Male 8-week-old Sprague-Dawley (SD) rats were purchased form Charles River Laboratories Japan (Yokohama, Japan). The rats were housed in a light- and temperature-controlled room in the Laboratory Animal Center of Hiroshima University (Hiroshima, Japan) with free access to food and water. The Animal Care and Use Committee at Hiroshima University approved all experimental protocols (permit number: A19–127), which were performed in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals.
MSCs and human peritoneal mesothelial cells (HPMCs)
Human MSCs derived from the bone marrow were obtained from Riken BRC (Ibaraki, Japan). MSCs at passage 4 were used in all experiments. MSCs cultured in STK2 and Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (Sigma-Aldrich) were designated as “SF-MSCs” and “10%MSCs,” respectively. HPMCs were isolated from human omentum as previously described  and cultured in M199 medium (Life Technologies, New York City, NY, USA), including 10% FBS and penicillin/streptomycin (Nacalai Tesque, Inc., Kyoto, Japan). The Medical Ethics Committee of Hiroshima Graduate School of Biomedical Science permitted harvesting the omentum (E-84). Each patient provided written informed consent.
Induction of peritoneal fibrosis and MSC treatment
After a 2-week acclimation, the SD rats were injected intraperitoneally with 3 mL of 0.1% chlorhexidine gluconate (CG) in 15% ethanol dissolved in saline. Thirty minutes after the CG injection, the rats were injected intraperitoneally with MSCs (5.0 × 106 cells) suspended in 1 mL of phosphate-buffered saline.
Peritoneal equilibrium test
Ten days after the CG and MSC injections, we performed peritoneal equilibrium testing before killing the rats. The rats were instilled intraperitoneally with PD solution (4.25% Dianeal; Baxter HealthCare, Deerfield, IL, USA) at 100 mL/kg body weight. After 30 min, peritoneal fluid and blood samples were collected via laparotomy and cardiac puncture, respectively, and the glucose and urea nitrogen concentrations were measured. The dialysate-to-plasma concentration ratio (D/P) of urea nitrogen (UN) and the dialysate-to-baseline dialysate concentration ratio (D/D0) of glucose represent the peritoneal permeabilities of blood urea nitrogen (BUN) and glucose.
Histology and immunohistochemistry
Histological and immunohistochemical staining of paraffin-embedded 4-μm-thick tissue sections were performed as described previously . The following primary antibodies were used: mouse monoclonal anti-α-SMA antibody (A2547; Sigma-Aldrich), rabbit polyclonal anti-TGF-β1 antibody (SAB4502954; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-collagen I antibody (ab34710; Abcam, Cambridge, UK), rabbit polyclonal anti-collagen III antibody (ab7778; Abcam), rabbit polyclonal anti-CD3 antibody (ab5690; Abcam), rabbit polyclonal anti-CD68 antibody (ab125212; Abcam), and rabbit polyclonal anti-CD163 antibody (ab182422; Abcam). Images of the microscopic sections were captured and measured using NIS-Elements (Nikon Corporation, Tokyo, Japan; × 200). The areas containing α-SMA, TGF-β1, collagen I, and collagen III were assessed in predetermined fields of the submesothelial compact zone, and the stained area was determined using Lumina Vision (Mitani, Osaka, Japan) in 50 fields from 5 rats.
Cell cultures and treatments
To prepare the conditioned medium (CM) from both the 10%MSCs and SF-MSCs, cells were seeded into 10-cm dishes. When the cells reached 60–80% confluence, the medium was substituted with DMEM containing 0.1% FBS, followed by 48 h incubation. Then, each medium sample was collected and used as “CM from 10%MSCs” or “CM from SF-MSCs”. HPMCs were seeded into six-well plates and grown to subconfluence in M199 medium containing 10% FBS. The medium was then replaced with DMEM containing 0.1% FBS, CM from the 10%MSCs or CM from the SF-MSCs. After 12 h, HPMCs were treated with 2.5 ng/mL TGF-β1 (R&D Systems, Minneapolis, MN, USA) for 30 min or 24 h. Whole-cell lysates were prepared and subjected to western blot analysis.
Water-soluble tetrazolium salts (WST)-1
MSCs (2.5 × 103 cells/100 μL) were seeded into 96-well microplates and cultured in DMEM containing 10% FBS or STK2. After incubating for 0, 12, 24, and 48 h, 10 μL of WST-1 reagent (Takara Bio, Shiga, Japan) was added to each well and then incubated for 4 h. The absorbance was determined using a microplate reader at a test wavelength of 450 nm and a reference wavelength of 620 nm.
Western blot analysis
Sample collection and western blotting were performed as previously described . The following primary antibodies were used: rabbit polyclonal anti-phosphorylated Smad2 antibody (#3108; Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-Smad2 antibody (#3103; Cell Signaling Technology), anti-phosphorylated Smad3 antibody (#9520; Cell Signaling Technology), anti-Smad3 antibody (#9523; Cell Signaling Technology), mouse monoclonal anti-α-SMA antibody (A2547; Sigma-Aldrich), and mouse monoclonal anti-α-tubulin antibody (T9026; Sigma). The intensity of each band was quantified using ImageJ software (version 1.48p; National Institutes of Health, Bethesda, MD, USA).
Quantitative real-time reverse-transcription PCR
RNA extraction and real-time reverse-transcription PCR were performed in accordance with previously described methods . Specific oligonucleotide primers and probes for human TSG-6 (assay ID: Hs00200180_m1) and β-actin (assay ID: Hs01060665_g1) were obtained as TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). The mRNA levels were standardized by the level of β-actin.
Statistical analysis was performed using the Mann–Whitney U test and Kruskal–Wallis test. P < 0.05 was considered statistically significant.