Project description:Here, we have undertaken comparative label-free quantitative (LFQ) proteomic profiling of the proteins expressed in BeWo and HTR8/SVneo cell lines using a mass spectrometry approach. We have identified 1557 proteins in total. 915 proteins were expressed in both cell lines including various transcription factors, chaperones and transport proteins. A further 338 and 304 proteins were uniquely expressed in BeWo and HTR8/SVneo cells respectively. Our bioinformatics analysis reflects the known epithelial and mesenchymal phenotypes of these cell models with principal differences in GO observed in ‘Cell junction’, ‘Catenin complex’ as well as ‘Cell adhesion’ and ‘Cell differentiation’. Our novel comparative proteomic analysis of these trophoblastic cell lines will set the stage for the use of trophoblastic cell lines for the study of pregnancy disorders and further research focusing on placental function.
Project description:The term gestational trophoblastic disease (GTD) describes a range of pathologies derived from the villous trophoblasts of the placenta. These include benign entities such as partial and complete hydatidiform mole as well as invasive cancers such as gestational choriocarcinoma, placental site trophoblastic tumors, and epithelioid trophoblastic tumors. Collectively, the malignant forms of GTD are known as gestational trophoblastic neoplasia (GTN). The risk of GTN following a complete molar pregnancy ranges between 8-25%. Low risk patients are expected to have a high likelihood of response to single agent chemotherapy with methotrexate or actinomycin D, but the incidence of resistance to single agent chemotherapy among low risk patients remains 25-50%. We used gene expression microarrays to compare methotrexate sensitive trophoblastic cell lines to sublines that were conditioned to become methotrexate resistant.
Project description:The STOX1 transcription factor has been involved in a complex human disease of pregnancy, preeclampsia, in human families, and mouse models. However, its mode of action is still largely unknown. Overexpression of either the long (STOX1A) or the short (STOX1B) isoform was obtained in the BeWo villous trophoblast model, a cell line able to fuse in syncytiotrophoblast following induction by forskolin treatment. The effects at the transcriptional level are evaluated in every condition.
Project description:To gain more insight in the expression and role of microRNAs in trophoblastic cells, we have assessed the miRNA expression pattern of four trophoblastic cell lines and have compared them with the miRNA signature of human isolated 3rd trimester cytotrophoblast cells. Total RNA was reverse-transcribed, pre-amplified with miRNA-specific primers, and loaded into TLDA cards. Quantitative real-time PCR was performed for 768 miRNAs. For each of two cards per sample (A and B), 9 ul of pre-amplified diluted sample was mixed with TaqMan reaction mix (Applied Biosystems) and loaded into miRNA TLDA cards (Applied Biosystems) by centrifugation. Cards were sealed, and qRT-PCR was performed at a real-time thermocycler (ABI-7900, Applied Biosystems) following the manufacturer's recommendations. MiRNA signatures of human primary 3rd trimester trophoblast cells were compared with those of four trophoblastic cell lines
Project description:The human placenta is covered by a single multinucleated fetal cell, the syncytiotrophoblast, which is bathed in maternal blood. During all pregnancies, membrane enclosed extracellular vesicles derived from the syncytiotrophoblast are extruded into the maternal blood.The large size of these extracellular vesicles (diameter larger than 10µm) is referred to as trophoblastic debris in this study. We have shown in the past that endothleial cells are involved in clearence of this trophoblastic debris and induction of immune tolerence by trophoblastic debris.This study aimed to characterise the transcriptional changes that occur in human vascular endothelial cells following exposure to trophoblastic debris from normal first trimester placentae. Microarrays were used to probe transcriptomic changes 2 and 21 hours after exposure of endothelial cells (Human microvascular endothelial cell line,HMEC-1) to trophoblastic debris from normal first trimester placentae Trophoblastic debris were isolated by low speed centrifugation from three individual first trimester human placentae (three biological replicates). The protein content in trophoblastic debris was measured by BCA assay. HMEC-1 was co-cultured with trophoblastic debris (60ug/ml total debris protein contents) for either 2 or 21 hours before RNA extraction. Untreated HMEC-1 at 2 and 21 hours were used as controls.In total, 12 samples were analyzed.
Project description:The human placenta is covered by a single multinucleated fetal cell, the syncytiotrophoblast, which is bathed in maternal blood. During all pregnancies, membrane enclosed extracellular vesicles derived from the syncytiotrophoblast are extruded into the maternal blood.The large size of these extracellular vesicles (diameter larger than 10µm) is referred to as trophoblastic debris in this study. We have shown in the past that endothleial cells are involved in clearence of this trophoblastic debris and induction of immune tolerence by trophoblastic debris.This study aimed to characterise the transcriptional changes that occur in human vascular endothelial cells following exposure to trophoblastic debris from normal first trimester placentae. Microarrays were used to probe transcriptomic changes 2 and 21 hours after exposure of endothelial cells (Human microvascular endothelial cell line,HMEC-1) to trophoblastic debris from normal first trimester placentae
Project description:Aim of this study was to identify miRNAs which are regulated by LIF in different trophoblastic cell lines Total RNA was reverse-transcribed, pre-amplified with miRNA-specific primers, and loaded into TaqMan Arrays. Quantitative real-time PCR was performed for 768 miRNAs. For each of two cards per sample (A and B), 9 ul of pre-amplified diluted sample was mixed with TaqMan reaction mix (Applied Biosystems) and loaded into miRNA TLDA cards (Applied Biosystems) by centrifugation. Cards were sealed, and qRT-PCR was performed with a real-time thermocycler (ABI-7900, Applied Biosystems) per manufacturer's recommendations.
Project description:Two trophoblastic cell lines, CRL-1584 (3A-Sub E) and JEG-3 were purchased from American Type Culture collection (ATCC) (Manassas, VA). CRL-1584 (3A-Sub E) was originally derived from human term primary placental cells and then immortalized using SV40 virus. JEG-3 is a clonal human choriocarcinoma. The cell lines were serially passaged in complete medium supplemented with each cell lineâs respective IC50 concentration of methotrexate until the cell lines were able to proliferate normally. Subsequently, the concentration of methotrexate was increased by one half-log concentration. The process was repeated iteratively until the cells became senescent. At each log concentration of methotrexate resistance, cell line stocks were frozen to establish a separate subline of each cell line. Based on their relative level of resistance to methotrexate, these were designated JEG-3-R7, JEG-3-R6, and JEG-3-R5 (for 10-7 M through 10-5 M methotrexate resistance). Over the same time period (approximately 11 months), we were able to induce only one order of magnitude of resistance in the normal placenta cell line; that line was designated NP-R7. In parallel, the parent cell lines were passaged in normal medium to control for any physiologic changes induced by prolonged culture in vitro.
Project description:Gene expressions obtained from the total RNA sequencing of 2 choriocarcinomas, 4 epithelioid trophoblastic tumors, and 4 placental site trophoblastic tumors were evaluated for differential gene expression, pathway alteration, fusion gene, infiltrating immune cell type, and PD-L1 expression level, and mutation analysis was performed using the RNA sequencing data.