The trophoblast cell lineage is essential for the survival of the mammalian embryo in utero. This lineage is specified before implantation into the uterus and is restricted to form the fetal portion of the placenta. A culture of mouse blastocysts or early postimplantation trophoblasts in the presence of fibroblast growth factor 4 (FGF4) permitted the isolation of permanent trophoblast stem cell lines. These cell lines differentiated to other trophoblast subtypes in vitro in the absence of FGF4 and exclusively contributed to the trophoblast lineage in vivo in chimeras.
Members of the Ets family of transcription factors mediate transcriptional responses of multiple signaling pathways in diverse cell types and organisms. Targeted deletion of the conserved DNA binding domain of the Ets2 transcription factor results in the retardation and death of homozygous mouse embryos before 8.5 days of embryonic development. Defects in extraembryonic tissue gene expression and function include deficient expression of matrix metalloproteinase-9 (MMP-3, gelatinase B), persistent extracellular matrix, and failure of ectoplacental cone proliferation. Mutant embryos were rescued by aggregation with tetraploid mouse embryos, which complement the developmental defects by providing functional extraembryonic tissues. Rescued Ets2-deficient mice are viable and fertile but have wavy hair, curly whiskers, and abnormal hair follicle shape and arrangement, resembling mice with mutations of the EGF receptor or its Ligands. However, these mice are not deficient in the production of TGF alpha or the EGF receptor. Homozygous mutant cell lines respond mitogenically to TGF alpha, EGF, FGF1, and FGF2 However, FGF fails to induce MMP-13 (collagenase-3) and MMP-3 (stromelysin-1) in the Ets2-deficient fibroblasts. Ectopic expression of Ets2 in the deficient fibroblasts restores expression of both matrix metalloproteinases. Therefore, Ets2 is essential for placental function, mediating growth factor signaling to key target genes including MMP-3, MMP-9, and MMP-13 in different cell types, and for regulating hair development.
During early development, a subset of fetal (placental) cytotrophoblasts exhibits tumor-like behavior and invades the uterus. To access a supply of maternal blood, they invade arterioles and form heterotypic interactions with, and replace, resident maternal endothelium, creating a hybrid uterine vasculature. Recently, it has become clear that invading cytotrophoblasts transform their adhesion receptor phenotype to resemble the endothelial cells they replace. Furthermore, they express vasculogenic factors and receptors. Is this a form of vasculogenesis?.
Trophoblast growth and invasion of the uterus are tightly regulated by locally produced factors. Since insulin-like growth factor (IGF)-II is produced by the invasive human extravillous trophoblast (EVT) cells and IGF-binding protein (IGFBP)-1 by the decidual cellsin situthat are in proximity to each other, we examined the possible influence of these molecules on proliferation, migration, and invasiveness of first-trimester EVT cells in culture. These EVT cell functions were respectively measured by3H-TdR uptake,in vitromigration, and invasion assays. Secretion of invasion-associated enzymes was assessed by zymography, and IGF-binding moieties on the EVT cell were examined by affinity cross-linking. Proliferation of serum-starved EVT cells was stimulated by addition of serum but unaffected by a wide range of IGF-I, IGF-II, and IGFBP-1 concentrations. IGF-II and IGFBP-1 or their combination stimulated EVT cell invasiveness and migration, when assays were conducted in serum-reduced media. Affinity cross-linking studies failed to detect the type 1 IGF receptor, although several IGF-II-specific and IGF-II-preferring binding molecules including type 2 IGF receptor were identified on the EVT cell surface. IGF-II enhancement of invasion was unaffected in the presence of IGF-1 receptor-blocking antibody and IGF-1 failed to influence EVT cell invasion, indicating that type 1 IGF receptor was not responsible for the IGF-II effects. Secretion of gelatinases or plasminogen activators was unaltered by IGF-II or IGFBP-1. We conclude that trophoblast-derived IGF-II and decidua-derived IGFBP-1 provide autocrine/paracrine enhancement of trophoblast invasiveness largely by stimulating migration, an essential step in invasion.Copyright 1998 Academic Press.
The human trophoblast differentiates from proximal cell column cytotrophoblasts into two lineages: a villous phenotype that results in cell fusion and formation of syncytium and an extravillous phenotype that adopts an invasive behavior and displays cell surface markers of an endothelial cell. Both phenotypes develop spontaneously in in vitro cultured cytotrophoblasts, but there is a clear gestational regulation by unknown genetic and/or maternal environmental factors that results in first trimester villous cytotrophoblasts entering the invasive pathway and term villous cytotrophoblasts entering the syncytial pathway. No genetic factors are known that induce the invasive pathway. First trimester cytotrophoblasts are induced to enter the invasive pathway by activin A, LIF and IL-1β but inhibited from differentiating in this direction by TGFβ1, TGFβ3, glucocorticoids and hypoxia. Term villous cytotrophoblasts are stimulated by EGF, EGF-II, IGFBP-1, α1β1 integrin (laminin receptor) and hypoxia. Term villous cytotrophoblasts are stimulated to form a syncytium by EGF, GM-CSF, CSF-1, dexamethasone, hCG, fibronectin, collagen I and PL48 and inhibited by TGFβ1. As well, there is evidence that TNFα and interferon γ induce and EGF inhibits apoptosis. This provides a mechanism for trophoblast turnover and renewal. Further research will be likely to uncover additional genetic, cytokine, extracellular matrix and physicochemical factors that regulate this complex process.
It has been suggested previously that phagocytic activity in the human placenta is confined to cells of the macrophage lineage. However, earlier studies were hampered by the paucity and poor viability of cells inherent in primary trophoblast cell cultures, contamination by other cell types which themselves have phagocytic activity, lack of reliable markers of trophoblasts, and by limitations of methods available to demonstrate unequivocally the internalization of particulate material. We have overcome these limitations by using: (i) DNA transfection to provide unlimited supplies of pure trophoblast cell lines; (ii) human placental lactogen as a marker unique to trophoblast; and (iii) confocal microscopy to demonstrate unequivocally the intracellular locality of phagocytosed material. We found that both untransfected primary culture extravillous trophoblast cells, as well as the cell lines, had the capacity to phagocytose sheep red blood cells, Staphylococcus aureus and baker's yeast cells, and that this activity was inhibited by cytochalasin B and by culture at 4 degrees C. Phagocytic activity in trophoblast cells was less avid than that seen in a professional phagocyte. In physiological and pathological situations where tissue remodelling occurs, such as the rapid turnover in the periodontal ligament or during inflammation, epithelial cells and other cells that are not considered professional phagocytes actively phagocytose components of the extracellular matrix. We postulate that phagocytosis by human trophoblasts may play an important role in the extensive tissue remodelling that occurs during trophoblastic invasion of the decidua.
Placenta growth factor (PlGF) is a homodimeric glycoprotein, 46-50 kDa in size, belonging to the vascular endothelial growth factor (VEGF) sub-family. It exists as two isoforms, PlGF-1 and -2, the latter having a heparin-binding domain. Like VEGF, it is a potent angiogenic factor; however, PlGF homodimers interact with the VEGF receptor Flt-1 (fms-like tyrosine kinase), but not with the kinase domain-containing region (KDR). Since PlGF is made by the human placenta and extravillous trophoblast (EVT) cells of the human placenta express Flt-1 in situ, these cells may be responsive to PlGF. Therefore, this study examined whether first trimester EVT cells propagated in vitro expressed the mRNA or the protein of Flt-1 and PlGF, and whether exogenous PlGF-1 had any effect on EVT cell proliferation, migration or invasiveness. Immunocytochemical and RT-PCR analyses revealed that both normal and SV40 Tag-immortalized EVT cells expressed the protein and mRNA for Flt-1, but not for PlGF-1 or -2. Exogenous PlGF-1 stimulated proliferation (measured by H-thymidine uptake) of normal EVT cells in a concentration-dependent manner, but only in the presence of excess heparan sulphate proteoglycans (HSPGs). These results raise two possibilities: that exogenous PlGF-1 (in spite of having a low affinity for heparin) was sequestered away from its receptor because of binding to heparan sulphate proteoglycans on the EVT cell surface or the ECM, or that HSPGs could modify the interaction between Flt-1 and PlGF. PlGF-1, in the presence or absence of HSPGs, however, had no effect on EVT migration or invasiveness, when measured with a transwell invasion (in the presence of Matrigel®) or migration (in the absence of Matrigel®) assay. These findings place PlGF amongst a large group of growth factors that promote EVT cell proliferation without influencing their migratory or invasive behaviours, and suggest that P1GF-Flt-1 interactions may be regulated by HSPGs in situ.
At embryo implantation, it is postulated that the initial contact between blastocyst and maternal tissues is by adhesion of the trophoblast to the uterine epithelium. This cell-to-cell interaction is thought to be critical for implantation, although the actual adhesive forces have never been determined. In the present study, the atomic force microscope (AFM) was used to study the adhesion between human uterine epithelial cell lines (HEC-1-A; RL95-2) and human trophoblast-type cells (JAR). Specific interaction forces of these epithelia via their apical cell poles were determined on the basis of approach-and-separation cycles. For this purpose, the AFM tip was functionalized with JAR cells, then brought to the surface of uterine epithelial monolayers and was kept in contact for different periods of time (ms, 1, 10, 20, 40 min). The approach force curves displayed repulsive interactions for both HEC-1-A and RL95-2 cells. However, RL95-2 cells (with a smooth surface structure and a thin glycocalyx) showed lower values of the repulsive regime than HEC-1-A cells (with a rough surface structure and a thick glycocalyx). After having overcome repulsive interactions, the initial contact was followed by adhesive interactions. For contact times of 20 and 40 min, RL95-2 cells, but not HEC-1-A cells, showed specific JAR binding, i.e. the separation force curves displayed repeated rupture events in the range of 1-3 nN with a distance between 7-15 microm and, thereafter, a final rupture event at a distance of up to 45 microm. These features point to the formation of strong cell-to-cell bonds. Collectively, these studies provide the first definition of interaction forces between the trophoblast and the uterine epithelium, and are consistent with the hypothesis that an RL95-2-like architecture of uterine epithelial cells, i.e. an non-polarized phenotype, is essential for apical adhesiveness for the human trophoblast.
The invasion of extravillous trophoblast cells into the maternal endometrium is one of the key events in human placentation. The ability of these cells to infiltrate the uterine wall and to anchor the placenta to it as well as their ability to infiltrate and to adjust utero-placental vessels to pregnancy depends, among other things, on their ability to secrete enzymes that degrade the extracellular matrix. Most of the latter enzymes belong to the family of matrix metalloproteinases. Their activity is regulated by the tissue inhibitors of matrix metalloproteinases. We have studied the distribution patterns of matrix metalloproteinases-1, -2, -3, and -9 and their inhibitors TIMP-1 and TIMP-2 as compared to the distribution of their substrates along the invasive pathway of extravillous trophoblast of 1st, 2nd, and 3rd trimester placentas by means of light microscopy on paraffin and cryostat sections as well as at the ultrastructural level (only 3rd trimester placenta). The comparison of different methods proved to be necessary, since the immunohistochemical distribution patterns of these soluble enzymes are considerably influenced by the pretreatment of tissues. All three methods revealed immunoreactivities of both, proteinases and their inhibitors, not only intracellularly in the extravillous trophoblast but also extracellularly in its surrounding matrix, the distribution patterns depending on the stage of pregnancy and on the degree of differentiation of trophoblast cells along their invasive pathway. Within the extracellular matrix, immunolocalization of matrix metalloproteinases as well as their inhibitors showed a specific relation to certain extracellular matrix molecules.
Human trophoblast cells offer a unique in vitro model for the study of aspects of the dynamic processes occurring during cell fusion and syncytium formation. In the human placenta, mononuclear cytotrophoblasts aggregate and fuse to form a multinucleated syncytiotrophoblast. In vitro, the addition of cyclic AMP analogs, 8-bromo-cyclic-AMP or Sp-8-bromo-cyclic AMPS, promotes syncytiotrophoblast formation, as shown by the disappearance of immunostained E-cadherin and desmoplakin, and increased numbers of nuclei per syncytium. An antagonist of cyclic AMP, Rp-8-bromo-cyclic AMPS, and an inhibitor of the cyclic All IP-dependent protein kinase catalytic subunit, H-89, impair cell fusion, This led us to study the pattern of expression and subcellular localization of cyclic-AMP-dependent protein kinase subunits during syncytium formation. Cytotrophoblasts expressed the RI alpha and RII alpha regulatory subunits and the C alpha and C beta catalytic subunits, RI alpha was down-regulated during syncytium formation, No change in RII omega. protein levels was observed, but there was a drastic subcellular redistribution. RII alpha located in the Golgi-centrosomal area of cytotrophoblasts was scattered throughout the cytoplasm of the syncytiotrophoblast. Interestingly, an accumulation of RII alpha was observed underneath the epical membrane of syncytiotrophoblast in vitro and in situ This suggests a key role of cyclic AMP-dependent protein kinase type II alpha during cell fusion and microvilli formation, both of which are essential for the secretory and transfer functions of the syncytiotrophoblast.