UCMSCs were isolated from the umbilical cord as previously described (21). Briefly, the umbilical cord was diced into sections following disinfection in 75% ethanol for 10–20 min. The small sections of mesenchymal tissues were placed into 25 mm² plates. The culture medium was added when the tissues tightly adhered to the bottom of the plates. The dissociated MSCs were collected approximately 5–8 days after the mesenchymal tissues were removed. The dissociated MSCs were either maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both from Invitrogen, Carlsbad, CA, USA) at 1×10⁵ cells/well in a 6-well plate (Falcon, Bedford, MA, USA) or stored in liquid nitrogen for later use. This study was approved by the Ethics Committee of West China Hospital, Chengdu, China.

The TLR3 agonist, polyinosinic:polycytidylic acid [poly(I:C); P1530], and the TLR4 agonist, LPS (L2880), were both purchased from Sigma-Aldrich (St. Louis, MO, USA) and prepared as recommended by the manufacturer.

Total RNA was isolated using an RNeasy kit (Qiagen, Dusseldorf, Germany). cDNA was synthesized using the ReverTra Ace qPCR RT kit (FSQ-101; Toyobo, Kagoshima, Japan), and the reverse transcription conditions were as follows: 65°C (5 min), 37°C (15 min) and 98°C (5 min). Reactions were performed using RealMaster Mix (SYBR-Green; FP202; Tiangen Biotech, Beijing, China). RT-PCR was performed in an iCycler iQ™ Optical Module (Beckman Coulter, Fullerton, CA, USA) under the following conditions: 95°C for 30 sec, 95°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec, followed by a melt curve from 55 to 95°C in 0.5°C increments and 10 sec intervals for 40 cycles. The primers used are listed in Table I. All amplifications were performed 3 times.

Supernatants were collected from the TLR3 and TLR4 agonist-treated and untreated UCMSCs at 4 h post-treatment and screened for secreted protein using RayBio Human Antibody Array C Series 1000 (RayBiotech, Norcross, GA, USA) according to the manufacturer’s instructions.The blots were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). A total of 20 molecules were selected for detection: alpha-fetoprotein (AFP), albumin, E-selectin, intercellular adhesion molecule 1 (ICAM-1), interferon (IFN)-α, IFN-γ, interleukin (IL)-10, IL-12, IL-18, IL-1β, IL-4, IL-5, IL-6, IL-8, monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1α, Notch-1, transforming growth factor (TGF)-β and vascular endothelial growth factor (VEGF).

Peripheral blood leukocytes (PBLs) were isolated from healthy volunteers (after obtaining written informed consent) by density centrifugation gradient and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) at 37°C for 10 min. The reaction was terminated by the addition of ice-cold DMEM supplemented with 10% FBS, and excess the CFSE was removed with ice-cold phosphate-buffered saline (PBS). The labeled PBLs (1×10⁶) were then co-cultured with the isolated UCMSCs for 72 h and then analyzed by FACS. All the experiments were approved by the Human Research Protection Program at our institution.

The PBLs were co-cultured with the UCMSCs and stimulated with the TLR3 and TLR4 agonists. Supernatants were then collected from the treated and untreated groups 72 h post co-culture and the LDH levels in the cell supernatants were measured using a cytotoxicity detection kit (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer’s specifications. Cytotoxicity (percent lysis) was calculated using the following formula: 100 x (E-M)/(T-M), where E is the experimental release, M is the spontaneous release in the presence of medium alone, and T is the maximum release in the presence of 5% Triton X-100.

The TLR3 and TLR4 agonist-treated and untreated UCSMCs were treated with either TLR-3 or TLR-4 or left untreated for 72 h. At this point, cells were harvested and stained for the co-stimulator and surface markers listed in Table II using monoclonal antibodies. Following staining, the cells were analyzed by flow cytometry using a FACScan flow cytometer (Beckman Coulter). The cells were gated according to the fluorescence intensity of the control group.

The UCMSCs were cultured in conditioned medium specific for osteocytes (A10072-01; Gibco, Carlsbad, CA, USA) and the TLR3 and TLR4 agonists according to the manufacturer’s specifications. Media was replaced every 3 days and treated and untreated cells were stained after 3, 10 and 14 days in culture using Alizarin red.

The TLR3 and TLR4 agonist-treated UCMSCs were washed twice with cold PBS and proteins were extracted using a standard mammalian protein extraction reagent (Pierce, Rockford, IL, USA) containing protease inhibitors (Roche Applied Science). Lysates were clarified by centrifugation at 14,000 rpm for 10 min at 4°C. The protein concentration was measured using the Micro BCA Protein Assay kit (Pierce). Protein lysates (20 μg) were resolved on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred onto nitrocellulose membranes (Invitrogen). The blocked membranes were incubated with primary antibodies (Table III), washed and then incubated with a secondary antibody conjugated to horseradish peroxidase (1:5,000; Abcam, Cambridge, UK) for 1 h at room temperature. Antigen-antibody complexes were visualized using an enhanced chemiluminescence reagent (Amersham Biosciences, Fairfield, CT, USA).

The RT-qPCR and antibody chip data were analyzed using Bio-Rad iQ5 software (Bio-Rad, Hercules, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used as an internal control. The results are expressed as the mean values ± standard error of the mean using SPSS 16.0 software (IBM SPSS Statistics, New York, NY, USA). Values of P<0.05 and P<0.001 were considered to indicate statistically significant differences compared to the control group. The figures were completed using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA).

The UCMSCs were treated with the TLR3 agonist, poly(I:C), and the TLR4, agonist LPS, and the expression of pro-inflammatory cytokines [IFN-β, TGF-β, IP-10 and CDC2], chemokines (CCL1, CCL2, CCL4 and CCL13) and interleukins (IL-1β, IL-8, IL-10 and IL-12) was examined at 4, 12, 24, 72 and 12 h following treatment with the ligands. We found that treatment with TLR3 agonist induced the expression of IFN-β, TGF-β, CCL1, CCL4, IL-10 and IL-12 to varying extents different kinetics as shown in Fig. 1A–C. In addition, treatment with the TLR3 agonist inhibited the expression of CDC2, CCL2 and IL-8 at different time points. Lastly, treatment with the TLR3 agonist induced the expression of IP-10 and IL-1β, but the expression levels decreased at later time points as shown in Fig. 1A–C.

By contrast, treatment with the TLR4 agonist downregulated the expression of CDC2 and IFN-β. However, the expression of TGF-β, IL-1β, IL-8, IL-10 and IL-12 increased significantly at different time points post-treatment (4, 12, 24, 72 and 120 h). Other markers, such as IP-10, CCL1, CCL2, CCL4 and CCL13 showed a slight variation in their expression levels following treatment with LPS (TLR4 agonist; Fig. 1A–C).

Stem cell marker expression was measured to confirm whether treatment with poly(I:C) or LPS influences the stemness of UCMSCs. We found that the expression of stem cell markers [Krüppel-like factor 4 (Klf4), muscle segment homeobox (MSX), Nanog and Sox2] increased following treatment with poly(I:C). In addition, treatment with LPS treatment markedly inhibited the expression of Klf4, MSX and Nanog, but increased the expression of Sox2 (Fig. 1D). Therefore, the activation of TLR3 and TLR4 induced the expression of pro-inflammatory molecules and TLR3 increased the stemness of UCMSCs, but TLR4 inhibited it.

Immune-related molecules in the supernatants of the treated UCMSCs were measured using the RayBio antibody chip. We then observed increased levels of IL-1β, IL-10 and IFN-γ in the supernatants of the UCMSCs treated with poly(I:C) or LPS (Fig. 2). The levels of TGF-β, MCP-3 and VEGF were increased only following treatment with LPS. In addition, the levels of MIP-1α and IFN-α were increased only following treatment with poly(I:C) (Fig. 2). Together, these results confirm that the activation TLR3 and TLR4 induces the secretion of pro-inflammatory molecules in UCMSCs.

The majority of the MSC-related clinical trials have been based on the assumption that MSCs can inhibit the immune response. In order to determine whether the immune suppressive function of MSCs can be compromised following treatment with TLR3 and TLR4, CFSE-labeled allogeneic human PBLs were co-cultured with UCMSCs treated or not with TLR3 and TLR4 ligands. The results revealed that the proliferation of the PBLs was not significantly affected following incubation with either the TLR3 or TLR4 agonist (Fig. 3A).

At this point, we wished to determine whether PBLs exert an immune attack against UCMSCs by measuring the levels of LDH in the cell supernatants from the co-culture of PBLs and UCMSCs. We found that the LDH levels remained relatively unaltered in both the poly(I:C)- and the LPS-treated groups at both 24 and 48 h post-treatment (Fig. 3B). However, the activation of TLR3 and TLR4 induced the leakage of LDH in the PBL-UCMSC co-cultures compared to the control at 72 h (Fig. 3B). Therefore, the activation of the TLR3 and TLR4 pathways did not promote the rejection of UCMSCs through the TLR3 and TLR4 dependent pathway.

The data from our experiments suggested that the TLR3 and TLR4 agonists did not induce immune rejection of the UCMSCs, although they induced the expression of pro-inflammatory molecules. Therefore, we then assessed whether the activation of TLR3 and TLR4 upregulates the expression of co-stimulatory molecules, which play an important role in immune responses. We found that 2 major co-stimulatory proteins, CD80 and CD86, were unaffected following treatment with the TLR3 agonist (Fig. 4A). However, the TLR4 ligand increased the expression of CD86 (21.6%) compared to the control (3.1%), but did not affect CD80 expression (Fig. 4A). Similarly, the levels of the MSC-specific surface markers, CD74 and CD105, in the UCMSC following the activation of TLR3 and TLR4 were unaffected (Fig. 4B).

Since TLR3 increased the expression of stem cell markers, but TLR4 inhibited them, we hypothesized that TLR3 and TLR4 play a role in the differentiation of UCMSCs. Poly(I:C) and LPS were added to the conditioned medium and osteocyte differentiation was measured by Alizarin red staining. We found that the TLR3 agonist markedly inhibited the differentiation of UCMSCs into osteocytes, as indicated by the calcium deposition (Fig. 5). By contrast, the TLR4 agonist promoted the differentiation of USMSCs into osteocytes (Fig. 5).

To elucidate the signaling pathways that are activated by the TLR3 and TLR4 agonists, we probed for signaling pathway proteins in the UCMSCs following treatment with poly(I:C) and LPS. We found that treatment with the TLR3 agonist markedly increased β-catenin and interleukin-1 receptor-associated kinase 4 (IRAK4) levels in the UCMSCs. By contrast, the activation of TLR4 by LPS activated the phosphorylated signal transducer and activator of transcription 3 (pSTAT3) and p38 pathways (Fig. 6).