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. 2013 Nov 11;8(11):e79636.
doi: 10.1371/journal.pone.0079636. eCollection 2013.

Hypoxia-induced Changes in the Bioactivity of Cytotrophoblast-Derived Exosomes

Free PMC article

Hypoxia-induced Changes in the Bioactivity of Cytotrophoblast-Derived Exosomes

Carlos Salomon et al. PLoS One. .
Free PMC article


Migration of extravillous trophoblasts (EVT) into decidua and myometrium is a critical process in the conversion of maternal spiral arterioles and establishing placenta perfusion. EVT migration is affected by cell-to-cell communication and oxygen tension. While the release of exosomes from placental cells has been identified as a significant pathway in materno-fetal communication, the role of placental-derived exosomes in placentation has yet to be established. The aim of this study was to establish the effect of oxygen tension on the release and bioactivity of cytotrophoblast (CT)-derived exosomes on EVT invasion and proliferation. CT were isolated from first trimester fetal tissue (n = 12) using a trypsin-deoxyribonuclease-dispase/Percoll method. CT were cultured under 8%, 3% or 1% O2 for 48 h. Exosomes from CT-conditioned media were isolated by differential and buoyant density centrifugation. The effect of oxygen tension on exosome release (µg exosomal protein/10(6)cells/48 h) and bioactivity were established. HTR-8/SVneo (EVT) were used as target cells to establish the effect (bioactivity) of exosomes on invasion and proliferation as assessed by real-time, live-cell imaging (Incucyte™). The release and bioactivity of CT-derived exosomes were inversely correlated with oxygen tension (p<0.001). Under low oxygen tensions (i.e. 1% O2), CT-derived exosomes promoted EVT invasion and proliferation. Proteomic analysis of exosomes identified oxygen-dependent changes in protein content. We propose that in response to changes in oxygen tension, CTs modify the bioactivity of exosomes, thereby, regulating EVT phenotype. Exosomal induction of EVT migration may represent a normal process of placentation and/or an adaptive response to placental hypoxia.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Detection and characterization of Cytotrophoblast cell-derived exosomes.
Cytotrophoblast cells were isolated from chorionic villi obtained from first trimester pregnancies and cultured under different oxygen tension (see Methods). Exosomes were isolated from CTs culture media and characterized morphologically and using specific marker for exosome proteins. (A) Representative Western blot for exosome markers: CD63, CD9, CD81 and PLAP. Sample loading was normalized by protein content. Fractions 1 to 10, represent fractions collected after buoyant density centrifugation. (B) Protein profile of exosomal proteins and cytotrophoblast cells proteins. Exosomal proteins derived from fraction 5 to 8 (positive for exosomal marker) and cellular proteins of trophoblast cells (cells) were separated by SDS-PAGE and stained with SimplyBlueTM SafeStain. (C) Electron micrograph of exosomes isolated by ultracentrifuge and purified with a sucrose gradient (pooled exosomal pellet) from cytotrophoblast cells. In B, Scale bar 100 nm. In A, B and C, none of the experiments performed were significantly different using different oxygen tension.
Figure 2
Figure 2. Exosomes released from cytotrophoblast cell exposed to different oxygen tension.
Effects of oxygen tension on the release of exosomes from cytotrophoblast cells are presented as ug exosomal protein/106/48 h. Data are presented as a scatter plot with mean ± SEM (n = 6 biological samples and 2 independent duplicate cultures per placenta duplicate). ***p<0.001 versus 8% O2; **p<0.01 versus 1% O2; †p<0.05 versus 3% O2.
Figure 3
Figure 3. Cytotrophoblast cell-derived exosomes increased EVT invasion.
EVT cells were grown to confluence in complete media. A wound was made using 96 well WoundMaker and then overlaid to form a 3D matrix-gel (see Methods). EVT invasion was measured in absence (white circles) or presence (black circles) of 100 ug/ml of exosomes from cytotrophoblast cells exposed to 1% O2 (exo-CTs-1%) for 24 h. (A) Top: a, Wound imaged immediately after wounding; b, Graphical representation from a showing the calculation of initial wound width (black); c and e, Image at the midpoint of the experiment; d and f, Graphical representation from c and e of cell invasion (gray) at the midpoint of the experiment. (B) Time course of wound closure for HTR8/SVneo expressed as relative wound density (%). Data are presented as mean ± SEM for control (no exosomes, open circles) and treatment (100 µg/ml exosomal protein, closed circles). (C) Dose response curve for the effect of CT-derived exosomes on HTR8/SVneo invasion. Data are presented as a non-linear regression analysis (curve fit) and mean ± SEM.
Figure 4
Figure 4. Effect of oxygen tension on exosome bioactivity.
EVT cell invasion was measurement in presence of exosomes isolated from cytotrophoblast cells exposed to three different oxygen tension (1%, 3% and 8% O2). (A) The graph represents the changes of half-maximal effective concentration (EC50) and (B) half-maximal stimulatory time (ST50) exosomes on EVT invasion in response to oxygen tension (source). Values are mean ± SEM. *p<0.01 versus all conditions; †p<0.05 versus 8% O2.
Figure 5
Figure 5. Effect of cytotrophoblast cell-derived exosomes on EVT proliferation.
EVT cell proliferation was measurement in presence of exosomes (100 µg/ml) isolated from cytotrophoblast cells exposed to three different oxygen tension (1%, 3% and 8% O2). Values are mean ± SEM. *p<0.01 versus control (-exo-CTs) for each time point.
Figure 6
Figure 6. Analysis of cytotrophoblast cell-derived exosome proteins.
(A) The Venn diagram represents the distribution of common and unique proteins identified by nanospray LC-MS/MS (ABSciex 5600) in exosomes released from trophoblast cells exposed to 1%, 3% and 8% of oxygen tension. Comparison of canonical pathways: (B) HIFα, and (C) IL-8 signaling identified by IPA core analysis. Values are mean ± SEM. In B and C, *p<0.005 versus all condition; †p<0.05 versus 8% O2.
Figure 7
Figure 7. Ingenuity Pathway Analysis of Exosomal Proteins.
Unique proteins identified in exosomes isolated from cytotrophoblast cells exposed to 1% oxygen were submitted to IPA network analysis. Green: signaling involving in cellular movement.

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Grant support

CS holds a Postdoctoral Fellowship at The University of Queensland Centre for Clinical Research, Brisbane, Australia. GER was in receipt of an NHMRC Principal Research Fellowship. The work described herein was partially funded by a CIEF grant (University of Queensland), a Smart Futures Fund grant (Department of Employment, Economic Development and Innovation, Queensland Government) and a Translating Health Discovery into Clinical Applications SuperScience Award (Department of Industry, Innovation, Science, Research and Tertiary Education, Australian Government). This investigation was supported by CONICYT (ACT-73 PIA, Pasantía Doctoral en el Extranjero BECAS Chile), FONDECYT (1110977). CS holds CONICYT-PhD fellowships and Faculty of Medicine/PUC-PhD fellowships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.