Dataset Information

Activation of the interferon pathway in trophoblast cells infected with SARS-CoV-2

ABSTRACT: Infection with SARS-CoV-2 in pregnancy has been associated with poor maternal and neonatal outcomes and placental defects. The placenta, which acts as a physical and immunological barrier at the maternal/fetal interface, is not established until the end of the first trimester. Therefore, localized viral infection of the trophoblast compartment early in gestation could trigger an inflammatory response resulting in altered placental function and consequent suboptimal conditions for fetal growth and development. In this study, we investigated the effect of SARS-CoV-2 infection in early gestation placentae using placenta-derived human trophoblast stem cells (TSC), a novel in vitro model, and their extra-villous trophoblast (EVT) and syncytiotrophoblast (STB) derivatives. Consistent with host viral entry protein expression, SARS-CoV-2 was able to productively replicate in TSC-derived STB and EVT but not undifferentiated TSC. Both early EVT and STB elicited an interferon-mediated innate immune response similar to other cells infected with this virus. Therefore, we have also shown that placenta derived TSCs are a robust in vitro model to investigate the effect of this viral infection in the trophoblast compartment of the early placenta. Overall, these results suggest that SARS-CoV-2 infection in early gestation can adversely affect placental development and that might occur via directly infecting the differentiated trophoblast compartment, thus posing a higher risk for poor pregnancy outcomes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE216484 | GEO | 2023/10/14

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:Trophoblast is the primary epithelial cell type in the placenta, a transient organ required for proper fetal growth and development. We have identified a CDX2+/p63+ cytotrophoblast (CTB) subpopulation in the early post-implantation human placenta, which is significantly reduced later in gestation. CTB differentiate into different trophoblast subtypes, which are responsible for gas/nutrient exchange (syncytiotrophoblast/STB) and invasion and maternal vascular remodeling (extravillous trophoblast/EVT). Study of early human placental development is severely hampered by lack of a representative trophoblast stem cell (TSC) model, with the capacity for self-renewal and the ability to differentiate into both STB and EVT. We describe a reproducible protocol, using defined media containing BMP4, by which human embryonic stem cells (hESC) can be differentiated into CDX2+/p63+ CTB-like cells. These cells can be replated to further differentiate into STB- and EVT-like cells, based on marker expression, hormone secretion and invasive ability. Differentiation of hPSC-derived CTB in hypoxia leads to reduced hCG secretion and STB-associated gene expression, instead inducing EVT differentiation in a hypoxia-inducible factor-dependent manner. Human embryonic stem cells (hESC) (WA09/H9) were maintained on Geltrex-coated plates (BD Biosciences ) in StemPro (Thermo Fisher) + bFGF (12 ng/ml). Undifferentiated hESC (D-2) were switched to minimal media (EMIM (Erb et al., 2011) containing KO DMEM/F12 (Gibco), 1% Insulin-Transferrin-Selenium Mix (Sigma-Aldrich), 1% NEAA (Gibco), 2mM L-Glutamine (Corning), 0.1mM 2-mercaptoethanol (Gibo), and 2% BSA (Gemini Bio Products)) for 2 days then treated with BMP4 (StemRD, 10ng/ml) for 3 days in minimal media+BMP4 (D3). At D3, cells were replated onto Geltrex in Feeder Conditioned Media +BMP4 and cultured in normoxia (20% O2) or hypoxia (2% O2) for 2 days (D3+2) to assess the effect of hypoxia. Cells were infected with lentiviral shRNA for shScramble (control) or shARNT, the beta subunit of the Hypoxia Inducible Factor (HIF) complex to assess the effect of ARNT knockdown. Each sample includes biological triplicates.

Project description:Early placenta development involves cytotrophoblast differentiation into extravillous trophoblast (EVT) and syncytiotrophoblast (STB). Defective trophoblast development and function may result in severe pregnancy complications, including fetal growth restriction and pre-eclampsia. The incidence of these complications is increased in pregnancies of fetuses affected by Rubinstein–Taybi syndrome, a developmental disorder predominantly caused by heterozygous mutations in CREB-binding protein (CREBBP) or E1A-binding protein p300 (EP300). Although the acetyltransferases CREBBP and EP300 are paralogs with many overlapping functions, the increased incidence of pregnancy complications is specific for EP300 mutations. We hypothesized that these complications have their origin in early placentation and that EP300 is involved in that process. Therefore, we investigated the role of EP300 and CREBBP in trophoblast differentiation, using human trophoblast stem cells (TSCs) and trophoblast organoids. We found that pharmacological CREBBP/EP300 inhibition blocks differentiation of TSCs into both EVT and STB lineages, and results in an expansion of TSC-like cells under differentiation-inducing conditions. Specific targeting by RNA interference or CRISPR/Cas9-mediated mutagenesis demonstrated that knockdown of EP300 but not CREBBP, inhibits trophoblast differentiation, consistent with the complications seen in Rubinstein–Taybi syndrome pregnancies. By transcriptome sequencing, we identified transforming growth factor alpha (TGFA, encoding TGF-α) as being strongly upregulated upon EP300 knockdown. Moreover, supplementing differentiation medium with TGF-α, which is a ligand for the epidermal growth factor receptor (EGFR), likewise affected trophoblast differentiation and resulted in increased TSC-like cell proliferation. These findings suggest that EP300 facilitates trophoblast differentiation by interfering with at least EGFR signaling, pointing towards a crucial role for EP300 in early human placentation.

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.

Dataset Information

Activation of the interferon pathway in trophoblast cells infected with SARS-CoV-2

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure