Animals
Male Sprague-Dawley rats sacrificed for this study. All experimental procedures were approved by the Animal Ethics Committee of Zhejiang University and followed the National Institutes of Health guidelines strictly. Animals were housed under a 12-hour light/dark cycle with free access to food and water. All efforts were made to minimize the number of animals used and their suffering. The individual mouse was considered the experimental unit within the studies.
Primary BMSCs isolation, culture and characterization
Primary BMSCs were isolated from the femurs of 3-4 week old Sprague-Dawley male rats following our previous study. BMSCs were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) with 10% fetal bovine serum, 100U/ml penicillin and 100U/ml streptomycin, and medium were changed every two days. The fluorescence-activated cell sorting (FACS) analysis was adopted to characterize BMSCs in our previous study [31].
Construction of adenovirus and infection of BMSCs
The CD157 over expression vector was constructed with pHBAD-EF1-MCS-3flag-CMV-GFP vector. The MCS segment was inserted bst-1 gene, and the EF1 promoter regulated the expression of bst-1 gene and the CMV promoter regulated EGFP. Meanwhile, pHBAd-U6-MCS-CMV-GFP was used to construct CD157 interference vector that contained a U6 promoter regulated the expression of shRNA of bst-1 inserted in the EcoR I and BamH I sites and a CMV promoter regulated the expression of GFP gene. A vector only expressing GFP gene was used to be the control. All vectors were synthesized by the Han Bio Co. LTD (Shanghai, China).
The BMSCs were infected at a 50 multiplicity of infection with the adenovirus vectors after the polybrene (5µg/ml) treatment for 30 min. 48 h later, the infected BMSCs were observed with fluorescence microscope (Olympus Corp., Tokyo, Japan) and the Western blotting analysis was applied to detect the expression of CD157.
Oxygen-glucose deprivation (OGD) and re-oxygenation of VSC4.1 motor neurons
The ventral spinal cord 4.1 (VSC4.1) motor neuron cells were cultured in RPMI 1640 medium with 10% (V/V) fetal bovine serum and 100U/m penicillin and streptomycin at 37°C with 5% CO2 in a fully humidified incubator. OGD and re-oxygenation models were used to mimic ischaemia and hypoxia in SCI. In brief, VSC4.1 motor neurons were cultured in D-Hanks' balanced salt solution without glucose in a sealed hypoxic GENbag fitted with a AnaeroPack (MGC,Japan) to scavenge free oxygen and the Non-OGD group was cultured in Hanks’ balanced salt solution containing the normal concentration of glucose with 5% CO2 for 8 hours. Later, all cells were re-oxygenated and cultured in normal complete medium or were co-cultured with BMSCs. All duration were determined by our previous study [32].
SCI model and BMSCs transplantation
24 Sprague-Dawley male rats weighing 200-220g were divided into four groups randomly, by using the standard = RAND() function in Microsoft Excel. SCI was performed with Allen’s method in accordance with our previous study. In brief, all rats were anesthetized with pentobarbital (40 mg/kg, i.p.). Then, their vertebral columns were exposed, and laminectomy were operated at the T10 spinal vertebra. A weight of 10g was dropped from a height of 50mm on the exposed spinal cord. The impounder was left for 20 s to produce a moderate contusion. Immediately, the 10 µl culture medium containing 106 BMSC+MOCK, BMSC+Over or BMSC+shRNA were injected into the epicenter of the injured spinal cord using an electrode microneedle as the SCI +MOCK group, SCI+Over group, and SCI+shRNA group respectively. Meanwhile, the control rats received the sham operation with the same surgical procedure without injury, while the SCI group received the same dose of DMEM.
Collection of BMSCs-conditioned medium (BCM)
BMSCs were planted at 1×105 cells/dish and cultured in DMEM/F12 complete medium. When attached, the cells were washed with phosphate-buffered saline (PBS) for three times and incubated in high glucose Dulbecco's Modified Eagle Medium (DMEM) without serum to stimulate the production of extracellular mitochondria particles. The medium was collected 24 h later. BMSCs-conditioned medium (BCM) was treated by filtering through a 1.2 µm syringe filter or by spinning cell debris down with centrifuging at 2000g for 10 min. Meanwhile, BCM was filtrated through a 0.22 µm syringe filter to prepare mitochondria deleted medium (Md-BCM) that contains no extracellular mitochondria particles.
MitoTracker Red staining
BMSCs were stained with 200 nM MitoTracker Red CMXRos (Molecular Probes, M7512, Invitrogen, USA) for 30 min at 37°C to label the intracellular mitochondria. The cells were washed three times with PBS to exclude the interference of excessive dye.
Carboxyfluorescein succinimidyl ester (CFSE)-fluorescent label
Attached VSC4.1 motor neurons were stained by 10 μM carboxyfluorescein succinimidyl ester (CFSE, #C1031, green colour, Beyotime Institute of Biotechnology, China) for 30 min in 37°C and washed by PBS for three times.
Co-culture of post-OGD VSC4.1 motor neurons with BMSCs
VSC4.1 motor neurons were cultured directly with BMSCs in 10 cm dishes or in the 6-well 8 µm transwell system (Corning, USA) in a 1:1 ration. All co-culture system last for 24 h.
Microscope observation
The observation of extracellular mitochondria particles derived from BMSCs was carried out by transmission electron microscopy in accordance with previous study [33]. VSC4.1 motor neurons were cultured in 1×105 cell/well with round coverslips and fixed with 0.5% paraformaldehyde after co-culturing. 4’,6-diamidino-2-phenylindole (DAPI, blue colour, #C1002, Beyotime Institute of Biotechnology, China) was applied to all cells to label nucleus after fixing. The internalization of extracellular mitochondria (red colour) derived from BMSCs in co-cultured VSC4.1 motor neurons (green colour) was captured by fluorescence microscope. To detect the regeneration of motor neuron axons, optical microscope was used to observe the length and the number of axons.
Western blot analysis
Western blot was performed to determine the transfection efficiency of vectors and the expression level of proteins related to cellular apoptosis and mitochondrial apoptosis. Each sample was detected concentration and loaded onto 4–12% Bis-Tris gels (M00653, GeneScript, China). After the electorophoresis and transferred to PVDF membranes, the membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% skim milk (232100, BD, USA) for 90 min at room temperature. Membranes were washed with TBST and then incubated overnight at 4°C with anti-GAPDH (1:2000, 10494-1-AP, Proteintech, USA) and anti-Bone marrow stromal cell antigen 1 (CD157) antibody (1:1000, ab208442, Abcam, USA). After washed with TBST, membranes were incubated with infrared-labelled peroxidase-conjugated secondary antibodies for 1 h at room temperature. Bands were captured and quantificated by Odyssey CLx Image Studio (Gene Ltd, USA).
Immunofluorescence staining
0.5 % paraformaldehyde fixed cells or frozen spinal cord sections were washed 3 times in 1×PBS and blocked in blocking buffer for 60 min. Then cells or spinal cord sections were incubated overnight at 4°C with primary antibody as following: Rabbit anti-Grp 78 antibody (1:200, ER40402, HuaBio, CN), Rabbit Anti-NF-kB p65 antibody (1:200, ab16502, Abcam, USA), GAP 43 (D9C8) Rabbit mAb (1:200, #8945, CST, USA), Bcl-xL (54H6) Rabbit mAb (1:200, #2764, CST, USA), AKT (phospho Thr308) antibody (1:50, om238718, Omnimabs, USA). After washing by PBS 3 times, fluorescent secondary antibodies incubated for 2 hours at room temperature. Finally, DAPI was added to visualize the nucleus, and coverslips were placed
Potential of mitochondrion measurement
The mitochondrial membrane potential (MMP) assay kit with JC-1 (C2006, Beyotime Institute of Biotechnology , China) was used to assess mitochondrial membrane potential to detect whether the extracellular mitochondrion still had function. BCM and Md-BCM were collected and mixed with JC1 (5 μM) for 30 min at 37 °C. When the membrane potential of mitochondria completely lost, green fluorescence (Ex 485 nm Em 516 nm) would be observed and the normal cells stained should show red fluorescence (Ex 579 nm/Em 599 nm). MMP was determined by the Varioskan Flash microplate reader.
ATP measurement
ATP level was determined by CellTiter-Glo luminescence (G7570, Promega, USA). For intracellular ATP content, cells were incubated by 200 µl reagent buffer each well, standing for 30 min at room temperature for lysing cells. For extracellular ATP, the culture medium was collected and centrifuged at 2,000 g for 10 min. Then the supernatant was collected and centrifuged at 20,000 g for 20 min at 4 °C, remaining the lower half to use. CellTiter-Glo luminescence test solution (50 μl) was added into culture media (50 μl) and incubated for 30 min at room temperature in opaque-walled 96-well plates. Luminescent signal was determined by the microplate reader.
FACS analysis on the content of extracellular mitochondria particles
FACS analysis were performed by BD Fortessa or CytoFLEX LX. BCM were prepared and collected as described as before. 200 nm and 300 nm diameter calibration Particles, DMEM media incubating unstained BMSCs and DMEM media were used to control for determining appropriate gates, voltages, and compensations required in multivariate flow cytometry.
Determination of CD157/ADPR-cyclase activity
ADPR cyclase activity was determined with nicotinamide guanine dinucleotide (NGD+) (N5131, Sigma, USA) as the substrate as described before [34]. Briefly, BMSCs were cultured in 96-well plate with 200 µl complete DMEM/F12 medium. After attaching, BMSCs were incubated with 500 μM NGD+ in 0.1 M PBS (pH 7.2) at 37 °C for 10 min, and the production was determined at excitation/emission wavelengths of Ex 290 nm/Em 410 nm with the microplate reader.
Ca2+ signal detection
To explore whether the CD157/cADPR mechanism was Ca2+ dependent, the level of Ca2+ in BMSCs was determined by Fluo-4 AM (S1060, Beyotime Institute of Biotechnology, China). BMSCs were incubated in 96-well plates with 2 µM Fluo-4 AM for 30 min at room temperature. After washing with PBS for three times, fluorescence intensity was determined by the Varioskan Flash microplate reader at excitation/emission wavelengths of Ex 488 nm/Em 516 nm.
Assessment of motor function
Basso, Beattie, and Bresnahan (BBB) locomotor scales were used to assess the recovery of rats’ motor function. The scores were obtained by two independent examiners who were blind to the four groups. All rats were observed and assessed the relevant indicators of hind limb motor function and physical control function in an open field for 3 min at 1, 7, 14, 21 and 28 days post-surgery.
Statistical analysis
All data were expressed as mean ± SEM. Statistical analysis was analyzed by GraphPad Prism 6 software. Unpaired t-test was used for the analysis of two groups. Multiple comparisons were evaluated by one-way ANOVA or two-way ANOVA. P<0.05 was considered to be statistically significant.