Human DPSCs isolation and culture
Dental pulp tissues were obtained from healthy human orthodontic molars of adult patients (19-29 years old) at the Dental Clinic of Beijing Stomatological Hospital. This study was conducted under approved guidelines set by the Research Ethics Committee of Capital Medical University, China. All patients gave their written informed consent to participate in the study. The pulp tissues were cut into about 1 mm3 and digested in a solution of 3 mg/ml collagenase type I enzyme (Worthington Biochem, USA) and 4 mg/ml dispase type Ⅱenzyme (Boehringer Mannheim, German) for 1 h at 37℃ with vigorous shaking. Afterward, the digested tissue precipitations by centrifugation were seeded into culture flasks (NEST, USA) with α-MEM supplemented with 20% fetal bovine serum (Every Green, China), 100 units/ml of penicillin and streptomycin (Sigma, USA), and then incubated in 5% CO2 atmosphere at 37℃. Upon reaching 80% confluence, hDPSCs were detached with 0.05 % trypsin (Sigma, USA), and passed with α-MEM supplemented with 10% fetal bovine serum (Every Green, China). The cells at passage 6 (P6) were used in this study, while extra cells were cryopreserved in liquid nitrogen for further use ( -196 ℃).
Adipogenic differentiation of DPSCs
Human DPSCs (P6) were seeded in six-well plates (NEST, USA) at a density of 1×105 cells/well. Then, cultured for 14 days with adipogenic induction medium, supplemented with 0.5mM 3-isobuty-1-1-Methylxanthine, 1μM dexamethasone, and 0.1mM indomethacin (Sigma, USA). Then the cells were fixed in 4% (v/v) phosphate-buffered paraformaldehyde and stained with 0.5% Oil Red O (Sigma, USA) solution. To examine the adipogenic differentiation, the stained cells were observed and photographed under a phase-contrast inverted microscope (Olympus, Japan).
Osteogenic differentiation of DPSCs
Human DPSCs (P6) were seeded in six-well plates (NEST, USA) at a density of 1×105 cells/well. Next, cells were cultured for 21 days with osteogenic induction medium, supplemented with 100nM dexamethasone, 50nM ascorbic acid, and 10 mM β-glycerophosphate (Sigma, USA). Then the cells were fixed in 4% (v/v) phosphate-buffered paraformaldehyde and stained with 1% alizarin red solution. To examine the osteogenic differentiation, the stained cells were observed and photographed under a phase-contrast inverted microscope (Olympus, Japan).
Transfection of DPSCs by Ad-HGF
Adenovirus vector lack of exogenous genes (Ad-Null) and Ad-HGF were used in this study. hDPSCs were infected with 150 multiplicities of infection (MOI) of Ad-Null or Ad-HGF. The Ad-Null transfected DPSCs (Null-DPSCs) and HGF-DPSCs were collected 48 hours post-transfection for use. HGF expression was analyzed by quantitative real time-polymerase chain reaction (Q-PCR) and enzyme linked immunosorbent assay (ELISA).
Immunophenotype analysis of DPSCs and HGF-DPSCs
To characterize the immunophenotype of DPSCs and HGF-DPSCs, their expression of surface markers was analyzed by flow cytometry. Briefly, the tested cells were trypsinized and washed with phosphate buffer saline (PBS). 2×105 cells/tube were harvested and incubated with various antibodies, including human CD73, CD90, CD105, CD45, CD19, CD34, CD11b, HLA-DR in the dark for 30min at room temperature, mouse IgG1 FITC and mouse IgG1 PE were used as isotype controls (BD Bioscience, USA). The analysis was performed with a FACS Calibur flow cytometer (BD Bioscience, USA) and FlowJo software (FlowJo, Ashland, USA).
Imiquimod induced psoriasis like skin inflammation in mice and treatment
All animal experiments were approved by the ethics committee of the Beijing Institute of Radiation Medicine, and all procedures were carried out in accordance with the relevant guidelines and regulations. Female BALB/C mice (18-22 g, 8 weeks old) were held in a specific pathogen free (SPF) animal laboratory house for one week before the experiment. Then, 62.5 mg of imiquimod cream (Med Shine pharmaceutical, China) was applied on the shaved back skin of the mice for 6 consecutive days with or without intravenous tail injection of DPSCs or HGF-DPSCs on day −1. The Blank group was tail intravenous injected of normal saline (Shijiazhuang No.4 pharmaceutical, China). While the Control group was applied with IMQ treatment and tail intravenous injected of normal saline (Shijiazhuang No.4 pharmaceutical, China). The mice were euthanized on day 6, psoriasis like back skin lesions were collected for histological and immunohistochemical analysis and Q-PCR analysis; the blood serums were collected for MILLIPLEX analysis.
Histological and immunohistochemical analysis of the back skins
Sections from the mice back skins were stained with hematoxylin-eosin (H&E) stain for histological evaluation as previously described (34). Briefly, the skin samples were fixed and embedded in paraffin. Tissue sections of 5 μm thick were deparaffinized with xylene and rehydration with a graded ethanol solution, then stained with H&E. All sections were observed and photographed under a phase-contrast inverted microscope (Olympus, Japan), the epidermal thickness of psoriasis like back skin lesions were measured by Image J software (National Institutes of Health, USA)
The above skin sections were analyzed by immunohistochemical stains. Briefly, the sections were incubated with the following primary antibodies at 4 °C overnight: rabbit anti-mouse CD8, CD83, CK6, and CK17 (Bioss, China). The biotinylated goat anti-rabbit secondary antibodies were conjugated to streptavidin-peroxidase, and DAB solution were used for the visualization of immunoreactivity (Bioss, China). All the sections were observed and photographed with a light microscope (Olympus, Japan).
Q-PCR analysis
Q-PCR analysis was performed to measure gene expression of HGF in DPSCs, Null-DPSCs or HGF-DPSCs; T-box transcription factor 21 (T-bet), Interferon-gamma (IFN-γ), GATA binding protein 3 (GATA3), Interleukin-4 (IL-4), Retinoic acid-related orphan receptor-γt (RORγt), Interleukin 17A (IL-17A), Interleukin 17F (IL-17F), Interleukin 23 (IL-23), Forkhead box protein 3 (FOXP3), Interleukin 10 (IL-10) in the skin lesions of all mice. Briefly, total ribose nucleic acid (RNA) from all samples was extracted by using TRIzol Reagent (Invitrogen, USA), according to the manufacturer’s instructions. First-strand complementary deoxyribonucleic acid (cDNA) was synthesized, and the quantification of messenger RNA (mRNA) expression was performed on ABI PRISM 7500 FAST sequence detector (Applied Biosystems). The expression of the above genes was evaluated by Q-PCR using the human primers listing in Table 1 and mouse primers listing in Table 2, with GAPDH as internal control, respectively. The relative gene expression levels were calculated by the 2-ΔΔCT method.
MILLIPLEX analysis
The concentrations of inflammatory cytokines IFN-γ, TNF-α, and IL-17A in the mice blood serum were measured by using a MILLIPLEX kit, according to the manufacturer’s instructions (Mouse Th17 Magnetic Bead Panel, Merck Millipore, USA). Briefly, the background, standard, or sample were mixed with chemically dyed antibody bound beads in the plate wells, incubate with agitation overnight at 4°C, and then incubated with biotinylated detection antibodies for 1 hour and Streptavidin-Phycoerythrin reporter molecules for 30 minutes at room temperature. The data was collected by MAGPI and analyzed with Milliplex Analyst software (Merck Millipore, USA).
Peripheral blood mononuclear cells (PBMCs) isolation
Human PBMCs were isolated from the buffy coats of healthy volunteers by a protocol approved by the Beijing Red Cross Blood Center at Beijing. Briefly, the buffy coat samples were diluted with an equal volume of PBS, then slowly layered upon human lymphocyte separation medium (specific gravity 1.077, TBD sciences, China), and centrifuged at 600 × g for 20 min. PBMCs were collected from the interphase and washed with PBS supplemented with 5% fetal bovine serum (Every Green, China). The isolated PBMCs were cultured in RPMI-1640 medium (Gibco, Germany) supplemented with 10% fetal bovine serum (Every Green, China), 100 units/ml of penicillin, and streptomycin (Sigma, USA).
CD4+ T cell subpopulations detection
To investigate the immunomodulatory effects of DPSCs or HGF-DPSCs on CD4+ T cell differentiation, Mitomycin C (Selleck Chemicals, USA) treated DPSCs or HGF-DPSCs were cocultured with PBMCs and stimulated with CD3/CD28 Streptamers (IBA Lifesciences, Germany) and recombinant human interleukin 2 (IL-2, Roche, USA). Briefly, 2×105 DPSCs or HGF-DPSCs were plated in six-well plates (NEST, USA), and treated with 25 μg/ml Mitomycin C for 30 min at 37°C. PBMCs were suspended in RPMI-1640 medium (Gibco, Germany) supplemented with 10% fetal bovine serum (Every Green, China), 15 μl/ml CD3/CD28 Fab-Strep, 200 units/ml interleukin 2, 100 units/ml penicillin and streptomycin (Sigma, USA) at a concentration of 1×105 cells/ml. The PBMCs were added to six-well plates with or without DPSCs or HGF-DPSCs at a ratio of 1:1, then incubated for 5 days.
The supernatants were collected to measure the production of CD4+ T cell-related cytokines IFN-γ, IL-4, TGF-β1, and IL-6 by ELISA. The PBMCs were harvested and the percentages of CD4+IFN-γ+ Th1 cells, CD4+IL-4+ Th2 cells, CD4+IL-17A+ Th17 cells and CD4+CD25+CD127- Treg cells were analyzed by flow cytometry. Briefly, to detect Th1, Th2, and Th17 cells, the PBMCs were stimulated for 4h with a leukocyte activation cocktail in the presence of GolgiStop (BD Bioscience, USA). Then, the PBMCs were incubated with BB700 conjugated anti-CD4 antibody (BD Bioscience) for 30 min at 4℃ in the dark. Afterward, the cells were fixed and permeabilized, and stained with FITC conjugated anti-IFN-γ antibody, APC conjugated anti-IL-4 antibody, PE conjugated anti-IL-17A antibody (BD Bioscience, USA) for 30 min in the dark at 4℃; to detect Treg cells, the PBMCs were stained with BB700 conjugated anti-CD4 antibody, BB515 conjugated anti-CD25 antibody, and Alexa Fluor 647 conjugated anti-CD125 (BD Bioscience, USA) for 30 min in the dark at 4℃. The stained cells were analyzed by FACS Calibur flow cytometer (BD Bioscience, USA) and FlowJo software (FlowJo, Ashland, USA).
PBMCs proliferation assay
To investigate the immunomodulatory effects of DPSCs or HGF-DPSCs on lymphocyte proliferation, Mitomycin C (Selleck Chemicals, USA) treated DPSCs or HGF-DPSCs were cocultured with Dye 670 (Thermo Fisher Scientific, USA) labeled PBMCs under the stimulation of phytohemagglutinin (PHA, Sigma, USA). Briefly, 2×105 DPSCs or HGF-DPSCs were plated in six-well plates (NEST, USA), and treated with 25 μg/ml Mitomycin C for 30 min at 37°C. PBMCs were labeled with 5μM Dye 670 for 10 min at 37°C, and suspended in RPMI-1640 medium (Gibco, Germany) supplemented with 10% fetal bovine serum (Every Green, China), 10μg/ml PHA (Sigma, USA), 100 units/ml of penicillin and streptomycin (Sigma, USA) at a concentration of 1×105 cells/ml. The PBMCs were added to six-well plates with or without DPSCs or HGF-DPSCs at a ratio of 1:1, then incubated for 3 days. The proliferation index of PBMCs was analyzed by FACS Calibur flow cytometer (BD Bioscience, USA) and ModFit software (Verity Software House, USA).
ELISA analysis
The concentrations of HGF in the culture supernatants of DPSCs or Null-DPSCs or HGF-DPSCs; IFN-γ, IL-4, TGF-β1, and IL-6 in the coculture supernatants of PBMCs with or without DPSCs or HGF-DPSCs were quantitated by anti-human ELISA kits according to the manufacturer’s instructions (Neobioscience Biotech, China). Briefly, all samples were centrifuged at 400 × g for 10 min, then diluted or stimulated respectively. The pretreated samples were added to the test wells and incubated for 90 min at 37℃ to capture the antibodies. Afterward, the test wells were incubated with biotinylated antibodies and avidin HRP successively. The color was developed and stopped by using tetramethylbenzidine substrate solution and stop solution. The plates were read by a microplate manager (Bio-Rad, USA) at 450 nm.
Statistical analysis
All data were expressed as the mean ± SD. The significant difference was assessed by unpaired t-test (p<0.05).