Project description:The study was designed to expolre the differential gene expression after peroxiredoxin2 (Prdx2) downregulation by siRNAs transfection in human trophoblast cell line HTR8 compared with negative control.
Project description:Cells in culture will be exposed to S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a relevant metabolite of TCE. The first cell line used will be HTR-8/SVneo, originally derived from first trimester female human cytotrophoblast cells and immortalized with simian virus 40 large T antigen, this cell line models first-trimester placental extravillous trophoblasts in vitro. HTR-8/SVneo cells will be cultured for 24 hours followed by exposure to cell culture media (control) or 20 µM DCVC for 6 or 12 hours. Cells (cells frozen in cell culture dish) will then be collected (n=5) In the TCE aim of this study, we will utilize one cell model that phenotypically represents an important placental cell population.
Project description:Comparison of genes associated with the EMT between cytotrophoblast cells (CTB) and extravillous trophoblast cells (EVT) from normal third trimester placenta and abnormally invasive placenta (AIP)
Project description:We report the single nuclear RNA sequencing analysis for the profiling of trophoblast lineages generated from human embryoinc stem cells (hESCs) in vitro under 20 % and 5 % O2 concentration conditions. The hESCs were exposed to bone morphogenetic protein 4 (BMP4) in presence of inhibitors of ACTIVIN/TGFB (A83–01) and FGF2 (PD1730) signaling formerly called BAP treatment for 8 days after passaging, which generated a mixture of cytotrophoblast, syncytiotrophoblast, and HLA-G positive cells with similarities to extravillous trophoblast under both high and low O2 conditions (20 % and 5 %, respectively). A single nuclei RNA sequence approach from both O2 conditions determined two major groupings of cell clusters, one comprised of five and the second of four subcluster cell populations respectively. Of these, two subclusters resembled extravillous trophoblast, two carried the hallmark transcripts of syncytiotrophoblast, while the remaining five were likely different kinds of mononucleated cytotrophoblast.
Project description:Comparison of genes associated with the EMT between undifferentiated cytotrophoblast cells (CTB) and differentiated extravillous trophoblast cells (EVT) from third trimester human placenta. Cells isolated from control (placenta previa) and cases (preeclampsia). Cells isolated by immunomagnetic separation using anti-integrin beta4 antibody to purify CTB and anti-HLA-G antibody to purify EVT.
Project description:5 days after fertilisation the human embryo forms a blastocyst, comprising an outer layer of trophectoderm (TE), that gives rise to placental trophoblast cells, and the inner cell mass (ICM) which later segregates into epiblast (EPI) and primitive endoderm (PE). After implantation TE differentiates into cytotrophoblast cells (CTBs) which give rise to the multinucleated syncytiotrophoblast (STB) and invasive extravillous trophoblast cells (EVTs). A conserved molecular cascade regulates TE initiation (Gerri et al. Nature 2020), but subsequent TE development is poorly defined. To study this in greater detail we performed RNA-seq analysis of human blastocysts cultured to different stages of pre-implantation TE development: Day 5: TE appearance, Day 6: TE expansion and Day 7: TE hatching.
Project description:Recent reports suggested human embryonic development is not always consistent with mouse. However, experimental approach to in vivo human embryos is extremely limited. Therefore, it is important to develop an appropriate in vitro cell culture system to analyse human pre-implantation development. In this report, we use human naive pluripotent stem cells (PSCs), which share features with pre-implantation epiblasts and show in vitro lineage specification of human trophoblast lineages. We successfully differentiated naive PSCs into trophectoderm, cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast. Cytotrophoblasts could be cultured for long term as stem cells.
Project description:Dysregulated extravillous trophoblast invasion and proliferation are known to increase the risk of recurrent spontaneous abortion (RSA); however, the underlying mechanism remains unclear. Herein, in our retrospective observational case-control study we show that villous samples from RSA patients, compared to healthy controls, display reduced succinate dehydrogenase complex iron sulfur subunit (SDHB) DNA methylation, elevated SDHB expression, and reduced succinate levels, indicating that low succinate levels correlate with RSA. Moreover, we find high succinate levels in early pregnant women are correlated with successful embryo implantation. SDHB promoter methylation recruited MBD1 and excluded c-Fos, inactivating SDHB expression and causing intracellular succinate accumulation which mimicked hypoxia in extravillous trophoblasts cell lines JEG3 and HTR8 via the PHD2-VHL-HIF-1α pathway; however, low succinate levels reversed this effect and increased the risk of abortion in mouse model. This study reveals that abnormal metabolite levels inhibit extravillous trophoblast function and highlights an approach for RSA intervention.
