Evaluation of the technique of immunosurgery for the isolation of inner cell masses from mouse blastocysts

Development ◽  
1977 ◽  
Vol 37 (1) ◽  
pp. 217-226
Author(s):  
A. H. Handyside ◽  
S. C. Barton
Keyword(s):  
Rabbit Antiserum ◽  
Trophoblast Cell ◽  
Trophoblast Cells ◽  
Rabbit Igg ◽  
Developmental Capacity ◽  
Synthetic Profile

Inner cell masses (ICMs) immunosurgically-isolated from 3½-day mouse blastocysts were examined for trophoblast cell contamination and developmental capacity. Blastocysts were preincubated in rabbit anti-mouse antiserum, washed thoroughly and then incubated in complement. The ICMs were then easily dissected by drawing through a fine pipette. Various experiments confirmed that the trophectoderm had been completely removed by this treatment. Firstly, the ICMs did not bind a fiuorescein-conjugated antibody directed against rabbit IgG, indicating the absence of cells exposed to the rabbit antiserum during the immunosurgical procedure. Secondly, ICMs dissected from blastocysts preincubated in a suspension of melanin granules did not include any of the trophoblast cells that had phagocytosed the granules. And, thirdly, the protein synthetic profile of these ICMs was similar to microsurgically dissected ICMs, and in particular, trophoblast specific spots were absent. The developmental capacity of immunosurgically-isolated ICMs was tested by injecting them into blastocysts and transferring to the uterus of 2½-day pseudopregnant recipients. Extensive chimaerism was detected in the majority of implants, 5–6 days after transfer, but only in ICM-derived tissues. This demonstrates both the lack of trophoblast cell contamination and functional viability of these ICMs.

scholarly journals YB-1 Is Altered in Pregnancy-Associated Disorders and Affects Trophoblast in Vitro Properties via Alternation of Multiple Molecular Traits

2021 ◽  
Vol 22 (13) ◽  
pp. 7226
Author(s):  
Violeta Stojanovska ◽  
Aneri Shah ◽  
Katja Woidacki ◽  
Florence Fischer ◽  
Mario Bauer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Progression ◽  
Cell Function ◽  
Trophoblast Cell ◽  
Mrna Levels ◽  
Trophoblast Cells ◽  
Invasion And Migration ◽  
And Migration ◽  
Apoptosis And Inflammation

Cold shock Y-box binding protein-1 (YB-1) coordinates several molecular processes between the nucleus and the cytoplasm and plays a crucial role in cell function. Moreover, it is involved in cancer progression, invasion, and metastasis. As trophoblast cells share similar characteristics with cancer cells, we hypothesized that YB-1 might also be necessary for trophoblast functionality. In samples of patients with intrauterine growth restriction, YB-1 mRNA levels were decreased, while they were increased in preeclampsia and unchanged in spontaneous abortions when compared to normal pregnant controls. Studies with overexpression and downregulation of YB-1 were performed to assess the key trophoblast processes in two trophoblast cell lines HTR8/SVneo and JEG3. Overexpression of YB-1 or exposure of trophoblast cells to recombinant YB-1 caused enhanced proliferation, while knockdown of YB-1 lead to proliferative disadvantage in JEG3 or HTR8/SVneo cells. The invasion and migration properties were affected at different degrees among the trophoblast cell lines. Trophoblast expression of genes mediating migration, invasion, apoptosis, and inflammation was altered upon YB-1 downregulation. Moreover, IL-6 secretion was excessively increased in HTR8/SVneo. Ultimately, YB-1 directly binds to NF-κB enhancer mark in HTR8/SVneo cells. Our data show that YB-1 protein is important for trophoblast cell functioning and, when downregulated, leads to trophoblast disadvantage that at least in part is mediated by NF-κB.

Intersection of regulatory pathways controlling hemostasis and hemochorial placentation

2021 ◽  
Vol 118 (50) ◽  
pp. e2111267118
Author(s):  
Masanaga Muto ◽  
Damayanti Chakraborty ◽  
Kaela M. Varberg ◽  
Ayelen Moreno-Irusta ◽  
Khursheed Iqbal ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Cell Differentiation ◽  
Cell Invasion ◽  
Cell Development ◽  
Trophoblast Cell ◽  
Extravillous Trophoblast ◽  
Cell Phenotype ◽  
Trophoblast Cells ◽  
Rat Models ◽  
Endovascular Trophoblast

Hemochorial placentation is characterized by the development of trophoblast cells specialized to interact with the uterine vascular bed. We utilized trophoblast stem (TS) cell and mutant rat models to investigate regulatory mechanisms controlling trophoblast cell development. TS cell differentiation was characterized by acquisition of transcript signatures indicative of an endothelial cell-like phenotype, which was highlighted by the expression of anticoagulation factors including tissue factor pathway inhibitor (TFPI). TFPI localized to invasive endovascular trophoblast cells of the rat placentation site. Disruption of TFPI in rat TS cells interfered with development of the endothelial cell-like endovascular trophoblast cell phenotype. Similarly, TFPI was expressed in human invasive/extravillous trophoblast (EVT) cells situated within first-trimester human placental tissues and following differentiation of human TS cells. TFPI was required for human TS cell differentiation to EVT cells. We next investigated the physiological relevance of TFPI at the placentation site. Genome-edited global TFPI loss-of-function rat models revealed critical roles for TFPI in embryonic development, resulting in homogeneous midgestation lethality prohibiting analysis of the role of TFPI as a regulator of the late-gestation wave of intrauterine trophoblast cell invasion. In vivo trophoblast-specific TFPI knockdown was compatible with pregnancy but had profound effects at the uterine–placental interface, including restriction of the depth of intrauterine trophoblast cell invasion while leading to the accumulation of natural killer cells and increased fibrin deposition. Collectively, the experimentation implicates TFPI as a conserved regulator of invasive/EVT cell development, uterine spiral artery remodeling, and hemostasis at the maternal–fetal interface.

110. EFFECTS OF cAMP AND SERUM ON HUMAN TROPHOBLAST CELL VIABILITY AND DIFFERENTIATION

2009 ◽  
Vol 21 (9) ◽  
pp. 29
Author(s):  
Y. Chen ◽  
M. Allars ◽  
C. Abou-Seif ◽  
R. C. Nicholson
Keyword(s):  
Cell Viability ◽  
Syncytium Formation ◽  
Culture Conditions ◽  
Trophoblast Cell ◽  
Bewo Cell ◽  
Trophoblast Cells ◽  
Human Trophoblast ◽  
Serum Free ◽  
Bewo Cells ◽  
Free Media

The formation of syncytium is a pivotal event for trophoblast cells to interact with the placental bed. While cAMP is regarded as an inducer of syncytialisation, the affect of different culture conditions on this cAMP effect has not been explored. Therefore, the effects of cAMP on cell differentiation and viability in the presence or absence of serum were investigated in the human choriocarcinoma cell lines, BeWo and JEG-3. We observed that in the absence of cAMP, BeWo cells grew best in media containing 10% FCS, followed by media containing charcoal-stripped 10% FCS (10%CCS), and less well in serum-free media. In the presence of cAMP ( 0.25~1.5 mM ), our observations suggest different cellular programmes may be in play. Treatment of BeWo cells with 0.75 mM cAMP for 24h and 48h, in the absence of serum, increased cell viability (MTT assay) by 25.1% and 46.1% respectively, compared to the control cells. Interestingly, this cAMP effect on cell viability was not observed in the JEG-3 cell line. In contrast, BeWo cell viability was decreased by 49.5% and 25.2%, and by 27.5% and 31.1% in JEG-3 cells, when the cAMP stimulated cells were cultured for 48h in 10% CCS and 10% FCS media, respectively. In addition, we observed a change in BeWo, but not JEG-3, cell morphology to a spindle-like shape with pseudopodia when cAMP stimulated cells were cultured in media containing 10% CCS or 10% FCS for greater than 24h. Since the process of syncytialisation may involve apoptotic events, we speculate that the different effects of cAMP on cell viability in trophoblast cells may be related to syncytialising factors contained in serum media. Further study will clarify whether serum promotes syncytium formation, while the lack of serum based factors could switch the cellular programme from one of syncytialisation toward a more proliferative type.

Development Of A Rapid Immunometric Assay For The Factor VIII Antigens In Plasma

1981 ◽  
Author(s):  
P J Gaffney ◽  
M Mahmoud ◽  
T W Barrowcliffe ◽  
C Kemball-Cook
Keyword(s):  
Factor Viii ◽  
Rabbit Antiserum ◽  
Response Curves ◽  
Standard Curve ◽  
Immunometric Assay ◽  
Freeze Dried ◽  
Coagulant Activity ◽  
Rabbit Igg ◽  
Alternative Methodology ◽  
Purified Antigen

Immunoradiometric assays (IRMA) for both Factor VIII related antigen (VIII R:Ag) and Factor VIII clotting antigen (VIII C:Ag) have been described, involving the preparation of specific radiolabeled antibodies. The alternative methodology described in this report for F VIII R:Ag involves three steps:1. the immobilization of a crude preparation of F VIII R:Ag (0.05 units/ml) on polyvinyl plates (96-well); 2. the incubation of known amounts (defined by a freeze-dried plasma standard) of F VIII R:Ag with a predetermined excess (1/2000) of a monospecific rabbit antiserum to F VIII R:Ag (called first antibody, ab'); 3. the binding of the various residual amounts of ab' (from step 2) to the immobilized F VIII R:Ag. The residual ab' bound to the coated polyvinyl plates is measured using a radiolabeled (125I) goat IgG to rabbit IgG (called second antibody, ab"). Thus the radioactivity in each well of the plate is inversely related to the amount of F VIII R:Ag in the incubation mixture. A linear standard curve was established for pooled plasma between 0.04-0.002 units of F VIII R:Ag per ml. Test plasmas gave parallel dose-response curves with this standard curve. Thus the F VIII R:Ag levels in a variety of plasmas and concentrates were established and found to correlate with data obtained by Laurell ‘rocket’ immunoelectrophoresis. A similar assay to that described above has been attempted for Factor VIII C:Ag in plasma. The antiserum was obtained from a patient with an elevated level of ‘inhibitor’ (antibody) to F VIII C:Ag. A standard curve has been established using a highly purified preparation of F VIII C:Ag between 0.3-0.01 u of coagulant activity. This form of immunometric assay has the following advantages:1. a source of highly purified antigen is not necessary; 2. no isolation or labelling of the F VIII antibodies is required; 3. the assay is sensitive, technically simple and rapid.

microRNA-29b contributes to pre-eclampsia through its effects on apoptosis, invasion and angiogenesis of trophoblast cells

2012 ◽  
Vol 124 (1) ◽  
pp. 27-40 ◽  
Author(s):  
Pengfei Li ◽  
Wei Guo ◽  
Leilei Du ◽  
Junli Zhao ◽  
Yaping Wang ◽  
...  
Keyword(s):  
Target Genes ◽  
Inverse Correlation ◽  
Myeloid Cell ◽  
Trophoblast Cell ◽  
Trophoblast Invasion ◽  
Cell Leukaemia ◽  
Trophoblast Cells ◽  
Computer Based ◽  
Induced Apoptosis ◽  
Endothelial Growth Factor

PE (pre-eclampsia), a pregnancy-specific disorder, is characterized by increased trophoblast cell death and deficient trophoblast invasion and reduced trophoblast-mediated remodelling of spiral arteries. The present study was performed to determine the function of miR-29b (microRNA-29b) in trophoblast cells and its underlying role in the pathogenesis of PE. The prediction of miR-29b target genes was performed using computer-based programs, including Targetscan, Pictar and miRBase. The function of these target genes was analysed further by gene ontology (GO). The effects of miR-29b on apoptosis, and invasion and angiogenesis of trophoblast cell lines (HTR-8/SVneo, BeWo and JAR) were examined by flow cytometry and Matrigel assay respectively. We found that miR-29b induced apoptosis and inhibited invasion and angiogenesis of trophoblast cells. Further studies confirmed that miR-29b regulated the expression of MCL1 (myeloid cell leukaemia sequence 1), MMP2 (encoding matrix metallproteinase 2), VEGFA (vascular endothelial growth factor A) and ITGB1 (integrin β1) genes by directly binding to their 3′-UTRs (untranslated regions). Moreover, we identified that there was an inverse correlation between miR-29b and its target genes in subjects with PE. Taken together, these findings support a novel role for miR-29b in invasion, apoptosis and angiogenesis of trophoblast cells, and miR-29b may become a new potential therapeutic target for PE.

scholarly journals Uterine Natural Killer Cells Are Targets for a Trophoblast Cell-Specific Cytokine, Prolactin-Like Protein A*

Endocrinology ◽  
1999 ◽  
Vol 140 (6) ◽  
pp. 2711-2720 ◽  
Author(s):  
Heiner Müller ◽  
Bing Liu ◽  
B. Anne Croy ◽  
Judith R. Head ◽  
Joan S. Hunt ◽  
...  
Keyword(s):  
Natural Killer ◽  
Nk Cell ◽  
Specific Interaction ◽  
Protein A ◽  
Trophoblast Cell ◽  
Surface Marker ◽  
Trophoblast Cells ◽  
New Paradigm ◽  
Uterine Natural Killer Cells ◽  
Chorioallantoic Placenta

Abstract PRL-like protein A (PLP-A) is a member of the PRL family expressed in trophoblast cells coincident with establishment of the chorioallantoic placenta. The purpose of this investigation was to identify targets for PLP-A. Using an alkaline phosphatase-tagging strategy, we show that PLP-A specifically interacts with a population of natural killer (NK) lymphocytes within the mesometrial compartment of decidua from pregnant and pseudopregnant rats. These observations are supported by the codistribution of PLP-A targets with cells expressing the rat NK cell surface marker, gp42, the absence of PLP-A binding in conceptuses from NK cell-deficient tgε26 mice, and the specific interaction of PLP-A with a rat NK cell line, RNK-16. We have further demonstrated that PLP-A effectively suppresses RNK-16 cell cytolytic activities. Our results provide evidence for a new paradigm of embryonic-maternal communication involving a PLP-A signaling pathway between trophoblast cells and uterine NK lymphocytes.

scholarly journals The Cytokine Gene CXCL14 Restricts Human Trophoblast Cell Invasion by Suppressing Gelatinase Activity

Endocrinology ◽  
2009 ◽  
Vol 150 (12) ◽  
pp. 5596-5605 ◽  
Author(s):  
HaiBin Kuang ◽  
Qi Chen ◽  
Ying Zhang ◽  
Li Zhang ◽  
HongYing Peng ◽  
...  
Keyword(s):  
Trophoblast Cell ◽  
Trophoblast Invasion ◽  
Trophoblast Cells ◽  
Human Trophoblast ◽  
Gelatinase Activity ◽  
Invasion And Migration ◽  
Human Pregnancy ◽  
Decidual Cells ◽  
And Migration ◽  
Maternal Fetal Interface

Abstract Well-controlled trophoblast invasion into uterine decidua is a critical process for the normal development of placenta, which is tightly regulated by various factors produced within the trophoblast-endometrial microenvironment. CXCL14 is involved in tumor growth and metastasis, and its expression in placenta is temporally regulated during pregnancy. However, the role of CXCL14 in trophoblast function during human pregnancy is not clear. In this study, by using RT-PCR through human pregnancy, we found that CXCL14 was selectively expressed at early but not late pregnancy. Immunostaining revealed that CXCL14 proteins were strongly expressed in villous cytotrophoblasts and moderately in decidualized stromal cells but very weakly in syncytiotrophoblasts and extravillous trophoblasts. The effect of CXCL14 on trophoblast invasion were examined by using human villous explants cultured on Matrigel and further proved by invasion and migration assay of primary trophoblast cells and trophoblast cell line HTR-8/SVneo. Our data showed that CXCL14 significantly inhibited outgrowth of villous explant in vitro; this effect is due to suppression of trophoblast invasion and migration through regulating matrix metalloproteinases activities, whereas the trophoblast proliferation was not affected. Moreover, because a receptor for CXCL14 has not been identified, we performed further cell-specific CXCL14 binding activities with regard to different cell types within the maternal-fetal interface. Our data revealed that CXCL14 could specifically bind to trophoblast cells but not decidual cells from the maternal-fetal interface. These results suggest that CXCL14 plays an important role in regulating trophoblast invasion through an autocrine/paracrine manner during early pregnancy.

scholarly journals Effects of Human Umbilical Cord Mesenchymal Stem Cells on Human Trophoblast Cell Functions In Vitro

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Yajing Huang ◽  
Yanming Wu ◽  
Xinwen Chang ◽  
Yan Li ◽  
Kai Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Cell Function ◽  
Trophoblast Cell ◽  
Migration And Invasion ◽  
Human Umbilical Cord ◽  
Trophoblast Cells ◽  
Human Trophoblast ◽  
Cell Functions

Trophoblast cell dysfunction is involved in many disorders during pregnancy such as preeclampsia and intrauterine growth restriction. Few treatments exist, however, that target improving trophoblast cell function. Human umbilical cord mesenchymal stem cells (hUCMSCs) are capable of self-renewing, can undergo multilineage differentiation, and have homing abilities; in addition, they have immunomodulatory effects and paracrine properties and thus are a prospective source for cell therapy. To identify whether hUCMSCs can regulate trophoblast cell functions, we treated trophoblast cells with hUCMSC supernatant or cocultured them with hUCMSCs. Both treatments remarkably enhanced the migration and invasion abilities of trophoblast cells and upregulated their proliferation ability. At a certain concentration, hUCMSCs also modulated hCG, PIGF, and sEndoglin levels in the trophoblast culture medium. Thus, hUCMSCs have a positive effect on trophoblast cellular functions, which may provide a new avenue for treatment of placenta-related diseases during pregnancy.

scholarly journals The depletion of MARVELD1 leads to placenta accreta via integrin β4-dependent trophoblast cell invasion

2017 ◽  
Author(s):  
Yue Chen ◽  
Hui Zhang ◽  
Fang Han ◽  
Lei Yue ◽  
Chunxiao Qiao ◽  
...  
Keyword(s):  
Cell Invasion ◽  
Placenta Accreta ◽  
Wild Type Mouse ◽  
Trophoblast Cell ◽  
Trophoblast Invasion ◽  
Trophoblast Cells ◽  
Integrin Β4 ◽  
Underlying Mechanisms

AstractThe mammalian placenta is a remarkable organ. It serves as the interface between the mother and the fetus. Proper invasion of trophoblast cells into the maternal decidua is required for a successful pregnancy. Previous studies have found that the adhesion molecule integrin β4 plays important roles during trophoblast cell invasion. Here, we found that the overall birth rate of the MARVELD1 knockout mouse is much lower than that of the wild-type mouse (P<0.001). In E18.5 MARVELD1 knockout mice, we observed an over-invasion of trophoblast cells, and indeed, the pregnant mice had a partial placenta accreta phenotype. The HTR8/SVneo cell line was used as an in vitro model to elucidate the underlying mechanisms of MARVELD1-mediated trophoblast invasion. We detected a diminished expression of integrin β4 upon the downregulation of MARVELD1 and enhanced migration and invasive abilities of trophoblast cells both in vivo and in vitro. The integrin β4 rescue assay also supported the results. In conclusion, this study found that MARVELD1 mediated the invasion of trophoblast cells via regulating the expression of integrin β4.

scholarly journals PM10 Alters Trophoblast Cell Function and Modulates miR-125b-5p Expression

2022 ◽  
Vol 2022 ◽  
pp. 1-11
Author(s):  
Wittaya Chaiwangyen ◽  
Komsak Pintha ◽  
Payungsak Tantipaiboonwong ◽  
Piyawan Nuntaboon ◽  
Orawan Khantamat ◽  
...  
Keyword(s):  
Mirna Expression ◽  
Cell Function ◽  
Premature Mortality ◽  
Trophoblast Cell ◽  
Migration And Invasion ◽  
Trophoblast Cells ◽  
High Concentration ◽  
Particulate Pollution ◽  
Cell Functions ◽  
Pollution Exposure

Air pollution is one of the largest global environmental health hazards that threaten premature mortality or morbidity. Particulate matter 10 (PM10) has been demonstrated to contribute to several human diseases via dysregulated miRNA expression. Trophoblast cells play a key role in implantation and placentation for a successful pregnancy. Nonetheless, the PM10 associated trophoblast cell functions during pregnancy and miRNA expression are still unknown. Our study showed that PM10 affected HTR-8/SVneo cell viability and also decreased cell proliferation, migration, and invasion. A high concentration of PM10 caused an increase in HTR-8/SVneo cell apoptosis. Treatment with PM10 induced inflammation through the upregulated IL-1β, IL-6, and TNF-α expression in trophoblast cells. In PM10-treated HTR-8/SVneo cells, miR-125b-5p expression was considerably increased and TXNRD1 was found to be negatively related to miR-125b-5p. Collectively, our findings revealed that PM10 could alter miR-125b-5p expression by targeting TXNRD1 and suppressing trophoblast cell functions. Additional investigations relating to the function of miR-125b-5p and its target on particulate pollution exposure in trophoblast are warranted for future biomarker or effective therapeutic approaches.

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