Culture and isolation of cells
Newborn UC were collected from full-term infants delivered by cesarean section at the Department of Obstetrics. UC sample collection was authorized by the Clinical Research Ethics Committee, Third Affiliated Hospital, Soochow University. UC samples were rinsed 3 times with PBS (GIBCO, Grand Island, NY, USA) to remove blood. One human UCV and two UCAs were mechanically removed from the UC . The WJ, UCAs and UCV were dissected into 2-3 mm3 sections. The fragments were placed in a humidified environment at 37 ℃ and 5% CO2. The sections were cultured in low glucose DMEM with streptomycin (100 μg/ml, GIBCO), penicillin (100 IU/ml, GIBCO) and fetal bovine serum (FBS, 10%, GIBCO). Colonies of WJ-MSCs, UCV-PSCs and UCA-PSCs were observed after approximately 15 days. The UCA-PSCs, UCV-PSCs and WJ-MSCs were detected regularly as mycoplasma-free cells.
Flow cytometry analysis
Cell suspensions were incubated with various phycoerythrin (PE)-conjugated antibodies against CD34 (BD Pharmingen, San Diego, CA) and CD90 (eBioscience, San Diego, CA), an Allophycocyanin (APC)-conjugated antibody against human CD45 (eBioscience), and fluorescein isothiocyanate (FITC)-conjugated antibodies against human CD29 (eBioscience), CD73 (BD Pharmingen) and HLA-DR (eBioscience).
Differentiation of cells
The multipotency of three kinds of cells was evaluated by osteogenesis, adipogenesis and neuroid differentiation experiments. The three kinds of cells were cultured with adipogenesis induction medium (GIBCO) to promote adipogenesis differentiation. Two weeks later, oil red staining (Sigma, Steinheim, Germany) showed intracellular lipid droplets. The three kinds of cells were treated with osteogenic induction medium (GIBCO) to induce osteogenesis. After four weeks, the three kinds of cells were stained with alizarin (Sigma) to detect the calcified extracellular matrix in the cells. For neural differentiation, three kinds of cells were cultured for 24 h in pre-medium, then cultured in modified neuronal medium for one and half day. The cells were observed by immunofluorescence for NFM and NSE described as previously .
Tube formation assays
Ninety-six-well plates were inoculated with 1× 104 cells per well. Then, the cells were coated with liquid Matrigel (50 μl) (Cat. No. 356234, BD Matrigel Matrix, BD Biosciences, USA) in serum-free DMEM, where the liquid Matrigel was pretreated for 30 min at 37 ℃. The tubes and branching points were photographed with a microscope (Leica), and the magnification was 100x. Branching points and tube formation were then examined in 6 random fields. Using ImageJ software, the tubular length from 6 random fields at 100x magnification per well was measured and computed as the mean.
Transwell migration assay
There were five groups: WJ-MSC group, UCV-PSC group, UCA-PSC group, negative control group and positive control group. WJ-MSCs, UCV-PSCs, UCA-PSCs, VEGF (positive control) or human endometrial stromal cells (hESCs) (negative control) (1 × 106 cells/well) were seeded into the lower chamber in twenty-four-well plates with Transwell inserts (Corning, NY, USA). The cells in the lower chamber were cultured with 1300 μl of DMEM-LG medium or DMEM-F12 medium containing 10% FBS, while HUVECs (1 × 105 cells/well) in the upper chamber were cultured with serum-free medium. Cells that migrated after 24 h of coculture, which were fixed with 4% PFA, were stained with crystal violet (0.1% w/v) for 0.5 h at 37 °C. The average number of migrated cells was determined by examining 6 random fields.
The three kinds of cells were harvested in cell lysis buffer (CST Biological Reagents Co., Ltd., MA, USA). The cell supernatant was collected after the cells were lysed for 0.5 h at 4 °C and centrifuged for 15 min at 12000 rpm. Protein concentration analysis was performed by a BCA assay (Beyotime Biotechnology, Shanghai, CN). SDS-PAGE gel electrophoresis (10%) was used to separate the same amount (25 μg) of protein. The membranes were incubated with antibodies against GSK3β (1:5000, ab93926, Abcam, Cambridge, UK), p-GSK3β (1:10000, ab75814), β-catenin (1:5000, ab32572), CD146 (1:100, ab75769), Sp1 (1:1000, Proteintech, Manchester, U.K.), GAPDH (1:10000, AP0063, Bioworld Technology, Inc., St. Louis Park, MN, USA), goat anti-rabbit (1:10000, A0545, Sigma) or goat anti-mouse antibody (1:10000, BS12478, Bioworld Technology).
The pGL3-basic luciferase reporter plasmids loaded with the CD146 (Gene ID 4162) promoter were employed in this experiment. The construction of Sp1 plasmid (Gene ID 6667) was purchased by Genscript. Pre-confluent (60%) UCA-PSCs were infected with the plasmids using the Lipo3000(Invitrogen, Tokyo, Japan). Using the Dual-Luciferase Assay kit (Promega, Madison, WI, USA) to detect the luciferase activities. Luciferase activity was analyzed by the fluorescence microplate reader. The firefly luciferase activity was normalized to the Renilla luciferase activity to analysis transfection efficiency.
Chromatin immunoprecipitation assay
UCA-PSCs (60% confluence) were transfected with PCMV-Flag or PCMV-Flag-Sp1. After 48 h, the UCA-PSCs cells were collected for ChIP as described previously . The recovered DNA using Flag beads was assessed by PCR. 2 μl DNA was used to conduct the PCR, with primers (CD146-F 5′- TTGATCAATGTGCTGGGCTG-3′ and CD146-R 5′- GTCTTGGCTAGGCTGGTCTT-3′).
Small interfering RNA transfection
UCA-PSCs were transfected with control siRNA, Sp1 siRNA (50 nM) or CD146 siRNA (50 nM) separately, which were purchased from Ribo Life Science Co.,Ltd., Suzhou, CN. Transfection was mediated by Lipo 3000 (Invitrogen).
The data are shown as the mean ± SEM. To assess the difference between two unpaired groups, the unpaired Student's t test was used. To evaluate the differences among more than two groups, nonparametric one-way ANOVA was performed. All statistical analyses were performed by GraphPad 8.0. P < 0.05 was considered statistically significant.