Enhanced cell survival and therapeutic benefits of IL-10-expressing multipotent mesenchymal stromal cells for muscular dystrophy

doi:10.21203/rs.3.rs-73149/v2

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Enhanced cell survival and therapeutic benefits of IL-10-expressing multipotent mesenchymal stromal cells for muscular dystrophy

https://doi.org/10.21203/rs.3.rs-73149/v2

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published 04 Feb, 2021

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Background:

Multipotent mesenchymal stromal cells (MSCs) are potentially therapeutic for muscle disease because they can accumulate at the sites of injury and are immunosuppressive. MSCs are attractive candidates for cell-based strategies that target diseases with chronic inflammation, such as Duchenne muscular disease (DMD). We focused on the IL-10 based anti-inflammatory properties and hypothesized that IL-10 could increase the typically low survival of MSCs by exerting a paracrine effect after transplantation.

Methods:

We developed a continuous IL-10 expression system of MSCs using an adeno-associated virus (AAV) vector. To investigate the potential benefits of AAV/IL-10 vector-transduced MSCs (IL-10-MSCs), we examined the cell survival rates in the skeletal muscles after intramuscular injection into mice and dogs. The systemic treatment of IL-10-MSCs derived from dental pulp (DPSCs) was comprehensively analyzed using the canine X-linked muscular dystrophy model in Japan (CXMD_J), which has a severe phenotype similar to DMD patients.

Results:

In vivo bioluminescence imaging analysis revealed higher retention of IL-10-MSCs injected into the hind-limb muscle of mice. In the muscles of dogs, myofiber-like tissue was formed after the stable engraftment of IL-10-MSCs. Repeated systemic administration of IL-10-DPSCs into CXMD_J model resulted in long-term engraftment of cells, slightly increased serum levels of IL-10. IL-10-hDPSCs showed remarkably reduced expression of pro-inflammatory MCP-1, and upregulated stromal-derived factor-1 (SDF-1). In fact, MRI and histopathology of the hDPSC-treated CXMD_J indicated the regulation of inflammation in the muscles, but not myogenic differentiation from treated cells. hDPSC-treated CXMD_Jshowed improved running capability, and recovery in tetanic force with concomitantincrease in physical activity. Serum creatine kinase levels, which increased immediately after exercise, were suppressed in the IL-10-hDPSC-treated CXMD_J.

Conclusions:

In case of local injection, IL-10-MSCs could maintain the long-term engraftment status and facilitate associated tissue repair. In case of repeated systemic administration, IL-10-MSCs facilitated the long-term retention of the cells in the skeletal muscle and also protected muscles with physical damage-induced injury, which improved muscle dysfunction in DMD. We can conclude that the local and systemic administration of IL-10-producing MSCs offers potential benefits for DMD therapy, through the beneficial IL-10 paracrine effects involving SDF-1.

Stem Cell & Developmental Cell Biology

Mesenchymal stromal cells

IL-10

DMD

Multipotent mesenchymal stromal cells (MSCs) derived from the bone marrow are conventionally termed adherent non-hematopoietic cells. The cells express several cell-surface antigenic markers, including CD44, CD73, CD90, and CD105 [1]. MSCs can self-renew and differentiate into several different cell types. These include cells of mesodermal origin, such as osteoblasts, chondrocytes, adipocytes, and myocytes [2-4], as well as cells of non-mesodermal origin, such as hepatocytes [5], neural cells [6], and epithelial cells [7].

The multi-lineage potential of MSCs has been exploited for prospective use in therapies for various diseases. The cells can be easily expanded in culture and are non-tumorigenic. Furthermore, the use of MSCs as third-party materials in cell therapy reflects that MSCs are immune-privileged, unlike other stem cells or induced pluripotent stem cells (iPS), as they do not express human leukocyte antigen (HLA) class II, CD40, CD80, or CD86 molecules [8], and express only low levels of HLA class I. These cells are not lysed by natural killer cells or cytotoxic T lymphocytes [9]. MSCs can influence immune effector cell development, maturation, and function as well as reactive T-cell responses through the production of bioactive cytokines and proteins [10, 11]. The mechanism underlying the immunosuppressive effects of MSCs is unclear. Nonetheless, their immunosuppressive properties have been exploited in clinical applications. MSCs are commercially authorized for the treatment of acute graft-versus-host disease (GVHD). MSCs are attractive candidates for cell-based strategies that target diseases with chronic inflammation, such as Duchenne muscular dystrophy (DMD) [11].

Interleukin-10 (IL-10) is an anti-inflammatory cytokine with anti-apoptotic properties [12] that modulates the inflammatory immune response. IL-10 reduces M1 macrophage activation and inhibits the production of pro-inflammatory cytokines such as interferon-gamma (IFN-g), tumor necrosis factor-alpha (TNF-a), IL-1b, and IL-6 in inflamed tissues [13, 14]. IL-10 also reduces the expression of CD54, CD80, CD86, and major histocompatibility complex class II molecules, resulting in incomplete T-cell signaling and the induction of antigen-specific antibody. We hypothesized that IL-10 could increase the typically low survival of MSCs by exerting a paracrine effect after transplantation. IL-10-expressing MSCs were previously developed using retroviral [15, 16], lentiviral vectors [17][18], and transcription activator-like effector nuclease (TALEN)-mediated gene editing [19]. They have therapeutic benefits in collagen-induced inflammatory arthritis [20], as well as preventing lung ischemia-reperfusion injury [15], endotoxin-induced acute injury [21], GVHD [16], traumatic brain injury [17], and left ventricular remodeling after myocardial infarction [19].

We developed a continuous IL-10 expression system of MSCs using an adeno-associated virus (AAV) vector. AAV vectors are safe and do not integrate into the host cell genome, and the risk of insertional mutagenesis is low. To investigate the potential benefits of using MSCs for treating muscle disease, we examined whether the MSCs transduced with the IL-10-expressing AAV vector enhances the survival rates of themselves in skeletal muscles, and the potential advantages offered by MSC transplantation. The systemic treatment using MSCs derived from dental pulp (dental pulp stem cells, DPSCs) was comprehensively analyzed using the canine X-linked muscular dystrophy model in Japan (CXMD_J), which has a severe phenotype similar to DMD in humans [22, 23]. DPSCs are similar to bone marrow MSCs, which showed high expression of the surface markers CD29, CD73, CD90, and CD105, as common stem cell markers in MSCs, but not CD34 or CD45. We previously reported DPSCs also highly expressed IL-10 and vascular endothelial growth factor (VEGF) and had immunosuppressive activities [24, 25]. We evaluated whether DPSCs expressing IL-10 play an important role as a cell source for DMD therapy.

Animals

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 CXMD_J 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.

Cell preparation

MSCs derived from rat bone marrow were isolated and expanded as previously described [26]. 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 Micro Beads for cell separation (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), as previously reported [27]. 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) 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% CO₂atmosphere.

Cell culture and gene transduction

To generate luciferase-expressing MSCs, the MSCs isolated from Sprague-Dawley rat bone-marrow[26] were transduced with vesicular stomatitis virus-glycoprotein (VSV-G)-pseudotyped retroviral vector encoding firefly luciferase [28]. 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 we previously reported [27]. 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 the cells were maintained in DMEM supplemented with 10% FBS as well as 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO). For preparation of transplantation, cells were washed with PBS to remove the culture medium containing vectors, completely.

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 side hind-limb muscle of NOD/SCID mice. AAV1/IL-10- or eGFP-vector-transduced Luc-MSCs (1.0 × 10⁷ cells, Figure 1) were intramuscularly injected into the right side (eGFP-MSCs) and the left side (IL-10-MSCs) hind-limb muscle 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% isofluorane 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) 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 calculated using the Living Image^® 3.2 software package (Xenogen Corp.).

Transplantation into dogs

IL-10-transduced Luc-CD271⁺MSCs derived from healthy dog (2.4–2.7 × 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, St. Louis, MO) under the maintenance of anesthesia. For analgesia treatment, 0.02 mg/kg of buprenorphine hydrochloride (Lepetan, Otsuka Pharmaceutical, Tokyo, Japan) was injected intramuscularly before the dogs awoke from general anesthesia. On day 0 and day 50 of the experiment, 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 IL-10-transduced hDPSCs (4.0 × 10⁶ cells/mL/kg body weight at a rate of 1 mL/min) were given via intravenous injection into CXMD_J that were pretreated with polaramine (chlorpheniramine maleate, 0.15 mg/kg) by nine injections at 2-week intervals (Table I). 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).

Blood test

The dogs underwent periodic veterinary examinations at 1–2 week intervals until sampling. Hematological and serum biochemical testing for 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 8μm 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 with 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 incubated with 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 coverslips slides were washed with PBS and mounted in Vectashield (Vector Laboratories Inc., Burlingame, CA) with 4′,6′ -diamidino-2-phenylindole (DAPI). Immunofluorescence 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% H₂O₂to block endogenous peroxidase. The nonspecific binding sites were blocked with 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 subjected to DAB chromogen staining to determine the form of the brown-antigen reaction product. The tissue sections were visualized using an IX71 microscope (Olympus).

ELISA

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 collagen type III immunoassay (Cloud-clone corp.), according to the manufacturers’ recommendations. The final values were normalized to the protein concentrations, which were determined 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-Glo^TM Luciferase Assay System (Promega Corporation, Madison, WI) according to the manufacturer’s instructions. Luciferase levels were measured on 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). The experiments were performed in duplicates in three independent experiments.

Biodistribution of MSCs

The tissue samples were disrupted in a Multi-Beads Shocker (Yasui Kikai Co., Ltd., Osaka, Japan). DNA was extracted from tissue suspensions using a DNeasy Blood and Tissue kit (QIAGEN, Valencia, CA) and quantified with a NanoDrop spectrophotometer (Thermos Fisher Scientific). Real-time qPCR was performed using 125 ng of DNA in a total volume (20ml) of 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); for murine Gapdh, 5′-GATGACATCAAGAAGGTGGTGA-3′ (forward) and 5′-TGCTGTAGCCGTATTCATTGTC-3′ (reverse). PCR conditions were as follows: 95°C for 2 min, followed by 40 cycles at 95°C for 15 s and 68°C for 30 s, and at 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 reaction 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 Profiler^TM Array (Mouse Cytokine Array, Panel A; R&D Systems Inc.), as previously described [28]. 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

Physical activity levels of CXMD_Jand 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 [31]. 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. Further, we measured the 15-m running time of normal and CXMD_Jlittermates during the experimental period. The running speed was averaged four times.

Magnetic resonance imaging (MRI)

CXMD_Janesthetized 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. Heart rate and oxygen saturation were monitored continuously. Images of the T2-weighted and fat-saturated T2-weighted series were captured using the same method as described in a previous study [32]. 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 our previous report [33]. The analysis was performed on the right and left side TA muscle, EDL, gastrocnemius medial head, GL, flexor digitorum superficialis, flexor digitorum longus, and flexor hallucis longus muscle.

Hind-limb extensor strength test

The functional status of the two hind limbs in the CXMD_J was evaluated by measuring the flexion and extension strengths of the wrist using a customized torque measurement device. Stimulation frequencies from 60 Hz can activate muscles that extend or push the hind paw against the ground. A transducer captures the torque generated when the paw pushes against the force plate. The maximal torque was expressed as a percentage of predicted values computed using a model based on control values with respect to the animal weight. P < 0.05 was considered statistically significant [34].

Statistical analyses

Data are presented as mean ± SD. Differences between the two groups were assessed using unpaired two-tailed t-tests. Multiple comparisons between three or more groups were performed using one-way ANOVA (n = 3–6). Statistical significance was defined by ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, and ^****P < 0.0001, and was calculated using Excel (Microsoft, Redmond, WA) and GraphPad Prism 8 (GraphPad, La Jolla, CA).

Enhanced engraftment of IL-10-expressing MSCs

In vivo bioluminescence imaging analysis revealed a 1.7-fold higher retention of IL-10-expressing MSCs injected into the hind-limb muscle of NOD/SCID mice compared to the retention of MSCs alone 4 days after injection (Figures S1A, B). When luciferase (Luc)-MSCs were injected together with AAV1/IL-10 or AAV/Lac Z control vectors, which were not transduced into the cells, there was no significant difference in cell survival of the Luc-MSCs-treated mice (Figures S1C, D). IL-10 plasmid-transfected Luc-MSCs expressed higher levels of IL-10 and were more effective at enhancing post-transplantation retention. We also confirmed a maximum 5.03-fold stronger signal intensity of luciferase in muscles transplanted with IL-10-expressing MSCs compared to the control MSC-treated muscles at day 7 (See also Figure S2), although most of these cells disappeared within 12 days. We developed a continuous IL-10 expression system of MSCs using an AAV vector to verify the expected high and prolonged cell survival rates following transplantation. IL-10-Luc-MSCs displayed a higher cell survival rate immediately after administration (luciferase signal at 3 days after transplantation, 4.41 ± 1.78 × 10⁵counts) with a maximum of 6.5-fold at 24 days (3.46 ± 1.12 × 10⁴counts) compared to the signal of GFP-Luc-MSCs (2.98 ± 1.04 × 10⁵counts, p < 0.0001; 5.35 ± 2.93 × 10³counts, p = 0.002, respectively), as observed in vivo in imaging analysis (Figure 1A, B, Figure S3). Significantly higher levels of IL-10 expression from AAV1/IL-10-transduced Luc-MSCs (IL-10-Luc-MSCs) were confirmed in vitro (Figure 1C) and in treated muscles (See also Figure S4). The findings suggested that the higher retention in the early stage had a significant effect on long-term engraftment. Notably, IL-10-Luc-MSCs were retained more than 67 days after transplantation (Figure 1A, B).

Successful long-term engraftment of IL-10-MSCs in injured muscle tissue

We also investigated the effects of IL-10 overexpression in CD271⁺MSCs derived from dog bone-marrow, using Beagle dogs as a larger animal model (Figure 2A, Table I). CD271⁺MSCs transduced with AAV1/IL-10 (IL-10-Luc-CD271⁺MSCs) showed overexpression of IL-10 in the culture medium (MyoD-MSCs, 3.1 pg/mL; IL-10-MSCs, 93.6 pg/mL). Four weeks after intramuscular injection, accumulation of IL-10-Luc-CD271⁺MSCs was observed in immunochemical analysis of the cardiotoxin-injured tibialis anterior (TA) muscle. Immunofluorescence analysis revealed the accumulation of luciferase-positive IL-10-Luc-CD271⁺MSCs around the inflammatory region in MSC-treated TA muscle 4 weeks after the secondary injection (Figure 2B). Luciferase-positive muscle-like tissue was detected in the IL-10-Luc-CD271⁺MSC-treated muscle, similar to that observed in the muscle after treatment with Luc-CD271⁺ MSCs transduced with MyoD (MyoD-Luc-CD271⁺MSC), which is a key factor for myogenic determination, as described in our previous report [27]. These findings suggest that the Luc-CD271⁺MSCs transduced with AAV1/IL-10 formed myofibers. Luciferase activity, which was correlated to the number of MSCs, was twice as high in IL-10-Luc-CD271⁺MSC-treated TA muscle compared to that in MyoD-Luc-CD271⁺MSC-treated muscle (Figure 2C). The IL-10 levels in IL-10-Luc-CD271⁺MSC-treated TA muscles increased, while those in MyoD- Luc-CD271⁺MSC-treated muscles did not (Figure 2D). These data suggest that IL-10-expressing CD271⁺MSCs could survive long-term and engraft after intramuscular injection during muscle regeneration.

Safety and efficacy of systemic transplantation of IL-10-DPSCs in the DMD model

Next, we evaluated the efficacy of IL-10-expressing MSCs by systemic transplantation using hDPSCs (Figure 3A). We confirmed that extracellular secretion of IL-10 from AAV1/IL-10-transduced-human DPSCs (IL-10-hDPSCs) remarkably reduced the expression of pro-inflammatory monocyte chemotactic protein-1 (MCP-1), and conversely upregulated stromal-derived factor-1 (SDF-1) in IL-10-hDPSCs compared with the levels in hDPSCs (Figures 3B, C). IL-10-hDPSCs or hDPSCs were intravenously injected nine times at biweekly intervals in the acute phase in the CXMD_J model with the DMD phenotype (Table I). No obvious abnormalities related to liver damage, kidney damage, or anemia were induced in response to the systemic administration in the hDPSC-treated CXMD_J. Transient increases in ALT, AST, and BUN levels was observed occasionally in the CXMD_Jmodel, independent of treatment with hDPSCs (See also Figure S5). During the experiment, the IL-10-hDPSC-treated CXMD_J showed better growth compared to the untreated littermates, CXMD_J (p < 0.0001) and the hDPSC-treated CXMD_J (< 0.0001), in terms of body weight (Figure 3D). Although the serum levels of IL-10 were increased transiently 6 h after IL-10-hDPSC injection (104.0 pg/mL vs. control DMD, vs. hDPSC-DMD, p < 0.0001), the levels reduced rapidly within 24 h of transplantation (32.6 pg/mL vs. control DMD, p < 0.0067; vs. hDPSC-DMD, p < 0.0278), and did not differ significantly from the control CXMD_J7 days after injection (21.6 pg/mL) (Figure 3E). The levels of IL-6 in blood increased transiently in CXMD_J(maximum 742.9 pg/mL). In contrast, the levels in IL-10-hDPSC-treated CXMD_J were within the normal range (0–9.85 pg/mL) during the experiments. High levels of IFN-g were also observed in CXMD_J (maximum 103.9 pg/mL) and hDPSC-treated CXMD_J(114.8 pg/mL), whereas they tended to be marginally lower in the IL-10-hDPSC-treated CXMD_J(68.4 pg/mL) compared to that in other groups. Cell retention in blood was similar to that of hDPSCs (17.9 ± 19.5 pg/100 ng genomic DNA) and IL-10-hDPSCs 24 h after transplantation (79.4 ± 7.89 pg/100 ng genomic DNA), as revealed by human-specific Alu-PCR. The long-term engraftment in tissue was investigated. No hDPSCs were detected in the skeletal muscle, lung, or liver tissues of the hDPSC-treated CXMD_J. IL-10-hDPSCs could only survive and maintain the engraftment status in the TA muscle (56.1 pg/100 ng genomic DNA) 4 months after treatment, and were not detected in other organs. On the other hand, no dystrophin expression was detectable in the muscle tissue of the hDPSC-treated CXMD_J (Figure S6).

Morphological improvement in IL-10-hDPSC-treated DMD dog

The high-intensity T2-signals in MRI, which were detected in the necrotic/edematous and inflammatory lesions in the dystrophic muscle, were significantly reduced in the cross-sectional muscles of the IL-10-hDPSC-treated dog (82.5 ± 16.9 average signal-to-noise ratios, SNRs) compared to the signals in hDPSC-treated CXMD_J(97.4 ± 13.3, p = 0.008) after transplantation (Figure 4A, B). The weight of each muscle obtained from IL-10-hDPSC-treated CXMD_J (3.73 ± 1.43 g) was improved compared to untreated (2.16 ± 1.04 g, vs. IL-10-hDPSC- CXMD_J, p = 0.0014) and hDPSC-treated CXMD_J(2.33 ± 1.04 g, p = 0.0048) (Figure 4C). We also confirmed that the muscle fiber size in the extensor digitorum longus (EDL) muscle from IL-10-hDPSC-treated CXMD_J (935.7 ± 601.9 mm₂) was increased compared with untreated (537.6 ± 439.2 mm₂, vs. IL-10-hDPSC- CXMD_J, p < 0.0001) and hDPSC-treated CXMD_J(629.3 ± 352.8 mm₂, p < 0.0001) (Figure 4D). The gastrocnemius lateral (GL), EDL, and flexor digitorum superficialis (FDS) muscles from CXMD_J displayed smaller (regenerating fibers) and larger (hypertrophic fibers) fiber diameter in the dystrophic muscles, spread muscle interstitium, and cell infiltration interspersed in the muscle interstitium (Figure 4E). In contrast, the histopathological observations of the hDPSC- and IL-10-hDPSC-treated CXMD_J muscles revealed significantly limited infiltration of nuclei, which indicated a milder phenotype compared to untreated CXMD_J. These data suggest that the repeated systemic administration of IL-10-hDPSCs produces morphological improvement, including regulated inflammation in CXMD_J. In addition, we performed quantitative analysis of collagen-3 expression at 8 muscle tissue sites to investigate fibrosis. However, we could not confirm a significant difference between groups (control-DMD, 2.27 ± 2.18 pg/mg protein; hDPSC- and IL-10-hDPSC-DMD, 3.91 ± 1.66, and 6.17 ± 2.50 pg/mg protein, respectively, p = 0.317).

Long-term maintenance of muscle function in IL-10-hDPSC-treated DMD dog

CXMD_J developed progressive general weakness owing to the reduced strength in the skeletal muscles. The tetanic force on hind limbs in CXMD_J (2.55 ± 0.42 N·m/s) was 41.2 ± 5.1% of that in normal dogs (p < 0.0001) (Figure S7). Conversely, significantly stronger torque values were observed in the IL-10-hDPSC-treated CXMD_J (4.17 ± 1.28 N·m/s, 66.5 ± 12.2% of the value in the normal dog, p < 0.0001) than in CXMD_J (p < 0.0008), similar to that in the hDPSC-treated CXMD_J (3.68 ± 0.57 N·m/s). These results suggest that the progressive loss of limb muscle strength is ameliorated upon treatment with hDPSCs and IL-10-hDPSCs.

Additionally, the physical activity of CXMD_J in the home cage also underwent a dramatic reduction with age compared to that in normal dogs (Figure 5A) [31]. Improved activity was confirmed in both groups, the hDPSC-treated (8340.4 ± 1090.3 counts; vs. control DMD, p = 0.0006) and IL-10-hDPSC-treated CXMD_J (8531.6 ± 1146.5 counts; vs. control DMD, p < 0.0001), which was observed upon the comparison of one-year-old CXMD_J littermates with advanced symptoms (3954.4 ± 792.0 counts) (Figure 5A). The IL-10-hDPSC-treated CXMD_J exhibited persistent and predominantly higher activity (6 months; 13008.8 ± 1367.1 counts) than CXMD_J (9926.0 ± 1436.8 counts, p = 0.0268) as well as hDPSC-treated CXMD_J (12605.8 ± 1756.3 counts). Furthermore, hDPSC- or IL-10-hDPSC-treated CXMD_J maintained their 15-m running speed, and were active at 3 to 12 months of age (Figure 5B, see also Figure S8, and Table S1; normal vs. control DMD, p < 0.0001−0.0062; control DMD vs. hDPSC-DMD, p = 0.0025−0.0104; control DMD vs. IL-10-hDPSC-DMD, p = 0.0012−0.0113). There was no significant difference in the running speed between hDPSC-, and IL-10-hDPSC-treated CXMD_J. However, the increased serum creatine kinase (CK) levels after running exercise (50595 ± 67255 unit/L) were recovered immediately until 20 min in IL-10-hDPSC-treated CXMD_J(Figure 5C, 16490 ± 4850 unit/L; vs. hDPSC-treated DMD, p = 0.0134). Conversely, a persistent and significant increase in the serum CK levels was observed in hDPSC-treated CXMD_J (0 min, 96075 ± 24311 unit/L; 20 min, 95300 ± 16835 unit/L) as well as in the untreated CXMD_J (0 min, 76650 ± 46995 unit/L; 20 min, 81425 ± 47458 unit/L, p = 0.9277) after exercise. The CXMD_J also showed higher concentration of lactic acid before and after exercise compared to normal dogs. However, a significant change in the levels of lactic acid was not observed in hDPSC-treated CXMD_J (Figure 5D). These findings suggest that IL-10-hDPSCs exert a protective effect against dystrophic damage caused by exercise.

Overall, we observed that IL-10-expressing hDPSCs were able to ameliorate the dystrophic phenotype upon systemic repeated administration to dogs with DMD by the immunomodulation of hDPSCs.

To improve the functionality of MSCs as a cell source, we focused on the overexpression of IL-10 based on its anti-apoptotic and anti-inflammatory properties. In this study, bone marrow MSCs derived from rat and CD271⁺MSCs isolated from healthy dogs were injected intramuscularly into NOD/SCID mice, or healthy Beagle dogs to study the cell survival and engraftment in the injured muscle (Figure 1 and 2). hDPSCs show very similar cellular properties to bone-marrow MSCs. Human-origin DPSCs or IL-10-transduced hDPSCs were also given via intravenous injection into CXMD_J to investigate their systemic therapeutic effects (Figure 3-5). Our results indicate an improvement in survival rate, engraftment, and protective effects in the muscle tissues by different types of MSCs. This property is considered to be highly common to MSCs.

The higher survival rate of IL-10-MSCs in the early stage immediately after transplantation, as seen in in vivo imaging analysis, is thought to allow long-term cell retention (Figure 1), suggesting stable IL-10 expression enabled the long-term survival and engraftment of MSCs after transplantation. Our findings suggest that during skeletal muscle regeneration, prolonged engraftment of IL-10-expressing CD271⁺MSCs eased the formation of new myofiber-like tissue and the preservation of a functional contractile apparatus, following exposure to the muscle stem cell niche/microenvironment (Figure 2). Rarely, MSCs differentiate into cells of myogenic lineage in the absence of triggers, such as MyoD [35], 5-azacytidine [36], and Notch I intracellular domain [4]. Although we couldn’t confirm myogenic differentiation of MSCs themselves in this study, IL-10 is also thought to play a role in the long-term engraftment and survival of MSCs in muscle tissue, resulting from association with surrounding myogenic stem/progenitor cell populations, such as satellite cells, skeletal muscle-MSCs, and fibro-adipogenic progenitors (FAB) for muscle repair. Furthermore, myocardial infarction with CD271⁺bone-marrow MSCs lowered expression of inflammatory cytokines and significantly increased pro-angiogenic VEGF [37]. CD271⁺ MSCs expressing IL-10 are also thought to enhance pro-angiogenic differentiation in injured tissue as a result of prolonged survival.

It was reasoned that IL-10-expressing hDPSCs had enhanced anti-inflammatory and protective effects on damaged tissue, because of the downregulation of MCP-1, and up-regulation of SDF-1 in IL-10-hDPSCs (Figure 3). SDF-1 is crucial factor that supports tissue regeneration, and the roles of SDF-1 in MSC paracrine-mediated tissue repair have been reported [38]. Furthermore, it was also reported that multiple pro-angiogenic factors such as SDF-1, FGF-2, IGF-1, and VEGF-A are enhanced in IL-10-MSC-treated cardiac-muscle [19], suggesting changes in the surrounding microenvironment.

In the dog model, repeated systemic transplantation of hDPSCs and IL-10-expressing hDPSCs was safe and effective as DMD therapy, as indicated by blood tests, growth, spontaneous activity, and running function (Figures 3 and 4). Long-term engraftment of hDPSCs was only confirmed in the dystrophic muscles in IL-10-DPSC-treated DMD, which suggests that the engraftment of hDPSCs was enhanced through IL-10 paracrine effects. These facts provide evidence of the accumulation of hDPSCs at the site of inflammation after the systemic administration, similar to MSCs. The functional recovery in the dystrophic skeletal muscle was attributed to the alleviation of the morphological pathologies, such as MRI findings and the histopathological appearance in the hDPSC-, and IL-10-hDPSC-treated CXMD_J, but did not prevent fibrosis (Figure 4). Indeed, both hDPSC-, and IL-10-hDPSC-treated CXMD_J showed improved limb strength as evidenced by the tetanic force, revealed an improvement in the spontaneous activity and running speed, while no significant difference was observed in the treated CXMD_J on maintaining apparent function. We observed that the IL-10-hDPSC-treated CXMD_J was more stable than the hDPSC-treated CXMD_J, because the increase in serum CK levels after exercise was rapidly stabilized in the IL-10-hDPSC-treated CXMD_J (Figure 5C). These facts suggest that treatment with IL-10-hDPSCs protects from physical damage-induced muscle injury in CXMD_J, as opposed to the effect observed in dogs injected with untreated or non-transgenic cells, which is further evidenced by the effects of the modified characteristics of IL-10-hDPSCs involved in SDF-1 and VEGF. However, it was not clear whether the protective effects of IL-10-hDPSCs on the dystrophic muscle were caused by IL-10 directly or indirectly. The molecular mechanisms of IL-10-hDPSCs are expected to be elucidated by further study.

This is the first report of increased cell survival, engraftment, and possible tissue formation of MSCs in muscle tissue by secreting IL-10. In our previous report, myogenic lineage-MSCs were successfully engrafted in muscle tissue [27]. However, a more efficient transplantation strategy is required for functional improvement of muscle dystrophy. This study evaluates the possibility of improving survival, engraftment, and immune-modulation of MSCs by AAV vector-mediated stable expression of IL-10.

We previously provided evidence that severe phenotypes in IL-10 knockout mdx mice, such as increased M1-macrophage infiltration, high inflammatory factor levels, and progressive cardiorespiratory dysfunction, show a predisposition toward inflammation [39]. Glucocorticoids are widely used in patients to interrupt and improve muscle strength during early stages, which may also act directly on muscle fibers by stabilizing the sarcolemma [40, 41], but this is frequently associated with severe side effects. In our strategy, MSCs appeared to show inflammatory regulation effects and a protective effect in the dystrophic muscle through the suppression of M1-macrophage infiltration by secreting IL-10. In addition, our stable IL-10 expression system is safe, with a low risk of genome insertion owing to the use of an AAV vector. Random integration, off-target effects, and poor specificity are associated with the use of other viruses and techniques of genome engineering. IL-10-expressing MSCs are expected to have potential applicability in muscle regeneration and treatment of muscle disease. We previously reported that the overexpression of IL-10 promotes neuroprotection in an experimental acute ischemic stroke model [42]. There is clinical interest in the applicability of IL-10-MSCs in ex vivo cell therapy owing to their anti-inflammatory properties and ability to release cytokines into the surrounding environment, which mediates the paracrine effects and thereby modifies the developmental fate of neighboring cells. We expect that the multiple characteristics and various regenerative effects of MSCs alone as well as in combination with CD271⁺ IL-10 MSCs will result in an improved therapeutic impact on DMD.

Our methods were developed to enhance MSCs survival and improve their therapeutic effects using the anti-inflammatory properties of IL-10 for DMD. In the case of local injection, IL-10-expressing MSCs could maintain long-term engraftment status and facilitate associated tissue repair. In the case of repeated systemic administration, IL-10-MSCs also protected muscles with physical damage-induced injury, which improved muscle dysfunction in DMD. We can conclude that the local and systemic administration of IL-10-expressing MSCs may exert beneficial IL-10 paracrine effects, which has potential value in DMD therapeutics.

AAV: Adeno-associated virus

ALT: Alanine aminotransferase

BSA: Bovine serum albumin

BUN: Blood urea nitrogen

CD: Cluster of differentiation

CK: Creatine kinase

CXMD_J: Canine X-linked muscular dystrophy model in Japan (

DAB: 3,3'-diaminobenzidine

DAPI: 4′,6′-diamidino-2-phenylindole

DMD: Duchenne muscular disease

DMEM: Dulbecco's Modified Eagle Medium

DPSCs: Derived from dental pulp

eGFP: Enhanced green fluorescent protein

EDL: Extensor digitorum longus

FAB: Fibro-adipogenic progenitors

FBS: Fetal bovine serum

FDS: Flexor digitorum superficialis

GAPDH: Glyceraldehyde 3-phosphate dehydrogenase

GL: Gastrocnemius lateral

GVHD: Graft-versus-host disease

HLA: Human leukocyte antigen

HRP: Horseradish peroxidase

IFN-g: Interferon-gamma

IL-10: Interleukin-10

iPS: Induced pluripotent stem cells

MCP-1: Monocyte chemotactic protein-1

MSCs: Multipotent mesenchymal stromal cells

MRI: Magnetic resonance imaging

PBS: Phosphate-buffered saline

PCR: Polymerase chain reaction

SD: Standard deviation

SDF-1: Stromal-derived factor-1

SNRs: Signal-to-noise ratios,

TA: Tibialis anterior

TALEN: Transcription activator-like effector nuclease

TNF-a: Tumor necrosis factor-alpha

VEGF: Vascular endothelial growth factor

VSV-G: Vesicular stomatitis virus-glycoprotein

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

The authors express their gratitude to Akihiko Umezawa, Akinori Nakamura, Tetsuya Nagata, Masanori Kobayashi, Yuko Shimizu-Motohashi, Jun Tanihata, Michihiro Imamura, and Kazuhiro Yamamoto for their technical advice, support, and helpful discussions; Tomoko Chiyo, Chiaki Masuda, Kazue Kinoshita, Ryoko Nakagawa, and Rie Ogawa for technical assistance; Hideki Kita, Shinichi Ichikawa, Yumiko Yahata-Kobayashi, Aya Kuriyama, Akane Hanaoka-Hayashi, Namiko Ogawa, and other staff members of JAC Co. for caring for the dogs (General Animal Research Facility, NCNP). We are thankful to JCR Pharmaceuticals Co., Ltd for their generous contribution of the DPSCs and MSCs. We also thank Posadas Herrera Guillermo for checking the quality of English in the manuscript.

Funding

This work was supported by a research grant from JCR Pharmaceuticals Co., Ltd., Health Sciences Research Grants for Research on Human Genome and Gene Therapy from the Ministry of Health, Labor and Welfare of Japan, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (grant number: 22390284, 22-40134).

Contributions

Y. N-K. and T. O. conceived and planned the experiments. Y. N-K. and M.K. performed the experiments, derived the models, and analyzed the data. H. H-K. Y.O. and M.K. contributed to sample preparation and assisted with experiments involving animal models. Y. N-K. wrote the manuscript in consultation, and T.O. and S.T. helped supervise the project. T.O. supervised the project.

Ethics approval and consent to participate

Animal experiments using MSCs were conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee of Nippon Medical School (27-199) and National Center of Neurology and Psychiatry (NCNP) Animal Ethics Committees (2012011, 2015004, and 19–30-06).

Consent for publication

Not applicable.

Conflicts of Interest

N-K., T. O. and S.T. have patent applications related to MSCs expressing IL-10. H. H-K., Y. N-K. and T. O. were members of the Division of Cell and Gene Therapy, Nippon Medical School, which is an endowment department, supported with a grant from JCR Pharmaceuticals Co., Ltd and Kaneka corporation.

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Table I. Summary of transplantation experiments

Dog ID	Sex	Age^a	BW^b	Cell	Cell numbers	Interval	Injection numbers	Route
4502MN	M	51	11.3	IL-10-CD271⁺MSCs, MyoD-CD271⁺MSCs	2.5 ×10⁷ cells	-	1	i.m.^c
5601MN	M	40	11.2	IL-10-CD271⁺MSCs, MyoD-CD271⁺MSCs	4.0-10.0×10⁶cells	-	2	i.m.^c
14103MN	M	3	5.0	-	-	-	-	-
14102MA	M	3	3.3	-	-	-	-	-
14105MA	M	3	3.4	hDPSCs	4.0 x10⁶ cells/kg	2 weeks	9	i,v.^d
14108MA	M	3	3.5	IL-10-hDPSCs	4.0 x10⁶ cells/kg	2 weeks	9	i,v.^d

M, male; ^aAge at injection (months); ^bBW, body weight at first injection (kg); ^ci.m, intramuscular injection; ^di.v., intravenous injectio

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Enhanced cell survival and therapeutic benefits of IL-10-expressing multipotent mesenchymal stromal cells for muscular dystrophy

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