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.
Critical roles for DNA methylation in embryonic development are well established, but less is known about its roles during trophoblast development, the extraembryonic lineage that gives rise to the placenta. We dissected the role of DNA methylation in trophoblast development by performing mRNA and DNA methylation profiling of mutants. We find that oocyte-derived methylation plays a major role in regulating trophoblast development but that imprinting of the key placental regulator is only partially responsible for these effects. We have identified several methylation-regulated genes associated with trophoblast differentiation that are involved in cell adhesion and migration, potentially affecting trophoblast invasion. Specifically, trophoblast-specific DNA methylation is linked to the silencing of , a Polycomb Repressive Complex 1 protein that drives loss of cell adhesion in methylation-deficient trophoblast. Our results reveal that maternal DNA methylation controls multiple differentiation-related and physiological processes in trophoblast via both imprinting-dependent and -independent mechanisms. Branco et al. dissect the role of DNA methylation in mouse trophoblast development through genome-wide profiling of methylation-deficient mutants. DNA methylation marks carried over from the oocyte play a major role in trophoblast development and cell adhesion, which is partly dependent on silencing of the Polycomb gene .
In pregnancy, trophoblast invasion and uterine spiral artery remodelling are important for lowering maternal vascular resistance and increasing uteroplacental blood flow. Impaired spiral artery remodelling has been implicated in pre-eclampsia, a major complication of pregnancy, for a long time but the underlying mechanisms remain unclear(1,2). Corin (also known as atrial natriuretic peptide-converting enzyme) is a cardiac protease that activates atrial natriuretic peptide (ANP), a cardiac hormone that is important in regulating blood pressure(3). Unexpectedly, corin expression was detected in the pregnant uterus(4). Here we identify a new function of corin and ANP in promoting trophoblast invasion and spiral artery remodelling. We show that pregnant corin-or ANP-deficient mice developed high blood pressure and proteinuria, characteristics of pre-eclampsia. In these mice, trophoblast invasion and uterine spiral artery remodelling were markedly impaired. Consistent with this, the ANP potently stimulated human trophoblasts in invading Matrigels. In patients with pre-eclampsia, uterine Corin messenger RNA and protein levels were significantly lower than that in normal pregnancies. Moreover, we have identified Corin gene mutations inpre-eclamptic patients, which decreased corin activity in processing pro-ANP. These results indicate that corin and ANP are essential for physiological changes at the maternal-fetal interface, suggesting that defects in corin and ANP function may contribute to pre-eclampsia.
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.
BACKGROUND Routes of trophoblast invasion seem to be clear, whereas specific invasive pathways need further elucidation. Extravillous trophoblasts (EVTs) transform spiral arteries to guarantee appropriate blood flow to the placenta in the second trimester. Embryo nutrition during the first trimester is thought to be histiotrophic, whereas proof that EVTs also invade uterine glands is lacking. We developed novel three-dimensional confrontation co-culture models to elucidate invasion of EVTs into uterine glands. METHODS First trimester decidua parietalis and placental villous explants were directly confronted and co-cultured for 72 h, or confronted indirectly after 72 h pre-culture for re-epithelialization of decidua pieces. Cryosections were stained by immunohistochemistry or immunofluorescent/immunohistochemical double labelling and compared with first trimester placentation sites in situ. RESULTS EVTs deeply invaded decidual tissues in direct confrontation assays and were found between the decidual epithelial cells and epithelial basement membrane. EVTs were also detected in the decidual stroma in direct proximity to glands, sometimes even replacing glandular epithelial cells. Similar observations were made in sections from the first trimester decidua/placental bed. In the invaded parts of sections of decidua basalis, 55% ± 7% (mean ± SEM; n = 10, range 6–11 weeks) of glandular cross sections were associated with or infiltrated by EVTs. CONCLUSIONS Using novel confrontation co-culture assays, a potential new route of EVT invasion was detected. EVTs appear to break through the basement membrane of uterine glands to open their lumen towards the intervillous space. These data support the hypothesis of histiotrophic nutrition of the embryo prior to onset of maternal blood flow within the placenta.
Abstract Trophoblast (TB) comprises the outer cell layers of the mammalian placenta that make direct contact with the maternal uterus and, in species with a highly invasive placenta, maternal blood. It has its origin as trophectoderm, a single epithelial layer of extra-embryonic ectoderm that surrounds the embryo proper at the blastocyst stage of development. Here, we briefly compare the features of TB specification and determination in the mouse and the human. We then review research on a model system that has been increasingly employed to study TB emergence, namely the BMP4 (bone morphogenetic protein-4)-directed differentiation of human embryonic stem cells (ESCd), and discuss why outcomes using it have proved so uneven. We also examine the controversial aspects of this model, particularly the issue of whether or not the ESCd represents TB at all. Our focus here has been to explore similarities and potential differences between the phenotypes of ESCd, trophectoderm, placental villous TB, and human TB stem cells. We then explore the role of BMP4 in the differentiation of human pluripotent cells to TB and suggest that it converts the ESC into a totipotent state that is primed for TB differentiation when self-renewal is blocked. Finally we speculate that the TB formed from ESC is homologous to the trophectoderm-derived, invasive TB that envelopes the implanting conceptus during the second week of pregnancy. BMP4-driven differentiation of human embryonic and induced pluripotent stem cells provides a useful model for studying the specification and early development of early placental trophoblast.
Problem Macrophages are one of the first immune cells observed at the implantation site. Their presence has been explained as the result of an immune response toward paternal antigens. The mechanisms regulating monocyte migration and differentiation at the implantation site are largely unknown. In the present study, we demonstrate that trophoblast cells regulate monocyte migration and differentiation. We propose that trophoblast cells ‘educate’ monocytes/macrophages to create an adequate environment that promote trophoblast survival. Method of study CD14+ monocytes were isolated from peripheral blood using magnetic beads. Co‐culture experiments were conducted using a two‐chamber system. Monocytes were stimulated with lipopolysaccharide (LPS) and cytokine levels were determined using multiplex cytokine detecting assay. Results Trophoblast cells increase monocyte migration and induce a significant increase in the secretion and production of the pro‐inflammatory cytokines [interleukin‐6 (IL‐6), IL‐8, tumor necrosis factor‐α] and chemokines (growth‐related oncogen‐α, monocyte chemoattractant protein‐1, macrophage inflammatory protein‐1β, RANTES). Furthermore, the response of monocytes to LPS was different in monocytes pre‐exposed to trophoblast cells. Conclusion The results of this study suggest that trophoblast cells are able to recruit and successfully educate monocytes to produce and secrete a pro‐inflammatory cytokine and chemokine profile supporting its growth and survival. Furthermore we demonstrate that trophoblast cells can modulate monocytes response to bacterial stimuli.
Normal implantation depends on appropriate trophoblast growth and invasion. Inadequate trophoblast invasion results in pregnancy-related disorders, such as early miscarriage and pre-eclampsia, which are dangerous to both the mother and fetus. Msh Homeobox 2 (MSX2), a member of the MSX family of homeobox proteins, plays a significant role in the proliferation and differentiation of various cells and tissues, including ectodermal organs, teeth, and chondrocytes. Recently, MSX2 was found to play important roles in the invasion of cancer cells into adjacent tissues via the epithelial-mesenchymal transition (EMT). However, the role of MSX2 in trophoblastic invasion during placental development has yet to be explored. In the present study, we detected MSX2 expression in cytotrophoblast, syncytiotrophoblast, and extravillous cytotrophoblast cells of first or third trimester human placentas via immunohistochemistry analysis. Furthermore, we found that the in vitro invasive ability of HTR8/SVneo cells was enhanced by exogenous overexpression of MSX2, and that this effect was accompanied by increased protein expression of matrix metalloproteinase-2 (MMP-2), vimentin, and β-catenin. Conversely, treatment of HTR8/SVneo cells with MSX2-specific siRNAs resulted in decreased protein expression of MMP-2, vimentin, and β-catenin, and reduced invasion levels in a Matrigel invasion test. Notably, however, treatment with the MSX2 overexpression plasmid and the MSX2 siRNAs had no effect on the mRNA expression levels of β-catenin. Meanwhile, overexpression of MSX2 and treatment with the MSX2-specific siRNA resulted in decreased and increased E-cadherin expression, respectively, in JEG-3 cells. Lastly, the protein expression levels of MSX2 were significantly lower in human pre-eclamptic placental villi than in the matched control placentas. Collectively, our results suggest that MSX2 may induce human trophoblast cell invasion, and dysregulation of MSX2 expression may be associated with pre-eclampsia.
Until recently, trophoblast invasion during human placentation was characterized by and restricted to invasion into uterine connective tissues and the uterine spiral arteries. The latter was explained to connect the arteries to the intervillous space of the placenta and to guarantee the blood supply of the mother to the placenta. Today, this picture has dramatically changed. Invasion of endoglandular trophoblast into uterine glands, already starting at the time of implantation, enables histiotrophic nutrition of the embryo prior to perfusion of the placenta with maternal blood. This is followed by invasion of endovenous trophoblasts into uterine veins to guarantee the drainage of fluids from the placenta back into the maternal circulation throughout pregnancy. In addition, invasion of endolymphatic trophoblasts into the lymph vessels of the uterus has been described. Only then, invasion of endoarterial trophoblasts into spiral arteries takes place, enabling hemotrophic nutrition of the fetus starting with the second trimester of pregnancy. This new knowledge paves the way to identify changes that may occur in pathological pregnancies, from tubal pregnancies to recurrent spontaneous abortions.