Microarray data obtained:
Three human 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 human samples, of which 3 were undifferentiated stem cells and 3 were differentiated stem cells. 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.
Cells sacrificed
The rabbit adipose derived stem cells were sacrificed in our laboratory from an eight-week-old Newzealand white male rabbit. The subpatellar fat pad was harvested for the further step. The remaining tissues were minced finely into the shape of chyle, mixed with collagenase typeⅠ(Tianjin Haoyang Biotechnology Comparison, China) and moved to a 50 ml centrifuge tube. At the end of digestion, the tissues were transferred into a centrifuge machine (Sigma Centrifuge, Germany), and the rotator speed was set at 1700 r/min, which lasted 10 minutes. The top content and supernatant were discarded, leaving the lower white block mass content in the tube. After centrifuging the liquid for 3 minutes at 800 r/min and discarding the supernatant, the lower content containing the stem cells was obtained finally. Subsequently, we inoculated the stem cells into the flask filled with 5 ml DMEM of fetal bovine serum (FBS, Gibco Company, St Louis, MO, USA) and observed the morphological characteristics under the invert microscope (Leica, Germany). The cells were cultured with the DMEM of fetal bovine serum subjected to culture and change every three days.
Flow cytometry(FCM)
The cells were trypsinized, washed, and re-suspended in phosphate-buffered saline (PBS) and blocked with 3% fetal bovine serum for 15 minutes before the flow cytometry analysis. We divided the cells into 3 ADSCs tubes, labeling the three ADSCs tubes as blank, CD90 testing, and CD45 testing (Invitrogen, USA, and Gibco, USA). To every tube was added the corresponding fluorescein-labeled antibody whose concentration was set at a paralleled level of 5μl/ml, and the tubes were incubated at 37℃ in a 5% CO2-saturated humidity float tank for 30 minutes. We centrifuged the tubes and its contents at 1200 r/min for 5 minutes following the last incubation. We then discarded the supernatant, washed the remaining products twice with PBS, added appropriate amounts of 4% paraformaldehyde for 30 minutes, and assessed the mixture on the flow cytometry machine (BD FACSVerse, USA).
Tetrazolium method (MTT)
The passage-3 ADSCs were digested and diluted, and the mixture was transferred to a 96-well culture plate (Thermo Scientific, USA) at 100μl cell suspension per well. 5-Aza was then added to each well containing ADSCs and the BMSCs at concentrations of 0, 10, 20, 30, 40 μmol/l. The absorbance of each well was measured at 550 nm (OD) by a microplate reader (Biotek, USA) to detect the cell viability under the induction of different concentrations of 5-Aza.
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 9 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 9 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. 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.