ADSCs isolation and culture

This research was approved by the Institutional Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology, China, and followed the principles set forth in the Declaration of Helsinki regarding the handling of patient samples. Consent was obtained from the parents of young donors and old donors before they took part in the study. The detailed information of these donors has been described in our previous study [5]. Samples of adipose tissue were taken from patients who had skin-flap transplantation surgery and did not have any additional systemic illnesses. The ADSCs were cultured in Human Adipose-derived Mesenchymal Stem Cells Medium (OriCell, China), following a set procedure. Cells from passages 2 to 7 were used for the experiments.

Multilineage differentiation assay

ADSCs subjected to gene transfection were employed to evaluate both chondrogenic and adipogenic differentiation, in accordance with the standardized protocol provided by the manufacturer (Oricell, China). The capacity for adipogenesis was evaluated following a two-week adipogenic culture period through oil red staining, whereas the capacity for chondrogenesis was assessed after a three-week chondrogenic culture period using alcian blue staining.

RNA sequencing

Total RNA was isolated from ADSCs at passage 3 or 4 using the Tissue & Cell Total RNA Kit from HY Cezmbio in China. The quality and quantity of RNA were assessed through the Bioanalyzer 2100 from Agilent in CA, USA, and the NanoDrop ND-1000 from NanoDrop in Wilmington, DE, USA. mRNA containing poly(A) tails was enriched using dynabeads oligo (dT) from Thermo Fisher in the USA, followed by fragmentation with the NEBNextR Magnesium RNA Fragmentation Module from NEB in the USA. Subsequently, a standard cDNA library was prepared and sequenced on the Illumina NovaseqTM 6000 platform. Raw data underwent filtration to obtain clean data, which was then aligned to the reference genome using Hisat2 [19]. The expression levels of mRNAs were quantified using the Fragments Per Kilobase Million (FPKM) value calculated by StringTie [20]. Genes showing significant dysregulation were identified based on the criteria of fold changes ≥ 2 or ≤ -2 and a p value < 0.05.

GO and KEGG analyses

The identified genes exhibiting differential expression were subjected to additional analysis using the Gene Ontology (GO) database (http://geneontology.org) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.genome.jp/kegg). The GO categories, encompassing cellular component, molecular function, and biological process information of the candidate genes, were utilized for further characterization. Furthermore, the biological pathways associated with these genes were investigated through the KEGG database. Significantly enriched gene sets were identified using the hypergeometric distribution test, with a p-value of less than 0.05 considered as statistically significant.

CUT & tag assay

The CUT & Tag assay was carried out utilizing a Hyperactive Universal CUT & Tag Assay Kit (TD903, Vazyme, China) in accordance with the provided guidelines. In summary, 60,000 ADSCs were collected, rinsed, and subsequently exposed to Concanavalin A-coated magnetic beads. Subsequently, the cells were treated with either an anti-flag antibody (Cat. 14793 S, Cell Signaling Technology, USA) or an isotype control antibody (Cat. 2729 S, Cell Signaling Technology, USA) for a duration of 2 h, followed by an additional hour with a secondary antibody at ambient temperature. The hyperactive pG-Tn5 transposonase was then introduced to facilitate tagmentation. Subsequently, DNA fragments were isolated and amplified using indexed P5 and P7 primers. The DNA library was sequenced on the Illumina NovaseqTM 6000 platform employing the PE150 model. The initial data underwent filtration to acquire clean data, which was subsequently aligned to the reference genome utilizing bowtie2 (version 2.2.6) with default settings [21]. The distribution of reads upstream and downstream of TSS was depicted using DeepTools (version 2.4.1) [22]. Peaks annotation and peak distribution analysis were conducted using bedtools (version 2.30.0) [23]. Differential peaks were discerned utilizing csaw (version 1.24.3) [24]. To further validate the outcomes of DNA-seq, the DNA fragments were amplified using primers specific to the binding sites of HES1 with STAT1 genome, visualized through agarose gel electrophoresis, and sequenced via Sanger’s sequencing.

Lentiviral transfection

A lentivirus vector containing green fluorescence protein was utilized to create a stable overexpression system for HES1, along with a negative control. These vectors were subsequently transfected into O-ADSCs for research purposes. In short, O-ADSCs at passage 3 were seeded in a six-well culture plate at a density of 1 × 105 cells per well. The next day, the ADSCs medium was supplemented with the virus stock solution and cotransfection reagent, with an appropriate multiplicity of infection. After 12 h of transfection, the medium containing the virus was swapped with ADSCs medium. When the cells reached 70% confluence, puromycin-containing ADSCs medium was employed to isolate the cells that were successfully transfected.

siRNA transfection

The si-HES1, si-STAT1 and negative control si-NC were synthesized by RiboBio, China, and the sequences of these oligonucleotides are listed in Table S1. In short, O-ADSCs at passage 3 were seeded in a six-well culture plate at a density of 1 × 105 cells per well. The next day, the cells were transfected with the oligonucleotides using the riboFECT CP Transfection Kit (RiboBio, China), in accordance with the provided guidelines. The effectiveness of transfection was verified through qRT-PCR and western blot analysis. Following a 72-hour incubation period, the ADSCs with different treatments were gathered for the subsequent experiment.

Real-time PCR analysis

In this study, total RNA was isolated using the Tissue & cell RNA Kit (HYCEZMBIO, China), and the RNA samples were converted into complementary DNA (cDNA) through reverse transcription using the HiScript III RT SuperMix for qPCR (Vazyme, China). Then the qRT-PCR was conducted was carried out in accordance with the manufacturer’s guidelines, using the ChamQ SYBR qPCR Master Mix (Vazyme, China). The qRT-PCR primer sequences are available in Table S2. Gene expression levels were calculated using the 2−ΔΔCt method and were standardized against GAPDH.

Western blot

Total protein was extracted by using RIPA (Epizyme, China) with protease inhibitor, and the protein concentration was measured using the BCA protein assay (HYCEZMBIO, China). 20 µg of total protein was separated using a 10% SDS-PAGE gel, then transferred to PVDF membranes. The membranes were blocked with blocking buffer and left to incubate overnight with primary antibodies specific to HES1((Abcam, ab108937, USA), STAT1 (Abcam, ab109320, USA), β-tubulin (Proteintech, 10094-1-AP, China), GAPDH (Proteintech, 60004-1-Ig, China). Afterward, the membranes were exposed to a secondary antibody tagged with HRP for one hour. Then, they were treated with the immobilon ECL substrate kit for one minute and photographed using an imaging system. Original blots were exhibited in Figure S3.

Cell proliferation and migration assay

In the cell proliferation test, ADSCs with different treatment were grown in 96-well plates with 7000 cells per well, and each group had 5 replicated wells. After incubating overnight, the cells were exposed to the BeyoClick™ EdU-594 kit (Beyotime, China) for 3 h as per the manufacturer’s guidelines. Afterwards, the EdU-labeled ADSCs (colored red) and all cells (colored blue) were photographed with a fluorescent microscope. The rate of cell proliferation was assessed by dividing the count of red-stained cells by the count of blue-stained cells, then multiplying by 100%.

In the cell migration experiment, ADSCs with different treatment were placed in the upper chamber of 24-well transwell insert with 8.0 μm pores (Corning, USA), with each well was seeded 3 × 104 cells in DMEM medium without fetal bovine serum, and the lower chamber contained 650 µl of ADSCs medium. There were 5 replicated wells in each experimental group. After 24 h, the cells that moved to the lower chamber were stained with crystal violet and photographed under a microscope. The quantity of migrated cells was then measured for each group.

β-galactosidase staining assay

ADSCs with different treatment were grown in 48-well plates with 1.5 × 104 cells per well. There were 5 replicated wells in each experimental group. After being left to incubate overnight, the cells were stained with Senescence β-Galactosidase Staining Kit (Beyotime, China). The appearance of blue-stained cells indicated the presence of SA-β-gal, which was visible through a light microscope. The percentage of cells testing positive for SA-β-gal was then calculated for each group in the experiment.

Enzyme-linked immunosorbent assay (ELISA)

The levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α) in cell culture supernates were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Bioswamp, China) according to the manufacturers’ protocols. The ELISA plates were measured using the plate reader at a wavelength of 450 nm; With the concentration of the standard product as the horizontal coordinate and OD value as the vertical coordinate, the standard curve was obtained use four parameter logistics curve fitting, and then plug the OD value of a sample into the fitting equation to calculate the concentrations of the sample.

In vivo wound-healing model

In this study, all animal tests were approved by the Animal Care Committee of Tongji Medical College and followed the ARRIVE guidelines. The experiment was conducted by separate individuals for the design and operation tasks in order to minimize the influence of subjective factors, ensuring strict adherence to the experimental methodology. Twenty-eight healthy male Sprague Dawley rats, 10 weeks old and weighing around 180–200 g, were sourced from the Experimental Animal Center at Tongji Medical College, Huazhong University of Science and Technology. The rats were equipped with numbered ear tags and subsequently assigned to four groups through a digital randomization process: the PBS group (PBS), the oe-NC O-ADSCs group (oe-NC), the HES1 O-ADSCs group (oe-HES1), and the Y-ADSCs group (Y-ADSCs). Based on past experience, we allocated 7 rats per group. Before the experiments, the rats were given anesthesia through intraperitoneal injections of ketamine (0.025 mg/kg) and xylazine (0.25 mg/kg). Then, a circular full-thickness skin wound with 18 mm diameter was created on the back of each rat. Around 1.0 × 106 ADSCs were mixed in 100 µl of PBS and injected at four points around the wound, while the control group was injected with 100 µl of PBS only. To prevent wound infection, all rats were administered intramuscular injections of veterinary antibiotics following the modeling procedure. To minimize potential confounding factors, such as wound bites, each rat was housed individually in a cage and given uniform laboratory food, following a 12-hour day and night cycle. The endpoint of the wound healing experiment was set at 18 days, as previous experience indicates that re-epithelialization can be achieved by this time for most wounds, thereby facilitating analysis of the internal healing process. Images of the injuries were captured on days 0, 3, 7, 10, 14 and 18 after the wounds were inflicted, and the size of the wounds was determined in each image using Image J software. In order to obtain more accurate statistics, the maximum and minimum values of each group’s data were excluded, leaving 5 data points for statistical analysis. The percentage of wound closure was determined by dividing the reduced area by the original wound area and multiplying by 100%. On the 18th day, the rats were euthanized while under anesthesia through cervical dislocation after all samples were collected.

Histological analysis

On the 18th day, the wounds were gathered and then processed through tissue fixation, embedding, and sectioning. The sections were later stained with hematoxylin and eosin (H&E) to examine the wound healing. Furthermore, Masson staining was performed to assess the collagen accumulation.

Immunofluorescence analysis

To assess the density of blood vessels in wound beds, the tissue samples were treated with α-SMA (Abcam, ab7817, USA) and CD31 (Abcam, ab182981) antibody overnight at 4 °C, then exposed to a fluorescein-conjugated secondary antibody (Invitrogen, 710,369, USA) for 1 h, at room temperature. The cell nuclei were stained with DAPI (Sigma-Aldrich, D9542-5MG, USA). Subsequently, the sections were scanned under a fluorescence microscope, and the quantity of blood vessels in the dermal layer of each sample was measured using Image J software.

Immunohistochemistry analysis

To assess cell growth in wound areas, the tissue samples were treated with Proliferating Cell Nuclear Antigen (PCNA) antibody (Abcam, ab7817, USA) overnight at 4 °C. Next, a second antibody linked to horseradish peroxidase (Beyotime, A0181, China) was applied to the samples for 1 h at room temperature. The samples were then scanned and the percentage of PCNA-positive cells in each sample was determined using Image J software.

Statistics analysis

The data analysis was conducted using GraphPad Prism software, version 8.0.2. Unpaired Student’s t-test was used to compare data between two groups, while one- and two-way analysis of variance (ANOVA) test was used to compare three or more groups. Results are shown as mean ± standard deviation (SD), and statistical significance was considered at p < 0.05.