NOD/SCID mice were purchased from Nihon CLEA (Tokyo, Japan) and were housed at the National Center of Neurology and Psychiatry (Tokyo, Japan). All experiments using mice were performed in accordance with the guidelines approved by the Nippon Medical School and National Center of Neurology and Psychiatry (NCNP) Animal Ethics Committees. Beagle dogs and CXMDJ colony dogs were maintained according to the NCNP standard protocol for animal care. Experiments were performed in accordance with the guidelines approved by the Ethics Committee for the Treatment of Laboratory Animals at NCNP.
MSCs derived from rat bone marrow were isolated and expanded as previously described . For the experiments on dogs, healthy donor Beagle dogs were anesthetized using thiopental and isoflurane, and 1.0 mL of bone marrow fluid was collected. The CD271+ MSCs were enriched and cultivated using the MSC Research Tool Box-CD271 (LNGFR) containing CD271 (LNGFR)-PE and anti-PE microbeads for cell separation (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), as previously reported . Human DPSCs were provided by JCR Pharmaceuticals (Hyogo, Japan). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% antibiotic-antimycotic solution (FUJIFILM Wako Pure Chemical Industries, Osaka, Japan) at 37 °C in a 5% CO2 atmosphere.
Cell culture and gene transduction
To generate luciferase-expressing MSCs, MSCs isolated from Sprague-Dawley rat bone-marrow were transduced with vesicular stomatitis virus-glycoprotein (VSV-G)-pseudotyped retroviral vector encoding firefly luciferase . Canine CD271+ MSCs were transduced with a luciferase-expressing retroviral vector, followed by transduction with enhanced green fluorescent protein (eGFP) or MyoD-expressing adenoviral vector (Ad C2-eGFP or Ad C2-MyoD), as reported previously . To assess the long-term effects of IL-10 expression, MSCs or DPSCs were transduced with AAV1/eGFP or control AAV1/IL-10 vectors developed according to methods described previously [29, 30]. All cells were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). For transplantation, the cells were washed with PBS to completely remove the culture medium containing vectors.
Transplantation of MSCs into mice
Luciferase-expressing rat MSCs (Luc-MSCs, 5.0−10.0 × 106 cells) were injected intramuscularly into the right or left hind-limb muscle of NOD/SCID mice pretreated with cardiotoxin (10 µM, Merck KGaA, Darmstadt, Germany) 1 day before the treatment. AAV1/IL-10- or eGFP-vector-transduced Luc-MSCs (1.0 × 107 cells, Figure 1) were intramuscularly injected into the right (eGFP-MSCs) and left (IL-10-MSCs) hind-limb muscles of NOD/SCID mice.
In vivo imaging analysis
After the injection of luciferase expressing rat-MSCs on day 0 of the experiment, in vivo luminescence images were acquired periodically to assess the engraftment efficiency and cell survival in the transplanted mice. Prior to imaging, the mice were anesthetized by inhalation of 2.0% isoflurane and oxygen and injected intraperitoneally with 150 mg luciferin (Summit Pharmaceuticals International Corp., Tokyo, Japan.) per kg body weight. In vivo images were acquired using the IVIS charge-coupled-device camera system (Xenogen Corp., Alameda, CA, USA) at multiple time points (0, 3, 7, 18, 27, 31, 34, 42, 49, 54, and 67 days after transplantation). The region of interest (ROI) luminescence signals from individual MSC-injected sites were measured using the Living Image® 3.2 software package (Xenogen Corp.).
Transplantation into dogs
AAV1/IL-10-transduced Luc-CD271+ MSCs derived from a healthy dog (22.214.171.124 × 107 cells/2 mL) were injected into the muscles of healthy Beagle dogs. Five days before the treatment, muscle degeneration and regeneration cycles were induced in the tibialis anterior (TA) muscles by injecting 10 nmol/kg cardiotoxin (C9759, Sigma-Aldrich) under anesthesia. For analgesia treatment, 0.02 mg/kg of buprenorphine hydrochloride (Lepetan, Otsuka Pharmaceutical, Tokyo, Japan) was injected intramuscularly before the effect of general anesthesia wore off. On days 0 and 50 of the experiment, the MSCs were injected into pretreated muscles without using immunosuppressants. The injected muscles were then biopsied at 4 weeks after the treatment, or the animals were sacrificed at 8 weeks after transplantation. The dogs underwent periodic veterinary examinations during the experiments.
hDPSCs or AAV1/IL-10-transduced hDPSCs (4.0 × 106 cells/mL/kg body weight at a rate of 1 mL/min) were administered via intravenous injection into CXMDJ that were pretreated with polaramine (chlorpheniramine maleate, 0.15 mg/kg) using nine injections at 2-week intervals (Table 1). After each injection, the activity, heart rate, respiratory rate, and signs of abnormalities were carefully monitored. Weight measurement and blood tests were performed weekly to examine the side effects of repeated cell treatment.
For biopsy and necropsy, the individual muscles were sampled for tendon-to-tendon dissection, divided into several fragments, and immediately frozen in liquid nitrogen-cooled isopentane for histological analysis. Whole muscle tissue homogenates were prepared using a POLYTRON homogenizer (150–180 min-1) and Multi-Beads Shocker (Yasui Kikai Corp. Osaka, Japan).
The dogs underwent periodic veterinary examinations at 1-2 week intervals until sampling. Hematological and serum biochemical testing for creatine kinase (CK) was performed using a model F-820 semi-automated hematology analyzer (Sysmex, Hyogo, Japan). The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) were determined using a DRI-CHEM3506 automated analyzer (Fuji Film, Tokyo, Japan).
Histopathological and immunohistochemical analyses
Samples from MSC-treated TA muscles were collected and immediately frozen in liquid nitrogen-cooled isopentane. Five mice from each group were used for analysis at each time point. Transverse cryosections 8 μm in thickness prepared from the skeletal muscles were stained with H&E using standard procedures. For immunohistochemical analyses, thick cryosections were fixed in acetone for 5 min at –20 °C. The tissue sections were then blocked using 0.5% bovine serum albumin (BSA) in PBS. The following antibodies were used for antigen detection at 1:40–1:50 dilutions: rabbit anti-firefly luciferase (ab21176; Abcam Plc., Cambridge, UK) and mouse anti-dystrophin (NCL-DYS3, Leica, Wetzlar, Germany). These antibodies were diluted using 0.5% BSA in PBS and used to treat the cells or tissue sections overnight at 4 °C. The tissue sections were washed with PBS and then probed with Alexa 568-conjugated anti-rabbit IgG antibodies (Thermo Fisher Scientific) and Alexa 488-conjugated anti-mouse IgG antibodies (Thermo Fisher Scientific) at 1:250–1:100 dilution for 1 h at 4 °C. The coverslip slides were washed with PBS and mounted in Vectashield (Vector Laboratories Inc., Burlingame, CA, USA) with 4,6 -diamidino-2-phenylindole (DAPI). Immunofluorescence analysis was performed using an IX71 fluorescence microscope (Olympus, Tokyo, Japan).
To confirm the presence of transplanted cells at the injection sites, the MSCs were labeled with luciferase or eGFP. The tissue sections were incubated in a solution of 3% H2O2 to block endogenous peroxidase. The nonspecific binding sites were blocked using a 2% BSA solution. The tissue sections were probed with primary antibodies for 1 h and then treated using the 3,3'-diaminobenzidine (DAB) substrate kit (Vector Laboratories Inc.) containing horseradish peroxidase (HRP) as an enzyme indicator. The slices were then stained with DAB chromogen to determine the form of the brown-antigen reaction product. The tissue sections were visualized using an IX71 microscope (Olympus).
The IL-10 expression levels were measured in the FBS-free MSC culture medium after 2 days of incubation, in the TA muscle lysate, and in the serum obtained from animals using the Quantikine ELISA mouse or canine IL-10 Immunoassay (Thermo Fisher Scientific) and canine IL-6 (R&D Systems Inc.) and the collagen type III Immunoassay (Cloud-clone Corp. TX, USA), according to the manufacturers’ recommendations. The final values were normalized to the protein concentrations, which were measured using the Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific).
Luciferase reporter assays
Luciferase reporter assays were performed to evaluate the retention of Luc-MSCs in the TA muscle. Firefly luciferase activity was tested in whole tissue homogenates using the Bright-GloTM Luciferase Assay System (Promega Corporation, Madison, WI) according to the manufacturer’s instructions. Luciferase levels were measured using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). Protein concentrations were measured using a Pierce® BCA Protein Assay Kit (Thermo Scientific Pierce, Rockford, IL, USA). Three independent experiments were performed in duplicate.
Biodistribution of MSCs
The tissue samples were disrupted in a Multi-Beads Shocker (Yasui Kikai Co., Ltd., Osaka, Japan). DNA was extracted from the tissue suspensions using a DNeasy Blood and Tissue kit (QIAGEN, Valencia, CA) and quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific). Real-time qPCR was performed using 125 ng of DNA in a total volume (20 µL) containing DNA Master SYBR Green I kit (Roche Diagnostics, Basel, Switzerland) and primers for Alu or murine glyceraldehyde 3-phosphate dehydrogenase (Gapdh). The primer sequences used were as follows: human Alu, 5’-GTCAGGAGATCGAGACCATCCC-3’ (forward) and 5’-TCCTGCCTCAGCCTCCCAAG-3’ (reverse); murine Gapdh, 5’-GATGACATCAAGAAGGTGGTGA-3’ (forward) and 5’-TGCTGTAGCCGTATTCATTGTC-3’ (reverse). The PCR conditions were as follows: 95 °C for 2 min, followed by 40 cycles at 95 °C for 15 s, 68 °C for 30 s, and 72 °C for 30 s. The standard was generated by adding 10-fold serial dilutions of human DPSCs to determine the number of human DPSCs in 125 ng of DNA that was used in the real-time PCR for each organ sample. We extrapolated the quantity of DNA isolated from each organ to determine the number of human DPSCs per organ.
Proteome cytokine/cytokine array
The FBS-free DPSC culture medium was collected after 2 days of incubation for array analysis. The relative expression of cytokines and chemokines in the culture medium was quantified using the Proteome ProfilerTM Array (Mouse Cytokine Array, Panel A; R&D Systems Inc.), as previously described . To achieve maximum assay sensitivity, the blots were incubated overnight with plasma. Enhanced chemiluminescence incubation was performed for 5 min using the Super Signal West Femto Chemiluminescence Kit (Thermo Scientific Pierce), and the samples were imaged and analyzed using the Image Quant LAS 4000 coupled with Image Quant TL software (GE Healthcare Japan, Tokyo, Japan) and Image J software (NIH, Bethesda, MD).
Locomotor activity analyses
The physical activity levels of CXMDJ and littermate normal dogs used as controls were monitored during the experimental period using an infrared sensor system (Supermex, Muromachi Kikai Co., Ltd., Tokyo, Japan), as previously described . These systems monitor and enumerate all spontaneous movements. The average of all counts of spontaneous locomotor activity in animals determined over 5 days and nights (12 h light/dark cycles) was calculated. Furthermore, we measured the 15-m running time of normal and CXMDJ littermates during the experimental period. The running speed was averaged four times.
Magnetic resonance imaging (MRI)
CXMDJ anesthetized by injection (20 mg/kg) were intubated using an endotracheal tube, and general anesthetization was maintained using an inhalational mixture of 2% to 3% isofluorane and oxygen. The heart rate and oxygen saturation levels were monitored continuously. Images of the T2-weighted and fat-saturated T2-weighted series were captured using a method described in a previous study . We examined the crus muscles of the lower limbs using a superconducting 3.0-Tesla MRI device (MAGNETOM Trio; Siemens Medical Solutions, Erlanger, Germany) with an 18-cm diameter/18-cm length human extremity coil. The images were analyzed quantitatively using the Syngo MR2004A software (Siemens Medical Solutions), as previously reported [32, 33]. Briefly, the ROIs were selected to avoid flow artifacts and large vessels, and the signal intensities were measured for these ROIs. The SNRs for each ROI were calculated using the following equation: SNR = signal intensity/SDair, where SDair is the standard deviation (SD) of the background noise. The average SNR (Ave SNR) was calculated using the equation described in a previous report . The right and left TA muscles, EDL, gastrocnemius medial head, GL, flexor digitorum superficialis, flexor digitorum longus, and flexor hallucis longus muscle were examined.
Data are presented as the mean ± SD. Differences between two groups were assessed using unpaired two-tailed t-tests. Multiple comparisons between three or more groups were performed using ANOVA (n = 3–6). Statistical significance was defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, and was calculated using Microsoft Excel (Microsoft, Redmond, WA, USA) and GraphPad Prism 8 (GraphPad, La Jolla, CA, USA).