Differentiation Of Trophoblast Stem Cells Research Articles

KDM5B decommissions the H3K4 methylation landscape of self-renewal genes during trophoblast stem cell differentiation.

ABSTRACTTrophoblast stem (TS) cells derived from the trophectoderm (TE) of mammalian embryos have the ability to self-renew indefinitely or differentiate into fetal lineages of the placenta. Epigenetic control of gene expression plays an instrumental role in dictating the fate of TS cell self-renewal and differentiation. However, the roles of histone demethylases and activating histone modifications such as methylation of histone 3 lysine 4 (H3K4me3/me2) in regulating TS cell expression programs, and in priming the epigenetic landscape for trophoblast differentiation, are largely unknown. Here, we demonstrate that the H3K4 demethylase, KDM5B, regulates the H3K4 methylome and expression landscapes of TS cells. Depletion of KDM5B resulted in downregulation of TS cell self-renewal genes and upregulation of trophoblast-lineage genes, which was accompanied by altered H3K4 methylation. Moreover, we found that KDM5B resets the H3K4 methylation landscape during differentiation in the absence of the external self-renewal signal, FGF4, by removing H3K4 methylation from promoters of self-renewal genes, and of genes whose expression is enriched in TS cells. Altogether, our data indicate an epigenetic role for KDM5B in regulating H3K4 methylation in TS cells and during trophoblast differentiation.

Impact of Galectin-1 on Trophoblast Stem Cell Differentiation and Invasion in In Vitro Implantation Model.

Trophoblast stem cells (TSCs) differentiate in an orderly manner, which plays an important role in the process of embryo implantation, placentation, and early pregnancy maintenance. At the maternal-fetal interface, the dialogue is crucial between trophoblast cells and endometrial epithelial cells. Previous studies suggested that galectin-1 (Gal-1) may play an important role in placental development. In this study, we used Ishikawa (IK) cells-TSC coculture model to simulate the maternal-fetal interface and induce the differentiation of TSCs by differentiation media. The messenger RNA level of each cell type markers, fusion markers, and Gal-1 was detected by quantitative reverse transcription polymerase chain reaction during the differentiation of TSCs. Wound healing and transwell invasion assays were used to detect the migration and invasion ability in each group. We found that coculture with IK cells or conditioned media from IK cells could promote the differentiation and invasion of TSCs and increase Gal-1 expression in TSCs. Furthermore, recombinant Gal-1 could also promote the differentiation and invasion of TSCs, suggesting that some of IK cells secretion increase the expression of Gal-1 in TSCs during implantation, which then induced trophoblast differentiation and invasion in vitro. These findings provide significant insights into the biology of embryo-maternal interactions with the importance of Gal-1 in TSCs for the successful establishment and maintenance of pregnancy.

Nucleosomes of polyploid trophoblast giant cells mostly consist of histone variants and form a loose chromatin structure

Trophoblast giant cells (TGCs) are one of the cell types that form the placenta and play multiple essential roles in maintaining pregnancy in rodents. TGCs have large, polyploid nuclei resulting from endoreduplication. While previous studies have shown distinct gene expression profiles of TGCs, their chromatin structure remains largely unknown. An appropriate combination of canonical and non-canonical histones, also known as histone variants, allows each cell to exert its cell type-specific functions. Here, we aimed to reveal the dynamics of histone usage and chromatin structure during the differentiation of trophoblast stem cells (TSCs) into TGCs. Although the expression of most genes encoding canonical histones was downregulated, the expression of a few genes encoding histone variants such as H2AX, H2AZ, and H3.3 was maintained at a relatively high level in TGCs. Both the micrococcal nuclease digestion assay and nucleosome stability assay using a microfluidic device indicated that chromatin became increasingly loose as TSCs differentiated. Combinatorial experiments involving H3.3-knockdown and -overexpression demonstrated that variant H3.3 resulted in the formation of loose nucleosomes in TGCs. In conclusion, our study revealed that TGCs possessed loose nucleosomes owing to alterations in their histone composition during differentiation.

Sirtuin1 is required for proper trophoblast differentiation and placental development in mice

IntroductionPlacental insufficiency, arising from abnormal trophoblast differentiation and function, is a major cause of fetal growth restriction. Sirtuin-1 (Sirt1) is a ubiquitously-expressed NAD-dependent protein deacetylase which plays a key role in numerous cellular processes, including cellular differentiation and metabolism. Though Sirt1 has been widely studied, its role in placentation and trophoblast differentiation is unclear. MethodSirt1-heterozygous mice were mated and evaluated at various points during embryogenesis. In situ hybridization and immunohistochemistry were used to further characterize the placental phenotype of Sirt1-null mice. Wild-type (WT) and Sirt1-null mouse trophoblast stem cell (TSC) lines were derived from e3.5 littermate blastocysts. These cells were then evaluated at various points following differentiation. Differentiation was evaluated by expression of lineage specific markers using qPCR and flow cytometry, as well as Matrigel invasion assays. Global gene expression changes were evaluated using microarray-based RNA profiling; changes in specific pathways were validated using qPCR and western blot. ResultsIn the absence of Sirt1, both embryos and placentas were small, with placentas showing abnormalities in both the labyrinthine layer and junctional zone. Sirt1-null TSCs exhibited an altered phenotype in both undifferentiated and differentiated states, phenotypes which corresponded to changes in pathways relevant to both TSC maintenance and differentiation. Specifically, Sirt1-null TSC showed blunted differentiation, and appeared to be suspended in an Epcamhigh trophoblast progenitor state. DiscussionOur results suggest that Sirt1 is required for proper TSC differentiation and placental development.

Impact of Xist RNA on chromatin modifications and transcriptional silencing maintenance at different stages of imprinted X chromosome inactivation in vole Microtus levis.

In vole Microtus levis, cells of preimplantation embryo and extraembryonic tissues undergo imprinted X chromosome inactivation (iXCI) which is triggered by a long non-coding nuclear RNA, Xist. At early stages of iXCI, chromatin of vole inactive X chromosome is enriched with the HP1 heterochromatin-specific protein, trimethylated H3K9 and H4K20 attributable to constitutive heterochromatin. In the study, using vole trophoblast stem (TS) cells as a model of iXCI, we further investigated chromatin of the inactive X chromosome of M. levis and tried to find out the role of Xist RNA. We demonstrated that chromatin of the inactive X chromosome in vole TS cells also contained the SETDB1 histone methyltransferase and KAP1 protein. In addition, we observed that Xist RNA did not contribute significantly to maintenance of X chromosome inactive state during iXCI in vole TS cells. Xist repression affected neither transcriptional silencing caused by iXCI nor maintenance of trimethylated H3K9 and H4K20 as well as HP1, KAP1, and SETDB1 on the inactive X chromosome. Moreover, the unique repertoire of chromatin modifications on the inactive X chromosome in vole TS cells could be disrupted by a chemical compound, DZNep, and then restored even in the absence of Xist RNA. However, Xist transcript was necessary for recruitment of an additional repressive histone modification, trimethylated H3K27, to the inactive X chromosome during vole TS cell differentiation.

A potential role of galectin-1 in promoting mouse trophoblast stem cell differentiation.

Galectin-1 is highly expressed in blastocysts and trophoblast giant cells during implantation, and dysregulated galectin-1 is associated with many pregnancy-related abnormalities. Elevated galectin-1 contributes to cancer cells invasion. Here, we found that galectin-1 is expressed in mouse oocytes, preimplantation embryos (all stages), and trophoblast stem (TS) cells. Peak levels of galectin-1 mRNA and protein were detected on day 4 and day 5 after the induction of TS cells differentiation. Overexpression of galectin-1 increased TS cells migration and invasion, whereas knockdown of galectin-1 attenuated these effects. Additionally, knockdown of galectin-1 in TS cells decreased the expression of matrix metalloproteinase (MMP) 2/9, ZEB-1, Snail, N-cadherin, TGF-β, Nodal, and phospho-Smad2/3, whereas the expression of E-cadherin was increased. In contrast, overexpression of galectin-1 in TS cells increased the expression of MMP2/9, ZEB-1, and N-cadherin, whereas the expression of E-cadherin was decreased. These findings suggest a potential role of galectin-1 in the differentiation of mouse TS cells.

Hypoxic Stress Forces Adaptive and Maladaptive Placental Stress Responses in Early Pregnancy

This review focuses on hypoxic stress and its effects on the placental lineage and the earliest differentiation events in mouse and human placental trophoblast stem cells (TSCs). Although the placenta is a decidual organ at the end of pregnancy, its earliest rapid growth and function at the start of pregnancy precedes and supports growth and function of the embryo. Earliest function requires that TSCs differentiate, however, "hypoxia" supports rapid growth, but not differentiation of TSCs. Most of the literature on earliest placental "hypoxia" studies used 2% oxygen which is normoxic for TSCs. Hypoxic stress happens when oxygen level drops below 2%. It decreases anabolism, proliferation, potency/stemness and increases differentiation, despite culture conditions that would sustain proliferation and potency. Thus, to study the pathogenesis due to TSC dysfunction, it is important to study hypoxic stress below 2%. Many studies have been performed using 0.5 to 1% oxygen in cultured mouse TSCs. From all these studies, a small number has examined human trophoblast lines and primary first trimester placental hypoxic stress responses in culture. Some other stress stimuli, aside from hypoxic stress, are used to elucidate common and unique aspects of hypoxic stress. The key outcomes produced by hypoxic stress are mitochondrial, anabolic, and proliferation arrest, and this is coupled with stemness loss and differentiation. Hypoxic stress can lead to depletion of stem cells and miscarriage, or can lead to later dysfunctions in placentation and fetal development. Birth Defects Research 109:1330-1344, 2017. © 2017 Wiley Periodicals, Inc.

Mapping the chromatin landscape and Blimp1 transcriptional targets that regulate trophoblast differentiation

Trophoblast stem cells (TSCs) give rise to specialized cell types within the placenta. However, the regulatory mechanisms that guide trophoblast cell fate decisions during placenta development remain ill defined. Here we exploited ATAC-seq and transcriptional profiling strategies to describe dynamic changes in gene expression and chromatin accessibility during TSC differentiation. We detect significantly increased chromatin accessibility at key genes upregulated as TSCs exit from the stem cell state. However, downregulated gene expression is not simply due to the loss of chromatin accessibility in proximal regions. Additionally, transcriptional targets recognized by the zinc finger transcriptional repressor Prdm1/Blimp1, an essential regulator of placenta development, were identified in ChIP-seq experiments. Comparisons with previously reported ChIP-seq datasets for primordial germ cell-like cells and E18.5 small intestine, combined with functional annotation analysis revealed that Blimp1 has broadly shared as well as cell type-specific functional activities unique to the trophoblast lineage. Importantly, Blimp1 not only silences TSC gene expression but also prevents aberrant activation of divergent developmental programmes. Overall the present study provides new insights into the chromatin landscape and Blimp1-dependent regulatory networks governing trophoblast gene expression.

Sex differences in rat placental development: from pre-implantation to late gestation

BackgroundA male fetus is suggested to be more susceptible to in utero and birth complications. This may be due in part to altered morphology or function of the XY placenta. We hypothesised that sexual dimorphism begins at the blastocyst stage with sex differences in the progenitor trophectoderm (TE) and its derived trophoblast lineages, as these cells populate the majority of cell types within the placenta. We investigated sex-specific differences in cell allocation in the pre-implantation embryo and further characterised growth and gene expression of the placental compartments from the early stages of the definitive placenta through to late gestation.MethodsNaturally mated Sprague Dawley dams were used to collect blastocysts at embryonic day (E) 5 to characterise cell allocation; total, TE, and inner cell mass (ICM), and differentiation to downstream trophoblast cell types. Placental tissues were collected at E13, E15, and E20 to characterise volumes of placental compartments, and sex-specific gene expression profiles.ResultsPre-implantation embryos showed no sex differences in cell allocation (total, TE and ICM) or early trophoblast differentiation, assessed by outgrowth area, number and ploidy of trophoblasts and P-TGCs, and expression of markers of trophoblast stem cell state or differentiation. Whilst no changes in placental structures were found in the immature E13 placenta, the definitive E15 placenta from female fetuses had reduced labyrinthine volume, fetal and maternal blood space volume, as well as fetal blood space surface area, when compared to placentas from males. No differences between the sexes in labyrinth trophoblast volume or interhaemal membrane thickness were found. By E20 these sex-specific placental differences were no longer present, but female fetuses weighed less than their male counterparts. Coupled with expression profiles from E13 and E15 placental samples may suggest a developmental delay in placental differentiation.ConclusionsAlthough there were no overt differences in blastocyst cell number or early placental development, reduced growth of the female labyrinth in mid gestation is likely to contribute to lower fetal weight in females at E20. These data suggest sex differences in fetal growth trajectories are due at least in part, to differences in placenta growth.

Mouse prolyl oligopeptidase plays a role in trophoblast stem cell differentiation into trophoblast giant cell and spongiotrophoblast

IntroductionProlyl oligopeptidase (prolyl endopeptidase, Prep), a multifunctional protease hydrolyzing -Pro-X- peptide bonds, is highly expressed in the mouse placenta, but the function during development is not known. We explored the possibility of Prep's involvement in placental differentiation. MethodsWe cultured trophoblast stem cells (TSCs) derived from the E6.5 mouse embryo and investigated the detailed expression pattern of Prep during their differentiation. Prep-specific inhibitors were added to the TSC culture, and the effect on the differentiation was assessed by microscopic observation and the expression of marker gene for each placental cell. ResultsDuring TSC differentiation for 6 days, Prep was constantly detected at mRNA, protein, and activity levels, and the protein was found mainly in the cytoplasm. The addition of 30 μM and 10 μM SUAM-14746, a Prep-specific inhibitor, effectively inhibited the differentiation into spongiotrophoblasts (SpTs) and trophoblast giant cells (TGCs), while the TSC viability was not affected. 5 μM SUAM-14746 impaired the differentiation into SpTs, and 1 μM SUAM-14746 exhibited no effects. Another Prep-specific inhibitor, KYP-2047, did not affect the differentiation. We confirmed efficient inhibition of Prep enzymatic activity in TSCs by both inhibitors. ConclusionThe dose-dependent effect of SUAM-14746 on TSCs suggests that Prep plays an important role in the differentiation into SpTs and TGCs in the mouse placenta.

Suppression of Fgf2 by ETS2 repressor factor (ERF) is required for chorionic trophoblast differentiation

ETS2 repressor factor (ERF) is a ubiquitous transcriptional repressor regulated by Extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Homozygous deletion of Erf in mice blocks chorionic trophoblast differentiation, resulting in the failure of chorioallantoic fusion and subsequent embryo death. Fibroblast growth factor (FGF) signaling is important for proper trophoblast stem cell (TSC) differentiation and development of the hemochorial placenta. Lack of Fgf2 promotes TSC differentiation, while FGF4 or FGF2 is required for murine TSC maintenance. Here, we show that low in vivo Fgf2 mRNA abundance occurs in patches of placental chorion cells and ex vivo in TSCs. This expression is repressed via direct interaction of ERF with the Fgf2 transcription unit is increased in the absence of ERF, and is decreased in the presence of an ERF mutant resistant to ERK phosphorylation. Thus, FGF2 inhibition by ERF appears to be necessary for proper chorionic TSC differentiation, and may account for the block of chorionic trophoblast differentiation in Erf-knockout animals. The differentiation of ERF-overexpressing TSC lines also suggests that ERF may have an FGF2-independent effect during the commitment towards syncytiotrophoblasts.

Plac1 Expression Pattern at the Mouse Fetomaternal Interface and Involvement in Trophoblast Differentiation

Background/Aims: It is well known that Plac1 is a placenta-specific gene; however, its spatiotemporal expression pattern and exact role at t h e mouse fetomaternal interface r e m a i n s unclear. Methods: In situ hybridization (ISH) was used to localize the Plac1 mRNA at the mouse fetomaternal interface. A trophoblast stem cell (TS) differentiation model with Plac1 shRNA-overexpressing lentivirus was employed to investigate the possible role of Plac1 in placentation. Real-time RT-PCR was used to detect changes in gene expression. Results: Plac1 was exclusively expressed in the ectoplacental cone (EPC) as well as in 8.5 and 9.5 days post-coitum (dpc) embryos. Subsequently, Plac1 expression was abundant in the spongiotrophoblast layer and moderately in the labyrinth layer until 13.5 dpc, and declined thereafter. Interestingly, Plac1 was also expressed by secondary trophoblast giant cells and glycogen trophoblast cells, but not in primary trophoblast giant cells. Plac1 transcription was increased during the TS differentiation (P < 0.01), and knockdown of Plac1 significantly impaired TS differentiation. Conclusion: Plac1 is abundantly expressed at the fetomaternal interface and in all trophoblast subtypes except in primary trophoblast giant cells. Plac1 knockdown retarded the progress of TS differentiation, indicating that Plac1 is necessary for normal trophoblast differentiation into various trophoblast subpopulations.

Nicotinamide benefits both mothers and pups in two contrasting mouse models of preeclampsia

Preeclampsia (PE) complicates ∼5% of human pregnancies and is one of the leading causes of pregnancy-related maternal deaths. The only definitive treatment, induced delivery, invariably results in prematurity, and in severe early-onset cases may lead to fetal death. Many currently available antihypertensive drugs are teratogenic and therefore precluded from use. Nonteratogenic antihypertensives help control maternal blood pressure in PE, but results in preventing preterm delivery and correcting fetal growth restriction (FGR) that also occurs in PE have been disappointing. Here we show that dietary nicotinamide, a nonteratogenic amide of vitamin B3, improves the maternal condition, prolongs pregnancies, and prevents FGR in two contrasting mouse models of PE. The first is caused by endotheliosis due to excess levels in the mothers of a soluble form of the receptor for vascular endothelial growth factor (VEGF), which binds to and inactivates VEGF. The second is caused by genetic absence of Ankiryn-repeat-and-SOCS-box-containing-protein 4, a factor that contributes to the differentiation of trophoblast stem cells into the giant trophoblast cells necessary for embryo implantation in mice; its absence leads to impaired placental development. In both models, fetal production of ATP is impaired and FGR is observed. We show here that nicotinamide decreases blood pressure and endotheliosis in the mothers, probably by inhibiting ADP ribosyl cyclase (ADPRC), and prevents FGR, probably by normalizing fetal ATP synthesis via the nucleotide salvage pathway. Because nicotinamide benefits both dams and pups, it merits evaluation for preventing or treating PE in humans.

From the stem of the placental tree: trophoblast stem cells and their progeny.

Trophoblast stem cells (TSCs) retain the capacity to self-renew indefinitely and harbour the potential to differentiate into all trophoblast subtypes of the placenta. Recent studies have shown how signalling cascades integrate with transcription factor circuits to govern the fine balance between TSC self-renewal and differentiation. In addition, breakthroughs in reprogramming strategies have enabled the generation of TSCs from fibroblasts, opening up exciting new avenues that may allow the isolation of this stem cell type from other species, notably humans. Here, we review these recent advances in light of their importance for understanding placental pathologies and developing personalised medicine approaches for pregnancy complications.

Sox21 deletion in mice causes postnatal growth deficiency without physiological disruption of hypothalamic-pituitary endocrine axes

The hypothalamic-pituitary axes are the coordinating centers for multiple endocrine gland functions and physiological processes. Defects in the hypothalamus or pituitary gland can cause reduced growth and severe short stature, affecting approximately 1 in 4000 children, and a large percentage of cases of pituitary hormone deficiencies do not have an identified genetic cause. SOX21 is a protein that regulates hair, neural, and trophoblast stem cell differentiation. Mice lacking Sox21 have reduced growth, but the etiology of this growth defect has not been described. We studied the expression of Sox21 in hypothalamic-pituitary development and examined multiple endocrine axes in these mice. We find no evidence of reduced intrauterine growth, food intake, or physical activity, but there is evidence for increased energy expenditure in mutants. In addition, despite changes in pituitary hormone expression, hypothalamic-pituitary axes appear to be functional. Therefore, SOX21 variants may be a cause of non-endocrine short stature in humans.

Role of decorin in trophoblast stem cell self-renewal and differentiation

Role of decorin in trophoblast stem cell self-renewal and differentiation

Characterizing the contribution of extracellular matrix (ECM) to mouse trophoblast stem and progenitor cell differentiation

All eggs have an extracellular coat (EC) that plays unique roles during reproduction and development. ECs are designed to protect eggs and support their growth, regulate fertilization of eggs, and protect early embryos. ECs of mammalian and nonmammalian eggs consist of only a few proteins that are closely related to one another. All these proteins possess regulatory elements and a structural domain responsible for processing and assembly of the proteins into ECs. They also possess regions responsible for their functional roles during and after fertilization. Our essay addresses these and other aspects of EC biology.

A quiet death; hypoxic stress forces diminished growth and potency and large, irreversible trophoblast stem cell differentiation

A quiet death; hypoxic stress forces diminished growth and potency and large, irreversible trophoblast stem cell differentiation

Alcohol exposure impairs trophoblast survival and alters subtype-specific gene expression in vitro

Maternal alcohol consumption is common prior to pregnancy recognition and in the rat results in altered placental development and fetal growth restriction. To assess the effect of ethanol (EtOH) exposure on the differentiation of trophoblast stem (TS) cells, mouse TS lines were differentiated in vitro for 6 days in 0%, 0.2% or 1% EtOH. This reduced both trophoblast survival and expression of labyrinth and junctional zone trophoblast subtype-specific genes. This suggests that fetal growth restriction and altered placental development associated with maternal alcohol consumption in the periconceptional period could be mediated in part by direct effects on trophoblast development.

Plet1 is an epigenetically regulated cell surface protein that provides essential cues to direct trophoblast stem cell differentiation.

Gene loci that are hypermethylated and repressed in embryonic (ESCs) but hypomethylated and expressed in trophoblast (TSCs) stem cells are very rare and may have particularly important roles in early developmental cell fate decisions, as previously shown for Elf5. Here, we assessed another member of this small group of genes, Placenta Expressed Transcript 1 (Plet1), for its function in establishing trophoblast lineage identity and modulating trophoblast differentiation. We find that Plet1 is tightly repressed by DNA methylation in ESCs but expressed on the cell surface of TSCs and trophoblast giant cells. In hypomethylated ESCs that are prone to acquire some trophoblast characteristics, Plet1 is required to confer a trophoblast-specific gene expression pattern, including up-regulation of Elf5. Plet1 displays an unusual biphasic expression profile during TSC differentiation and thus may be pivotal in balancing trophoblast self-renewal and differentiation. Furthermore, overexpression and CRISPR/Cas9-mediated knockout in TSCs showed that high Plet1 levels favour differentiation towards the trophoblast giant cell lineage, whereas lack of Plet1 preferentially induces syncytiotrophoblast formation. Thus, the endogenous dynamics of Plet1 expression establish important patterning cues within the trophoblast compartment by promoting differentiation towards the syncytiotrophoblast or giant cell pathway in Plet1-low and Plet1-high cells, respectively.