Isolation of rat ESCs
One six-week-old rat was sacrificed by cervical dislocation. The back skin of the rat was taken and placed in a 15 ml centrifuge tube with 1% phosphate buffered saline (PBS; 10010023; Gibco), and then transported in an ice box to the ultra-clean bench in the laboratory. Remove the muscle layer in the ultra-clean table, cut the skin to a size of 1 × 1 cm2 and place it in a sterile 15 ml centrifuge tube, add 2 ml 10xTryple (A1217702; Gibco), digest in a constant temperature water bath at 37 °C for 15–30 minutes, shaking every 3 minutes. Take several T25 culture flasks, evenly coat the culture flask (about 5ug/cm2) with fibronectin (FN; Shanghai Fibronectin Biotechnology, Shanghai, China) solution 1 ml (0.5 mg/ml) before planting the basal cell suspension, and leave it in a 37 °C incubator for 20 minutes. After the skin is completely digested, rinse the skin with 1% PBS to stop the digestion, scrape the basal cells with a sterile scalpel, rinse and collect the cells with keratinocyte serum-free medium (K-SFM; 17005042; Gibco), filter the cell suspension in a 50 ml centrifuge tube with a 200-mesh filter. Transfer the cell suspension to a 15 ml centrifuge tube, centrifuge at 1000r/min for 10 minutes, discard the supernatant, and resuspend in 4 ml complete medium by pipetting. Add the above basal cell suspension to the coated culture flask and let it stand in a 37 °C incubator for 20 minutes. It can be seen that about 10% of the cells adhere to the wall first. These cells are regarded as ESCs. ESCs were cultured in K-SFM medium at 37 °C incubator, and changed the medium every 2 days.
Immunofluorescence and confocal microscopy
The third-generation rat ESCs were digested, then resuspended and cultured in confocol glass bottom petri dishes. After the cells adhered, they were washed 3 times with PBS, 5 minutes each time, fixed with 4% paraformaldehyde (P0099-500 ml; Beyotime Biotechnology, Shanghai, China) for 20 minutes, and washed with PBS 3 times, then 0.5% Triton X-100 (P0096-500 ml; Beyotime Biotechnology, Shanghai, China) permeated at room temperature for 20 minutes. Dip in PBS three times, add 5% goat serum (C0265; Beyotime Biotechnology, Shanghai, China) onto the glass bottom of the petri dish, and block at room temperature for 40 minutes. Aspirate the 5% goat serum blocking solution, add 100 ul of diluted primary antibodies: p63 (1:200, ab735; Abcam), α6-integrin (1:200, ab235905; Abcam), CD31 (1:200, ab24590; Abcam) and CD34 (1:200, ab81289; Abcam), and incubate at 4 °C overnight. Wash the petri dish three times with PBS for 5 minutes each time. After absorbing excess liquid from the petri dish, add diluted fluorescent secondary antibodies: goat anti-rabbit IgG labeled with Alex Fluor 488 (1:200, ab150077; Abcam) and goat anti-mouse IgG labeled with Alexa Fluor 594 (1:200, ab150116; Abcam), and incubate for 1 hour at room temperature in the dark. DAPI (D9542; Sigma-Aldrich) was added dropwise and incubated for 5 minutes in the dark, and rinsed 4 times with PBS for 5 minutes each time. The liquid on the dish was sucked off, mounted with anti-fluorescence quencher, observed under a fluorescence microscope and collected images.
Flow Cytometry
Collect the third-generation rat ESCs by centrifugation and aspirate the supernatant. Resuspend cells briefly in 0.5 ml PBS. To fix and permeabilize cells by BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit. Adding 2–3 ml of incubation buffer to each tube, then rinse by centrifugation. Resuspending cells in 100 µl of incubation buffer per test tube. Blocking in incubation buffer for 10 minutes at room temperature. Adding the primary antibodies (p63, Abcam, ab124762, 1:200; α6-integrin, Abcam, ab77906, 1:200) to the test tube at the appropriate dilution. Incubating at room temperature for 60 minutes. Following the instructions and rinsing cells in incubation buffer by centrifugation. Resuspending the cells in the fluorescent-labeled secondary antibodies, and diluting in incubation buffer as recommended by the manufacturer. Incubating at room temperature for 30 minutes, then rinsing in incubation buffer by centrifugation. Resuspending the cells in 0.5 ml PBS and analyzing by flow cytometry.
Lentivirus and transfection
To generate ESCs with stable enhanced green fluorescent protein (EGFP) expression, 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 puromycin, and the expression level of EGFP was determined by Immunofluorescence.
Animal experiment
To explore the function of ESCs on full-thickness wound bed in vivo, the rats’ dorsal wound model was adopted. Twenty rats were anesthetized by inhaling isoflurane (INH), and a diameter of 2-cm full-thickness wound was made on the dorsal skin of each rat. The wounds were divided into 2 groups randomly: control group and ESCs group. The ESCs can stably express EGFP and evenly spray to the wound bed by a 2-ml syringe. For the ESCs group, 1 ml cell suspension with a cell density of 1 × 105/ml were evenly sprayed to the wound bed, while in control group, sprayed 1 ml PBS. The rats and wounds were observed, photographed, and measured daily until the rats were sacrificed. 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 at 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 analysis, and the other was rapidly frozen in liquid nitrogen for western blots analysis.
Immunohistochemistry staining analysis
The paraffin-embedded fixed tissue sections of each group were deparaffinized and rehydrated in xylene and graded ethanol. Antigen retrieval was performed by using Proteinase K solution(20 µg/ml) at 37 °C for 15 minutes. After Bloxall blocking, the sections were blocked with goat serum for 30 minutes and then incubated with primary antibodies: 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 microscope.
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. For western blotting, cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Cell Signaling Technology) containing PMSF (100:1, v/v) (Cell Signaling Technology) for 30 minutes. A 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).
Tissues immunofluorescence analysis
Formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated through graded ethanol. 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, then adding primary anti-rats antibody (CD31, Abcam, ab24590, 1:200). After incubating at 4 °C overnight, the sections were washed with PBST and incubated with the following secondary antibody for 1 hour: goat anti-mouse IgG labeled with Alexa Fluor 594 (1:200, ab150116; Abcam). DAPI was added dropwise and incubated for 5 minutes in the dark, and rinsed 4 times with PBS for 5 minutes each time. The liquid on the Petri dish was sucked off, mounted with anti-fluorescence quencher. Sections were documented with a fluorescence microscope (OLYMPUS, Japan).
Statistical analysis
Values were expressed as the mean ± standard deviation (SD) unless otherwise indicated. Comparisons of expression difference between control and experimental groups were conducted by Student’s t test. All statistical analyses were performed by SPSS 20.0 software (SPSS, Chicago, IL, USA), and P < 0.05 indicates that the difference was statistically significant.