Canine adipose tissue was obtained from a healthy dog < 1 year old during routine spaying at Seoul National University Veterinary Medicine Teaching Hospital (SNU VMTH), and MSCs were isolated as previously described [36]. Briefly, the tissue was washed three times with phosphate-buffered saline (PBS; PAN-Biotech, Germany) containing 100 U/mL penicillin and 100 g/mL streptomycin, then cut into small pieces and digested for 1 h at 37℃ with collagenase type IA (1 mg/mL; Sigma-Aldrich, USA). The enzymatic activity was inhibited with Dulbecco's Modified Eagle's Medium (DMEM; PAN-Biotech) containing 10% fetal bovine serum (FBS; PAN-Biotech). Following centrifugation at 1,200 × g for 5 min, the pellet was filtered through a 70 µm Falcon cell strainer (Fisher Scientific, USA) to remove debris, then incubated in DMEM containing 10% FBS at 37℃ in a humidified atmosphere of 5% CO₂. After 48 h, cultures were washed with PBS to remove non-adherent cells and incubated with fresh medium, which was changed every 48 h until cells reached 70% to 80% confluence. The cells were then repeatedly subcultured under standard conditions. Before their use in experiments, cells were characterized for the expression of several stem cell markers by flow cytometry using fluorescein isothiocyante (FITC)-, phycoerythrin (PE)-, or allophycocyanin (APC)-conjugated antibodies against the following proteins: cluster of differentiation (CD)29-FITC, CD31-FITC, CD34-PE, and CD73-PE (BD Biosciences, USA), and CD44-FITC, CD45-FITC, and CD90-APC (eBiosciences, USA). Cells were analyzed using a FACSAria II system (BD Biosciences). Cellular differentiation was evaluated using the kits (Gibco, USA) of the StemPro Adipogenesis Differentiation, StemPro Osteogenesis Differentiation, and StemPro Chondrogenesis Differentiationaccording to the manufacturer's instructions, followed by Oil Red O staining, Alizarin red staining, and Alcian blue staining, respectively.

Male Sprague-Dawley rats (Nara Biotech, Korea) weighing 190 to 220 g were used for experiments. All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of SNU (protocol No. SNU-150820-1). Rats were randomly divided into the following four groups: SHAM+PBS (n = 4), SHAM+MSC (n = 4), SAP+PBS (n = 8), and SAP+MSC (n = 8). SAP was induced by retrograde injection of sodium taurocholate (TCA; Sigma-Aldrich) into the pancreatic duct as previously described [16]. Briefly, rats were anesthetized and laparotomized at the midline. After exposing the first loop of the duodenum and the pancreas, the proximal common bile duct was clipped with a microclamp. A needle catheter was then inserted through the middle common bile duct via the duodenal papilla and ligated, after which a solution of 3% TCA in saline (1 mL/kg) was injected into the common bile-pancreatic duct over a 60 sec period. The microclamp and ligature were subsequently removed and the abdomen closed. Rats in SHAM groups were also anesthetized and laparotomized at the midline, but their abdomens were closed without additional manipulations. Following surgery, cAT-MSCs labeled with chloromethylbenzamido-1,1′-dioctadecyl-3,3,3′-tetramethyli ndo-carbocyanine perchlorate (CM-DiI; Invitrogen, USA) were administered via the tail vein to rats in the MSC groups (1 × 10⁷ cells/kg in 200 µL PBS), while an identical volume of PBS was administered via the same route to PBS groups. Rats were sacrificed 3 days after these procedures. Blood samples were centrifuged to obtain serum, which was stored at −80℃ until use. Pancreatic tissue was collected and washed in cold PBS. After weighing the pancreas, half of the tissue was fixed in 10% formaldehyde and embedded in paraffin, while the other half was stored at −80℃ until use.

Paraffin-embedded tissue samples were cut into 4 µm sections that were stained with hematoxylin and eosin and examined under a light microscope. A total of 20 randomly selected fields per group were scored in a blinded manner. Pancreatic acinar cell injury was scored on a scale from 0 to 4 based on the degree of edema (0, absent; 1, expanded interlobar septa; 2, expanded interlobular septa; 3, expanded interacinar septa; 4, expanded intercellular septa), infiltration (0, 0–1 white blood cells [WBCs]/high-power field [HPF]; 1, 2–10 WBCs/HPF; 2, 11–20 WBCs/HPF; 3, 21–30 WBCs/HPF; 4, > 30 WBCs/HPF), and necrosis (0, absent; 1, 1–4 necrotic cells/HPF; 2, 5–10 necrotic cells/HPF; 3, 11–15 necrotic cells/HPF; 4, > 15 necrotic cells/HPF).

Serum amylase and lipase activities were measured using the EnzyChrom α-Amylase Assay and QuantiChrom Lipase Assay kits (both from BioAssay Systems, USA) according to the manufacturer's instructions. Serum IFN-γ and IL-10 levels were measured using rat-specific Quantikine enzyme-linked immunosorbent assay kits (R&D Systems, USA) according to the manufacturer's instructions.

Approximately 100 mg of pancreas tissue were homogenized and RNA was extracted using the Easy-BLUE Total RNA Extraction kit (iNtRON Biotechnology, Korea). Next, cDNA was synthesized using LaboPass M-MuLV Reverse Transcriptase (Cosmogenetech, Korea) according to the manufacturer's instructions. Samples were assayed in duplicate in 10 µL AMPIGENE qPCR Green Mix Hi-ROX with SYBR Green dye (Enzo Life Sciences, USA) using 1 µL cDNA and 400 nM forward primer and reverse primers (Cosmogenetech). The cycling conditions were 95℃ for 2 min, followed by 40 cycles of 95℃ for 5 s and 60℃ for 25 sec. Expression levels were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

The blood of a healthy canine donor was obtained from SNU VMTH and diluted with an equivalent volume of PBS, then layered over Ficoll-Paque PLUS (GE Healthcare Life Sciences, Sweden) in conical tubes. Following centrifugation at 400 × g for 30 min, the cell layer was collected and washed twice with PBS. cPBMCs were then resuspended in Roswell Park Memorial Institute (RPMI)-1640 medium (PAN-Biotech) containing 10% FBS.

Splenocytes were isolated from rats as previously described [3]. Briefly, rat spleens were removed and cut into small pieces in a cell strainer with PBS. The tissue was then crushed using the plunger from a 1 mL syringe. Next, the homogenized cell suspension was transferred to a tube and the cell pellet was obtained by centrifugation and resuspended in Red Blood Cell Lysis Buffer (Sigma-Aldrich). After two washes with PBS, splenocytes were resuspended in RPMI-1640 containing 10% FBS.

Isolated canine PBMCs and rat splenocytes were used in the MLR. Briefly, cAT-MSCs were treated with 25 µg/mL mitomycin C (Sigma-Aldrich) for 1 h at 37℃. After five washes, cAT-MSCs were seeded in a 96-well plate at 1 × 10⁴, 1 × 10³, and 1 × 10² per well to determine the cAT-MSC-to-cPBMC ratio dependency. After 6 h, mitomycin C-treated cAT-MSCs were attached, and cPBMCs stimulated with 2 µg/mL concanavalin A (ConA; Sigma-Aldrich) were added to each well of cAT-MSCs cultured at 1 × 10⁵/well. After 5 days, cPBMC proliferation was assessed with the BrdU Cell Proliferation Assay Kit (BioVision, USA) according to the manufacturer's instructions. For MLR with conditioned medium (CM), cAT-MSCs were seeded in 6-well plates at 3 × 10⁵/well, and the medium was changed after 24 h. After 5 days, the CM was harvested and centrifuged to remove debris. The MLR was carried out in CM as described above. The procedure was then repeated using rat splenocytes.

Paraffin sections were cut at a thickness of 4 µm for immunolabeling. Sections were deparaffinized and rehydrated, and antigen retrieval was carried out in 10 mM citrate buffer. Sections were then washed and blocked with blocking buffer containing 5% normal goat serum (Gibco) and 0.3% Triton X-100 (Sigma-Aldrich) for 1 h, then incubated overnight at 4℃ with mouse monoclonal anti-CD3 (1 : 100) and anti-forkhead box (Fox)P3 (1 : 100) antibodies (both from Santa Cruz Biotechnology, USA). After three washes, the sections were incubated with secondary antibody (1 : 200; Santa Cruz Biotechnology) for 1 h at room temperature in the dark, then washed three times and mounted in Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (Vector Laboratories, USA). Finally, the samples were visualized with an epifluorescence microscope. Immunoreactive cells were counted in 20 random fields per group.

Data are expressed as the mean ± standard deviation. Group means were compared by one-way analysis of variance and an unpaired Student's t-test using the GraphPad Prism 6.01 (GraphPad Software, USA). A p < 0.05 was considered statistically significant.

Cells isolated from canine adipose tissue were identified by immunophenotyping and multilineage differentiation. The cells had a high expression of known MSC markers such as CD29, CD44, CD73, and CD90, and did not express CD31, CD34, or CD45 (panel A in Fig. 1). The cAT-MSCs had multilineage plasticity, as demonstrated by their potential for adipogenic, osteogenic, and chondrogenic differentiation (panel B in Fig. 1).

Pancreatic tissue samples from SAP groups exhibited edema, inflammatory cell infiltration, and necrosis of acinar cells compared to those from SHAM animals. The severity of pancreatic injury was markedly diminished in the SAP+MSC relative to the SAP+PBS group (p < 0.001; panel A in Fig. 2). After cAT-MSC infusion, serum amylase and lipase activities, which are important markers of pancreatic function, decreased by 45% (panel B in Fig. 2) and 32% (panel C in Fig. 2), respectively, relative to the SAP+PBS group. In addition, the pancreas-to-body weight ratio, a measure of pancreatic edema, was reduced in the SAP+MSC relative to the SAP+PBS group (p < 0.05; panel D in Fig. 2).

The capacity of infused cAT-MSCs to migrate to the injured pancreas was evaluated by fluorescence microscopy and reverse transcription PCR analysis. CM-DiI-labeled cAT-MSCs were identified by their red fluorescence. There was a greater number of CM-DiI-labeled cAT-MSCs in the pancreatic tissue of the SAP+MSC than the SHAM+MSC group, whereas no fluorescent cells were detected in the PBS groups (panel A in Fig. 3). Consistent with this observation, the mRNA level of canine ribosomal protein S5 (cRPS5) was higher in the SAP+MSC than in the SHAM+PBS group (panel B in Fig. 3), confirming the presence of cAT-MSCs.

The mRNA expression of pro- and anti-inflammatory cytokines in pancreatic tissue was evaluated by quantitative real-time PCR. The levels of TNF-α, IL-1β, -6, -12, -17, and -23 and IFN-γ were markedly increased after SAP induction. cAT-MSC infusion resulted in downregulation of these pro-inflammatory cytokines (p < 0.05 or p < 0.01; panel A in Fig. 4). Conversely, the expression of the anti-inflammatory cytokines IL-4 and -10 decreased by SAP induction, but increased in the SAP+MSC group relative to the SAP+PBS group (p < 0.05; panel A in Fig. 4). Furthermore, serum levels of IFN-γ and IL-10 were decreased by 30% and increased by 21%, respectively, in the SAP+MSC relative to the SAP+PBS group (panel B in Fig. 4).

We next evaluated the effects of cAT-MSCs on T cells, which release inflammatory cytokines. cAT-MSCs suppressed the proliferation of cPBMCs treated with ConA in a cAT-MSC:cPBMC ratio-dependent manner. At ratios of 1 : 10 and 1 : 100, cPBMCs proliferation was markedly suppressed (p < 0.001 and p < 0.05, respectively) relative to cells grown without cAT-MSCs. However, this was not observed at a ratio of 1 : 1,000. To assess the effects of soluble factors from cAT-MSCs on T cells, CM from cAT-MSC cultures was used as described above. The proliferation of cPBMCs stimulated with ConA in CM was suppressed (p < 0.05) relative to cells treated with control medium (panel A in Fig. 5). cAT-MSCs also inhibited the proliferation of rat splenocytes stimulated with ConA in a cAT-MSCs:rat splenocyte ratio-dependent manner. This effect was apparent at a 1 : 10 ratio and in CM (p < 0.001 and p < 0.05, respectively), but was not observed at ratios of 1 : 100 and 1 : 1,000 relative to splenocytes grown without cAT-MSCs or in control medium (panel B in Fig. 5). In addition, quantitative analysis of T cells detected in pancreatic tissue by immunocytochemistry revealed that the percentage of CD3⁺ T cells was decreased (p < 0.05; panel C in Fig. 5), whereas that of FoxP3⁺ regulatory T cells was increased (p < 0.01; panel D in Fig. 5) in the SAP+MSC relative to the SAP+PBS group.