Documentation of DNA by flow cytometry in exosomes using propidium iodide (PI).

<p>There was no significant difference in DNA content in exosomes untreated with DNase (top) and those pretreated with DNase (bottom). There was also no significant difference in DNA content between CD9, CD63 and CD81 positive exosomes. The average DNA content was similar for both control (4.7 ng/μL) and CSE (5.1 ng/μL) derived exosomes. AEC-amnion epithelial cell, CSE-cigarette smoke extract.</p

Proteomic analysis of AEC-derived exosome proteins.

<p>(A) The Venn diagram represents the distribution of common and unique proteins identified by nanospray LC-MS/MS in exosomes released from AEC cultured under normal or stress conditions. List contain 221 unique proteins. (B and C) Proteins identified in exosomes isolated from AEC under normal (B) or oxidative stress (C) conditions were submitted to IPA network analysis. Red circle: central molecules involved in the signaling pathways.</p

Colocalization of exosome marker P-p38MAPK and CD9 in AECs.

<p>Colocalization of pro-senescence marker P-p38MAPK and CD9: Immunofluorescence imaging of control (top panel) and CSE treated amnion cells (bottom panel) show colocalization differences of HSP70 (green) and CD9 (red). Significantly higher colocalization (line graph and bar graph) of H3 was seen after CSE treatment compared to control (Pearson’s Coefficient P<0.0001) AEC-amnion epithelial cell, CSE-cigarette smoke extract.</p

Cytokeratin staining of primary AECs.

<p>Primary AECs were fluorescently labeled for cytokeratin 18 to show cultures were predominantly epithelial cells. AEC-amnion epithelial cell.</p

Colocalization of exosome marker HSP70 and CD9 in AECs.

<p>Colocalization of DAMP, HSP70, and CD9: Immunofluorescence imaging of control (top panel) and CSE treated amnion cells (bottom panel) show colocalization differences of HSP70 (green) and CD9 (red). Significantly higher colocalization (line graph and bar graph) of H3 was seen after CSE treatment compared to control (Pearson’s Coefficient P<0.001) AEC-amnion epithelial cell, CSE-cigarette smoke extract; DAMP–Damage associated molecular pattern.</p

Cell cycle analysis and p38 MAPK activation of control and CSE treated amnion cells.

<p>Cell cycle analysis and p38 MAPK activation of control and CSE treated amnion cells.</p

Colocalization of exosome marker H3 and CD9 in AECs.

<p>Immunofluorescence imaging of control (top panel) and CSE treated amnion cells (bottom panel) show colocalization differences of H3 (green) and CD9 (red). Significantly higher colocalization (line graph and bar graph) of H3 was seen after CSE treatment compared to control (Pearson’s Coefficient P<0.0001) AEC-amnion epithelial cell, CSE-cigarette smoke extract.</p

Amnion-Epithelial-Cell-Derived Exosomes Demonstrate Physiologic State of Cell under Oxidative Stress

<div><p>At term, the signals of fetal maturity and feto-placental tissue aging prompt uterine readiness for delivery by transitioning quiescent myometrium to an active stage. It is still unclear how the signals reach the distant myometrium. Exosomes are a specific type of extracellular vesicle (EVs) that transport molecular signals between cells, and are released from a wide range of cells, including the maternal and fetal cells. In this study, we hypothesize that <i>i</i>) exosomes act as carriers of signals in utero-placental compartments and <i>ii</i>) exosomes reflect the physiologic status of the origin cells. The primary aims of this study were to determine exosomal contents in exosomes derived from primary amnion epithelial cells (AEC). We also determined the effect of oxidative stress on AEC derived exosomal cargo contents. AEC were isolated from amniotic membrane obtained from normal, term, not in labor placentae at delivery, and culture under standard conditions. Oxidative stress was induced using cigarette smoke extract for 48 hours. AEC-conditioned media were collected and exosomes isolated by differential centrifugations. Both growth conditions (normal and oxidative stress induced) produced cup shaped exosomes of around 50 nm, expressed exosomes enriched markers, such as CD9, CD63, CD81 and HSC70, embryonic stem cell marker Nanog, and contained similar amounts of cell free AEC DNA. Using confocal microscopy, the colocalization of histone (H) 3, heat shock protein (HSP) 70 and activated form of pro-senescence and term parturition associated marker p38 mitogen activated protein kinase (MAPK) (P-p38 MAPK) co-localized with exosome enrich marker CD9. HSP70 and P-p38 MAPK were significantly higher in exosomes from AEC grown under oxidative stress conditions than standard conditions (p<0.05). Finally, mass spectrometry and bioinformatics analysis identified 221 different proteins involved in immunomodulatory response and cell-to-cell communication. This study determined AEC exosome characteristics and their cargo reflected the physiologic status of the cell of origin and suggests that AEC-derived exosomal p38 MAPK plays a major role in determining the fate of pregnancy. Understanding the propagation of fetal signals and their mechanisms in normal term pregnancies can provide insights into pathologic activation of such signals associated with spontaneous preterm parturitions.</p></div

Characterization of AEC exosomes.

<p>The exosomes isolated from untreated (control) and CSE treated primary AEC carry cargo representative of the state of the origin cell. AEC exosomes contain tetraspnins (CD9, CD63 and CD81), Nanog, HSC70, HSP70 and DNA regardless of treatment, while CSE exosomes contain significantly increased amounts of P-p38MAPK and H3 compared with control exosomes.</p

Ingenuity pathway analysis of AEC-derived-exosomes proteins.

<p>Exosomal protein identified under normal (control) or oxidative stress conditions were analyzed using the IPA software. Comparison of canonical pathways: (A) ERK/MAPK, (B) PI3K/AKT, (C) epithelial adherens junctions, (D) LPS/IL-1 mediated inhibition of RXR function and (E) IL-6 signaling. Diseases and functions analysis: (F) eosinophilic inflammation. Values are mean ± SD. In A and F, **p < 0.001. In B, C and D, *p < 0.005.</p