Ethical approval was obtained from the Institutional Review Board of Shantou University Medical College, Shantou, China. Human umbilical cords from consenting patients (full-term caesarian sections) were collected immediately into a sterilized 50 ml tube, washed with phosphate-buffered saline (PBS) and cut into small 2–3-cm-thick sections. After dissecting the arteries and veins, the remaining tissue, the Wharton’s jelly, was diced into smaller fragments and transferred to a 75 cm² flask in DMEM/F12 (Sigma-Aldrich, St. Louis, MO, USA) culture medium supplemented with 10% fetal bovine serum (FBS; Gibco, Sydney, Australia), 100 μg/ml penicillin/streptomycin (Shanghai Bioscience, Shangai, China), 1 g/ml amphotericin B (Gilead Sciences, Inc., San Dimas, CA, USA), 5 ng/ml epidermal growth factor (EGF; Invitrogen Life Technologies, Carlsbad, CA, USA) and 5 ng/ml basic fibroblast growth factor (bFGF; Sigma-Aldrich). The cultures were left undisturbed for 5–7 days at 37°C, 5% CO₂ to allow the migration of cells from the explants, after which the medium was replaced.

Approximately 1×10⁶ HUMSCs at passage 3 were dispersed with trypsin and resuspended in PBS containing phycoerythrin (PE)-conjugated antibodies against CD40, CD40L, CD80 and CD86 (BD Biosciences, Franklin Lakes, NJ, USA) for 60 min at 4°C. The cells were washed 3 times with PBS and incubated with PE-conjugated rabbit anti-mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, USA) or FITC-conjugated goat anti-rat IgG (Santa Cruz Biotechnology) for 30 min at room temperature. After 3 washes, the cells were resuspended in 0.5 ml PBS and analyzed by flow cytometry with the use of Epics XL flow cytometer (Beckman Coulter, Brea, CA, USA).

Human peripheral blood lymphocytes (PBMCs) were isolated from healthy donors by Ficoll-Paque (1.077 g/ml) density gradient centrifugation. The cell concentration was adjusted to 1×10⁶/ml with RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS. HUMSCs at passage 3 were harvested and adjusted to 1×10³ cells/ml, 1×10⁴ cells/ml, or 1×10⁵ cells/ml in L-DMEM containing 10% FBS. A 100 μl suspension of HUMSCs was plated into 96-well plates. The plates were incubated for 72 h at 37°C, 5% CO₂. After the cells reached 70–80% confluence, the medium was removed and 100 μl of fresh medium containing 2.5 μl of mitomycin C (1 μg/μl; Sigma-Aldrich) were added for 30 min at 37°C to mitotically inactivate the HUMSCs. After the medium was removed, the inactivated HUMSCs were washed twice with PBS. HUMSCs were resuspended in 100 μl of lymphocyte medium (RPMI-1640 containing 10% FBS), co-cultured with 1×10⁵ cells/l PBMC, and stimulated by PHA (10 mg/l) (Sigma-Aldrich) for 72 h at 37°C, 5% CO₂. The cells were divided into the following groups: PBMCs + PHA (positive control); HUMSCs (1×10⁵) + PBMCs + PHA; HUMSCs (1×10⁴) + PBMCs + PHA; and HUMSCs (1×10³) + PBMCs + PHA. Three ratios of HUMSCs to PBMCs were used: 1:1, 1:10 and 1:100. Each trial was repeated in triplicate. The CCK-8 kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to assess the immunomodulatory impact of HUMSCs on PBMCs following stimulation with PHA. The procedure was carried out according to the manufacturer’s instructions. The inhibitory effects of HUMSCs on lymphocyte proliferation were evaluated by comparing the optical density (OD) in cells co-cultured with inactivated HUMSCs with the OD of lymphocytes cultured alone.

Passage 3 HUMSCs were trypsinized, the concentrations adjusted to 1×10⁵ cells/ml, and the cells were plated (1 ml) in 24-well plates. After the cells reached 70% confluence, 10 μl mitomycin-C (1 μg/μl) were added into each well. Following incubation for 1 h at 37°C, 5% CO₂, the medium was removed and the cells were washed twice with PBS. PBMCs (1×10⁵) in 1 ml of lymphocyte medium were added and co-cultured in the presence of 10 μl of PHA for 72 h at 37°C, 5% CO₂. The groups were as follows: PBMCs + PHA (positive control); unstimulated PBMCs (negative control); and HUMSCs (1×10⁵ cells) + PBMCs + PHA. The supernatants of each group were collected and IFN-γ expression was evaluated using the ELISA detection kit according to the manufacturer’s instructions (Invitrogen).

Total RNA was isolated from the HUMSCs. In addition, we extracted the pancreas of the diabetic rats after HUMSC transplantation, as well as the pancreas of diabetic rats without HUMSC transplantation using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was prepared using the Prime Script RT Reagent kit (Takara Bio, Inc., Shiga, Japan). cDNA samples were analyzed by quantitative PCR using SYBR premix (Takara) in an ABI 7300 system. The primers used for qRT-PCR analyses were as follows: human pancreatic and duodenal homeobox 1 (PDX1; 148 bp) forward, 5′-ttcacgagccagtatgaccttcac-3′ and reverse, 5′-gaagacagacctgggatgcaca-3′; human insulin (221 bp) forward, 5′-acccagccgcagcctttgtg-3′ and reverse, 5′-ttccacaatgcc acgcttctgc-3′; human glucagon (161 bp) forward, 5′-cagagctta ggacacagagcacatc-3′ and reverse, 5′-acgttgccagctgccttgta-3′; HLA-I (293 bp) forward, 5′-gcagacacggaatgtgaagg-3′ and reverse, 5′-gtaggctctcaactgctccg-3′; HLA-DR (350 bp) forward, 5′-tcttgtctgttctgcctcactc-3′ and reverse, 5′-ttccaggttggctttgtcc-3′; and β-actin (396 bp) forward, 5′-tggcaccacaccttctacaatgagc-3′ and reverse, 5′-gcacagcttctccttaatgtcacgc-3′.

The E1-deleted adenovirus (serotype 5) carrying the CMV promoter/EGFP hybrid gene was purchased from Vector Gene Technology Company (Beijing, China). For amplification of the adenoviruses, 1×10⁸ infection units/ml (IU/ml) of viruses was added into a 10-cm dish pre-seeded with 1×10⁶ Ad293 cells (Stratagene, La Jolla, CA, USA) overnight. Following incubation for 30–48 h, the cells were harvested by scraping and centrifugation at 3,000 rpm for 10 min while the supernatant was saved for the following round of virus amplification. The harvested cells underwent 4 freeze/thaw cycles and were centrifuged at 12,000 × g for 10 min to obtain cell lysates. Serial dilutions of the supernatant and cell lysates were used to transduce Ad293 cells in a 96-well plate pre-seeded with 5,000 cells overnight. The viral titers (IU/ml) were determined by counting the EGFP-positive cells under a fluorescence microscope after 30 h of culture. HUMSCs at passages 3–5 were seeded at a density of 1×10⁵ cells/well in 6-well plates. Following 24 h of culture, the medium was replaced with 1 ml of serum-free medium containing indicated adenoviruses at a multipicity of infection (MOI) of 50 for 4 h.

Ethical approval for the animal experiments was obtained from the Institutional Review Board of Shantou University Medical College. A total of 20 rats received an intraperitoneal injection of streptozotocin (STZ, Sigma-Aldrich) at 70 mg/kg in order to induce diabetes. Blood glucose levels were monitored every 3 days. Rats with blood glucose levels >16.7 mmol/l for 3 measurements were diagnosed with type 1 diabetes.

The rats were divided into 3 groups, with 6–8 rats/group. After blood glucose spontaneously increased to 16.7 mmol/l, the rats were restrained and 5×10⁶ HUMSCs suspended in 0.1 ml of normal saline were injected through the tail vein. The control group underwent the same procedure, but was only injected with PBS. Body weight, blood glucose and serum insulin levels were recorded before and after cell transplantation. Blood was collected from the tail vein and blood glucose levels were measured using a blood glucose meter (Bayer, Leverkusen, Germany).

The results are expressed as the means ± standard deviation (SD). The statistical significance of the differences was assessed by the analysis of variance. In all comparisons, a value of P<0.05 was considered to indicate a statistically significant difference.

The HUMSCs derived from Wharton’s Jelly grew as a flat monolayer after being cultured in vitro for 7–10 days. After 2 weeks, some adherent cells had dissociated around the adherent tissue sections and were visible under an inverted microscope (Fig. 1A). Cells gradually multiplied and grew into a radial-like array around the adherent tissue sections. After the HUMSCs were passaged, they showed strong proliferative ability. These cells proliferated with a doubling time of approximately 24 h (Fig. 1B), but this proliferation rate decreased after the 9th passage (Fig. 1C). Ad293-EGFP was then transfected into the HUMSCs at passages 3–5 for transplantation (Fig. 1D).

Flow cytometry analysis revealed that the HUMSCs expressed low levels of CD80, CD86, CD40 and CD40L (Fig. 2A). qRT-PCR indicated that HUMSCs expressed the HLA-I gene (MHC-I), but not HLA-DR (MHC-II), which is closely related to graft-versus-host disease (Fig. 2B). To investigate the mechanisms responsible for the immunosuppressive effects mediated by HUMSCs, we co-cultured PHA-stimulated human PBMCs with various concentrations of HUMSC culture supernatant. The PHA-induced proliferation of human PBMCs was significantly suppressed by co-culture with different numbers of mitotically inactivated HUMSCs. The OD value of each group was as follows: PBMCs + PHA (positive control), 1.90±0.25; HUMSCs (1×10⁵) + PBMCs + PHA, 1.37±0.024 (P<0.05, n=3); HUMSCs (1×10⁴) + PBMCs + PHA, 1.81±0.31 (P>0.05, n=3); HUMSCs (1×10³) + PBMCs + PHA, 1.71±0.28 (P>0.05, n=3); (Fig. 2C). ELISA analysis of the PBMCs revealed that the secretion of IFN-γ was 12.88±4.22 IU/ml in the absence of HUMSCs, but decreased to 9.48±1.98 IU/ml when the cells were co-cultured with HUMSCs (P<0.05, n=3) (Fig. 2D).

STZ was used to induce diabetes in rats, with a single administration of 70 mg/kg. After 9 days, the blood glucose levels of the STZ-treated rats increased from the normal levels (7.14±2.08 mM) to severe hyperglycemic levels (24.04±2.84 mM). EGFP-positive HUMSCs were infused into diabetic rats on the 10th day. To avoid the aggregation of these cells and to ensure reproducible delivery, the cells were injected into the rats through the caudal vein. The rat pancreas and liver sections were analyzed by fluorescence microscopy 4 weeks after transplantation to observe whether the HUMSCs colonized in vivo. Green fluorescence was detected by laser scanning microscopy of frozen sections of the rat pancreas and liver. The pancreas and liver of the rats transplanted with HUMSCs stained positive for PDX1 and glucagon, but the control groups showed negative staining (Fig. 3). The PDX1 and glucagon gene were also detected in the pancreas of the group transplanted with HUMSCs, although insulin expression was not detected (Fig. 4). Blood glucose levels in the diabetic rats transplanted with HUMSCs (Fig. 5A) decreased significantly. Serum insulin levels (Fig. 5D), body weight (Fig. 5B) and the survival ratio increased (Fig. 5C), and islet repair was also improved (Fig. 6) compared with the diabetic rats not transplanted with HUMSCs.