This study was approved by the ethics committee of The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University (Wenzhou, China).
ADSC isolation culture and identification
Adipose tissue was obtained from patients undergoing liposuction and digested with an equal volume of type I collagenase (GIBCO, USA) on a shaker at 37 °C for 45 min. Undigested tissue was removed with 100-mesh and 200-mesh screens; the remaining tissue was centrifuged at 1200 rpm for 5 min, and then the final pellet resuspended in complete medium (CM, Cyagen, USA). The cells were seeded at 1×104/mL density in culture flasks and incubated at 5% CO2 at 37 °C. The cells were passaged when they reached 80%–90% confluence, and third- to fifth-generation cells were used for subsequent experiments. The CM was changed every three days. Differentiation of ADSCs was induced using Human Adipose-derived Stem Cell Osteogenic Differentiation Medium (Cyagen), Chondrogenic Differentiation Medium (Cyagen), Adipogenic Differentiation Medium (Cyagen) and detected by alizarin red, alizarin blue, and oil red O staining.
Flow cytometry
Third generation ADSCs were washed three times with phosphate buffer saline (PBS), adjusted to 1×106/mL, and incubated with PE anti-CD29 (Biolegend, USA), PE anti-CD31 (Biolegend), PE anti- CD44 (Biolegend), PE anti-CD45-, PE (Multi Sciences, China) and anti-CD90 (Biolegend). PE-coated non-specific mouse IgG1 (Multi Sciences) was used as a negative control. Samples were processed with a flow cytometer, and the data were analyzed with FlowJo v10 software.
Induction of differentiation of ADSCs into SMCs and Wnt/β-catenin pathway inhibition assay
Third generation ADSCs were inoculated into 6-well plates and reached 80% confluence. Cells were then incubated in SMC-induction medium (5 ng/mL TGF-β1 (PeproTech, USA) in CM). Cells were cultured in the induction medium over 12 days, and the medium was changed every 2 days. The evolution of cells during induction was observed under the microscope. Proteins were extracted every three days (12 days in total) and stored at -80°C, and the protein levels of the different time-period groups were compared by western blotting.
The Wnt pathway was analyzed using TGF-β1 inhibitor LY2109761 (MCE, USA) or Wnt/β-catenin pathway inhibitor KYA1797K (MCE, USA). Briefly, at 80% confluence of the cells, the medium was replaced with SMC-induction medium supplemented with LY2109761 (10 μM) or KYA1797K (10 μM). An equal volume of DMSO (Solarbio, China) was added to the control group. Ten days after incubation, the protein levels were compared with those of the control and blank groups by western blotting.
Immunofluorescence
To authenticate that SMCs can be differentiated from ADSCs, immunofluorescence staining of intracellular molecules was performed. After fixation in 4% paraformaldehyde for 15 min, permeabilization with 0.5% Triton X-100 (dissolved in PBS) for 20 min, and blocking with 10% FBS for 30 min at 37 °C, the cells were incubated with antibodies including mouse anti-desmin (1:100, Abcam, USA), mouse anti-α-SMA (1:100, Abcam), rabbit anti- SMMHC (1:100, Affinity, USA), or rabbit anti-calponin (1:100, Affinity) at 4 °C overnight and then incubated with immunofluorescence-labeled secondary antibodies (Affinity) for 2 h at 37 °C, protected from light. Nuclei were reverse dyed with DAPI (Beyotime Biotechnology, China). We observed protein expression under a fluorescence microscope.
Western blotting
Total protein was harvested from the cells using RIPA (Beyotime Biotechnology). Equivalent proteins were separated by 6%–10% SDS-PAGE and transfected onto PVDF membranes. The protein-transfected PVDF membranes were blocked with TBST (Tris buffered saline with Tween) containing 5% skim milk for 90 min, then incubated with primary antibody on a shaker at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (1:5000, Solarbio) for 45 min. The primary antibodies were rabbit monoclonal β-catenin (1:10000, Abcam), calponin (1:2000, Affinity), α-SMA (1:500, Affinity), desmin (1:500, Affinity), SMMHC (1:1000, Affinity), or GAPDH (1:3000, Affinity). Protein expression was assessed using ECL reagent (Affinity). Quorum relative protein expression intensity was determined by optical density using ImageJ software.
Statistical analysis
All data are represented as mean ± standard deviation (SD) from three independently conducted experiments (unless otherwise noted). Differences between the experimental and control groups were determined using one-way ANOVA. P <0.05 indicated statistical significance. Statistical tests were performed by utilizing the GraphPad Prism 8 software.