The human iPS cell line was provided by Stem Cell Bank, Chinese Academy of Sciences. hiPSCs were cultured on Matrigel (BD Biosciences, CA) in mTeSRTM1 medium (Stemcell Technologies, CA) and passaged every 4-7 days. hiPSCs were induced to keratinocytes as described previously . Briefly, hiPSCs were switched to unconditioned medium (UM) supplemented with retinoic acid (RA): DMEM/F12 containing 20% knockout serum replacer, 1mM L-glutamine, 1× MEM nonessential amino acids, 0.1 mM b-mercaptoethanol (all from Life Technologies, USA) and 1 mM all-trans RA (Sigma-Aldrich, USA). Cells were cultured in UM+RA for 7 days, changing medium daily. On day 8, cells were passaged onto gelatin-coated plates in Keratinocyte Defined Serum-free Medium (K-DSFM, Life Technologies). After 4 weeks, differentiated cells were subcultured using trypsin to obtain high purity keratinocytes populations. Terminal differentiation was induced by adding 1.5 mM CaCl2 in K-DSFM for 10 days. HaCaT cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA), supplemented with 10% fetal bovine serum (Gibico, USA). Human umbilical cord samples were collected from patients with informed consent. The middle part of the human umbilical cord vein was cut into small segments, and the residual blood was cleaned with PBS. Collagenase Ⅰ was added into the segments. After digestion for 15 minutes, the lysis solution was sucked out and added into ECM medium for primary culture. Human umbilical vein endothelial cells (HUVECs) from the third passages were used. HaCaT cells and HUVECs were transfected with PML overexpression plasmid (pcDNA3.1-PML), PML siRNA, and miR-762 mimics using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions.
Extraction and identification of iPSCs-KCs-Exos
iPSCs-KCs were cultured in K-DSFM for 48 h. After collecting the supernatant, exosomes were isolated and purified by serial ultracentrifugation, using ultracentrifuge (Beckman, Germany). The exosomes was suspended in PBS. Concentration of exosomes was measured by Enhanced BCA Protein Assay Kit (Beyotime, China). Then, transmission electron microscopy (TEM) was used to observe the morphological structure of exosomes. Size distribution and concentration of iPSCs-KCs-Exos were analyzed by nano-particle tracking analysis (NTA) using Nanosight LM 10 (Malvern Panalytical, UK).
Treatment of deep second-degree burns
The 8-week-old C57BL/6 mice were purchased from the Experimental Animal Center of Southern Medical University. After the hair removal from the back of mice, the opening at one end of a cylindrical plastic tube with a diameter of 1.5 cm was affixed to the back skin of mice. Then pour boiling water into the tube at a height of 2 cm and then the tube was removed after 18 seconds. After burn, local subcutaneous injections of PBS or exosomes were given to the wound site.
The skin tissues were fixed with 4% paraformaldehyde for 72 h, embedded in paraffin, and then cut into 5 mm thick sections. The sections were stained with hematoxylin eosin (H&E) staining (Leagene, China) and Masson's tri-chrome staining (MXB Biotechnologies, China). The re-epithelialization was calculated in the accordance with the formula [distance of the re-epithelium]/ [distance of the wound] ×100.
At room temperature, cells were fixed with 4% paraformaldehyde for 30 min and then drilled with 0.5% Triton X-100 for 15 minutes. The cells were incubated overnight at 4°C with primary antibodies against K14, loricrin or involucrin (Proteintech, USA) and then incubated with Alexa Fluor 647-conjugated sheep anti-rabbit second antibody (Abcam, UK) for 1 h at room temperature. DAPI (500 ng/mL) was used to stain nuclei. Fluorescence was observed under an inverted fluorescence microscope (Leica, Germany)
After cells were treated for 48 h, EdU was added to the culture medium and incubated for 4 h. The cells were fixed with 4% paraformaldehyde and then drilled with 0.5% Triton X-100 for 15 minutes. Then, the cells were stained using Beyoclick™ EDU cell proliferation kit Alexa Fluor 488 (Beyotime, China) and imaged using an inverted fluorescence microscope (Leica, Germany).
Wound healing assay
Cells were seeded into 24-well plates and cultured overnight. After each well was filled with 95% monolayer of cells, the tip of the 200 µL pipette was used to form linear scratch wounds. At 0 h and 24 h, the wound healing was recorded by a microscope (Leica, Germany). Image J software was used to quantitatively evaluate the wound healing rate.
200 µL cell suspension (serum-free DMEM) was added to the upper chamber of Transwell, and 600 µL DMEM containing 2% FBS was added to the lower chamber. After 24 h incubation at 5% CO2 and 37℃, the cells were fixed with 4% paraformaldehyde. The cells at the lower side of the membrane were stained with Crystal Violet Staining Solution (Beyotime China) for 15 min. An inverted microscope (Leica, Germany) randomly collected 10 images from each sample.
miRNA microarray analysis
miRNA microarray analysis was performed by Epibiotek (Guangzhou, China) as previously described .
Western blot analysis
The Western blot assay was performed as previously described . Antibodies used were described as follow: anti-PML, anti-ITGB1or anti β-actin (all from Abcam, UK).
Reverse transcription quantitative polymerase chain (qRT-PCR)
Trizol™ reagent (Invitrogen, USA) was used to extract the total RNA from cells, according to the manufacturer's instructions. RNA was reverse-transcribed using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). qRT-PCR analysis was using ACEQ Universal SYBR qPCR Master Mix (Vazyme, China). Midtect A TrackTM miRNA qRT-PCR Starter Kit (RiboBio, China) was used for synthesis of miRNA cDNA. Then, Midtect A TrackTM miRNA qRT-PCR Starter Kit (RiboBio, China) were used for qRT-PCR.
Luciferase report assay
The wild type and mutant miR-762 binding sites of PML 3′UTR sequence were subcloned into psiCHECK-2 vector. MiR-762 mimics or negative control mimics were co-transfected with recombinant plasmid into HEK-293 T cells using Lipofectamine 2000 (Invitrogen, USA). According to the manufacturer’s instructions, the Firefly and Renilla luciferase activities were detected by Dual Luciferase Reporter Gene Assay Kit (Beyotime, China).
Data were presented as mean ± standard deviation (SD) of at least three independent experiments (n ≥ 3). Statistical analysis was performed by independent samples t-test for comparison between two groups or one-way ANOVA among the groups. p < 0.05 was considered statistically significant.