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Healthy pregnancy depends on proper placentation-including proliferation, differentiation, and invasion of trophoblast cells-which, if impaired, causes placental ischemia resulting in intrauterine growth restriction and preeclampsia. Mechanisms regulating trophoblast invasion, however, are unknown. We report that reduction of ( alters intracellular trafficking and significantly impairs invasion in a model of human extravillous trophoblasts. Furthermore, global loss of in mice recapitulates maternal and fetal phenotypes of placental insufficiency. dams have reduced spiral artery numbers and late gestational hypertension with resolution following delivery. fetuses are growth restricted and demonstrate changes in umbilical artery Doppler consistent with poor placental perfusion and fetal distress. Loss of increases fetal vascular density in the placenta and dysregulates trophoblast expression of angiogenic factors. Our data support a critical regulatory role for in trophoblast invasion-a necessary process for placentation-representing a possible future target for improving placentation and fetal outcomes.
Various stem cells have been explored for the purpose of cardiac repair. However, any individual stem cell population has not been considered as the ideal source. Recently, trophoblast stem cells (TSCs), a newly described stem cell type, have demonstrated extensive plasticity. The present study evaluated the therapeutic effect of TSCs transplantation for heart regeneration in a mouse model of myocardial infarction (MI) and made a direct comparison with the most commonly used mesenchymal stem cells (MSCs). Transplantation of TSCs and MSCs led to a remarkably improved cardiac function in contrast with the PBS control, but only the TSCs exhibited the potential of differentiation into cardiomyocytes in vivo. In addition, a significantly high proliferation level of both transplanted stem cells and resident cardiomyocytes was observed in the TSCs group. These findings primary revealed the therapeutic potential of TSCs in transplantation therapy for MI.
INTRODUCTION - We performed RNA sequencing with the primary goal of discovering key placental villous trophoblast (VT) and decidua basalis (DB) transcripts differentially expressed in intra-amniotic infection (IAI)-induced preterm birth (PTB).
METHODS - RNA was extracted from 15 paired VT and DB specimens delivered of women with: 1) spontaneous PTB in the setting of amniocentesis-proven IAI and histological chorioamnionitis (n = 5); 2) spontaneous idiopathic PTB (iPTB, n = 5); and 3) physiologic term pregnancy (n = 5). RNA sequencing was performed using the Illumina HiSeq 2500 platform, and a spectrum of computational tools was used for gene prioritization and pathway analyses.
RESULTS - In the VT specimens, 128 unique long transcripts and 7 mature microRNAs differed significantly between pregnancies complicated by IAI relative to iPTB (FDR<0.1). The up-regulated transcripts included many characteristic of myeloblast-derived cells, and bioinformatic analyses revealed enrichment for multiple pathways associated with acute inflammation. In an expanded cohort including additional IAI and iPTB specimens, the expression of three proteins (cathepsin S, lysozyme, and hexokinase 3) and two microRNAs (miR-133a and miR-223) was validated using immunohistochemistry and quantitative PCR, respectively. In the DB specimens, only 11 long transcripts and no microRNAs differed significantly between IAI cases and iPTB controls (FDR<0.1). Comparison of the VT and DB specimens in each clinical scenario revealed signatures distinguishing these placental regions.
DISCUSSION - IAI is associated with a transcriptional signature consistent with acute inflammation in the villous trophoblast. The present findings illuminate novel signaling pathways involved in IAI, and suggest putative therapeutic targets and potential biomarkers associated with this condition.
Copyright © 2016. Published by Elsevier Ltd.
Cathepsin P is a member of a family of placentally expressed cathepsins (PECs). The closest human homolog of cathepsin P is cathepsin L, a broad specificity enzyme that has functions in many tissues in addition to placenta. The gene duplications that gave rise to the PECs provide a rare opportunity to define proteolytic functions in placenta, a transient organ unique to mammals. Peptidyl substrate and inhibitor libraries have shown that cathepsin P has evolved an unusually restricted preference for substrates containing hydrophobic amino acids. Proteomic techniques were used to probe for substrates of this enzyme. Recombinant cathepsin P was incubated with rat choriocarcinoma (Rcho-1) cell proteins to identify substrates using two-dimensional difference gel electrophoresis. Substrate proteins were excised from gels and characterized by trypsin digestion and MALDI MS/MS. Two endoplasmic reticulum (ER) proteins, gp96 and calreticulin, emerged as potential substrates, and western blotting showed that these proteins are processed by cathepsin P from their C-terminus, removing the KDEL ER retention signal. Immunohistochemistry showed that a portion of cathepsin P co-localizes with calreticulin in Rcho-1 cells. Extracellular calreticulin induces differentiation of Rcho-1 cells, indicating a potential role of cathepsin P in processing and secretion of calreticulin during differentiation of trophoblast giant cells.
The murine blastocyst contains two nonoverlapping pools of progenitor cells: the embryonic component contributes to the fetus and generates embryonic stem cells in vitro, whereas the extraembryonic pool contributes to the placenta and generates trophoblast stem cells in vitro. The transcriptional repressor Foxd3 is required for maintenance of the epiblast and the in vitro establishment of embryonic stem cell lines. Here, we demonstrate that Foxd3 is also required in the trophoblast lineage. Trophoblast progenitors in Foxd3-/- embryos do not self-renew and are not multipotent, but instead give rise to an excess of trophoblast giant cells. Injection of Foxd3-/- blastocysts with wild type ES cells fails to rescue Foxd3-/- placentas and such chimeras die around 10 days of embryogenesis. These results indicate an essential role for Foxd3 in two nonoverlapping progenitor cell populations that require different secreted factors to maintain their multipotent properties in vitro and give rise to divergent tissues in vivo. Moreover, this provides support for the hypothesis that there are conserved molecular mechanisms for maintaining the self-renewing properties of diverse progenitor cell types.
Trophoblast invasion of the uterine extracellular matrix, a critical process of human implantation and essential for fetal development, is a striking example of controlled invasiveness. To identify molecules that regulate trophoblast invasion, mRNA signatures of trophoblast cells isolated from first trimester (high invasiveness) and term placentae (no/low invasiveness) were compared using U95A GeneChip microarrays yielding 220 invasion/migration-related genes. In this 'invasion cluster', KiSS-1 and its G-protein-coupled receptor KiSS-1R were expressed at higher levels in first trimester trophoblasts than at term of gestation. Receptor and ligand mRNA and protein were localized to the trophoblast compartment. In contrast to KiSS-1, which is only expressed in the villous trophoblast, KiSS-1R was also found in the extravillous trophoblast, suggesting endocrine/paracrine activation mechanisms. The primary translation product of KiSS-1 is a 145 amino acid polypeptide (Kp-145), but shorter kisspeptins (Kp) with 10, 13, 14 or 54 amino acid residues may be produced. We identified Kp-10, a dekapeptide derived from the primary translation product, in conditioned medium of first trimester human trophoblast. Kp-10, but not other kisspeptins, increased intracellular Ca(2+) levels in isolated first trimester trophoblasts. Kp-10 inhibited trophoblast migration in an explant as well as transwell assay without affecting proliferation. Suppressed motility was paralleled with suppressed gelatinolytic activity of isolated trophoblasts. These results identified Kp-10 as a novel paracrine/endocrine regulator in fine-tuning trophoblast invasion generated by the trophoblast itself.
The events that regulate trophoblast invasion need to be characterized at the transcriptional level. Several types of gene products may be involved in various stages oftrophoblast infiltration, including integrins, matrix metalloproteases (MMPs) and extracellular matrix (ECM) proteins. Autocrine or paracrine regulators of cytotrophoblast proliferation or differentiation in vitro (e.g. growth factors and cytokines, as well as oxygen tension) could be characterized mechanistically at the transcriptional level. Large-scale gene expression profiling of trophoblasts of distinct invasive stages could be carried out on fixed tissue obtained by laser-directed microdissection. This information may shed light on physiological implantation and placentation, as well as on the interpretation of pathological processes such as pre-eclampsia. The applications of DNA microarrays are ideal for studies of genomic structure (e.g. mutation and polymorphism analyses) and monitoring of gene expression. The ultimate goal is to understand the critical events underlying growth, development, homeostasis, 'behaviour and the onset of disease at a genomic level. Microarrays detect gene expression levels in parallel by measuring the hybridization of labelled, single-stranded DNA to many thousands of partial or whole gene sequences immobilized on a glass surface (the 'chip'). Microarrays are available both commercially and can be manufactured in house.
Oct4 is a mammalian POU transcription factor expressed by early embryo cells and germ cells. We report that the activity of Oct4 is essential for the identity of the pluripotential founder cell population in the mammalian embryo. Oct4-deficient embryos develop to the blastocyst stage, but the inner cell mass cells are not pluripotent. Instead, they are restricted to differentiation along the extraembryonic trophoblast lineage. Furthermore, in the absence of a true inner cell mass, trophoblast proliferation is not maintained in Oct4-/- embryos. Expansion of trophoblast precursors is restored, however, by an Oct4 target gene product, fibroblast growth factor-4. Therefore, Oct4 also determines paracrine growth factor signaling from stem cells to the trophectoderm.
Maternal-facing brush border membrane vesicles isolated from normal term human placentas were found to accumulate norepinephrine in a concentrative manner in the presence of an inwardly directed NaCl gradient. Both Na+ and Cl- were obligatory for maximal uptake. The NaCl-dependent norepinephrine uptake was further stimulated by the presence of K+ or an acidic pH in the intravesicular medium. The uptake process was electrogenic, being stimulated by an inside-negative membrane potential, and this characteristic was observed in the absence as well as in the presence of K+ inside the vesicles. Kinetic analyses revealed that one Na+ and one Cl- were involved per transport of one norepinephrine molecule. The apparent Michaelis-Menten constant for norepinephrine was 104 +/- 5nM. The uptake process exhibited higher affinity for dopamine than for norepinephrine but had low affinity for serotonin and histamine. The uptake of norepinephrine was inhibited very effectively by nomifensine, desipramine, imipramine, and cocaine, but much less effectively by bupropion and GBR 12909. Northern blot analysis with the cDNA of the human (SK-N-SH cell) norepinephrine transporter as the probe revealed that the human placenta contained two mRNAs, 5.8 and 3.6 kb in size, which hybridized to the probe. The JAR human placental choriocarcinoma cells were found unable to accumulate norepinephrine in a NaCl-dependent manner. These cells were also found not to contain mRNAs which hybridized to the norepinephrine cDNA probe in northern blot. It is concluded that the human placental syncytiotrophoblast expresses a cocaine-sensitive norepinephrine transporter and that these findings may be directly relevant and important to the clinical complications of maternal cocaine abuse during pregnancy.