Background: The trophoblast compartment of the placenta comprises various subpopulations with distinct functions. They interact among each other by secreted signals thus forming autocrine or paracrine regulatory loops. We established a first trimester trophoblast cell line (ACH-3P) by fusion of primary human first trimester trophoblasts (week 12 of gestation) with a human choriocarcinoma cell line (AC1-1). Results: Expression of trophoblast markers (cytokeratin-7, integrins, matrix metalloproteinases), invasion abilities and transcriptome of ACH-3P closely resembled primary trophoblasts. Morphology, cytogenetics and doubling time was similar to the parental AC1-1 cells. The different subpopulations of trophoblasts e. g., villous and extravillous trophoblasts also exist in ACH-3P cells and can be immuno-separated by HLA-G surface expression. HLA-G positive ACH-3P display pseudopodia and a stronger expression of extravillous trophoblast markers. Higher expression of insulin-like growth factor II receptor and human chorionic gonadotropin represents the basis for the known autocrine stimulation of extravillous trophoblasts. Conclusion: We conclude that ACH-3P represent a tool to investigate interaction of syngeneic trophoblast subpopulations. These cells are particularly suited for studies into autocrine and paracrine regulation of various aspects of trophoblast function. As an example a novel effect of TNF-alpha on matrix metalloproteinase 15 in HLA-G positive ACH-3P and explants was found.
Abstract During human pregnancy the semi-allogeneic/allogeneic fetal graft is normally accepted by the mother's immune system. Initially the contact between maternal and fetal cells is restricted to the decidua but during the 2nd trimester it is extended to the entire body. Two contrary requirements influence the extent of invasion of extravillous fetal trophoblast cells (EVT) in the maternal decidua: anchorage of the placenta to ensure fetal nutrition and protection of the uterine wall against over-invasion. To establish the crucial balance between tolerance of the EVT and its limitation, recognition of the semi-allogeneic/allogeneic fetal cell by maternal leukocytes is prerequisite. A key mechanism to limit EVT invasion is induction of EVT apoptosis. Apoptotic bodies are phagocytosed by antigen-presenting cells (APC). Peptides from apoptotic cells are presented by APC cells and induce an antigen-specific tolerance against the foreign antigens on EVT cells. These pathways, including up-regulation of the expression of IDO, IFNγ and CTLA-4 as well as the induction of Tregulatory cells, are general immunological mechanisms which have developed to maintain peripheral tolerance to self-antigens. Together these data suggest that the mother extends her “definition of self” for 9 months on the foreign antigens of the fetus.
The trophoblast, i.e. the peripheral part of the human conceptus, exerts a crucial role in implantation and placentation. Both processes properly occur as a consequence of an intimate dialogue between fetal and maternal tissues, fulfilled by membrane ligands and receptors, as well as by hormone and local factor release. During blastocyst implantation, generation of distinct trophoblast cell types begins, namely the villous and the extravillous trophoblast, the former of which is devoted to fetal-maternal exchanges and the latter binds the placental body to the uterine wall. Physiological placentation is characterized by the invasion of the uterine spiral arteries by extravillous trophoblast cells arising from anchoring villi. Due to this invasion, the arterial structure is replaced by amorphous fibrinoid material and endovascular trophoblastic cells. This transformation establishes a low-resistance, high-capacity perfusion system from the radial arteries to the intervillous space, in which the villous tree is embedded. The physiology of pregnancy depends upon the orderly progress of structural and functional changes of villous and extravillous trophoblast, whereas a derangement of such processes can lead to different types of complications of varying degrees of gravity, including possible pregnancy loss and maternal life-threatening diseases. In this review we describe the mechanisms which regulate trophoblast differentiation, proliferation, migration and invasiveness, and the alterations in these mechanisms which lead to pathological conditions. Furthermore, based on the growing evidence that proper inflammatory changes and oxidative balance are needed for successful gestation, we explain the mechanisms by which agents able to influence such processes may be useful in the prevention and treatment of pregnancy disorders.
This study examined the placentation in the degu, the origin of the extrasubplacental trophoblast (EST) (extravillous trophoblast in human), and the activity of Na /K ATPase in the placental barrier during different gestational ages, as part of a wider effort to understand the reproductive biology of this species. Fifteen degus at the first stage of gestation, midgestation and at term of pregnancy were studied. At day 27 of gestation, the subplacenta is formed under the wall of the central excavation. Simultaneously, the outermost trophoblast of the ectoplacental cone differentiated into secondary trophoblast giant cells that lie on the outside of the placenta, forming an interface with the maternal cells in the decidua. These giant cells immunostained positive for cytokeratin (CK) and placental lactogen (hPL) until term. During this period, the EST merged from the subplacenta to the decidua and immunostained negative for CK, but at term, immunostained for CK and hPL in the maternal vessels. The vascular mesenchyme of the central excavation invaded the chorioallantoic placenta during this period, forming two fetal lobules of labyrinthine-fine syncytium, the zone of the placental barrier. The activity of Na /K ATPase in the placental barrier was constant during the gestational period. The residual syncytium at the periphery of the placental disc and between the lobules was not invaded by fetal mesenchyme and formed the marginal and interlobular labyrinthine syncytium that immunostained first for CK, and later for hPL, as in the labyrinthine fine syncytium. The presence of intracytoplasmic electron-dense material in the interlobular labyrinthine syncytium suggested a secretory process in these cells that are bathed in maternal blood. Placentas obtained from vaginal births presented a large, single lobe, absence of the subplacenta, and a reduced interlobular labyrinthine syncytium. At day 27, the inverted visceral yolk sac is observed and its columnar epithelium immunostained for CK and hPL. This suggests that the yolk sac is an early secretory organ. The epithelium of the parietal yolk sac covers the placenta. The origin of the EST in the degu placenta and its migration to maternal vessels allows us to present this animal model for the study of pregnancy pathologies related to alterations in the migration of the extravillous 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.
In healthy individuals, the non‐classical MHC molecule HLA‐G is only expressed on fetal trophoblast cells that invade the decidua during placentation. We show that a significant proportion of HLA‐G at the surface of normal human trophoblast cells is present as a disulphide‐linked homodimer of the conventional β2m‐associated HLA‐I complex. HLA‐G is a ligand for leukocyte immunoglobulin‐like receptors (LILR), which bind much more efficiently to dimeric HLA‐G than to conventional HLA‐I molecules. We find that a LILRB1‐Fc fusion protein preferentially binds the dimeric form of HLA‐G on trophoblast cells. We detect LILRB1 expression on decidual myelomonocytic cells; therefore, trophoblast HLA‐G may modulate the function of these cells. Co‐culture with HLA‐G+ cells does not inhibit monocyte‐derived dendritic cell up‐regulation of HLA‐DR and costimulatory molecules on maturation, but did increase production of IL‐6 and IL‐10. Furthermore, proliferation of allogeneic lymphocytes was inhibited by HLA‐G binding to LILRB1/2 on responding antigen‐presenting cells (APC). As HLA‐G is the only HLA‐I molecule that forms β2m‐associated dimers with increased avidity for LILRB1, this interaction could represent a placental‐specific signal to decidual APC. We suggest that the placenta is modulating maternal immune responses locally in the uterus through HLA‐G, a trophoblast‐specific, monomorphic signal present in almost every pregnancy. See accompanying commentary: http://dx.doi.org/10.1002/eji.200737515
Trophoblast giant cells (TGCs) are the first terminally differentiated subtype to form in the trophoblast cell lineage in rodents. In addition to mediating implantation, they are the main endocrine cells of the placenta, producing several hormones which regulate the maternal endocrine and immune systems and promote maternal blood flow to the implantation site. Generally considered a homogeneous population, TGCs have been identified by their expression of genes encoding placental lactogen 1 or proliferin. In the present study, we have identified a number of TGC subtypes, based on morphology and molecular criteria and demonstrated a previously underappreciated diversity of TGCs. In addition to TGCs that surround the implantation site and form the interface with the maternal deciduas, we demonstrate at least three other unique TGC subtypes: spiral artery-associated TGCs, maternal blood canal-associated TGCs and a TGC within the sinusoidal spaces of the labyrinth layer of the placenta. All four TGC subtypes could be identified based on the expression patterns of four genes: , , (encoded by genes of the prolactin/prolactin-like protein/placental lactogen gene locus), and (from a placental-specific cathepsin gene locus). Each of these subtypes was detected in differentiated trophoblast stem cell cultures and can be differentially regulated; treatment with retinoic acid induces / TGCs preferentially. Furthermore, cell lineage tracing studies indicated unique origins for different TGC subtypes, in contrast with previous suggestions that secondary TGCs all arise from ectoplacental cone precursors.
During the invasive phase of implantation, trophoblasts and maternal decidual stromal cells secrete products that regulate trophoblast differentiation and migration into the maternal endometrium. Paracrine interactions between the extravillous trophoblast and the maternal decidua are important for successful embryonic implantation, including establishing the placental vasculature, anchoring the placenta to the uterine wall, and promoting the immunoacceptance of the fetal allograph. To our knowledge, global crosstalk between the trophoblast and the decidua has not been elucidated to date, and the present study used a functional genomics approach to investigate these paracrine interactions. Human endometrial stromal cells were decidualized with progesterone and further treated with conditioned media from human trophoblasts (TCM) or, as a control, with control conditioned media (CCM) from nondecidualized stromal cells for 0, 3, and 12 h. Total RNA was isolated and processed for analysis on whole-genome, high-density oligonucleotide arrays containing 54â600 genes. We found that 1374 genes were significantly upregulated and that 3443 genes were significantly downregulated after 12 h of coincubation of stromal cells with TCM, compared to CCM. Among the most upregulated genes were the chemokines CXCL1 ( GRO1 ) and IL8 , CXCR4 , and other genes involved in the immune response ( CCL8 [SCYA8] , pentraxin 3 ( PTX3) , IL6 , and interferon-regulated and -related genes) as well as TNFAIP6 ( tumor necrosis factor alpha-induced protein 6 ) and metalloproteinases ( MMP1 , MMP10 , and MMP14 ). Among the downregulated genes were growth factors, e.g., IGF1 , FGF1 , TGFB1, and angiopoietin-1, and genes involved in Wnt signaling ( WNT4 and FZD ). Real-time RT-PCR and ELISAs, as well as immunohistochemical analysis of human placental bed specimens, confirmed these data for representative genes of both up- and downregulated groups. The data demonstrate a significant induction of proinflammatory cytokines and chemokines, as well as angiogenic/static factors in decidualized endometrial stromal cells in response to trophoblast-secreted products. The data suggest that the trophoblast acts to alter the local immune environment of the decidua to facilitate the process of implantation and ensure an enriched cytokine/chemokine environment while limiting the mitotic activity of the stromal cells during the invasive phase of implantation.
The Ets2 transcription factor is essential for the development of the mouse placenta and for generating signals for embryonic mesoderm and axis formation. Using a conditional targeted Ets2 allele, we show that Ets2 is essential for trophoblast stem (TS) cells self-renewal. Inactivation of Ets2 results in TS cell slower growth, increased expression of a subset of differentiation-associated genes and decreased expression of several genes implicated in TS self-renewal. Among the direct TS targets of Ets2 is , a key master regulator of TS cell state. Thus Ets2 contributes to the regulation of multiple genes important for maintaining the undifferentiated state of TS cells and as candidate signals for embryonic development.
Expression of T‐box family member Eomesodermin (Tbr2) is spatiotemporally restricted in the mouse embryo; initially expressed in extraembryonic lineages in the sequential progression from the trophectoderm of the blastocyst, its derivatives the extraembryonic ectoderm, and thereafter the chorion, in addition to the visceral endoderm and primitive streak at gastrula stages, and the telencephalon at later stages. We describe the spatiotemporal expression of GFP in embryos of a Tg(Eomes::GFP) BAC transgenic strain, and have compared it with the localization of endogenous Eomes transcripts and protein. Our analysis reveals the following: (1) robust easily visualized reporter expression in live hemizygous transgenic embryos, (2) increased levels of expression in live homozygous transgenic embryos that are compatible with embryo viability, and (3) a close correlation between endogenous Eomes and GFP reporter expression in BAC transgenic embryos. These features establish the Tg(Eomes::GFP) BAC transgenic strain as a novel reagent for both live imaging and the isolation of Eomes expressing cells from specific locations within the embryo. genesis 45:208–217, 2007. Published 2007 Wiley‐Liss, Inc.