Cell Culture
Mouse MSCs (mMSCs) isolated from the bone marrow of C57BL/6mice (mBM-MSC) were purchased from Cyagen Biosciences, Inc. (Guangzhou, China), and 293T cells were supplied by Zoonbio Biotechnology Co., Ltd. (Nanjing, China). The mBM-MSC were identified by detecting cell surface phenotypes and their multipotent potential for differentiation along the adipogenic, osteogenic, and chondrogenic lineages as previously described 13, 14.
Either mBM-MSC or 293T cells were cultured in a 1:1 mix of Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12) (Wisent, Inc., St-Bruno, Montreal, Quebec, Canada) containing 10% FBS (Wisent, Inc.) and 1% antibiotic-antimycotic (streptomycin, penicillin and amphotericin B; Wisent, Inc.), incubated at 37 °C in a humidified atmosphere of 5% CO2 and passaged every 3–4 days by 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA; Gibco, Carlsbad, CA, USA) when they reached about 80% confluence. Passages between 5 and 10 were used for the experimental protocols.
Recombinant Lentivirus Vector Construction And Package
The full-length coding sequences (CDS) of TGFβ1was transferred into CMV promoter-dependent lentivirus vector PDS159_pL6.3-CMV-GFPa1-IRES-MCS (Zoonbio Biotechnology Co., Ltd.). Subsequently, the lentivectors CL721-pL6.3-CMV-GFPa1-IRES-mus-TGF-β (overexpressing TGFβ1) which co-expressed the enhanced green fluorescent protein (eGFP) and TGFβ1 was obtained, and the empty vector CL721-pL6.3-CMV-GFPa1-IRES was used as an empty vector control. Then, the recombinant plasmid, CL721-pL6.3-CMV-GFPa1-IRES-mus-TGF-β and CL721-pL6.3-CMV-GFPa1-IRES were separately co-transfected with packaging plasmids into 293T cells at the indicated concentrations using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions, producing lentivirus LV402-pL6.3-CMV-GFPa1-IRES-mus-TGF-β and the negative control PDS019.
Lentiviral Vector Transduction And eGFP Reporter Gene Detection
The mBM-MSC (1 × 106/well seeded in six-well cell culture plates) were transduced with viral supernatant at a multiplicity of infection (MOI) value of 160:1 for 24 hours. Then the stable cell lines were harvested after selection using blasticidin (BSD; InvivoGen) at the minimal lethal concentration (6 µg/mL) as previously described 15 and cultured in normal culture media for 20 passages after transduction. Finally, the transduction efficiency of mBM-MSC and the percentage of eGFP positive cells were evaluated by fluorescence microscopy and flow cytometry analysis using a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA).
RNA Isolation And Quantitative Real-time Pcr (qRT-PCR)
Total RNA was isolated from the cells and tissues using TRIzol reagent (Invitrogen, Austin, TX, USA) according to the manufacturer’s protocol, and the purity of the RNA (260/280 nm absorbance ratio of 1.8–2.2) was assessed by a spectrophotometer (Tecan, Switzerland). Reverse transcription was completed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) with 1 mg of RNA according to the manufacturer’s instructions. The qRT-PCR reaction was performed by a CFX96™ Real-Time system (Bio-Rad). Relative changes in gene expression were normalized to the expression of actin and calculated by the 2(-△△Ct) method. The primer sequences used for PCR amplification in our study were designed based on the sequences of the genomic clones and are as follows:
Gene | Primer | Primer sequence | PCR amplified products (bp) |
Actin | Forward | 5’-AGAGGGAAATCGTGCGTGAC-3’ | 195 |
Reverse | 5’-CCATACCCAAGAAGGAAGGCT-3’ |
GAPDH | Forward | 5’-TGTGTCCGTCGTGGATCTGA-3’ | 150 |
Reverse | 5’-TTGCTGTTGAAGTCGCAGGAG-3’ |
TGFβ1 | Forward | 5’-GACTCTCCACCTGCAAGACC-3’ | 100 |
Reverse | 5’-GGACTGGCGAGCCTTAGTTT-3’ |
Collagen I | Forward | 5’-GTGTTTCCTGTGCTACTG-3’ | 132 |
Reverse | 5’-TCTTTCTCCTCTCTGACC-3’ |
α-SMA | Forward | 5’-CCTCGCCTCTACCCCTTA-3’ | 120 |
Reverse | 5’-ATTCGCTTGCCTTTGCTT-3’ |
Western Blot Analysis
To evaluate the TGFβ1 concentration in mBM-MSC, the total cellular protein was extracted by RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing an antiprotease cocktail (1 mmol/L PMSF, 1 mmol/L NaF and 1 mmol/L Na3VO4; US Biological Inc., Swampscott, MA, USA) according to the manufacturer’s instructions, quantified by a BCA protein assay kit (Beyotime), separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) (10%), electro-transferred to PVDF membranes (Millipore, Bedford, MA, USA) and then incubated with primary antibodies against TGFβ1 (1:5000 dilution; Abcam Incorporated, Cambridge, MA) or β-actin (1:3000 dilution; Abcam Incorporated, Cambridge, MA) at 4℃ overnight. The blots were washed three times with TBST and then incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (1:5000 dilution; Zoonbio Biotechnology) for 1 h at room temperature. Immunoreactive complexes were visualized by chemiluminescence reagents (Thermo Fisher Scientific Inc., Waltham, MA, USA) and immunoreactive bands were obtained using a chemiluminescence imaging system (BioshineChemiQ4800 mini; Ouxiang, Shanghai, China). Finally, intensity of those bands was analysed by Image J software (NIH, USA).
Protein Concentration In Culture Medium
mBM-MSC, mBM-MSC-NC, and mBM-MSC-TGFβ1 were seeded in a 12-well plate at a density of 1 × 105 cells per well. After 12 h the culture medium was changed, and mBM-MSC were cultured in an incubator at 37 °C, 5% CO2 for 24 h. The culture medium was then collected and TGFβ1 protein levels in the culture medium were quantified using an enzyme-linked immunosorbent assay (ELISA) kit (Abcam, USA) according to the manufacturer’s instructions.
Cell Proliferation Assay
Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology, Haimen, China) assay was used to further investigate the effects of overexpressing TGFβ1 on mBM-MSC proliferation according to the manufacturer's instructions. Briefly, cells were seeded in 96-well plates at 2 × 103 cells per well in 100 µL of growth medium. After staining with CCK-8 (10 µL per well), the cells were incubated for 4 h at 37 °C. Absorbance was assessed at 450 nm with a microplate reader (Tecan, Switzerland).
Multi-differentiation Of mMSCs After Gene Transduction
For osteogenic differentiation, the cells were seeded in 6-well plates and cultured in 2 mL of DMEM/F12 supplemented with 10% FBS. When reached approximately 80–90% confluence, the cells were switched to C57BL/6 mouse MSC osteogenic differentiation medium (Cyagen Biosciences, Inc., Guangzhou, China) for 2–3 weeks. The calcium deposition was assessed by staining the cells with 40 mM Alizarin Red S solution at room temperature for 10 min.
For adipocytic differentiation, when reached confluence, the cells were treated with mouse MSC adipogenic differentiation basal medium A (Cyagen Biosciences, Inc., Guangzhou, China) for 3 days, followed by exchanged with mouse MSC adipogenic differentiation basal medium B (Cyagen Biosciences, Inc., Guangzhou, China) for 24 h and then switched back to basal medium A. After five to six cycles, the cells were cultured in basal medium B for 3 days until lipid vacuoles enlarged. To assess the accumulation of neutral lipid vacuoles, the cells were stained with filtered Oil red O solution for 10 min at room temperature, and the incorporated Oil red O was extracted by adding 1 mL of isopropanol to each well at room temperature for 15 min.
For chondrogenic differentiation, 2.5 × 105 cells were centrifuged in a 15 mL tube at 150 g for 5 min to form a pellet. Chondrogenic differentiation was processed by the three-dimensional culture method and C57BL/6 mouse MSC chondrogenic differentiation medium (Cyagen Biosciences, Inc., Guangzhou, China). After 28 days, the pellets were embedded in paraffin and then fixed in dimethylbenzene and ethanol. Five micrometer slides were cut and stained with Alcian Blue to determine polysaccharide amine combination.
In vitro scratch assay
The horizontal migration of cells was determined by the in vitro scratch assay. Cells were seeded in six-well culture plates. After reaching approximately 100% confluence, a scratch was made with a 10 µl sterile pipette tip. And then the cells were cultured in serum-free DMEM/F12 for another 12 h. The images of the wound area were recorded by a light microscope immediately after scratching and 12 h later. The horizontal migration ability of the cells was quantified by measuring the wound area in each group by Image J analysis software 13.
Transwell Migration Assay
The vertical migration of cells was determined by the Transwell migration assay. Transwell inserts (6.5 mm diameter and 8 mm pore size; Millipore) which were seeded with 2 × 104 cells in 100µL of serum-free DMEM/F12 were loaded into lower chambers with 600 µL of DMEM/F12 supplemented with 10% FBS. After incubated for 12 h, the cells remaining on the upper surface of the inserts were removed with cotton swabs, and the cells that had migrated to the lower surface were stained with crystal violet (Beyotime Institute of Biotechnology, Haimen, China) for 20 min. The stained cells from four randomly chosen areas were measured under a light microscope 13.
Ethics Statement
All animal experiments in this study were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Southeast University. Wild-type (WT) C57BL/6 mice aged 6–8 weeks were purchased from the Laboratory Animal Centre (Shanghai, China). And mice were housed in individual microisolator cages under specific pathogen-free conditions, with free access to water and chow.
Murine Model Of Lipopolysaccharide-induced Acute Respiratory Distress Syndrome
After anaesthetised with an intra-peritoneal injection of pentobarbital at 50 mg/kg, mice were subjected to intratracheal (i.t.) administration of LPS (2 mg/kg, Escherichia coli serotype 0111: B4; Sigma-Aldrich, St Louis, MO, USA) dissolved in 20 µL sterile normal saline (NS). Sham operation was performed in a similar manner with same volume of only 0.9% NS instead of LPS. Then, the mice recovered until fully awake in a 100% oxygen chamber.
Experimental Protocol
The mice were randomly divided into five groups as follows: Control group, mice received phosphate-buffered saline (PBS) intratracheally4 hours after i.t. administration of 0.9% NS; ARDS group, mice received PBS intratracheally 4 hours after LPS challenge; the LPS + mBM-MSC group received WT mBM-MSC (2 × 105 cells per mouse) intratracheally; the LPS + mBM-MSC-NC group received mBM-MSC-NC (normal control, 2 × 105 cells per mouse) intratracheally; and the LPS + mBM-MSC-TGFβ1 group received mBM-MSC-TGFβ1 (overexpressing TGFβ1, 2 × 105 cells per mouse) intratracheally. The mice were sacrificed at 1d, 3d or 7d after mBM-MSC injection, and the lung lobes were collected for further analysis.
Lung Histopathology Analysis
The right lung lobes (n = 3 for each group at each time-point) were collected and fixed in 4% paraformaldehyde, embedded in paraffin and sliced into 5 µm sagittal sections. After stained with a haematoxylin and eosin staining kit (Beyotime Institute of Biotechnology, Haimen, China), the slices were then viewed by a pathologist based on ten randomly selected high-power fields (400×) in each section according to oedema, alveolar and interstitial inflammation, alveolar and interstitial haemorrhage, atelectasis and necrosis, which was graded on a 0- to 4-point scale (0, no injury; 1, injury in 25% of the field; 2, injury in 50% of the field; 3, injury in 75% of the field; and 4, injury throughout the entire field). The total lung injury score was calculated as the sum of these scores, which has been described previously 14−16.
Preparation Of Lung Tissue Lymphocytes And Flow Cytometry Analysis
After the mice were sacrificed, 5 mL PBS/0.6 mm EDTA was injected into the right ventricular cannula for lung perfusion. The lung was then isolated from the surrounding tissue, and added to the medium containing digestive enzymes (RPMI 1640, 20 mM HEPES, 10% FCS, 175 U/mL collagenase, 75 U/mL DNAse I, 0.2 U/mL pancreatic elastase, 35 U/mL hyaluronidase, 100 IU/mL penicillin, and 100 mg/mL streptomycin) for incubation of 45 minutes at 37 °C. The resulting suspension was passed three times through a 19-gauge needle to break up the clumps and then through a 40 mm filter to remove debris. The leukocytes were enriched by discontinuous Percoll gradient centrifugation and recovered at the interface between 40% Percoll and 70% Percoll layers17.
The following antibodies (Miltenyi, USA) were used for surface and nuclear staining: FITC-labeled anti-CD4, APC-labeled anti-CD25, PE-labeledanti-Foxp3, and PE-labeled anti-RORγt. For the analysis of Tregs, cells were incubated with surface marker antibodiesFITC-anti-CD4 and APC-anti-CD25, followed by fixation and permeabilization with Foxp3 staining buffer (Miltenyi) and intracellular staining with PEanti-Foxp3. To detect the phenotypes of Th17 in lungs, cells were incubated with surface marker antibodiesFITC-anti-CD4 and APC-anti-CD25. After then, the cells were fixed and permeabilized using RORγt staining buffer (Miltenyi), followed by intracellular staining with PE anti-RORγt.
Protein Concentration In Lungs And Bronchoalveolar Lavage Fluid
To analyse the expression of IL-17A, IL-10 and occludin in the lungs after transplantation, total protein lysates were extracted by RIPA lysis buffer (Beyotime) from left lung lobes (n = 3 per group at each time-point) and measured by western blot as previously described. The PVDF membranes were incubated with primary antibodies against IL-17A (1:5000 dilution, Abcam), IL-10 (1:5000 dilution, Abcam), occludin (1:5000 dilution; Abcam) or β-actin (1:3000 dilution; Abcam Incorporated, Cambridge, MA).
Bronchoalveolar lavage fluid (BALF) was collected by flushing 1 mL ice-cold PBS back and forth three times through a tracheal cannula as previously described 14, 15. After centrifugation at 800 × g for 10 minutes, total protein (TP) and albumin (ALB) concentrations in the BALF were measured by ELISA kits (Cusabio Biotech, Wuhan, China).
Labelling And Tracing Of Mesenchymal Stem Cells
WT mBM-MSC, mBM-MSC-NC and mBM-MSC-TGFβ1 were labelled with CellVue NIR815 dye (eBioscience Inc., San Diego, CA, USA) according to the manufacturer’s instructions. Then, NIR815-labelled cells (5 × 105 cells) were directly administered into the trachea of the mice in different groups according to the protocol. After 1d, 3dand 7d post-transplantation, three mice at each time-point were sacrificed, and ex vivo lungs were imaged using a Maestro in vivo optical imaging system (excitation = 786 nm, emission = 814 nm, and 4,000 ms exposure time; Caliper Life Sciences, MA, Boston, USA)15, 16. The autofluorescence spectra were then unmixed based on their spectral patterns using Maestro 2.4 software (Caliper Life Sciences). The fluorescence intensity of the lungs was measured by placing the regions of interest (ROIs) on the lungs, and the signals were analysed based on the total fluorescence counts of the ROIs.
Evaluation Of Lung Oedema
Lung oedema was evaluated using the ratio of lung wet weight to body weight (LWW/BW) that was measured as previously described 16. Briefly, the whole lung was removed and cleared of all extrapulmonary tissues, and the LWW/BW was calculated based on the values of the lung wet weight and the body weight (mg/g).
Lung Fibrosis Analysis
The lung sections were stained sequentially with Weigert’s iron haematoxylin solution, Biebrich scarlet-acid fuchsin solution and aniline blue solution, and a blue signal indicated positive staining for collagen. The criteria of Ashcroft were used 15, 16 to assess lung fibrosis, which was quantified based on the findings in ten randomly selected high-power fields (400×) for each slide by histopathologists blinded to the protocol. Collagen-I and α-SMA mRNA expression in lung tissues was measured by RT-PCR.
Statistical analysis
The data are presented as the means ± standard deviation (SD). Statistical analyses were performed using SPSS 26.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 8 (GraphPad Software, La Jolla California USA). Comparisons among multiple groups were performed by one-way ANOVA followed by Bonferroni’s post hoc test if the data were normally distributed. Kaplan–Meier curves were used to describe the survival rate of mice in each group, and log-rank tests were performed to analyze the significance of differences. A p-value < 0.05 was considered statistically significant.