Placental Trophoblast Cell Isolation from the Term Placenta.

Multiple cell lines have been utilized over time in studying placental biology. Still, most of them rely on choriocarcinoma cells or immortalized trophoblast cells that may not be entirely comparable with actual human placental trophoblast cells. Term placentas can be a source of primary villous trophoblasts. However, challenges remain in isolating them and maintaining them in extended culture. This manuscript describes our three-phase protocol utilizing enzymatic/mechanical digestion, modified Percoll gradient density separation, and immunopurification using magnetic beads. The resulting trophoblast culture remains viable for an extended period and highly pure after initial passaging.

Establishment and comparison of human term placenta-derived trophoblast cells.

Research on the biology of fetal-maternal barriers has been limited by access to physiologically relevant cells, including trophoblast cells. In this study, we describe the development of a human-term placenta-derived cytotrophoblast immortalized cell line (hPTCCTB) derived from the basal plate. hPTCCTB cells are comparable to their primary cells of origin in terms of morphology, marker expression, and functional responses. We demonstrate that these can transform into syncytiotrophoblast and extravillous trophoblasts. We also compared the hPTCCTB cells to immortalized chorionic trophoblasts (hFM-CTC), trophoblasts of the chorionic plate, and BeWo cells, choriocarcinoma cell lines of conventional use. hPTCCTB and hFM-CTCs displayed more similarity to each other than to BeWos, but these differ in syncytialization ability. Overall, this study (1) demonstrates that the immortalized hPTCCTB generated are cells of higher physiological relevance, and (2) provides a look into the distinction between the spatially placental and fetal barrier trophoblasts cells, hPTCCTB and hFM-CTC, respectively.

mRNA and Protein Expression in Human Fetal Membrane Cells: Potential Biomarkers for Preterm Prelabor Rupture of the Fetal Membranes?

Clinically, unique markers in fetal membrane cells may contribute to the search for biomarkers for preterm prelabor rupture of the fetal membranes (pPROM) in maternal blood. pPROM is associated with overwhelming inflammation and premature cellular senescence causing "biological microfractures" of the fetal membranes. We hypothesize that these pathological processes are associated with the shedding of fetal membrane cells into the maternal circulation. The aim of this study was to identify markers expressed exclusively in fetal membrane cells to facilitate their isolation, characterization, and determination of biomarker potential in maternal blood. We have (1), by their transcriptomic profile, identified markers that are upregulated in amnion and chorion tissue compared to maternal white blood cells, and (2), by immunohistochemistry, confirmed the localization of the differentially expressed proteins in fetal membranes, placenta, and the placental bed of the uterus. RNA sequencing revealed 31 transcripts in the amnion and 42 transcripts in the chorion that were upregulated. Among these, 22 proteins were evaluated by immunohistochemistry. All but two transcripts were expressed both on mRNA and protein level in at least one fetal membrane cell type. Among these remaining 20 proteins, 9 proteins were not significantly expressed in the villous and extravillous trophoblasts of the placenta.

Amplification of microbial DNA from bacterial extracellular vesicles from human placenta.

Introduction: The placenta is essential for fetal growth and survival and maintaining a successful pregnancy. The sterility of the placenta has been challenged recently; however, the presence of a placental microbiome has been controversial. We tested the hypothesis that the bacterial extracellular vesicles (BEVs) from Gram-negative bacteria as an alternate source of microbial DNA, regardless of the existence of a microbial community in the placenta. Methods: Placentae from the term, not in labor Cesareans deliveries, were used for this study, and placental specimens were sampled randomly from the fetal side. We developed a protocol for the isolation of BEVs from human tissues and this is the first study to isolate the BEVs from human tissue and characterize them. Results: The median size of BEVs was 130-140 nm, and the mean concentration was 1.8-5.5 × 1010 BEVs/g of the wet placenta. BEVs are spherical and contain LPS and ompA. Western blots further confirmed ompA but not human EVs markers ALIX confirming the purity of preparations. Taxonomic abundance profiles showed BEV sequence reads above the levels of the negative controls (all reagent controls). In contrast, the sequence reads in the same placenta were substantially low, indicating nothing beyond contamination (low biomass). Alpha-diversity showed the number of detected genera was significantly higher in the BEVs than placenta, suggesting BEVs as a likely source of microbial DNA. Beta-diversity further showed significant overlap in the microbiome between BEV and the placenta, confirming that BEVs in the placenta are likely a source of microbial DNA in the placenta. Uptake studies localized BEVs in maternal (decidual) and placental cells (cytotrophoblast), confirming their ability to enter these cells. Lastly, BEVs significantly increased inflammatory cytokine production in THP-1 macrophages in a high-dose group but not in the placental or decidual cells. Conclusion: We conclude that the BEVs are normal constituents during pregnancy and likely reach the placenta through hematogenous spread from maternal body sites that harbor microbiome. Their presence may result in a low-grade localized inflammation to prime an antigen response in the placenta; however, insufficient to cause a fetal inflammatory response and adverse pregnancy events. This study suggests that BEVs can confound placental microbiome studies, but their low biomass in the placenta is unlikely to have any immunologic impact.

Development of oxidative stress-associated disease models using feto-maternal interface organ-on-a-chip.

Oxidative stress (OS) and inflammation arising from cellular derangements at the fetal membrane-decidual interface (feto-maternal interface [FMi]) is a major antecedent to preterm birth (PTB). However, it is impractical to study OS-associated FMi disease state during human pregnancy, and thus it is difficult to develop strategies to reduce the incidences of PTB. A microfluidic organ-on-chip model (FMi-OOC) that mimics the in vivo structure and functions of FMi in vitro was developed to address this challenge. The FMi-OOC contained fetal (amnion epithelial, mesenchymal, and chorion) and maternal (decidua) cells cultured in four compartments interconnected by arrays of microchannels to allow independent but interconnected co-cultivation. Using this model, we tested the effects of OS and inflammation on both fetal (fetal → maternal) and maternal (maternal → fetal) sides of the FMi and determined their differential impact on PTB-associated pathways. OS was induced using cigarette smoke extract (CSE) exposure. The impacts of OS were assessed by measuring cell viability, disruption of immune homeostasis, epithelial-to-mesenchymal transition (EMT), development of senescence, and inflammation. CSE propagated (LC/MS-MS analysis for nicotine) over a 72-hour period from the maternal to fetal side, or vice versa. However, they caused two distinct pathological effects on the maternal and fetal cells. Specifically, fetal OS induced cellular pathologies and inflammation, whereas maternal OS caused immune intolerance. The pronounced impact produced by the fetus supports the hypothesis that fetal inflammatory response is a mechanistic trigger for parturition. The FMi disease-associated changes identified in the FMi-OOC suggest the unique capability of this in vitro model in testing in utero conditions.

A Microphysiological Device to Model the Choriodecidual Interface Immune Status during Pregnancy.

During human pregnancy the chorion (fetal) lines decidua (maternal) creating the feto-maternal interface. Despite their proximity, resident decidual immune cells remain quiescent during gestation and do not invade the chorion. Infection and infiltration of activated immune cells toward the chorion are often associated with preterm birth. However, the mechanisms that maintain choriodecidual immune homeostasis or compromise immune barrier functions remain unclear. To understand these processes, a two-chamber microphysiological system (MPS) was created to model the human choriodecidual immune interface under normal and infectious conditions in vitro. This MPS has outer (fetal chorion trophoblast cells) and inner chambers (maternal decidual + CD45+ cells [70:30 ratio]) connected by microchannels. Decidual cells were treated with LPS to mimic maternal infection, followed by immunostaining for HLA-DR and HLA-G, immune panel screening by imaging cytometry by time of flight, and immune regulatory factors IL-8 and IL-10, soluble HLA-G, and progesterone (ELISA). LPS induced a proinflammatory phenotype in the decidua characterized by a decrease in HLA-DR and an increase in IL-8 compared with controls. LPS treatment increased the influx of immune cells into the chorion, indicative of chorionitis. Cytometry by time of flight characterized immune cells in both chambers as active NK cells and neutrophils, with a decrease in the abundance of nonproinflammatory cytokine-producing NK cells and T cells. Conversely, chorion cells increased progesterone and soluble HLA-G production while maintaining HLA-G expression. These results highlight the utility of MPS to model choriodecidual immune cell infiltration and determine the complex maternal-fetal crosstalk to regulate immune balance during infection.

Generation and characterization of human Fetal membrane and Decidual cell lines for reproductive biology experiments†.

Human fetal membrane and maternal decidua parietalis form one of the major feto-maternal interfaces during pregnancy. Studies on this feto-maternal interface is limited as several investigators have limited access to the placenta, and experience difficulties to isolate and maintain primary cells. Many cell lines that are currently available do not have the characteristics or properties of their primary cells of origin. Therefore, we created, characterized the immortalized cells from primary isolates from fetal membrane-derived amnion epithelial cells, amnion and chorion mesenchymal cells, chorion trophoblast cells and maternal decidua parietalis cells. Primary cells were isolated from a healthy full-term, not in labor placenta. Primary cells were immortalized using either a HPV16E6E7 retroviral or a SV40T lentiviral system. The immortalized cells were characterized for the morphology, cell type-specific markers, and cell signalling pathway activation. Genomic stability of these cells was tested using RNA seq, karyotyping, and short tandem repeats DNA analysis. Immortalized cells show their characteristic morphology, and express respective epithelial, mesenchymal and decidual markers similar to that of primary cells. Gene expression of immortalized and primary cells were highly correlated (R = 0.798 to R = 0.974). Short tandem repeats DNA analysis showed in the late passage number (>P30) of cell lines matched 84-100% to the early passage number (

Extracellular vesicles from maternal uterine cells exposed to risk factors cause fetal inflammatory response.

BACKGROUND: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. METHODS: Decidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 h to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 h. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined. RESULTS: Exosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells. CONCLUSION: Various pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response. Video Abstract.

Extracellular vesicle mediated feto-maternal HMGB1 signaling induces preterm birth.

Preterm birth (PTB; <37 weeks of gestation) impacts ∼11% of all pregnancies and contributes to 1 million neonatal deaths worldwide annually. An understanding of the feto-maternal (F-M) signals that initiate birthing (parturition) at term is critical to design strategies to prevent their premature activation, resulting in PTB. Although endocrine and immune cell signaling are well-reported, fetal-derived paracrine signals capable of transitioning quiescent uterus to an active state of labor are poorly studied. Recent reports have suggested that senescence of the fetal amnion membrane coinciding with fetal growth and maturation generates inflammatory signals capable of triggering parturition. This is by increasing the inflammatory load at the feto-maternal interface (FMi) tissues (i.e., amniochorion-decidua). High mobility group box 1 protein (HMGB1), an alarmin, is one of the inflammatory signals released by senescent amnion cells via extracellular vesicles (exosomes; 40-160 nm). Increased levels of HMGB1 in the amniotic fluid, cord and maternal blood are associated with term and PTB. This study tested the hypothesis that senescent amnion cells release HMGB1, which is fetal signaling capable of increasing FMi inflammation, predisposing them to parturition. To test this hypothesis, exosomes from amnion epithelial cells (AECs) grown under normal conditions were engineered to contain HMGB1 by electroporation (eHMGB1). eHMGB1 was characterized (quantity, size, shape, markers and loading efficiency), and its propagation through FMi was tested using a four-chamber microfluidic organ-on-a-chip device (FMi-OOC) that contained four distinct cell types (amnion and chorion mesenchymal, chorion trophoblast and decidual cells) connected through microchannels. eHMGB1 propagated through the fetal cells and matrix to the maternal decidua and increased inflammation (receptor expression [RAGE and TLR4] and cytokines). Furthermore, intra-amniotic injection of eHMGB1 (containing 10 ng) into pregnant CD-1 mice on embryonic day 17 led to PTB. Injecting carboxyfluorescein succinimidyl ester (CFSE)-labeled eHMGB1, we determined in vivo kinetics and report that eHMGB1 trafficking resulting in PTB was associated with increased FMi inflammation. This study determined that fetal exosome mediated paracrine signaling can generate inflammation and induce parturition. Besides, in vivo functional validation of FMi-OOC experiments strengthens the reliability of such devices to test physiologic and pathologic systems.

Characterizing the immune cell population in the human fetal membrane.

PROBLEM: This study localized CD45+ immune cells and compared changes in their numbers between term, not in labor (TNIL) and term, labor (TL) human fetal membranes. METHOD OF STUDY: Fetal membranes (amniochorion) from normal TNIL and TL subjects were analyzed by immunohistochemistry (IHC), immunofluorescence (IF), and flow cytometry for evidence of total (CD45+ ) immune cells as well as innate immune cells (neutrophils, macrophages and NK cells) using specific markers. Fetal origin of immune cells was determined using polymerase chain reaction (PCR) for SRY gene in Y chromosome. RESULTS: CD45+ cells were localized in human fetal membranes for both TNIL and TL. A threefold increase in CD45+ cells was seen in TL fetal membranes of (7.73% ± 2.35) compared to TNIL (2.36% ± 0.78). This increase is primarily contributed by neutrophils. Macrophages and NK cells did not change in the membranes between TNIL and TL. Leukocytes of fetal origin are present in the fetal membranes. CONCLUSION: The fetal membranes without decidua contain a small proportion of immune cells. Some of these immune cells in the fetal membrane are fetal in origin. There is a moderate increase of immune cells in the fetal membranes at term labor; however, it is unclear whether this is a cause or consequence of labor. Further functional studies are needed to determine their contribution to membrane inflammation associated with parturition.

Isolation and characterization human chorion membrane trophoblast and mesenchymal cells.

INTRODUCTION: To develop protocols for isolation and culture of human chorionic mesenchymal and trophoblast cells and test their differential responsiveness to oxidative stress. METHODS: Chorion trophoblast cells (CTC) and chorion mesenchymal cells (CMC) were isolated from term fetal membranes by modifying current protocols. Their purity and characteristics were tested using bright field microscopy and after staining for cytokeratin (CK)-7 and vimentin. Cigarette smoke extract (CSE) was used to stimulate cells, and we determined reactive oxygen species (ROS) production using 2'7'-dichlorodihydro-fluorescein assay, stress signaler p38MAPK activation (Western blot) and senescence by flow cytometry. Co-treatment with antioxidant N-acetyl cystine (NAC) either alone or in combination with SB203580 (p38MAPK inhibitor) was used to test oxidative stress (OS)- and p38MAPK-mediated effects. RESULTS: The isolation and cell culture protocol used in this study yielded 92% pure CTC and 100% pure CMC. CSE treatment significantly induced ROS production, P-p38MAPK activation, and senescence in both cell types compared to controls. Cotreatment with NAC reduced ROS production and p38MAPK activation, and co-treatment with both NAC and SB203580 reduced senescence. ROS response in CMC was higher than CTC; however, senescence of CTC was 10-fold higher than CMC. CONCLUSIONS: We introduce approaches for proper isolation and culture of CTC and CMC without any influence or overgrowth of one specific type cell that can confound results. Using this approach, we determined differential effects of CTC and CMC to OS condition seen at term labor. Both CTC and CMC undergo p38MAPK-mediated senescence; however, the rate of senescence is higher in CTC.

Inflammation, but not infection, induces EMT in human amnion epithelial cells.

A non-reversible state of epithelial to mesenchymal transition (EMT) at term accumulates proinflammatory mesenchymal cells and predisposes fetal membrane to weakening prior to delivery at term. We investigated the induction of EMT in amnion epithelial cells (AEC) in response to inflammation and infection associated with spontaneous preterm birth (SPTB). For this, membranes from SPTB were screened for EMT markers. Primary AEC in culture were treated with TNF-α (10 and 50 ng/mL) and LPS (50 and 100 ng/mL) for 72 h. Cell shape index (SI) was determined based on morphological shift (microscopy followed by ImageJ software analysis). Immunocytochemistry and Western blot assessed changes in epithelial markers (cytokeratin-18 and E-cadherin) and mesenchymal markers (vimentin and N-cadherin). Involvement of transforming growth factor beta (TGF-ß) in EMT induction and EMT associated inflammation was tested using specific markers (Western blot) and by measuring MMP9 (ELISA), respectively. We report that PTB is associated with fetal membrane EMT. TNF-α produced dose- and time-dependent induction of EMT; within 24 h by 50 ng/mL and after 72 h by 10 ng/mL. AEC showed mesenchymal morphology, lower E-cadherin, higher vimentin and N-cadherin and higher MMP9 compared to control. TNF-α-induced EMT was not associated with canonical TGF-ß pathway. LPS, regardless of dose or time, did not induce EMT in AEC. We conclude that PTB with intact membranes is associated with EMT. Our data suggest that inflammation, but not infection, is associated with non-canonical activation of EMT and inflammation that can predispose membrane to undergo weakening.

Stretch, scratch, and stress: Suppressors and supporters of senescence in human fetal membranes.

INTRODUCTION: Throughout gestation, amnion membranes undergo mechanical and or physiological stretch, scratch, or stress which is withstood by repairing and remodeling processes to protect the growing fetus. At term, increased oxidative stress (OS) activates p38MAPK, induces senescence, and inflammation contributing to membrane dysfunction to promote labor. However, the signaling initiated by stretch and scratch is still unclear. This study compares the induction of p38MAPK mediated senescence by stretch, scratch, and stress in human amnion epithelial cells (AECs). METHODS: Primary AECs from term, not-in-labor, fetal membranes were cultured using the following conditions (N = 3); 1) CellFlex chambers with or without 20% biaxial stretch, 2) 8-well coverslips with or without scratch, and 3) cells exposed to cigarette smoke extract (CSE) inducing OS. p38MAPK (Western blot or immunocytochemistry), senescence activation, and inflammation (matrix metalloproteinases 9 [MMP9] activity-ELISA) were determined in cells exposed to various conditions. T-test and One-Way ANOVA was used to assess significance. RESULTS: Biological membrane extension, mimicked by 20% biaxial stretch of AEC, maintained an epithelial morphology and activated P-p38MAPK (P = 0.02) compared to the non-stretch controls, but did not induce senescence or MMP9 activation. AEC scratches were healed within 40-hrs, which included proliferation, migration, and cellular transitions aided by p38MAPK activation but not senescence. CSE induced OS increased p38MAPK (P = 0.018) activation, senescence (P = 0.019), and MMP9 (P = 0.02). CONCLUSION: Physiologic stretch and scratch experienced during gestation can cause p38MAPK activation without causing senescence or inflammation. This may be indicative of p38MAPK's role in tissue remodeling during pregnancy. Overwhelming OS, experienced at term, results in P-p38MAPK mediated senescence and inflammation to disrupt membrane remodeling.

Fetal membrane extracellular vesicle profiling reveals distinct pathways induced by infection and inflammation in vitro.

PROBLEM: Fetal inflammatory signals can be propagated to maternal tissues to initiate labor via exosomes (extracellular vesicles; 30-150 nm). We tested the hypothesis that fetal membrane cells exposed to infectious and inflammatory mediators associated with preterm birth (PTB) produce exosomes with distinct protein cargo contents indicative of underlying pathobiology. METHODS OF STUDY: Fetal membrane explants (FM) as well as primary amnion epithelial (AEC) and mesenchymal cells (AMC), and chorion cells (CC) from term deliveries were maintained in normal conditions (control) or exposed to LPS 100 ng/mL or TNF-α 50 ng/mL for 48 hours. Exosomes were isolated from media by differential centrifugation and size exclusion chromatography and characterized using cryo-electron microscopy (morphology), nanoparticle tracking analysis (size and quantity), Western blot (markers), and mass spectroscopy (cargo proteins). Ingenuity pathway analysis (IPA) determined pathways indicated by differentially expressed proteins. RESULTS: Irrespective of source or treatment, exosomes were spherical, had similar size, quantities, and markers (ALIX, CD63, and CD81). However, exosome cargo proteins were different between FM and individual fetal membrane cell-derived exosomes in response to treatments. Several common proteins were seen; however, there are several unique proteins expressed by exosomes from different cell types in response to distinct stimuli indicative of unique pathways and physiological functions in cells. CONCLUSIONS: We demonstrate collective tissue and independent cell response reflected in exosomes in response to infectious and inflammatory stimuli. These cargoes determined underlying physiology and their potential in enhancing inflammation in a paracrine fashion.

Oxidative stress-induced downregulation of glycogen synthase kinase 3 beta in fetal membranes promotes cellular senescence†.

OBJECTIVE: Oxidative stress (OS)-induced stress signaler p38 mitogen-activated protein kinase (p38MAPK) activation and fetal membrane senescence are associated with parturition. This study determined changes in glycogen synthase kinase 3 beta (GSK3ß) and its regulation by p38MAPK in effecting senescence to further delineate the molecular mechanism involved in senescence. METHODS: Primary human amnion epithelial cells and amnion mesenchymal cells were treated with cigarette smoke extract (CSE, OS inducer). Expression of total and phosphorylated GSK3ß and p38MAPK, and that of GSK3ß's downstream targets: beta-catenin (ß-Cat) and nuclear factor erythroid 2-related factor 2 (Nrf2) (western blot analysis), cell cycle regulation and senescence (flow cytometry) were determined. The specificity of GSK3ß and p38MAPK's mechanistic role was tested by co-treating cells with their respective inhibitors, CHIR99021 and SB203580. Exosomal secretion of ß-Cat from OS-induced cells was confirmed by immunofluorescence confocal microscopy and western blot. RESULTS: OS induced by CSE resulted in phosphorylation of GSK3ß (inactivation) and p38MAPK (activation) that was associated with cell cycle arrest and senescence. Inhibitors to GSK3ß and p38MAPK verified their roles. Glycogen synthase kinase 3 beta inactivation was associated with nuclear translocation of antioxidant Nrf2 and exosomal secretion of ß-Cat. CONCLUSIONS: OS-induced P-p38MAPK activation is associated with functional downregulation of GSK3ß and arrest of cell cycle progression and senescence of amnion cells. Lack of nuclear translocation of ß-Cat and its excretion via exosomes further supports the postulation that GSK3ß down-regulation by p38MAPK may stop cell proliferation preceding cell senescence. A better understanding of molecular mechanisms of senescence will help develop therapeutic strategies to prevent preterm birth.

Damage-Associated molecular pattern markers HMGB1 and cell-Free fetal telomere fragments in oxidative-Stressed amnion epithelial cell-Derived exosomes.

Term labor in humans is associated with increased oxidative stress (OS) -induced senescence and damages to amnion epithelial cells (AECs). Senescent fetal cells release alarmin high-mobility group box 1 (HMGB1) and cell-free fetal telomere fragments (cffTF) which can be carried by exosomes to other uterine tissues to produce parturition-associated inflammatory changes. This study characterized AEC-derived exosomes under normal and OS conditions and their packaging of HMGB1 and cffTF. Primary AECs were treated with either standard media or oxidative stress-induced media (exposure to cigarette smoke extract for 48h). Senescence was determined, and exosomes were isolated and characterized. To colocalize HMGB1 and cffTF in amnion exosomes, immunofluorescent staining and in situ hybridization were performed, followed by confocal microscopy. Next generation sequencing (NGS) determined exosomal cffTF and other cell-free amnion cell DNA specificity. Regardless of condition, primary AECs produce exosomes with a classic size, shape, and markers. OS and senescence caused the translocation of HMGB1 and cffTF from AECs' nuclei to cytoplasm compared to untreated cells, which was inhibited by antioxidant N-acetyl cysteine (NAC). Linescans confirmed colocalization of HMGB1 and cffTF in exosomes were higher in the cytoplasm after CSE treatment compared to untreated AECs. NGS determined that besides cffTF, AEC exosomes also carry genomic and mitochondrial DNA, regardless of growth conditions. Sterile inflammatory markers HMGB1 and cffTF from senescent fetal cells are packaged inside exosomes. We postulate that this exosomal cargo can act as a fetal signal at term and can cause labor-associated changes in neighboring tissues.

A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus Infection.

Currently there are no approved vaccines or specific therapies to prevent or treat Zika virus (ZIKV) infection. We interrogated a library of FDA-approved drugs for their ability to block infection of human HuH-7 cells by a newly isolated ZIKV strain (ZIKV MEX_I_7). More than 20 out of 774 tested compounds decreased ZIKV infection in our in vitro screening assay. Selected compounds were further validated for inhibition of ZIKV infection in human cervical, placental, and neural stem cell lines, as well as primary human amnion cells. Established anti-flaviviral drugs (e.g., bortezomib and mycophenolic acid) and others that had no previously known antiviral activity (e.g., daptomycin) were identified as inhibitors of ZIKV infection. Several drugs reduced ZIKV infection across multiple cell types. This study identifies drugs that could be tested in clinical studies of ZIKV infection and provides a resource of small molecules to study ZIKV pathogenesis.

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

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) exosomes act as carriers of signals in utero-placental compartments and ii) 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.

Senescence of primary amniotic cells via oxidative DNA damage.

OBJECTIVE: Oxidative stress is a postulated etiology of spontaneous preterm birth (PTB) and preterm prelabor rupture of the membranes (pPROM); however, the precise mechanistic role of reactive oxygen species (ROS) in these complications is unclear. The objective of this study is to examine impact of a water soluble cigarette smoke extract (wsCSE), a predicted cause of pregnancy complications, on human amnion epithelial cells. METHODS: Amnion cells isolated from fetal membranes were exposed to wsCSE prepared in cell culture medium and changes in ROS levels, DNA base and strand damage was determined by using 2'7'-dichlorodihydro-fluorescein and comet assays as well as Fragment Length Analysis using Repair Enzymes (FLARE) assays, respectively. Western blot analyses were used to determine the changes in mass and post-translational modification of apoptosis signal-regulating kinase (ASK1), phospho-p38 (P-p38 MAPK), and p19(arf). Expression of senescence-associated ß-galectosidase (SAß-gal) was used to confirm cell ageing in situ. RESULTS: ROS levels in wsCSE-exposed amnion cells increased rapidly (within 2 min) and significantly (p<0.01) at all-time points, and DNA strand and base damage was evidenced by comet and FLARE assays. Activation of ASK1, P-p38 MAPK and p19(Arf) correlated with percentage of SAß-gal expressing cells after wsCSE treatment. The antioxidant N-acetyl-L-cysteine (NAC) prevented ROS-induced DNA damage and phosphorylation of p38 MAPK, whereas activation of ASK1 and increased expression of p19(Arf) were not significantly affected by NAC. CONCLUSIONS: The findings support the hypothesis that compounds in wsCSE induces amnion cell senescence via a mechanism involving ROS and DNA damage. Both pathways may contribute to PTB and pPROM. Our results imply that antioxidant interventions that control ROS may interrupt pathways leading to pPROM and other causes of PTB.