Embryonic development is accompanied by a series of cell differentiation, by which highly specialised cell types are developed from a single undifferentiated, fertilised egg. One of the earliest lineages to form in mamalian conceptus is the trophoblast, which only contributes to extraembryonic constructs and is the essential component of the placenta. Trophoblast stem cells derived from early trophectoderm and later placental villous tissue were pluripotent cells which can undergo selfrenewal in supporting medium and give rise to trophoblast lineages in differentiation conditions. Abnormal development of trophoblast is related to primary infertility, recurrent abortion and newborn infant diseases. Aberrant placental expression of apoptosis and inflammation-related genes in early pregnancy was found in recurrent miscarriage and hydatidiform mole samples. Altered placental transcription of metabolic regulatory genes has been associated to affected fetal growth and maternal pregnancy complications. The successful establishment of trophoblast stem cell line benefits the in vitro research of trophoblast development. This study aimed to detect RhoGDIα fonction in rabbit trohoblast cells maintenance and differentiation. RhoGDI is the binding protein of RhoGTPase, which inhibits RhoGTPase activation. In our previous work, we found the apoptosis of trophoblast cells was accompanied by RhoGDIα up-regulation, suggesting the possible functions of RhoGDIα in regulating trophoblast stem cell maintenance. In this research, we further reveal that RhoGDIα took part in the regulation of trophoblast stem cell self renewal and its over expression led to apoptosis in trophoblast cells. we also found that knocking down of RhoGDIαpromoted trophoblast stem cells migration without affecting differentiation. Our results verify RhoGDIα has a function in trophoblast stem cell maintenance and migration, but has no obvious influence on trophoblast cell differentiation.
In the early development of the placenta, some highly proliferative and undifferentiated primitive cytotrophoblast cells that are derived from the trophectoderm give rise to different subtype of trophoblast cells. The highly proliferative extravillous trophoblasts (EVTs) migrate into the deep layer of the maternal endometrium and even into the inner third of the myometrium, thereby anchoring the fetus to the mother. They also can penetrate into the uterine spiral arteries and replace maternal endothelial cells. In this way, the uterine spiral arteries are remodeled into low-resistance, high-capacity utero-placental arteries that provide the increased blood flow towards the placenta that is needed to meet the requirements of the growing fetus. Trophoblast differentiation during placental development is precisely regulated by the the factors from themselves and within the maternal-fetal interface. The dysregulation of trophoblast activities, especially defects in the invasion of EVTs into the uterus and the subsequent failure in the remodeling of the maternal spiral arteries, are the key mechanisms that underlie the development of preeclampsia. The large number of the lymphocytes in the maternal-fetal microenvioment which have deep adhension with trophoblast cells, may affect trophoblast differenciation process directly or indirecly. Decidual NK cells (dNK) account for approximately 70% of the lymphocytes in decidua during early pregnancy. The dNK cells, generally identified by CD56+CD16-CD3-, are much different from the NK cells in the peripheral blood. Though they aquire a strong cytokines secretion ability, their cytotoxicity ability reduced greatly. The previous reports shows that dNK cells and trophoblast cells closely surround the spiral arteries in the endometrium. They all participate in the process of the placental development through the interaction mediated by the cytokines or the direct recognizition between them. However, the mechanism of the interaction between these two cells remains to be explained in-depth. Here we mainly focus on revealing the mechanism that dNK cells regulate the abilities of invasion and the spiral arteries remodeling of the trophoblast cells by the secretion of cytokines. We isolated and purifed the CD56+CD16-CD3- dNK cells from the decidual tissues at early pregnancy. After culture of the cells for 48h in vitro, the dNK-CM was collected. The human trophoblast cells HTR8/SVneo were treated by dNK-CM. The invasion ability of the treated cells were analyzed by Transwell invasion assay. Meanwhile, the ability of the trophoblast cells to differentiate towards the endothelial phenotype was studied using HTR8/SVneo and HUVEC co-culture model, Matrigel tube formation assay and the Live Cell imaging. Our results showed that dNK cells could promote trophoblast cell invasion and the tube formation with HUVEC. Treatment of dNK-CM in HTR8/SVneo cells could also reduce the expression of the epithelial cell marker E-cadherin, and promote the expression of VE-cadherin and integrinβ1 which represent the features of endothelial cells. This indicates that dNK-CM may induce the trophoblast cells to differentiate towards the endovascular phenotype. Further studies proved that dNK cells promote trophoblast cells invasion through the production of IL-8 and HGF, and induce trophoblast cells to form tubes with HUVEC via the production of VEGF-C and HGF. On the other hand, we found that dNK cells coud express ActivinA receptors including ActRIIA, ActRIIB, ALK4 and ALK7. ActivinA could activate Smad2 in dNK cells, and dose-dependently regulate the expressions of the cytokines such as VEGF-A, VEGF-C, PLGF, IL-8 and HGF. It could also reduce the expressions of the cytotoxicity markers in dNK cells. These data indicate that trophoblast cells may affect dNK cell functions through the secretion of ActivinA. In conclusion, this study proves that dNK cells and trophoblast cells have active interaction at the maternal-fe
(1) Combinatorial signals of TGF-β/activin/Nodal and Bone Morphogenic Protein regulate the early lineage segregation of human embryonic stem cellsCell fate commitment of pre-implantation blastocysts, to either the inner cell mass or trophoblast, is the first step in cell lineage segregation of the developing human embryo. However, the intercellular signals that control fate determination of these cells remain obscure. Human embryonic stem cells (hESCs) provide a unique model for studying human early embryonic development. We have previously shown that TGF-β/activin/Nodal signaling contributes to maintaining pluripotency of hESCs, which are derivatives of the inner cell mass. Here we further demonstrate that the inhibition of TGF-β/activin/Nodal signaling results in the loss of hESC pluripotency and trophoblast differentiation, similar to BMP4-induced trophoblast differentiation from hESCs. We also show that the trophoblast induction effect of BMP4 correlates with and depends on the inhibition of TGF-β/activin/Nodal signaling. However,the activation of BMP signaling is still required for trophoblast differentiation when TGF-β/activin/Nodal signaling is inhibited. These data reveal that the early lineage segregation of hESCs is determined by the combinatorial signals of TGF-β/activin/Nodal and BMP.
Placenta forms the interface between the maternal and fetal circulation, facilitating metabolic and gas exchange as well as fetal waste disposal, and forms a barrier against the maternal immune system. The placental development originates at the recognition and attachment of the extraembryonic trophoblast and the luminal epithelia of the uterus, involving the trophoblast cell invasion to the endometrial stromal and the reconstruction, the establishment of the maternal and fetal blood circulation, the maintain of the endocrine function of the trophoblast cell and so on. Abnormalities in any of these events can be associated with poor pregnancy outcome, such as spontaneous abortion, preeclampsia, the intrauterine growth restriction and severe pregnancy diseases even embryonic death. Recently, increasing studies have been focused on revealing the genes governing the process of placental development, among which, many targeted mutations of Wnt family genes have pointed toward to the content that Wnt signalling plays an important role in placental development. In this study, we applied the Cre-loxp strategy to investigate the consequence of the conditional deletion or overactivation of ß-catenin encoded by Ctnnb1 (a key mediator of canonical Wnt signalling) on placentation. The birth ratio of the mutant mice was recorded and the morphological changes of the null placentas were analyzed by H&E and immunohistochemistry staining. We observed that the conditional depletion or overactivation of Ctnnb1 in placentas induced by cross-breeding with trophoblast specific Cyp19-Cre mice showed apparently normal placental formation. For example, the fusion of the allantois with the chorion, and subsequent branching of the allantochorionic plate occur in null placentas at E9.0-9,5. At E13.5 the fetus capillaries can develop apparently normally and then successfully invade into the maternal blood sinuses. Since the knockout efficiency of Cyp19-Cre needs to be further revealed, detailed analysis of the phenotypic alterations and underlying causes and mechanisms warrant future investigation.
Cullin-RING ligase multisubunit complexes are the largest E3 ubiquitin ligase family. Cullins are a family of scaffold proteins that assemble RING finger type E3 ligase complexes. Seven members of the Cullin family of proteins, including CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, and CUL7, have been identified in human beings. Cullin 3 (CUL3), a scaffold protein, assembles a large number of protein complex which is similar to Skp1-Cullin 1-F-box (SCF). CUL3 associates with Bric-a-brac-Tramtrack-Broad (BTB) complex and Rbx1 to form a BTB-CUL3-Rbx1 (BCR) ubiquitin ligase complex. Growing evidence has revealed that BCR ubiquitin ligase complex serves as a major regulator of cell cycle and developmental processes.Most Cullins are crucial for early embryonic development. Mutations in CUL4B and CUL7 are associated with growth retardation syndromes. CUL1, CUL3 and CUL4A knockout mice fail to develop beyond 7.5 days postcoitum. Embryos from CUL4B null mice show severe developmental arrest and usually die before embryonic day 9.5. We have previously reported that CUL7 triggers epithelial–mesenchymal transition of choriocarcinoma JEG-3 cells. In addition, we have also demonstrated that CUL1 promotes the invasion of human trophoblast cells and is significantly downregulated in the placentas from pre-eclampsia (PE) patients. Combined with the studies from other labs like the implication of Cullin 7 in 3-M syndrome and the Yakut short stature syndrome, characterized by pre- and post-natal growth retardation, we conclude that the Cullin family of proteins plays very special roles during human placentation. Several genetic models have shown that CUL3 is crucial for early embryonic development. Nevertheless, the role of CUL3 in human trophoblast function needs to be further investigated.In this study, immunostaining revealed that CUL3 was strongly expressed in the villous cytotrophoblast cells, the trophoblast column, and the invasive extravillous trophoblast cells. Silencing CUL3 significantly inhibited the outgrowth of villous explant ex vivo and decreased invasion and migration of trophoblast HTR8/SVneo cells. Furthermore, CUL3 siRNA decreased pro-MMP9 activity and increased the levels of TIMP1 and 2. We also found that the level of CUL3 in the placental villi from pre-eclamptic patients was significantly lower as compared to that from their gestational age-matched controls. Moreover, in the lentiviral-mediated placenta-specific CUL3 knockdown mice, lack of CUL3 in the placenta resulted in less invasive trophoblast cells in the maternal decidua. Taken together, these results suggest an essential role for CUL3 in the invasion and migration of trophoblast cells, and dysregulation of its expression may be associated with the onset of pre-eclampsia.
Normal proliferation and differentiation of human placental trophoblast cells contribute to the proper function of placenta. Dysregulated differentiation of trophoblast cells, including abnormal invasion and syncytialization, leads to pregnancy-related diseases, such as pre-eclampsia (PE). PE, a major cause of maternal mortality, morbidities, perinatal deaths, preterm birth, and intrauterine growth restriction, affects 3-5% of pregnancies worldwide. MicroRNAs (miRNAs) are a conserved group of approximately 22-nucleotide regulatory RNAs that play important roles in regulating gene expression by binding to 3’-untranslated region (3’-UTR) of mRNAs for either degradation or translation repression. MiRNAs have been shown by oligonucleotide microarrays to be highly enriched in the placenta. Furthermore, miRNAs are differentially expressed in the human placentas of patients with PE, indicating that miRNAs may have important roles in the pathogenesis of PE. Circulating miRNAs have emerged as potential novel diagnostic biomarkers for cancer, tissue injury and other pathological conditions. MiRNAs specifically expressed in human placentas were also detected in sera from pregnant women and found to be significantly elevated compared with those from non-pregnant women; their levels increased with gestational age and decreased after delivery, indicating potential utility of circulating miRNAs in monitoring process of pregnancy or pregnancy-related diseases. Using miRNA microarray assay and real-time stem-loop qRT-PCR analysis, the present study demonstrated a maternally differential circulating miRNA expression profile in plasma samples from severe pre-eclamptic pregnancies compared with those from term-matched normal pregnancies, and seven microRNAs, namely miR-24, miR-26a, miR-103, miR-130b, miR-181a, miR-342-3p, and miR-574-5p, were found to be elevated (2.0-fold changes or more) in plasma from severe pre-eclamptic pregnancies. Gene ontology and pathway enrichment analyses revealed that these microRNAs were involved in specific biological process categories (including regulation of metabolic processes, regulation of transcription, and cell cycle) and signaling pathways (including the mitogen-activated protein kinase signaling pathway, the transforming growth factor-β signaling pathway, and pathways in cancer metastasis). We further explored the role of miR-181a in the proliferation and differentiation of human placental trophoblast cells. When assessed using MTT assays, overexpressed miR-181a significantly accelerated the proliferation of HTR-8/SVneo cells, while the proliferation of HTR-8/SVneo cells was significantly impaired when introducing the inhibitor of miR-181a. Through transwell migration assay and matrigel invasion assay, the migration and invasion abilities of HTR-8/SVneo cells were enhanced by overexpressed miR-181a. However, only the migration ability of HTR-8/SVneo cells was weakened by introducing the inhibitor of miR-181a. Furthermore, the level of miR-181a was found to be significantly reduced during syncytialization. All the above suggested that miR-181a was involved in the proliferation and differentiation of human placental trophoblast cells, and further exploration of the molecular mechanisms underlying is needed. In summary, the present study demonstrated a maternally differential circulating miRNA expression profile in plasma samples from severe pre-eclamptic pregnancies compared with those from normal pregnancies. MiR-181a, one of the seven circulating miRNAs elevated in severe pre-eclamptic pregnancies, was involved in the proliferation and differentiation of human placental trophoblast cells.
The placenta is critical for the health of the mother and the growing fetus during pregnancy. The trophoblastic cells are the major cell types in the placenta, and one of their subtypes is the invasive extravillous trophoblast (EVTs), which can invade into uterine stroma and remodel uterine spiral arteries. In this way, an appropriate uterine-placental-fetal circulation is established to meet the need for nutrient exchange between the fetus and the mother. The abnormalities in trophoblast differentiation are tightly associated with various pregnancy diseases.Preeclampsia (PE), a leading cause of maternal and perinatal morbidity and mortality worldwide, is characterized by a newly onset of hypertension, proteinuria or dysfunctions in multiple maternal organs after the 20th week of gestation. Accumulating evidences indicated the direct contribution of the placenta defects to the development of this pregnancy disorder. It has been believed that in preeclampsia, the shallow invasion of uterine stroma and the insufficient remodeling of spiral arteries by EVTs lead to an impaired blood perfusion into the placenta, which further hampers trophoblast differentiation. The malfunctioning trophoblasts can release cell debris and many factors into maternal circulation that can interfere the normal functions of multiple maternal organs. Hepatocyte growth factor (HGF) and its receptor Met are essential for the placental development in mammals, playing important roles in regulating trophoblast cell migration, invasion and blood vessel remodeling. Knocking out in either HGF or Met in mice led to fetal death in utero, as well as severe defects in trophoblast cell differentiation and blood vessel formation within the feto-maternal interface. Our previous investigation identified an over production of soluble Met (sMet) in severe PE placenta, which could inhibit the invasion-promoting effect of HGF in trophoblast cells. Although no change in the levels of HGF or Met was found in severe PE placenta, the findings in sMet indicated the contribution of HGF/Met signaling insufficiencies to the placental defects in this disease.Our further investigation demonstrated the remarkably lowered levels in phosphorylated Met and downstream Erk in the placentas derived from severe PE when compared with the controls, especially in the early onset ones whose clinical manifestations occurred before the 34th gestational week. What’s more, a large number of immunofluroscent signals for punctate aggregation of Met was observed in the placentas of early onset severe PE (E-PE). The findings indicated a seriously impaired balance of Met activation and inactivation in E-PE placenta. Physiologically, Met receptor can rapidly get into cytoplasm via endocytosis upon the activation by its ligand, and can be subsequently subjected to the process of protein ubiquitination and degradation. This mechanism will ensure the appropriate signaling transduction, avoiding cell damage due to continuous activation in Met signaling. On the other hand, there has been studies demonstrating the cross activation of Met by sema4D/Plexin-B1 signaling in human endothelial cells and tumor cells. Based on these evidences, we hypothesized that in E-PE placental trophoblast cells, the HGF/Met signaling may be hampered due to the excessive endocytosis, abnormal degradation and insufficient corss-activation in Met, which resulted in the blockage of HGF/Met effects on trophoblast cell invasion. To address the hypothesis, we analyzed the distribution of Met protein and the endocytosis vesicles, the expressions of endocytosis-associated proteins (Clathrin and CAV-1), the E3 ubiquitin ligase for Met (Cbl) and sema4D/Plexin-B1 in the placentas from E-PE and late onset PE (L-PE). The in vitro trophoblast cell line, HTR8/SVneo, and human umbilical vessel endothelial cells (HUVEC) were used as in vitro models to investigation the endocytosis, ubiquitination degradation and sema4D transactivation of Met upon h