UCMSCs were purchased from the Sichuan Umbilical Cord Blood Stem Cell Bank (Chengdu, China). The cells were cultured in an incubator at 37°C, with 5% CO₂ and saturated humid air, and the medium was replaced three times each week. Once the cell density exceeded 70%, TrypLE Express (Gibco-BRL, Grand Island, N Y, USA) was used to digest the cells, following which the cells were subcultured. The present study was approved by the ethics committee of West China Hospital, Sichuan University (Chengdu, China).

The cells were seeded onto coverslips and fixed with neutral-buffered formalin for 15 min. The cells were then permeabilized in 0.1% Triton X-100 for 10 min and incubated with monoclonal antibodies against stage-specific embryonic antigen 4 (SSEA-4; cat. no. Ab16287; Abcam Cambridge, UK). Immunofluorescence analysis was then performed and the immunostained cells were visualized using a Bio-Rad A1S1 laser confocal microscope (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The TLR9 reagent, CpG (Hycult Biotech, Inc., Plymouth Meeting, PA, USA), was dissolved in DMSO at a storage concentration of 25 mg/ml. This was added to the UCMSC culture medium at 5 µg/ml, the final stimulation concentration, following which the UCMSCs were then seeded into a 6-well plate at 1.5×10⁵ concentration in 2 ml of the medium.

Following the provision of informed consent, human peripheral blood leukocytes (PBLs) were acquired from two healthy donors (aged 25 and 26 years-old; male) and were labeled with carboxyfluorescein succinimidyl ester. The UCMSCs were co-cultured with the labeled PBLs (1:106) for 72 h at 37°C, and cells were collected to detect the proliferation of PBLs using fluorescence-activated cell sorting (FACS; FC500 Cytomics; Beckman Coulter, Inc., Brea, CA, USA).

To detect the surface markers of the MSCs, CD80, CD86, HLA-E, CD90, CD59 and CD29 (eBioscience, San Diego, CA, USA) were used. The UCMSCs treated with the TLR9 agonist and the control group were collected following 72 h stimulation at room temperature and incubated with specific antibodies for 30 min at room temperature. The cells were then analyzed using flow cytometry, gating cells according to fluorescence intensity, also performed in the control group.

Total RNA was extracted from confluent UCMSCs using TRIzol^® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and reverse-transcribed using a ReverTraAce qPCR RT kit (FSQ-101; Toyobo, Co. Ltd., Kagoshima, Japan) to synthesize cDNA under the following conditions: 65°C for 5 min, 37°C for 15 min and 98°C for 5 min. RealMaster mix (SYBR Green; FP202; Tiangen Biotech, Co., Ltd., Beijing, China) was used to perform qPCR on 500 ng cDNA under the following cycling conditions: 95°C for 30 sec, 40 cycles at 95°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec, followed by a melt curve between 55 and 95°C in 0.5°C increments and 10 sec intervals. Primers for IL-1β, IL-6, IL-8, L-10, IL-12, IL-16, IFN-α, IFN-β, IFN-γ, TGF-β, TNF-α, iNOS, IP-10, MCP-1, MIP-1α, M I P-1β, RANTES, MCP-3, P53, c-myc, cdc2, TCAM-1, Selectin-E, VCAM-1, CCL21, CCL24, CCL26 and GAPDH were used. The primer sequences were synthesized by Tsingke Biotech Company (Chengdu, China).

From the two groups of UCMSCs, the supernatant was collected following 4 h stimulation, according to the manufacturer's instructions of the RayBio Human Antibody Array C Series 1000 (RayBiotech Norcross, GA, USA), and the expression of proteins were analyzed.

According to the manufacturer's instructions, a Cell-Light EdU Apollo 567 in vitro kit (RiboBio, Guangzhou, China) was used to assess UCMSC proliferation. The immunostained cells were visualized using a Bio-Rad A1S1 laser confocal microscope (Bio-Rad Laboratories, Inc.). UCMSC migration was examined using a Transwell assay/modified Boyden chamber. Briefly, 1×10⁶ cells were seeded on the upper chamber in 1% serum-free Dulbecco's modified Eagle's medium (DMEM; Invitrogen Life Technologies), with or without the agonists. DMEM (100 ml) supplemented with 10% fetal bovine serum (Invitrogen, Waltham, MA, USA) was added to the lower chamber. Subsequently, non-migratory cells were removed from the upper chamber, and the migratory cells were visualized using a Leica inverted microscope (DM4000B) after staining with crystal violet, and Leica Application Suite Advanced Fluorescence software (Leica Microsystems, Inc., Buffalo Grove, IL, USA).

The cells were harvested with 200 µl radioimmunoprecipitation assay lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS), supplemented with a protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA), following which the immunoreactive protein was detected using electrochemilu-minescence on an X-ray film. Using ImageJ 1.48u software (National Institutes of Health, Bethesda, MD, USA), the resulting autoradiographs were scanned and quantified. The primary antibodies used for western blotting were as follows: Rabbit monoclonal CD29 (1:1,000; cat. no. 1798-1; Epitomics, Burlingame, CA, USA), rabbit monoclonal mitogen-activated protein kinase (MAPK)-p38 (1:1,000; cat. no. 1544-1; Epitomics) rabbit monoclonal β-catenin (1:5,000; cat. no. 1247-1; Epitomics) and mouse monoclonal GAPDH (1:2,000; cat. no. ab8245; Abcam).

To analyze UCMSC differentiation, conditioned media specific for chondrocytes (cat. no. A10071-01; Gibco-BRL), osteocytes (cat. no. A10072-01l; Gibco-BRL) and adipocytes (cat. no. A10070-01; Gibco-BRL) were added to induce osteogenic and adipogenic differentiation of hUCMSCs. The two groups were treated with Oil Red O (Baso Diagnostics, Inc., Zhuhai, China) staining, Alizarin red staining (MeiLianShengWu, Shanghai, China) and safranine staining (Shanghai Kayon Biotechnology Co., Ltd., Shanghai, China) respectively after 3, 10 and 14 days to detect adipogenesis, osteogenesis and chondrogenesis.

Data are expressed as the mean ± standard error of the mean and were analyzed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance was used for multiple comparisons, followed by a Dunnett's test to analyze the significance between two groups. P<0.05 was considered to indicate a st atistically significant difference.

UCMSCs were stimulated by the TLR9-specific ligand CpG in order to detect changes in immune-associated molecule expression. From the UCMSCs treated with TLR9 agonist for varied durations (4, 12, 24, 72 and 120 h), mRNA was isolated, which was assayed using RT-qPCR. The data indicated an increase following TLR9 exposure of several important expressed cytokines and chemokines, including mucosa-associated epithelial chemokine, thymus-expressed chemokine, Leuk, nuclear factor-κB, monocyte chemoat-tractant protein 2, transforming growth factor (TGF-β) and other important immune factors (Fig. 1A). In addition, TLR9 stimulation markedly inhibited the expression of specific stem cell markers, and certain important tumor-associated factors were also induced by CpG.

To examine downstream signaling capabilities, the UCMSCs were stimulated between 4 and 120 h with TLR9 ligands and the results were assessed using western blot analysis. Several important kinase signal proteins, including β-catenin and CD29, as well as two members of the MAPK family, P38, were assessed. Treatment with the TLR9 agonist appeared to enhance the protein level of CD29, particularly at 24 and 72 h post-stimulation (Fig. 1A). Previous studies have confirmed that phosphorylated signal transducer and activator of transcription 3, as the downstream signal pathway of interleukin (IL)-6 (16), MAPK-P38 and β-catenin decreases at different intervals. In MAPK detection in the present study, one important member, p38, exhibited no significant variation following TLR9 stimulation. This observation is significantly different from that of a previous study, which reported that the protein level of MAPK is promoted by TLR agonist treatment (17). The present study also found that the protein level of β-catenin first decreased at the 12- and 24-h intervals, but increased at 72 h (Fig. 1B). This suggested that treatment with the TLR9 agonist treatment did not affect these pathways.

A number of ongoing clinical trials are based on the observation that MSCs can inhibit immune response (21). Therefore, the present study assessed whether this function of MSCs is compromised following addition of the TLR9 agonist. Initially, human PBLs were labelled with carboxyfluorescein succinimidyl ester (CFSE; Multiscience, Hangzhou, China) and co-cultured with UCMSCs, with or without TLR9 activation. The proliferation of PBLs was measured 72 h after co-culture, based on CFSE dilution. These experiments revealed that PBL proliferation did not vary significantly in the presence of the TLR9 agonist, compared with those without TLR9 ligands (Fig. 2A). The expression of co-stimulatory molecules in the UCMSCs exhibited almost no change in response to the TLR9 agonist (Fig. 2B). Therefore, the present results suggested that one of the mechanisms by which MSCs are not rejected or injured in vivo is their contact with TLR9 ligands. Antibody measurement confirmed the variation of secreted proteins (Fig. 2C), which were observed in Fig. 1.

To examine the effect of TLR9 on UCMSC differentiation, TLR9 agonist was added to the culture medium, as UCMSCs were induced by the conditioned medium to differentiate into osteocytes, chondrocytes and adipocytes. Osteocyte differentiation was detected, following supplementation of the cell culture media with osteogenic inducing medium via Alizarin red staining. Adipogenic differentiation, induced by adipogenic medium, appeared under Oilred O staining. UCMSCs grown in culture with chondrocyte-inducing media resulted in chondrogenic differentiation, which was detected by safranine. As shown in Fig. 3, the presence of the TLR9 agonist in the osteogenic medium markedly enhanced the level of calcium deposition, compared with conditioned medium without CpG, particularly 10 and 14 days after differentiation. The control groups exhibited no marked differences in the presence of CpG in adipogenic and chondrogenic media (data not shown).

The Cell-Light EdU Apollo 567 In Vitro kit and the transwell/modified Boyden chamber were used to investigate the proliferation and migration of UCMSCs in the present study. The data suggested that proliferation and migration were inhibited following stimulation by the TLR9 ligands (Fig. 4).