体外诱导人胚胎干细胞向滋养层细胞分化的研究进展

目前在体外研究中,主要通过分离胚体和骨形态发生蛋白4（bone morphogenetic protein4,BMP4）诱导这2种途径从人胚胎干细胞（hEsc）分化获取滋养层细胞（TB）。胚体途径可基于细胞的黏附性和培养基中β-hCG的含量从胚体中分离获得TB,进而在三维培养体系中可检测细胞的侵袭性及细胞与基质的相互作用。BMP4途径通过去除外源性成纤维细胞生长因子及提高氧含量均可促进BMP4诱导TB的分化。虽然hESC分化TB模型目前还存在一些争议,但是相关研究可为探讨人类胚胎的植入及胎盘的形成提供重要的理论依据。     

Stimulation of hCG and inhibition of hPL in isolated human trophoblast cells in vitro by glycodelin A

The immunosuppressive protein glycodelin A (formerly named PP14) is produced by human decidua and secreted in the maternal circulation. Glycodelin A concentrations in serum have been used as indicators of endometrial function. The purpose of this study was to investigate the effect of glycodelin A on human chorionic gonadotropin (hCG) and human placental lactogen (hPL) release by freshly isolated cytotrophoblasts (in vitro). Cytotrophoblasts have been prepared from human term placenta by the three-step trypsin-DNase dispersion method of villous tissue followed by a percoll gradient centrifugation step. When placed in culture, the isolated mononuclear trophoblasts differentiated into syncytial counterparts within 12-72 h after plating. Trophoblasts were incubated with varying concentrations (60-300 microg/ml) of glycodelin A. Glycodelin A was isolated and purified by chromatographic methods from amnion fluid. Supernatants of the trophoblast cell cultures were assayed for hCG and hPL by immunological methods. The release of hCG is increased in glycodelin A-treated trophoblast cell cultures compared to untreated trophoblast cells. Glycodelin A inhibits the production of hPL in vitro. Differences in Glycodelin A stimulated cells and untreated controls are statistical significant. hCG and hPL are markers for the differentiation process of trophoblast cells to syncytial trophoblasts. The results imply that glycodelin A secreted by decidualised endometrium modulates endocrine function, as well as the differentiation of trophoblasts in culture.     

Endometrial and placental CRH as regulators of human embryo implantation

Epithelial cells of the human endometrium and differentiated endometrial stromal cells express the corticotropin-releasing hormone (CRH) gene. CRH is also produced by human placental cytotrophoblast. Endometrial and placental CRH are under the endocrine control of gonadal steroids as well as under autocrine/paracrine regulation by prostanoids and interleukins. Human endometrium, myometrium and placenta express the relevant receptors. Human trophoblast and decidualized endometrial cells also express Fas ligand (FasL), a pro-apoptotic molecule. These findings suggest that intra-uterine CRH may participate in local inflammatory phenomena associated with blastocyst implantation, while FasL may assist with maternal immune tolerance to the semi-allograft embryo. A nonpeptidic CRH receptor type 1 (CRH-R1)-specific antagonist decreased the expression of FasL by human trophoblasts, suggesting that CRH regulates the pro-apoptotic potential of these cells in an auto-paracrine fashion. Invasive trophoblasts promoted apoptosis of activated Fas-expressing human T lymphocytes, an effect potentiated by CRH and inhibited by the CRH antagonist. Female rats treated with the CRH antagonist in the first 6 days of gestation had a dose-dependent decrease of endometrial implantation sites and live embryos as well as markedly diminished endometrial FasL expression. However, embryos of mothers lacking T cells (nude rats) and embryos of syngeneic matings were not rejected when mothers were treated with antalarmin, suggesting that the effect of antalarmin on embryonic implantation is not due to a nonspecific toxicity of this compound but a specific effect on T cells. Our data suggest important physiological roles of endometrial and placental CRH in the regulation of blastocyst implantation and early maternal tolerance.     

Expression and regulation of vascular endothelial growth factor in a first trimester trophoblast cell line

Embryo implantation and development are critically dependent upon the spatial and temporal regulation of angiogenesis and localized vascular permeability. A key mediator of these effects is the endothelial cell mitogen vascular endothelial growth factor (VEGF). VEGF has been shown to promote endometrial vascular permeability, fetal vasculogenesis and placental, fetal and maternal angiogenesis. However, the mechanism through which this regulation occurs in the placenta is poorly understood. This study was conducted to determine if the pro-angiogenic cytokines, TNF-alpha and TGF-beta1, affect VEGF expression in human first trimester trophoblasts. Culture of a first trimester trophoblast cell line (HTR-8/SVneo), in the presence of either TNF-alpha or TGF-beta1, resulted in the expression of significant levels of VEGF in culture. The trophoblast cell line also showed a time-dependent and a dose-dependent increase in VEGF mRNA levels when cultured in the presence of either TNF-alpha or TGF-beta1. These results suggest that both TNF-alpha and TGF-beta1 may regulate the production of VEGF in early gestational trophoblasts and may therefore serve to modulate placental vascular permeability and angiogenesis that are necessary for embryo implantation and placentation.     

Role of nuclear receptors and their ligands in human trophoblast invasion

Human implantation involves a major invasion of the uterine wall and complete remodelling of uterine arteries by extravillous cytotrophoblasts (EVCT). Abnormality in these early steps of placental development leads to poor placentation, fetal growth defects and is often associated with preeclampsia, a major and frequent complication of human pregnancy. To study the mechanisms that control trophoblast invasion during early placental development and provide new insight in the understanding of preeclampsia, we have developed in vitro models of human invasive trophoblasts. We have shown that activation of the ligand-activated nuclear receptor PPARgamma with synthetic (rosiglitazone) or natural (15deoxyPGJ(2)) agonists inhibits the trophoblastic invasion process. Analysis of PPARgamma-target genes revealed that placental growth hormone and the protease PAPP-A might be involved in the PPARgamma-mediated effect in an autocrine manner. We next investigated PPARgamma ligands at the materno-fetal interface and have shown that oxidized LDLs are present in EVCT in situ and decrease trophoblast invasion in vitro. Analysis of oxidized LDLs revealed that they contain potent PPARgamma agonists such as eicosanoids and also high levels of oxysterols, which are specific ligands for the liver X receptor (LXR). The isoform beta of LXR was found in EVCT in situ, and activation of LXRbeta with synthetic or natural ligands inhibits trophoblast invasion in vitro. Together, our data underscore a major role for PPARgamma and LXRbeta in the control of human trophoblast invasion and suggest that excess ligands such as oxidized LDLs at the implantation site might contribute to the development of preeclampsia.     

Expression and function of placenta growth factor: implications for abnormal placentation

OBJECTIVE: Essential requirements for successful gestation include the coordinated growth and differentiation of the placenta and the development of a functional placental vasculature. However, relatively little is known about factors that are responsible for regulating these functions. One angiogenic growth factor that might be involved in regulating both vascular endothelial cell and trophoblast function is placental growth factor (PGF). METHODS: Current published reports were surveyed and our own work was reviewed to highlight the expression, function, and potential significance of PGF at the human maternal-fetal interface. RESULTS: PGF is highly expressed in trophoblasts during normal pregnancy, and its expression is significantly decreased in preeclampsia, an obstetric complication presumed to be associated with placental bed hypoxia and ischemia. In agreement with this, in vitro trophoblast expression of PGF can be down-regulated by low oxygen tension. The cognate receptor for PGF, fms-like tyrosine kinase receptor, is expressed on trophoblasts as well as vascular endothelial cells, suggesting that it has autocrine and paracrine functions. Accordingly, PGF can regulate proliferation in first trimester trophoblasts, apoptosis in term trophoblasts, and it can directly or indirectly regulate vascular growth, maturation, and permeability. CONCLUSIONS: Many obstetric complications, most notably preeclampsia, are associated with aberrant trophoblast function and inadequate or dysfunctional vasculature within the developing placenta. The ability of PGF to influence trophoblast and vascular endothelial cells provides clear impetus for further studies to investigate the biological and clinical significance of PGF in normal and abnormal human pregnancies.     

Punicalagin promotes human villous trophoblast differentiation

Poor differentiation of trophoblasts is associated with placental dysfunction, predisposing women to multiple pregnancy disorders. Punicalagin, a prominent ellagitannin in pomegranate juice has been shown to exert anti-apoptosis and anti-oxidative effects in human trophoblasts. We hypothesized that punicalagin modulates trophoblast differentiation. We found that punicalagin-treated primary trophoblast showed reduced E-cadherin, higher Syncytin 1, more β−hCG, and increased GCM1, an upstream regulator of β−hCG. Punicalagin exposure of villous explants enhanced the number of cytotrophoblasts expressing the proliferation marker Ki67. We conclude that punicalagin enhances trophoblast differentiation and speculate that punicalagin might be used therapeutically in pregnancies at risk for placental dysfunction.     

Oxysterols inhibit differentiation and fusion of term primary trophoblasts by activating liver X receptors

Oxygenated cholesterol metabolites known as oxysterols display potent biological activities ranging from regulation of lipid homeostasis to cytotoxicity. Oxysterols have previously been shown to inhibit the invasion of first trimester trophoblasts, an effect which involves activation of the nuclear liver X receptors (LXRs). In the present study, we investigated the effects of several oxysterols on syncytialisation (differentiation and fusion) in term placental trophoblasts. Treatment of cultured term primary trophoblast cells with oxysterols [25-hydroxycholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol] and the synthetic LXR agonist T0901317 at non-toxic doses decreased expression of GCM-1 and HERV-W mRNA and reduced hCG secretion and placental alkaline phosphatase activity, indicative of diminished trophoblast differentiation. Furthermore, treatment with these compounds also decreased cell fusion measured by E-cadherin immunostaining and quantification of syncytialised nuclei. Treatment with an LXR antagonist (geranylgeranyl diphosphate) abrogated the inhibitory effects of oxysterols and T0901317 on trophoblast syncytialisation indicating that these effects are mediated by LXR. These findings suggest that oxysterols impair differentiation and fusion of term trophoblast cells via an LXR-dependent mechanism.     

Complex interactions between hypoxia inducible factors, insulin-like growth factor-II and oxygen in early murine trophoblasts

The human first trimester placenta experiences a low oxygen environment. The hypoxia inducible factors (HIFs) mediate the response to low oxygen, inducing genes such as insulin-like growth factor (IGF)-II. Interestingly, IGF-II has been shown to promote placental growth and function. Currently, the interaction between oxygen, IGF-II and HIFs in the regulation of trophoblast behaviour are unclear. Murine implantation sites from days 5.5-10.5 were collected for immunohistochemical analyses. Use of the hypoxia marker pimonidazole indicated that the early mouse implantation site is exposed to low oxygen levels similar to those seen in the early human placenta. HIF-1alpha protein immunostaining was also observed in the implantation site. Culturing murine ectoplacental cones in decreasing oxygen concentrations (20%, 5% and 1% O(2)), either with or without the addition of IGF-II, induced complex responses by trophoblasts in terms of their migration and differentiation. Following 3 days exposure to low oxygen there was reduced EPC outgrowth, reduced Igf2 and increased Tpbp mRNA levels, suggesting commitment to the spongiotrophoblast lineage. In addition, Hif-1alpha mRNA levels were decreased, whilst Hif-2alpha mRNA was unchanged. This decrease in Hif-1alpha may be due to the observed increase in antisense (as) Hif-1alpha mRNA levels in 1% cultures. Furthermore, expression of Hif-2alpha and the HIF target genes: asHif-1alpha, Vegf and Slc2a1 were reduced under low oxygen with the addition of IGF-II. In conclusion, Hif-1alpha and Hif-2alpha are differentially regulated by oxygen and IGF-II in cultured trophoblast cells and asHif-1alpha may mediate the response to prolonged hypoxia in murine trophoblasts.     

Human placentation from nidation to 5 weeks of gestation. Part II: Tools to model the crucial first days

Human pregnancy is unusual with respect to monthly spontaneous decidualisation as well as the degree of placental invasion and interaction with the decidualised endometrial stroma. This review covers in?vivo animal models and in?vitro cell culture models that have been used to study the earliest stages of human implantation and placentation from nidation to 5 weeks of gestation. The field has expanded rapidly in recent years due to the generation of human embryonic stem cell lines and the ability of some scientists to culture human blastocysts. These models have enabled researchers to begin to elucidate the interactions involved in human blastocyst apposition, adhesion and implantation. However, we still understand very little about the differentiation processes involved in the formation of the placenta. Continued improvements to current models, including the potential isolation of a human trophoblast stem cell, will significantly enhance our ability to define the molecular and structural events occurring during human implantation and early placental development.     

The contribution of apoptosis-inducing factor (AIF) to villous trophoblast differentiation

Apoptosis is postulated to be a delayed but important part of the differentiation of placental villous cytotrophoblasts (CT) into functional syncytiotrophoblast (ST). This hypothesis is based on the observation that the externalization of phosphatidylserine and the activation of caspase 8 are required for trophoblast differentiation. In contradiction to this hypothesis we have previously found that differentiation occurs in the presence of both broad spectrum and caspase 8 specific inhibitors. Apoptosis-inducing factor (AIF) is a mitochondria-associated protein which is known to translocate to the nucleus and induce caspase-independent nuclear condensation, phosphatidylserine externalization and cell death. Thus AIF nuclear translocation may result in the apoptotic-like features associated with trophoblast differentiation and may be an obligatory event for differentiation to proceed. AIF translocation was assessed in isolated primary trophoblasts by optical section microscopy of antibody stained cells. We found AIF to be strongly expressed in the villous trophoblast and that small amounts of AIF were localized to the nucleus of the cells. Significantly, inhibitors of AIF translocation (calpeptin and zFA-fmk) blocked translocation but not differentiation of the cells. We conclude that AIF translocation is not involved in CT differentiation in isolated cell culture.     

Involvement of EAT/mcl-1, a bcl-2 related gene, in the apoptotic mechanisms underlying human placental development and maintenance

The EAT/mcl-1 gene was isolated during the early differentiation of a retinoic acid-induced human embryonal carcinoma cell line to the trophectoderm lineage. EAT/mcl-1, a bcl-2 related gene, is involved in the genetic pathway of apoptosis; this suggests a role for apoptosis and the involvement of this gene in early placental development. In the current investigation, we analysed expression of EAT/mcl-1 at the mRNA level by Northern blot analysis and in situ hybridization, as well as at the protein level, by immunoblot analysis and immunohistochemistry. Our results identified constant expression of this gene in the placenta throughout pregnancy as well as a shift in its localization from the cytotrophoblast in the first trimester to the syncytiotrophoblast in the third trimester. In addition, there was an inverse correlation between EAT/mcl-1 expression and TaT-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) reactivity in trophoblasts in the first trimester. These results suggest a role for EAT/mcl-1 in both early placental development in regulating trophoblast differentiation as well as a role for this gene in placental maintenance in regulating the process of trophoblast turnover.     

Impaired function of trophoblast cells derived from translocated hESCs may explain pregnancy loss in women with balanced translocation (11;22)

Purpose

The aim of the study was to study whether the trophoblasts carrying unbalanced translocation 11,22 [t(11;12)] display abnormal expression of trophoblastic genes and impaired functional properties that may explain implantation failure.

Methods

t(11;22) hESCs and control hESCs were differentiated in vitro into trophoblast cells in the presence of BMP4, and trophoblast vesicles (TBVs) were created in suspension. The expression pattern of extravillous trophoblast (EVT) genes was compared between translocated and control TBVs. The functional properties of the TBVs were evaluated by their attachment to endometrium cells (ECC1) and invasion through trans-well inserts.

Results

TBVs derived from control hESCs expressed EVT genes from functioning trophoblast cells. In contrast, TBVs differentiated from the translocated hESC line displayed impaired expression of EVT genes. Moreover, the number of TBVs that were attached to endometrium cells was significantly lower compared to the controls. Correspondingly, invasiveness of trophoblast-differentiated translocated cells was also significantly lower than that of the control cells.

Conclusions

These results may explain the reason for implantation failure in couple carriers of t(11;22). They also demonstrate that translocated hESCs comprise a valuable in vitro human model for studying the mechanisms underlying implantation failure.

     

Comparison of cell cycle regulatory gene mRNA in three different types of human trophoblasts and effect of transforming growth factor

AIM: Identifying the factors responsible for reducing the proliferation, syncytialization, and invasiveness of trophoblast tissues, as seen with preeclampsia, intrauterine growth restriction, and spontaneous miscarriage, is a current challenge in reproductive biology. These factors, transforming growth factor (TGF)-beta as an example, can work by altering trophoblast differentiation or proliferation. We therefore investigated and compared specific markers of trophoblast proliferation and differentiation in three commonly used trophoblast tissue cell models, and also investigated the influence of TGF-beta on these markers. METHODS: In this study, we isolated human trophoblasts from first trimester and term placentas, and additionally used human choriocarcinoma cells (JEG-3). Baseline values of human chorionic gonadotropin (hCG) secretion and relative mRNA levels of cell cycle regulators (cyclin E, p21, p27, and p57) were investigated for each cell type. We also investigated the influence of TGF-beta on these parameters. RESULTS: Quantitative and longitudinal production of hCG differed between the three cell types. Significantly different amounts of cyclin E, p21, p27, and p57 mRNA were demonstrated within each cell type, as well as between all the cell types, throughout the culture time period. Each trophoblast type demonstrated a reduction of hCG secretion in response to TGF-beta. TGF-beta did not show a consistent effect on the cell cycle mRNA of any of the cell types. CONCLUSION: We were able to characterize and compare the differential production of hCG, as well as the differential expression of cell cycle-associated mRNA of early trophoblasts, term trophoblasts, and choriocarcinoma cells. The production of hCG was altered by TGF-beta, although mRNA levels were not markedly altered by TGF-beta.