ABSTRACT: We compare two groups of transcriptome profiles. They are 1) primary human trophoblasts (PHT) and 2) syncytiotrophoblast from human pluripotent stem cells. This data set represents the former data set and the latter has been deposited separately at GSE72712. Primary human trophoblasts (PHT) were purified cytotrophoblast populations from whole term placentas and then cultured in vitro. The cytotrophoblasts were cultured for short (9 h) or long (48 h) term that show undifferentiated (PHTu) or differentiated (PHTd) phenotype that led syncytialization, respectively. Transcriptome profiles of PHTu and PHTd were compared with three size fractioned syncytiotrophoblasts and trophoblasts from human pluripotent stem cells that are named ESCd_gt70 (greater than 70 µm), ESCd_40-70 (size between 40 and 70 µm), ESCd_lt40 (smaller than 40 µm) and undifferentiated progenitor (ESCu; see GSE72712). Primary human trophoblasts (PHT) were derived and cultured from three term human placentas obtained by the Obstetrical Specimen Procurement Unit from women after a healthy pregnancy, labor, and delivery at Magee-Womens Hospital of the University of Pittsburgh Medical Center. Isolated PHT lines obtained from three individual (1 female and 2 male) placentas were cultured separately.
Project description:Primary term human trophoblasts were derived from placentas after a healthy pregnancy, and exposed to ionizing irradiation (vs sham) in vitro Primary human trophoblasts were irradiated 24 h after initial plating, defined as time zero. Cells were irradiated at 10 Gy using a Clinac 600C (Varian Medical Systems, Palo Alto, CA) with a 6 MV photon beam and a dose rate of 250 cGy/min. The flasks containing the cells were placed on 1.5 cm of bolus (a tissue equivalent material) since the maximum irradiation depth was 1.5 cm, which corresponded to the plated cell layer. Cells were analyzed 4, 8, and 24 h after irradiation or sham.
Project description:Analyze gene expression levels in primary trophoblasts, derived from term human placenta and cultured under standard or hypoxic conditions Human placental trophoblasts were dispersed using a trypsin-deoxyribonuclease-dispase/Percoll method, plated in 6-well plates, and maintained in standard culture conditions (O2=20%). After 4 h (defined as time 0), the plates were divided to those in continued standard culture conditions, or to culture in hypoxia (O2=0%). Cells were then harvested at 6 h, 12 h, 24 h, 48 h and 72 h, and processed for mRNA arrays
Project description:Analyze miRNA expression levels in primary trophoblasts, derived from term human placenta and cultured under standard or hypoxic conditions Human placental trophoblasts were dispersed using a trypsin-deoxyribonuclease-dispase/Percoll method, plated in 6-well plates, and maintained in standard culture conditions (O2=20%). After 4 h (defined as time 0), the plates were divided to those in continued standard culture conditions, or to culture in hypoxia (O2=0%). Cells were then harvested at 6 h, 12 h, 24 h, 48 h and 72 h, and processed for miRNA arrays
Project description:YY1 is a sequence-specific DNA-binding transcription factor that has many important biological roles. However, its function in trophoblasts at the maternal-foetal interface remains to be elucidated. In this study, we used an mRNA microarray and quantitative reverse transcription-PCR and compared the YY1 mRNA expression level in trophoblasts between patients with recurrent miscarriage (RM) and healthy control subjects. Our results revealed that YY1 mRNA expression was significantly lower in the trophoblasts of the RM group compared with the healthy control group. Furthermore, immunofluorescence and immunohistochemical data showed that YY1 was highly expressed in human placental villi during early pregnancy, especially in cytotrophoblast cells and invasive extravillous trophoblasts, and it was expressed at a much lower level in the placental villi of term pregnancy. YY1 overexpression enhanced the invasion and proliferation of trophoblasts, while knockdown of YY1 repressed these effects. Antibody array screening revealed that YY1 significantly promoted MMP2 expression in trophoblasts. Bioinformatics analysis identified three YY1-binding sites in the MMP2 promoter region, and chromatin immunoprecipitation analysis verified that YY1 binds directly to its promoter region. Importantly, inhibition of YY1 by siRNA clearly decreased trophoblast invasion in an ex vivo explant culture model. Overall, our findings revealed a new regulatory pathway of YY1/MMP2 in trophoblast cells invasion during early pregnancy, and indicated that YY1 may be involved in the pathogenesis of RM. Total RNA was isolated using Trizol from trophoblast cells from three healthy controls (HC) and three recurrent miscarriage (RM) patients. Total RNA were extracted and used for hybridizing Affymetrix chips (GeneChip® Human Transcriptome Array 2.0(HTA2.0)). Data were normalised by gcRMA method and raw p-values adjusted by Bonferroni procedure (1%).
Project description:Primary cultures of human trophoblasts were established from normal, term placenta. Cells were treated five hours after plating. This sample was treated with 1 uM GW7845. RNA was extracted after 48 hours of treatment using Tri-reagent. Each sample was pooled from three individual placentas prior to target generation. Targets were produced using standard Affymetrix procedures from 30ug of total RNA.
Project description:The goal of this study was to identify the gene expression signatures of two closely related types of trophoblast in human placentas: villous cytotrophoblasts (vCTB) and proximal column extravillous trophoblasts (pcEVT). The two populations were isolated from first trimester placentas and identified using the specific surface markers, EGFR (vCTB) and HLA-G (pcEVT). Overall design: First trimester trophoblasts were isolated from 6-9 week gestational age placentas and sorted into EGFR+/HLAG- vCTB and EGFR-/HLAG+ pcEVT. Whole genome expression analysis was performed on purified RNA of the two sorted populations.
Project description:Decidual spiral arteriole (SpA) remodelling is essential to ensure optimal uteroplacental blood flow during human pregnancy. Decidual uterine natural killer cells and macrophages infiltrate the SpA and are proposed to initiate remodelling before colonisation by extravillous trophoblasts. Microarrays were used to measure the effect of extravillous trophoblasts conditioned medium on the transcriptome of human uterine microvascular endothelial cells.
Project description:During human pregnancy, placental cytotrophoblasts invade the uterus and its blood vessels, anchoring the progeny and rerouting maternal blood to the embryo/fetus. In preeclampsia, cytotrophoblast invasion is restricted and blood flow to the placenta is reduced. The causes of restricted cytotrophoblast invasion are unknown. Here, preeclampsia and control cytotrophoblasts were cultured for 48 h to allow differentiation/invasion. In various severe forms of preeclampsia ± intrauterine growth restriction, global transcriptional profiling revealed common aberrations in cytotrophoblast gene expression that resolved with culture. Villous cytotrophoblasts were isolated from preeclampsia placentas (PRE, n=5) and placentas of preterm labor patients without signs of infection (PTL, n=5), which served as gestation-matched controls. To better understand the CTB phenotype in the context of PE variants, we included patients with the most clinically significant forms of this condition that necessitated preterm delivery: women with severe PE ± intrauterine growth restrictions, PE with superimposed hypertension and HELLP syndrome (hemolysis, elevated liver enzymes; low platelet count). RNA was purified immediately after the cells were isolated (0 h) and after 12, 24 and 48 h in culture. The relative gene expression across the whole genome was profiled using the Affymetrix HG-U133Plus 2.0 GeneChip platform. Array quality was assesed using RMAExpress. One sample of preterm labor collected at 48h was omitted (39 arrays total). We used both LIMMA and maSigPro (R/Bioconductor) to determine differentially expressed genes.
Project description:Analyze miRNA expression levels in HTR-8/SVeo cells stably transfected with a BAC plasmid containing the entire C19MC miRNA cluster HTR-8/SVeo cells were transfected with a modified BAC plasmid containing the entire C19MC miRNA cluster and carrying a zeocin selection cassette. Several independent clones and a mixed population of transfected cells were analyzed and compared to non-transfected HTR-8/SVeo cells and primary human trophoblasts that express the C19MC miRNAs endogenously
Project description:Analyze gene expression profiles in HTR-8/SVeo cells stably transfected with a BAC plasmid containing the entire C19MC miRNA cluster HTR-8/SVeo cells were transfected with a modified BAC plasmid containing the entire C19MC miRNA cluster and carrying a zeocin selection cassette. Several independent clones and a mixed population of transfected cells were analyzed and compared to non-transfected HTR-8/SVeo cells and primary human trophoblasts that express the C19MC miRNAs endogenously