The template of IVT mRNA consisted of the T7 promoter (5′-TAATACGACTCACTATAGGG-3′), 5′-UTR, ORF, and 3′-UTR. Three canine globin 5′- and 3′-UTRs were used to identify the UTR sequence that most effectively stabilized mRNA: canine hemoglobin subunit alpha-like 1 (HBA1), HBA2, and hemoglobin subunit beta-like (HBB). The ORF was designed to encode green fluorescence protein (GFP) to evaluate expression efficiency with relative fluorescence, and a stop codon was placed downstream of the GFP-encoding sequence.

To produce TSG-6 mRNA, the 5′- and 3′-UTRs of HBA1 were used based on the relative fluorescence results. The designed DNA templates were synthesized using the Gene in Fragment form synthesis service provided by BIONICS (Korea). All the UTRs and ORF sequences were obtained from the National Center for Biotechnology Information (NCBI, USA).

Fragment DNA (0.2 μg) was used as a template for IVT. mRNA was produced using an mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Briefly, linear template DNA was transcribed with T7 enzyme and 5′ capped with Anti-Reverse Cap Analog (ARCA) during transcription; DNase I treatment and poly (A) tailing were then performed. The synthesized mRNA was purified using the lithium chloride (LiCl) precipitation method and quantified using a Nanophotometer Pearl (Implen, München, Germany).

Canine adipose tissue-derived (cAT)-MSCs were cultured in Dulbecco's modified Eagle's medium (DMEM; Welgene, Gyeongsan-si, Korea), supplemented with 10% fetal bovine serum (FBS; Gibco, Billings, MT, USA) and 1% penicillin/streptomycin (PS; PAN-Biotech, Aidenbach, Germany). DH82, a canine macrophage cell line, was purchased from the American Type Culture Collection (ATCC number: CRL-10389; Manassas, VA, USA) and maintained in DMEM with 15% FBS and 1% PS. All cells were incubated at 37°C and 5% CO₂. The medium was changed every 2–3 days, and the cells were subcultured when they reached 70–80% confluence. cAT-MSCs from the third passage were used for all subsequent experiments.

Canine adipose tissues were obtained from three healthy adult female dogs undergoing ovariohysterectomy at the Seoul National University Veterinary Medical Teaching Hospital (SNU VMTH) under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of SNU (protocol no. SNU-220818-1). MSC isolation and culture were performed as previously described (29). Adipose tissue samples were washed with Dulbecco's phosphate-buffered saline (DPBS; Welgene) supplemented with 1% PS, disintegrated using sterilized scissors, treated with collagenase type IA (0.1%; Gibco), and incubated at 37°C for 1 h. After incubation, tissue sample neutralization was performed using DMEM supplemented with 10% FBS. The neutralized samples were centrifuged at 1,200 × g for 5 min, and the pellet was passed through a 70-μm cell strainer (Thermo Fisher Scientific, Waltham, MA, USA) and again centrifuged at 1,200 × g for 10 min. To remove the remaining red blood cells, the pellet was resuspended in RBC lysis buffer (Sigma-Aldrich, St. Louis, MO, USA), incubated at room temperature for 5 min, washed with DPBS, and centrifuged at 1,200 × g for 5 min. After removing the supernatant, the pellets were resuspended in DMEM with 10% FBS and 1% PS and seeded at a density of 3,000 cells/cm² in 100 mm cell culture dishes. Cells were maintained at 37°C in 5% CO₂ and the medium was replaced every 2–3 days. Subculture was performed when cells exhibiting fibroblast-like morphology reached 70–80% confluence.

To assess their differentiation capacities, cAT-MSCs were cultured using three differentiation media (StemPro Adipogenesis Differentiation, Stem Pro Osteogenesis Differentiation, and StemPro Chondrogenesis Differentiation kits; Gibco). Differentiation into adipocytes, osteocytes, and chondrocytes was identified by staining the cells with Oil Red O, 1% alizarin red, and alcian blue (Sigma-Aldrich), respectively. Additionally, the expression of specific surface markers was evaluated with flow cytometry using FACS Aria II system (BD Biosciences). Cells were stained with monoclonal antibodies against CD34-PE conjugated, CD45-FITC conjugated, CD29-FITC conjugated, and CD90-PE conjugated (BD Biosciences, San Jose, CA, USA), and the resulting fluorescence was analyzed using Flowjo software (Tree Star, Woodburn, OR, USA).

We compared the translation efficacy of the GFP mRNAs with the UTRs HBA1, HBA2, and HBB in transfected cAT-MSCs. Cells (1 × 10⁵) were seeded in confocal dishes and transfected with 0.5 μg of each mRNA for 24 h with Lipofectamine MessengerMAX Reagent (Thermo Fisher Scientific) and Opti-MEM I Reduced Serum Medium (Gibco) according to the manufacturers' instructions. The fluorescence levels of transfected cells were measured using a confocal laser scanning microscope (LSM710; Zeiss, Germany) at 6, 12, 24, 36, 48, 60, 72, 84, and 96 h post-transfection. At each time point, three images per confocal dish were randomly acquired. The relative fluorescence was calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Briefly, we evaluated the area and intensity of green fluorescence using the ImageJ as the cells were evenly seeded. The fluorescence area was relatively calculated by assuming the total area as 1.

Total RNA of TSG-6 mRNA-transfected cAT-MSCs and co-cultured DH82 cells was extracted using an Easy-Blue total RNA extraction kit (iNtRON Biotechnology, Seongnam, Korea). The concentration and the purity of samples were measured using a Nanophotometer Pearl (Implen). CellScript All-in-One 5 × 1st cDNA Strand Synthesis Master Mix (CellSafe, Bucheon-si, Korea) was used for cDNA synthesis. qPCR was conducted in duplicate using AMPIGENE qPCR Green Mix Hi-Rox with SYBR green dye (Enzo Life Sciences, Farmingdale, NY, USA), 400 nM forward and reverse primers, and 1 μL of cDNA. Thermocycler settings were 95°C for 2 min, followed by 40 cycles at 95°C for 5 s and 60°C for 25 s. Expression levels were analyzed using the 2^−ΔΔ/Cts method and normalized to that of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primer sequences used in this study are listed in Table 1.

Total protein of TSG-6 mRNA-transfected cAT-MSCs was extracted using Pro-Prep protein extraction solution (iNtRON Biotechnology). The concentration of extracted protein was quantified using a DC Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). We separated 20 μg of protein by SDS-PAGE, which was then transferred to polyvinylidene difluoride membranes (EMD Millipore, Burlington, MA, USA). The membranes were blocked with 5% non-fat dry milk and exposed to primary antibodies against TSG-6 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and β-actin (1:1,000, Santa Cruz Biotechnology) at 4°C for 24 h. Membranes were washed and then incubated with secondary antibodies at room temperature for 1 h. We detected the immunoreactive bands of the proteins using a chemiluminescence kit (Advansta, San Jose, CA, USA); the protein expression levels were normalized to that of β-actin.

We measured TSG-6 content in the cell culture supernatant of TSG-6 mRNA-transfected cAT-MSCs using a commercial TSG-6 ELISA kit (MyBioSource, San Diego, CA, USA) according to the manufacturer's instructions.

cAT-MSCs (4 × 10⁵) were seeded onto 0.4 μM pore-sized Transwell inserts (SPL Life Sciences, Pocheon-si, Korea) and transfected with 2.5 μg TSG-6 mRNA per well for 24 h (or without, as a control), using Lipofectamine MessengerMAX Reagent and Opti-MEM I Reduced Serum Medium. After 24 h, the inserts were washed twice with DPBS and the medium was changed to DMEM supplemented with 10% FBS and 1% PS.

DH82 cells were seeded in 6-well plates at a density of 1 × 10⁶ cells/well and incubated with or without 200 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich) for 24 h. After changing the medium to DMEM with 10% FBS and 1% PS, DH82 macrophages were co-cultured with TSG-6 mRNA-transfected cAT-MSCs, naïve cAT-MSCs, or treated with 10 μM dexamethasone (a well-known anti-inflammatory drug) for 24 h. This concentration of dexamethasone (24-h treatment) was selected because it effectively reduced inflammatory cytokine (IL-1β and IL-6) expression in LPS-stimulated DH82 cells without affecting viability, based on a Cell Counting Kit-8 (CCK-8) assay (Donginbio, Seoul, Korea) and qPCR (Supplementary Figure 1).

Each experiment was conducted at least three times. Data are shown as the mean ± standard deviation. Mean values from different groups were compared using the Mann–Whitney t-test and one-way analysis of variance using GraphPad Prism v.7.01 software (GraphPad Software, La Jolla, CA, USA). Statistical significance was set at P < 0.05.

We evaluated the stem cell identity of the cAT-MSCs using differentiation assays and flow cytometry. The cells were successfully differentiated into adipocytes, osteocytes, and chondrocytes (Figures 1A–C). Flow cytometry analysis showed high expression of the stem cell markers CD29 and CD90, while no expression of the hematopoietic markers CD34 and CD25 was detected (Figures 1D–G).

cAT-MSCs were transfected with GFP mRNA containing three different UTRs (HBA1, HBA2, and HBB) and monitored for 96 h (Figures 2A, B). We observed significantly different fluorescence levels depending on the UTR, indicating variation in the translation efficacy of each mRNA. HBA1-, HBA2-, and HBB-GFP mRNA reached a peak fluorescence level at 24, 12, and 72 h, respectively, of which HBA1 had the highest fluorescence level and HBB had the lowest (Figure 2C). At 24 h post-transfection, HBA1 showed a significantly higher level of fluorescence than HBA2 and HBB did. HBA1 maintained a higher fluorescence level than the other two mRNAs for about 36 h. Fluorescence expression of HBA1 was observed up to 96 h post-transfection (Figure 3). Overall, HBA1 most effectively enhanced mRNA stability and translation efficacy.

TSG-6 mRNA was produced with the HBA1 UTR and transfected into cAT-MSCs for 24 h. The coding sequence of TSG-6 mRNA is described in Supplementary Data 1. The expression of TSG-6 mRNA was more than 10,000-times higher in the MSC^TSG-6 than in the MSC group, indicating the successful transfection of TSG-6 mRNA (Figure 4A, Supplementary Tables 1, 2). Western blot confirmed that the expression of TSG-6 protein was significantly higher in the MSC^TSG-6 group than that in the MSC group (Figure 4B). TSG-6 ELISA also confirmed that the secretion of TSG-6 was enhanced in the MSC^TSG-6 group (Figure 4C). These results demonstrate that TSG-6 mRNA transfection could produce high levels of TSG-6 in cAT-MSCs.

We evaluated the immunomodulatory effect of MSC^TSG-6 in the co-culture system with DH82 macrophages (Figure 5A). In LPS-stimulated DH82 cells, the mRNA expression of inflammatory cytokines demonstrating the M1 polarization significantly increased compared to the naïve group. In contrast, in DH82 cells co-cultured with MSCs or MSCs^TSG-6, the expression of inflammatory cytokines was significantly decreased compared with that in the LPS-stimulated group. Compared to the MSC-co-cultured group, the mRNA expression levels of all inflammatory cytokines were significantly lower in the MSC^TSG-6-co-cultured group (Figures 5B–D, Supplementary Tables 3–6).

The anti-inflammatory effect of MSC^TSG-6 was compared with that of dexamethasone. We compared the expression levels of inflammatory cytokines between LPS-stimulated DH82 cells treated with 10 μM dexamethasone to those co-cultured with MSCs or MSCs^TSG-6. The concentration of IL-6 was higher in the dexamethasone-treated group than in the MSC- and MSC^TSG-6-co-cultured groups (Figure 5B). In contrast, the mRNA expression level of IL-1β was lower in the dexamethasone-treated group than in the MSC- and MSC^TSG-6-co-cultured groups (Figure 5C). TNF-α mRNA expression levels did not vary between groups (Figure 5D).