Isolation and Identification of USC-Exo
Human urine stem cells-exosome (USC-Exo) were isolatedusing ultracentrifugation as previously described.Briefly, after human USCwere obtained, it was washed with PBS and cultured with exosome-free FBSUSC medium at 37 °C and 5 % CO2. The conditioned medium was collected and centrifuged at 2000 ×g for 30 min.After centrifugation, the supernatant was filtered using a 0.22 µm filter (Millipore, Billerica, USA) to remove the cellular debris. Then, the supernatant was added to an Ultra-clear tube (Millipore, USA) and were ultra-centrifuged at 100,000 × g twice, each for 2h, to precipitate exosome pellets. The USC-Exo pellets were resuspended in 200 μL of PBS and was stored in a - 80 °C refrigerator to be used in the following experiments. USC-Exo fraction was examined and photographed by a transmission electron microscope,TEM (Hitachi H-7650, Hitachi, Japan). The size distribution and concentration of USC-Exo were determined by Nanoparticle Tracking Analysis (NTA) using the Nanosight NS3000 system (Nanosight, Amesbury, United Kingdom) according to the manufacturer’s instruction,USC-Exo protein contents were determined using the BCA™ Protein Assay Kit (Thermo Fisher, USA). The specific exosome surface makers CD63, CD81, and TSG101 were identified by Western blot (WB).
ANGPTL3 deletion using shRNA plasmid
The transfection was performed using Lipofectamine LTX with Plus reagents (Invitrogen, Carlsbad, CA, USA). We established stable ANGPTL3 knockdown USC using ANGPTL3 shRNA (shANGPTL3) and the control shRNA (shMock) vectors, respectively (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer's protocol.
USC-Exo internalization assays
USC-Exo were collected and labeled using PKH26red Fluorescent Cell Linker Kit (Sigma Aldrich; Saint Louis, MO, USA) according to the manufacturer's instruction.USC-Exo pellets were resuspended in 1ml of Diluent C. 1μl PKH26 dye was diluted in 250 μl of Diluent C and co-incubated with USC-Exo then gently mixed for 4 min. An equal volume of 1% BSA was added into the reaction system to stopped the reaction. Then the PKH26 labeled exosomes were ultracentrifuge at 100,000 ×g for 70 minutes, washed with PBS, and ultracentrifuge again to remove excess dye.HUVECs were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).The PKH26-labeled USC-Exowere incubated with cultured HUVEC cells and stain with 4′,6-diamidino-2-phenylindole (DAPI) (Vectashield, Vector Laboratories, Burlingame, CA, USA) to visualize nuclear structures and further analysis by a fluorescence microscope (BIOREVO BZ7000; Keyence; Osaka, Japan).
Tube Formation Assay
To investigate the effect of USC-Exo on the activity of HUVEC, the formation of capillary-like structures assay was performed as previously reported. 50 μl Cold growth factor-reduced Matrigel (BD Biosciences) was added onto a 96-well plate. HUVECs (2 × 104 cells/well) were seeded on top of Matrigel and treated with medium in the presence or absence of USC-Exoin different conditions:1)treated with PBS group; 2) treated with USC-Exo50 μg/ml from shMock transfected USC groups); 3) treated with USC-Exo50 μg/ml from ANGPTL3 silenced human USC.After 12 hours coculture, the tube formation was captured under the bright-field microscope (BZ-8000, Keyence, Osaka, Japan). The ability to form capillary-like structures was quantified by the number of branch points and tubule lengths in five randomly chosen microscopic fields using ImageJ software.
Cignal Finder reporter array
To determine what signaling pathways were affected by USC-Exo treatment in HUVEC, a Cignal Finder Reporter Array plate (Qiagen, US) was performed according to the manufacturer’s instructions. Briefly, attractene(0.6 µl /well) was distributed into a 96-well Cignal Finder Multi-Pathway Reporter Array plate. HUVECs were seeded in each well and incubated to allow complex formation. Then, the cell treated with 200μg exosomes derived from USC.After 48 h of treatment, Dual-Glo Luciferase Reagent (75 µl) was then added to designated wells and luciferase activities were measured according to the manufacturer’s recommendations. The luminescence signal was quantified by normalizing the ratios of cellular responses to USC-Exo treatment (firefly luminescence signal) to the nonspecific responses to PBS (Renilla luminescence signal). The USC-Exotreated cells were compared with the PBS-treated control cells, and the results were plotted as log2-fold change. If log2>1, was considered significant difference. To investigate the PI3K/Akt signaling involved in the USC-Exomediated effects on HUVECs, the specific inhibitors of the PI3K/AKT signaling (LY294002 and L-NAME) on tube formation were tested.
Western blotting analysis
Protein extracts from the celllysates or exosomeby RIPA buffer (R0278, Sigma). The protein concentration was determined by Bradford microassay (Bio-Rad Laboratories, Hercules, CA)and separated by SDS-PAGE gel and transferred to iBlot nitrocellulose membranes (Invitrogen, Carlsbad, CA, USA). Membranes were blotted with primary antibodies toCD63 (Santa Cruz Biotechnology), CD81 (Santa Cruz Biotechnology), TSG101 (ProteinTech, Chicago, USA), ANGPTL3, GAPDH (Abcam,Cambridge, UK), PI3K, Akt, and phosphorylated PI3K(p-PI3K), Akt (p-Akt) (Cell Signal Technology).After washing with TBST, the membranes were incubated with appropriate HRP-conjugated secondary antibodies (Amersham Pharmacia). The immunoreactive bands were detected using enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, USA) and imaged by the ChemiDoc XRS Plus luminescent image analyser (Bio-Rad).
RNA isolation and quantitative reverse transcriptase–PCR
Total RNA was extracted from the cultured cells and tissues using TRIzol Reagent (Invitrogen, Carlsbad, USA) and reverse transcribed into cDNA with a Revert Aid First Strand cDNA Synthesis kit (Fermentas, Burlington, Canada). qRT-PCR was performed using FastStart Universal SYBR Premix ExTaq (Takara Biotechnology, Japan) and analyzed on ABI VII7 Real-Time RT-PCR system (Bio-Rad Laboratories Inc., Hercules, CA, USA). Primer sequences used for qRT-PCR were listed in Supplementary Table1.The fold changes of target mRNA expression relative to GAPDHwere calculated based on the threshold cycle (CT) as r = 2−Δ(ΔCT), where ΔCT = CT (target) − CT(GAPDH) and Δ(ΔCT) = ΔCT (experimental) − ΔCT (control).
Mouse model of spinal cord contusion trauma and USC-Exo treatment
Animal experiment procedures were conductedaccording to the Guideline of Animal Care and Use Committee of Central South University (Permit numbers: 20170101).Mice received spinal contusion trauma as previously described. Briefly, after the mice were fully anaesthetized with 1 mg kg −1 ketamine intramuscularly and anesthetized with 1.5–2.5% isoflurane. A vertebral laminectomy at thoracic T9-10 was performed to expose the spinal cord. A clinically relevant spinal cord contusion trauma was performed using Horizons Impactor (Precision Scientific) (60 kdyn force) according previously described.Mice were placed in a temperature and humidity-controlled chamber. Manual bladder emptying was performed three times daily until reflex bladder emptying was established. The SCI mice were randomly divided into three groups(n = 8/group), and respectively treated with PBS containing PKH26, 200 μL, 200μg exosomes labeled with PKH26 from shMock transfected human USCin 200 μL of PBS, 200μg exosomelabeled with PKH26 from ANGPTL3 silenced USCin 200 μL of PBS all embedding with hydrogel respectivelypost-SCI atlocal intrathecal injection. The functional recovery of spinal cord injury was measured at different time-point through locomotor behavioral, electro-physiological, morphological and immunofluorescence and 3D vessel visualization.
In vivo imaging of DiR labeled USC-Exo in spinal cord injury models
To track the USC-Exo in vivo, we labeled USC-Exo with 1 μM fluorescent lipophilic tracer DiR (Invitrogen, Life Technologies) according to previous described, and embedded with hydrogel. Then transplanted DiR-labeled USC-Exo with hydrogel complex onto the surface of injured spinal cord area. Animals were euthanized and placed in the Xenogen IVIS Imaging System (Caliper Life Sciences) to detect red fluorescence for biodistribution analysis.
Locomotor functional recovery Assessment
Mouse locomotor function was evaluated at 1,3,7,14,21,28,56days post SCI by two examiners who were blinded to the experimental design according to the Basso Mouse Scale (BMS) system.The BMS scores is a 10‐point locomotor rating scale, range from 0 (complete hindlimb paralysis) to 9 (normal locomotion) points. Briefly, Mice were allowed to walk in an open field, and the examiners took notes on the ankle movement, plantar placement, weight support, stepping, coordination, paw position and trunk stability. The mean score of both examiners for the two hind limbs was used as the BMS score of each mice.
HE staining and cavity analysis
The mice were sacrificed at 28days after SCI (n = 8/group), the injured spinal cords were carefully removed and fixed in 4 % paraformaldehyde, then embedded in paraffin after dehydration.Eight transverse sections (5-μm thickness) at 100μm from the injury epicenter of the spinal cord were produced and stained with HEstaining to assess the tissue morphology and determine the cavity site. The identified lesion areas in each sectionwere measured using ImageJ software (National Institutes of Health).
Motor evoked potentials (MEPs) recoding
The MEPSs of hindlimb were assessed by elec-tromyography at 8weeks post-surgery as previously described. After effective anesthesia, the stimulating electrodes secured onto the surface of the skull corresponding to the motor cortex area, and recording electrodes were inserted into the tibialis anterior muscle in contralateral hindlimb. The reference electrodes were insert into subcutaneous tissue between the stimulating and recording electrodes for the record in the target muscles.Mean MEPs values including latencyand amplitude period were record. The latency period was measured as the length of time from the stimulus to the onset of the first response wave. The amplitude period was measured from the initiation point of the first response wave to its peak point.
For immunofluorescence staining, the sections were rehydrated and blocked with 5%BSA then incubated with primary antibodies anti-ki67 (Abcam) or anti-CD31 (Abcam).Then sections were incubated with the respective Alexa Fluor 488 goat anti-mouse IgG(H + L) antibody/Alexa Fluor 594 horse anti-rabbit secondary antibody. A DAPI solution was applied for nuclear staining. Images were examined under fluorescence microscope (Leica). Vessel density and ki67/CD31 double positive cells were quantified from fives random selected visual fields per section using ImageJ software (National Institutes of Health).
3D vessel analysis using synchrotron radiation micro-CT
The effects of USC-Exo on the angiogenesis of rats was assessed by novelsynchrotron radiation micro-CT(SRμCT) at the BL13W beamline in the Shanghai Synchrotron Radiation Facility (SSRF). The imaging for SRμCT scanning has been setup according to previously described. Briefly, animals at 28 days post-SCI were anesthetized. Heparinized saline was perfused into the circulatory system, allowing an effective drain of blood flow, followed by 10% buffered formalin perfusion for vessel fixation. The mixed contrast agent Microfil (Flow Tech, CA) was infused into the spinal cord microvasculature system via a perfusion pump according previous described. A5-mm-longspinalcordsegment at the T10 thoracic cord including the injury site was harvested and prepared for SRμCT scanning. After image acquisition, the Image J 3D Skeletonization plugin was used to extract the vascular 3D skeleton. The morphological parameters of the vascularnetwork, including VesselVolumeFraction (VVF),VesselSegmentNumbers(VSN) and Vessel Bifurcation Numbers (VBN), could be calculated by the Image Pro Analyzer 3D software (Version 7.0; Media Cybernetics, Rockville, MD, USA). according to previously reported.
Results were statistically analyzed with the use of the SPSS 22.0. software (SPSS, Inc.). All data were expressed as mean ± standard deviation (SD). Statistical analysis of multiple-group comparisons was performed by one-way analysis of variance (ANOVA), followed by the Bonferroni post hoc test. Values of P less than 0.05 were considered statistically significant.