Analysis of cytotrophoblast cell-derived exosome proteins.

<p>(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.</p

List of proteins identified in exosomes from CT exposed to different oxygen level.

<p>All mass spectra were analysed using the Mascot and Protein Pilot search engines against the Swissprot-swissprot database with the species set as human (score over 30). Exosomal proteins identified by mass-spectrometry were analyzed with the Ingenuity Pathway Analysis (Ingenuity Systems, <a href="http://www.ingenuity.com" target="_blank">www.ingenuity.com</a>). False discovery rate (FDR) was estimated using a reversed sequence database. List of total exosomal protein from cytotrophoblast cells exposed to different oxygen level are presented as Protein ID, Symbol, Entrez Gene Name, Location and type.</p

Detection and characterization of Cytotrophoblast cell-derived exosomes.

<p>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.</p

Ingenuity Pathway Analysis of Exosomal Proteins.

<p>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.</p

Exosomes released from cytotrophoblast cell exposed to different oxygen tension.

<p>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.</p

Effect of oxygen tension on exosome bioactivity.

<p>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.</p

Characterisation of exosomes from placental mesenchymal stem cell (pMSC).

<p>Cells were isolated from chorionic villi obtained from first trimester pregnancy and cultured under standard conditions. Exosomes were isolated from pMSC supernatant as was indicated in Methods. (A) Representative flow cytometry histogram of pMSC labeled with positive markers such as CD29, CD44, CD73, CD90 and CD105 (top panel) or negative markers such as CD11b, CD14, CD31, CD34 and CD45 (bottom panel). Black solid peaks represent the isotype controls and the red solid peak represents the marker indicated. (B) Mulit differntiation potential of first trimester placental chorionic villi. 1, Adipogenesis was determined using oil red O staining of lipid droplets after 21 days in adipogenic media. 2, Osteogenesis was determined using alizarin red staining for the mineral matrix deposition after 21 days in osteogenic media. (C) Electron micrograph of exosomes isolated by ultracentrifuge from pMSC. (D) pMSC were exposed to 1%, 3% or 8% O2 during 48 hours and then exosomes proteins were isolated. Samples in each condition were analyzed by western blot after the separation of 20 ug of exosomes protein (same amount of exosome protein lead) for the presence of CD63, CD9 and CD81. In B, <i>Scale bar</i> 100 nm.</p

List of proteins identified in exosomes from pMSC exposed to different oxygen level.

<p>List of proteins identified in exosomes from pMSC exposed to different oxygen level.</p

The level of pMSC-derived exosomes compared to low oxygen tension.

<p>Exosomes were isolated from pMSC supernatant exposed to 1%, 3% or 8% oxygen per 48 h. (A) Levels of exosomes are presented as protein concentration from 1×10⁶ pMSC cell. (B) Same volume of exosome pellet loaded and analyzed by western blot for CD63 and β-actin in exosome from pMSC and cells, respectively. Lower panel: CD63/β-actin ratio densitometries from data in top panel normalized to 1 in 1% O₂. (C) Effect of low oxygen tension on pMSC proliferation. (D) Trypan blue dye exclusion test to show residual pMSC cell viability exposed to 1%, 3% or 8% O₂. Values are mean ± SEM. In A and B, <i>*P</i><0.001 versus all condition; ^†<i>p</i><0.001 versus 8% O₂.</p

Ingenuity pathway analysis of pMSC derived-exosomes proteins.

<p>(A) The Venn diagram depicts the distribution of common and unique proteins identified by nanospray LC-MS/MS (ABSciex 5600) in exosomes released from pMSC exposed to 1%, 3% and 8% oxygen. Comparison of canonical pathways: (B) actin cytoskeleton signaling, (C) growth hormone signaling, (D) VEGF signaling, and (E) clathrin-mediated endocytosis signaling identified by IPA core analysis. Values are mean ± SEM. In B, C, D and E, <i>*p</i><0.005 versus all condition.</p