Microarray data obtained:
Three gene expression profiles, GSE131125(GPL 20844, SurePrint G3 Human GE v3 8x60K Microarray 039494), GSE148994 and GSE149055(GPL16686, Affymetrix Human Gene 2.0 ST Array) were obtained from the GEO database. Both the GSE149055 and GSE148994 contained 6 samples, of which 3 were undifferentiated stem cells and 3 were differentiated cells after 30 days differentiation. GSE133125 contained 24 samples which include different time-point of the differentiation. We choose the 3 undifferentiated stem cells and 3 differentiated for 25 days into our analysis.
Identification of differently expressed genes (DEGs)
The downloaded platform files were matched to the gene expression profiles by the “VLOOKUP” function of Excel 2010. Gene differential analysis was determined to summarized the differentially expressed genes (DEGs). The DEGs threshold of our study was |logFC|>1 and adj.P-value<0.01. Heatmaps of DEGs from 3 groups were generated by graphpad 8.0.2. Online tool Venn, version 2.1(bioinformatics.psb.ugent.be/webtools/Venn;version 2.1) was used to determine the common DEGs among the three profiles.
(Protein-protein interaction) PPI network construction and module selection
Search Tool for the Retrieval Interacting Genes (STRING) database was used to construct the network of differentially expressed genes and proteins and Molecular Complex Detection (MCODE; version 1.31) in the Cytoscape (version 3.8.0) was used to analysis modules in the network.
GO and pathway enrichment analysis construction
Both the GO and KEGG analysis was applied under the online program Database for Annotation, Visualization and Integrated Discovery (DAVID, version 6.8) whose subgroup of functional annotation tools can help the researchers to understand the biological meanings about the selected genes. Gene Set Enrichment Analysis (version 4.0.3) was used to verify whether DEGs Showed statistical significance in one phenotype or pathway based on the expression profiles.
Isolation and cultivation of ADSCs
An eight-week-old New Zealand white rabbit (Animal Experiment Center of Jiangsu University) weighing 2.0 kg was sacrificed under the guidelines of the Institutional Animal Care and Use Committee of Jiangsu University, China. The rabbit was kept and fed in a single cage in housing conditions. Housing was controlled in temperature (25°C)，humidity (40– 60% ) and light (12 h, light–dark cycle). Animals were observed for one week before surgery to confirm that they were healthy and disease-free.
0.6 % sodium pentobarbital (4 mg/ kg) was injected into the rabbits’ ear veins for general anesthesia. Then 2 % lidocaine hydrochloride was injected into the planned skin incision to enhance the effect of anesthesia. Priority to placing the animals in a laminar flow chamber, the hair was clipped at the abdominal area. An incision was made along with the linea alba to expose the peritoneum, and the inguinal fat was removed.
The adipose tissue was washed three times with phosphate-buffered saline (PBS) to remove red blood cells. The collected adipose tissue was cut into small pieces and transferred into one 20 mL centrifuge tube, and an equal volume of a 0.25% trypsin (Gibco, USA) and 0.1% type I collagenase (Sigma, USA) mixture was added. The tissue was incubated on shaking tables at 37°C with constant agitation for approximately 15 minutes. Afterwards, the liquid was separated into three layers: the upper layer contained yellow oily lipocytes, the intermediate layer contained adipose tissue, and the bottom layer contained mononuclear cells. The bottom layer was extracted and transferred into a centrifuge tube containing 15% fetal bovine serum (FBS, Gibco, USA) and high-glucose DMEM (Sigma, USA). The remaining stromal fractions were treated with 3 mL red blood cell lysis buffer (Sigma, USA) for 10 minutes at room temperature, filtered through a 100-mm nylon mesh, and centrifuged at 1200 ×g for 10 minutes; then, the supernatant was removed. The cell pellets were then suspended in high-glucose DMEM containing 15% FBS (Gibco Company, St. Louis, MO, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco Company, St. Louis, MO, USA). The cells were cultured at 37°C and 5.0% CO2 in a humidified incubator, with full media replacement every 3 days. When the cells reached 80% confluence, they were digested with a mixture of 0.25% trypsin and 0.04% EDTA (Shanghai Reagent, China) and passaged for later use.
Flow cytometry（FCM）analysis of ADSCs
Passage-3 adherent cells were treated with 0.25% trypsin (Gibco, USA) and washed twice with PBS. The cells were incubated with rabbit anti-CD45, anti-CD90 antibodies (Invitrogen, USA, and Gibco, USA) overnight at 4°C. Unbound antibodies were removed by washing three times with PBS. After washing, the cells were incubated for 45 minutes at room temperature in the dark with Cy3-labeled secondary anti-goat/anti-rabbit antibody and resuspended in PBS for FACS analysis. At least 1×106 cells per sample were analyzed with a flow cytometer (BD FACSVerse, USA). CELLQuest software was used for the analysis.
Assessment of cell viability by tetrazolium method （MTT）
The cell viability was quantitatively determined by the Tetrazolium (MTT) method. MTT is a yellow tetrazolium dye which responds to metabolic activity. The reductases in living cells reduce MTT from a pale-yellow compound to dark-blue formazan crystals. The passage-3 ADSCs were digested and diluted, and the mixture was transferred to a 96-well culture plate (Thermo Scientific, USA) at 1×105 cells per well. 5-Aza was then added to each well at concentrations of 0, 10, 20, 30, 40 μmol/l. Then, at 24h, 48 and 72 hours after induction, the absorbance which represent cell viability was tested in each group. Firstly, the supernatant was removed. Then, 200 μL of dimethyl sulfoxide (DMSO, Merck, Germany) was added to each well to dissolve the blue substance. Finally, the absorbance (OD) at 570 nm was read using a microplate reader (Biotek, USA).
Induction of differentiation of ADSCs by 5-Azacytidine(5-Aza)
The passage 3 ADSCs were digested by a mixture of trypsin and EDTA and dilutaed to single-cell suspension of 104 cells/mL and then seeded into cell culture flasks. Group A, B and C were induced by 0, 10, and 20 µmol/L 5-Aza (Sigma, USA) for 24hours and washed with D-Hanks balanced salt solution (HBSS, Gibco Company, St. Louis, MO, USA). Then, the medium was replaced with low-glucose DMEM containing 10% FBS. The cells in each group were incubated at 37°C with 5% CO2 in conventional incubator. The medium was replaced with fresh DMEM and FBS every 3 days until the test begin after 25 days cultivation.
Immunohistochemical staining and Semi-quantitative analysis
We determined the KEGG pathway of actin cytoskeleton about expression of actin by immunohistochemical staining. The cells were digested and diluted 25 days after induction and added 150μl 4% paraformaldehyde fixative to every slide and left them undisturbed for 30 minutes before adding 150μl 0.1% Triton x-100microplate reader (Biotek, USA). Primary antibody α-SMA (1:200) (Proteintech, USA), secondary antibody (1:200) (Proteintech, USA) and Hoechst33258 stain (C1011 Beyotine, China) was added to each slide in a dark environment at room temperature. Finally, we observed the cells under a fluorescence microscop e(Leica, Germany), photographed, and stored them. ImageJ (Rawak Software, Germany) software was used for photography and Prism Demo software for data statistics (GraphPad Software, USA).
Quantitative real-time PCR
Total RNA was extracted from the ADSCs after induction of 25 days using Trizol lysate （invitrogen）. The schizolytic cells were then transferred into another tube without RNA enzymes, and 200 μl pre-cooling chloroform (Sigma Centrifuge, Germany) was added per milliliter of Trizol. The ccentrifugation yielded RNA sediments that were preserved in a -20℃ surrounding for 30 minutes. The sediments were washed with 75% ethyl alcohol and centrifuged for 5 minutes, and the supernatant was discarded after washing and centrifuging the sediments twice. The reverse transcription system was prepared using a reverse transcription kit (Thermo Scientific, USA) according to instructions provided in the protocol of the kit.
Statistical analysis
Statistical analysis was performed on Graphpad 8.0.2 and R 4.0.0. Expressed data were shown as mean±SD. Student’s t test was used to evaluate the statistical significance of different 3 groups. P value less than 0.05 was considered as significant.