is: GCCTGTTCTGTGTGGTCAT; siRNA2 is: GCATCGAGTGGAACTTCAT; siRNA3 is: GCGTGACTGGTCTCCTTAA.
Pull-down (group goat and group Ch). Cell lysate was added to OPCs to extract total cellular protein. The proteins were mixed with goat IgG (group goat) (Beyotime, A7007), Chi3l1 antibodies (group Ch) (Santa Cruz, sc-393484) and incubated overnight at 4 °C with shaking on a shaker. Then, the agarose beads (Santa Cruz, sc-2344) were added to the protein, incubated overnight at 4 °C, and collected. The loading buffer was added to agarose beads and boiled for 10 min, followed by gel electrophoresis. The gel after electrophoresis was subjected to Coomassie blue staining, then the protein bands obtained after the staining were cut out and subjected to mass spectrometry to identify the protein species.
Co-immunoprecipitation (Co-IP, group goat and group My). The total cellular protein of OPCs was extracted, mixed with goat IgG and Myh9 antibodies (PRB-440P, Biolegend), incubated overnight at 4 °C with shaking on a shaker. Then, the agarose beads were added to the protein, incubated overnight at 4 °C, and collected. The loading buffer was then added to the beads and boiled for 10 min. Next, the final obtained protein was analyzed by western blot.
Flow cytometry. OPCs were digested with 0.01% EDTA and fixed with 75% alcohol overnight at 4 °C. The cell cycle was detected by flow cytometry.
Immunofluorescence. After the cells were washed with PBS, they were fixed with 4% paraformaldehyde for 30 min and then incubated with 0.5% Triton X-100 for 10 min. After a PBS washed, goat serum (Boster, AR0009) was blocked for 1 h. The cells were incubated with the primary antibody (PDGFRα, 1:100, Santa Cruz, sc-338; GFAP, 1:100, Santa Cruz, sc-33673; Cx47, 1:50, Bioworld, BS7447; Chi3l1, 1:50; Myh9, 1:200) overnight at 4 °C, and then with the fluorescent secondary antibody (FITC-labelled goat anti-rabbit IgG (H + L), Beyotime, A0562; Cy3-labelled goat anti-mouse IgG (H + L) Beyotime, A0521) for 2 h in the dark. The cells were subjected to nuclear staining (DAPI, Beyotime, C1002). Fluorescence was measured by fluorescence microscopy, and the fluorescence intensity of the cells was analyzed by ImageJ.
Transmission electron microscopy (TEM). The exosomes of groups were resuspended in PBS and dropped onto the copper mesh grid of electron microscopy in the form of water droplets. The droplets were retained on the copper grid for 1 min and fixed in 2% uranyl acetate for 1–10 min. Then it were dried naturally at room temperature and observed and photographed under 120 kV biotransmission electron microscope.
Nanoparticle tracking analysis (NTA). The exosome samples were appropriately diluted using 1X PBS buffer to measure the particle size and concentration. The exosomes particle size and concentration using NTA at VivaCell Biosceinces with Zeta View PMX 110 (Particle Metrix, Meerbusch, Germany) and corresponding software Zeta View 8.04.02. NTA measurement was recorded and analyzed at 11 positions. The Zeta View system was calibrated using 110 nm polystyrene particles. Temperature was maintained around 23 °C -30 °C.
Western blot. After the total protein of OPCs was extracted, the loading buffer was added and boiled for 10 min. The protein was then subjected to gel electrophoresis, and the strips were cut and electrotransferred. After the strips were blocked with 5% skim milk powder for 2 h, they were incubated with the primary antibody (Cx47, 1:200; Chi3l1, 1:200; Myh9, 1:500, Cyclin D1, 1:500, Bioss, bs-20596R; GAPDH, Bioss, 1:2000) overnight at 4 °C. After incubation with the secondary antibody for 2 h, the machine captured and developed the strip.
EdU. EdU incubation and fluorescent staining were performed according to the protocol for the EdU kit (RiboBio, C10310-1, C10310-3). Fluorescence microscopy was used to detect cellular fluorescence.
Gene Ontology (GO) analysis. GO analysis of differentially expressed genes was performed using DAVID 6.8 (https://david.ncifcrf.gov), |log2 FC|≥1.5, p < 0.05.
Quantification of exosomes. Exosomes of each group were isolated from 50 ml OPC proliferation medium during a 36 h culture period and were suspended in 500 µl PBS. NTA and western blot analysis Alix of exosomal marker protein for exosomes quantification.
Cellular uptake of exosomes. The exosomes were labeled with PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich, USA). The OPC was coincubated with labeled exosomes at 37 °C for 24 h. Images were detected via confocal microscopy after mounting using Antifade Mounting Medium with DAPI.
Statistical analysis. One-way analysis of variance was used in this study. All data were processed using SPSS17.