Using a Multi-Stage hESC Model to Characterize BDE-47 Toxicity During Neurogenesis.

Although the ramifications associated with polybrominated diphenyl ethers (PBDEs) exposures during human pregnancy have yet to be determined, increasing evidence in humans and animal models suggests that these compounds cause neurodevelopmental toxicity. Human embryonic stem cells (hESCs) models can be used to study the effects of environmental chemicals throughout the successive stages of neuronal development. Here, using a hESC differentiation model, we investigated the effects of common PBDE congeners (BDE-47 or -99) on the successive stages of early neuronal development. First, we determined the points of vulnerability to PBDEs across 4 stages of in vitro neural development by using assays to assess for cytotoxicity. Differentiated neural progenitors were identified to be more sensitive to PBDEs than their less differentiated counterparts. In follow-up investigations, we observed BDE-47 to inhibit functional processes critical for neurogenesis (eg, proliferation, expansion) in hESC-derived neural precursor cells (NPCs) at sub-lethal concentrations. Finally, to determine the mechanism(s) underlying PBDE-toxicity, we conducted global transcriptomic and methylomic analyses of BDE-47. We identified 589 genes to be differentially expressed due to BDE-47 exposure, including molecules involved in oxidative stress mediation, cell cycle, hormone signaling, steroid metabolism, and neurodevelopmental pathways. In parallel analyses, we identified a broad significant increase in CpG methylation. In summary our results suggest, on a cellular level, PBDEs induce human neurodevelopmental toxicity in a concentration-dependent manner and sensitivity to these compounds is dependent on the developmental stage of exposure. Proposed mRNA and methylomic perturbations may underlie toxicity in early embryonic neuronal populations.

An Investigation of the Single and Combined Phthalate Metabolite Effects on Human Chorionic Gonadotropin Expression in Placental Cells.

BACKGROUND: Observational studies have reported associations between maternal phthalate levels and adverse outcomes at birth and in the health of the child. Effects on placental function have been suggested as a biologic basis for these findings. OBJECTIVE: We evaluated the effects of phthalates on placental function in vitro by measuring relevant candidate genes and proteins. MATERIALS AND METHODS: Human trophoblast progenitor cells were isolated at 7-14 wk of pregnancy (two female and three male concepti), and villous cytotrophoblast cells (vCTBs) were isolated at 15-20 wk (three female and four male concepti). Cells were cultured in vitro with four phthalate metabolites and their combination at concentrations based on levels found previously in the urine of pregnant women: mono-n-butyl (MnBP, 200 nM), monobenzyl (MBzP, 3µM), mono-2-ethylhexyl (MEHP, 700 nM), and monoethyl (MEP, 1.5µM) phthalates. mRNA levels of CGA, CGB, PPARG, CYP19A1, CYP11A1, PTGS2, EREG, and the intracellular ß subunit of human chorionic gonadotropin (hCGß) and peroxisome proliferator activated receptor γ (PPARγ) were measured in the cellular extracts, and protein levels for four forms of secreted hCG were measured in the conditioned media. RESULTS: Previously reported associations between maternal phthalates and placental gene expression were reproduced experimentally: MnBP with CGA, MBzP with CYP11A1, and MEHP with PTGS2. CGB and hCGß were up-regulated by MBzP. In some cases, there were marked, even opposite, differences in response by sex of the cells. There was evidence of agonism in female cells and antagonism in male cells of PPARγ by simultaneous exposure to multiple phthalates. CONCLUSIONS: Concentrations of MnBP, MBzP and MEHP similar to those found in the urine of pregnant women consistently altered hCG and PPARγ expression in primary placental cells. These findings provide evidence for the molecular basis by which phthalates may alter placental function, and they provide a preliminary mechanistic hypothesis for opposite responses by sex. https://doi.org/10.1289/EHP1539.

Human stem cells from single blastomeres reveal pathways of embryonic or trophoblast fate specification.

Mechanisms of initial cell fate decisions differ among species. To gain insights into lineage allocation in humans, we derived ten human embryonic stem cell lines (designated UCSFB1-10) from single blastomeres of four 8-cell embryos and one 12-cell embryo from a single couple. Compared with numerous conventional lines from blastocysts, they had unique gene expression and DNA methylation patterns that were, in part, indicative of trophoblast competence. At a transcriptional level, UCSFB lines from different embryos were often more closely related than those from the same embryo. As predicted by the transcriptomic data, immunolocalization of EOMES, T brachyury, GDF15 and active ß-catenin revealed differential expression among blastomeres of 8- to 10-cell human embryos. The UCSFB lines formed derivatives of the three germ layers and CDX2-positive progeny, from which we derived the first human trophoblast stem cell line. Our data suggest heterogeneity among early-stage blastomeres and that the UCSFB lines have unique properties, indicative of a more immature state than conventional lines.

Human cytomegalovirus infection interferes with the maintenance and differentiation of trophoblast progenitor cells of the human placenta.

UNLABELLED: Human cytomegalovirus (HCMV) is a major cause of birth defects that include severe neurological deficits, hearing and vision loss, and intrauterine growth restriction. Viral infection of the placenta leads to development of avascular villi, edema, and hypoxia associated with symptomatic congenital infection. Studies of primary cytotrophoblasts (CTBs) revealed that HCMV infection impedes terminal stages of differentiation and invasion by various molecular mechanisms. We recently discovered that HCMV arrests earlier stages involving development of human trophoblast progenitor cells (TBPCs), which give rise to the mature cell types of chorionic villi-syncytiotrophoblasts on the surfaces of floating villi and invasive CTBs that remodel the uterine vasculature. Here, we show that viral proteins are present in TBPCs of the chorion in cases of symptomatic congenital infection. In vitro studies revealed that HCMV replicates in continuously self-renewing TBPC lines derived from the chorion and alters expression and subcellular localization of proteins required for cell cycle progression, pluripotency, and early differentiation. In addition, treatment with a human monoclonal antibody to HCMV glycoprotein B rescues differentiation capacity, and thus, TBPCs have potential utility for evaluation of the efficacies of novel antiviral antibodies in protecting and restoring placental development. Our results suggest that HCMV replicates in TBPCs in the chorion in vivo, interfering with the earliest steps in the growth of new villi, contributing to virus transmission and impairing compensatory development. In cases of congenital infection, reduced responsiveness of the placenta to hypoxia limits the transport of substances from maternal blood and contributes to fetal growth restriction. IMPORTANCE: Human cytomegalovirus (HCMV) is a leading cause of birth defects in the United States. Congenital infection can result in permanent neurological defects, mental retardation, hearing loss, visual impairment, and pregnancy complications, including intrauterine growth restriction, preterm delivery, and stillbirth. Currently, there is neither a vaccine nor any approved treatment for congenital HCMV infection during gestation. The molecular mechanisms underlying structural deficiencies in the placenta that undermine fetal development are poorly understood. Here we report that HCMV replicates in trophoblast progenitor cells (TBPCs)-precursors of the mature placental cells, syncytiotrophoblasts and cytotrophoblasts, in chorionic villi-in clinical cases of congenital infection. Virus replication in TBPCs in vitro dysregulates key proteins required for self-renewal and differentiation and inhibits normal division and development into mature placental cells. Our findings provide insights into the underlying molecular mechanisms by which HCMV replication interferes with placental maturation and transport functions.

Establishment of human trophoblast progenitor cell lines from the chorion.

Placental trophoblasts are key determinants of in utero development. Mouse trophoblast (TB) stem cells, which were first derived over a decade ago, are a powerful cell culture model for studying their self-renewal or differentiation. Our attempts to isolate an equivalent population from the trophectoderm of human blastocysts generated colonies that quickly differentiated in vitro. This finding suggested that the human placenta has another progenitor niche. Here, we show that the chorion is one such site. Initially, we immunolocalized pluripotency factors and TB fate determinants in the early gestation placenta, amnion, and chorion. Immunoreactive cells were numerous in the chorion. We isolated these cells and plated them in medium containing fibroblast growth factor which is required for human embryonic stem cell self-renewal, and an inhibitor of activin/nodal signaling. Colonies of polarized cells with a limited lifespan emerged. Trypsin dissociation yielded continuously self-replicating monolayers. Colonies and monolayers formed the two major human TB lineages-multinucleate syncytiotrophoblasts and invasive cytotrophoblasts (CTBs). Transcriptional profiling experiments revealed the factors associated with the self-renewal or differentiation of human chorionic TB progenitor cells (TBPCs). They included imprinted genes, NR2F1/2, HMGA2, and adhesion molecules that were required for TBPC differentiation. Together, the results of these experiments suggested that the chorion is one source of epithelial CTB progenitors. These findings explain why CTBs of fully formed chorionic villi have a modest mitotic index and identify the chorionic mesoderm as a niche for TBPCs that support placental growth.

Human embryonic stem cells as a model system for studying the effects of smoke exposure on the embryo.

Human embryonic stem cells (hESCs) share many characteristics including pluripotency with cells of the early embryo and so are potentially useful tools for studying the harmful effects of xenobiotics during early development. Here, we used hESCs as a model system to test the effects of nicotine on the pluripotent population of cells that forms the whole body. Specifically, we exposed hESCs (H7 and H9) to various concentrations of nicotine ranging from 0.1 to 6microM. We evaluated the effects in terms of cell adhesion, integrin expression, hESC colony morphology, markers of pluripotency and survival. The results revealed a significant negative impact of nicotine in the dose range between 1.8 and 3.7microM on all the endpoints analyzed. The observed effects were reversed by the addition of the nicotine antagonist d-tubocurarine, suggesting that the effects are receptor mediated. Together these results offer new explanations in terms of embryo toxicity for the large negative impact of cigarette smoke exposure on a woman's reproductive capacity.

Nuclear FAK promotes cell proliferation and survival through FERM-enhanced p53 degradation.

FAK is known as an integrin- and growth factor-associated tyrosine kinase promoting cell motility. Here we show that, during mouse development, FAK inactivation results in p53- and p21-dependent mesodermal cell growth arrest. Reconstitution of primary FAK-/-p21-/- fibroblasts revealed that FAK, in a kinase-independent manner, facilitates p53 turnover via enhanced Mdm2-dependent p53 ubiquitination. p53 inactivation by FAK required FAK FERM F1 lobe binding to p53, FERM F2 lobe-mediated nuclear localization, and FERM F3 lobe for connections to Mdm2 and proteasomal degradation. Staurosporine or loss of cell adhesion enhanced FERM-dependent FAK nuclear accumulation. In primary human cells, FAK knockdown raised p53-p21 levels and slowed cell proliferation but did not cause apoptosis. Notably, FAK knockdown plus cisplatin triggered p53-dependent cell apoptosis, which was rescued by either full-length FAK or FAK FERM re-expression. These studies define a scaffolding role for nuclear FAK in facilitating cell survival through enhanced p53 degradation under conditions of cellular stress.

Focal adhesion kinase controls pH-dependent epidermal barrier homeostasis by regulating actin-directed Na+/H+ exchanger 1 plasma membrane localization.

Ubiquitously expressed focal adhesion kinase (FAK), linked to multiple intracellular signaling pathways, has previously been shown to control cell motility, invasion, proliferation, and survival. Using mice with a keratinocyte-restricted deletion of fak (FAK(K5 KO)), we report here a novel role for FAK: maintenance of adult epidermal permeability barrier homeostasis. Abundant lacunae of unprocessed lipids in stratum corneum (SC) of FAK(K5 KO) mice and delayed barrier recovery pointed to malfunction of pH-dependent enzymes active in extracellular space of SC. Measuring the SC pH gradient showed significantly more neutral pH values in FAK(K5 KO) mice, suggesting the importance of FAK for acidification. Moreover, normal functions were restored when FAK(K5 KO) mice were exposed to a surface pH typical of mouse SC (pH = 5.5). Baseline levels and response to barrier disruption of secretory phospholipase A2 isoforms, enzymes that mediate generation of free fatty acids in epidermis, appeared similar in both FAK(K5 KO) and control littermates. We found that the critical SC acidification regulator Na(+)/H(+) exchanger 1 failed to localize to the plasma membrane in FAK-deficient keratinocytes both in vivo and in vitro. Thus, for plasma membrane localization in terminally differentiated keratinocytes, Na(+)/H(+) exchanger 1 requires an intact actin cytoskeleton, which is impaired in FAK-deficient cells.