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Transcriptomic analysis of placenta and its released extracellular vesicles in normal and preeclampsia pregnancies: insigths into novel biomarkers and mechanisms of disease

ABSTRACT: Purpose: The goals of this study is to compare and profile the transcriptome of the placenta in preeclamptic and normal patients using RNA sequencing. Methods: Placental and Placental vesicles (STB-EVs) mRNA profiles of normal and preeclamptic patients were generated by deep sequencing using Illumina HISEQ. The sequence reads that passed quality filters were analyzed at the gene level HISAT2 followed by featureCounts. Quantitative PCR validation was performed using TaqMan gene expression assays Results: This contains a set of three parallel mRNA sequencing experiments involving placenta tissue, medium/large STB-EVs and small STB-EVs. Comparison between PE and normal pregnancy placental tissue revealed 18,070 genes from which 580 were upregulated and 563 were downregulated (p-value of <0.05 ) while in medium/large STB-EVs, revealed 14,216 genes from which 1,128 were upregulated and 833 were downregulated. In small STB-EVs, out of 23,322 genes, 232 were upregulated and 106 were downregulated) (adjusted P-value of <0.05) We identified a number of mechanistic and biomarker targets, which were validated with qRT–PCR and confirmed to be signifficantly DE. The differentially expressed analysis identified potential yet undescribed genes that may contribute to the pathogenesis of preeclampsia or/and may act as biomarkers of the disease. Conclusions: Our study represents the first detailed analysis of combined Placenta and placental extracellular vesicles (STB-EVs) transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results have identified potential STB-EV based small RNA biomarkers in preeclampsia.

ORGANISM(S): Homo sapiens

PROVIDER: GSE190971 | GEO | 2021/12/17

REPOSITORIES: GEO

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Project description:Prenatal development is a critical period for programming of neurological disease. Preeclampsia, a pregnancy complication involving oxidative stress in the placenta, has been associated with long-term health implications for the child, including an increased risk of developing schizophrenia and autism spectrum disorders in later life. We have shown previously, in a rodent model of placental oxidative stress, that culture medium conditioned by the placenta alters neuronal characteristics when applied to primary cortical cultures in vitro and mimics many of the neurodevelopmental changes observed in the offspring brain. To further investigate if molecules released by the placenta may be important mediators in foetal programming of the brain, we analysed if placental tissue delivered from patients with preeclampsia secreted molecules that could affect cortical cells in culture. Application of culture medium conditioned by preeclamptic placentae to mixed cortical cultures caused changes in neurons and astrocytes that were related to key changes observed in brains of patients with schizophrenia and autism, including effects on dendrite lengths, astrocyte number as well as glutamate and GABA receptors. Treatment of the placental explants with an antioxidant prevented neuronal abnormalities. Furthermore, we identified that bidirectional communication between neurons and astrocytes, potentially via glutamate, is required to produce the effects of preeclamptic placenta medium on cortical cells. Analysis of possible signalling molecules in the placenta conditioned medium showed that the secretion profile of extracellular microRNA, small posttranscriptional regulators, was altered in preeclampsia and partially rescued by antioxidant treatment of the placental explants. Predicted targets of these differentially abundant microRNAs were linked to neurodevelopment and the placenta. The presented study provides further evidence that the diseased placenta may release factors that damage cortical cells in the brain and suggests the possibility of targeted antioxidant treatment of the placenta to prevent neurodevelopmental disorders.

Project description:Purpose: Syncytiotrophoblast (STB) is a multi-nucleated, terminally differentiated syncytium that covers the surface of the villous placenta and forms the major interface with maternal blood. It releases placental hormones and plays a primary role in exchange of gases, nutrients and waste products. Alterations in STB development and turnover have been implicated in placental diseases, including preeclampsia (PE). In vitro cell models are badly needed to study STB development and physiology due to inaccessibility to placental tissues during gestation. To establish in vitro STB model system, we generate STB and its mononucleated precursors from human embryonic stem cells (hESC) and profile for RNA content by RNAseq. Methods: H1 Human ESC (WA01) were treated with BMP4, the ALK4/5/7 inhibitor (A83-01), and the FGF2 signaling inhibitor (PD173074) (BAP) to direct them to the trophoblast lineage and provided both STB and extravillous trophoblast. Syncytial areas emerged at day 8 BAP treatment ranged in diameter from ~40 Âµm to > 100 Âµm. The intact syncytial areas were isolated by sieving successively through 70 Î¼m and 40 Î¼m mesh cell strainers. The captured cells are recovered by inverting the strainer and rinsing with culture medium to separate large (>70 Î¼m) and middle size cell sheets (40-70 Î¼m). The fraction that passes through both sieves represents cells of smallest diameter (< 40 Î¼m), presumably cytotrophoblast. Total 12 RNA samples from triplicate three size-fractioned BAP treated and three untreated hESC cultured in a FGF2 supplemented medium in parallel were analyzed. Results: The larger > 70 Î¼m areas stained positively for STB markers while ultrastructural analysis clearly revealed multi-nuclear cells with an extensive cytoplasm containing many prominent secretion granules. The larger STB areas also had a larger DNA content that > 70 Î¼m fraction contained 37 times more nuclear content and 40-70 Âµm fraction did 16 times more. Compared to the < 40 Î¼m cell fraction, these larger cells over-expressed a full repertoire of genes characteristic of STB, e.g. CGA, CGB, PGF, ERVW1, GCM1. The smallest cell fraction had a DNA content consistent with mononuclear diploid cells, contained few secretory granules, and were only weakly positive for STB markers. Conclusion: The data are consistent with the > 70 Î¼m cells being mature STB, while the intermediate fraction may represent a precursor population. Human ESC directed along the trophoblast lineage by BAP treatment offers a useful model for following STB formation in vitro and suggest that this protocol may have utility in studying the basis of certain placental diseases, especially preeclampsia, where placental tissue isolated at term or after pregnancy terminations can only offer limited information. Three size fraction mRNA profiles of syncytial areas emerged at day 8 BAP treatment of hESC were generated by deep sequencing along with untreated hESC, in triplicate, using Illumina HiSeq 2500.

Project description:Purpose: Syncytiotrophoblast (STB) is a multi-nucleated, terminally differentiated syncytium that covers the surface of the villous placenta and forms the major interface with maternal blood. It releases placental hormones and plays a primary role in exchange of gases, nutrients and waste products. Alterations in STB development and turnover have been implicated in placental diseases, including preeclampsia (PE). In vitro cell models are badly needed to study STB development and physiology due to inaccessibility to placental tissues during gestation. To establish in vitro STB model system, we generate STB and its mononucleated precursors from human embryonic stem cells (hESC) and profile for RNA content by RNAseq. Methods: H1 Human ESC (WA01) were treated with BMP4, the ALK4/5/7 inhibitor (A83-01), and the FGF2 signaling inhibitor (PD173074) (BAP) to direct them to the trophoblast lineage and provided both STB and extravillous trophoblast. Syncytial areas emerged at day 8 BAP treatment ranged in diameter from ~40 µm to > 100 µm. The intact syncytial areas were isolated by sieving successively through 70 μm and 40 μm mesh cell strainers. The captured cells are recovered by inverting the strainer and rinsing with culture medium to separate large (>70 μm) and middle size cell sheets (40-70 μm). The fraction that passes through both sieves represents cells of smallest diameter (< 40 μm), presumably cytotrophoblast. Total 12 RNA samples from triplicate three size-fractioned BAP treated and three untreated hESC cultured in a FGF2 supplemented medium in parallel were analyzed. Results: The larger > 70 μm areas stained positively for STB markers while ultrastructural analysis clearly revealed multi-nuclear cells with an extensive cytoplasm containing many prominent secretion granules. The larger STB areas also had a larger DNA content that > 70 μm fraction contained 37 times more nuclear content and 40-70 µm fraction did 16 times more. Compared to the < 40 μm cell fraction, these larger cells over-expressed a full repertoire of genes characteristic of STB, e.g. CGA, CGB, PGF, ERVW1, GCM1. The smallest cell fraction had a DNA content consistent with mononuclear diploid cells, contained few secretory granules, and were only weakly positive for STB markers. Conclusion: The data are consistent with the > 70 μm cells being mature STB, while the intermediate fraction may represent a precursor population. Human ESC directed along the trophoblast lineage by BAP treatment offers a useful model for following STB formation in vitro and suggest that this protocol may have utility in studying the basis of certain placental diseases, especially preeclampsia, where placental tissue isolated at term or after pregnancy terminations can only offer limited information.

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Transcriptomic analysis of placenta and its released extracellular vesicles in normal and preeclampsia pregnancies: insigths into novel biomarkers and mechanisms of disease

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