This review summarises current knowledge about the specification, commitment and maintenance of the trophoblast lineage in mice and cattle. Results from gene expression studies, in vivo loss-of-function models and in vitro systems using trophoblast and embryonic stem cells have been assimilated into a model seeking to explain trophoblast ontogeny via gene regulatory networks. While trophoblast differentiation is quite distinct between cattle and mice, as would be expected from their different modes of implantation, recent studies have demonstrated that differences arise much earlier during trophoblast development. Reproduction (2012) 143 231-246
Trophoblast stem cells (TSC) are the precursors of the differentiated cells of the placenta. In the mouse, TSC can be derived from outgrowths of either blastocyst polar trophectoderm (TE) or extraembryonic ectoderm (ExE), which originates from polar TE after implantation. The mouse TSC niche appears to be located within the ExE adjacent to the epiblast, on which it depends for essential growth factors, but whether this cellular architecture is the same in other species remains to be determined. Mouse TSC self-renewal can be sustained by culture on mitotically inactivated feeder cells, which provide one or more factors related to the NODAL pathway, and a medium supplemented with FGF4, heparin, and fetal bovine serum. Repression of the gene network that maintains pluripotency and emergence of the transcription factor pathways that specify a trophoblast (TR) fate enables TSC derivation in vitro and placental formation in vivo. Disrupting the pluripotent network of embryonic stem cells (ESC) causes them to default to a TR ground state. Pluripotent cells that have acquired sublethal chromosomal alterations may be sequestered into TR for similar reasons. The transition from ESC to TSC, which appears to be unidirectional, reveals important aspects of initial fate decisions in mice. TSC have yet to be derived from domestic species in which remarkable TR growth precedes embryogenesis. Recent derivation of TSC from blastocysts of the rhesus monkey suggests that isolation of the human equivalents may be possible and will reveal the extent to which mechanisms uncovered by using animal models are true in our own species.
At the time of implantation, the trophoblast cells of the embryo adhere and then invade into the maternal endometrium and eventually establish placentation. The endometrium at the same time undergoes decidualization, which is essential for successful pregnancy. While the NK cells of the decidua have been implicated to play a key role in trophoblast invasion, few evidence are now available to demonstrate a pro‐invasive property of decidual stromal cells. Secretions from decidualized endometrial stromal cells promote invasion of primary trophoblasts and model cell lines by activating proteases and altering expression of adhesion‐related molecules. The decidual secretions contain high amounts of pro‐invasive factors that include IL‐1β, IL‐5, IL‐6, IL‐7, IL‐8, IL‐9, IL‐13, IL‐15, Eotaxin CCL11, IP‐10 and RANTES, and anti‐invasive factors IL‐10, IL‐12 and VEGF. It appears that these decidual factors promote invasion by regulating the protease pathways and integrin expression utilizing the STAT pathways in the trophoblast cells. At the same time the decidua also seem to secrete some anti‐invasive factors that are antagonist to the matrix metalloproteinases and/or are activators of tissue inhibitors of metalloproteinases. This might be essential to neutralize the effects of the invasion‐promoting factors and restrain overinvasion. It is tempting to propose that during the course of pregnancy, the decidua must balance the production of these pro and anti‐invasive molecules and such harmonizing production would allow a timely and regulated invasion.
Abstract Oxygen is necessary for life yet too much or too little oxygen is toxic to cells. The oxygen tension in the maternal plasma bathing placental villi is <20 mm Hg until 10–12 weeks’ gestation, rising to 40–80 mm Hg and remaining in this range throughout the second and third trimesters. Maldevelopment of the maternal spiral arteries in the first trimester predisposes to placental dysfunction and sub-optimal pregnancy outcomes in the second half of pregnancy. Although low oxygen at the site of early placental development is the norm, controversy is intense when investigators interpret how defective transformation of spiral arteries leads to placental dysfunction during the second and third trimesters. Moreover, debate rages as to what oxygen concentrations should be considered normal and abnormal for use in vitro to model villous responses in vivo. The placenta may be injured in the second half of pregnancy by hypoxia, but recent evidence shows that ischemia with reoxygenation and mechanical damage due to high flow contributes to the placental dysfunction of diverse pregnancy disorders. We overview normal and pathologic development of the placenta, consider variables that influence experiments in vitro , and discuss the hotly debated question of what in vitro oxygen percentage reflects the normal and abnormal oxygen concentrations that occur in vivo . We then describe our studies that show cultured villous trophoblasts undergo apoptosis and autophagy with phenotype-related differences in response to hypoxia.
Trophoblast cells play an essential role in the interactions between the fetus and mother. Mouse trophoblast stem (TS) cells have been derived and used as the best model for molecular and functional analysis of mouse trophoblast lineages, but attempts to derive human TS cells have so far been unsuccessful. Here we show that activation of Wingless/Integrated (Wnt) and EGF and inhibition of TGF-β, histone deacetylase (HDAC), and Rho-associated protein kinase (ROCK) enable long-term culture of human villous cytotrophoblast (CT) cells. The resulting cell lines have the capacity to give rise to the three major trophoblast lineages, which show transcriptomes similar to those of the corresponding primary trophoblast cells. Importantly, equivalent cell lines can be derived from human blastocysts. Our data strongly suggest that the CT- and blastocyst-derived cell lines are human TS cells, which will provide a powerful tool to study human trophoblast development and function. Trophoblast cells are specialized cells in the placenta that mediate the interactions between the fetus and mother. Okae et al. report the derivation of human trophoblast stem cells from blastocysts and early placentas, which will provide a powerful tool to study human placental development and function.
Extravillous trophoblasts (EVTs) migrate into uterine decidua and induce vascular smooth muscle cell (VSMC) loss through mechanisms thought to involve migration and apoptosis, achieving complete spiral artery remodeling. Long noncoding RNA maternally expressed gene 3 (MEG3) can regulate diverse cellular processes, such as proliferation and migration, and has been discovered highly expressed in human placenta tissues. However, little is known about the role of MEG3 in modulating EVT functions and EVT‐induced VSMC loss. In this study, we first examined the location of MEG3 in human first‐trimester placenta by in situ hybridization. Then, exogenous upregulation of MEG3 in HTR‐8/SVneo cells was performed to investigate the effects of MEG3 on EVT motility and EVT capacity to displace VSMCs. Meanwhile, the molecules mediating EVT‐induced VSMC loss, such as tumor necrosis factor‐α (TNF‐α), Fas ligand (FasL), and tumor necrosis factor‐α‐related apoptosis‐inducing ligand (TRAIL) were detected at transcriptional and translational levels. Finally, VSMCs were cocultured with MEG3‐upregulated HTR‐8/SVneo to explore the role of MEG3 on EVT‐mediated VSMC migration and apoptosis. Results showed that MEG3 was expressed in trophoblasts in placental villi and decidua, and MEG3 enhancement inhibited HTR‐8/SVneo migration and invasion. Meanwhile, the displacement of VSMCs by HTR‐8/SVneo and the expression of TNF‐α, FasL and TRAIL in HTR‐8/SVneo were reduced following MEG3 overexpression in HTR‐8/SVneo. Furthermore, HTR‐8/SVneo with MEG3 upregulation impaired VSMC migration and apoptosis. The PI3K/Akt pathway, which is possibly downstream, was inactivated in MEG3‐upregulated HTR‐8/SVneo. These findings suggest that MEG3 might be a negative regulator of spiral artery remodeling via suppressing EVT invasion and EVT‐mediated VSMC loss. Maternally expressed gene 3 (MEG3) suppressed trophoblast‐mediated vascular smooth muscle cell (VSMC) migration and apoptosis, which might be associated with reduced expression of tumor necrosis factor‐α (TNF‐α), Fas ligand (FasL), and tumor necrosis factor‐α‐related apoptosis‐inducing ligand (TRAIL) in trophoblast with MEG3 enhancement.
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 Placental stress has been implicated in the pathophysiology of complications of pregnancy, including growth restriction and pre-eclampsia. Initially, attention focused on oxidative stress, but recently mitochondrial and endoplasmic reticulum stress have been identified. Complex molecular interactions exist among these different forms of stress, making it unlikely that any occurs in isolation. In part, this is due to close physiological connections between the two organelles principally involved, mitochondria and the endoplasmic reticulum (ER), mediated through Ca2+ signalling. Here, we review the involvement of the mitochondria-ER unit in the generation of stress within the trophoblast, and consider consequences for obstetric outcome. Mild stress may induce adaptive responses, including upregulation of antioxidant defences and autophagy, while moderate levels may affect stem cell behaviour and reduce cell proliferation, contributing to the growth-restricted phenotype. High levels of stress can stimulate release of pro-inflammatory cytokines and anti-angiogenic factors, increasing the risk of pre-eclampsia. In addition, chronic stress may promote senescence of the trophoblast, which in other cell types leads to a pro-inflammatory senescence-associated secretory phenotype. Evidence from rodents suggests that a degree of trophoblastic stress develops with increasing gestational age in normal pregnancies. The increase in maternal concentrations of soluble fms-like tyrosine kinase-1 (sFlt-1) and reduction in placental growth factor (PlGF) suggest the same may occur in the human, starting around 30 weeks of pregnancy. Placental malperfusion, or co-existing maternal conditions, such as diabetes, will exacerbate that stress. Amelioration of trophoblastic stress should remain a research priority, but will be difficult due to the complexity of the molecular pathways involved.
Abstract At the tips of anchoring villi, cytotrophoblast (CTB) proliferation leads to a process of multilayering in which cells lose their attachment to the villous basement membrane and develop into columns, within which they adhere to one another using desmosomes, with associated intermediate filament bundles. Non-desmosomal cadherins, tight junction proteins and other adhesion molecules are also present, suggesting that actin-associated adhesions contribute to placental anchorage. In the distal columns, cell–cell interactions diminish, cells upregulate β1 integrins and bind to a provisional fibrinoid extracellular matrix, eventually detaching to migrate into the decidual stroma and myometrium, where interstitial and endovascular extravillous trophoblast (EVT) populations show distinct repertoires of adhesion molecules.