This experiment was approved by the Ethics Committee of the Maternal and Child Health Hospital of Foshan, and informed consent was obtained from the donors. Pediatric urethral tissues were collected from patients with hypospadias. The urethral tissues were cut into small pieces using surgical scissors and transferred to centrifuge tubes. A five-fold volume of 1 mg/ml type II collagenase (Sigma) was added into the tubes, and the tissue was digested at 37°C for 4 h. After filtration through a 70-mesh cell sieve (NEST, China), the cells were cultured in SMC complete medium (ScienCell, United States). When the cells were cultured to 80% confluency, they were digested with 0.25% trypsin (Gibco, United States) and seeded into a new culture dish. After the cells adhered to the dishes for 0.5–1 h, the supernatant was then transferred to a new culture dish, and the above process was repeated. Multiple passages of purified PUSMCs were obtained through this procedure.

UCMSCs were purchased from Cyagen Biosciences Inc. (Guangzhou, China) and were cultured in the complete MSC medium (Cyagen, China) supplemented with 10% fetal bovine serum (FBS). Cells were passaged at a ratio of 1:2.

The third and sixth passages of PUSMCs were seeded into 96-well plates. Six wells were stained with cell counting kit-8 (CCK-8) at the same time every day and then analyzed on a microplate reader (MK3, Thermo). The results were recorded, and a growth curve was plotted. After the sixth passage, the PUSMCs were washed, permeabilized and blocked with goat serum, and fluorescein isothiocyanate (FITC)-labeled α-smooth muscle actin (α-SMA) monoclonal antibody (Bioss, China) was added; the cells were incubated overnight in a 4°C refrigerator in the dark. Then, the cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The expression of SMA-α, a marker for SMCs, was observed under an inverted fluorescence microscope.

Surface markers of UCMSCs were detected by a flow cytometry assay. UCMSCs were incubated with fluorescently labeled CD29, CD44, CD45, CD73, CD90, and human leucocyte antigen DR (HLA-DR) monoclonal antibodies (Biolegend, United States) for half an hour, and the results were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, United States).

The FBS used for cell culture was spun at 100,000 g overnight to remove the existing serum EVs. UCMSCs at passages four to eight were cultured in sEV-free medium for 48 h, and the cell supernatant was collected. sEV were isolated using ultracentrifugation and purified using a sEV purification reagent and concentration system (Exojuice, WeinaBio, China). Cell conditioned medium was collected and centrifuged at 300 g for 20 min to remove cells and centrifuged at 10,000 g for 20 min to remove cell debris. Supernatants were transferred into ultracentrifugation tubes and centrifuged at 100, 000 g for 70 min in order to obtain a pellet enriched in sEV. The precipitates were suspended in PBS and purified by Exojuice Kit at 100,000 g for 70 min in accordance with the protocol provided by the manufacturer. The resultant sEV was stored at −80°C.

sEV characterization was performed following the International Society for Extracellular Vesicles guidelines (Thery et al., 2018; Witwer et al., 2019). Briefly, the morphology of isolated sEV samples were visualized using transmission electron microscopy (TEM), and the particle size and concentration of sEV were analyzed by NanoFCM analysis. The sEV specific proteins, including CD9, CD63, CD81 and tumor susceptibility 101 (TSG101), were analyzed by Western blot.

PUSMCs were seeded in 96-well plates at a density of 5,000 cells/well/100 µL. After 12 h of culture, medium containing UCMSC-sEV with or without PSB12379 (a CD73 inhibitor) was added to cells. After 24 and 48 h of culture, 10 μL of CCK-8 reagent was added, and the cells were incubated at 37°C for 1.5 h. Then a microplate reader (Thermo MK3, United States) was used to measure the absorbance at 450 nm.

PUSMCs were seeded in 6-well plates at a density of 5×10⁵ cells/well. After 12 h of culture, medium containing UCMSC-sEV was added, and the culture was continued for 12 h. Then, the cells were treated with 5-ethynyl-2′-deoxyuridine (EdU) solution (Beyotime, China) for 2 h and then incubated in click reaction solution at 37°C for 30 min in the dark. The cells were stained following the instructions provided with the EdU solution and then imaged under a fluorescence microscope to calculate the percentage of EdU-positive cells.

A culture insert (Ibidi, Martinsried, Germany) was placed in the middle of a 24-well culture plate. Subsequently, PUSMCs were seeded at a density of 5×10⁵/ml per well (70 μL volume). After 24 h, the culture insert was carefully removed, and UCMSC-sEV were added to the PUSMCs for 12 h. Cell migration was imaged using an inverted microscope (Olympus IX 71, 100 × magnification, Olympus, Japan) and analyzed using ImageJ software v1.8 (National Institutes of Health, United States).

PUSMCs were resuspended in serum-free medium and then were seeded in the upper chamber of a Transwell at a density of 5×10⁵ cells/ml (100 μL volume), and culture medium containing UCMSC-sEV with or without PSB12379 was added to the lower chamber of the Transwell. After 24 h of culture, the cells that had migrated to the lower chamber were counted after fixation and crystal violet staining.

Total RNA was extracted using TRIzol reagent (Thermo, United States), and cDNA was synthesized using HiScript^® III RT SuperMix for qPCR (+gDNA wiper) (Novvia, China). Quantitative PCR was performed using ChamQ universal SYBR qPCR Master Mix (Novenza, China) to detect the mRNA level of CD73. All gene sequences were obtained through GenBank, and primers were designed using Primer Premier five and synthesized by Sangong Biotech (Shanghai, China). The primer sequences are provided in Table 1.

The protein concentration was determined using a bicinchoninic acid (BCA) reagent kit. Total protein (30 mg) was separated by 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, United States). The membrane was blocked with 5% skim milk for 2 h and then incubated with a primary antibody (dilution ratio of 1:1,000) at 4°C overnight. After washing with 0.1% Tween^® 20 (TBST) 3 times, the corresponding horseradish peroxidase-labeled secondary antibody (dilution ratio of 1:5,000) was added, and the membrane was incubated for 1 h. After washing with TBST 3 times, a chemiluminescence reagent (Tanon, China) was added dropwise to the membrane. Finally, the protein expression was observed using a chemiluminescent imaging system (Tanon, China). The monoclonal antibodies for CD9 and CD81 were from Affinity Biosciences (Affinity, United States). The monoclonal antibodies for TSG101 and CD63 were from Santa Cruz Biotechnology (Santa Cruz, United States). The monoclonal antibodies for CD73 were from Abcam (United Kingdom). The monoclonal antibodies for phospho-AKT, phospho-PI3K and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Bioss Biotechnology (Bioss, China).

The extracted UCMSC-sEV were dissolved in phosphate buffered saline (PBS) containing 2% bovine serum albumin (BSA), followed by incubation with CD73 antibody to obtain phycoerythrin (PE)-conjugated CD73 sEV. Subsequently, the PE-conjugated CD73 sEV were incubated with PUSMCs at 37°C for 3 h, after which the cells were fixed in 4% paraformaldehyde for 15 min. The cytoskeleton was stained with FITC-phalloidin for 45 min, and the nuclei were stained with DAPI. Fluorescence microscopy was used to detect fluorescence signals in the cells.

PUSMCs were seeded at a density of 50,000 cells/well and cultured in medium with or without 5′AMP (Sigma) and UCMSC-sEV for 4 h. Then, the supernatant was collected and quickly frozen at −80°C. Adenosine concentrations were analyzed using an Adenosine Assay Kit (BioVision) in accordance with the protocol provided by the manufacturer. The fluorescent intensity was measured at Ex/Em 535/587 by Thermo Scientific Varioskan Flash Multimode Reader (Thermo, United States).

Shapiro-Wilk normality tests were performed for each set of data, in the case of normal distribution, the data are presented as mean ± SD. The data were assessed using the SPSS 11.0 program for Windows (SPSS Co., United States). Significance was evaluated at p-value of 0.05 and 0.01 using t test.

After passaging, the PUSMCs grew in bundles and exhibited a cascade arrangement and a “peak-to-valley” phenomenon (Figure 1A). The immunofluorescence assay results showed that the cultured cells expressed α-SMA, a smooth muscle surface marker (Figure 1B). The proliferation of PUSMCs was vigorous, and the growth curve (Figure 1C) trend was S-shaped, with lag, logarithmic and plateau phases.

Flow cytometry analysis of the surface markers of MSCs indicated that UCMSCs expressed high levels of the CD29, CD44, CD73, and CD90 but were negative for the CD45 and HLA-DR (Figure 1D), findings that are consistent with the general characteristics of MSCs.

Transmission electron microscopy revealed that the UCMSC-sEV were cup-shaped vesicles (Figure 1E). NanoFCM analysis indicated that the average diameter of the sEV was 78.91 nm and that the concentration was 2.53E+10 particles/mL (Figure 1F). Western blot results showed that the extracted sEV expressed CD81, CD63, CD9 and TSG101 (Figure 1G).

Results of CCK-8 assays (Figure 2A) and EdU assays (Figure 2B) indicated that UCMSC-sEV promoted the proliferation of PUSMCs in a dose- and time-dependent manner.

As shown in Figures 2C,D, both scratch wound assay and Transwell assay results indicated that low concentrations of UCMSC-sEV (10 μmol/L) had no effect on the migration of PUSMCs, and high concentrations of UCMSC-sEV (50 μmol/L) significantly promoted the migration of PUSMCs.

CD73, also known as ecto-5′-nucleotidase, is a cell surface enzyme that is highly expressed on the surface of UCMSCs (Figure 1D). To verify whether UCMSC-sEV also expressed CD73, qRT-PCR and Western blotting were used to assess the mRNA and protein levels of CD73 in UCMSCs and UCMSC-sEV, respectively. The results showed that compared with HEK293T cell-derived sEV (HEK293T-sEV), UCMSCs and UCMSC-sEV highly expressed CD73 mRNA and protein (Figure 3A and Figure 3B).

To confirm internalization of CD73-positive UCMSC-sEV by PUSMCs, a PE-conjugated anti-CD73 monoclonal antibody was used to label UCMSC-sEV. The labeled sEV were cocultured with PUSMCs for 3 h, and the internalization of UCMSC-sEV was observed under a fluorescence microscope. The red fluorescence of CD73 on the surface of UCMSC-sEV was localized to PUSMCs, indicating that CD73-positive UCMSC-sEV were endocytosed by PUSMCs (Figure 3C).

To further investigate whether the proliferation- and migration-promoting effects of UCMSC-sEV were related to the transmission of CD73 molecules, we added a CD73 inhibitor (PSB12379, MCE) to the medium contained with UCMSC-sEV. The results indicated that the addition of PSB12379 inhibited CD73 activity and blocked the proliferation- and migration-promoting effects of UCMSC-sEV on PUSMCs (Figures 3D,E).

The expression of adenosine was detected in the cell culture supernatant containing 5′AMP (Sigma) and UCMSC-sEV (Figure 4A), indicating that CD73 on the surface of UCMSC-sEV used exogenous 5′AMP to produce adenosine. Moreover, the addition of exogenous adenosine to the culture medium of PUSMCs significantly promoted the proliferation and migration of PUSMCs (Figures 4B,C). These results demonstrate that the proliferation- and migration-promoting effects of UCMSC-sEV were related to the CD73/adenosine signaling axis.

The PI3K/AKT signaling pathway is an important pathway for cell proliferation and survival and is closely related to adenosine metabolism. Therefore, we investigated the downstream PI3K/AKT signaling pathway by which CD73 affects the cellular functions of PUSMCs through Western blotting analysis. The protein expression levels of phospho-AKT and phospho-PI3K in PUSMCs increased after treated with adenosine or UCMSC-sEV (Figure 4D), indicating that UCMSC-sEV activated the PI3K/AKT signaling pathway in PUSMCs.