UCs (n=10, male newborns, clinically normal pregnancies) were dissected and the blood vessels were removed. The remaining tissues were cut into small pieces (1–2 mm³) and placed in plates with α-minimum essential medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) 2 ng/ml vascular endothelial growth factor (VEGF; R&D Systems, Inc., Minneapolis, MN, USA), 2 ng/ml epidermal growth factor (EGF; R&D Systems, Inc.) and 2 ng/ml fibroblast growth factor (FGF; R&D Systems, Inc.). Cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂ in air. The culture medium was changed every 3–4 days. Adherent cells proliferated from individual explanted tissues 7–12 days after incubation. These cells were subsequently trypsinized using 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc.) and cultured at a density of 1×10⁴ cells/cm² in the medium described. The cells were used at ≤5 passages. The present study was approved by the Human Research Ethics Committee of Qilu Hospital (Jinan, China). Informed consent was obtained from all donors.

The proliferation of UC-MSCs at passage 4 was measured. The cells (6,000 cells/cm²) were seeded into 24-well culture plates and allowed to adhere. Different concentrations (0.1, 0.5, 1, 5, 10 and 20 ng/ml) of TGF-β1 (R&D Systems, Inc.) were then added to the plates. The plates were maintained at 37°C in a humidified atmosphere of 5% CO₂ and 95% air for 24, 48 or 72 h. Proliferation was measured after these incubations using a Cell Counting kit-8 assay (Beyotime Institute of Biotechnology, Haimen, China). The average optical density values of triplicate measurements were calculated and used for comparisons.

MSCs were cultured in 6-well plates at a density of 3×10⁵ cells/well in 2 ml culture medium. Following cell adherence, the cells were incubated for 24 h in different concentrations of TGF-β1 (0.1, 1 or 10 ng/ml). The total RNA was extracted from each sample using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to analyze the expression levels of ECM genes, including Col-I, Col-IV, FN, LN, integrin and tenascin-C, and ECM-associated genes, including matrix metalloproteinase (MMP) -1, MMP-2 and MMP-9, and tissue inhibitor of metalloproteinase (TIMP) -1 and TIMP-2. cDNA was generated from the total RNA via RT using an Omniscript cDNA Synthesis kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's instructions. Typically, 0.2 µg total RNA was reverse transcribed in a 20 µl final reaction volume containing the following components: 1X RT buffer, deoxynucleotide triphosphate mix (5 mM), RNase inhibitor (RNaseOUT; 10 U/µl; Invitrogen; Thermo Fisher Scientific, Inc.), oligo dT primers and 4 units Omniscript RT. The samples were incubated at 37°C for 60 min, and the resulting cDNA was stored at −80°C prior to RT-qPCR analysis using an ABI 500 PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and SYBR green I dye (Toyobo Co., Ltd., Osaka, Japan). The primers used are listed in Table I. All reagents and primers were obtained from Bioasi Co., Ltd. (Shanghai, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The expression of each gene was determined using the 2^−ΔΔCq method. The qPCR conditions were as follows: 1 cycle at 95°C for 4 min; 1 cycle at 94°C for 15 sec; and 1 cycle at 60°C for 1 min; for a total of 40 cycles.

The data were analyzed using Sequence Detection software (version 1.4; Applied Biosystems; Thermo Fisher Scientific, Inc.). The data are presented as the mean ± standard deviation of ≥3 independent experiments. mRNA expression is presented as the fold difference with respect to the untreated control groups and the control group values are set at a fold change equal a value of 1.

Based on the above experimental results, 0.1 ng/ml was the concentration of TGF-β1 most effective at promoting the proliferation of MSCs and ECM gene transcription in MSCs. Thus, all subsequent experiments assessing MSC gene expression used TGF-β1 at a concentration of 0.1 ng/ml.

The cells were collected and treated with 0.25% trypsin. The cells were blocked with phosphate-buffered saline (PBS) containing 1% FBS for 10 min at room temperature in the dark. Subsequently, the cells were incubated with either fluorescein isothiocyanate- or phycoerythrin-conjugated monoclonal antibodies (single labeling experiments) in 100 µl phosphate buffer, for either 15 min at room temperature or 30 min at 4°C, as recommended by the manufacturers. Antibodies against human cluster of differentiation (CD)45 (cat. no. 10894), CD31 (cat. no. 16284), CD34 (cat. no. 28906), CD29 (cat. no. 05556), CD44 (cat. no. 16070), CD73 (cat. no. 21589), CD90 (cat. no. 15421) and CD105 (cat. no. 17813) were purchased from AbD SeroTec (Raleigh, NC, USA) and used at a 1:10 dilution. Immunofluorescence was detected using flow cytometry (Guava easyCyte 6HT; EMD Millipore, Billerica, MA, USA) and the data were analyzed using Guava Incyte (version 2.8; EMD Millipore). Positive cells were counted and compared with the signal of corresponding immunoglobulin isotypes.

To investigate the differentiation potential of MSCs in response to administration of TGF-β1, MSCs at passage 4 were treated with 0.1 ng/ml TGF-β1 for 24 h prior to inducing the differentiation of each lineage. The cell density was adjusted to 2×10⁴ cells/cm², and the inducing medium was changed every 3–4 days for differentiation assays. The osteogenic differentiation medium consisted of low-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 0.1 µM dexamethasone, 50 mM β-glycerol phosphate and 0.2 mM ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA). Adipogenic differentiation medium consisted of high-glucose DMEM supplemented with 0.25 mM 3-isobutyl-1-methyl-xanthine, 0.1 µM dexamethasone, 0.1 mM indomethacin (Sigma-Aldrich), 6.25 µg/ml insulin (PeproTech EC Ltd., London, UK) and 10% FBS. Cells were maintained in regular culture medium served as a control.

Mineralized matrix was evaluated by fixing the cells with 4% formaldehyde and staining with 1% alizarin-red S (Sigma-Aldrich) solution in water for 10 min. Alkaline phosphatase (ALP) staining was performed using an ALP staining kit (Tuo Yang Biotechnology Co., Ltd., Shanghai, China), according to the manufacturer's recommendations. Briefly, the cells were fixed in 4% paraformaldehyde and incubated with a substrate buffer at room temperature for 30 min. For oil red O staining, the cells were fixed with 4% formaldehyde, stained with oil red O (Sigma-Aldrich) for 10 min, and then counterstained with Mayer's hematoxylin (Sigma-Aldrich) for 1 min. Then, images were obtained with a light microscope (Olympus BX53; Olympus Corporation, Tokyo, Japan) fitted with a digital camera (Olympus cellSens Standard; Olympus Corporation).

For immunocytochemical analyses, the cells were cultured in 6-well plates at a density of 3×10⁵ cells/well in 2 ml culture medium. Following cell adherence, the cells were incubated for 24 h with 0.1 ng/ml TGF-β1. Subsequently, the cells were fixed in 4% paraformaldehyde and washed in 0.01 M PBS (pH 7.2–7.4). Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in absolute methanol for 30 min. Prior to the immunocytochemical procedure, proteolytic treatment was applied using 0.4% pepsin in 0.01 M HCl for 30 min at 37°C. Non-spicific binding was blocked using 5% goat serum (Beijing Kang Century Biotechnology Co., Ltd., Beijing, China). Primary anti-Col-I (cat. no. ab34710), -Col-IV (cat. no. ab6586), -FN (cat. no. ab45688) and -integrin (cat. no. ab15459) antibodies (all obtained from Abcam, Cambridge, UK) were added to two wells of each group at 1:300 dilution, and allowed to bind for 1 h at 37°C. Peroxidase-conjugated secondary antibody (cat. no. CW0220S; Beijing Kang Century Biotechnology Co., Ltd.) were added for 30 min at room temperature. The cells were washed three times in PBS, incubated with diaminobenzidine chromogen (Biocare Medical, LLC., Concord, CA, USA) for 10 min at room temperature, and then counterstained with hematoxylin.

Following stimulation with 0.1 ng/ml TGF-β1 for 24 h, the cells were washed twice with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 8.0); 150 mM NaCl; 1% Nonidet P-40; 0.5% deoxycholate and 0.1% sodium dodecyl sulphate (SDS)], containing protease and phosphatase inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). Protein concentrations in cleared cell lysates were determined using a Bradford assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein extracts (3 µg/µl) were subjected to 7% SDS-polyacrylamide gel electrophoresis and transferred onto Immobilon-P membranes (EMD Millipore). The membranes were blocked with 5% milk in Tris-buffered saline Tween-20 [20 mM Tris-HCl (pH 7.6); 137 mM NaCl and 0.1% Tween-20] at room temperature for 1 h. Subsequently, the membranes were incubated with the following primary antibodies in blocking buffer (1:1,000) at 4°C for 16 h: Monoclonal rabbit anti-RhoA (clone 67B9; cat. no. 2117), anti-SMAD3 (clone C69H7; cat. no. 9523), monclonal rabbit anti-phospho-SMAD3 (Ser423/425; clone C25A9; cat. no. 9520) and anti-GAPDH (clone D16H11; cat. no. 5174), all from Cell Signaling Technology, Inc. Danvers, MA, USA. Following incubation with horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. CW0103S; Beijing Kang Century Biotechnology Co., Ltd.), immunoreactive bands were detected by electrochemiluminescence (Beyotime Institute of Biotechnology) and quantified using a C-DiGit blot scanner (model 3600; Li-cor, Lincoln, NE, USA) and Image Studio software (version 4.0; Li-cor).

Female Sprague-Dawley rats (weighing 240–280 g) obtained from Shandong University Experimental Animal Centre (Jinan, China) were randomly divided into four groups (n=16/group) as follows: i) Control; ii) ALI/PBS; iii) ALI/MSC; iv) ALI/MSC + TGF-β1. The animals were maintained on a standard 12-h light/dark cycle, at a constant temperature (22±2°C) and a relative humidity of 55±10% with free access to water ad libitum and standard laboratory rodent food. ALI was induced by intraperitoneal injection of LPS (10 mg/kg) from Escherichia coli O111:B4 (Sigma-Aldrich). After 1 h, the rats were administered with either MSCs (MSCs or MSC + TGF-β1; 5×10⁵ cells in 300 µl PBS) or 300 µl PBS (as the control) via injection into the tail vein. For the MSC + TGF-β1 group, MSCs were stimulated with 0.1 ng/ml TGF-β1 for 24 h prior to injection. At each time point (6, 24 and 48 h post-injection of LPS), three rats from each group were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg) and sacrificed to assess lung injury. All procedures were approved by the Animal Care and Use Committee of Shandong University (Jinan, China).

The rats were sacrificed, the trachea was isolated and the right bronchial tube was ligated. BALF was obtained by placing a 20-gauge catheter into the trachea, through which 3 ml cold PBS was flushed back and forth three times. BALF was centrifuged at 2,400 × g for 20 min at 4°C. A counter (Beckman Coulter, Inc., Brea, CA, USA) was used to determine the total cell count in the resulting cell pellet. The cells were smeared with Wright-Giemsa stain to confirm the neutrophil percentage. The protein concentration of cell-free BALF from all groups was measured using a Bio-Rad protein assay kit (Bio-Rad Laboratories), and was used to indicate endothelial and epithelial permeabilities.

The right upper lung lobes were used for wet-dry analysis to quantify the magnitude of pulmonary edema. The lungs were placed into previously weighed microcentrifuge tubes and were subsequently weighed. Subsequently, tissues were desiccated under a vacuum overnight at 80°C and weighed again. The wet lung mass was divided by the dry lung mass to obtain the wet-dry ratio. The right middle lung lobes were stored in TRIzol at −20°C to detect male UC-MSC survival in vivo. The right lower lobes were used for histological evaluation. Paraffin-embedded lungs were cut into 5 µm thick sections and were subsequently stained with hematoxylin and eosin for histological analysis.

To measure male MSC survival in vivo, 27 female Sprague-Dawley rats were randomly divided into three groups (n=9/group) as follows: i) ALI/PBS, ii) ALI/MS; iii) ALI/MSC + TGF-β1. ALI was induced as described, and the rats were administered with either male MSCs (MSC or MSC + TGF-β1; 5×10⁵ cells in 300 µl PBS) or 300 µl PBS via injection into the tail vein. At each time point (days 1, 7 and 14 post-injection of LPS), three rats from each group were anesthetized and sacrificed, as previously, to assess MSC survival. The survival of male donor human MSCs, which expressed the sex-determining region Y (Sry) gene, in the female recipient rat lung was calculated using RT-qPCR. Different numbers of male MSCs (100, 50, 25, 10, 5, 1 and 0×10³ MSCs/10 mg lung tissue) were added to female DNA (from rat lung tissue) as standards, to allow the construction of a calibration curve. Sry expression levels were quantified by normalizing the values relative to the rat housekeeping gene, GAPDH. The number of stem cells was calculated according to the calibration curve, which plotted the cycle threshold (Cq) of Sry expression against the number of serially diluted MSCs (26).

The data were analyzed using SPSS software (version 14.0; SPSS Inc., Chicago, IL, USA). Quantitative data are presented as the mean ± standard deviation. Analysis of variance with Fisher's protected least significant difference as a post hoc analysis was used for multigroup comparisons. P<0.05 was considered to indicate a statistically significant difference.

Adherent cells with fibroblastic morphology were observed as early as 48 h using the tissue-explant adherent method (Fig. 1A). The cells formed a monolayer of homogeneous bipolar spindle-like cells with a whirlpool-like array within 2 weeks. Following several passages, adherent cells from the UC formed a monolayer of typical fibroblastic cells (Fig. 1B).

The concentration-dependent effects of TGF-β1 on UC-MSC proliferation were further investigated (Fig. 1C). The results suggested that exposure to 0.1 ng/ml TGF-β1 was optimal for enhancing UC-MSC proliferation from 24 h after treatment, with a significant effect on proliferation after treatment for 72 h compared with control (P=0.0241). Higher concentrations of TGF-β1 demonstrated minimal effects on UC-MSC proliferation. Thus, all subsequent experiments investigating the effects of TGF-β1 on MSCs utilized a TGF-β1 concentration of 0.1 ng/ml, and the TGF-β1 treatment group was denoted as MSC + TGF-β1.

Flow cytometry results (Fig. 2) demonstrated that the UC-derived cells exhibited a similar immunophenotype to MSCs, including positive expression of stromal markers (CD29, CD44, CD73, CD90 and CD105), and negative expression of endothelial (CD31) and hematopoietic (CD34 and CD45) markers. Treatment with 0.1 ng/ml TGF-β1 demonstrated no significant effect on the percentage of cells expressing each marker (Table II).

MSCs treated with TGF-β1 retained the ability to differentiate into osteoblasts and adipocytes. When induced to differentiate under osteogenic conditions, the TGF-β1-treated MSCs began to grow in clusters with increasing induction time, and the cells formed a mineralized matrix, confirmed by alizarin red staining (Fig. 3A). The majority of the cells became alkaline phosphatase-positive by day 14 (Fig. 3B). No mineralized matrix was observed in cells maintained in regular growth medium (Fig. 3E). The spindle shape of TGF-β1-treated MSCs flattened and broadened after 1 week of adipogenic induction (Fig. 3C). Small oil droplets gradually appeared in the cytoplasm, and after 2 weeks, almost all cells contained numerous oil red O-positive lipid droplets (Fig. 3D). The observed morphological changes and staining characteristics indicated that treatment with TGF-β1 caused no affect on the ability of UC-MSCs to undergo osteogenic and adipogenic differentiation.

The effect of TGF-β1 on the mRNA expression levels of ECM components and ECM-associated molecules in cultured UC-MSCs was also investigated. RT-qPCR assays were performed following treatment of UC-MSCs with various concentrations of TGF-β1 for 24 h. A low concentration of TGF-β1 (0.1 ng/ml) significantly increased the expression level of five ECM genes (Col-I, Col-IV, FN, integrin and tenascin-C; P=0.0387, 0.0219, 0.0274, 0.0402 and 0.0336, respectively), whereas TGF-β1 at 1 ng/ml significantly increased only Col-I expression (P= 0.0057; Fig. 4A). Furthermore, higher concentrations of TGF-β1 (1 and 10 ng/ml) significantly inhibited LN (P=0.0004 and 0.0023, respectively) and integrin (P=0.0011 and 0.0048, respectively) expression levels (Fig. 4A). Regarding the ECM-associated genes, including MMP-1, MMP-2 and MMP-9, the mRNA expression levels were maximally increased by TGF-β1 at a concentration of 1 ng/ml (Fig. 4A). Treatment with 0.1 ng/ml TGF-β1 significantly inhibited MMP-1 expression in UC-MSCs (P=0.0012). The mRNA expression levels of TIMP-1 and -2 were not significantly altered by TGF-β1 at any concentration (Fig. 4A). Notably, high concentrations of TGF-β1 (10 ng/ml) did not increase the mRNA expression levels of various ECM components and ECM-associated molecules, except MMPs.

Based on these data, a TGF-β1 concentration of 0.1 ng/ml was selected to examine the effects of TGF-β1 on ECM protein expression. The expression levels of four ECM components (Col-I, Col-IV, FN and integrin) were examined by immunocytochemistry prior to and following treatment with TGF-β1 (Fig. 4B). UC-MSCs in the TGF-β1 group maintained their morphology compared with the control group. MSCs in the untreated control group demonstrate no immunoreactivity toward Col-I, Col-IV or integrin. Furthermore, 0.1 ng/ml TGF-β1 increased the protein expression levels of Col-I, Col-IV and, in particular, FN compared with the untreated control. However, TGF-β1 failed to observably increase the protein expression of integrin.

To further investigate the mechanisms underlying the effects of TGF-β1 described above, the expression levels of RhoA and SMAD3, which are important genes in FN synthesis, were analyzed. Western blot analysis demonstrated that 0.1 ng/ml TGF-β1 significantly increased the expression level of RhoA compared with control MSCs (P=0.0215), whereas the total expression level of SMAD3 was not significantly altered. However, the levels of phosphorylated SMAD3 were significantly higher in TGF-β1-treated MSCs compared with the control MSCs (P=0.0240; Fig. 5).

Animals receiving LPS exhibited physical signs of systemic illness, including lethargy, piloerection and diarrhea. Following intraperitoneal injection of LPS, the capillaries in the lung tissue expanded and became congested by a significant increase in neutrophils compared with untreated rats (Fig. 6A and B). Histological assessment of the lungs of animals injected with LPS demonstrated various pathological changes associated with ALI, including lung tissue hyperemia, hemorrhage, infiltration of inflammatory cells and neutrophil accumulation. These results indicated that the rat model of ALI was successfully developed. Additionally, the lung septa were observably thickened, and exhibited no improvement at the 48 h time-point (Fig. 6A and B). The MSC + LPS and MSC + TGF-β1 + LPS groups also exhibited signs of moderate injury, however, the severity of the injury was observably reduced compared with the LPS alone group at the same time points (Fig. 6C and D).

The lung wet-dry weight ratio was significantly elevated at 24 and 48 h after LPS administration compared with the normal group (P<0.05), with the ratio marginally lower at 48 h compared with 24 h. However, the administration of MSC or MSC + TGF-β1 significantly attenuated these increases in lung wet-dry weight ratio at 24 h compared with the ALI group (P=0.0148 and 0.0102, respectively), while at 48 h only MSC + TGF-β1 administration significantly attenuated the wet-dry weight ratio compared with the ALI group (P=0.0144; Fig. 6E). LPS injection also resulted in significant increases in the BALF neutrophil count (P=0.0207, 0.0153 and 0.0119 for 6h, 24h and 48h, respectively; Fig. 6F) and protein concentration (an indicator of endothelial and epithelial permeability; P=0.0298, 0.0131 and 0.0243 for 6h, 24h and 48h, respectively; Fig. 6G) compared with the normal group, both of which peaked at 24 h. The increase in BALF neutrophil count at 24 h was significantly reduced in the MSC-treated group compared with the ALI group (P<0.05), and although the count was also numerically lower in the MSC + TGF-β1-treated group, this did not reach statistical significance (Fig. 6F). Furthermore, BALF protein concentration at 24 h was significantly lower in the MSC-treated and MSC + TGF-β1-treated groups compared with the ALI group (P=0.0155 and 0.0130, respectively; Fig. 6G).

To characterize the survival of transplanted MSCs in damaged lungs, a calibration curve was established to calculate the number of MSCs in the recipient lung compared with Sry gene expression (Fig. 7A). Cq values of the Sry gene were plotted against a semi-logarithmic scale of MSC number to obtain a balanced contribution of all reference dilutions. Subsequently, lungs (3/group) were harvested from rats at 1, 7 and 14 days after their respective treatment. The number of MSCs was calculated from the above calibration curve. The expression of the Sry gene in the lungs at each time-point was significantly higher in the MSC + TGF-β1 group compared with the MSC group. TGF-β1 pre-treatment (0.1 ng/ml) significantly increased MSC survival in damaged lungs at day 14 compared with untreated MSCs (P=0.0326; Fig. 7B). Sry expression was not detected in the control animals that were administered with medium without MSCs.