Extracellular vesicles generated by placental tissues ex vivo: A transport system for immune mediators and growth factors

Abstract

Problem: To study the mechanisms of placenta function and the role of extracellular vesicles (EVs) in pregnancy, it is necessary to develop an ex vivo system that retains placental cytoarchitecture and the primary metabolic aspects, in particular the release of EVs and soluble factors. Here, we developed such a system and investigated the pattern of secretion of cytokines, growth factors, and extracellular vesicles by placental villous and amnion tissues ex vivo.

Methods of study: Placental villous and amnion explants were cultured for 2 weeks at the air/liquid interface and their morphology and the released cytokines and EVs were analyzed. Cytokines were analyzed with multiplexed bead assays, and individual EVs were analyzed with recently developed techniques that involved EV capture with magnetic nanoparticles coupled to anti-EV antibodies and flow cytometry.

Results: Ex vivo tissues (i) remained viable and preserved their cytoarchitecture; (ii) maintained secretion of cytokines and growth factors; (iii) released EVs of syncytiotrophoblast and amnion epithelial cell origins that contain cytokines and growth factors.

Conclusion: A system of ex vivo placental villous and amnion tissues can be used as an adequate model to study placenta metabolic activity in normal and complicated pregnancies, in particular to characterize EVs by their surface markers and by encapsulated proteins. Establishment and benchmarking the placenta ex vivo system may provide new insight in the functional status of this organ in various placental disorders, particularly regarding the release of EVs and cytokines. Such EVs may have a prognostic value for pregnancy complications.

Keywords: 3D cultures; amnion; cytokine; growth factors; pregnancy; syncytiotrophoblast.

Figure 1. Placental villous and amnion tissue explants maintain their cytoarchitecture

H&E sections of placental villous explants at (a) day 1, (b) 7, and (c) 14 of culture (one representative tissue out of 10). Villi maintained normal morphology with well-preserved syncytiotrophoblasts and blood vessels with some focal degenerative changes. H&E sections of amnion explants at (d) day 1, (e) 7, and (f) 14 of culture also show well-preserved tissue with focal degenerative changes at day 14.

Figure 2. Placental villous and amnion tissue explants maintain cytokine and growth factor production throughout culture period

Soluble cytokines, growth factors, angiogenic and anti-angiogenic factors are produced by explants over the entire 14-day culture period (presented are average productions, mean ± SEM) as measured by multiplexed bead assays. Culture medium is replaced at each sampling time point. (a) Placental villous explants: amounts of cytokines released at day 1, 4, 7, 10, and 14, n=10; (b) Placental villous explants: amounts of growth factors released at day 1, 4, 7, 10, and 14, n=10; (c) Amnion explants: amounts of cytokines released at day 1, 4, 7, 10, and 14, n=10; (d) Amnion explants: amounts of growth factors released at day 1, 4, 7, 10, and 14, n=10.

Figure 3. Placental villous tissues release a variety of EVs carrying different surface markers

Placental villous explants release EVs that (a) carry surface markers that are representative of syncytiotrophoblast cells throughout culture and (b) are of a variety of sizes (average % of total EVs for each time point ± SEM, n=10). EVs carrying (c) CD51, (d) CD63, (e) CD105, (f) CD200, (g) CD274, and (h) syncytin-1 maintain similar patterns of expression over time and some are preferentially on EVs of certain sizes (Average % of total EVs for each size range. Mean ± SEM, n=10).

Figure 4. Amnion tissues release a variety of EVs carrying different surface markers

Amnion explants release EVs that (a) carry numerous surface markers that are representative of amnion epithelial and mesenchymal cells throughout culture and (b) are of a variety of sizes (average % of total EVs for each time point ± SEM, n=10). EVs carrying (c) CD29, (d) CD44, (e) CD105, (f) CD140b, (g) CD324, and (h) CD326 maintain similar patterns of expression over time and some are preferentially on EVs of certain sizes (average % of total EVs for each size range. Mean ± SEM, n=10).

Figure 5. Distribution of cytokines between the surface and inner volume of EVs from placental villous tissues

Distribution between encapsulated and surface cytokines is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™(b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP-captured EVs. Free and EV-associated cytokines are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated cytokines, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).

Figure 6. Distribution of cytokines between the surface and inner volume of EVs from amnion tissues

Distribution between encapsulated and surface cytokines is shown for amnion cultures (a) Total EVs isolated by Exoquick™; (b) anti-CD90 MNP-captured EVs; (c) anti-HLA-G MNP-captured EVs. Free and EV-associated cytokines are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated cytokines, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).

Figure 7. Distribution of growth factors between the surface and inner volume of EVs from placental villous tissues

Distribution between encapsulated and surface growth factors is shown for placental villous cultures. (a) Total EVs isolated by Exoquick™; (b) anti-PLAP MNP-captured EVs; (c) anti-CD31 MNP-captured EVs; (d) anti-HLA-G MNP- captured EVs. Free and EV-associated growth factors are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated growth factors, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).

Figure 8. Distribution of growth factors between the surface and inner volume of EVs from amnion tissues

Distribution between encapsulated and surface growth factors is shown for amnion cultures. (a) Total EVs isolated by Exoquick™; (b) anti-CD90 MNP-captured EVs (c) anti-HLA-G MNP-captured EVs. Free and EV-associated growth factors are expressed as percent of total (Mean ± SEM, n=5). Blue bars: surface-associated growth factors, red: EV-encapsulated. Multiplexed bead assay measurements on samples collected at day 4 (cumulative amount for days 1–4 of culture).