Trophoblast stem cells (TSC) are the precursors of the differentiated cells of the placenta. In the mouse, TSC can be derived from outgrowths of either blastocyst polar trophectoderm (TE) or extraembryonic ectoderm (ExE), which originates from polar TE after implantation. The mouse TSC niche appears to be located within the ExE adjacent to the epiblast, on which it depends for essential growth factors, but whether this cellular architecture is the same in other species remains to be determined. Mouse TSC self-renewal can be sustained by culture on mitotically inactivated feeder cells, which provide one or more factors related to the NODAL pathway, and a medium supplemented with FGF4, heparin, and fetal bovine serum. Repression of the gene network that maintains pluripotency and emergence of the transcription factor pathways that specify a trophoblast (TR) fate enables TSC derivation in vitro and placental formation in vivo. Disrupting the pluripotent network of embryonic stem cells (ESC) causes them to default to a TR ground state. Pluripotent cells that have acquired sublethal chromosomal alterations may be sequestered into TR for similar reasons. The transition from ESC to TSC, which appears to be unidirectional, reveals important aspects of initial fate decisions in mice. TSC have yet to be derived from domestic species in which remarkable TR growth precedes embryogenesis. Recent derivation of TSC from blastocysts of the rhesus monkey suggests that isolation of the human equivalents may be possible and will reveal the extent to which mechanisms uncovered by using animal models are true in our own species.
Trophoblast cells play an essential role in the interactions between the fetus and mother. Mouse trophoblast stem (TS) cells have been derived and used as the best model for molecular and functional analysis of mouse trophoblast lineages, but attempts to derive human TS cells have so far been unsuccessful. Here we show that activation of Wingless/Integrated (Wnt) and EGF and inhibition of TGF-β, histone deacetylase (HDAC), and Rho-associated protein kinase (ROCK) enable long-term culture of human villous cytotrophoblast (CT) cells. The resulting cell lines have the capacity to give rise to the three major trophoblast lineages, which show transcriptomes similar to those of the corresponding primary trophoblast cells. Importantly, equivalent cell lines can be derived from human blastocysts. Our data strongly suggest that the CT- and blastocyst-derived cell lines are human TS cells, which will provide a powerful tool to study human trophoblast development and function. Trophoblast cells are specialized cells in the placenta that mediate the interactions between the fetus and mother. Okae et al. report the derivation of human trophoblast stem cells from blastocysts and early placentas, which will provide a powerful tool to study human placental development and function.
Extravillous trophoblasts (EVTs) migrate into uterine decidua and induce vascular smooth muscle cell (VSMC) loss through mechanisms thought to involve migration and apoptosis, achieving complete spiral artery remodeling. Long noncoding RNA maternally expressed gene 3 (MEG3) can regulate diverse cellular processes, such as proliferation and migration, and has been discovered highly expressed in human placenta tissues. However, little is known about the role of MEG3 in modulating EVT functions and EVT‐induced VSMC loss. In this study, we first examined the location of MEG3 in human first‐trimester placenta by in situ hybridization. Then, exogenous upregulation of MEG3 in HTR‐8/SVneo cells was performed to investigate the effects of MEG3 on EVT motility and EVT capacity to displace VSMCs. Meanwhile, the molecules mediating EVT‐induced VSMC loss, such as tumor necrosis factor‐α (TNF‐α), Fas ligand (FasL), and tumor necrosis factor‐α‐related apoptosis‐inducing ligand (TRAIL) were detected at transcriptional and translational levels. Finally, VSMCs were cocultured with MEG3‐upregulated HTR‐8/SVneo to explore the role of MEG3 on EVT‐mediated VSMC migration and apoptosis. Results showed that MEG3 was expressed in trophoblasts in placental villi and decidua, and MEG3 enhancement inhibited HTR‐8/SVneo migration and invasion. Meanwhile, the displacement of VSMCs by HTR‐8/SVneo and the expression of TNF‐α, FasL and TRAIL in HTR‐8/SVneo were reduced following MEG3 overexpression in HTR‐8/SVneo. Furthermore, HTR‐8/SVneo with MEG3 upregulation impaired VSMC migration and apoptosis. The PI3K/Akt pathway, which is possibly downstream, was inactivated in MEG3‐upregulated HTR‐8/SVneo. These findings suggest that MEG3 might be a negative regulator of spiral artery remodeling via suppressing EVT invasion and EVT‐mediated VSMC loss. Maternally expressed gene 3 (MEG3) suppressed trophoblast‐mediated vascular smooth muscle cell (VSMC) migration and apoptosis, which might be associated with reduced expression of tumor necrosis factor‐α (TNF‐α), Fas ligand (FasL), and tumor necrosis factor‐α‐related apoptosis‐inducing ligand (TRAIL) in trophoblast with MEG3 enhancement.
Abstract Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2016 there were twelve themed workshops, four of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of decidual-trophoblast interaction, regulation of trophoblast invasion, immune cells at the maternal-fetal interface, and placental inflammation.
Our understanding of how cells communicate has undergone a paradigm shift since the recent recognition of the role of exosomes in intercellular signaling. In this study, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT) cells, modifying their bioactivity on endothelial cells (EC). Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE) and spontaneous preterm birth (SPTB). HTR-8/SVneo cells were used as an EVT model. The effect of oxygen tension (i.e. 8% and 1% oxygen) on exosome release was quantified using nanocrystals (Qdot (R)) coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte (TM)) was used to establish the effect of exosomes on EC. Plasma samples were obtained at early gestation (< 18 weeks) and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly higher in EVT cultured under 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, whilst exosomes cultured at 1% oxygen decreased EC migration. These changes were inversely proportional to TNF-alpha released from EC. Finally, we have identified a set of unique miRNAs in exosomes from EVT cultured at 1% oxygen and exosomes isolated from the circulation of mothers at early gestation, who later developed PE and SPTB. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically relevant biomarker of pregnancy complications.
Infection of pregnant women by Asian lineage strains of Zika virus (ZIKV) has been linked to brain abnormalities in their infants, yet it is uncertain when during pregnancy the human conceptus is most vulnerable to the virus. We have examined two models to study susceptibility of human placental trophoblast to ZIKV: cytotrophoblast and syncytiotrophoblast derived from placental villi at term and colonies of trophoblast differentiated from embryonic stem cells (ESC). The latter appear to be analogous to the primitive placenta formed during implantation. The cells from term placentas, which resist infection, do not express genes encoding most attachment factors implicated in ZIKV entry but do express many genes associated with antiviral defense. By contrast, the ESC-derived trophoblasts possess a wide range of attachment factors for ZIKV entry and lack components of a robust antiviral response system. These cells, particularly areas of syncytiotrophoblast within the colonies, quickly become infected, produce infectious virus and undergo lysis within 48 h after exposure to lowtiters (multiplicity of infection > 0.07) of an African lineage strain (MR766 Uganda: ZIKV(U)) considered to be benignwith regards to effects on fetal development. Unexpectedly, lytic effects required significantly higher titers of the presumed more virulent FSS13025 Cambodia (ZIKV(C)). Our data suggest that the developing fetus might be most vulnerable to ZIKV early in the first trimester before a protective zone of mature villous trophoblast has been established. Additionally, MR766 is highly trophic toward primitive trophoblast, which may put the early conceptus of an infected mother at high risk for destruction.
Difficulties associated with long-term culture of primary trophoblasts have proven to be a major hurdle in their functional characterization. In order to circumvent this issue, several model cell lines have been established over many years using a variety of different approaches. Due to their differing origins, gene expression profiles and behaviour in vitro, different model lines have been utilized to investigate specific aspects of trophoblast biology. However, generally speaking, the molecular mechanisms underlying functional differences remain unclear. In this study, we profiled genome-scale DNA methylation in primary first trimester trophoblast cells and seven commonly used trophoblast-derived cell lines in an attempt to identify functional pathways differentially regulated by epigenetic modification in these cells. We identified a general increase in DNA promoter methylation levels in four choriocarcinoma (CCA)-derived lines and transformed HTR-8/SVneo cells, including hypermethylation of several genes regularly seen in human cancers, while other differences in methylation were noted in genes linked to immune responsiveness, cell morphology, development and migration across the different cell populations. Interestingly, CCA-derived lines show an overall methylation profile more similar to unrelated solid cancers than to untransformed trophoblasts, highlighting the role of aberrant DNA methylation in CCA development and/or long-term culturing. Comparison of DNA methylation and gene expression in CCA lines and cytotrophoblasts revealed a significant contribution of DNA methylation to overall expression profile. These data highlight the variability in epigenetic state between primary trophoblasts and cell models in pathways underpinning a wide range of cell functions, providing valuable candidate pathways for future functional investigation in different cell populations. This study also confirms the need for caution in the interpretation of data generated from manipulation of such pathways in vitro.
The placenta is the extraembryonic organ that supports the fetus during intrauterine life. Although placental dysfunction results in major disorders of pregnancy with immediate and lifelong consequences for the mother and child, our knowledge of the human placenta is limited owing to a lack of functional experimental models(1). After implantation, the trophectoderm of the blastocyst rapidly proliferates and generates the trophoblast, the unique cell type of the placenta. In vivo, proliferative villous cytotrophoblast cells differentiate into two main sub-populations: syncytiotrophoblast, the multinucleated epithelium of the villi responsible for nutrient exchange and hormone production, and extravillous trophoblast cells, which anchor the placenta to the maternal decidua and transform the maternal spiral arteries(2). Here we describe the generation of long-term, genetically stable organoid cultures of trophoblast that can differentiate into both syncytiotrophoblast and extravillous trophoblast. We used human leukocyte antigen (HLA) typing to confirm that the organoids were derived from the fetus, and verified their identities against four trophoblast-specific criteria(3). The cultures organize into villouslike structures, and we detected the secretion of placental-specific peptides and hormones, including human chorionic gonadotropin (hCG), growth differentiation factor 15 (GDF15) and pregnancy-specific glycoprotein (PSG) by mass spectrometry. The organoids also differentiate into HLA-G(+) extravillous trophoblast cells, which vigorously invade in three-dimensional cultures. Analysis of the methylome reveals that the organoids closely resemble normal first trimester placentas. This organoid model will be transformative for studying human placental development and for investigating trophoblast interactions with the local and systemic maternal environment.
Summary Human trophoblast cells express an unusual repertoire of human leucocyte antigen (HLA) molecules which has been difficult to define. Close homology between and extreme polymorphism at the classical HLA class‐I (HLA‐I) loci has made it difficult to generate locus‐specific monoclonal antibodies (mAbs). The problem of defining an antibody’s reactivity against the thousands of existing HLA‐I allotypes has often made it impossible to determine the HLA bound by a mAb in biological samples from a normal outbred population. Here we have used commercially available beads coated with individual HLA‐I to characterize experimentally the reactivity of nine mAb against 96 common HLA‐I allotypes. In conjunction with donor HLA‐I genotyping, we could then define the specific HLA molecules bound by these antibodies in normal individuals. We used this approach to analyse the HLA expression of primary trophoblast cells from normal pregnancies; the choriocarcinoma cells JEG‐3 and JAR; and the placental cell lines HTR‐8/SVneo, Swan‐71 and TEV‐1. We confirm that primary villous trophoblast cells are HLA null whereas extravillous trophoblast cells express HLA‐C, HLA‐G and HLA‐E, but not HLA‐A, HLA‐B or HLA‐DR molecules in normal pregnancy. Tumour‐derived JEG‐3 and JAR cells reflect extravillous and villous trophoblast HLA phenotypes, respectively, but the HLA repertoire of the in vitro derived placental cell lines is not representative of either in vivo trophoblast phenotype. This study raises questions regarding the validity of using the placental cell lines that are currently available as model systems for immunological interactions between fetal trophoblast and maternal leucocytes bearing receptors for HLA molecules.