Background: Vascular smooth muscle cells (VSMCs) migration is a critical process during human uterine spiral artery (SpA) remodeling and a successful pregnancy. Extravillous trophoblast cells (EVT) interact with VSMC and enhance their migration, however, the mechanisms by which EVT remodel SpA remain to be fully elucidated. We hypothesize that exosomes released from EVT promote VSMC migration. Methods: JEG-3 and HTR-8/SVneo cell lines were used as models for EVT. Cells were cultured at 37 0C and humidified under an atmosphere of 5% CO2-balanced N2 to obtain 8% O2. Cell-conditioned media were collected and exosomes (exo-JEG-3 and exo- HTR-8/SVneo) isolated by differential and buoyant density centrifugation. The effects of exo-EVT on VSMC migration were established using a real-time, live-cell imaging system (Incucyte™). Exosomal proteins where identified by mass spectrometry and submitted to bioinformatic pathway analysis (Ingenuity software) . Results: HTR-8/SVneo cells were significantly more (~30%) invasive than JEG-3 cells. HTR-8/SVneo cells released 2.6-fold more exosomes (6.39 x 108 ± 2.5 x108 particles/106 cells) compared to JEG-3 (2.86 x 108 ± 0.78 x108 particles/106 cells). VSMC migration was significantly increased in the presence of exo-JEG-3 and exo-HTR-8/SVneo compared to control (-exosomes) (21.83 ± 0.49 h and 15.57 ± 0.32, respectively, versus control 25.09 ± 0.58 h, p<0.05). Sonication completely abolished the effect of exosomes on VSMC migration. Finally, mass spectrometry analysis identified unique exosomal proteins for each EVT cell line-derived exosomes. Conclusion: The data obtained in this study are consistent with the hypothesis that the release, content and bioactivity of exosomes derived from EVT-like cell lines is cell origin-dependent and differentially regulates VSMC migration. Thus, an EVT exosomal signaling pathway may contribute to SpA remodeling by promoting the migration of VSMC out of the vessel walls.

/pdf/extravillous-trophoblast-cells-derived-exosomes-promote-2ntfknfma7.pdf

Extravillous trophoblast cells-derived exosomes promote vascular smooth muscle cell migration.

Background The human placenta is vital for fetal development, yet little is understood about how it forms successfully to ensure a healthy pregnancy or why this process is inadequate in 1 in 10 pregnancies, leading to miscarriage, intrauterine growth restriction or preeclampsia. Trophoblasts are placenta-specific epithelial cells that maximize nutrient exchange. All trophoblast lineages are thought to arise from a population of trophoblast stem cells (TSCs). However, whilst the isolation of murine TSC has led to an explosion in understanding murine placentation, the isolation of an analogous human TSC has proved more difficult. Consequently, alternative methods of studying human trophoblast lineage development have been employed, including human embryonic stem cells (hESCs), induced pluripotent stem cells (iPS) and transformed cell lines; but what do these proxy models tell us about what is happening during early placental development? Objective and rationale In this systematic review, we evaluate current approaches to understanding human trophoblast lineage development in order to collate and refine these models and inform future approaches aimed at establishing human TSC lines. Search methods To ensure all relevant articles were analysed, an unfiltered search of Pubmed, Embase, Scopus and Web of Science was conducted for 25 key terms on the 13th May 2016. In total, 47 313 articles were retrieved and manually filtered based on non-human, non-English, non-full text, non-original article and off-topic subject matter. This resulted in a total of 71 articles deemed relevant for review in this article. Outcomes Candidate human TSC populations have been identified in, and isolated from, both the chorionic membrane and villous tissue of the placenta, but further investigation is required to validate these as 'true' human TSCs. Isolating human TSCs from blastocyst trophectoderm has not been successful in humans as it was in mice, although recently the first reported TSC line (USFB6) was isolated from an eight-cell morula. In lieu of human TSC lines, trophoblast-like cells have been induced to differentiate from hESCs and iPS. However, differentiation in these model systems is difficult to control, culture conditions employed are highly variable, and the extent to which they accurately convey the biology of 'true' human TSCs remains unclear, particularly as a consensus has not been met among the scientific community regarding which characteristics a human TSC must possess. Wider implications Human TSC models have the potential to revolutionize our understanding of trophoblast differentiation, allowing us to make significant gains in understanding the underlying pathology of pregnancy disorders and to test potential therapeutic interventions on cell function in vitro. In order to do this, a collaborative effort is required to establish the criteria that define a human TSC to confirm the presence of human TSCs in both primary isolates and to determine how accurately trophoblast-like cells derived from current model systems reflect trophoblast from primary tissue. The in vitro systems currently used to model early trophoblast lineage formation have provided insights into early human placental formation but it is unclear whether these trophoblast-like cells are truly representative of primary human trophoblast. Consequently, continued refinement of current models, and standardization of culture protocols is essential to aid our ability to identify, isolate and propagate 'true' human TSCs from primary tissue.

/pdf/stem-cell-insights-into-human-trophoblast-lineage-437716y411.pdf

Stem cell insights into human trophoblast lineage differentiation

Connexin43 (Cx43)-mediated gap junctional intercellular communication (GJIC) are required for human trophoblast differentiation. To date, whether Cx43 mediates TGF-β1-induced trophoblast differentiation has not been determined. We showed that treatment with TGF-β1 increased Cx43 expression and GJIC in HTR-8/SVneo human trophoblast cells. In addition, Smad and ERK1/2 signaling pathways were involved in TGF-β1-induced up-regulation of Cx43. Moreover, TGF-β1 increased the expression of the syncytiotrophoblast marker, β-hCG. Importantly, knockdown of Cx43 abolished the TGF-β1-induced up-regulation of β-hCG. Furthermore, overexpression of Cx43 up-regulated β-hCG expression. These results provide evidence that Cx43 and GJIC activity are up-regulated by TGF-β1 in human trophoblast cells, which subsequently contributes to TGF-β1-induced trophoblast differentiation. J. Cell. Physiol. 230: 1558–1566, 2015. © 2015 Wiley Periodicals, Inc., A Wiley Company

TGF-β1 up-regulates connexin43 expression: A potential mechanism for human trophoblast cell differentiation

Aberrant DNA methylation is frequently observed in disease, including many cancer types, yet the underlying mechanisms remain unclear. Because germline and somatic mutations in the genes that are responsible for DNA methylation are infrequent in malignancies, additional mechanisms must be considered. Mycoplasmas spp., including Mycoplasma hyorhinis, efficiently colonize human cells and may serve as a vehicle for delivery of enzymatically active microbial proteins into the intracellular milieu. Here, we performed, for the first time, genome-wide and individual gene mapping of methylation marks generated by the M. hyorhinis CG- and GATC-specific DNA cytosine methyltransferases (MTases) in human cells. Our results demonstrated that, upon expression in human cells, MTases readily translocated to the cell nucleus. In the nucleus, MTases selectively and efficiently methylated the host genome at the DNA sequence sites free from pre-existing endogenous methylation, including those in a variety of cancer-associated genes. We also established that mycoplasma is widespread in colorectal cancers, suggesting that either the infection contributed to malignancy onset or, alternatively, that tumors provide a favorable environment for mycoplasma growth. In the human genome, ∼ 11% of GATC sites overlap with CGs (e.g., CGAT(m)CG); therefore, the methylated status of these sites can be perpetuated by human DNMT1. Based on these results, we now suggest that the GATC-specific methylation represents a novel type of infection-specific epigenetic mark that originates in human cells with a previous exposure to infection. Overall, our findings unveil an entirely new panorama of interactions between the human microbiome and epigenome with a potential impact in disease etiology.

https://www.tandfonline.com/doi/pdf/10.1080/15592294.2015.1020000

Mycoplasma CG- and GATC-specific DNA methyltransferases selectively and efficiently methylate the host genome and alter the epigenetic landscape in human cells

Trichloroethylene (TCE) is a common environmental pollutant associated with adverse reproductive outcomes in humans. TCE intoxication occurs primarily through its biotransformation to bioactive metabolites, including S-(1,2-dichlorovinyl)-l-cysteine (DCVC). TCE induces oxidative stress and inflammation in the liver and kidney. Although the placenta is capable of xenobiotic metabolism and oxidative stress and inflammation in placenta have been associated with adverse pregnancy outcomes, TCE toxicity in the placenta remains poorly understood. We determined the effects of DCVC by using the human extravillous trophoblast cell line HTR-8/SVneo. Exposure to 10 and 20 μM DCVC for 10 h increased reactive oxygen species (ROS) as measured by carboxydichlorofluorescein fluorescence. Moreover, 10 and 20 μM DCVC increased mRNA expression and release of interleukin-6 (IL-6) after 24-h exposure, and these responses were inhibited by the cysteine conjugate beta-lyase inhibitor aminooxyacetic acid and by treatments with antioxidants (alpha-tocopherol and deferoxamine), suggesting that DCVC-stimulated IL-6 release in HTR-8/SVneo cells is dependent on beta-lyase metabolic activation and increased generation of ROS. HTR-8/SVneo cells exhibited decreased mitochondrial membrane potential at 5, 10, and 20 μM DCVC at 5, 10, and 24 h, showing that DCVC induces mitochondrial dysfunction in HTR-8/Svneo cells. The present study demonstrates that DCVC stimulated ROS generation in the human placental cell line HTR-8/SVneo and provides new evidence of mechanistic linkage between DCVC-stimulated ROS and increase in proinflammatory cytokine IL-6. Because abnormal activation of cytokines can disrupt trophoblast functions necessary for placental development and successful pregnancy, follow-up investigations relating these findings to physiologic outcomes are warranted.

Reactive Oxygen Stimulation of Interleukin-6 Release in the Human Trophoblast Cell Line HTR-8/SVneo by the Trichlorethylene Metabolite S-(1,2-Dichloro)-l-Cysteine.

HTR8/SVneo, but not JEG3, cells display trophoblast progenitor cell-like characteristics indicative of self-renewal, repopulation activity and expression of “stemness”- associated transcription factors

Introduction. JEG3 is a choriocarcinoma—and HTR8/SVneo a transformed extravillous trophoblast—cell line often used to model the physiologically invasive extravillous trophoblast. Past studies suggest that these cell lines possess some stem or progenitor cell characteristics. Aim was to study whether these cells fulfill minimum criteria used to identify stem-like (progenitor) cells. In summary, we found that the expression profile of HTR8/SVneo (CDX2

/pdf/htr8-svneo-cells-display-trophoblast-progenitor-cell-like-1pjlk81coj.pdf

HTR8/SVneo Cells Display Trophoblast Progenitor Cell-Like Characteristics Indicative of Self-Renewal, Repopulation Activity, and Expression of “Stemness-” Associated Transcription Factors

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HTR8/SVneo, but not JEG3, cells display trophoblast progenitor cell-like characteristics indicative of self-renewal, repopulation activity and expression of “stemness”- associated transcription factors

HTR8/SVneo Cells Display Trophoblast Progenitor Cell-Like Characteristics Indicative of Self-Renewal, Repopulation Activity, and Expression of “Stemness-” Associated Transcription Factors