Isolation of rat ESCs
One six-week-old rat was sacrificed by cervical dislocation. The back skin of the rat was removed and placed in a 15-ml centrifuge tube with 1% phosphate-buffered saline (PBS; 10010023; Gibco), and then was placed on ice. The muscle layer was removed, and the skin was cut to a size of 1 × 1cm2 and placed in a sterile 15-ml centrifuge tube. Next, 2 ml of 10× Tryple (A1217702; Gibco) was added, and the sample was digested in a constant-temperature water bath at 37°C for 15–30 minutes with shaking every 3 minutes. Several T25 culture flasks were evenly coated with 1 ml (0.5 mg/ml) of fibronectin (FN; ~5 µg/cm2; Shanghai Fibronectin Biotechnology, Shanghai, China) solution before planting the basal cell suspension, and then were incubated in a 37°C incubator for 20 minutes. After the skin was completely digested, it was rinsed with 1% PBS to stop the digestion. Next, the basal cells were scraped off using a sterile scalpel, rinsed and collected into keratinocyte serum-free medium (K-SFM; 17005042; Gibco), and then the cell suspension was filtered in a 50-ml centrifuge tube using a 200-mesh filter. The cell suspension was transferred to a 15-ml centrifuge tube and centrifuged at 1000 r/min for 10 minutes. The supernatant was discarded, and the pellet was resuspended in 4 ml of complete medium by pipetting. The basal cell suspension was then used to coat the culture flask, which was then incubated in a 37°C incubator for 20 minutes. Approximately 10% of the cells adhered to the wall first; these cells were regarded as ESCs. The ESCs were cultured in K-SFM medium at 37°C, and the medium was changed every 2 days.
Immunofluorescence and confocal microscopy
Cell passaging was performed when the cell density of the second generation of ESCs reached ~90%. Briefly, 1 mL of 0.25% trypsin was added to the flask, which was gently shaken so that the trypsin covered the cell surface evenly and then placed in a cell culture incubated for 3 minutes. After the cells dissociated from the flask wall, 3 mL of complete medium was added to stop digestion. The samples were then centrifuged at 1000 r/min for 5 minutes, the supernatants were removed, and then the pellets were resuspended and cultured in confocal-grade glass-bottomed Petri dishes. After the cells adhered, they were washed 3 times with PBS for 5 minutes each, fixed with 4% paraformaldehyde (P0099-500ml; Beyotime Biotechnology, Shanghai, China) for 20 minutes, washed again with PBS 3 times, and then incubated with 0.5% Triton X-100 (P0096-500ml; Beyotime Biotechnology, Shanghai, China) at room temperature for 20 minutes. The samples were washed with PBS three times, and then 5% goat serum blocking solution (C0265; Beyotime Biotechnology, Shanghai, China) was added to the glass-bottomed Petri dishes, followed by incubation at room temperature for 40 minutes. The blocking solution was aspirated, and then the samples were incubated at 4°C overnight with 100 µl of the following diluted primary antibodies: p63 (1:200; ab735; Abcam), a6-integrin (1:200; ab235905; Abcam), CD71 (1:200; ab22391; Abcam), CK15 (1:200; ab80522; Abcam), CK19 (1:200; ab84632; Abcam) CD31 (1:200; ab24590; Abcam) and CD34 (1:200; ab81289; Abcam). Next, the samples were washed three times with PBS for 5 minutes each. After aspirating the excess liquid from the Petri dishes, the samples were incubated for 1 hour at room temperature in the dark with the following diluted fluorescent secondary antibodies: goat anti-rabbit IgG labeled with Alex Fluor 488 (1:200; ab150077; Abcam), goat anti-mouse IgG labeled with Alexa Fluor 594 (1:200; ab150116; Abcam) and goat anti-rat IgG H&L DyLight® 594 (1:200; ab96889; Abcam). DAPI (D9542; Sigma-Aldrich) was added dropwise to the samples, followed by incubation for 5 minutes in the dark, and then the samples were rinsed 4 times with PBS for 5 minutes each. The supernatant was aspirated, and then the samples were mounted with an anti-fluorescence quencher and observed under a fluorescence microscope, followed by image analysis.
Third-generation rat ESCs were collected by centrifugation, and the supernatant was aspirated. The cells were then resuspended in 0.5 ml of PBS and then were fixed and permeabilized using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit. Next, 2–3 ml of incubation buffer was added to the cells, followed by rinsing by centrifugation. The cells were resuspended in 100 μl of incubation buffer per test tube and were blocked by incubation for 10 minutes at room temperature. Next, the primary antibodies (p63, Abcam, ab124762, 1:200; a6-integrin, Abcam, ab77906, 1:200) were added to the tubes at the appropriate dilution and incubated at room temperature for 60 minutes. Next, the cells were rinsed with incubation buffer by centrifugation. They then were incubated at room temperature for 30 minutes with fluorescent-labeled secondary antibodies diluted in incubation buffer as recommended by the manufacturer. Next, the cells were rinsed in incubation buffer by centrifugation, resuspended in 0.5 ml of PBS, and then subjected to flow cytometry analysis.
Lentivirus and transfection
To generate ESCs with stable enhanced green fluorescent protein (EGFP) expression, the cells were infected with a lentiviral vector encoding the full-length human EGFP gene or empty lentiviral vector as the control (OBiO Technology, Shanghai, China). Stable clones were selected after 2 weeks using 1 µg/ml of puromycin, and the EGFP expression level was determined by immunofluorescence.
To explore the function of ESCs in full-thickness wound beds in vivo, the rat dorsal wound model was adopted. Twenty rats were anesthetized by inhaling isoflurane (INH), and a 2-cm-diameter, full-thickness wound was made on the dorsal skin of each rat. The wounds were divided into 2 groups randomly: control group and ESC group. ESCs that stably expressed EGFP were evenly sprayed on the wound bed using a 2-ml syringe. For the ESC group, 1 ml of cell suspension at a cell density of 1×105/ml was evenly sprayed on the wound bed; for the control group, 1 ml of PBS was sprayed. The rats and wounds were observed, photographed, and measured daily until the rats were sacrificed. The wound healing time was recorded, and the residual wound area rate was calculated as [(day n area)/(day 0 area)] × 100% (n = 0, 3, 7, 14 or 21). Six rats of each group were sacrificed on days 0, 3, 7, 14, and 21, respectively, and the wound tissues were harvested and separated into two halves across the center: one half was processed for histological and immunohistochemistry analyses, and the other was rapidly frozen in liquid nitrogen for western blot analysis.
The paraffin-embedded fixed tissue sections of each group were deparaffinized and rehydrated in xylene and graded ethanol. Antigen retrieval was performed using Proteinase K solution (20 μg/ml) at 37°C for 15 minutes. After blocking with Bloxall, the sections were blocked with goat serum for 30 minutes and then were incubated with primary antibody anti-CD31 (1:100; ab24590; Abcam) overnight at 4°C. After washing in PBST, the sections were then incubated with an HRP conjugated secondary antibody (1:2000; ab97051; Abcam) for 1 hour at room temperature. The sections were further incubated with 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin and observed by microscopy.
Western blot analysis
Western blotting was performed using antibodies directed against CD31 (1:1000; ab24590; Abcam) and GAPDH (Sigma-Aldrich; SAB1405848; 1:6000). GAPDH served as an internal control. The cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Cell Signaling Technology) containing PMSF (1:100; v/v) (Cell Signaling Technology) for 30 minutes. The BCA Protein Assay Kit (Pierce, Thermo Scientific) was used to measure the total protein concentrations. Aliquots (40 µg) of total cellular protein were resolved by SDS-PAGE (10%~12%), electrotransferred to PVDF membranes, and blocked with 5% skim milk (w/v) at room temperature for 1 hour. The membranes were then incubated with primary antibodies on an orbital shaker at 4°C overnight, and secondary antibodies (HRP-conjugated goat anti-mouse and HRP-conjugated goat anti-rabbit) were added and incubated for 1 hour at room temperature. Protein-antibody complexes were then detected by chemiluminescence (Pierce ECL Western Blotting Substrate, Thermo, USA).
Tissue immunofluorescence analysis
Formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed using citrate buffer in a pressure cooker at 95°C for 30 minutes. The 4-μm sections of each group were blocked in 10% goat serum (16210064; Gibco) for 30 minutes at 37°C and then were incubated with the primary anti-rat CD31 antibody (Abcam; ab24590; 1:200). After incubating at 4°C overnight, the sections were washed with PBST and incubated for 1 hour with a goat anti-mouse IgG secondary antibody labeled with Alexa Fluor 594 (1:200; ab150116; Abcam). DAPI was added dropwise and incubated with the sections for 5 minutes in the dark, and then the sections were rinsed 4 times with PBS for 5 minutes each. The remaining liquid in the Petri dish was aspirated, and the sections were mounted with an anti-fluorescence quencher. The sections were analyzed by fluorescence microscopy (OLYMPUS, Japan).
The values were expressed as means ± standard deviation (SD) unless otherwise indicated. Comparisons of the expression difference between the control and experimental groups were conducted by Student’s t test. All statistical analyses were performed using SPSS 20.0 software (SPSS, Chicago, IL, USA), and P < 0.05 indicated that the difference was statistically significant.