Human placenta samples were collected from women undergoing surgical abortion at 7th to 10th weeks with written consent. This study was approved by the Institutional Review Board (IRB) of The University of Hong Kong/Hospital Authority Hong Kong West Cluster (IRB no.: UW 22-013). Placental explant was cultured according to previously described methods (Bai et al., 2022; Bai et al., 2023). Briefly, placenta villi were isolated and cultured in 40 μm cell strainer at 37°C, 2% oxygen environment. The culture medium was replaced 3 h after culture to remove cell debris and apoptotic bodies. The villi explant was cultured for 40 h for conditioned medium collection. The conditioned medium was first centrifuged at 300 g for 10 min, followed by 2,000 g for 20 min to remove cell debris. The supernatant was then centrifuged at 120,000 g for 120 min at 4°C for pEV isolation and washed in PBS twice. Protein concentrations of the isolated pEVs were determined by bicinchoninic acid (BCA) assay. Characterization of pEVs was conducted by Western blot analysis, electron microscopy and nanoparticle tracking analysis (NTA) according to previously published methods (Bai et al., 2022; Bai et al., 2023).

Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza (Lonza, C2517A) and cultured in F12K (Sigma Aldrich) supplemented with 10% FBS, 30 μg/mL of endothelial cell growth supplements and 0.1 mg/mL of heparin at 37°C, 5% CO₂ environment.

Human TSC (hTSC) line was established from human expanded potential stem cells (hEPSCs) according to previously described method, namely, TSC-like cells (Zhang et al., 2023). TSC-like cells were cultured in 1% Geltrex-coated plate at 37°C in TSC medium (Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 50 μM β-mercaptoethanol, 0.2% FBS, 0.5% penicillin-streptomycin-glutamine (PSG), 0.3% Bovine serum albumin (BSA), 1% Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) supplement, 50 μg/ml L-ascorbic acid, 50 ng/mL epidermal growth factor (EGF), 2 μM CHIR99021, 0.5 μM A83-01, 1 μM SB431542, 10 μM Valproic acid (VPA), and 5 μM Y27632). TSC-like cells were subcultured every 3–4 days with 1 mL of TrypLE.

After TSC-like cells reached 80% confluency, they were digested with TrypLE for 5 min at 37°C for induction of differentiation into EVTs (TSC-EVTs). TSC-like cells were seeded at a density of 1 × 10⁵ cells per well in 1% Matrigel-coated 6-well plate in 2 mL of EVT medium (DMEM/F12 supplemented with 50 μM β-mercaptoethanol, PSG, 0.3% BSA, 1% ITS- X, 7.5 μM A83-01, 10 μM Y27632, 4% KnockOut Serum Replacement (KSR), 100 ng/mL neuregulin 1 (NRG1) and 2% Matrigel). The differentiation medium was changed on day 3 (DMEM/F12 supplemented with 50 μM β-mercaptoethanol, PSG, 0.3% BSA, 1% ITS- X, 7.5 μM A83-01, 10 μM Y27632, 4% KSR and 0.5% Matrigel). TSC-EVTs were harvested on day 6 for downstream analysis.

For pEV internalization by TSC-EVTs, pEVs were labeled with PKH26-Red Fluorescent cell linker kit (Sigma-Aldrich) according to the manufacturer’s protocol. In brief, pEVs were resuspended with Diluent C solution after washing. Protein concentration was determined with BCA assay. pEVs were stained with fluorescent dye for 5 min and washed by PBS. TSC-EVTs were incubated with 10 μg/mL of pEVs diluted in the complete medium for 4 h, followed by washing with PBS and fixed with 4% PFA. The negative control group was treated with Diluent C at the same volume. TSC-EVTs were permeabilized with 1x Permeabilizing buffer, blocked with 10% goat serum and incubated with primary antibody against CK7 (1:200, CBL194F, Sigma) overnight at 4°C, followed by Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor^® 488 conjugate (1:600, A28175, Invitrogen) for 1 h at room temperature. The cells were counterstained with DAPI for 5 min and visualized under fluorescence microscopy.

For pEV internalization by HUVECs, pEVs were labeled with PKH67-Green Fluorescent cell linker kit (Sigma-Aldrich) according to the protocol mentioned above. HUVECs were stained with CellTrackerRed (Thermo Scientific) by incubation in 500 μL of working solution (2.5 μL CellTrackerRed diluted in 2.5 mL blank medium) for 30 min at 37°C. HUVECs were treated with 10 μg/mL of pEVs diluted in the complete culture medium for 4 h, while the negative control group was treated with Diluent C at the same volume. After 4 h of incubation, the culture medium was removed and HUVECs were rinsed with cold PBS three times. Cells were fixed with 4% PFA, counterstained with DAPI for 5 min and visualized under the fluorescence microscope.

HUVECs were treated with 10 μg/mL of pEVs for 24 h. After that, pEV-treated HUVECs were seeded at a density of 10,000 cells on μ-slide angiogenesis chamber with Matrigel coating (Corning) and cultured for 6 h for tube formation. Images were captured under inverted microscope and tube formation was analyzed using Image-Pro-Plus software (Media cybernetics) with the angiogenesis plugin.

Conditioned medium of pEV-treated HUVECs was collected after 24 h for cytokine array using a human cytokine antibody array kit (ARY005B, R&D) according to manufacturer’s instructions. Briefly, the membrane was blocked with 2 mL of blocking reagent for 1 h, and 1 mL of diluted conditioned medium was added to the membranes and incubated at 4°C overnight. The membrane was washed in wash buffers and incubated in biotin-conjugated detection antibody for 2 h, followed by incubation in HRP-conjugated streptavidin for 30 min. The spots were developed with Chemi Reagent Mix using X-ray films, and the intensity of each cytokine-representing spot was measured by ImageJ software with normalization to positive control spots.

The concentration of IL-6 in conditioned medium of pEV-treated HUVECs was analyzed using ELISA kit (eBioscience) under manufacturer’s instruction. In brief, 96-well culture plate was coated with primary antibody against IL-6 and incubated at 4°C overnight, then blocked with assay buffer for 2 h after washing. One-hundred microliters of conditioned medium was added to the plates with 50 μL of detection antibody and incubated for 16 h at 4°C, followed by incubation with HRP-conjugated streptavidin for 30 min after washing. The plates were then incubated with 100 μL of 3, 3′, 5, 5′-tetramethylbenzidine (TMB, BD Biosciences) substrate for 15 min, stopped by 50 μL of 1.8 N sulfuric acid, and read at 450 nm with a reference wavelength of 595 nm.

To conjugate pEVs with antibodies, pEVs were first incubated with anti-placental alkaline phosphatase (PLAP, ab133602, Abcam)-conjugated magnetic beads (20 μL/mL, Dynabeads™, Invitrogen) for 2 h to ensure their placental origin. AF647-labelled antibodies, including isotype antibody, siglec-6 antibody (ab262851, Abcam) and CD147 antibody (ab666, Abcam), were incubated with anti-PLAP bead-linked pEVs (10 μg) for 2 h. Antibody-conjugated pEVs were examined by flow cytometry to confirm the successful binding of siglec-6 and CD147 antibody. Fluorescence signal of labelled pEVs was detected with CytoFLEX flow cytometer (Beckman Coulter). Data was analyzed with FlowJo software (Tree Star).

TSC-like cells were treated with pEVs in the presence of isotype, siglec-6 and CD147 antibodies at 10 μg/mL on day 0 and day 3 during differentiation into TSC-EVTs. Total RNA of TSC-like cells, TSC-EVTs and TSC-EVTs treated with pEVs was extracted by the QuickPrep RNA extraction kit (GE Healthcare) and reversely transcribed by the TaqMan Reverse Transcription Reagent (Takara). qPCR was conducted with QuantStudio 5 Real-Time PCR System (Applied Biosystems) using TaqMan qPCR assay probes including EVT markers Human leukocyte antigen G (HLAG, Hs00365950_g1), Matrix metallopeptidase 2 (MMP2, Hs01548727_m1) and Integrin alpha 5 (ITGA5, Hs01547673_m1), pan-trophoblast marker Cytokeratin 7 (KRT7, Hs00559840_m1), and 18S ribosomal RNA (Hs99999901_s1) as internal control. Calculation of relative expression level was conducted with the threshold cycle (CT) method (2^−△△CT method).

TSC-EVTs were collected for transwell invasion assay using Matrigel Invasion Chamber 24-well plate 8.0 Micron (Corning), and for transwell migration assay using 8 μm culture insert (Merck) and 24-well culture plate. TSC-EVTs were seeded at a density of 10,000 cells in 300 μL of blank medium supplemented with 10 μg/mL of pEVs in the presence of isotype, siglec-6 and CD147 antibodies in the upper chamber for invasion assay, and at a density of 8,000 cells for migration assay. A control group was set with identical cell number but supplemented with blank medium in the upper chamber. The lower chamber was filled with 10% FBS diluted in blank medium. The invasion and migration system were cultured at 37°C for 20 h separately. The chambers were stained in 0.5% Crystal Violet for 15 min, followed by washing and removal of non-invaded cells using cotton sticks. Images of the lower chamber were captured, and the mean number of invaded/migrated cells was analyzed using ImageJ software (US National Institute of Health). The relative invasion/migration ratio was calculated by dividing the invasion/migration ratio of control group without pEV treatment.

All the data was presented as means ± standard deviation. All the data were analyzed by the Kolmogrov–Smirnov normality test followed by unpaired Student’s t-test or one-way analysis of variance (ANOVA) with multiple comparison using Graph Pad Prism 9 software (Graph Pad Software Inc.). P < 0.05 was considered as a statistically significant difference.

To isolate pEVs, an ex vivo culture system of placental explant was established (Bai et al., 2022; Bai et al., 2023) (Figure 1A). The isolated pEVs showed the positive expression of placental marker PLAP and EV marker CD63 (Figure 1B). Furthermore, NTA and electron microscopy result confirmed the size and morphology of pEVs, which showed majority of the particles fell into the size range from 100 to 500 nm (Figures 1C, D), together demonstrating the successful isolation of pEVs.

To study the ability of pEV uptake, TSC-EVTs and HUVECs were incubated with pEVs supplemented in the culture medium for 4 h followed by fluorescence signal capture. TSC-EVTs showed prominent fluorescence signals of pEVs labelled with PKH26, and the positive expression of CK7 demonstrated their trophoblast origin, while the negative control group incubated with blank medium did not show fluorescence signal of pEVs (Figure 1E). Likewise, HUVECs showed positive fluorescence signals for PKH67-labelled pEVs, while the negative control group only showed the red signal for CellTracker-red (Figure 1F). Together, pEVs were capable to be internalized by TSC-EVTs and HUVECs from the culture medium.

To understand the role of pEVs on endothelial cell functions, HUVECs were treated with 10 μg/mL of pEVs for 24 h. The pEVs treatment significantly inhibited the tube formation ability of HUVECs, with a reduced number of nodes, junctions, meshes, and total length of tubular structures compared to the control group (Figures 2A, B).

To further assess the effect of pEVs on cytokine production by HUVECs, the conditioned medium from pEV-treated HUVECs showed lower levels of IL-6 and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) compared to the ones from the control group (Figures 2C, D). This reduced expression of IL-6 was further confirmed by ELISA (Figure 2E). These findings suggested that pEVs inhibit endothelial cell tube formation and IL-6 production, potentially playing a role in maintaining homeostasis during the early stages for endothelial function regulation.

To investigate the presence of CD147 and siglec-6 on pEVs, we first conjugated pEVs with PLAP antibody-linked magnetic beads to confirm their placental origin. Subsequently, fluorescence-conjugated antibodies against CD147 and siglec-6 were attached, along with an isotype antibody as a negative control. Flow cytometry result confirmed the successful loading of antibodies with the PLAP bead (Figure 3A).

To investigate the impact of pEVs on TSC differentiation, TSC-like cells were treated with pEVs conjugated with antibodies against siglec-6/CD147 or isotypic antibodies during their differentiation into EVTs (TSC-EVTs). Treatment with pEV at a concentration of 10 μg/mL promoted the differentiation of TSC-like cells into TSCs-EVTs, with an increased expression of HLAG when compared to TSC-like cells without pEV treatment. This stimulatory effect was abolished when CD147 in pEVs was blocked by antibodies, but not by antibodies against siglec-6. There were no significant differences in the expression levels of MMP2 and ITGA5 between pEV-treated TSC-EVTs and TSC-EVTs, nor in the expression of the pan-trophoblast marker KRT7 (Figures 3B, C). Furthermore, to study the invasion and migration ability of TSC-EVTs upon pEV treatment, treating TSC-EVTs with 10 μg/mL of pEVs significantly enhanced their cell migration and invasiveness compared to the control group incubated with identical volume of blank medium. However, this effect was abolished when CD147 was blocked in the pEVs (Figures 3D, E). In summary, pEVs promote the differentiation, migration, and invasion of TSC-EVTs in vitro, likely through the action of CD147 on the pEVs.