Modulation of trophoblast stem cell and giant cell phenotypes: analyses using the Rcho-1 cell model

Trophoblast giant cells are located at the maternal-embryonic interface and have fundamental roles in the invasive and endocrine phenotypes of the rodent placenta. In this report, we describe the experimental modulation of trophoblast stem cell and trophoblast giant cell phenotypes using the Rcho-1 trophoblast cell model. Rcho-1 trophoblast cells can be manipulated to proliferate or differentiate into trophoblast giant cells. Differentiated Rcho-1 trophoblast cells are invasive and possess an endocrine phenotype, including the production of members of the prolactin (PRL) family. Dimethyl sulfoxide (DMSO), a known differentiation-inducing agent, was found to possess profound effects on the in vitro development of trophoblast cells. Exposure to DMSO, at non-toxic concentrations, inhibited trophoblast giant cell differentiation in a dose-dependent manner. These concentrations of DMSO did not significantly affect trophoblast cell proliferation or survival. Trophoblast cells exposed to DMSO exhibited an altered morphology; they were clustered in tightly packed colonies. Trophoblast giant cell formation was disrupted, as was the expression of members of the PRL gene family. The effects of DMSO were reversible. Removal of DMSO resulted in the formation of trophoblast giant cells and expression of the PRL gene family. The phenotype of the DMSO-treated cells was further determined by examining the expression of a battery of genes characteristic of trophoblast stem cells and differentiated trophoblast cell lineages. DMSO treatment had a striking stimulatory effect on eomesodermin expression and a reciprocal inhibitory effect on Hand1 expression. In summary, DMSO reversibly inhibits trophoblast differentiation and induces a quiescent state, which mimics some but not all aspects of the trophoblast stem cell phenotype.     

Control of trophoblast cell differentiation: Lessons from the genetics of early pregnancy loss and trophoblast neoplasia

Trophoblast cell differentiation is crucial to the morphogenesis of the placenta and thus the establishment of pregnancy and the growth and development of the embryo/fetus. In the present review, we discuss current evidence for the existence of regulatory genes crucial to trophoblast cell differentiation and placental morphogenesis. The elucidation of regulatory pathways controlling normal differentiation of trophoblast cells will facilitate the identification of sensitive junctures in the regulatory pathways leading to various developmental disorders, including those associated with the initiation of pregnancy, fetal growth retardation and gestational trophoblast disease.     

Alteration of inhibitory and activating NK cell receptor expression on NK cells in HIV-infected Chinese

Natural killer (NK) cell function, based on the expression of activating and inhibitory natural killer receptors (NKRs), may become abnormal during human immunodeficiency virus (HIV) infection. In this study, we investigated changes in receptor expression with individual and combinational analysis on NK cell subsets in HIV-infected Chinese. The results showed that natural killer group 2 member D (NKG2D) expression on total NK cells decreased significantly in HIV infection, while the expressions of natural killer group 2 member A (NKG2A) and killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail 1 (KIR3DL1) on total NK cells were not significantly different between any of the groups including HIV-positive treatment-naïve group, AIDS treatment-naïve group, HAART-treatment AIDS group and HIV-negative control group. Individual analysis of NKG2A⁺ and KIR3DL1⁺ cells revealed no significant differences in expression in any NK cell subsets between any of the groups, but the combinational analysis of NKG2D⁻NKG2A⁺, and NKG2D⁻KIR3DL1⁺ on the NK CD56^dim cell subset in the AIDS group were increased compared to the HIV-negative control group. On the contrary, NKG2D⁻NKG2A⁺ expression on the CD56^bright subset decreased in the AIDS group compared to the control group. Highly active antiretroviral therapy (HAART) treatment almost completely restored the levels of these receptor expressions. The results indicate that the distinct alteration of activating and inhibitory NKR expression on NK cells and its subsets occurred during HIV progression. Moreover, the imbalanced change of activating and inhibitory NKRs on NK cells and its subsets may explain the impaired NK cell immunity in HIV infected individuals.     

Maternal hypoxia activates endovascular trophoblast cell invasion

Oxygen is a critical regulator of placentation. Early placental development occurs in a predominantly low oxygen environment and is, at least partially, under the control of hypoxia signaling pathways. In the present study, in vivo hypobaric hypoxia was used as an experimental tool to delineate hypoxia-sensitive events during placentation. Pregnant rats were exposed to the equivalent of 11% oxygen between days 6.5 and 13.5 of gestation. Pair-fed pregnant animals exposed to ambient conditions were included as a control group. Uterine mesometrial blood vessels in the hypoxia-exposed animals were greatly expanded and some contained large cuboidal cells that were positive for cytokeratin and other markers characteristic of invasive trophoblast cells. Unlike later in gestation, the route of trophoblast cell invasion in the hypoxia-exposed animals was restricted to endovascular, with no interstitial invasion observed. Hypoxia-activated endovascular trophoblast invasion required exposure to hypoxia from gestation day 8.5 to day 9.5. Activation of the invasive trophoblast lineage was also associated with an enlargement of the junctional zone of the chorioallantoic placenta, a source of invasive trophoblast cell progenitors. In summary, maternal hypoxia during early stages of placentation activates the invasive endovascular trophoblast cell lineage and promotes uterine vascular remodeling.     

Irisin induces trophoblast differentiation via AMPK activation in the human placenta

Irisin, an adipokine, regulates differentiation and phenotype in various cell types including myocytes, adipocytes, and osteoblasts. Circulating irisin concentration increases throughout human pregnancy. In pregnancy disorders such as preeclampsia and gestational diabetes mellitus, circulating irisin levels are reduced compared to healthy controls. To date, there are no data on the role and molecular function of irisin in the human placenta or its contribution to pathophysiology. Aberrant trophoblast differentiation is involved in the pathophysiology of preeclampsia. The current study aimed to assess the molecular effects of irisin on trophoblast differentiation and function. First-trimester placental explants were cultured and treated with low (10 nM) and high (50 nM) physiological doses of irisin. Treatment with irisin dose-dependently increased both in vitro placental outgrowth (on Matrigel™) and trophoblast cell-cell fusion. Adenosine monophosphate-activated protein kinase (AMPK) signaling, an important regulator of cellular energy homeostasis that is involved in trophoblast differentiation and pathology, was subsequently investigated. Here, irisin exposure induced placental AMPK activation. To determine the effects of irisin on trophoblast differentiation, two trophoblast-like cell lines, HTR-8/SVneo and BeWo, were treated with irisin and/or a specific AMPK inhibitor (Compound C). Irisin-induced AMPK phosphorylation in HTR-8/SVneo cells. Additionally, as part of the differentiation process, integrin switching from α6 to α1 occurred as well as increased invasiveness. Overall, irisin promoted differentiation in villous and extravillous cell-based models via AMPK pathway activation. These findings provide evidence that exposure to irisin promotes differentiation and improves trophoblast functions in the human placenta that are affected in abnormal placentation.     

Interplay between neutrophils and trophoblast cells conditions trophoblast function and triggers vascular transformation signals

Normal placentation entails highly regulated interactions of maternal leukocytes with vascular and trophoblast cells to favor vascular transformation. Neutrophil activation and neutrophil extracellular trap (NET) formation associate with poor placentation and severe pregnancy complications. To deepen into the mechanisms of trophoblast–neutrophil interaction, we explored the effects of NETs on trophoblast cell function and, conversely, whether trophoblast cell-derived factors condition neutrophils to favor angiogenesis and anti-inflammatory signals required for fetal growth. NETs isolated from activated neutrophils hindered trophoblast cell migration. Trophoblast conditioned media prevented the effect as well as the vasoactive intestinal peptide (VIP) known to regulate trophoblast and neutrophil function. On the other hand, factors released by trophoblast cells and VIP shaped neutrophils to a proangiogenic profile with increased vascular endothelial growth factor synthesis and increased capacity to promote vascular transformation. Results presented here provide novel clues to reconstruct the interaction of trophoblast cells and neutrophils in vivo during placentation in humans.     

Tumour‐experienced T cells promote NK cell activity through trogocytosis of NKG2D and NKp46 ligands

Natural killer (NK) cells trigger cytotoxicity and interferon (IFN)‐γ secretion on engagement of the natural‐killer group (NKG)2D receptor or members of the natural cytotoxicity receptor (NCR) family, such as NKp46, by ligands expressed on tumour cells. However, it remains unknown whether T cells can regulate NK cell‐mediated anti‐tumour responses. Here, we investigated the early events occurring during T cell–tumour cell interactions, and their impact on NK cell functions. We observed that on co‐culture with some melanomas, activated CD4⁺ T cells promoted degranulation, and NKG2D‐ and NKp46‐dependent IFN‐γ secretion by NK cells, probably owing to the capture of NKG2D and NKp46 ligands from the tumour‐cell surface (trogocytosis). This effect was observed in CD4⁺, CD8⁺ and resting T cells, which showed substantial amounts of cell surface major histocompatibility complex class I chain‐related protein A on co‐culture with tumour cells. Our findings identify a new, so far, unrecognized mechanism by which effector T cells support NK cell function through the capture of specific tumour ligands with profound implications at the crossroad of innate and adaptive immunity.     

Impaired autophagy by soluble endoglin,under physiological hypoxia in early pregnant period,is involved in poor placentation in preeclampsia

In early pregnancy, trophoblasts and the fetus experience hypoxic and low-nutrient conditions; nevertheless, trophoblasts invade the uterine myometrium up to one third of its depth and migrate along the lumina of spiral arterioles, replacing the maternal endothelial lining. Here, we showed that autophagy, an intracellular bulk degradation system, occurred in extravillous trophoblast (EVT) cells under hypoxia in vitro and in vivo. An enhancement of autophagy was observed in EVTs in early placental tissues, which suffer from physiological hypoxia. The invasion and vascular remodeling under hypoxia were significantly reduced in autophagy-deficient EVT cells compared with wild-type EVT cells. Interestingly, soluble endoglin (sENG), which increased in sera in preeclamptic cases, suppressed EVT invasion by inhibiting autophagy. The sENG-inhibited EVT invasion was recovered by TGFB1 treatment in a dose-dependent manner. A high dose of sENG inhibited the vascular construction by EVT cells and human umbilical vein endothelial cells (HUVECs), meanwhile a low dose of sENG inhibited the replacement of HUVECs by EVT cells. A protein selectively degraded by autophagy, SQSTM1, accumulated in EVT cells in preeclamptic placental biopsy samples showing impaired autophagy. This is the first report showing that impaired autophagy in EVT contributes to the pathophysiology of preeclampsia.     

Dynamic change of Adamalysin 19 (ADAM19) in human placentas and its effects on cell invasion and adhesion in human trophoblastic cells

Human ADAM19 is a recently identified member of the ADAM family. It is highly expressed in human placentas, but its dynamic change and function at the human feto-maternal interface during placenta-tion remain to be elucidated. In this present study, the spatial and temporal expression and cellular localization of ADAM19 in normal human placentas were first demonstrated, and the effects of ADAM19 on trophoblast cell adhesion and invasion were further investigated by using a human choriocarcinoma cell line (JEG-3) as an in vitro model. The data demonstrated that ADAM19 was widely distributed in villous cytotrophoblast cells, syncytiotrophoblast cells, column trophoblasts, and villous capillary endothelial cells during early pregnancy. The mRNA and protein level of ADAM19 in placentas was high at gestational weeks 8—9, but diminished significantly at mid- and term pregnancy. In JEG-3 cells, the overexpression of ADAM19 led to diminished cell invasion, as well as increases in cell adhesiveness and the expression of E-cadherin, with no changes in b-catenin expression observed. These data in-dicate that ADAM19 may participate in the coordinated regulation of human trophoblast cell behaviors during the process of placentation.     

低氧促进神经干细胞向多巴胺能神经元分化

神经干细胞（neural stem cells,NSCs）作为具有多向分化潜能的神经前体细胞,被广泛应用于细胞移植等研究,而低氧不但调节干细胞的体外增殖,在干细胞分化中也具有重要的作用。本文着重探讨了低氧对NSCs分化的调节作用。采用Wistar孕大鼠（E13．5d）,分离胚胎中脑NSCs,加入无血清DMEM／F12培养液（含20ng／mL EGF、20ng／mL bFGF、1％ N2和B27）,3～5d后传代,细胞培养至第三代进行诱导分化,分别在低氧（3％O2）和常氧（20％O2）条件下诱导分化3d,然后在常氧条件下分化成熟5～7d（DMEM／F12含1％FBS、N2和B27）后进行检测。Nestin、NeuN以及TH免疫组织化学鉴定NSCs;流式细胞术分析测定NSCs向TH阳性神经元方向的分化;高效液相色谱测定细胞培养上清液中多巴胺（dopamine,DA）含量。结果显示,分离培养的NSCs均为nestin阳性细胞;低氧可明显促进NSCs向神经元方向的分化;TH阳性神经元比例在常氧和低氧组分别为（10．25±1．03）％和（19．88±1．44）％。NSCs诱导分化7d后,低氧组细胞培养上清液中DA浓度明显增加,约为常氧组的2倍（P〈0．05,n=8）。上述结果表明,3％低氧可促进NSCs向神经元方向,特别是向DA能神经元方向分化。这为NSCs应用于临床治疗帕金森病提供了基础。     

Accurate control of oxygen level in cells during culture on silicone rubber membranes with application to stem cell differentiation

Oxygen level in mammalian cell culture is often controlled by placing culture vessels in humidified incubators with a defined gas phase partial pressure of oxygen (pO_2gas). Because the cells are consuming oxygen supplied by diffusion, a difference between pO_2gas and that experienced by the cells (pO_2cell) arises, which is maximal when cells are cultured in vessels with little or no oxygen permeability. Here, we demonstrate theoretically that highly oxygen‐permeable silicone rubber membranes can be used to control pO_2cell during culture of cells in monolayers and aggregates much more accurately and can achieve more rapid transient response following a disturbance than on polystyrene and fluorinated ethylene‐propylene copolymer membranes. Cell attachment on silicone rubber was achieved by physical adsorption of fibronectin or Matrigel. We use these membranes for the differentiation of mouse embryonic stem cells to cardiomyocytes and compare the results with culture on polystyrene or on silicone rubber on top of polystyrene. The fraction of cells that are cardiomyocyte‐like increases with decreasing pO₂ only when using oxygen‐permeable silicone membrane‐based dishs, which contract on silicone rubber but not polystyrene. The high permeability of silicone rubber results in pO_2cell being equal to pO_2gas at the tissue‐membrane interface. This, together with geometric information from histological sections, facilitates development of a model from which the pO₂ distribution within the resulting aggregates is computed. Silicone rubber membranes have significant advantages over polystyrene in controlling pO_2cell, and these results suggest they are a valuable tool for investigating pO₂ effects in many applications, such as stem cell differentiation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

人胚胎干细胞向滋养层分化的研究进展

哺乳动物胚胎发育产生的第一个细胞系的分离是内细胞团和滋养层的分离,不同哺乳动物之间胚胎干细胞向滋养层细胞分化不同,滋养层细胞对胚胎的植入、促进胚胎在子宫内的生存和生长至关重要.人胚胎干细胞为研究人类胚胎发育及向滋养层分化提供了一个独特的模型.人胚胎干细胞可以在实验室条件下保持无限期稳定的培养,用于最初胚胎和滋养外胚层发生的机制研究.目前人胚胎干细胞分化为滋养层细胞在体外可以通过自发分化、基因敲除、分离EB小体和BMP4诱导等几种途径实现.不同哺乳动物之间胚胎干细胞向滋养层分化机制,主要通过信号通路如BMP4,LIF等以及某些标志基因如OCT4,CDX2,Eomes等的变化调节.人胚胎干细胞向滋养层分化的研究为临床应用提供了一定的基础.