Human umbilical cords from full-term Caesarean section patients were collected upon delivery, stored in Dulbecco’s modified Eagle medium (DMEM)/F12 (1:1) culture medium, which was supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (GIBCO, Invitrogen Inc., Carlsbad, CA, USA), and transferred immediately for cell isolation, according to a previously described protocol . Briefly, the cord was cut into pieces that were 4–5 cm long, and the vessels were pulled away to isolate Wharton’s Jelly (WJ). WJ was cut into 1–2-mm3 pieces and digested with 1 mg/ml collagenase II (Millipore Sigma, St. Louis, MO, USA) with phosphate-buffered saline (PBS) at 37 °C for 45 min. The digested mixture was then passed through a 100-μm filter (BD Biosciences, Franklin Lakes, NJ, USA) to obtain cell suspensions. The cells were washed with PBS solution and then cultured in DMEM/F12 medium containing 10 % fetal bovine serum, 2 mmol/L glutamine, 1 % nonessential amino acids, and 1 % penicillin/streptomycin (GIBCO, Invitrogen Inc., Carlsbad, CA, USA) at 37 °C and 5 % CO2. Nonadherent cells were removed by changing the medium after 3 days. Cells were expanded and identified according to the current statement of the International Society for Cellular Therapy (ISCT) . Briefly, a minimal set of three standard criteria was used as the uniform definition of multipotent MSCs: adherence to plastic, specific surface antigen expression, and multipotent differentiation potential. The phenotype of multipotent MSCs is defined to be, at a minimum, the cell surface co-expression of antigens (CD105, CD73, and CD90 [≥95 % positive]) and the absence of hematopoietic lineage markers (CD45, CD34, CD14, CD19, and HLA-DR [≤2 % positive]). The surface marker was defined by the BD Stemflow hMSC Analysis Kit (BD Biosciences, Franklin Lakes, NJ, USA) containing pre-conjugated and pre-titrated cocktails of ISCT-defined positive expression markers (CD105 PerCP-Cy™5.5/CD73 APC/CD90 FITC) and negative expression markers (CD45/CD34/CD11b/CD19/HLA-DR PE). The multipotent differentiation potential of the isolated cells was identified using the Human Mesenchymal Stem Cell Functional Identification Kit (R&D, Minneapolis, MN, USA). Briefly, hUC-MSCs were seeded at 2 × 104 cells/cm2 in StemXVivo Osteogenic/Adipogenic Base Media. And after 24 hours, the medium was replaced with adipogenic differentiation medium to induce adipogenesis. HUC-MSCs were seeded at 4.2 × 103 cells/cm2 in StemXVivo Osteogenic/Adipogenic Base Media. When cells were to 50–70 % confluency, the medium was replaced by osteogenic differentiation medium. Differentiation medium was replaced every 3 days, and after 3 weeks cells were fixed in 10 % formalin and processed for histochemical analysis. Adipogenic differentiation was detected by oil red staining, and osteogenic differentiation was analyzed by alizarin red staining. This project was approved by the Human Ethics Committee of The First Affiliated Hospital at Sun Yat-sen University, and written informed consent was obtained for umbilical cord collections.
Induction of colitis and cell transplantation
Colitis was induced in specific pathogen-free male BALB/c mice (6–8 weeks old), according to a previously described method . All experiments were performed according to the Institutional Guidelines for the Care and Use of Laboratory Animals in Research and were approved by the Ethics Committee at Sun Yat-sen University. Briefly, mice were pre-sensitized with the trinitrobenzenesulfonic acid (TNBS) pre-sensitization solution on day 1. The pre-sensitization solution was prepared by mixing acetone and olive oil in a 4:1 volume ratio by rigorous vortexing and then mixing 4 volumes of acetone/olive oil with 1 volume of 5 % TNBS solution to obtain 1 % (w/v) TNBS. Control mice were treated with the pre-sensitization solution without TNBS. BALB/c mice were lightly anesthetized after a 24-hour fast on day 8. To induce colitis, 5 % TNBS (Millipore Sigma, St. Louis, MO) in 50 % ethanol (2.5 mg/kg TNBS) was administered intrarectally via a 3.5 French (F) catheter equipped with a 1-ml syringe. The catheter was inserted into the rectum until the tip was advanced to 4 cm proximal to the anal verge. Control mice received 50 % ethanol alone. Passages 3–5 of hUC-MSCs were used for cell transplantation. BALB/c mice were treated intraperitoneally either with the medium as the control or with 106 hUC-MSCs/mouse 2 hours after instillation of TNBS.
Assessment of colitis severity
Animals were monitored for the appearance of diarrhea, body weight loss, and survival every day for a total of 14 days. The baseline data were collected before instillation of TNBS. Disease activity and histologic scores were evaluated as previously described . For disease activity, a score system containing percentage of weight loss, stool consistency, and fecal occult blood test was used [16, 17]. For histopathology analysis, a colon specimen from the middle part (1 cm to the anus to cecum) was fixed in 10 % buffered formalin phosphate and then embedded in paraffin. Sections were stained with hematoxylin and eosin, and inflammation was graded from 0–4 as follows, in a blinded fashion: 0, no signs of inflammation; 1, low leukocyte infiltration; 2, moderate leukocyte infiltration; 3, high leukocyte infiltration, moderate fibrosis, high vascular density, thickening of the colon wall, moderate goblet cell loss and focal loss of crypts; and 4, transmural infiltrations, massive loss of goblet cells, extensive fibrosis, and diffuse loss of crypts. Myeloperoxidase (MPO) activity was assessed by the MPO kit (Jiancheng, Nanjing, China), according to the manufacturer’s instructions. For survival and colitis score analysis, there were 20 mice in the model and treatment groups and 10 mice for the control and naïve groups. For histological and immunological analysis, mice were sacrificed at day 3 after colitis induction, at the peak of inflammation (n = 9 for each group).
In vivo imaging
MSCs were traced in vivo with the Renilla Luciferase Assay System (Promega, Madison, WI, USA). First, the EGFP-luciferase system was conducted and transferred to MSCs. Cell transplantation was conducted as described above. Renilla luciferase substrate was intraperitoneally injected after cell transfer at different time points (day 1, day 3, and day 5). Using the Xenogen IVIS Spectrum in vivo visible light system (Caliper Life Sciences, Hopkinton, MA, USA), cell tracing was performed at about 10 min after substrate injection.
Immunologic analysis of T- and B-cell subsets in the mesenteric lymph node (MLN) and spleen
Colitis mice were sacrificed at day 3 after colitis induction, at the peak of inflammation. Lymphocytes of MLN cells and the spleen were isolated through a 100-μm filter (BD Biosciences, Franklin Lakes, NJ, USA). Lymphocytes were then suspended at a density of 2 × 106 cells/ml in RPMI 1640 culture medium, which was supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mmol/L glutamine, and 10 % heat-inactivated fetal calf serum (GIBCO, Invitrogen Inc., Carlsbad, CA, USA). To identify Tregs, 2 × 106 lymphocytes were surface-labeled with phycoerythrin (PE)-labeled anti-CD4 and allophycocyanin (APC)-cyanine (Cy)7-labeled Foxp3. For Th1/Th2/Th17 cell subgroup analyses, 2 × 106 cells were stimulated with 50 ng/ml phorbol myristate acetate and 1 mmol/L ionomycin (Millipore Sigma, St. Louis, MO, USA) for 4 hours in the presence of monensin (BD Biosciences, Franklin Lakes, NJ, USA). The incubator was set at 37 °C under a 5 % CO2 atmosphere. After 4 hours, intracellular staining was performed with APC-labeled anti-CD4, PE-labeled anti-interleukin (IL)-4, PE-labeled anti-IL-17, and FITC-labeled anti-interferon (IFN)-γ (BD Biosciences, Franklin Lakes, NJ, USA). For CD5 cell subgroup analysis, cells were surface-labeled with FITC-labeled CD5 and PE-CY7-labeled CD19. Flow cytometry was carried out using a BD FACScan (BD Biosciences, Franklin Lakes, NJ, USA), in which 300,000–500,000 events were collected, and lymphocytes were gated based on their forward and side light scatter properties. Data were analyzed using the Gallios Flow Cytometer (Beckman Coulter, Brea, CA, USA) and Kaluza Analysis software. The proportion of Tregs was determined based on CD4+Foxp3. CD4+IFN-γ+, CD4+IL-4+, CD4+IL-17+, and CD5 + CD19+ cells were defined as Th1 cells, Th2 cells, Th17 cells, and CD5+ Bregs, respectively. For cytokine expression, the serum was separated from peripheral blood collected through tail vein on day 3. Tumor necrosis factor (TNF)-α, IL-12, IL-6, IL-23, IL-21, IFN-γ, and IL-17A were detected by the ProcartaPlex™ Multiplex Immunoassays kit (eBioscience, Santa Clara, CA, USA) and Bio-plex system (Bio-Rad, Hercules, CA, USA). Transforming growth factor (TGF)-β was detected by measured by an enzyme-linked immunosorbent assay (ELISA) (eBioscience, Santa Clara, CA, USA), according to manufacturer instructions. All assays were performed in triplicate wells per condition in each experiment.
In vitro and in vivo study of CD5+ Bregs
CD5+ B cells have been shown to exist in the peritoneal cavity . Thus, CD5+ B cells were also analyzed in peritoneal lavage fluid to clarify its distribution in hUC-MSC-treated colitis mice. CD5+ B cells were isolated by flow cytometry and co-cultured with carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, Inc., Carlsbad, CA, USA)-labeled T cells. Cell proliferation was then detected by flow cytometry (Beckman Coulter, Brea, CA, USA). For the in vivo functional study, CD5+ B cells were isolated form spleen lymphocytes by flow cytometry. Isolated CD5+ B cells were transplanted through the tail vein of TNBS-induced colitis mice on day 3, in the peak of the inflammation. Disease severity and T-cell subgroups were analyzed according to the methods described above.