Human mesenchymal stem/stromal cells suppress spinal inflammation in mice with contribution of pituitary adenylate cyclase-activating polypeptide (PACAP)

Wild-type C57BL/6 mice were purchased from Sankyo Lab Service Corporation (Tokyo, Japan). Adcyap1 ^+/− mice on a C57BL/6 background were originally provided by Dr. Hashimoto of Osaka University [23]. All mice were housed in the specific pathogen-free animal facility at Showa University and had free access to food and water. In all experiments, adult male mice (8 to 12 weeks old, weighing 17 to 25 g) were used. All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of Showa University (#00168 and 01150).

The SCI mouse model was produced according to our previous report [24]. Anesthesia was induced in mice by inhalation of 4.0% sevoflurane and maintained with 3.0% sevoflurane. Under aseptic conditions, an incision was made along the midline of skin of the back, and the muscles, soft tissues, and yellow ligaments overlying the spinal column between T9 and T10 were removed. The intervertebral spinal cord between T9 and T10 was then transected with a thin-bladed razor (FEATHER, Osaka, Japan). After bleeding had stopped and coagulated blood was removed, the incision was closed and the animals were given 1.0-mL lactate Ringer’s solution (s.c., Otsuka, Tokyo, Japan) to avoid dehydration. Following recovery, foods were placed on the cage floor and the intake of the water bottle was lowered to allow for easy access. All mice were allowed to recover in a room maintained at 24°C ± 1°C during the experimental period. To support urination, the region of the lower abdomen in all mice was gently stimulated a few times a day.

Frozen vials of hMSCs from bone marrow were obtained from Dr. Prockop (The Center for the Preparation and Distribution of Adult Stem Cells (http://​medicine.​tamhsc.​edu/​irm/​msc-distribution.​html)) under the auspices of a National Institutes of Health (NIH)/National Center for Research Resources grant (P40 RR 17 447-06). The experiments were performed with hMSCs from donor 281L [11,25]. To expand hMSCs, a frozen vial of 1.0 × 10⁶ passage 3 cells was thawed and plated at 100 cells/cm² in multiple 150-mm plates (Nunclon, Thermo Fisher Scientific, Rochester, NY) with a 20-mL complete culture medium (CCM) that consisted of α-minimal essential medium (α-MEM; Invitrogen, Grand Island, NY), 20% heat-inactivated fetal bovine serum (FBS, Hyclone; Thermo Fisher Scientific), 100 units/mL penicillin, 100 μg/mL streptomycin (Invitrogen), and 2 mM L-glutamine (Invitrogen). The cultures were incubated, and the medium replaced every 3 days for approximately 8 days until cells were 70% to 80% confluent. The medium was then discarded, and the cultures plates were washed with PBS. Adherent cells were harvested with 0.25% trypsin and 1 mM EDTA (Invitrogen) for 5 min at 37°C and were resuspended at 5 × 10⁵ cells in 0.5 μL of sterile Hank’s balanced salt solution (HBSS; Invitrogen) for injection. Unviable hMSCs were prepared by repeated freezing and thawing (three times) of aliquots of these cells.

PKH26-labeled hMSCs were prepared according to instructions provided with the PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich, St Louis, MO). In brief, harvested hMSCs (approximately 1 × 10⁷ cells) were washed with α-MEM and centrifuged at 1,500 rpm for 7 min. The cells were then suspended for 3 min at 25°C in 1.0 mL of Diluent C with 1.0 mL of a PKH26 solution diluted 250-fold in Diluent C. Two mL of FBS was added to the suspension and incubated for 1 min at room temperature. A further 4.0 mL of CCM was added, and the suspension was centrifuged at 1,500 rpm for 6 min. After discarding the supernatant, the cells were washed three times with CCM and resuspended finally with HBSS at 5 × 10⁵ cells/mL. In a preliminary study, we confirmed that the cell suspension showed greater red fluorescence (Ex 544, Em580-10) than naive hMSCs or HBSS (Additional file 2: Figure S2). The red fluorescence of the hMSCs was also confirmed with fluorescence immunocytochemistry with CD59 (BD Bioscience) or HuNu (Chemicon) antibodies (Additional file 2: Figure S2).

The day following surgery to invoke SCI, mice were reanesthetized by inhalation of 4.0% sevoflurane. The animals were placed face-down and a 29G-needle (HAMILTON, Reno, NV) with a 5.0-μL glass syringe (HAMILTON) was inserted directly into the intervertebral spinal cord between T10 and T11. hMSCs (5 × 10⁵ cells/μL) or HBSS were infused at a rate of 0.5 μL/min with an Ultra Micro Pump (World Precision Instruments, Sarasota, FL). After infusion, the needle was left in place for 1 min to enable the solution to diffuse into the tissue. We have shown that the fate of hMSCs was not different between immunocompetent and immunodeficient animals after ischemia [11]. Therefore, the present study did not use any immunosuppressant after cell implantation.

Motor function after SCI was assessed by using an open-field behavior test that focused on hindlimb function according to the Basso Mouse Scale (BMS) [26]. The BMS consists of an open-field locomotor rating scale, ranging from 0 (complete paralysis) to 9 (normal mobility). Briefly, individual mice were placed in the center of the open field (e.g., 50 × 50 cm²) with a smooth, non-slip floor and monitored for 4 min. The hindlimb movements, trunk/tail stability, and forelimb-hindlimb coordination were assessed and graded. Mice were tested daily until post-operative (p.o.) day 7. Mice with peritoneal infection, hindlimb wounds, and/or tail or foot autophagia were excluded from the study. Scoring was done by randomly numbering the mice to ensure that the investigators were not aware of the treatment groups.

After anesthesia with sodium pentobarbital (50 mg/kg, i.p.), the animals were perfused transcardially on p.o. day 7 with 0.9% saline followed by 4% paraformaldehyde (PFA) in 50 mM phosphate buffer (pH 7.2) and the spinal cord removed (T7 to L1 vertebrae). Spinal cords were then post-fixed with 20% sucrose in 0.1 M phosphate buffer (pH 7.2) for two nights, and then embedded in an O.C.T. compound (Sakura Finetech, Tokyo, Japan) for subsequent preparation of frozen blocks. Five spinal cord sections (5-μm thickness) were obtained from each mouse: at the midline which included the central canal nearby to the core-injury site, and bilaterally at 30 μm and 60 μm lateral to the midline (total five sections from each mouse). The damaged area can be identified by glial fibrillary acidic protein (GFAP) immunostaining of the surrounding area, which is considered to be indicative of glial scarring [24]. The frozen sections were washed with phosphate-buffered saline (PBS) and incubated in 0.3% H₂O₂. The sections were blocked with 2.5% normal horse serum (NHS) in PBS for 1 h at room temperature. Subsequently, the sections were incubated overnight with rabbit anti-GFAP antibody (1:10, DAKO, Glostrup, Denmark). The sections were washed with PBS and immersed with goat anti-rabbit IgG (1:200, Santa Cruz, Santa Cruz, CA) for 2 h. They were then incubated in an avidin-biotin complex solution (Vector, Burlingame, CA) followed by diaminobenzidine (DAB; Vector) as a chromogen. Control staining involved carrying out the same steps without the incubation with the primary antibody. The injury area consisting of GFAP-immunopositive cells was measured by DP2-BSW image analysis software (Olympus, Tokyo, Japan), and the estimated injury volume was calculated by integration of the injured areas.

Immediately following, and then at 7 and 14 days after injection of hMSCs, mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and the spinal cord was dissected. The tissue was snap frozen in liquid nitrogen and stored at −80°C until use. Genomic DNA was extracted (n = 4; DNeasy, Qiagen, Valencia, CA) and total DNA was assayed by UV absorbance. Real-time PCR was performed with 100 ng of target DNA, hAlu-specific primers, and a fluorescent probe (Model 7700; Applied Biosystems, Foster City, CA). The primers were as follows: Alu forward, 5′-CAT GGT GAA ACC CCG TCT CTA-3′; Alu reverse, 5′-GCC TCA GCC TCC CGA GTA G-3′; Probe, 5′-FAM-ATT AGC CGG GCG TGG TGG CG-TAMRA-3′. Standard curves were prepared by adding 1 × 10² to 1 × 10⁶ hMSCs to samples of spinal cord from uninjured mice.

Spinal cords (T7 to L1 segment) from immediately after the injection of hMSCs and on p.o. day 7 were obtained and frozen sections prepared as described above. Microscope slides containing attached hMSCs in culture were washed three times with HBSS, fixed with 4% PFA for 15 min, and immersed in PBS containing 0.1% Tween 20 (PBST).

Tissue sections or microscope slides were washed several times with PBST and incubated in 2.5% NHS/PBST for 1 h. Subsequently, the sections were incubated overnight with primary antibodies. The sections were then rinsed with PBST and immersed with appropriate fluorescently labeled secondary antibodies for 2 h. Control staining involved carrying out the same procedures but without the incubation with primary antibodies. The primary antibodies were used as follows: rabbit anti-β2-microglobulin (B2M, 1:1000; LifeSpan Biosciences, Seattle, WA), rabbit anti-PACAP (1:1000; Peninsula Laboratories, San Carlos, CA), and rabbit anti-type1 PACAP receptor (PAC1R, 1:400). The rabbit anti-PAC1R antibody was raised by using the N-terminal residue as an antigen [15,27]. The secondary antibody used was goat anti-rabbit Alexa 488 (1:400; Invitrogen). Some sections were stained with 4,6-Diamidine-2-phenylindole dihydrochloride (DAPI, 1:10,000; Roche, Manheim, Germany) to identify cell nuclei. Fluorescence was detected using an Axio Imager optical sectioning microscope with ApoTome (Carl Zeiss, Inc.; Oberkochen, Germany).

Immediately after injection of hMSCs, or on p.o. days 3 or 7, mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and the spinal cord was dissected (T7 to L1 vertebrae). The excised tissue was snap frozen in liquid nitrogen and stored at −80°C until use. The total RNA was isolated from the cultured hMSCs or the spinal cord samples using the TRIZOL Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. In brief, cultured hMSCs (1 × 10⁶ cells) or spinal cord tissue samples (40 mg) in 1.0 mL of TRIZOL Reagent were homogenized using a Dounce tissue grinder (WHEATON, Millville, NJ). Added to the homogenized samples was 0.2 mL of chloroform per 1 mL of TRIZOL Reagent. Following centrifugation, the aqueous phase (containing RNA) was separated from the mixture. Added to this aqueous phase was 0.5 mL of isopropyl alcohol per 1 mL of TRIZOL Reagent. After centrifugation, RNA precipitate was formed on the bottom of sample tube. The RNA precipitate was washed with 75% ethanol and dried completely at room temperature. The RNA was dissolved in RNase-free water. The purity and concentration of extracted RNA were determined spectrophotometrically (NanoDrop, Wilmington, DE). Extracted RNA was stored at −80°C until use.

cDNA was then synthesized with a TaKaRa PrimeScript RT reagent Kit (TaKaRa BIO Inc., Shiga, Japan), using 3 μg of the total RNA. The synthesized cDNA was made up to a volume of 30 μL with sterile distilled water. Real-time PCR was performed as previously reported by our group [28], with minor modifications. Reverse transcription PCR (RT-PCR) for tumor necrosis factor α-induced protein 6 (TSG-6) was performed on a Taqman system [29], while for the others, SYBR Green was used. All human- or mouse-specific primers and probes were designed as described in Table 1.

Arginase is a marker for AAM, and its activity was measured according to our previous report [24]. Briefly, spinal cord sections containing the T5 and L1 vertebrae from p.o. day 7 animals were removed. The tissues were homogenized with a lysis buffer (10 mM Tris-HCl (pH 7.4), 0.15 M NaCl and 1% Triton X-100, 1 mM ethylene glycol tetraacetic acid (EGTA), 50 mM NaF, 2 mM sodium orthovanadate, 10 mM sodium pyruvate, and protease inhibitor cocktail (Sigma-Aldrich)) and centrifuged at 800 × g for 10 min at 4°C, and the supernatant was collected. Protein concentration in the samples was determined using the BCA protein assay kit (Thermo Fisher Scientific).

The homogenate was mixed with an equal volume of pre-warmed 50 mM Tris-HCl, pH 7.5 containing 10 mM MnCl₂ and incubated for 15 min at 55°C for activation. The mixture was then incubated in 0.25 M L-arginine for 60 min at 37°C to hydrolyze urea from L-arginine, and the reactions were stopped by adding Stop solution (H₂SO₄/H₃PO₄/H₂O, 1:3:7). A 1% (final concentration) solution 1-phenyl-1,2-propanedione-2-oxime (ISPF, Wako, Tokyo, Japan) in ethanol was then added to the solution, which was heated at 100°C for 45 min. The reaction between urea and ISPF produced a pink color, and absorption was measured at 540 nm. Data are presented as specific activity (nmol/min/mg of protein).

hMSCs were plated at 2 × 10⁵ cells/well in 6-well plates. The next day, the cells were washed twice with PBS (−) and incubated in an experimental medium (α-MEM supplemented with 1% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine). Then the cells (n = 3 plates for each phenotype) were exposed to a recombinant mouse IFNγ (10 ng/mL, PeproTech, Rocky Hill, NJ) or vehicle. Forty-eight hours later, the cells were collected and stored at −30°C until analysis.

Human monocyte-like cell line U937 was obtained from the RIKEN Cell Bank (Tsukuba, Japan). For routine maintenance, cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS and 1.0% penicillin/streptomycin in 5% CO₂ at 37°C in a humidified chamber. Cell concentrations were maintained between 2 × 10⁵ and 2 × 10⁶ cells/mL. For differentiation into MΦ, 5 × 10⁵ cells were placed into the wells of a 12-well plate and treated with 20 nM phorbol myristate acetate (PMA, Sigma-Aldrich) for 24 h to induce the MΦ-like adherent phenotype. Subsequently, the medium was replaced with fresh RPMI 1640 medium and cells cultured for a further 48 h. PMA-induced U937 cells were stimulated with 0.1 μg/mL of lipopolysaccharide (LPS, Sigma-Aldrich) for 24 hours as a positive control.

RT-PCR was performed as reported previously [30]. Briefly, the total RNA was extracted from cell pellets (hMSCs, hMSCs stimulated with IFNγ, MΦ-like differentiated U937, MΦ-like differentiated U937 stimulated with LPS) by TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. The purity and concentration of extracted RNA were determined spectrophotometrically (NanoDrop, Wilmington, DE). The cDNA was then synthesized with an AffinifyScript QPCR cDNA Synthesis Kit (Stratagene, Agilent Technologies, La Jolla, CA), using 1 μg of the total RNA. The synthesized cDNA was made up to a volume of 50 μL with sterile water supplied with the kit. The reaction mixture contained 0.6 μL of the first-strand cDNA, 7 pmols of each primer set and 6.0 μL of the Emerald Amp PCR Master Mix (2X premix) (TaKaRa) in a total volume of 12 μL. The primers were as follows: hPACAP forward, 5′-GAAACAAATGGCTGTCAAGAAA-3′; hPACAP reverse, 5′- TCTGTGCATTCTCTAGTGCTTTG-3′; hPACAPR1forward, 5′-GTTACTTCGCTGTGGACTTCAA-3′; hPACAPR1 reverse, 5′-GGACCAGTACCAAAACAAGGAG-3′; hGAPDH forward, 5′-GGTGGTCTCCTCTGACTTCAAC-3′; hGAPDH reverse, 5′-GTCTACATGGCAACTGTGAGGA-3′. Thermal-cycling parameters were set as follows: 97°C for 5 min for an initial denaturation, then a cycling regime of 40 cycles at 95°C for 45 s, 60°C for 45 s, and 72°C for 1 min. At the end of the final cycle, an additional extension step was carried out for 10 min at 72°C. Three microliters of each reaction mixture were loaded for 1.6% agarose gel electrophoresis and bands were visualized with ethidium bromide.

Each mouse was assigned a random number, and all data were collected and analyzed without investigator knowledge of group identities. Data are expressed as mean ± SEM for in vivo experiments and as mean ± SD for in vitro experiments. Statistical comparisons were made by Student’s t-test for two groups and by one-way ANOVA following non-parametric multiple comparison as indicated in each figure legend. A value of P < 0.05 was considered to indicate statistical significance.