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Sample records for human trophoblast differentiation

  1. Punicalagin promotes human villous trophoblast differentiation.

    PubMed

    Chen, Baosheng; Longtine, Mark S; Costa, Maria Laura; Nelson, D Michael

    2016-08-01

    Poor differentiation of trophoblasts is associated with placental dysfunction, predisposing women to multiple pregnancy disorders. Punicalagin, a prominent ellagitannin in pomegranate juice has been shown to exert anti-apoptosis and anti-oxidative effects in human trophoblasts. We hypothesized that punicalagin modulates trophoblast differentiation. We found that punicalagin-treated primary trophoblast showed reduced E-cadherin, higher Syncytin 1, more β-hCG, and increased GCM1, an upstream regulator of β-hCG. Punicalagin exposure of villous explants enhanced the number of cytotrophoblasts expressing the proliferation marker Ki67. We conclude that punicalagin enhances trophoblast differentiation and speculate that punicalagin might be used therapeutically in pregnancies at risk for placental dysfunction. PMID:27452441

  2. BROMODICHLOROMETHANE INHIBITS HUMAN PLACENTAL TROPHOBLAST DIFFERENTIATION

    EPA Science Inventory

    BROMODICHLOROMETHANE INHIBITS HUMAN PLACENTAL
    TROPHOBLAST DIFFERENTIATION
    Jiangang Chen, Twanda L. Thirkill, Peter N. Lohstroh, Susan R. Bielmeier, Michael
    G. Narotsky, Deborah S. Best, Randy A. Harrison, Kala Natarajan, Rex A. Pegram,
    Bill L. Lasley, and Gordon C. Do...

  3. Formaldehyde Crosses the Human Placenta and Affects Human Trophoblast Differentiation and Hormonal Functions.

    PubMed

    Pidoux, Guillaume; Gerbaud, Pascale; Guibourdenche, Jean; Thérond, Patrice; Ferreira, Fatima; Simasotchi, Christelle; Evain-Brion, Danièle; Gil, Sophie

    2015-01-01

    The chorionic villus of the human placenta is the source of specific endocrine functions and nutrient exchanges. These activities are ensured by the syncytiotrophobast (ST), which bathes in maternal blood. The ST arises and regenerates throughout pregnancy by fusion of underlying cytotrophoblasts (CT). Any anomaly of ST formation or regeneration can affect pregnancy outcome and fetal growth. Because of its direct interaction with maternal blood, the ST is sensitive to drugs, pollutants and xenohormones. Ex vivo assays of perfused cotyledon show that formaldehyde, a common pollutant present in furniture, paint and plastics, can accumulate in the human placenta and cross to the fetal compartment. By means of RT-qPCR, immunoblot and immunocytochemistry experiments, we demonstrate in vitro that formaldehyde exerts endocrine toxicity on human trophoblasts, including a decrease in the production of protein hormones of pregnancy. In addition, formaldehyde exposure triggered human trophoblast fusion by upregulating syncitin-1 receptor expression (ASC-type amino-acid transporter 2: ASCT2). Moreover, we show that formaldehyde-exposed trophoblasts present an altered redox status associated with oxidative stress, and an increase in ASCT2 expression intended to compensate for this stress. Finally, we demonstrate that the adverse effects of formaldehyde on trophoblast differentiation and fusion are reversed by N-acetyl-L-cysteine (Nac), an antioxidant.

  4. Formaldehyde Crosses the Human Placenta and Affects Human Trophoblast Differentiation and Hormonal Functions

    PubMed Central

    Pidoux, Guillaume; Gerbaud, Pascale; Guibourdenche, Jean; Thérond, Patrice; Ferreira, Fatima; Simasotchi, Christelle; Evain-Brion, Danièle; Gil, Sophie

    2015-01-01

    The chorionic villus of the human placenta is the source of specific endocrine functions and nutrient exchanges. These activities are ensured by the syncytiotrophobast (ST), which bathes in maternal blood. The ST arises and regenerates throughout pregnancy by fusion of underlying cytotrophoblasts (CT). Any anomaly of ST formation or regeneration can affect pregnancy outcome and fetal growth. Because of its direct interaction with maternal blood, the ST is sensitive to drugs, pollutants and xenohormones. Ex vivo assays of perfused cotyledon show that formaldehyde, a common pollutant present in furniture, paint and plastics, can accumulate in the human placenta and cross to the fetal compartment. By means of RT-qPCR, immunoblot and immunocytochemistry experiments, we demonstrate in vitro that formaldehyde exerts endocrine toxicity on human trophoblasts, including a decrease in the production of protein hormones of pregnancy. In addition, formaldehyde exposure triggered human trophoblast fusion by upregulating syncitin-1 receptor expression (ASC-type amino-acid transporter 2: ASCT2). Moreover, we show that formaldehyde-exposed trophoblasts present an altered redox status associated with oxidative stress, and an increase in ASCT2 expression intended to compensate for this stress. Finally, we demonstrate that the adverse effects of formaldehyde on trophoblast differentiation and fusion are reversed by N-acetyl-L-cysteine (Nac), an antioxidant. PMID:26186596

  5. Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease.

    PubMed

    Horii, Mariko; Li, Yingchun; Wakeland, Anna K; Pizzo, Donald P; Nelson, Katharine K; Sabatini, Karen; Laurent, Louise Chang; Liu, Ying; Parast, Mana M

    2016-07-01

    Trophoblast is the primary epithelial cell type in the placenta, a transient organ required for proper fetal growth and development. Different trophoblast subtypes are responsible for gas/nutrient exchange (syncytiotrophoblasts, STBs) and invasion and maternal vascular remodeling (extravillous trophoblasts, EVTs). Studies of early human placental development are severely hampered by the lack of a representative trophoblast stem cell (TSC) model with the capacity for self-renewal and the ability to differentiate into both STBs and EVTs. Primary cytotrophoblasts (CTBs) isolated from early-gestation (6-8 wk) human placentas are bipotential, a phenotype that is lost with increasing gestational age. We have identified a CDX2(+)/p63(+) CTB subpopulation in the early postimplantation human placenta that is significantly reduced later in gestation. We describe a reproducible protocol, using defined medium containing bone morphogenetic protein 4 by which human pluripotent stem cells (hPSCs) can be differentiated into CDX2(+)/p63(+) CTB stem-like cells. These cells can be replated and further differentiated into STB- and EVT-like cells, based on marker expression, hormone secretion, and invasive ability. As in primary CTBs, differentiation of hPSC-derived CTBs in low oxygen leads to reduced human chorionic gonadotropin secretion and STB-associated gene expression, instead promoting differentiation into HLA-G(+) EVTs in an hypoxia-inducible, factor-dependent manner. To validate further the utility of hPSC-derived CTBs, we demonstrated that differentiation of trisomy 21 (T21) hPSCs recapitulates the delayed CTB maturation and blunted STB differentiation seen in T21 placentae. Collectively, our data suggest that hPSCs are a valuable model of human placental development, enabling us to recapitulate processes that result in both normal and diseased pregnancies. PMID:27325764

  6. Direct Involvement of HERV-W Env Glycoprotein in Human Trophoblast Cell Fusion and Differentiation

    PubMed Central

    Frendo, Jean-Louis; Olivier, Delphine; Cheynet, Valérie; Blond, Jean-Luc; Bouton, Olivier; Vidaud, Michel; Rabreau, Michèle; Evain-Brion, Danièle; Mallet, François

    2003-01-01

    We recently demonstrated that the product of the HERV-W env gene, a retroviral envelope protein also dubbed syncytin, is a highly fusogenic membrane glycoprotein inducing the formation of syncytia on interaction with the type D mammalian retrovirus receptor. In addition, the detection of HERV-W Env protein (Env-W) expression in placental tissue sections led us to propose a role for this fusogenic glycoprotein in placenta formation. To evaluate this hypothesis, we analyzed the involvement of Env-W in the differentiation of primary cultures of human villous cytotrophoblasts that spontaneously differentiate by cell fusion into syncytiotrophoblasts in vitro. First, we observed that HERV-W env mRNA and glycoprotein expression are colinear with primary cytotrophoblast differentiation and with expression of human chorionic gonadotropin (hCG), a marker of syncytiotrophoblast formation. Second, we observed that in vitro stimulation of trophoblast cell fusion and differentiation by cyclic AMP is also associated with a concomitant increase in HERV-W env and hCG mRNA and protein expression. Finally, by using specific antisense oligonucleotides, we demonstrated that inhibition of Env-W protein expression leads to a decrease of trophoblast fusion and differentiation, with the secretion of hCG in culture medium of antisense oligonucleotide-treated cells being decreased by fivefold. Taken together, these results strongly support a direct role for Env-W in human trophoblast cell fusion and differentiation. PMID:12724415

  7. Human monocytes undergo functional re-programming during differentiation to dendritic cell mediated by human extravillous trophoblasts.

    PubMed

    Zhao, Lei; Shao, Qianqian; Zhang, Yun; Zhang, Lin; He, Ying; Wang, Lijie; Kong, Beihua; Qu, Xun

    2016-01-01

    Maternal immune adaptation is required for a successful pregnancy to avoid rejection of the fetal-placental unit. Dendritic cells within the decidual microenvironment lock in a tolerogenic profile. However, how these tolerogenic DCs are induced and the underlying mechanisms are largely unknown. In this study, we show that human extravillous trophoblasts redirect the monocyte-to-DC transition and induce regulatory dendritic cells. DCs differentiated from blood monocytes in the presence of human extravillous trophoblast cell line HTR-8/SVneo displayed a DC-SIGN(+)CD14(+)CD1a(-) phenotype, similar with decidual DCs. HTR8-conditioned DCs were unable to develop a fully mature phenotype in response to LPS, and altered the cytokine secretory profile significantly. Functionally, conditioned DCs poorly induced the proliferation and activation of allogeneic T cells, whereas promoted CD4(+)CD25(+)Foxp3(+) Treg cells generation. Furthermore, the supernatant from DC and HTR-8/SVneo coculture system contained significant high amount of M-CSF and MCP-1. Using neutralizing antibodies, we discussed the role of M-CSF and MCP-1 during monocyte-to-DCs differentiation mediated by extravillous trophoblasts. Our data indicate that human extravillous trophoblasts play an important role in modulating the monocyte-to-DC differentiation through M-CSF and MCP-1, which facilitate the establishment of a tolerogenic microenvironment at the maternal-fetal interface. PMID:26857012

  8. Human monocytes undergo functional re-programming during differentiation to dendritic cell mediated by human extravillous trophoblasts

    PubMed Central

    Zhao, Lei; Shao, Qianqian; Zhang, Yun; Zhang, Lin; He, Ying; Wang, Lijie; Kong, Beihua; Qu, Xun

    2016-01-01

    Maternal immune adaptation is required for a successful pregnancy to avoid rejection of the fetal–placental unit. Dendritic cells within the decidual microenvironment lock in a tolerogenic profile. However, how these tolerogenic DCs are induced and the underlying mechanisms are largely unknown. In this study, we show that human extravillous trophoblasts redirect the monocyte-to-DC transition and induce regulatory dendritic cells. DCs differentiated from blood monocytes in the presence of human extravillous trophoblast cell line HTR-8/SVneo displayed a DC-SIGN+CD14+CD1a− phenotype, similar with decidual DCs. HTR8-conditioned DCs were unable to develop a fully mature phenotype in response to LPS, and altered the cytokine secretory profile significantly. Functionally, conditioned DCs poorly induced the proliferation and activation of allogeneic T cells, whereas promoted CD4+CD25+Foxp3+ Treg cells generation. Furthermore, the supernatant from DC and HTR-8/SVneo coculture system contained significant high amount of M-CSF and MCP-1. Using neutralizing antibodies, we discussed the role of M-CSF and MCP-1 during monocyte-to-DCs differentiation mediated by extravillous trophoblasts. Our data indicate that human extravillous trophoblasts play an important role in modulating the monocyte-to-DC differentiation through M-CSF and MCP-1, which facilitate the establishment of a tolerogenic microenvironment at the maternal–fetal interface. PMID:26857012

  9. Trophoblast lineage cells derived from human induced pluripotent stem cells

    SciTech Connect

    Chen, Ying; Wang, Kai; Chandramouli, Gadisetti V.R.; Knott, Jason G.; Leach, Richard

    2013-07-12

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

  10. Hypericum caprifoliatum and Hypericum connatum affect human trophoblast-like cells differentiation and Ca2+ influx

    PubMed Central

    da Conceição, Aline O.; von Poser, Gilsane Lino; Barbeau, Benoit; Lafond, Julie

    2014-01-01

    Objective To study the effect of crude methanol and n-hexane extracts of Hypericum connatum (H. connatum) and Hypericum caprifoliatum on trophoblast-like cells. Methods BeWo and JEG-3 trophoblast-like cells were submitted to different extract concentrations (1, 5, 10 and 15 µg/mL) and evaluated in relation to cell viability and in vitro trophoblast differentiation and function. Cell viability was evaluated using WST-1 reagent. Differentiation was measured by luciferase production, hCG production/release, and mitogen-activated protein kinase signaling pathway activation. The function of the trophoblast-like cells was measured by 45Ca2+ influx evaluation. Results The results showed a decrease in cell viability/proliferation. Both plants and different extracts induced a significant decrease in hCG production/release and luciferase production. H. connatum did not cause mitogen-activated protein kinase signaling pathway disturbance; however, Hypericum caprifoliatum n-hexane extract at 15 µg/mL inhibited extracellular signal-regulated kinase 1/2 activation. The significant increase in Ca2+ influx by JEG-3 cells was seen after short and long incubation times with H. connatum methanolic extract at 15 µg/mL. Conclusions The results indicated that these two Hypericum species extracts can interfere on trophoblast differentiation and Ca2+ influx, according to their molecular diversity. Although in vivo experiments are necessary to establish their action on placental formation and function, this study suggests that attention must be paid to the potential toxic effect of these plants. PMID:25182721

  11. Identification of Epigenetic Factor Proteins Expressed in Human Embryonic Stem Cell-Derived Trophoblasts and in Human Placental Trophoblasts.

    PubMed

    Sarkar, Prasenjit; Mischler, Adam; Randall, Shan M; Collier, Timothy S; Dorman, Karen F; Boggess, Kim A; Muddiman, David C; Rao, Balaji M

    2016-08-01

    Human embryonic stem cells (hESCs) have been used to derive trophoblasts through differentiation in vitro. Intriguingly, mouse ESCs are prevented from differentiation to trophoblasts by certain epigenetic factor proteins such as Dnmt1, thus necessitating the study of epigenetic factor proteins during hESC differentiation to trophoblasts. We used stable isotope labeling by amino acids in cell culture and quantitative proteomics to study changes in the nuclear proteome during hESC differentiation to trophoblasts and identified changes in the expression of 30 epigenetic factor proteins. Importantly, the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B were downregulated. Additionally, we hypothesized that nuclear proteomics of hESC-derived trophoblasts may be used for screening epigenetic factor proteins expressed by primary trophoblasts in human placental tissue. Accordingly, we conducted immunohistochemistry analysis of six epigenetic factor proteins identified from hESC-derived trophoblasts-DNMT1, DNMT3B, BAF155, BAF60A, BAF57, and ING5-in 6-9 week human placentas. Indeed, expression of these proteins was largely, though not fully, consistent with that observed in 6-9 week placental trophoblasts. Our results support the use of hESC-derived trophoblasts as a model for placental trophoblasts, which will enable further investigation of epigenetic factors involved in human trophoblast development. PMID:27378238

  12. Gestational protein restriction affects trophoblast differentiation.

    PubMed

    Gao, Haijun; Yallampalli, Uma; Yallampalli, Chandra

    2013-01-01

    Whether and how gestational protein restriction (PR) affects placental development and function remain unknown. To test the hypothesis that PR can affect trophoblast differentiation in mid-and late pregnancy, rats were fed a 20% or an isocaloric 6% protein diet from Day 1 to 14 or 18 of pregnancy and effects of PR on trophoblast differentiation were determined by changes in expressions of marker gene(s) for trophoblast lineages. At Day 18 of pregnancy, PR increased expressions of Esrrb, Id1 andId2 (trophoblast stem cell markers), decreased expressions of Ascl2 (spongiotrophblast cell marker) and Prl2c1 (trophoblast giant cell marker), but did not alter expressions of Gjb3 and Pcdh12(glycogen cell markers) in the junctional zone (JZ). In the labyrinth zone (LZ), PR did not change expressions of Prl2b1 (trophoblast giant cell marker), Gcm1 and Syna (syncytiotrophoblast cell markers), but decrease expression of Ctsq (sinusoidal trophoblast giant cell marker). These results indicate that PR impairs the differentiation of trophoblast stem cell into spongiotrophoblast and trophoblast giant cells in JZ, and formation of sinusoidal trophoblast giant cells in LZ.

  13. Potential role of epigenetic mechanisms in regulation of trophoblast differentiation, migration, and invasion in the human placenta

    PubMed Central

    Kohan-Ghadr, Hamid-Reza; Kadam, Leena; Jain, Chandni; Armant, D. Randall; Drewlo, Sascha

    2016-01-01

    ABSTRACT The proper establishment and organogenesis of the placenta is crucial for intrauterine fetal growth and development. Endometrial invasion by the extravillous trophoblast cells, as well as formation of the syncytiotrophoblast (STB), are of vital importance for placental function. Trophoblast migration and invasion is often compared to tumor metastasis, which uses many of the same molecular mechanisms. However, unlike cancer cells, both initiation and the extent of trophoblast invasion are tightly regulated by feto-maternal cross-talk, which when perturbed, results in a wide range of abnormalities. Multiple factors control the trophoblast, including cytokines and hormones, which are subject to transcriptional regulatory networks. The relevance of epigenetics in transcriptional regulation of trophoblast differentiation and invasion, as well as in the onset of placenta-related pregnancy disorders, became recognized decades ago. Although, there has been tremendous progress in uncovering the molecular foundation of placental development, there is still much to be learned about the epigenetic machinery, and its role in trophoblast differentiation and invasion. This review will provide an overview of the epigenetic control of trophoblast differentiation and invasion. It will also highlight the major epigenetic mechanisms involved in pregnancy complications related to placental deficiencies. PMID:26745760

  14. Neuregulin-1-mediated ErbB2-ErbB3 signalling protects human trophoblasts against apoptosis to preserve differentiation.

    PubMed

    Fock, Valerie; Plessl, Kerstin; Draxler, Peter; Otti, Gerlinde Regina; Fiala, Christian; Knöfler, Martin; Pollheimer, Jürgen

    2015-12-01

    During placentation, foetal trophoblasts invade deeply into maternal tissue to establish a foeto-maternal circulation. We have previously shown that extravillous trophoblast (EVT) lineage cells express ErbB2 and ErbB3, of which the potential as an oncogenic unit is well established. However, a physiological function of this receptor combination in humans remains a puzzling question. Here, we demonstrate neuregulin 1 (NRG1) expression and secretion by human decidual stromal cells. Stimulation of human primary trophoblasts with exogenous NRG1 induced phosphorylation of ErbB2, ErbB3 and related downstream effectors. Co-immunoprecipitation experiments confirmed the formation of ErbB2-ErbB3 dimers upon ligand engagement. Along this line, receptor knockdown and ErbB3 neutralization strongly diminished NRG1-dependent activation of the signalling complex. Functional studies revealed that NRG1 promotes EVT formation in placental explant cultures. Although, in the presence of NRG1, basal and camptothecin-induced trophoblast apoptosis was significantly repressed, this effect was abolished upon ErbB3 inhibition. Notably, camptothecin provoked a strong reduction of trophoblast cell column size, whereas NRG1-treated explants were refractory to the compound. Taken together, our findings newly identify a physiological function of the NRG1-ErbB2-ErbB3 axis in trophoblast survival during human placental development. PMID:26490994

  15. What Is Trophoblast? A Combination of Criteria Define Human First-Trimester Trophoblast.

    PubMed

    Lee, Cheryl Q E; Gardner, Lucy; Turco, Margherita; Zhao, Nancy; Murray, Matthew J; Coleman, Nicholas; Rossant, Janet; Hemberger, Myriam; Moffett, Ashley

    2016-02-01

    Controversy surrounds reports describing the derivation of human trophoblast cells from placentas and embryonic stem cells (ESC), partly due to the difficulty in identifying markers that define cells as belonging to the trophoblast lineage. We have selected criteria that are characteristic of primary first-trimester trophoblast: a set of protein markers, HLA class I profile, methylation of ELF5, and expression of microRNAs (miRNAs) from the chromosome 19 miRNA cluster (C19MC). We tested these criteria on cells previously reported to show some phenotypic characteristics of trophoblast: bone morphogenetic protein (BMP)-treated human ESC and 2102Ep, an embryonal carcinoma cell line. Both cell types only show some, but not all, of the four trophoblast criteria. Thus, BMP-treated human ESC have not fully differentiated to trophoblast. Our study identifies a robust panel, including both protein and non-protein-coding markers that, in combination, can be used to reliably define cells as characteristic of early trophoblast. PMID:26862703

  16. What Is Trophoblast? A Combination of Criteria Define Human First-Trimester Trophoblast

    PubMed Central

    Lee, Cheryl Q.E.; Gardner, Lucy; Turco, Margherita; Zhao, Nancy; Murray, Matthew J.; Coleman, Nicholas; Rossant, Janet; Hemberger, Myriam; Moffett, Ashley

    2016-01-01

    Summary Controversy surrounds reports describing the derivation of human trophoblast cells from placentas and embryonic stem cells (ESC), partly due to the difficulty in identifying markers that define cells as belonging to the trophoblast lineage. We have selected criteria that are characteristic of primary first-trimester trophoblast: a set of protein markers, HLA class I profile, methylation of ELF5, and expression of microRNAs (miRNAs) from the chromosome 19 miRNA cluster (C19MC). We tested these criteria on cells previously reported to show some phenotypic characteristics of trophoblast: bone morphogenetic protein (BMP)-treated human ESC and 2102Ep, an embryonal carcinoma cell line. Both cell types only show some, but not all, of the four trophoblast criteria. Thus, BMP-treated human ESC have not fully differentiated to trophoblast. Our study identifies a robust panel, including both protein and non-protein-coding markers that, in combination, can be used to reliably define cells as characteristic of early trophoblast. PMID:26862703

  17. Triploid human embryonic stem cells derived from tripronuclear zygotes displayed pluripotency and trophoblast differentiation ability similar to the diploid human embryonic stem cells.

    PubMed

    Rungsiwiwut, Ruttachuk; Numchaisrika, Pranee; Ahnonkitpanit, Vichuda; Virutamasen, Pramuan; Pruksananonda, Kamthorn

    2016-04-22

    Because the diploid human embryonic stem cells (hESCs) can be successfully derived from tripronuclear zygotes thus, they can serve as an alternative source of derivation of normal karyotype hESC lines. The aim of the present study was to compare the pluripotency and trophoblast differentiation ability of hESCs derived from tripronuclear zygotes and diploid hESCs. In the present study, a total of 20 tripronuclear zygotes were cultured; 8 zygotes developed to the blastocyst stage and 1 hESC line was generated. Unlike the previous studies, chromosomal correction of tripronuclear zygotes during derivation of hESCs did not occur. The established line carries 3 sets of chromosomes and showed a numerical aberration. Although the cell line displayed an abnormal chromosome number, it was found the cell line has been shown to be pluripotent with the ability to differentiate into 3 embryonic germ layers both in vitro and in vivo. The expression of X inactive specific transcript (XIST) in mid-passage (passage 42) of undifferentiated triploid hESCs was detected, indicating X chromosome inactivation of the cell line. Moreover, when this cell line was induced to differentiate toward the trophoblast lineage, morphological and functional trophoblast cells were observed, similar to the diploid hESC line. PMID:26821869

  18. Cell Adhesion Minimization by a Novel Mesh Culture Method Mechanically Directs Trophoblast Differentiation and Self-Assembly Organization of Human Pluripotent Stem Cells

    PubMed Central

    Kurosawa, Osamu; Yamazaki, Satoshi; Oana, Hidehiro; Kotera, Hidetoshi; Nakauchi, Hiromitsu; Washizu, Masao

    2015-01-01

    Mechanical methods for inducing differentiation and directing lineage specification will be instrumental in the application of pluripotent stem cells. Here, we demonstrate that minimization of cell-substrate adhesion can initiate and direct the differentiation of human pluripotent stem cells (hiPSCs) into cyst-forming trophoblast lineage cells (TLCs) without stimulation with cytokines or small molecules. To precisely control cell-substrate adhesion area, we developed a novel culture method where cells are cultured on microstructured mesh sheets suspended in a culture medium such that cells on mesh are completely out of contact with the culture dish. We used microfabricated mesh sheets that consisted of open meshes (100∼200 μm in pitch) with narrow mesh strands (3–5 μm in width) to provide support for initial cell attachment and growth. We demonstrate that minimization of cell adhesion area achieved by this culture method can trigger a sequence of morphogenetic transformations that begin with individual hiPSCs attached on the mesh strands proliferating to form cell sheets by self-assembly organization and ultimately differentiating after 10–15 days of mesh culture to generate spherical cysts that secreted human chorionic gonadotropin (hCG) hormone and expressed caudal-related homeobox 2 factor (CDX2), a specific marker of trophoblast lineage. Thus, this study demonstrates a simple and direct mechanical approach to induce trophoblast differentiation and generate cysts for application in the study of early human embryogenesis and drug development and screening. PMID:25914965

  19. Trophoblast stem cells differentiate in vitro into invasive trophoblast giant cells.

    PubMed

    Hemberger, Myriam; Hughes, Martha; Cross, James C

    2004-07-15

    Trophoblast cells are characterized by an invasive behavior into the surrounding uterine tissue. In rodents, an early peri-/endovascular type of invasion exerted by trophoblast giant cells can be distinguished from a late interstitial type carried out by glycogen trophoblast cells. Analysis of the molecular mechanisms of trophoblast invasion has been hampered, however, by the complex temporal and spatial patterns of invasion. We utilized trophoblast stem (TS) cell lines to study trophoblast invasion in vitro and to establish a model that facilitates investigation of this process on the molecular level. Our results showed that trophoblast giant cells that differentiate from TS cells in vitro are capable of penetrating a reconstituted basement membrane matrix. Consequently, invasion rates were increased in various giant cell differentiation-promoting conditions. We also derived TS cell lines that are homozygous for a mutation of the Hand1 transcription factor. The Hand1-/- TS cells showed reduced levels of giant cell differentiation and exhibited an approximately 50% decrease in invasion rates. In summary, trophoblast giant cells that differentiate from TS cells in vitro recapitulate the invasive capacity of normal trophoblast cells in vivo. The TS cell system is a valuable tool to identify and quantitatively study regulators of trophoblast invasion.

  20. Roles of CDX2 and EOMES in human induced trophoblast progenitor cells

    SciTech Connect

    Chen, Ying; Wang, Kai; Gong, Yun Guo; Khoo, Sok Kean; Leach, Richard

    2013-02-08

    Highlights: ► CDX2 and EOMES play critical roles in human induced trophoblast progenitors (iTP). ► iTP cells directly transformed from fibroblasts. ► Differentiation of iTP cells into extravillous trophoblasts and syncytiotrophoblasts. -- Abstract: Abnormal trophoblast lineage proliferation and differentiation in early pregnancy have been associated with the pathogenesis of placenta diseases of pregnancy. However, there is still a gap in understanding the molecular mechanisms of early placental development due to the limited primary trophoblast cultures and fidelity of immortalized trophoblast lines. Trophoblasts stem (TS) cells, an in vitro model of trophectoderm that can differentiate into syncytiotrophoblasts and extravillous trophoblasts, can be an attractive tool for early pregnancy research. TS cells are well established in mouse but not in humans due to insufficient knowledge of which trophoblast lineage-specific transcription factors are involved in human trophectoderm (TE) proliferation and differentiation. Here, we applied induced pluripotent stem cell technique to investigate the human trophoblast lineage-specific transcription factors. We established human induced trophoblast progenitor (iTP) cells by direct reprogramming the fibroblasts with a pool of mouse trophoblast lineage-specific transcription factors consisting of CDX2, EOMES, and ELF5. The human iTP cells exhibit epithelial morphology and can be maintained in vitro for more than 2 months. Gene expression profile of these cells was tightly clustered with human trophectoderm but not with human neuron progenitor cells, mesenchymal stem cells, or endoderm cells. These cells are capable of differentiating into cells with an invasive capacity, suggesting extravillous trophoblasts. They also form multi-nucleated cells which secrete human chorionic gonadotropin and estradiol, consistent with a syncytiotrophoblast phenotype. Our results provide the evidence that transcription factors CDX2 and

  1. Effect of Microcystin-LR on human placental villous trophoblast differentiation in vitro

    EPA Science Inventory

    Microcystin-LR is a cyanobacterial toxin found in surface and recreational waters that inhibits protein phosphatases and may disrupt the cytoskeleton. Microcystins induce apoptosis in hepatocytes at ≤2.0 μM. Nothing is known about the effects of microcystins on human placental tr...

  2. Differential effect of DDT, DDE, and DDD on COX-2 expression in the human trophoblast derived HTR-8/SVneo cells.

    PubMed

    Dominguez-Lopez, Pablo; Diaz-Cueto, Laura; Olivares, Aleida; Ulloa-Aguirre, Alfredo; Arechavaleta-Velasco, Fabian

    2012-11-01

    The purpose of this study was to investigate the effect of 1,1,1-trichloro-2,2-bis-(chlorophenyl)ethane (DDT), 1,1-bis-(chlorophenyl)-2,2-dichloroethene (DDE), and 1,1-dichloro-2,2-bis(chlorophenyl)ethane (DDD) isomers on COX-2 expression in a human trophoblast-derived cell line. Cultured HTR-8/SVneo trophoblast cells were exposed to DDT isomers and its metabolites for 24 h, and COX-2 mRNA and protein expression were assessed by RT-PCR, Western blotting, and ELISA. Prostaglandin E₂ production was also measured by ELISA. Both COX-2 mRNA and protein were detected under control (unexposed) conditions in the HTR-8/SVneo cell line. COX-2 protein expression and prostaglandin E₂ production but not COX-2 mRNA levels increased only after DDE and DDD isomers exposure. It is concluded that DDE and DDD exposure induce the expression of COX-2 protein, leading to increased prostaglandin E2 production. Interestingly, the regulation of COX-2 by these organochlorines pesticides appears to be at the translational level.

  3. Evidence for Differential Glycosylation of Trophoblast Cell Types.

    PubMed

    Chen, Qiushi; Pang, Poh-Choo; Cohen, Marie E; Longtine, Mark S; Schust, Danny J; Haslam, Stuart M; Blois, Sandra M; Dell, Anne; Clark, Gary F

    2016-06-01

    Human placental villi are surfaced by the syncytiotrophoblast (STB), with a layer of cytotrophoblasts (CTB) positioned just beneath the STB. STB in normal term pregnancies is exposed to maternal immune cells in the placental intervillous space. Extravillous cytotrophoblasts (EVT) invade the decidua and spiral arteries, where they act in conjunction with natural killer (NK) cells to convert the spiral arteries into flaccid conduits for maternal blood that support a 3-4 fold increase in the rate of maternal blood flow into the placental intervillous space. The functional roles of these distinct trophoblast subtypes during pregnancy suggested that they could be differentially glycosylated. Glycomic analysis of these trophoblasts has revealed the expression of elevated levels of biantennary N-glycans in STB and CTB, with the majority of them bearing a bisecting GlcNAc. N-glycans terminated with polylactosamine extensions were also detected at low levels. A subset of the N-glycans linked to these trophoblasts were sialylated, primarily with terminal NeuAcα2-3Gal sequences. EVT were decorated with the same N-glycans as STB and CTB, except in different proportions. The level of bisecting type N-glycans was reduced, but the level of N-glycans decorated with polylactosamine sequences were substantially elevated compared with the other types of trophoblasts. The level of triantennary and tetraantennary N-glycans was also elevated in EVT. The sialylated N-glycans derived from EVT were completely susceptible to an α2-3 specific neuraminidase (sialidase S). The possibility exists that the N-glycans associated with these different trophoblast subpopulations could act as functional groups. These potential relationships will be considered. PMID:26929217

  4. Neuregulin-1-mediated ErbB2–ErbB3 signalling protects human trophoblasts against apoptosis to preserve differentiation

    PubMed Central

    Fock, Valerie; Plessl, Kerstin; Draxler, Peter; Otti, Gerlinde Regina; Fiala, Christian; Knöfler, Martin; Pollheimer, Jürgen

    2015-01-01

    ABSTRACT During placentation, foetal trophoblasts invade deeply into maternal tissue to establish a foeto–maternal circulation. We have previously shown that extravillous trophoblast (EVT) lineage cells express ErbB2 and ErbB3, of which the potential as an oncogenic unit is well established. However, a physiological function of this receptor combination in humans remains a puzzling question. Here, we demonstrate neuregulin 1 (NRG1) expression and secretion by human decidual stromal cells. Stimulation of human primary trophoblasts with exogenous NRG1 induced phosphorylation of ErbB2, ErbB3 and related downstream effectors. Co-immunoprecipitation experiments confirmed the formation of ErbB2–ErbB3 dimers upon ligand engagement. Along this line, receptor knockdown and ErbB3 neutralization strongly diminished NRG1-dependent activation of the signalling complex. Functional studies revealed that NRG1 promotes EVT formation in placental explant cultures. Although, in the presence of NRG1, basal and camptothecin-induced trophoblast apoptosis was significantly repressed, this effect was abolished upon ErbB3 inhibition. Notably, camptothecin provoked a strong reduction of trophoblast cell column size, whereas NRG1-treated explants were refractory to the compound. Taken together, our findings newly identify a physiological function of the NRG1–ErbB2–ErbB3 axis in trophoblast survival during human placental development. PMID:26490994

  5. Dynamic Regulation of AP-1 Transcriptional Complexes Directs Trophoblast Differentiation

    PubMed Central

    Kent, Lindsey N.; Rumi, M. A. Karim; Roby, Katherine F.

    2015-01-01

    Placentation is a process that establishes the maternal-fetal interface and is required for successful pregnancy. The epithelial component of the placenta consists of trophoblast cells, which possess the capacity for multilineage differentiation and are responsible for placenta-specific functions. FOS-like antigen 1 (FOSL1), a component of AP-1 transcription factor complexes, contributes to the regulation of placental development. FOSL1 expression is restricted to trophoblast giant cells and invasive trophoblast cells. In the present study, we characterized the FOSL1 regulatory pathway in rat trophoblast cells. Transcriptome profiling in control and FOSL1 knockdown cells identified FOSL1-dependent gene sets linked to endocrine and invasive functions. FOSL1 was shown to occupy AP-1 binding sites within these gene loci, as determined by chromatin immunoprecipitation (ChIP). Complementary in vivo experiments using trophoblast-specific lentiviral delivery of FOSL1 short hairpin RNAs (shRNAs) provided in vivo validation of FOSL1 targets. FOSL1 actions require a dimerization partner. Coimmunoprecipitation, coimmunolocalization, and ChIP analyses showed that FOSL1 interacts with JUNB and, to a lesser extent, JUN in differentiating trophoblast cells. Knockdown of FOSL1 and JUNB expression inhibited both endocrine and invasive properties of trophoblast cells. In summary, FOSL1 recruits JUNB to form AP-1 transcriptional complexes that specifically regulate the endocrine and invasive trophoblast phenotypes. PMID:26149388

  6. Antigens of human trophoblasts: A working hypothesis for their role in normal and abnormal pregnancies

    PubMed Central

    Faulk, W. Page; Temple, Anne; Lovins, R. E.; Smith, Nancy

    1978-01-01

    This report describes the preparation and characterization of antisera to human trophoblast membranes. Rabbit antisera were raised to trophoblast microvilli prepared by differential ultracentrifugation. Antibodies to serum proteins were removed by solid-phase immunoabsorption with normal human serum, and indirect immunofluorescence experiments with cryostat sections of human placentas showed that the absorbed anti-trophoblast sera reacted with trophoblasts as well as with stromal cells and endothelium of chorionic villi. The antisera also produced membrane fluorescence when studied on viable lymphocytes and certain human cell lines. These anti-trophoblast sera were also lymphocytotoxic, and this reaction was abolished by prior absorption of the antisera with leukocytes. The leukocyte-absorbed anti-trophoblast sera retained their ability to react with trophoblasts and certain human cell lines, but no longer reacted with lymphocytes or placental stromal cells and endothelium. Two categories of trophoblast membrane antigens are thus defined: one present on trophoblasts and certain human cells lines (tentatively designated TA1), and the other on trophoblasts and lymphocytes, villous fibroblasts, and endothelium (tentatively designated TA2). A working hypothesis is proposed stating that normal pregnancy involves the generation of anti-TA2 subsequent to blastocyst implantation and entrance of trophoblasts into the maternal circulation. This involves a mechanism similar to allogeneic cell stimulation and results in antibodies that block either the recognition or cytotoxicity of TA1. Failure to mount this response allows TA1 recognition and trophoblast immunopathology. Experimental and clinical studies in support of this working hypothesis, particularly involving abortion and toxemia, are cited from published reports. Images PMID:273921

  7. Expression of uPAR in human trophoblast and its role in trophoblast invasion

    PubMed Central

    Liu, Shuai; Zheng, Qin; Cui, Xin-Yuan; Dai, Kui-Xing; Yang, Xue-Song; Li, Fa-Sheng; Yan, Qiu

    2015-01-01

    Placental trophoblast cells differentiate into invasive trophoblasts or syncytiotrophoblasts. Abnormal trophoblast invasion results in pregnancy-associated disease and abortion. uPAR is a cell membrane-bound glycosylated protein, involved in physiological and pathological processes. However, uPAR expression in villi during threatened abortion and its role in trophoblast differentiation are unclear. We determined that, uPAR expression in the villi was reduced in threatened abortion patients than that in normal pregnancy. uPARsiRNA inhibited the potential for trophoblast migration and invasion in explants culture and HTR8/SVneo cells. It also enhanced forskolin-induced fusion of HTR8/SVneo cells. Overall, this study provides a possible reason for abortion. PMID:26823748

  8. OVO-like 1 regulates progenitor cell fate in human trophoblast development

    PubMed Central

    Renaud, Stephen J.; Chakraborty, Damayanti; Mason, Clifford W.; Rumi, M. A. Karim; Vivian, Jay L.; Soares, Michael J.

    2015-01-01

    Epithelial barrier integrity is dependent on progenitor cells that either divide to replenish themselves or differentiate into a specialized epithelium. This paradigm exists in human placenta, where cytotrophoblast cells either propagate or undergo a unique differentiation program: fusion into an overlying syncytiotrophoblast. Syncytiotrophoblast is the primary barrier regulating the exchange of nutrients and gases between maternal and fetal blood and is the principal site for synthesizing hormones vital for human pregnancy. How trophoblast cells regulate their differentiation into a syncytium is not well understood. In this study, we show that the transcription factor OVO-like 1 (OVOL1), a homolog of Drosophila ovo, regulates the transition from progenitor to differentiated trophoblast cells. OVOL1 is expressed in human placenta and was robustly induced following stimulation of trophoblast differentiation. Disruption of OVOL1 abrogated cytotrophoblast fusion and inhibited the expression of a broad set of genes required for trophoblast cell fusion and hormonogenesis. OVOL1 was required to suppress genes that maintain cytotrophoblast cells in a progenitor state, including MYC, ID1, TP63, and ASCL2, and bound specifically to regions upstream of each of these genes. Our results reveal an important function of OVOL1 as a regulator of trophoblast progenitor cell fate during human trophoblast development. PMID:26504231

  9. MSX2 Induces Trophoblast Invasion in Human Placenta

    PubMed Central

    Lu, Junjie; Yang, Genling; Tian, Na; Wang, Xiaojie; Tan, Yi; Tan, Dongmei

    2016-01-01

    Normal implantation depends on appropriate trophoblast growth and invasion. Inadequate trophoblast invasion results in pregnancy-related disorders, such as early miscarriage and pre-eclampsia, which are dangerous to both the mother and fetus. Msh Homeobox 2 (MSX2), a member of the MSX family of homeobox proteins, plays a significant role in the proliferation and differentiation of various cells and tissues, including ectodermal organs, teeth, and chondrocytes. Recently, MSX2 was found to play important roles in the invasion of cancer cells into adjacent tissues via the epithelial-mesenchymal transition (EMT). However, the role of MSX2 in trophoblastic invasion during placental development has yet to be explored. In the present study, we detected MSX2 expression in cytotrophoblast, syncytiotrophoblast, and extravillous cytotrophoblast cells of first or third trimester human placentas via immunohistochemistry analysis. Furthermore, we found that the in vitro invasive ability of HTR8/SVneo cells was enhanced by exogenous overexpression of MSX2, and that this effect was accompanied by increased protein expression of matrix metalloproteinase-2 (MMP-2), vimentin, and β-catenin. Conversely, treatment of HTR8/SVneo cells with MSX2-specific siRNAs resulted in decreased protein expression of MMP-2, vimentin, and β-catenin, and reduced invasion levels in a Matrigel invasion test. Notably, however, treatment with the MSX2 overexpression plasmid and the MSX2 siRNAs had no effect on the mRNA expression levels of β-catenin. Meanwhile, overexpression of MSX2 and treatment with the MSX2-specific siRNA resulted in decreased and increased E-cadherin expression, respectively, in JEG-3 cells. Lastly, the protein expression levels of MSX2 were significantly lower in human pre-eclamptic placental villi than in the matched control placentas. Collectively, our results suggest that MSX2 may induce human trophoblast cell invasion, and dysregulation of MSX2 expression may be associated

  10. Intrauterine trophoblast migration: A comparative view of humans and rodents.

    PubMed

    Silva, Juneo F; Serakides, Rogéria

    2016-03-01

    Trophoblast migration and invasion through the decidua and maternal uterine spiral arteries are crucial events in placentation. During this process, invasive trophoblast replace vascular endothelial cells as the uterine arteries are remodeled to form more permissive vessels that facilitate adequate blood flow to the growing fetus. Placentation failures resulting from either extensive or shallow trophoblastic invasion can cause pregnancy complications such as preeclampsia, intrauterine growth restriction, placenta creta, gestational trophoblastic disease and even maternal or fetal death. Consequently, the use of experimental animal models such as rats and mice has led to great progress in recent years with regards to the identification of mechanisms and factors that control trophoblast migration kinetics. This review aims to perform a comparative analysis of placentation and the mechanisms and factors that coordinate intrauterine trophoblast migration in humans, rats and mice under physiological and pathological conditions.

  11. Glial cells missing homologue 1 is induced in differentiating equine chorionic girdle trophoblast cells.

    PubMed

    de Mestre, Amanda M; Miller, Donald; Roberson, Mark S; Liford, Jenny; Chizmar, Lisay C; McLaughlin, Kristin E; Antczak, Douglas F

    2009-02-01

    The objective of this study was to identify transcription factors associated with differentiation of the chorionic girdle, the invasive form of equine trophoblast. The expression patterns of five transcription factors were determined on a panel of conceptus tissues from early horse pregnancy. Tissues from Days 15 through 46 were tested. Eomesodermin (EOMES), glial cells missing homologue 1 (GCM1), heart and neural crest derivatives expressed transcript 1 (HAND1), caudal type homeobox 2 (CDX2), and distal-less homeobox 3 (DLX3) were detected in horse trophoblast, but the expression patterns for these genes varied. EOMES had the most restricted distribution, while DLX3 CDX2, and HAND1 were widely expressed. GCM1 seemed to increase in the developing chorionic girdle, and this was confirmed by quantitative RT-PCR assays. GCM1 expression preceded a striking increase in expression of equine chorionic gonadotropin beta (CGB) in the chorionic girdle, and binding sites for GCM1 were discovered in the promoter region of the CGB gene. GCM1, CGB, and CGA mRNA were expressed preferentially in binucleate cells as opposed to uninucleate cells of the chorionic girdle. Based on these findings, it is likely that GCM1 has a role in differentiation and function of the invasive trophoblast of the equine chorionic girdle and endometrial cups. The equine binucleate chorionic girdle (CG) secreting trophoblast shares molecular, morphological, and functional characteristics with human syncytiotrophoblast and represents a model for studies of human placental function.

  12. Function and control of human invasive trophoblast subtypes: Intrinsic vs. maternal control.

    PubMed

    Velicky, Philipp; Knöfler, Martin; Pollheimer, Jürgen

    2016-03-01

    The establishment of a functional placenta is pivotal for normal fetal development and the maintenance of pregnancy. In the course of early placentation, trophoblast precursors differentiate into highly invasive trophoblast subtypes. These cells, referred to as extravillous trophoblasts (EVTs), penetrate the maternal uterus reaching as far as the inner third of the myometrium. One of the most fundamental functions of EVTs is the transformation of spiral arteries to establish the uteroplacental blood circulation assuring an adequate nutrient and gas supply to the developing fetus. To achieve this, specialized EVT subpopulations interact with maternal immune cells, provoke elastolysis in the arterial wall and replace the endothelial cells lining the spiral arteries to induce intraluminal vascular remodeling. These and other trophoblast-mediated processes are tightly controlled by paracrine signals from the maternal decidua and furthermore underlie an intrinsic cell-type specific program. Various severe pregnancy complications such as preeclampsia or intrauterine growth retardation are associated with abnormal EVT function, shallow invasion, and decreased blood flow to the placenta. Hence a better understanding of human trophoblast invasion seems mandatory to improve therapeutic intervention. This approach, however, requires a profound knowledge of the human placenta, its various trophoblast subtypes and in particular a better understanding of the regulatory network that controls the invasive phenotype of EVTs. PMID:26418186

  13. Human placental trophoblasts express the immunosuppressive cytokine IL-35.

    PubMed

    Mao, Haiting; Gao, Wenjuan; Ma, Chao; Sun, Jintang; Liu, Jia; Shao, Qianqian; Song, Bingfeng; Qu, Xun

    2013-07-01

    Studies of maternal-fetal tolerance focus on defining mechanisms for establishment of immunological privilege within the uterus during pregnancy. Fetal trophoblasts play a key role in maternal tolerance, in part through cytokines production. As a novel inhibitory cytokine, IL-35 is produced by Foxp3(+) regulatory T cells (Tregs) and mediates maximal suppression of Tregs. The purpose of the study is to analyze the expression of IL-35 in first-trimester human placental trophoblasts. IL-35 expression was detected at both protein and mRNA levels by immunohistochemical staining and quantitative real-time PCR method, respectively and secretion of IL-35 was measured by ELISA assay. Our results demonstrated that human trophoblasts constitutively expressed IL-35. Ebi3 and p35 (two subunits of IL-35) mRNA was shown to be co-expressed in trophoblast cells. Moreover, large amounts of secreted IL-35 were detected in the supernatants of trophoblast cells. But we did not detect the constitutive expression of IL-35 in decidual stromal cells. Our findings confirmed for the first time that first-trimester human trophoblast cells expressed and secreted IL-35, which might contribute to their suppressive capacity to maternal immune cells. Therefore, IL-35 may be an important factor of the cytokine network regulating local immune responses during human pregnancy.

  14. Iodine Affects Differentiation and Migration Process in Trophoblastic Cells.

    PubMed

    Olivo-Vidal, Zendy Evelyn; Rodríguez, Roció Coutiño; Arroyo-Helguera, Omar

    2016-02-01

    Iodine deficiency is associated with oxidative stress increase and preeclampsia during gestation, suggesting that iodine concentration plays an important role in the normal placenta physiology. The question raised is to analyze the effect of iodine deficiency on oxidative stress, viability, differentiation, and migration process and changes in the expression of differentiation and migration markers. Iodine deprivation was done using potassium perchlorate (KCLO4) to block sodium iodide symporter (NIS) transporter and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid DIDS to inhibit pendrine (PEN) transport for 3-48 h. Then trophoblast cells were treated with low iodine doses of 5-500 μM and high iodine doses of 100-5000 μM. Oxidative stress, viability, and human chorionic gonadotropin (hGC) were measured by colorimetric methods. Migration throphoblast cells were evaluated by both wound healing and Boyden chamber assays. Changes in mRNA expression were analyzed by real-time RT-PCR. Iodine deprivation induces a significant increase of reactive oxygen species (ROS), viability, and migration process vs control cells. We found a significant overregulation in the mRNA's peroxisome proliferator-activated receptor (PPAR-gamma), Snail, and matrix metalloproteinase-9 (MMP-9) mRNA's in cells deprived of iodine, as well as a down glial cell missing-1 (GCM-1) regulation, hGC, pregnancy-associated plasma protein-A (PAPP-A), and E-cadherin mRNA expression. The expression of hypoxic induction factor alpha (HIFα) mRNA does not change with iodine deprivation. In cells deprived of iodine, supplementing low iodine doses (5-500 μM) does not induce any significant changes in viability. However, ROS and migration process were decreased, although we found an increased human chorionic gonadotropin (hCG) secretion as a differentiation marker. In addition, we found that PPAR-gamma, Snail, and MPP-9 mRNAs expression are downregulated with low iodine doses, in contrast with GCM-1, PAPP

  15. Glucose metabolism in cultured trophoblasts from human placenta

    SciTech Connect

    Moe, A.J.; Farmer, D.R.; Nelson, D.M.; Smith, C.H. )

    1990-02-26

    The development of appropriate placental trophoblast isolation and culture techniques enables the study of pathways of glucose utilization by this important cell layer in vitro. Trophoblasts from normal term placentas were isolated and cultured 24 hours and 72 hours in uncoated polystyrene culture tubes or tubes previously coated with a fibrin matrix. Trophoblasts cultured on fibrin are morphologically distinct from those cultured on plastic or other matrices and generally resemble in vivo syncytium. Cells were incubated up to 3 hours with {sup 14}C-labeled glucose and reactions were stopped by addition of perchloric acid. {sup 14}CO{sub 2} production by trophoblasts increased linearly with time however the largest accumulation of label was in organic acids. Trophoblasts cultured in absence of fibrin utilized more glucose and accumulated more {sup 14}C in metabolic products compared to cells cultured on fibrin. Glucose oxidation to CO{sub 2} by the phosphogluconate (PG) pathway was estimated from specific yields of {sup 14}CO{sub 2} from (1-{sup 14}C)-D-glucose and (6-{sup 14}C)-D-glucose. Approximately 6% of glucose oxidation was by the PG pathway when cells were cultured on fibrin compared to approximately 1% by cells cultured in the absence of fibrin. The presence of a fibrin growth matrix appears to modulate the metabolism of glucose by trophoblast from human placenta in vitro.

  16. Differentiation-dependent expression of gelatinase B/matrix metalloproteinase-9 in trophoblast cells.

    PubMed

    Peters, T J; Albieri, A; Bevilacqua, E; Chapman, B M; Crane, L H; Hamlin, G P; Seiki, M; Soares, M J

    1999-02-01

    The purpose of this study was to evaluate the Rcho-1 trophoblast culture system as a model for studying trophoblast invasion and to examine stage-specific expression of enzyme(s) potentially participating in rat trophoblast giant cell invasive behavior. The invasive behavior of the differentiating Rcho-1 trophoblast cells was demonstrated using Matrigel invasion chambers. Gelatin zymography and Western blot analysis of conditioned medium from differentiating Rcho-1 trophoblast cell cultures and rat ectoplacental cone outgrowths revealed a differentiation-dependent increase in gelatinase B/matrix metalloproteinase (MMP-9). Nothern blot and reverse transcriptase polymerase chain reaction (RT-PCR) analyses of Rcho-1 trophoblast or ectoplacental cone cells also showed increasing expression of MMP-9 accompanying cell differentiation. Rcho-1 trophoblast cells stably transfected with MMP-9 promoter/luciferase reporter constructs exhibited a differentiation-dependent increase in MMP-9 promoter activation. In conclusion, trophoblast giant cell differentiation is characterized by transcriptional activation of the MMP-9 gene and appearance of the invasive phenotype.

  17. NLRP7 affects trophoblast lineage differentiation, binds to overexpressed YY1 and alters CpG methylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maternal-effect mutations in NLRP7 cause rare biparentally inherited hydatidiform moles (BiHMs), abnormal pregnancies containing hypertrophic vesicular trophoblast but no embryo. BiHM trophoblasts display abnormal DNA methylation patterns affecting maternally methylated germline differentially methy...

  18. EFFECT OF BROMODICHLOROMETHANE ON HUMAN TROPHOBLAST CHORIONIC GONADOTROPHIN SECRETION

    EPA Science Inventory

    Effect of Bromodichloromethane on Human Trophoblast Chorionic Gonadotrophin Secretion

    Jiangang Chen1, Twanda L. Thirkill1, Peter N. Lohstroh1, Susan R. Bielmeier2, Michael G. Narotsky3, Deborah S. Best3, Randy A. Harrison3, Kala Natarajan1, Rex A. Pegram3, Gordon C. Dougla...

  19. DNA methyltransferases and TETs in the regulation of differentiation and invasiveness of extra-villous trophoblasts

    PubMed Central

    Logan, Philip C.; Mitchell, Murray D.; Lobie, Peter E.

    2013-01-01

    Specialized cell types of trophoblast cells form the placenta in which each cell type has particular properties of proliferation and invasion. The placenta sustains the growth of the fetus throughout pregnancy and any aberrant trophoblast differentiation or invasion potentially affects the future health of the child and adult. Recently, the field of epigenetics has been applied to understand differentiation of trophoblast lineages and embryonic stem cells (ESC), from fertilization of the oocyte onward. Each trophoblast cell-type has a distinctive epigenetic profile and we will concentrate on the epigenetic mechanism of DNA methyltransferases and TETs that regulate DNA methylation. Environmental factors affecting the mother potentially regulate the DNA methyltransferases in trophoblasts, and so do steroid hormones, cell cycle regulators, such as p53, and cytokines, especially interlukin-1β. There are interesting questions of why trophoblast genomes are globally hypomethylated yet specific genes can be suppressed by hypermethylation (in general, tumor suppressor genes, such as E-cadherin) and how invasive cell-types are liable to have condensed chromatin, as in metastatic cancer cells. Future work will attempt to understand the interactive nature of all epigenetic mechanisms together and their effect on the complex biological system of trophoblast differentiation and invasion in normal as well as pathological conditions. PMID:24363660

  20. Glial Cell Missing 1 Regulates Placental Growth Factor (PGF) Gene Transcription in Human Trophoblast1

    PubMed Central

    Chang, Miao; Mukherjea, Debashree; Gobble, Ryan M.; Groesch, Kathleen A.; Torry, Ronald J.; Torry, Donald S.

    2008-01-01

    Placental growth factor (PGF, previously known as PlGF) is prominently expressed by trophoblasts in human placenta, whereas most nontrophoblast cells express low levels of PGF mRNA under normal physiological conditions. We have shown that hypoxia decreases PGF expression in the trophoblast, but little is known about transcriptional regulation of PGF gene expression. We sought to determine promoter regions of the human PGF gene that contribute to its restricted high constitutive expression in the trophoblast. Overlapping putative promoter regions of human PGF gene encompassing −1.5 kb were cloned into reporter vectors and co-transfected into trophoblast and nontrophoblast cell lines. Promoter activity generated by a −1.5-kb clone was significantly higher in trophoblasts than in nontrophoblasts. Selective deletion mutants showed that a clone encompassing the PGF (−828/+34) region generated promoter activity similar to the −1.5-kb region in the trophoblast. However, deletion of another 131 bp from this subclone (−698/+34) resulted in significantly less promoter activity in the trophoblast. The (−828/−698) region significantly enhanced activity of a minimal promoter construct in trophoblast but not in nontrophoblast cells, suggesting that this region contributes to regulating PGF transcription in the trophoblast. Site-directed mutagenesis of a glial cell missing 1 (GCM1) motif in the 131-bp region significantly decreased enhancer activity in the trophoblast. Furthermore, overexpression of GCM1 significantly increased PGF −1.5-kb promoter activity and PGF mRNA expression in trophoblast and nontrophoblast cells. Forced overexpression of GCM1 restored PGF expression in the hypoxic trophoblast. These data support a functional role for GCM1 contributing to constitutively high trophoblast PGF expression and is the first direct evidence of an oxygen-responsive, trophoblast-specific transcription factor contributing to the regulation of PGF expression. PMID

  1. Expression of PAPPA2 in human fetomaternal interface and involvement in trophoblast invasion and migration.

    PubMed

    Wang, H Y; Zhang, Z; Yu, S

    2016-01-01

    Pregnancy-associated plasma protein-A 2 (PAPPA2) is a placental-enriched gene that is important for normal human placentation and defects in the gene can cause complications in pregnancy. Yet the exact expression pattern and role of PAPPA2 in the human fetomaternal interface are not clear. In this study, in situ hybridization (ISH) and immunohistochemistry (IHC) were employed to examine the spatial and temporal expression of PAPPA2 in the human fetomaternal interface. IHC results exhibited wide expression of PAPPA2 in the fetomaternal interface, with placental syncytiatrophoblast (STB) and extravillous trophoblast (EVT) showing strong expression and the cytotrophoblast (CTB) showing weak expression of PAPPA2. These results were confirmed by ISH. Quantitative reverse transcription-polymerase chain reaction and western blot showed the elevation of PAPPA2 in first trimester EVT differentiation and term CTB spontaneous syncytialization. PAPPA2-siRNA transfection significantly depressed the invasion and migration ability of a trophoblast cell line (HTR8/SVneo) in a transwell migration and Matrigel invasion model compared to a negative control siRNA (P < 0.05), also revealing that matrix metalloproteinase 9 (MMP9) secretion is downregulated. This was confirmed using a human first trimester placental villi explant culture model. Our results reveal the spatial and temporal expression of PAPPA2 in the human fetomaternal interface and show the positive regulatory role of PAPPA2 in human trophoblast invasion and migration through the secretion of MMP9. PMID:27525857

  2. Regulation of metalloproteinases by nitric oxide in human trophoblast cells in culture.

    PubMed

    Novaro, V; Colman-Lerner, A; Ortega, F V; Jawerbaum, A; Paz, D; Lo Nostro, F; Pustovrh, C; Gimeno, M F; González, E

    2001-01-01

    The process of embryo implantation requires extensive remodelling of the endometrial extracellular matrix, a function largely performed by matrix-degrading metalloproteinases (MMPs). In the present study, we used trophoblast cells isolated from human term placentas to study the regulation of MMPs by nitric oxide (NO). Using a combination of zymography, Western blot and indirect immunofluorescence, we showed that MMP-2 and MMP-9 are increased during the conversion from low-motile cytotrophoblast cells to the highly motile and differentiated syncytiotrophoblast multinucleated cells. We also observed an increase in NO production and NO synthase (NOS) expression during this cellular differentiation process. In addition, we demonstrated a positive regulatory role of NO on the activity and protein expression of MMP-2 and MMP-9, because NO donors (NOC-18 and spermine-NONOate) or the NOS substrate (L-arginine) stimulate, whereas NOS inhibitors (N(G)-nitro-L-arginine methyl ester and N(G)-monomethyl-L-arginine) reduce the expression and gelatinolytic activity of MMP-2 and MMP-9 in isolated trophoblast cells. Taken together, these results suggest that, in differentiating trophoblasts, NO regulates the induction of matrix-degrading proteases required for invasion during embryo implantation.

  3. Elf5 counteracts precocious trophoblast differentiation by maintaining Sox2 and 3 and inhibiting Hand1 expression.

    PubMed

    Pearton, David J; Smith, Craig S; Redgate, Emma; van Leeuwen, Jessica; Donnison, Martyn; Pfeffer, Peter L

    2014-08-15

    In mice the transcription factor Elf5 is necessary for correct trophoblast development. Upon knockdown of Elf5, TS cells display neither a decrease in proliferation nor an increase in cell death but rather an increased propensity to differentiate. Such cells rapidly lose Sox2 and 3 expression, while transiently upregulating the giant cell differentiation determinant gene Hand1. Other genes affected within 24h of Elf5 knock-down, many of which have not previously been implicated in trophoblast development, exhibited in vivo expression domains and in vitro expression responses consistent with Elf5 having a role in counteracting trophoblast differentiation. In an ES to TS differentiation assay using Cdx2 overexpression with Elf5 loss of function cell lines, it was shown that Elf5 is necessary to prevent terminal trophoblast differentiation. This data thus suggest that Elf5 is a gatekeeper for the TS to differentiated trophoblast transition thereby preventing the precocious differentiation of the undifferentiated extraembryonic ectoderm.

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

    PubMed Central

    Murray, Alexander; Sienerth, Arnold R.; Hemberger, Myriam

    2016-01-01

    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. PMID:27121762

  5. The psychoactive compound of Cannabis sativa, Δ(9)-tetrahydrocannabinol (THC) inhibits the human trophoblast cell turnover.

    PubMed

    Costa, M A; Fonseca, B M; Marques, F; Teixeira, N A; Correia-da-Silva, G

    2015-08-01

    The noxious effects of cannabis consumption for fertility and pregnancy outcome are recognized for years. Its consumption during gestation is associated with alterations in foetal growth, low birth weight and preterm labor. The main psychoactive molecule of cannabis, Δ(9)-tetrahydrocannabinol (THC) impairs the production of reproductive hormones and is also able to cross the placenta barrier. However, its effect on the main placental cells, the trophoblasts, are unknown. Actually, the role of THC in cell survival/death of primary human cytotrophoblasts (CTs) and syncytiotrophoblasts (STs) and in the syncytialization process remains to be explored. Here, we show that THC has a dual effect, enhancing MTT metabolism at low concentrations, whereas higher doses decreased cell viability, on both trophoblast phenotypes, though the effects on STs were more evident. THC also diminished the generation of oxidative and nitrative stress and the oxidized form of glutathione, whereas the reduced form of this tripeptide was increased, suggesting that THC prevents ST cell death due to an antioxidant effect. Moreover, this compound enhanced the mitochondrial function of STs, as observed by the increased MTT metabolism and intracellular ATP levels. These effects were independent of cannabinoid receptors activation. Besides, THC impaired CT differentiation into STs, since it decreased the expression of biochemical and morphological biomarkers of syncytialization, through a cannabinoid receptor-dependent mechanism. Together, these results suggest that THC interferes with trophoblast turnover, preventing trophoblast cell death and differentiation, and contribute to disclose the cellular mechanisms that lead to pregnancy complications in women that consume cannabis-derived drugs during gestation. PMID:26070387

  6. The psychoactive compound of Cannabis sativa, Δ(9)-tetrahydrocannabinol (THC) inhibits the human trophoblast cell turnover.

    PubMed

    Costa, M A; Fonseca, B M; Marques, F; Teixeira, N A; Correia-da-Silva, G

    2015-08-01

    The noxious effects of cannabis consumption for fertility and pregnancy outcome are recognized for years. Its consumption during gestation is associated with alterations in foetal growth, low birth weight and preterm labor. The main psychoactive molecule of cannabis, Δ(9)-tetrahydrocannabinol (THC) impairs the production of reproductive hormones and is also able to cross the placenta barrier. However, its effect on the main placental cells, the trophoblasts, are unknown. Actually, the role of THC in cell survival/death of primary human cytotrophoblasts (CTs) and syncytiotrophoblasts (STs) and in the syncytialization process remains to be explored. Here, we show that THC has a dual effect, enhancing MTT metabolism at low concentrations, whereas higher doses decreased cell viability, on both trophoblast phenotypes, though the effects on STs were more evident. THC also diminished the generation of oxidative and nitrative stress and the oxidized form of glutathione, whereas the reduced form of this tripeptide was increased, suggesting that THC prevents ST cell death due to an antioxidant effect. Moreover, this compound enhanced the mitochondrial function of STs, as observed by the increased MTT metabolism and intracellular ATP levels. These effects were independent of cannabinoid receptors activation. Besides, THC impaired CT differentiation into STs, since it decreased the expression of biochemical and morphological biomarkers of syncytialization, through a cannabinoid receptor-dependent mechanism. Together, these results suggest that THC interferes with trophoblast turnover, preventing trophoblast cell death and differentiation, and contribute to disclose the cellular mechanisms that lead to pregnancy complications in women that consume cannabis-derived drugs during gestation.

  7. Dynamic Trk and G Protein Signalings Regulate Dopaminergic Neurodifferentiation in Human Trophoblast Stem Cells

    PubMed Central

    Lee, Tony Tung-Yin; Tsai, Cheng-Fang; Chen, Hung-Sheng; Lai, Feng-Jie; Yokoyama, Kazunari K.; Hsieh, Tsung-Hsun; Wu, Ruey-Meei; Lee, Jau-nan

    2015-01-01

    Understanding the mechanisms in the generation of neural stem cells from pluripotent stem cells is a fundamental step towards successful management of neurodegenerative diseases in translational medicine. Albeit all-trans retinoic acid (RA) has been associated with axon outgrowth and nerve regeneration, the maintenance of differentiated neurons, the association with degenerative disease like Parkinson's disease, and its regulatory molecular mechanism from pluripotent stem cells to neural stem cells remain fragmented. We have previously reported that RA is capable of differentiation of human trophoblast stem cells to dopamine (DA) committed progenitor cells. Intracranial implantation of such neural progenitor cells into the 6-OHDA-lesioned substantia nigra pars compacta successfully regenerates dopaminergic neurons and integrity of the nigrostriatal pathway, ameliorating the behavioral deficits in the Parkinson’s disease rat model. Here, we demonstrated a dynamic molecular network in systematic analysis by addressing spatiotemporal molecular expression, intracellular protein-protein interaction and inhibition, imaging study, and genetic expression to explore the regulatory mechanisms of RA induction in the differentiation of human trophoblast stem cells to DA committed progenitor cells. We focused on the tyrosine receptor kinase (Trk), G proteins, canonical Wnt2B/β-catenin, genomic and non-genomic RA signaling transductions with Tyrosine hydroxylase (TH) gene expression as the differentiation endpoint. We found that at the early stage, integration of TrkA and G protein signalings aims for axonogenesis and morphogenesis, involving the novel RXRα/Gαq/11 and RARβ/Gβ signaling pathways. While at the later stage, five distinct signaling pathways together with epigenetic histone modifications emerged to regulate expression of TH, a precursor of dopamine. RA induction generated DA committed progenitor cells in one day. Our results provided substantial mechanistic

  8. Dynamic Trk and G Protein Signalings Regulate Dopaminergic Neurodifferentiation in Human Trophoblast Stem Cells.

    PubMed

    Tsai, Eing-Mei; Wang, Yu-Chih; Lee, Tony Tung-Yin; Tsai, Cheng-Fang; Chen, Hung-Sheng; Lai, Feng-Jie; Yokoyama, Kazunari K; Hsieh, Tsung-Hsun; Wu, Ruey-Meei; Lee, Jau-Nan

    2015-01-01

    Understanding the mechanisms in the generation of neural stem cells from pluripotent stem cells is a fundamental step towards successful management of neurodegenerative diseases in translational medicine. Albeit all-trans retinoic acid (RA) has been associated with axon outgrowth and nerve regeneration, the maintenance of differentiated neurons, the association with degenerative disease like Parkinson's disease, and its regulatory molecular mechanism from pluripotent stem cells to neural stem cells remain fragmented. We have previously reported that RA is capable of differentiation of human trophoblast stem cells to dopamine (DA) committed progenitor cells. Intracranial implantation of such neural progenitor cells into the 6-OHDA-lesioned substantia nigra pars compacta successfully regenerates dopaminergic neurons and integrity of the nigrostriatal pathway, ameliorating the behavioral deficits in the Parkinson's disease rat model. Here, we demonstrated a dynamic molecular network in systematic analysis by addressing spatiotemporal molecular expression, intracellular protein-protein interaction and inhibition, imaging study, and genetic expression to explore the regulatory mechanisms of RA induction in the differentiation of human trophoblast stem cells to DA committed progenitor cells. We focused on the tyrosine receptor kinase (Trk), G proteins, canonical Wnt2B/β-catenin, genomic and non-genomic RA signaling transductions with Tyrosine hydroxylase (TH) gene expression as the differentiation endpoint. We found that at the early stage, integration of TrkA and G protein signalings aims for axonogenesis and morphogenesis, involving the novel RXRα/Gαq/11 and RARβ/Gβ signaling pathways. While at the later stage, five distinct signaling pathways together with epigenetic histone modifications emerged to regulate expression of TH, a precursor of dopamine. RA induction generated DA committed progenitor cells in one day. Our results provided substantial mechanistic

  9. Regulation of HBEGF by Micro-RNA for Survival of Developing Human Trophoblast Cells

    PubMed Central

    Jain, Chandni V.; Jessmon, Philip; Kilburn, Brian A.; Jodar, Meritxell; Sendler, Edward; Krawetz, Stephen A.; Armant, D. Randall

    2016-01-01

    Introduction The growth factor HBEGF is upregulated post-transcriptionally in the low O2 environment of the human placenta during the first 10 weeks of pregnancy. We have examined the possible roles of HBEGF turnover and micro-RNA (miRNA) in its regulation by O2 in human first trimester trophoblast. Methods HTR-8/SVneo trophoblast cells were cultured at 2% or 20% O2. The cells were transfected with a dual luciferase reporter construct (psiCHECK-2) containing no insert (control), the HBEGF 3’ untranslated region (3’UTR), or sub-regions of the 3’UTR, as well as with siRNA for DGCR8. RNA was extracted from trophoblast cells cultured at 2% O2 for 0–4 h for next-generation sequencing. HBEGF was quantified by ELISA. HBEGF, DGCR8, and β–actin were examined by western blotting. Results Protein turnover studies, using 10 μg/ml cyclohexamide, 1 μg/ml lactocystin, or 100 μg/ml MG132, demonstrated faster HBEGF degradation at 20% O2 than 2% O2, mediated by the proteasome. However, proteasome inhibition failed to initiate HBEGF accumulation at 20% O2. Reporter assays, comparing to empty vector, demonstrated that the intact HBEGF 3’ UTR inhibited expression (0.26), while fragments containing only its flanking regions increased reporter activity (3.15; 3.43). No differential expression of miRNAs was found in trophoblast cells cultured at 2% and 20% O2. Nevertheless, HBEGF upregulation at 2% O2 was blocked when the miRNA-processing protein DGCR8 was silenced, suggesting a role for miRNA. Conclusion Our findings suggest involvement of flanking regions of the 3’UTR in activating HBEGF protein synthesis in response to 2% O2, possibly through a miRNA-mediated mechanism. PMID:27701455

  10. FGF4-DEPENDENT STEM CELLS DERIVED FROM RAT BLASTOCYSTS DIFFERENTIATE ALONG THE TROPHOBLAST LINEAGE

    PubMed Central

    Asanoma, Kazuo; Rumi, M.A. Karim; Kent, Lindsey N.; Chakraborty, Damayanti; Renaud, Stephen J.; Wake, Norio; Lee, Dong-Soo; Kubota, Kaiyu; Soares, Michael J.

    2011-01-01

    Differentiated trophoblast cell lineages arise from trophoblast stem (TS) cells. To date such a stem cell population has only been established in the mouse. The objective of this investigation was to establish TS cell populations from rat blastocysts. Blastocysts were cultured individually on a feeder layer of rat embryonic fibroblasts (REFs) in fibroblast growth factor-4 (FGF4) and heparin supplemented culture medium. Once cell colonies were established REF feeder layers could be replaced with REF conditioned medium. The blastocyst-derived cell lines, in either proliferative or differentiated states, did not express genes indicative of ICM-derived tissues. In the proliferative state the cells expressed established stem cell-associated markers of TS cells. Cells ceased proliferation and differentiated when FGF4, heparin, and REF conditioned medium were removed. Differentiation was characterized by a decline of stem cell-associated marker gene expression, the appearance of large polyploid cells (trophoblast giant cells), and the expression of trophoblast differentiation-associated genes. Collectively, the data indicate that the rat blastocyst-derived cell lines possess many features characteristic of mouse TS cells but also possess some distinct properties. These rat TS cell lines represent valuable new in vitro models for analyses of mechanisms controlling TS cell renewal and differentiation. PMID:21215265

  11. Evidence for bystander signalling between human trophoblast cells and human embryonic stem cells.

    PubMed

    Jones, Anna J; Gokhale, Paul J; Allison, Thomas F; Sampson, Barry; Athwal, Sharan; Grant, Simon; Andrews, Peter W; Allen, Nicholas D; Case, C Patrick

    2015-01-01

    Maternal exposure during pregnancy to toxins can occasionally lead to miscarriage and malformation. It is currently thought that toxins pass through the placental barrier, albeit bi-layered in the first trimester, and damage the fetus directly, albeit at low concentration. Here we examined the responses of human embryonic stem (hES) cells in tissue culture to two metals at low concentration. We compared direct exposures with indirect exposures across a bi-layered model of the placenta cell barrier. Direct exposure caused increased DNA damage without apoptosis or a loss of cell number but with some evidence of altered differentiation. Indirect exposure caused increased DNA damage and apoptosis but without loss of pluripotency. This was not caused by metal ions passing through the barrier. Instead the hES cells responded to signalling molecules (including TNF-α) secreted by the barrier cells. This mechanism was dependent on connexin 43 mediated intercellular 'bystander signalling' both within and between the trophoblast barrier and the hES colonies. These results highlight key differences between direct and indirect exposure of hES cells across a trophoblast barrier to metal toxins. It offers a theoretical possibility that an indirectly mediated toxicity of hES cells might have biological relevance to fetal development.

  12. The diagnostic value of p27 in comparison to p57 in differentiation between different gestational trophoblastic diseases.

    PubMed

    Abdou, Asmaa; Kandil, Mona; El-Wahed, Moshira Abd; Shabaan, Mohammed; El-Sharkawy, Mona

    2013-12-01

    The histologic features that permit the identification of complete mole (CM) and partial mole (PM) as well as hydropic abortion (HA) may be overlapping. The cyclin-dependent kinase inhibitor p57Kip2 protein (p57) has been included among the paternally imprinted genes in humans that commonly used for diagnosis of CM. P27 is one of the cell-cycle controlling molecules that may be involved in the proliferation, differentiation and oncogenesis of trophoblastic cells. The current study tried to test the diagnostic validity of several histopathological parameters together with p57 and p27 immunostaining in differentiation between different gestational trophoblastic diseases. The current study was carried out on 13 cases product of conception, 13 cases PM, 25 cases CM and 8 cases choriocarcinoma. Maximal villous diameter at 1.5 mm cut-off point was found to be the most reliable factor in discrimination between PM and product of conception followed by presence of villous cistern, p57 expression by extravillous cytotrophoblasts at 1.5% cut-off point and p27 expression by villous cytotrophoblasts at 25% cut-off point. P27 expression by stromal cells and total trophoblastic population at 7.5% cut-off point could discriminate between PM and CM. P57 and p27 are co-parallely expressed in non-molar as well as partial molar gestations, but they did not show this coordination in choriocarcinoma. This study demonstrated diagnostic values for several cut-off points for p57 and p27 in discrimination between different categories of gestational trophoblastic diseases. The co-expression of p27 and p57 by extravillous cytotrophoblasts and their positive correlation in non-molar gestations may indicate its suppressive role on the proliferation of these cells to provide them the capacity for differentiation and invasion.

  13. Display of complete life cycle of human papillomavirus type 16 in cultured placental trophoblasts.

    PubMed

    Liu, Y; You, H; Chiriva-Internati, M; Korourian, S; Lowery, C L; Carey, M J; Smith, C V; Hermonat, P L

    2001-11-10

    Human papillomavirus (HPV) infection is threefold more prevalent in spontaneous abortion specimens compared to elective abortions preferentially targeting the placental trophoblasts in these specimens. Here by using infectious ceplar and Southern blot analysis, we demonstrate that the transfected HPV-16 genome de novo replicates in 3A trophoblasts in culture. Peak DNA replication occurred 9-24 days posttransfection, showing classic DNA forms I, II, and III and an 8-kb monomer band upon DpnI/BamHI digestion. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of mRNA expression revealed that E6 and E2 were significantly expressed by day 9, coinciding with HPV-16 DNA replication. However, significant L1 expression was delayed until day 18. L1 protein expression on day 18, but not day 9, was also confirmed by Western blot analysis. The production of HPV-16 virions was demonstrated by three techniques: the appearance of HPV-16 infectious units coinciding with L1 expression, the neutralization of these infectious units with known neutralizing anti-HPV-16 antibodies, and the appearance of spliced E1-E4 and E6-E7 transcripts (RT-PCR) in normal keratinocyte rafts infected with these trophoblast-produced HPV-16 infectious units. These data suggest that HPV-16 is carrying out its complete life cycle in trophoblasts. Previously, HPVs were known to productively replicate only in differentiating keratinocytes of skin. These findings expand HPV biology, support the hypothesis of a possible link between HPV and some spontaneous abortions, and present a new technology for studying HPV.

  14. Kisspeptin regulation of genes involved in cell invasion and angiogenesis in first trimester human trophoblast cells.

    PubMed

    Francis, Víctor A; Abera, Aron B; Matjila, Mushi; Millar, Robert P; Katz, Arieh A

    2014-01-01

    The precise regulation of extravillous trophoblast invasion of the uterine wall is a key process in successful pregnancies. Kisspeptin (KP) has been shown to inhibit cancer cell metastasis and placental trophoblast cell migration. In this study primary cultures of first trimester human trophoblast cells have been utilized in order to study the regulation of invasion and angiogenesis-related genes by KP. Trophoblast cells were isolated from first trimester placenta and their identity was confirmed by immunostaining for cytokeratin-7. Real-time quantitative RT-PCR demonstrated that primary trophoblast cells express higher levels of GPR54 (KP receptor) and KP mRNA than the trophoblast cell line HTR8Svneo. Furthermore, trophoblast cells also expressed higher GPR54 and KP protein levels. Treating primary trophoblast cells with KP induced ERK1/2 phosphorylation, while co-treating the cells with a KP antagonist almost completely blocked the activation of ERK1/2 and demonstrated that KP through its cognate GPR54 receptor can activate ERK1/2 in trophoblast cells. KP reduced the migratory capability of trophoblast cells in a scratch-migration assay. Real-time quantitative RT-PCR demonstrated that KP treatment reduced the expression of matrix metalloproteinase 1, 2, 3, 7, 9, 10, 14 and VEGF-A, and increased the expression of tissue inhibitors of metalloproteinases 1 and 3. These results suggest that KP can inhibit first trimester trophoblast cells invasion via inhibition of cell migration and down regulation of the metalloproteinase system and VEGF-A. PMID:24923321

  15. Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption.

    PubMed

    Sagrillo-Fagundes, Lucas; Clabault, Hélène; Laurent, Laetitia; Hudon-Thibeault, Andrée-Anne; Salustiano, Eugênia Maria Assunção; Fortier, Marlène; Bienvenue-Pariseault, Josianne; Wong Yen, Philippe; Sanderson, J Thomas; Vaillancourt, Cathy

    2016-01-01

    This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation. PMID:27500522

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

    PubMed Central

    Zdravkovic, Tamara; Nazor, Kristopher L.; Larocque, Nicholas; Gormley, Matthew; Donne, Matthew; Hunkapillar, Nathan; Giritharan, Gnanaratnam; Bernstein, Harold S.; Wei, Grace; Hebrok, Matthias; Zeng, Xianmin; Genbacev, Olga; Mattis, Aras; McMaster, Michael T.; Krtolica, Ana; Valbuena, Diana; Simón, Carlos; Laurent, Louise C.; Loring, Jeanne F.; Fisher, Susan J.

    2015-01-01

    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 β-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. PMID:26483210

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

    PubMed

    Zdravkovic, Tamara; Nazor, Kristopher L; Larocque, Nicholas; Gormley, Matthew; Donne, Matthew; Hunkapillar, Nathan; Giritharan, Gnanaratnam; Bernstein, Harold S; Wei, Grace; Hebrok, Matthias; Zeng, Xianmin; Genbacev, Olga; Mattis, Aras; McMaster, Michael T; Krtolica, Ana; Valbuena, Diana; Simón, Carlos; Laurent, Louise C; Loring, Jeanne F; Fisher, Susan J

    2015-12-01

    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 β-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.

  18. Galectin-4 expression is down-regulated in response to autophagy during differentiation of rat trophoblast cells.

    PubMed

    Arikawa, Tomohiro; Liao, Shengjun; Shimada, Hiroki; Inoue, Tomoki; Sakata-Haga, Hiromi; Nakamura, Takanori; Hatta, Toshihisa; Shoji, Hiroki

    2016-01-01

    Placental development and trophoblast invasion of the maternal endometrium establish the maternal-fetal interface, which is critical for the developing embryo and fetus. Herein we show that overexpression of Galectin-4 (Gal-4) during trophoblast differentiation inhibited the enlargement of Rcho-1 cells (a model for rat trophoblast differentiation) and promoted cell-cell adhesion, whereas trophoblast specific markers and MMP-9 activity were not affected. In the rat placenta, microtubule associated protein 1 light chain 3 alpha (LC3) protein, an autophagy marker, is highly expressed on the maternal side of the decidua where Gal-4 expression is weak. In vitro assays showed that the expression of trophoblast-specific differentiation markers was reduced by 3-Methyladenine (3-MA) and Bafilomycin A1, known as autophagy inhibitors, compared to control cells. Furthermore, Gal-4 expression in Rcho-1 cells, which is normally down-regulated during differentiation, was not attenuated in the presence of autophagy inhibitors, suggesting that autophagy is upstream of Gal-4 expression. We herein describe a possible mechanism by which autophagy regulates trophoblast differentiation via regulation of Gal-4 expression in order to establish the maternal-fetal interface. PMID:27572741

  19. Galectin-4 expression is down-regulated in response to autophagy during differentiation of rat trophoblast cells

    PubMed Central

    Arikawa, Tomohiro; Liao, Shengjun; Shimada, Hiroki; Inoue, Tomoki; Sakata-Haga, Hiromi; Nakamura, Takanori; Hatta, Toshihisa; Shoji, Hiroki

    2016-01-01

    Placental development and trophoblast invasion of the maternal endometrium establish the maternal-fetal interface, which is critical for the developing embryo and fetus. Herein we show that overexpression of Galectin-4 (Gal-4) during trophoblast differentiation inhibited the enlargement of Rcho-1 cells (a model for rat trophoblast differentiation) and promoted cell-cell adhesion, whereas trophoblast specific markers and MMP-9 activity were not affected. In the rat placenta, microtubule associated protein 1 light chain 3 alpha (LC3) protein, an autophagy marker, is highly expressed on the maternal side of the decidua where Gal-4 expression is weak. In vitro assays showed that the expression of trophoblast-specific differentiation markers was reduced by 3-Methyladenine (3-MA) and Bafilomycin A1, known as autophagy inhibitors, compared to control cells. Furthermore, Gal-4 expression in Rcho-1 cells, which is normally down-regulated during differentiation, was not attenuated in the presence of autophagy inhibitors, suggesting that autophagy is upstream of Gal-4 expression. We herein describe a possible mechanism by which autophagy regulates trophoblast differentiation via regulation of Gal-4 expression in order to establish the maternal-fetal interface. PMID:27572741

  20. Sox15 enhances trophoblast giant cell differentiation induced by Hand1 in mouse placenta.

    PubMed

    Yamada, Kayo; Kanda, Hiromi; Tanaka, Satoshi; Takamatsu, Nobuhiko; Shiba, Tadayoshi; Ito, Michihiko

    2006-06-01

    Some members of the Sry-type HMG box (Sox) protein family play important roles in embryogenesis as transcription factors. Here, we report that Sox15 transcripts were much more abundant in mouse placenta than in the fetus, the yolk sac, or several adult tissues. In situ hybridization analysis of the mouse E8.0 conceptus indicated that Sox15 mRNA was predominantly expressed in the trophoblast giant cells of the placenta. We also observed that the amount of Sox15 mRNA dramatically increased during the differentiation of mouse trophoblast stem cells. Ectopic expression of Sox15 in Rat choriocarcinoma cells enhanced the giant cell differentiation induced by a bHLH transcription factor, Hand1. Binding experiments in cotransfected 293 T cells and in vitro revealed that Sox15 interacted with Hand1. We next examined the effects of this interaction on the transcriptional activity of Hand1 and Sox15 using the luciferase reporter assay. Overexpression of Hand1 repressed the Sox15-driven reporter expression, but Sox15 enhanced the Hand1-driven transcription. This enhancement required both the Hand1-binding region and the transactivation domain of Sox15. These results may suggest that the increased transcriptional activity of Hand1 caused by Sox15 might promote the transcription of the target gene resulting in the trophoblast giant cell differentiation in the mouse placenta.

  1. Human HLA-G+ extravillous trophoblasts: Immune-activating cells that interact with decidual leukocytes.

    PubMed

    Tilburgs, Tamara; Crespo, Ângela C; van der Zwan, Anita; Rybalov, Basya; Raj, Towfique; Stranger, Barbara; Gardner, Lucy; Moffett, Ashley; Strominger, Jack L

    2015-06-01

    Invading human leukocyte antigen-G+ (HLA-G+) extravillous trophoblasts (EVT) are rare cells that are believed to play a key role in the prevention of a maternal immune attack on foreign fetal tissues. Here highly purified HLA-G+ EVT and HLA-G- villous trophoblasts (VT) were isolated. Culture on fibronectin that EVT encounter on invading the uterus increased HLA-G, EGF-Receptor-2, and LIF-Receptor expression on EVT, presumably representing a further differentiation state. Microarray and functional gene set enrichment analysis revealed a striking immune-activating potential for EVT that was absent in VT. Cocultures of HLA-G+ EVT with sample matched decidual natural killer cells (dNK), macrophages, and CD4+ and CD8+ T cells were established. Interaction of EVT with CD4+ T cells resulted in increased numbers of CD4+CD25(HI)FOXP3+CD45RA+ resting regulatory T cells (Treg) and increased the expression level of the Treg-specific transcription factor FOXP3 in these cells. However, EVT did not enhance cytokine secretion in dNK, whereas stimulation of dNK with mitogens or classical natural killer targets confirmed the distinct cytokine secretion profiles of dNK and peripheral blood NK cells (pNK). EVT are specialized cells involved in maternal-fetal tolerance, the properties of which are not imitated by HLA-G-expressing surrogate cell lines. PMID:26015573

  2. Methylation status and transcriptional expression of the MHC class I loci in human trophoblast cells from term placenta

    SciTech Connect

    Guillaudeux, T.; Rodriguez, A.M.; Girr, M.

    1995-04-01

    Of the various molecular regulatory mechanisms that may be used by human trophoblast cells to down-regulate expression of HLA class I genes, we chose to investigate the methylation of DNA, generally associated with inhibition of transcription. We analyzed the methylation status of different HLA class I loci in villous and extravillous cytotrophoblast cells and in vitro-differentiated syncytiotrophoblast, purified from human term placenta, as well as in the human trophoblast-derived JAR and JEG-3 cell lines. We then compared methylation status and transcriptional activity. An inverse relationship was established between JAR and JEG-3: HLA-A, -B, and -G are methylated and repressed in JAR, whereas in JEG-3, HLA-A is methylated and repressed but HLA-B and -G are partially methylated and transcribed. HLA-E is unmethylated and transcribed in both cell lines. Apart from HLA-E, which is always unmethylated and transcribed, no such relationship exists for the other class I loci in trophoblast cells. Whereas nonclassical HLA-G and classical HLA-A and -B class I genes are undermethylated in both cytotrophoblast and syncytiotrophoblast, they are clearly transcribed in the former but minimally transcribed in the latter subpopulation. Thus, the down-regulation of class I gene expression in the in vitro-differentiated synctiotrophoblast is unlikely to be caused by DNA methylation. Furthermore, there is no detectable expression of any class I molecule at the cell surface of either trophoblast cell subpopulation, suggesting a negative control on translation and/or on the secretory pathway to the plasma membrane. 50 refs., 11 figs., 1 tab.

  3. Somatic cell nuclear transfer alters peri-implantation trophoblast differentiation in bovine embryos.

    PubMed

    Arnold, Daniel R; Bordignon, Vilceu; Lefebvre, Réjean; Murphy, Bruce D; Smith, Lawrence C

    2006-08-01

    Abnormal placental development limits success in ruminant pregnancies derived from somatic cell nuclear transfer (SCNT), due to reduction in placentome number and consequently, maternal/fetal exchange. In the primary stages of an epithelial-chorial association, the maternal/fetal interface is characterized by progressive endometrial invasion by specialized trophoblast binucleate/giant cells (TGC). We hypothesized that dysfunctional placentation in SCNT pregnancies results from aberration in expression of genes known to be necessary for trophoblast proliferation (Mash2), differentiation (Hand1), and function (IFN-tau and PAG-9). We, therefore, compared the expression of these factors in trophoblast from bovine embryos derived from artificial insemination (AI), in vitro fertilization (IVF), and SCNT prior to (day 17) and following (day 40 of gestation) implantation, as well as TGC densities and function. In preimplantation embryos, Mash2 mRNA was more abundant in SCNT embryos compared to AI, while Hand1 was highest in AI and IVF relative to SCNT embryos. IFN-tau mRNA abundance did not differ among groups. PAG-9 mRNA was undetectable in SCNT embryos, present in IVF embryos and highest in AI embryos. In postimplantation pregnancies, SCNT fetal cotyledons displayed higher Mash2 and Hand1 than AI and IVF tissues. Allelic expression of Mash2 was not different among the groups, which suggests that elevated mRNA expression was not due to altered imprinting status of Mash2. The day 40 SCNT cotyledons had the fewest number of TGC compared to IVF and AI controls. Thus, expression of genes critical to normal placental development is altered in SCNT bovine embryos, and this is expected to cause abnormal trophoblast differentiation and contribute to pregnancy loss.

  4. Mst1 and Mst2 Are Essential Regulators of Trophoblast Differentiation and Placenta Morphogenesis

    PubMed Central

    Du, Xingrong; Dong, Yongli; Shi, Hao; Li, Jiang; Kong, Shanshan; Shi, Donghua; Sun, Ling V.; Xu, Tian; Deng, Kejing; Tao, Wufan

    2014-01-01

    The placenta is essential for survival and growth of the fetus because it promotes the delivery of nutrients and oxygen from the maternal circulation as well as fetal waste disposal. Mst1 and Mst2 (Mst1/2), key components of the mammalian hpo/Mst signaling pathway, encode two highly conserved Ser/Thr kinases and play important roles in the prevention of tumorigenesis and autoimmunity, control of T cell development and trafficking, and embryonic development. However, their functions in placental development are not fully understood, and the underlying cellular and molecular mechanisms remain elusive. Here, we investigated the functions of Mst1/2 in mouse placental development using both conventional and conditional (endothelial) Mst1/2 double knockout mice. We found that the number of trophoblast giant cells dramatically increased while spongiotrophoblast cells almost completely disappeared in Mst1/2 deficient placentas. We showed that Mst1/2 deficiency down regulated the expression of Mash2, which is required for suppressing the differentiation of trophoblast giant cells. Furthermore, we demonstrated that endothelial-specific deletion of Mst1/2 led to impaired placental labyrinthine vasculature and embryonic lethality at E11.5, but neither affected vasculature in yolk sac and embryo proper nor endocardium development. Collectively, our findings suggest that Mst1/2 regulate placental development by control of trophoblast cell differentiation and labyrinthine vasculature at midgestation and Mst1/2 control labyrinth morphogenesis in trophoblast- and fetal endothelial-dependent manners. Thus, our studies have defined novel roles of Mst1/2 in mouse placental development. PMID:24595170

  5. Lysine-specific demethylase 1 regulates differentiation onset and migration of trophoblast stem cells

    NASA Astrophysics Data System (ADS)

    Zhu, Dongmei; Hölz, Stefanie; Metzger, Eric; Pavlovic, Mihael; Jandausch, Anett; Jilg, Cordula; Galgoczy, Petra; Herz, Corinna; Moser, Markus; Metzger, Daniel; Günther, Thomas; Arnold, Sebastian J.; Schüle, Roland

    2014-01-01

    Propagation and differentiation of stem cell populations are tightly regulated to provide sufficient cell numbers for tissue formation while maintaining the stem cell pool. Embryonic parts of the mammalian placenta are generated from differentiating trophoblast stem cells (TSCs) invading the maternal decidua. Here we demonstrate that lysine-specific demethylase 1 (Lsd1) regulates differentiation onset of TSCs. Deletion of Lsd1 in mice results in the reduction of TSC number, diminished formation of trophectoderm tissues and early embryonic lethality. Lsd1-deficient TSCs display features of differentiation initiation, including alterations of cell morphology, and increased migration and invasion. We show that increased TSC motility is mediated by the premature expression of the transcription factor Ovol2 that is directly repressed by Lsd1 in undifferentiated cells. In summary, our data demonstrate that the epigenetic modifier Lsd1 functions as a gatekeeper for the differentiation onset of TSCs, whereby differentiation-associated cell migration is controlled by the transcription factor Ovol2.

  6. Lysine-specific demethylase 1 regulates differentiation onset and migration of trophoblast stem cells.

    PubMed

    Zhu, Dongmei; Hölz, Stefanie; Metzger, Eric; Pavlovic, Mihael; Jandausch, Anett; Jilg, Cordula; Galgoczy, Petra; Herz, Corinna; Moser, Markus; Metzger, Daniel; Günther, Thomas; Arnold, Sebastian J; Schüle, Roland

    2014-01-01

    Propagation and differentiation of stem cell populations are tightly regulated to provide sufficient cell numbers for tissue formation while maintaining the stem cell pool. Embryonic parts of the mammalian placenta are generated from differentiating trophoblast stem cells (TSCs) invading the maternal decidua. Here we demonstrate that lysine-specific demethylase 1 (Lsd1) regulates differentiation onset of TSCs. Deletion of Lsd1 in mice results in the reduction of TSC number, diminished formation of trophectoderm tissues and early embryonic lethality. Lsd1-deficient TSCs display features of differentiation initiation, including alterations of cell morphology, and increased migration and invasion. We show that increased TSC motility is mediated by the premature expression of the transcription factor Ovol2 that is directly repressed by Lsd1 in undifferentiated cells. In summary, our data demonstrate that the epigenetic modifier Lsd1 functions as a gatekeeper for the differentiation onset of TSCs, whereby differentiation-associated cell migration is controlled by the transcription factor Ovol2.

  7. Human trophoblast survival at low oxygen concentrations requires metalloproteinase-mediated shedding of heparin-binding EGF-like growth factor

    PubMed Central

    Armant, D. Randall; Kilburn, Brian A.; Petkova, Anelia; Edwin, Samuel S.; Duniec-Dmuchowski, Zophia M.; Edwards, Holly J.; Romero, Roberto; Leach, Richard E.

    2006-01-01

    Heparin-binding EGF-like growth factor (HBEGF), which is expressed in the placenta during normal pregnancy, is downregulated in pre-eclampsia, a human pregnancy disorder associated with poor trophoblast differentiation and survival. This growth factor protects against apoptosis during stress, suggesting a role in trophoblast survival in the relatively low O2 (∼2%) environment of the first trimester conceptus. Using a well-characterized human first trimester cytotrophoblast cell line, we found that a 4-hour exposure to 2% O2 upregulates HBEGF synthesis and secretion independently of an increase in its mRNA. Five other expressed members of the EGF family are largely unaffected. At 2% O2, signaling via HER1 or HER4, known HBEGF receptors, is required for both HBEGF upregulation and protection against apoptosis. This positive-feedback loop is dependent on metalloproteinase-mediated cleavage and shedding of the HBEGF ectodomain. The restoration of trophoblast survival by the addition of soluble HBEGF in cultures exposed to low O2 and metalloproteinase inhibitor suggests that the effects of HBEGF are mediated by autocrine/paracrine, rather than juxtacrine, signaling. Our results provide evidence that a post-transcriptional mechanism induced in trophoblasts by low O2 rapidly amplifies HBEGF signaling to inhibit apoptosis. These findings have a high clinical significance, as the downregulation of HBEGF in pre-eclampsia is likely to be a contributing factor leading to the demise of trophoblasts. PMID:16407398

  8. The HAND1 basic helix-loop-helix transcription factor regulates trophoblast differentiation via multiple mechanisms.

    PubMed

    Scott, I C; Anson-Cartwright, L; Riley, P; Reda, D; Cross, J C

    2000-01-01

    The basic helix-loop-helix (bHLH) transcription factor genes Hand1 and Mash2 are essential for placental development in mice. Hand1 promotes differentiation of trophoblast giant cells, whereas Mash2 is required for the maintenance of giant cell precursors, and its overexpression prevents giant cell differentiation. We found that Hand1 expression and Mash2 expression overlap in the ectoplacental cone and spongiotrophoblast, layers of the placenta that contain the giant cell precursors, indicating that the antagonistic activities of Hand1 and Mash2 must be coordinated. MASH2 and HAND1 both heterodimerize with E factors, bHLH proteins that are the DNA-binding partners for most class B bHLH factors and which are also expressed in the ectoplacental cone and spongiotrophoblast. In vitro, HAND1 could antagonize MASH2 function by competing for E-factor binding. However, the Hand1 mutant phenotype cannot be solely explained by ectopic activity of MASH2, as the Hand1 mutant phenotype was not altered by further mutation of Mash2. Interestingly, expression of E-factor genes (ITF2 and ALF1) was down-regulated in the trophoblast lineage prior to giant cell differentiation. Therefore, suppression of MASH2 function, required to allow giant cell differentiation, may occur in vivo by loss of its E-factor partner due to loss of its expression and/or competition from HAND1. In giant cells, where E-factor expression was not detected, HAND1 presumably associates with a different bHLH partner. This may account for the distinct functions of HAND1 in giant cells and their precursors. We conclude that development of the trophoblast lineage is regulated by the interacting functions of HAND1, MASH2, and their cofactors.

  9. IFPA Meeting 2010 Workshops Report II: Placental pathology; trophoblast invasion; fetal sex; parasites and the placenta; decidua and embryonic or fetal loss; trophoblast differentiation and syncytialisation.

    PubMed

    Al-Khan, A; Aye, I L; Barsoum, I; Borbely, A; Cebral, E; Cerchi, G; Clifton, V L; Collins, S; Cotechini, T; Davey, A; Flores-Martin, J; Fournier, T; Franchi, A M; Fretes, R E; Graham, C H; Godbole, G; Hansson, S R; Headley, P L; Ibarra, C; Jawerbaum, A; Kemmerling, U; Kudo, Y; Lala, P K; Lassance, L; Lewis, R M; Menkhorst, E; Morris, C; Nobuzane, T; Ramos, G; Rote, N; Saffery, R; Salafia, C; Sarr, D; Schneider, H; Sibley, C; Singh, A T; Sivasubramaniyam, T S; Soares, M J; Vaughan, O; Zamudio, S; Lash, G E

    2011-03-01

    Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 diverse topics were discussed in twelve themed workshops, six of which are summarized in this report. 1. The placental pathology workshop focused on clinical correlates of placenta accreta/percreta. 2. Mechanisms of regulation of trophoblast invasion and spiral artery remodeling were discussed in the trophoblast invasion workshop. 3. The fetal sex and intrauterine stress workshop explored recent work on placental sex differences and discussed them in the context of whether boys live dangerously in the womb.4. The workshop on parasites addressed inflammatory responses as a sign of interaction between placental tissue and parasites. 5. The decidua and embryonic/fetal loss workshop focused on key regulatory mediators in the decidua, embryo and fetus and how alterations in expression may contribute to different diseases and adverse conditions of pregnancy. 6. The trophoblast differentiation and syncytialisation workshop addressed the regulation of villous cytotrophoblast differentiation and how variations may lead to placental dysfunction and pregnancy complications.

  10. Type III Interferons Produced by Human Placental Trophoblasts Confer Protection against Zika Virus Infection.

    PubMed

    Bayer, Avraham; Lennemann, Nicholas J; Ouyang, Yingshi; Bramley, John C; Morosky, Stefanie; Marques, Ernesto Torres De Azeved; Cherry, Sara; Sadovsky, Yoel; Coyne, Carolyn B

    2016-05-11

    During mammalian pregnancy, the placenta acts as a barrier between the maternal and fetal compartments. The recently observed association between Zika virus (ZIKV) infection during human pregnancy and fetal microcephaly and other anomalies suggests that ZIKV may bypass the placenta to reach the fetus. This led us to investigate ZIKV infection of primary human trophoblasts (PHTs), which are the barrier cells of the placenta. We discovered that PHT cells from full-term placentas are refractory to ZIKV infection. In addition, medium from uninfected PHT cells protects non-placental cells from ZIKV infection. PHT cells constitutively release the type III interferon (IFN) IFNλ1, which functions in both a paracrine and autocrine manner to protect trophoblast and non-trophoblast cells from ZIKV infection. Our data suggest that for ZIKV to access the fetal compartment, it must evade restriction by trophoblast-derived IFNλ1 and other trophoblast-specific antiviral factors and/or use alternative strategies to cross the placental barrier. PMID:27066743

  11. Type III Interferons Produced by Human Placental Trophoblasts Confer Protection against Zika Virus Infection.

    PubMed

    Bayer, Avraham; Lennemann, Nicholas J; Ouyang, Yingshi; Bramley, John C; Morosky, Stefanie; Marques, Ernesto Torres De Azeved; Cherry, Sara; Sadovsky, Yoel; Coyne, Carolyn B

    2016-05-11

    During mammalian pregnancy, the placenta acts as a barrier between the maternal and fetal compartments. The recently observed association between Zika virus (ZIKV) infection during human pregnancy and fetal microcephaly and other anomalies suggests that ZIKV may bypass the placenta to reach the fetus. This led us to investigate ZIKV infection of primary human trophoblasts (PHTs), which are the barrier cells of the placenta. We discovered that PHT cells from full-term placentas are refractory to ZIKV infection. In addition, medium from uninfected PHT cells protects non-placental cells from ZIKV infection. PHT cells constitutively release the type III interferon (IFN) IFNλ1, which functions in both a paracrine and autocrine manner to protect trophoblast and non-trophoblast cells from ZIKV infection. Our data suggest that for ZIKV to access the fetal compartment, it must evade restriction by trophoblast-derived IFNλ1 and other trophoblast-specific antiviral factors and/or use alternative strategies to cross the placental barrier.

  12. Leptin receptor activation increases Sam68 tyrosine phosphorylation and expression in human trophoblastic cells.

    PubMed

    Sánchez-Jiménez, Flora; Pérez-Pérez, Antonio; González-Yanes, Carmen; Najib, Souad; Varone, Cecilia L; Sánchez-Margalet, Víctor

    2011-01-30

    Leptin is produced in placenta where it has been found to be an important autocrine signal for trophoblastic growth during pregnancy, promoting antiapoptotic and trophic effects. Leptin receptor is present in trophoblastic cells and leptin may fully activate signaling. We have previously implicated the RNA-binding protein Sam68 in leptin signal transduction in immune cells. In the present work, we have studied the possible role of Sam68 in leptin receptor signaling in trophoblastic cells (JEG-3 cells). Leptin dose-dependently stimulated Sam68 phosphorylation in JEG-3 cells, as assessed by immunoprecipitation and immunoblot with anti-phosphotyrosine antibodies. As previously observed in other systems, tyrosine phosphorylation of Sam68 in response to leptin inhibits its RNA binding capacity. Besides, leptin stimulation dose-dependently increases Sam68 expression in JEG-3 cells, as assessed by quantitative PCR. Consistently, the amount of Sam68 protein is increased after 24h of leptin stimulation of trophoblastic cells. In order to study the possible role of Sam68 on leptin receptor synthesis, we employed antisense strategy to knockdown the expression of Sam68. We have found that a decrease in Sam68 expression leads to a decrease in leptin receptor amount in JEG-3 cells, as assessed both by quantitative PCR and immunoblot. These results strongly suggest the participation of Sam68 in leptin receptor signaling in human trophoblastic cells, and therefore, Sam68 may mediate some of the leptin effects in placenta. PMID:21035519

  13. TCDD Induces the Hypoxia-Inducible Factor (HIF)-1α Regulatory Pathway in Human Trophoblastic JAR Cells

    PubMed Central

    Liao, Tien-Ling; Chen, Su-Chee; Tzeng, Chii-Reuy; Kao, Shu-Huei

    2014-01-01

    The exposure to dioxin can compromise pregnancy outcomes and increase the risk of preterm births. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated to induce placental hypoxia at the end of pregnancy in a rat model, and hypoxia has been suggested to be the cause of abnormal trophoblast differentiation and placental insufficiency syndromes. In this study, we demonstrate that the non-hypoxic stimulation of human trophoblastic cells by TCDD strongly increased hypoxia inducible factor-1 alpha (HIF-1α) stabilization. TCDD exposure induced the generation of reactive oxygen species (ROS) and nitric oxide. TCDD-induced HIF-1α stabilization and Akt phosphorylation was inhibited by pretreatment with wortmannin (a phosphatidylinositol 3-kinase (PI3K) inhibitor) or N-acetylcysteine (a ROS scavenger). The augmented HIF-1α stabilization by TCDD occurred via the ROS-dependent activation of the PI3K/Akt pathway. Additionally, a significant increase in invasion and metallomatrix protease-9 activity was found in TCDD-treated cells. The gene expression of vascular endothelial growth factor and placental growth factor was induced upon TCDD stimulation, whereas the protein levels of peroxisome proliferator-activated receptor γ (PPARγ), PPARγ coactivator-1α, mitochondrial transcription factor, and uncoupling protein 2 were decreased. Our results indicate that an activated HIF-1α pathway, elicited oxidative stress, and induced metabolic stress contribute to TCDD-induced trophoblastic toxicity. These findings may provide molecular insight into the TCDD-induced impairment of trophoblast function and placental development. PMID:25272228

  14. EFFECT OF BROMODICHLOROMETHANE ON CHORIONIC GONADOTROPHIN SECRETION BY HUMAN PLACENTAL TROPHOBLAST CULTURES

    EPA Science Inventory

    EFFECT OF BROMODICHLOROMETHANE ON CHORIONIC GONADOTROPHIN SECRETION BY HUMAN PLACENTAL TROPHOBLAST CULTURES

    Jiangang Chen1, Gordon C. Douglas1?,Twanda L. Thirkill1?, Peter N. Lohstroh1, Susan R. Bielmeier2, Michael G. Narotsky3, Deborah S. Best3, Randy A. Harrison3, Kala ...

  15. Galectin-1 confers immune privilege to human trophoblast: implications in recurrent fetal loss.

    PubMed

    Ramhorst, Rosanna E; Giribaldi, Laura; Fraccaroli, Laura; Toscano, Marta A; Stupirski, Juan C; Romero, Marta D; Durand, Edith S; Rubinstein, Natalia; Blaschitz, Astrid; Sedlmayr, Peter; Genti-Raimondi, Susana; Fainboim, Leonardo; Rabinovich, Gabriel A

    2012-10-01

    Mechanisms accounting for the protection of the fetal semi-allograft from maternal immune cells remain incompletely understood. In previous studies, we showed that galectin-1 (Gal1), an immunoregulatory glycan-binding protein, hierarchically triggers a cascade of tolerogenic events at the mouse fetomaternal interface. Here, we show that Gal1 confers immune privilege to human trophoblast cells through the modulation of a number of regulatory mechanisms. Gal1 was mainly expressed in invasive extravillous trophoblast cells of human first trimester and term placenta in direct contact with maternal tissue. Expression of Gal1 by the human trophoblast cell line JEG-3 was primarily controlled by progesterone and pro-inflammatory cytokines and impaired T-cell responses by limiting T cell viability, suppressing the secretion of Th1-type cytokines and favoring the expansion of CD4(+)CD25(+)FoxP3(+) regulatory T (T(reg)) cells. Targeted inhibition of Gal1 expression through antibody (Ab)-mediated blockade, addition of the specific disaccharide lactose or retroviral-mediated siRNA strategies prevented these immunoregulatory effects. Consistent with a homeostatic role of endogenous Gal1, patients with recurrent pregnancy loss showed considerably lower levels of circulating Gal1 and had higher frequency of anti-Gal1 auto-Abs in their sera compared with fertile women. Thus, endogenous Gal1 confers immune privilege to human trophoblast cells by triggering a broad tolerogenic program with potential implications in threatened pregnancies. PMID:22752006

  16. GATA3 inhibits GCM1 activity and trophoblast cell invasion

    PubMed Central

    Chiu, Yueh Ho; Chen, Hungwen

    2016-01-01

    Development of human placenta involves the invasion of trophoblast cells from anchoring villi into the maternal decidua. Placental transcription factor GCM1 regulates trophoblast cell invasion via transcriptional activation of HtrA4 gene, which encodes a serine protease enzyme. The GATA3 transcription factor regulates trophoblast cell differentiation and is highly expressed in invasive murine trophoblast giant cells. The regulation of trophoblastic invasion by GCM1 may involve novel cellular factors. Here we show that GATA3 interacts with GCM1 and inhibits its activity to suppress trophoblastic invasion. Immunohistochemistry demonstrates that GATA3 and GCM1 are coexpressed in villous cytotrophoblast cells, syncytiotrophoblast layer, and extravillous trophoblast cells of human placenta. Interestingly, GATA3 interacts with GCM1, but not the GCM2 homologue, through the DNA-binding domain and first transcriptional activation domain in GCM1 and the transcriptional activation domains and zinc finger 1 domain in GATA3. While GATA3 did not affect DNA-binding activity of GCM1, it suppressed transcriptional activity of GCM1 and therefore HtrA4 promoter activity. Correspondingly, GATA3 knockdown elevated HtrA4 expression in BeWo and JEG-3 trophoblast cell lines and enhanced the invasion activities of both lines. This study uncovered a new GATA3 function in placenta as a negative regulator of GCM1 activity and trophoblastic invasion. PMID:26899996

  17. BMP4 induction of trophoblast from mouse embryonic stem cells in defined culture conditions on laminin.

    PubMed

    Hayashi, Yohei; Furue, Miho Kusuda; Tanaka, Satoshi; Hirose, Michiko; Wakisaka, Noriko; Danno, Hiroki; Ohnuma, Kiyoshi; Oeda, Shiho; Aihara, Yuko; Shiota, Kunio; Ogura, Atsuo; Ishiura, Shoichi; Asashima, Makoto

    2010-05-01

    Because mouse embryonic stem cells (mESCs) do not contribute to the formation of extraembryonic placenta when they are injected into blastocysts, it is believed that mESCs do not differentiate into trophoblast whereas human embryonic stem cells (hESCs) can express trophoblast markers when exposed to bone morphogenetic protein 4 (BMP4) in vitro. To test whether mESCs have the potential to differentiate into trophoblast, we assessed the effect of BMP4 on mESCs in a defined monolayer culture condition. The expression of trophoblast-specific transcription factors such as Cdx2, Dlx3, Esx1, Gata3, Hand1, Mash2, and Plx1 was specifically upregulated in the BMP4-treated differentiated cells, and these cells expressed trophoblast markers. These results suggest that BMP4 treatment in defined culture conditions enabled mESCs to differentiate into trophoblast. This differentiation was inhibited by serum or leukemia inhibitory factor, which are generally used for mESC culture. In addition, we studied the mechanism underlying BMP4-directed mESC differentiation into trophoblast. Our results showed that BMP4 activates the Smad pathway in mESCs inducing Cdx2 expression, which plays a crucial role in trophoblast differentiation, through the binding of Smad protein to the Cdx2 genomic enhancer sequence. Our findings imply that there is a common molecular mechanism underlying hESC and mESC differentiation into trophoblast.

  18. Synthesis and release of fatty acids by human trophoblast cells in culture

    SciTech Connect

    Coleman, R.A.; Haynes, E.B.

    1987-11-01

    In order to determine whether placental cells can synthesize and release fatty acids, trophoblast cells from term human placentas were established in monolayer culture. The cells continued to secrete placental lactogen and progesterone and maintained specific activities of critical enzymes of triacylglycerol and phosphatidylcholine biosynthesis for 24 to 72 hr in culture. Fatty acid was rapidly synthesized from (/sup 14/C)acetate and released by the cells. Palmitoleic, palmitic, and oleic acids were the major fatty acids synthesized from (/sup 14/C)acetate and released. Small amounts of lauric, myristic, and stearic acids were also identified. (/sup 14/C)acetate was also incorporated into cellular triacylglycerol, phospholipid, and cholesterol, but radiolabeled free fatty acid did not accumulate intracellularly. In a pulse-chase experiment, cellular glycerolipids were labeled with (1-/sup 14/C)oleate; trophoblast cells then released /sup 14/C-labeled fatty acid into the media as the cellular content of labeled phospholipid and triacylglycerol decreased without intracellular accumulation of free fatty acid. Twenty percent of the /sup 14/C-label lost from cellular glycerolipid could not be recovered as a chloroform-extractable product, suggesting that some of the hydrolyzed fatty acid had been oxidized. These data indicate that cultured placenta trophoblast cells can release fatty acids that have either been synthesized de novo or that have been hydrolyzed from cellular glycerolipids. Trophoblast cells in monolayer culture should provide an excellent model for molecular studies of placental fatty acid metabolism and release.

  19. Vascular Endothelial Growth Factor-A (VEGF-A) Mediates Activin A-Induced Human Trophoblast Endothelial-Like Tube Formation.

    PubMed

    Li, Yan; Zhu, Hua; Klausen, Christian; Peng, Bo; Leung, Peter C K

    2015-11-01

    Remodeling of maternal spiral arteries during pregnancy requires a subpopulation of extravillous cytotrophoblasts (EVTs) to differentiate into endovascular EVTs. Activin A, which is abundantly expressed at the maternal-fetal interface, has been shown to promote trophoblast invasion, but its role in endovascular differentiation remains unknown. Vascular endothelial growth factor-A (VEGF-A) is well recognized as a key regulator in trophoblast endovascular differentiation. Whether and how activin A might regulate VEGF-A production in human trophoblasts and its relationship to endovascular differentiation have yet to be determined. In the present study, we found that activin A increased VEGF-A production in primary and immortalized (HTR8/SVneo) human EVT cells. In addition, activin A enhanced HTR8/SVneo endothelial-like tube formation, and these effects were attenuated by pretreatment with small interfering RNA targeting VEGF-A or the VEGF receptor 1/2 inhibitor SU4312. Pretreatment with the activin/TGF-β type 1 receptor (ALK4/5/7) inhibitor SB431542 abolished the stimulatory effects of activin A on phosphorylated mothers against decapentaplegic (SMAD)-2/3 phosphorylation, VEGF-A production, and endothelial-like tube formation. Moreover, small interfering RNA-mediated down-regulation of SMAD2, SMAD3, or common SMAD4 abolished the effects of activin A on VEGF-A production and endothelial-like tube formation. In conclusion, activin A may promote human trophoblast cell endothelial-like tube formation by up-regulating VEGF-A production in an SMAD2/3-SMAD4-dependent manner. These findings provide insight into the cellular and molecular events regulated by activin A during human implantation. PMID:26327470

  20. Human parvovirus B19 VP2 empty capsids bind to human villous trophoblast cells in vitro via the globoside receptor.

    PubMed Central

    Wegner, Carole C; Jordan, Jeanne A

    2004-01-01

    BACKGROUND: Pregnant women acutely infected with human parvovirus B19 (B19) may transmit the virus to the developing fetus. The mechanism whereby the virus interacts with the placenta is unknown. It is known that globoside receptor is required for successful infection of the target cells, which are the highly undifferentiated, actively dividing colony and burst-form units of the erythroid series. Globoside is present on trophoblast cells which have intimate contact with maternal blood, and may therefore serve as a potential route for B19 transmission into the fetal compartment. OBJECTIVES: The purpose of this study was to determine whether B19 VP2 capsids could bind to villous trophoblast cells in vitro and whether globoside was involved. METHODS: Binding of B19 VP2 empty capsid to first-trimester villous trophoblast cells was assessed by multiple approaches, including ICC using either biotinylated B19 VP2 empty capsid or unlabeled B19 VP2 empty capsid. Quantification of viral binding involved I125-labeled B19 VP2 empty capsid. Competition studies included excess unlabeled empty capsids or pretreatment with globoside-specific IgM antibody. RESULTS: Linear binding of B19 VP2 capsid to purified villous trophoblast cells in vitro was clearly demonstrated (R2= 0.9524). Competition studies revealed specificity of I125-labeled B19 VP2 capsid binding to villous trophoblast cells when pretreatment with either 60-fold excess unlabeled B19 capsid or globoside-specific IgM antibody took place. The results illustrated B19's ability to bind in a specific manner to globoside-containing villous trophoblast cells. CONCLUSION: We speculate that the globoside present on trophoblast cells may play a role in viral binding in vivo, which may facilitate B19 transmission across the maternal-fetal interface. PMID:15739820

  1. Placental melatonin system is present throughout pregnancy and regulates villous trophoblast differentiation.

    PubMed

    Soliman, Ahmed; Lacasse, Andrée-Anne; Lanoix, Dave; Sagrillo-Fagundes, Lucas; Boulard, Véronique; Vaillancourt, Cathy

    2015-08-01

    Melatonin is highly produced in the placenta where it protects against molecular damage and cellular dysfunction arising from hypoxia/re-oxygenation-induced oxidative stress as observed in primary cultures of syncytiotrophoblast. However, little is known about melatonin and its receptors in the human placenta throughout pregnancy and their role in villous trophoblast development. The purpose of this study was to determine melatonin-synthesizing enzymes, arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT), and melatonin receptors (MT1 and MT2) expression throughout pregnancy as well as the role of melatonin and its receptors in villous trophoblast syncytialization. Our data show that the melatonin generating system is expressed throughout pregnancy (from week 7 to term) in placental tissues. AANAT and HIOMT show maximal expression at the 3rd trimester of pregnancy. MT1 receptor expression is maximal at the 1st trimester compared to the 2nd and 3rd trimesters, while MT2 receptor expression does not change significantly during pregnancy. Moreover, during primary villous cytotrophoblast syncytialization, MT1 receptor expression increases, while MT2 receptor expression decreases. Treatment of primary villous cytotrophoblast with an increasing concentration of melatonin (10 pM-1 mM) increases the fusion index (syncytium formation; 21% augmentation at 1 mM melatonin vs. vehicle) and β-hCG secretion (121% augmentation at 1 mM melatonin vs. vehicle). This effect of melatonin appears to be mediated via its MT1 and MT2 receptors. In sum, melatonin machinery (synthetizing enzymes and receptors) is expressed in human placenta throughout pregnancy and promotes syncytium formation, suggesting an essential role of this indolamine in placental function and pregnancy well-being.

  2. MTA3 regulates differentiation of human cytotrophoblast stem cells

    PubMed Central

    Horii, Mariko; Moretto-Zita, Matteo; Nelson, Katharine K.; Li, Yingchun; Parast, Mana M.

    2015-01-01

    Introduction Early placental development depends on the correct balance of cytotrophoblast (CTB) proliferation and differentiation, into either syncytiotrophoblast (STB) involved in nutrient/gas exchange, or invasive extravillous trophoblast (EVT) involved in establishment of blood flow to the placenta. Metastasis associated protein-3 (MTA3) is a transcriptional co-repressor known to regulate cell migration. In addition, MTA3 is reportedly decreased in preeclampsia. We set out to investigate the role of MTA3 in human trophoblast differentiation. Methods We co-stained first and third trimester placental sections with antibodies to MTA3 and other trophoblast markers. We also evaluated MTA3 expression following in vitro differentiation of primary isolated CTB. In order to evaluate the role of MTA3 in trophoblast differentiation, we used lentiviral constructs to overexpress and knock down its expression. Trophoblast differentiation was assessed by a combination of marker expression and functional assays, including hCG ELISA and cell migration. Results MTA3 was abundantly expressed in CTB and proximal cell column EVT in the human placenta and decreased with further differentiation into STB and mature EVT. MTA3 knockdown in JEG3 resulted in a 2–3 fold decrease in STB markers, CGB and GCM1, as well as in hCG secretion. In terms of EVT differentiation, MTA3 knockdown led to a 1.5–2 fold increase in HLA-G and cell migration, but decreased the mature EVT marker ITGA1. Discussion Taken together, our data suggest a role for MTA3 in terminal trophoblast differentiation into both hCG-secreting STB and mature EVT. PMID:26198267

  3. Effect of Bisphenol A on invasion ability of human trophoblastic cell line BeWo

    PubMed Central

    Wang, Zi-Yi; Lu, Jing; Zhang, Yuan-Zhen; Zhang, Ming; Liu, Teng; Qu, Xin-Lan

    2015-01-01

    Bisphenol A (BPA) is a kind of environmental endocrine disruptors (EEDs) that interfere embryo implantation. Trophoblast invasion plays a crucial role during embryo implantation. In this study, the effects of BPA on invasion ability of human trophoblastic cell line BeWo and its possible mechanism were investigated. BeWo cells were exposed to BPA and co-cultured with human endometrial cells to mimic embryo implantation in transwell model. The proliferation and invasion capability of BeWo cells were detected. The expression of E-cadherin, DNMT1, MMP-2, MMP-9, TIMP-1 and TIMP-2 were also analyzed. The results showed that the invasion capability of BeWo was reduced after daily exposure to BPA. BPA had biphasic effect on E-cadherin expression level in BeWo cells and expression level of DNMT1 was decreased when treated with BPA. Moreover, BPA treatment also changed the balance of MMPs/TIMPs in BeWo cells by down-regulating MMP-2, MMP-9 and up-regulating TIMP-1, TIMP-2 with increasing BPA concentration. Taken together, these results showed that BPA treatment could reduce the invasion ability of BeWo cells and alter the expression level of E-cadherin, DNMT1, TIMP-1, TIMP-2, MMP-2, and MMP-9. Our study would help us to understand the possible mechanism of BPA effect on invasion ability of human trophoblastic cell line BeWo. PMID:26823751

  4. Activin Regulates Self-renewal and Differentiation of Trophoblast Stem Cells by Down-regulating the X Chromosome Gene Bcor*

    PubMed Central

    Zhu, Gaoyang; Fei, Teng; Li, Zhongwei; Yan, Xiaohua; Chen, Ye-Guang

    2015-01-01

    The development of a functional placenta is largely dependent upon proper proliferation and differentiation of trophoblast stem cells (TSCs). Activin signaling has long been regarded to play important roles during this process, but the exact mechanism is largely unknown. Here, we demonstrate that the X chromosome gene BCL-6 corepressor (Bcor) is a critical downstream effector of activin to fine-tune mouse TSC fate decision. Bcor was specifically down-regulated by activin A in TSCs in a dose-dependent manner, and immediately up-regulated upon TSC differentiation. Knockdown of Bcor partially compensated for the absence of activin A in maintaining the self-renewal of TSCs together with FGF4, while promoting syncytiotrophoblast differentiation in the absence of FGF4. Moreover, the impaired trophoblast giant cell and spongiotrophoblast differentiation upon Bcor knockdown also resembled the function of activin. Reporter analysis showed that BCOR inhibited the expression of the key trophoblast regulator genes Eomes and Cebpa by binding to their promoter regions. Our findings provide us with a better understanding of placental development and placenta-related diseases. PMID:26221038

  5. Proinflammatory Response of Human Trophoblastic Cells to Brucella abortus Infection and upon Interactions with Infected Phagocytes.

    PubMed

    Fernández, Andrea G; Ferrero, Mariana C; Hielpos, M Soledad; Fossati, Carlos A; Baldi, Pablo C

    2016-02-01

    Trophoblasts are targets of infection by Brucella spp. but their role in the pathophysiology of pregnancy complications of brucellosis is unknown. Here we show that Brucella abortus invades and replicates in the human trophoblastic cell line Swan-71 and that the intracellular survival of the bacterium depends on a functional virB operon. The infection elicited significant increments of interleukin 8 (IL8), monocyte chemotactic protein 1 (MCP-1), and IL6 secretion, but levels of IL1beta and tumor necrosis factor-alpha (TNF-alpha) did not vary significantly. Such proinflammatory response was not modified by the absence of the Brucella TIR domain-containing proteins BtpA and BtpB. The stimulation of Swan-71 cells with conditioned medium (CM) from B. abortus-infected human monocytes (THP-1 cells) or macrophages induced a significant increase of IL8, MCP-1 and IL6 as compared to stimulation with CM from non-infected cells. Similar results were obtained when stimulation was performed with CM from infected neutrophils. Neutralization studies showed that IL1beta and/or TNF-alpha mediated the stimulating effects of CM from infected phagocytes. Reciprocally, stimulation of monocytes and neutrophils with CM from Brucella-infected trophoblasts increased IL8 and/or IL6 secretion. These results suggest that human trophoblasts may provide a local inflammatory environment during B. abortus infections either through a direct response to the pathogen or through interactions with monocytes/macrophages or neutrophils, potentially contributing to the pregnancy complications of brucellosis.

  6. Proinflammatory Response of Human Trophoblastic Cells to Brucella abortus Infection and upon Interactions with Infected Phagocytes.

    PubMed

    Fernández, Andrea G; Ferrero, Mariana C; Hielpos, M Soledad; Fossati, Carlos A; Baldi, Pablo C

    2016-02-01

    Trophoblasts are targets of infection by Brucella spp. but their role in the pathophysiology of pregnancy complications of brucellosis is unknown. Here we show that Brucella abortus invades and replicates in the human trophoblastic cell line Swan-71 and that the intracellular survival of the bacterium depends on a functional virB operon. The infection elicited significant increments of interleukin 8 (IL8), monocyte chemotactic protein 1 (MCP-1), and IL6 secretion, but levels of IL1beta and tumor necrosis factor-alpha (TNF-alpha) did not vary significantly. Such proinflammatory response was not modified by the absence of the Brucella TIR domain-containing proteins BtpA and BtpB. The stimulation of Swan-71 cells with conditioned medium (CM) from B. abortus-infected human monocytes (THP-1 cells) or macrophages induced a significant increase of IL8, MCP-1 and IL6 as compared to stimulation with CM from non-infected cells. Similar results were obtained when stimulation was performed with CM from infected neutrophils. Neutralization studies showed that IL1beta and/or TNF-alpha mediated the stimulating effects of CM from infected phagocytes. Reciprocally, stimulation of monocytes and neutrophils with CM from Brucella-infected trophoblasts increased IL8 and/or IL6 secretion. These results suggest that human trophoblasts may provide a local inflammatory environment during B. abortus infections either through a direct response to the pathogen or through interactions with monocytes/macrophages or neutrophils, potentially contributing to the pregnancy complications of brucellosis. PMID:26792938

  7. HCG-Activated Human Peripheral Blood Mononuclear Cells (PBMC) Promote Trophoblast Cell Invasion

    PubMed Central

    Wang, Yaqin; Guo, Yue; Zhou, Danni; Xu, Mei; Ding, Jinli; Yang, Jing

    2015-01-01

    Successful embryo implantation and placentation depend on appropriate trophoblast invasion into the maternal endometrial stroma. Human chorionic gonadotropin (hCG) is one of the earliest embryo-derived secreted signals in the peripheral blood mononuclear cells (PBMC) that abundantly expresses hCG receptors. The aims of this study were to estimate the effect of human embryo–secreted hCG on PBMC function and investigate the role and underlying mechanisms of activated PBMC in trophoblast invasion. Blood samples were collected from women undergoing benign gynecological surgery during the mid-secretory phase. PBMC were isolated and stimulated with or without hCG for 0 or 24 h. Interleukin-1β (IL-1β) and leukemia inhibitory factor (LIF) expressions in PBMC were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction (PCR). The JAR cell line served as a model for trophoblast cells and was divided into four groups: control, hCG only, PBMC only, and PBMC with hCG. JAR cell invasive and proliferative abilities were detected by trans-well and CCK8 assays and matrix metalloproteinase (MMP)-2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 expressions in JAR cells were detected by western blotting and real-time PCR analysis. We found that hCG can remarkably promote IL-1β and LIF promotion in PBMC after 24-h culture. PBMC activated by hCG significantly increased the number of invasive JAR cells in an invasion assay without affecting proliferation, and hCG-activated PBMC significantly increased MMP-2, MMP-9, and VEGF and decreased TIMP-1 and TIMP-2 expressions in JAR cells in a dose-dependent manner. This study demonstrated that hCG stimulates cytokine secretion in human PBMC and could stimulate trophoblast invasion. PMID:26087261

  8. Effects of Alcohol, Lithium, and Homocysteine on Nonmuscle Myosin-II in the Mouse Placenta and Human Trophoblasts

    PubMed Central

    HAN, Mingda; NEVES, Ana Luisa; SERRANO, Maria; BRINEZ, Pilar; HUHTA, James C.; ACHARYA, Ganesh; LINASK, Kersti K.

    2012-01-01

    Mouse embryonic exposure to alcohol, lithium, and homocysteine results in intrauterine growth restriction (IUGR) and cardiac defects. Our present study focuses on the placental effects. Objectives We analyzed the hypothesis that expression of nonmuscle myosin (NMM)-II isoforms involved in cell motility, mechanosensing, and extracellular matrix assembly, are altered by the three factors in human trophoblast (HTR8/SVneo) cells in vitro and in the mouse placenta in vivo. Study Design After exposure during gastrulation to alcohol, homocysteine, or lithium, ultrasonography defined embryos exhibiting abnormal placental blood flow. Results NMM-IIA /NMM-IIB are differentially expressed in trophoblasts and in mouse placental vascular endothelial cells under pathological conditions. Misexpression of NMM-IIA/ NMM-IIB in the affected placentas continued stably to mid-gestation, but can be prevented by folate and myo-inositol supplementation. Conclusions It is concluded that folate and myo-inositol initiated early in mouse pregnancy can restore NMM-II expression, permit normal placentation/embryogenesis, and prevent IUGR induced by alcohol, lithium, and homocysteine. PMID:22704764

  9. C19MC MicroRNAs Regulate the Migration of Human Trophoblasts

    PubMed Central

    Xie, Lan; Mouillet, Jean-Francois; Chu, Tianjiao; Parks, W. Tony; Sadovsky, Elena; Knöfler, Martin

    2014-01-01

    Early in pregnancy, trophoblast invasion into the decidua and inner myometrium is essential for establishment of proper implantation, maternal-fetal exchange, and immunological tolerance of the feto-placental allograft. Unlike villous trophoblasts (VTs), extravillous trophoblasts (EVTs) are unique in their capacity to invade the maternal decidua and myometrium. The largest human microRNA (miRNA) gene cluster, the chromosome 19 miRNA cluster (C19MC), is expressed almost exclusively in the placenta and, rarely, in certain tumors and undifferentiated cells. In the work reported here, we found that the expression of C19MC miRNAs is higher in VTs than in EVTs. Using a bacterial artificial chromosome (BAC)-mediated overexpression of C19MC miRNAs in an EVT-derived cell line, which does not naturally express these miRNAs, we found that C19MC miRNAs selectively attenuate cell migration without affecting cell proliferation or apoptosis. A microarray analysis revealed that C19MC miRNAs regulate target transcripts related to cellular movement. Our data also implicated a specific C19MC member, miR-519d, indirectly regulating the EVT invasive phenotype by targeting CXCL6, NR4A2 and FOXL2 transcripts through a 3′UTR miRNA-responsive element. Together, our data suggest a role for C19MC miRNAs in modulating the migration of EVTs. PMID:25211593

  10. Glucose and metformin modulate human first trimester trophoblast function:A model and potential therapy for diabetes-associated uteroplacental insufficiency

    PubMed Central

    Han, Christina S.; Herrin, Melissa A.; Pitruzzello, Mary C.; Mulla, Melissa J.; Werner, Erika F.; Pettker, Christian M.; Flannery, Clare A.; Abrahams, Vikki M.

    2014-01-01

    Problem Diabetes confers an increased risk of preeclampsia, but its pathogenic role in preeclampsia is poorly understood. The objective of this study was to elucidate the effects of excess glucose on trophoblast function and whether any changes could be reversed by metformin. Method of Study The human first trimester trophoblast cell line (Sw.71) was treated with glucose at 5mM, 10mM, 25mM and 50mM, in the presence and absence of metformin. Trophoblast migration was quantified and supernatant cytokine, chemokine, and angiogenic factors measured. Results Increasing concentrations of glucose significantly increased trophoblast secretion of the inflammatory cytokines/chemokines: IL-1β, IL-6, IL-8, GRO-α, RANTES and G-CSF; significantly increased trophoblast secretion of the anti-angiogenic factors sFlt-1 and sEndoglin; and significantly decreased trophoblast migration. Excess glucose-induced trophoblast IL-1β production was inhibited by disabling the Nalp3/ASC inflammasome. Metformin partially reduced the glucose-induced inflammatory response, but had no effect on the anti-angiogenic or anti-migratory response. Conclusion Excess glucose induced a pro-inflammatory, anti-angiogenic, and anti-migratory state in first trimester trophoblast cells. Glucose-induced trophoblast IL-1β secretion was mediated by the inflammasome. Glucose-induced inflammation was partially reversed by metformin. These findings demonstrate the pleiotropic effects of hyperglycemia on the trophoblast, providing potential explanations for the strong link between diabetes and preeclampsia. PMID:25394884

  11. Calcitriol inhibits interleukin-10 expression in cultured human trophoblasts under normal and inflammatory conditions.

    PubMed

    Barrera, David; Noyola-Martínez, Nancy; Avila, Euclides; Halhali, Ali; Larrea, Fernando; Díaz, Lorenza

    2012-03-01

    Preeclampsia is associated with systemic inflammation and increased expression of placental Th1-cytokines. IL-10 and calcitriol inhibit proinflammatory cytokines expression in human placenta helping to fetal allograft toleration. Regulation of placental IL-10 by calcitriol and Th-1 cytokines has not yet been fully elucidated. Since it is believed that calcitriol promotes a shift from a Th1- to a Th2 profile, we hypothesized that it would stimulate IL-10 in a normal and an inflammatory scenario to conjointly restrain inflammation. Therefore, we investigated calcitriol effects upon IL-10 expression in cultured human trophoblasts obtained from normal (NT) and preeclamptic (PE) pregnancies. Similar studies in the presence of TNF-α (as an inflammatory stressor) were also performed. Calcitriol dose-dependently inhibited IL-10 expression in NT, PE and TNF-α-challenged trophoblasts (P<0.05). This effect was prevented by a vitamin D receptor (VDR) antagonist. IL-10 expression was significantly stimulated by TNF-α and IL-1β, inhibited by IFN-γ and was not affected by IL-6. Finally, calcitriol inhibited TNF-α and IL-1β stimulation upon IL-10. In summary, in cultured human trophoblasts, calcitriol down-regulates IL-10 expression under normal as well as under natural and experimental inflammatory conditions. This effect is mediated by the VDR and might involve direct inhibition of TNF-α. In view of these and previous results it seems that in placenta calcitriol suppresses both Th1- and Th2 cytokines while undertakes the anti-inflammatory effects of IL-10 by itself, since both factors exert this task redundantly. The regulation of IL-10 by IFN-γ suggests that this cytokine could be a viable candidate to explain low IL-10 levels in preeclampsia.

  12. Is Doppler ultrasound useful for evaluating gestational trophoblastic disease?

    PubMed Central

    Lin, Lawrence H; Bernardes, Lisandra S; Hase, Eliane A; Fushida, Koji; Francisco, Rossana P V

    2015-01-01

    Doppler ultrasound is a non-invasive method for evaluating vascularization and is widely used in clinical practice. Gestational trophoblastic neoplasia includes a group of highly vascularized malignancies derived from placental cells. This review summarizes data found in the literature regarding the applications of Doppler ultrasound in managing patients with gestational trophoblastic neoplasia. The PubMed/Medline, Web of Science, Cochrane and LILACS databases were searched for articles published in English until 2014 using the following keywords: “Gestational trophoblastic disease AND Ultrasonography, Doppler.” Twenty-eight articles met the inclusion criteria and were separated into the 4 following groups according to the aim of the study. 1 Doppler ultrasound does not seem to be capable of differentiating partial from complete moles, but it might be useful when evaluating pregnancies in which a complete mole coexists with a normal fetus. 2 There is controversy in the role of uterine artery Doppler velocimetry in the prediction of development of gestational trophoblastic neoplasia. 3 Doppler ultrasound is a useful tool in the diagnosis of gestational trophoblastic neoplasia because abnormal myometrial vascularization and lower uterine artery Doppler indices seem to be correlated with invasive disease. 4 Lower uterine artery Doppler indices in the diagnosis of gestational trophoblastic neoplasia are associated with methotrexate resistance and might play a role in prognosis. CONCLUSION: Several studies support the importance of Doppler ultrasound in the management of patients with gestational trophoblastic neoplasia, particularly the role of Doppler velocimetry in the prediction of trophoblastic neoplasia and the chemoresistance of trophoblastic tumors. Doppler findings should be used as ancillary tools, along with human chorionic gonadotropin assessment, in the diagnosis of gestational trophoblastic neoplasia. PMID:26735221

  13. Differentiation of first trimester cytotrophoblast to extravillous trophoblast involves an epithelial-mesenchymal transition.

    PubMed

    DaSilva-Arnold, Sonia; James, Joanna L; Al-Khan, Abdulla; Zamudio, Stacy; Illsley, Nicholas P

    2015-12-01

    The transformation of cytotrophoblast (CTB) to extravillous trophoblast (EVT) is an essential process for placental implantation. EVT generated at the tips of the anchoring villi migrate away from the placenta and invade the endometrium and maternal spiral arteries, where they modulate maternal immune responses and remodel the arteries into high-volume conduits to facilitate uteroplacental blood flow. The process of EVT differentiation has several factors in common with the epithelial-to-mesenchymal transition (EMT) observed in embryonic development, wound healing and cancer metastasis. We hypothesized that the generation of invasive EVT from CTB was a form of EMT. We isolated paired CTB and EVT from first trimester placentae, and compared their gene expression using a PCR array comprising probes for genes involved in EMT. Out of 84 genes, 24 were down-regulated in EVT compared to CTB, including epithelial markers such as E-cadherin (-11-fold) and occludin (-75-fold). Another 30 genes were up-regulated in EVT compared to CTB including mesenchymal markers such as vimentin (235-fold) and fibronectin (107-fold) as well as the matrix metalloproteinases, MMP2 and MMP9 (357-fold, 129-fold). These alterations also included major increases in the ZEB2 (zinc finger E-box binding homeobox 2, 198-fold) and TCF4 (transcription factor 4, 18-fold) transcription factors, suggesting possible stimulatory mechanisms. There was substantial up-regulation of the genes encoding TGFβ1 and TGFβ2 (48-fold, 115-fold), which may contribute to the maintenance of the mesenchymal-like phenotype. We conclude that transformation of CTB to EVT is consistent with an EMT, although the differences with other types of EMT suggest this may be a unique form. PMID:26545962

  14. Pomegranate juice and punicalagin attenuate oxidative stress and apoptosis in human placenta and in human placental trophoblasts.

    PubMed

    Chen, Baosheng; Tuuli, Methodius G; Longtine, Mark S; Shin, Joong Sik; Lawrence, Russell; Inder, Terrie; Michael Nelson, D

    2012-05-15

    The human placenta is key to pregnancy outcome, and the elevated oxidative stress present in many complicated pregnancies contributes to placental dysfunction and suboptimal pregnancy outcomes. We tested the hypothesis that pomegranate juice, which is rich in polyphenolic antioxidants, limits placental trophoblast injury in vivo and in vitro. Pregnant women with singleton pregnancies were randomized at 35∼38 wk gestation to 8 oz/day of pomegranate juice or apple juice (placebo) until the time of delivery. Placental tissues from 12 patients (4 in the pomegranate group and 8 in the control group) were collected for analysis of oxidative stress. The preliminary in vivo results were extended to oxidative stress and cell death assays in vitro. Placental explants and cultured primary human trophoblasts were exposed to pomegranate juice or glucose (control) under defined oxygen tensions and chemical stimuli. We found decreased oxidative stress in term human placentas from women who labored after prenatal ingestion of pomegranate juice compared with apple juice as control. Moreover, pomegranate juice reduced in vitro oxidative stress, apoptosis, and global cell death in term villous explants and primary trophoblast cultures exposed to hypoxia, the hypoxia mimetic cobalt chloride, and the kinase inhibitor staurosporine. Punicalagin, but not ellagic acid, both prominent polyphenols in pomegranate juice, reduced oxidative stress and stimulus-induced apoptosis in cultured syncytiotrophoblasts. We conclude that pomegranate juice reduces placental oxidative stress in vivo and in vitro while limiting stimulus-induced death of human trophoblasts in culture. The polyphenol punicalagin mimics this protective effect. We speculate that antenatal intake of pomegranate may limit placental injury and thereby may confer protection to the exposed fetus.

  15. Progesterone-induced blocking factor differentially regulates trophoblast and tumor invasion by altering matrix metalloproteinase activity.

    PubMed

    Halasz, Melinda; Polgar, Beata; Berta, Gergely; Czimbalek, Livia; Szekeres-Bartho, Julia

    2013-12-01

    Invasiveness is a common feature of trophoblast and tumors; however, while tumor invasion is uncontrolled, trophoblast invasion is strictly regulated. Both trophoblast and tumor cells express high levels of the immunomodulatory progesterone-induced blocking factor (PIBF), therefore, we aimed to test the possibility that PIBF might be involved in invasion. To this aim, we used PIBF-silenced or PIBF-treated trophoblast (HTR8/Svneo, and primary trophoblast) and tumor (HT-1080, A549, HCT116, PC3) cell lines. Silencing of PIBF increased invasiveness as well as MMP-2,-9 secretion of HTR8/SVneo, and decreased those of HT-1080 cells. PIBF induced immediate STAT6 activation in both cell lines. Silencing of IL-4Rα abrogated all the above effects of PIBF, suggesting that invasion-related signaling by PIBF is initiated through the IL-4Rα/PIBF-receptor complex. In HTR-8/SVneo, PIBF induced fast, but transient Akt and ERK phosphorylation, whereas in tumor cells, PIBF triggered sustained Akt, ERK, and late STAT3 activation. The late signaling events might be due to indirect action of PIBF. PIBF induced the expression of EGF and HB-EGF in HT-1080 cells. The STAT3-activating effect of PIBF was reduced in HB-EGF-deficient HT-1080 cells, suggesting that PIBF-induced HB-EGF contributes to late STAT3 activation. PIBF binds to the promoters of IL-6, EGF, and HB-EGF; however, the protein profile of the protein/DNA complex is different in the two cell lines. We conclude that in tumor cells, PIBF induces proteins, which activate invasion signaling, while-based on our previous data-PIBF might control trophoblast invasion by suppressing proinvasive genes.

  16. Decorin-Mediated Inhibition of Human Trophoblast Cells Proliferation, Migration, and Invasion and Promotion of Apoptosis In Vitro.

    PubMed

    Zou, Yanfen; Yu, Xiang; Lu, Jing; Jiang, Ziyan; Zuo, Qing; Fan, Mingsong; Huang, Shiyun; Sun, Lizhou

    2015-01-01

    Preeclampsia (PE) is a unique complication of pregnancy, the pathogenesis of which has been generally accepted to be associated with the dysfunctions of extravillous trophoblast (EVT) including proliferation, apoptosis, and migration and invasion. Decorin (DCN) has been proved to be a decidua-derived TGF-binding proteoglycan, which negatively regulates proliferation, migration, and invasiveness of human extravillous trophoblast cells. In this study, we identified a higher expression level of decorin in severe PE placentas by both real-time reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). And an inhibitory effect of decorin on proliferation, migration, and invasion and an enhanced effect on apoptosis in trophoblast cells HTR-8/SVneo and JEG-3 were validated in vitro. Also the modulations of decorin on trophoblast cells' metastasis and invasion functions were detected through regulating the matrix metalloproteinases (MMP2 and MMP9). Thus, we suggested that the contribution of decorin to the modulation of trophoblast cells might have implications for the pathogenesis of preeclampsia. PMID:26357650

  17. Spatiotemporal regulation of cAMP signaling controls the human trophoblast fusion

    PubMed Central

    Gerbaud, Pascale; Taskén, Kjetil; Pidoux, Guillaume

    2015-01-01

    During human placentation, mononuclear cytotrophoblasts fuse to form multinucleated syncytia ensuring hormonal production and nutrient exchanges between the maternal and fetal circulation. Syncytial formation is essential for the maintenance of pregnancy and for fetal growth. The cAMP signaling pathway is the major route to trigger trophoblast fusion and its activation results in phosphorylation of specific intracellular target proteins, in transcription of fusogenic genes and assembly of macromolecular protein complexes constituting the fusogenic machinery at the plasma membrane. Specificity in cAMP signaling is ensured by generation of localized pools of cAMP controlled by cAMP phosphodiesterases (PDEs) and by discrete spatial and temporal activation of protein kinase A (PKA) in supramolecular signaling clusters inside the cell organized by A-kinase-anchoring proteins (AKAPs) and by organization of signal termination by protein phosphatases (PPs). Here we present original observations on the available components of the cAMP signaling pathway in the human placenta including PKA, PDE, and PP isoforms as well as AKAPs. We continue to discuss the current knowledge of the spatiotemporal regulation of cAMP signaling triggering trophoblast fusion. PMID:26441659

  18. Differentiation of trophoblast stem cells into giant cells is triggered by p57/Kip2 inhibition of CDK1 activity

    PubMed Central

    Ullah, Zakir; Kohn, Matthew J.; Yagi, Rieko; Vassilev, Lyubomir T.; DePamphilis, Melvin L.

    2008-01-01

    Genome endoreduplication during mammalian development is a rare event for which the mechanism is unknown. It first appears when fibroblast growth factor 4 (FGF4) deprivation induces differentiation of trophoblast stem (TS) cells into the nonproliferating trophoblast giant (TG) cells required for embryo implantation. Here we show that RO3306 inhibition of cyclin-dependent protein kinase 1 (CDK1), the enzyme required to enter mitosis, induced differentiation of TS cells into TG cells. In contrast, RO3306 induced abortive endoreduplication and apoptosis in embryonic stem cells, revealing that inactivation of CDK1 triggers endoreduplication only in cells programmed to differentiate into polyploid cells. Similarly, FGF4 deprivation resulted in CDK1 inhibition by overexpressing two CDK-specific inhibitors, p57/KIP2 and p21/CIP1. TS cell mutants revealed that p57 was required to trigger endoreduplication by inhibiting CDK1, while p21 suppressed expression of the checkpoint protein kinase CHK1, thereby preventing induction of apoptosis. Furthermore, Cdk2−/− TS cells revealed that CDK2 is required for endoreduplication when CDK1 is inhibited. Expression of p57 in TG cells was restricted to G-phase nuclei to allow CDK activation of S phase. Thus, endoreduplication in TS cells is triggered by p57 inhibition of CDK1 with concomitant suppression of the DNA damage response by p21. PMID:18981479

  19. Elsevier Trophoblast Research Award Lecture: Unique properties of decidual T cells and their role in immune regulation during human pregnancy.

    PubMed

    Tilburgs, T; Claas, F H J; Scherjon, S A

    2010-03-01

    Maternal lymphocytes at the fetal-maternal interface play a key role in the immune acceptance of the allogeneic fetus. Most studies focus on decidual NK cells and their interaction with fetal trophoblasts, whereas limited data are available on the mechanisms of fetus specific immune recognition and immune regulation by decidual T cells at the fetal-maternal interface. The aim of this review is to describe the phenotypic characteristics of decidual T cell subsets present at the fetal-maternal interface, their interaction with HLA-C expressed by fetal trophoblasts and their role in immune recognition and regulation at the fetal-maternal interface during human pregnancy.

  20. The contribution of SNAT1 to system A amino acid transporter activity in human placental trophoblast

    SciTech Connect

    Desforges, M.; Greenwood, S.L.; Glazier, J.D.; Westwood, M.; Sibley, C.P.

    2010-07-16

    Research highlights: {yields} mRNA levels for SNAT1 are higher than other system A subtype mRNAs in primary human cytotrophoblast. {yields} SNAT1 knockdown in cytotrophoblast cells significantly reduces system A activity. {yields} SNAT1 is a key contributor to system A-mediated amino acid transport in human placenta. -- Abstract: System A-mediated amino acid transport across the placenta is important for the supply of neutral amino acids needed for fetal growth. All three system A subtypes (SNAT1, 2, and 4) are expressed in human placental trophoblast suggesting there is an important biological role for each. Placental system A activity increases as pregnancy progresses, coinciding with increased fetal nutrient demands. We have previously shown SNAT4-mediated system A activity is higher in first trimester than at term, suggesting that SNAT1 and/or SNAT2 are responsible for the increased system A activity later in gestation. However, the relative contribution of each subtype to transporter activity in trophoblast at term has yet to be evaluated. The purpose of this study was to identify the predominant subtype of system A in cytotrophoblast cells isolated from term placenta, maintained in culture for 66 h, by: (1) measuring mRNA expression of the three subtypes and determining the Michaelis-Menten constants for uptake of the system A-specific substrate, {sup 14}C-MeAIB, (2) investigating the contribution of SNAT1 to total system A activity using siRNA. Results: mRNA expression was highest for the SNAT1 subtype of system A. Kinetic analysis of {sup 14}C-MeAIB uptake revealed two distinct transport systems; system 1: K{sub m} = 0.38 {+-} 0.12 mM, V{sub max} = 27.8 {+-} 9.0 pmol/mg protein/20 min, which resembles that reported for SNAT1 and SNAT2 in other cell types, and system 2: K{sub m} = 45.4 {+-} 25.0 mM, V{sub max} = 1190 {+-} 291 pmol/mg protein/20 min, which potentially represents SNAT4. Successful knockdown of SNAT1 mRNA using target-specific si

  1. Elf5-centered transcription factor hub controls trophoblast stem cell self-renewal and differentiation through stoichiometry-sensitive shifts in target gene networks

    PubMed Central

    Latos, Paulina A.; Sienerth, Arnold R.; Murray, Alexander; Senner, Claire E.; Muto, Masanaga; Ikawa, Masahito; Oxley, David; Burge, Sarah; Cox, Brian J.; Hemberger, Myriam

    2015-01-01

    Elf5 is a transcription factor with pivotal roles in the trophoblast compartment, where it reinforces a trophoblast stem cell (TSC)-specific transcriptional circuit. However, Elf5 is also present in differentiating trophoblast cells that have ceased to express other TSC genes such as Cdx2 and Eomes. In the present study, we aimed to elucidate the context-dependent role of Elf5 at the interface between TSC self-renewal and the onset of differentiation. We demonstrate that precise levels of Elf5 are critical for normal expansion of the TSC compartment and embryonic survival, as Elf5 overexpression triggers precocious trophoblast differentiation. Through integration of protein interactome, transcriptome, and genome-wide chromatin immunoprecipitation data, we reveal that this abundance-dependent function is mediated through a shift in preferred Elf5-binding partners; in TSCs, Elf5 interaction with Eomes recruits Tfap2c to triply occupied sites at TSC-specific genes, driving their expression. In contrast, the Elf5 and Tfap2c interaction becomes predominant as their protein levels increase. This triggers binding to double- and single-occupancy sites that harbor the cognate Tfap2c motif, causing activation of the associated differentiation-promoting genes. These data place Elf5 at the center of a stoichiometry-sensitive transcriptional network, where it acts as a molecular switch governing the balance between TSC proliferation and differentiation. PMID:26584622

  2. Elf5-centered transcription factor hub controls trophoblast stem cell self-renewal and differentiation through stoichiometry-sensitive shifts in target gene networks.

    PubMed

    Latos, Paulina A; Sienerth, Arnold R; Murray, Alexander; Senner, Claire E; Muto, Masanaga; Ikawa, Masahito; Oxley, David; Burge, Sarah; Cox, Brian J; Hemberger, Myriam

    2015-12-01

    Elf5 is a transcription factor with pivotal roles in the trophoblast compartment, where it reinforces a trophoblast stem cell (TSC)-specific transcriptional circuit. However, Elf5 is also present in differentiating trophoblast cells that have ceased to express other TSC genes such as Cdx2 and Eomes. In the present study, we aimed to elucidate the context-dependent role of Elf5 at the interface between TSC self-renewal and the onset of differentiation. We demonstrate that precise levels of Elf5 are critical for normal expansion of the TSC compartment and embryonic survival, as Elf5 overexpression triggers precocious trophoblast differentiation. Through integration of protein interactome, transcriptome, and genome-wide chromatin immunoprecipitation data, we reveal that this abundance-dependent function is mediated through a shift in preferred Elf5-binding partners; in TSCs, Elf5 interaction with Eomes recruits Tfap2c to triply occupied sites at TSC-specific genes, driving their expression. In contrast, the Elf5 and Tfap2c interaction becomes predominant as their protein levels increase. This triggers binding to double- and single-occupancy sites that harbor the cognate Tfap2c motif, causing activation of the associated differentiation-promoting genes. These data place Elf5 at the center of a stoichiometry-sensitive transcriptional network, where it acts as a molecular switch governing the balance between TSC proliferation and differentiation. PMID:26584622

  3. Ceramide biosynthesis and metabolism in trophoblast syncytialization.

    PubMed

    Singh, Ambika T; Dharmarajan, Arunasalam; Aye, Irving L M H; Keelan, Jeffrey A

    2012-10-15

    Sphingolipid mediators such as ceramide are pleiotropic regulators of cellular growth, differentiation and apoptosis. We investigated the role of ceramide biosynthesis, metabolism and actions in term human cytotrophoblasts syncytialized over 7 days in culture. Intracellular C16 ceramide levels increased modestly after 3 days in culture, then declined. Ceramidase was present at particularly high levels in syncytialized trophoblasts; inhibition of ceramidase reduced the degree of cell fusion. Exposure to short chain C8 ceramide or aSMase enhanced secretion of the differentiation marker hCG without affecting fusion or cell viability. In contrast, pharmacological inhibition of ceramidase reduced the extent of fusion. Inhibition of the ceramide-responsive JNK and PP2A pathways did not abolish the effects of ceramide, and JNK phosphorylation was unresponsive to ceramide; however, ceramide significantly inhibited phosphorylation of Akt. This study suggests that changes in ceramide biosynthesis and metabolism play a differential role in the biochemical and morphological features of trophoblast differentiation.

  4. Expression and regulation of the ery operon of Brucella melitensis in human trophoblast cells

    PubMed Central

    Zhang, Hui; Dou, Xiaoxia; Li, Zhiqiang; Zhang, Yu; Zhang, Jing; Guo, Fei; Wang, Yuanzhi; Wang, Zhen; Li, Tiansen; Gu, Xinli; Chen, Chuangfu

    2016-01-01

    Brucellosis is primarily a disease of domestic animals in which the bacteria localizes to fetal tissues such as embryonic trophoblast cells and fluids containing erythritol, which stimulates Brucella spp. growth. The utilization of erythritol is a characteristic of the genus Brucella. The ery operon contains four genes (eryA, eryB, eryC and eryD) for the utilization of erythritol, and plays a major role in the survival and multiplication of Brucella spp. The objective of the present study was to conduct a preliminary characterization of differential genes expression of the ery operon at several time points after Brucella infected embryonic trophoblast cells (HPT-8 cells). The result showed that the ery operon expression was higher in HPT-8 cells compared with the medium. The relative expression of eryA, eryB and eryC peaked at 2 h post-infection in HPT-8 cells, and eryD expression peaked at 3 h post-infection. The expression of eryA, eryB and eryC may be inhibited by increased eryD expression. However, the expression of the ery operon was stable in the presence of erythritol in cells. 2308Δery and 027Δery mutants of the ery operon were successfully constructed by homologous recombination, which were attenuated in RAW 264.7 murine macrophages. The characterization of the ery operon genes and their expression profiles in response to Brucella infection further contributes to our understanding of the molecular mechanisms of infection and the pathogenesis of brucellosis. PMID:27698777

  5. Expression and regulation of the ery operon of Brucella melitensis in human trophoblast cells

    PubMed Central

    Zhang, Hui; Dou, Xiaoxia; Li, Zhiqiang; Zhang, Yu; Zhang, Jing; Guo, Fei; Wang, Yuanzhi; Wang, Zhen; Li, Tiansen; Gu, Xinli; Chen, Chuangfu

    2016-01-01

    Brucellosis is primarily a disease of domestic animals in which the bacteria localizes to fetal tissues such as embryonic trophoblast cells and fluids containing erythritol, which stimulates Brucella spp. growth. The utilization of erythritol is a characteristic of the genus Brucella. The ery operon contains four genes (eryA, eryB, eryC and eryD) for the utilization of erythritol, and plays a major role in the survival and multiplication of Brucella spp. The objective of the present study was to conduct a preliminary characterization of differential genes expression of the ery operon at several time points after Brucella infected embryonic trophoblast cells (HPT-8 cells). The result showed that the ery operon expression was higher in HPT-8 cells compared with the medium. The relative expression of eryA, eryB and eryC peaked at 2 h post-infection in HPT-8 cells, and eryD expression peaked at 3 h post-infection. The expression of eryA, eryB and eryC may be inhibited by increased eryD expression. However, the expression of the ery operon was stable in the presence of erythritol in cells. 2308Δery and 027Δery mutants of the ery operon were successfully constructed by homologous recombination, which were attenuated in RAW 264.7 murine macrophages. The characterization of the ery operon genes and their expression profiles in response to Brucella infection further contributes to our understanding of the molecular mechanisms of infection and the pathogenesis of brucellosis.

  6. IFPA Meeting 2011 workshop report III: Placental immunology; epigenetic and microRNA-dependent gene regulation; comparative placentation; trophoblast differentiation; stem cells☆

    PubMed Central

    Ackerman, W.E.; Bulmer, J.N.; Carter, A.M.; Chaillet, J.R.; Chamley, L.; Chen, C.P.; Chuong, E.B.; Coleman, S.J.; Collet, G.P.; Croy, B.A.; de Mestre, A.M.; Dickinson, H.; Ducray, J.; Enders, A.C.; Fogarty, N.M.E.; Gauster, M.; Golos, T.; Haider, S.; Heazell, A.E.; Holland, O.J.; Huppertz, B.; Husebekk, A.; John, R.M.; Johnsen, G.M.; Jones, C.J.P.; Kalionis, B.; König, J.; Lorenzon, A.R.; Moffett, A.; de Mello, J.C. Moreira; Nuzzo, A.M.; Parham, P.; Parolini, O.; Petroff, M.G.; Pidoux, G.; Ramírez-Pinilla, M.P.; Robinson, W.P.; Rolfo, A.; Sadovsky, Y.; Soma, H.; Southcombe, J.H.; Tilburgs, T.; Lash, G.E.

    2014-01-01

    Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells. PMID:22154501

  7. Prostaglandin E2-mediated migration of human trophoblast requires RAC1 and CDC42.

    PubMed

    Nicola, Catalin; Lala, Peeyush K; Chakraborty, Chandan

    2008-06-01

    The invasion of maternal decidua and uterine spiral arteries by a trophoblast subpopulation called extravillous trophoblast (EVT) is essential for the establishment of a normal placenta and an adequate blood flow toward the fetus. Derangements in these processes underlie pregnancy-related diseases like preeclampsia and intrauterine growth restriction. Many growth factors, growth factor binding proteins, and extracellular matrix components can positively or negatively regulate the proliferation, migration, and/or invasiveness of these EVT cells. RHO GTPases, including RHOA, RAC1, and CDC42, are ubiquitous proteins that control cytoskeletal changes by forming stress fibers and projecting lamellipodia and filopodia during cellular migration. We had previously shown that prostaglandin (PG) E(2) produced in abundance by the decidua promotes the migration of first-trimester human EVTs by increasing the intracellular concentration of calcium and activating calpain. Using our well-characterized immortalized EVT cell line, HTR-8/SVneo, as well as villus explants from first-trimester placentae, this study examined the role of RHO GTPases RAC1 and CDC42 in PGE(2)-mediated migratory responses of these cells. Though a RAC1 inhibitor, NSC23766 as well as RAC1 knockdown by siRNA decreased the migration of HTR-8/SVneo cells in a Transwell migration assay, this inhibition could not be restored by PGE(2) or 17-phenyl trinor PGE(2) (PGE receptor PTGER1 agonist) or PGE(1) Alcohol (PGE receptor PTGER4 agonist). Similar results were noted for EVT cell spreading in villus explants. Furthermore, CDC42 silencing using siRNA inhibited PGE(2)-induced migration of HTR-8/SVneo cells. Finally, the treatment of EVT cells with PGE(2), PTGER1 agonist, or PTGER4 agonist activated RAC1 and CDC42 at 10 min, suggesting that RAC1 and CDC42 play an essential role in PGE(2)-mediated migration of human EVTs.

  8. Fibulin-5 is upregulated in decidualized human endometrial stromal cells and promotes primary human extravillous trophoblast outgrowth.

    PubMed

    Winship, Amy; Cuman, Carly; Rainczuk, Katarzyna; Dimitriadis, Evdokia

    2015-12-01

    Interactions between the highly invasive trophoblasts and the maternal uterine decidual extracellular matrix (ECM) are crucial in the determination of a successful pregnancy. Fibulin-5 (FBLN5) is a member of the fibulin family that alters cell adhesive and invasive properties and is expressed in human villous cytotrophoblasts. We aimed to determine the expression and immunolocalization of FBLN5 in human first trimester decidua and examine the effect of FBLN5 in trophoblast invasion in vitro using a first trimester placental villous outgrowth assay. We demonstrated that FBLN5 mRNA expression is upregulated in response to cAMP-mediated decidualization of primary human endometrial stromal cells, although FBLN5 itself does not enhance decidualization. We reported for the first time, FBLN5 protein production in first trimester decidual cells and also co-localization to HLAG-positive EVTs in first trimester decidua. Consequently, we investigated the effects of exogenous FBLN5 on placental villous outgrowth in vitro and demonstrated that FBLN5 promotes EVT migration/invasion. This is the first study to identify FBLN5 in decidualized human endometrial stromal cells, first trimester decidua and EVT and determine a functional role for FBLN5 in human EVTs, suggesting that decidual and or EVT-derived FBLN5 regulates EVT invasion and placentation in women. PMID:26506560

  9. Hypoxic stress induces, but cannot sustain trophoblast stem cell differentiation to labyrinthine placenta due to mitochondrial insufficiency

    PubMed Central

    Xie, Yufen; Zhou, Sichang; Jiang, Zhongliang; Dai, Jing; Puscheck, Elizabeth E; Lee, Icksoo; Parker, Graham; Hüttemann, Maik; Rappolee, Daniel A

    2014-01-01

    Dysfunctional stem cell differentiation into placental lineages is associated with gestational diseases. Of the differentiated lineages available to trophoblast stem cells (TSC), elevated O2 and mitochondrial function are necessary to placental lineages at the maternal-placental surface and important in the etiology of preeclampsia. TSC lineage imbalance leads to embryonic failure during uterine implantation. Stress at implantation exacerbates stem cell depletion by decreasing proliferation and increasing differentiation. Implantation site O2 is normally ~2%. In culture, exposure to 2% O2 and fibroblast growth factor (FGF)4 enabled highest mouse TSC multipotency and proliferation. In contrast, hypoxic stress (0.5% O2) initiated the most TSC differentiation after 24 hr despite FGF4. However, hypoxic stress supported differentiation poorly after 4–7 days, despite FGF4 removal. At all tested O2 levels, FGF4 maintained Warburg metabolism; mitochondrial inactivity and aerobic glycolysis. However, hypoxic stress suppressed mitochondrial membrane potential, maintained low mitochondrial cytochrome c oxidase (oxidative phosphorylation/OxPhos), and high pyruvate kinase M2 (glycolysis) despite FGF4 removal. Inhibiting OxPhos inhibited differentiation at the differentiation optimum at 20% O2. Moreover, adding differentiation-inducing hyperosmolar stress failed to induce differentiation during hypoxia. Thus, differentiation depended on OxPhos at 20% O2; hypoxic and hyperosmolar stresses did not induce differentiation at 0.5% O2. Hypoxia-limited differentiation and mitochondrial inhibition and activation suggest that differentiation into two lineages of the labyrinthine placenta requires O2>0.5–2% and mitochondrial function. Stress-activated protein kinase increases an early lineage and suppresses later lineages in proportion to the deviation from optimal O2 for multipotency, thus it is the first enzyme reported to prioritize differentiation. PMID:25239494

  10. The molecular role of connexin 43 in human trophoblast cell fusion.

    PubMed

    Dunk, Caroline E; Gellhaus, Alexandra; Drewlo, Sascha; Baczyk, Dora; Pötgens, Andy J G; Winterhager, Elke; Kingdom, John C P; Lye, Steven J

    2012-04-01

    Connexin expression and gap junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43)/gap junction A1 (GJA1) are required for cytotrophoblast fusion into the syncytium, the outer functional layer of the human placenta. Cx43 also impacts intracellular signaling through protein-protein interactions. The transcription factor GCM1 and its downstream target ERVW-1/SYNCYTIN-1 are key players in trophoblast fusion and exert their actions through the ERVW-1 receptor SLC1A5/ASCT-2/RDR/ATB(0). To investigate the molecular role of the Cx43 protein and its interaction with this fusogenic pathway, we utilized stable Cx43-transfected cell lines established from the choriocarcinoma cell line Jeg3: wild-type Jeg3, alphahCG/Cx43 (constitutive Cx43 expression), JpUHD/Cx43 (doxycyclin-inducible Cx43 expression), or JpUHD/trCx43 (doxycyclin-inducible Cx43 carboxyterminal deleted). We hypothesized that truncation of Cx43 at its C-terminus would inhibit trophoblast fusion and protein interaction with either ERVW-1 or SLC1A5. In the alphahCG/Cx43 and JpUHD/Cx43 lines, stimulation with cAMP caused 1) increase in GJA1 mRNA levels, 2) increase in percentage of fused cells, and 3) downregulation of SLC1A5 expression. Cell fusion was inhibited by GJIC blockade using carbenoxylone. Neither Jeg3, which express low levels of Cx43, nor the JpUHD/trCx43 cell line demonstrated cell fusion or downregulation of SLC1A5. However, GCM1 and ERVW-1 mRNAs were upregulated by cAMP treatment in both Jeg3 and all Cx43 cell lines. Silencing of GCM1 prevented the induction of GJA1 mRNA by forskolin in BeWo choriocarcinoma cells, demonstrating that GCM1 is upstream of Cx43. All cell lines and first-trimester villous explants also demonstrated coimmunoprecipitation of SLC1A5 and phosphorylated Cx43. Importantly, SLC1A5 and Cx43 gap junction plaques colocalized in situ to areas of fusing cytotrophoblast, as demonstrated by the loss of E-cadherin staining in the plasma membrane in first

  11. Determination of hyperglycosylated human chorionic gonadotropin produced by malignant gestational trophoblastic neoplasias and male germ cell tumors using a lectin-based immunoassay and surface plasmon resonance

    PubMed Central

    Kelly, Lisa S.; Birken, Steven; Puett, David

    2007-01-01

    The ability to reliably detect aberrant glycosylation of human chorionic gonadotropin (hCG) may have profound implications for the diagnosis and monitoring of malignant gestational trophoblastic neoplasia, germ cell tumors, other malignancies, and pregnancy complications. To become a clinically useful assay, however, this discrimination of glycoforms should be possible on minimally treated biological specimens. Towards this end, we have developed a lectin-based sandwich-type immunoassay to compare the glycosylation patterns of hCG among urine specimens from patients presenting with a normal pregnancy, invasive mole, choriocarcinoma, and male germ cell tumors using carbohydrate-free antibody fragments as capture reagents and a panel of eight lectins, five recognizing neutral sugars and three recognizing sialic acid. There was no significant difference in the binding of any of the lectins to hCG in the urine of women over the gestational range of 6 – 38 weeks. Three lectins, however, exhibited differential binding to urinary hCG derived from these normal pregnant controls and that from patients with malignant forms of gestational trophoblastic disease and male germ cell tumors. Galanthus nivalis agglutinin and Maackia amurensis lectin, which bind terminal mannose and α(2–3)sialic acid, respectively, preferentially bound pregnancy-derived hCG, whereas the lectin, wheat germ agglutinin, which binds sialic acid and β(1–4)N-acetylglucosamine, exhibited decreased binding to pregnancy-derived hCG compared to that from patients with male germ cell tumors and malignant gestational trophoblastic neoplasia. The differential binding observed with these three promising lectins is most encouraging and warrants further examination. The experimental paradigm also holds promise for the development of comparable assays for other glycosylated tumor markers. PMID:17081681

  12. The histone variant H2A.Z is dynamically expressed in the developing mouse placenta and in differentiating trophoblast stem cells.

    PubMed

    Kafer, Georgia R; Carlton, Peter M; Lehnert, Sigrid A

    2015-11-01

    The histone variant H2A.Z is important in establishing new chromatin environments necessary for permitting changes in gene expression and thus differentiation in mouse embryonic stem (mES) cells. In this study we show that H2A.Z is highly expressed in the early mouse placenta, and is specifically limited to progenitor-like trophoblast cells. Using in vitro models, we revealed distinct differences in H2A.Z abundance between undifferentiated, differentiating and differentiated mouse trophoblast stem (mTS) cells. Our work supports the hypothesis that in addition to roles in differentiating mES cells, H2A.Z is also involved in the differentiation of extra-embryonic tissues.

  13. Expression of the human Hand1 gene in trophoblastic cells is transcriptionally regulated by activating and repressing specificity protein (Sp)-elements.

    PubMed

    Vasicek, Richard; Meinhardt, Gudrun; Haidweger, Eva; Rotheneder, Hans; Husslein, Peter; Knöfler, Martin

    2003-01-01

    The tissue-specific basic helix-loop-helix protein Hand1 is essential for the formation of trophoblast giant cells of the murine placenta. In humans, Hand1 is detectable in trophoblastic tumour cells suggesting an equivalent role in trophoblast differentiation. To understand its mode of expression we have cloned and characterized the human Hand1 gene promoter. Primer extension analyses suggest that transcription initiates 19 nucleotides downstream of the TATA element of the proximal 5' flanking region. Expression of luciferase reporter constructs harboring deletions of the 9.5 kb Hand1 5' flanking sequence defines a promoter region within 274 bp upstream of the transcriptional start site. Compared to a reporter bearing only the TATA box, the proximal promoter activates transcription up to 30-fold. However, transcriptional activity of the region was observed in both Hand1-expressing and non-expressing cell lines. Sequencing, DNAseI footprint analyses and electrophoretic mobility shift assays reveal the presence of four GC-rich sequences, which show different affinities to the endogenous specificity proteins (Sp), and a CCAAT box. In vitro, the Sp-elements mainly interact with Sp1 and Sp3 while the CCAAT element is recognized by the alpha CAAT binding factor protein. Mutant luciferase reporters bearing single active or inactive recognition sites demonstrate that two of the four Sp-binding sites (I and IV) contribute little to the overall transcription rate. The two other Sp-cognate sequences, II and III, downregulate and activate reporter expression 2.3- and 2.6-fold, respectively. Co-transfections of Sp1/Sp3 expression vectors and mutated reporter constructs in Sp-deficient SL2 cells indicate that the Sp-binding site II and III indeed function as repressing and activating enhancer sequences. In summary, the data suggest that constitutive expression of the Hand1 gene in cultured cells is regulated by a complex interplay of Sp-proteins interacting with activator and

  14. Transcriptional and functional studies of Human Endogenous Retrovirus envelope EnvP(b) and EnvV genes in human trophoblasts

    SciTech Connect

    Vargas, Amandine Thiery, Maxime Lafond, Julie Barbeau, Benoit

    2012-03-30

    HERV (Human Endogenous Retrovirus)-encoded envelope proteins are implicated in the development of the placenta. Indeed, Syncytin-1 and -2 play a crucial role in the fusion of human trophoblasts, a key step in placentation. Other studies have identified two other HERV env proteins, namely EnvP(b) and EnvV, both expressed in the placenta. In this study, we have fully characterized both env transcripts and their expression pattern and have assessed their implication in trophoblast fusion. Through RACE analyses, standard spliced transcripts were detected, while EnvV transcripts demonstrated alternative splicing at its 3 Prime end. Promoter activity and expression of both genes were induced in forskolin-stimulated BeWo cells and in primary trophoblasts. Although we have confirmed the fusogenic activity of EnvP(b), overexpression or silencing experiments revealed no impact of this protein on trophoblast fusion. Our results demonstrate that both env genes are expressed in human trophoblasts but are not required for syncytialization.

  15. Immune modulatory mesenchymal stem cells derived from human embryonic stem cells through a trophoblast-like stage.

    PubMed

    Wang, Xiaofang; Lazorchak, Adam S; Song, Li; Li, Enqin; Zhang, Zhenwu; Jiang, Bin; Xu, Ren-He

    2016-02-01

    Mesenchymal stem/stromal cells (MSCs) have great clinical potential in modulating inflammation and promoting tissue repair. Human embryonic stem cells (hESCs) have recently emerged as a potentially superior cell source for MSCs. However, the generation methods reported so far vary greatly in quality and efficiency. Here, we describe a novel method to rapidly and efficiently produce MSCs from hESCs via a trophoblast-like intermediate stage in approximately 11-16 days. We term these cells "T-MSCs" and show that T-MSCs express a phenotype and differentiation potential minimally required to define MSCs. T-MSCs exhibit potent immunomodulatory activity in vitro as they can remarkably inhibit proliferation of cocultured T and B lymphocytes. Unlike bone marrow MSCs, T-MSCs do not have increased expression of inflammatory mediators in response to IFNγ. Moreover, T-MSCs constitutively express a high level of the immune inhibitory ligand PD-L1 and elicit strong and durable efficacy in two distinct animal models of autoimmune disease, dextran sulfate sodium induced colitis, and experimental autoimmune encephalomyelitis, at doses near those approved for clinical trials. Together, we present a simple and fast derivation method to generate MSCs from hESCs, which possess potent immunomodulatory properties in vitro and in vivo and may serve as a novel and ideal candidate for MSC-based therapies.

  16. Immune modulatory mesenchymal stem cells derived from human embryonic stem cells through a trophoblast-like stage.

    PubMed

    Wang, Xiaofang; Lazorchak, Adam S; Song, Li; Li, Enqin; Zhang, Zhenwu; Jiang, Bin; Xu, Ren-He

    2016-02-01

    Mesenchymal stem/stromal cells (MSCs) have great clinical potential in modulating inflammation and promoting tissue repair. Human embryonic stem cells (hESCs) have recently emerged as a potentially superior cell source for MSCs. However, the generation methods reported so far vary greatly in quality and efficiency. Here, we describe a novel method to rapidly and efficiently produce MSCs from hESCs via a trophoblast-like intermediate stage in approximately 11-16 days. We term these cells "T-MSCs" and show that T-MSCs express a phenotype and differentiation potential minimally required to define MSCs. T-MSCs exhibit potent immunomodulatory activity in vitro as they can remarkably inhibit proliferation of cocultured T and B lymphocytes. Unlike bone marrow MSCs, T-MSCs do not have increased expression of inflammatory mediators in response to IFNγ. Moreover, T-MSCs constitutively express a high level of the immune inhibitory ligand PD-L1 and elicit strong and durable efficacy in two distinct animal models of autoimmune disease, dextran sulfate sodium induced colitis, and experimental autoimmune encephalomyelitis, at doses near those approved for clinical trials. Together, we present a simple and fast derivation method to generate MSCs from hESCs, which possess potent immunomodulatory properties in vitro and in vivo and may serve as a novel and ideal candidate for MSC-based therapies. PMID:26523849

  17. p63 Inhibits Extravillous Trophoblast Migration and Maintains Cells in a Cytotrophoblast Stem Cell-Like State

    PubMed Central

    Li, Yingchun; Moretto-Zita, Matteo; Leon-Garcia, Sandra; Parast, Mana M.

    2015-01-01

    Proper differentiation of placental epithelial cells, called trophoblast, is required for implantation. Early during placentation, trophoblast cell columns help anchor the developing embryo in the uterine wall. Although proximally continuous with villous cytotrophoblast (CTB) distally, these cells differentiate into invasive extravillous trophoblast. We previously reported that p63, a p53 family member, is highly expressed in proliferative villous CTB and required for induction of the trophoblast lineage in human pluripotent stem cells. We now further explore its function in human trophoblast by using both primary CTB from the early placenta and established trophoblast cell lines. We show that p63 is expressed in epidermal growth factor receptor-positive CTB and that its expression decreases with differentiation into HLA-G+ extravillous trophoblast. In trophoblast cell lines, p63 is expressed in JEG3 cells but absent from HTR8 cells. Overexpression of p63 in both cell lines enhances cell proliferation and significantly reduces cell migration; conversely, down-regulation of p63 in JEG3 cells reduces cell proliferation and restores cell migration. Analysis of epithelial-to-mesenchymal transition, cell adhesion, and matrix degradation pathways shows that p63 blocks epithelial-to-mesenchymal transition, promotes a CTB-specific cell adhesion profile, and inhibits expression of matrix metalloproteinases. Taken together, these data show that p63 maintains the proliferative CTB state, at least partially through regulation of epithelial-to-mesenchymal transition, cell adhesion, and matrix degradation pathways. PMID:25307348

  18. Claudin-3, claudin-7, and claudin-10 show different distribution patterns during decidualization and trophoblast invasion in mouse and human.

    PubMed

    Schumann, S; Buck, V U; Classen-Linke, I; Wennemuth, G; Grümmer, R

    2015-12-01

    Implantation of the mammalian embryo requires profound endometrial changes for successful pregnancy, including epithelial-mesenchymal transition of the luminal epithelium and stromal-epithelial transition of the stromal cells resulting in decidualization. Claudins (Cldn) determine the variability in tight junction paracellular permeability and may play a role during these epithelial and decidual changes. We here localized Cldn3, Cldn7 and Cldn10 proteins in the different compartments of murine endometrium up to day 8.5 of pregnancy (dpc) as well as in human endometrium and first trimester decidua. In murine estrous endometrium, luminal and glandular epithelium exhibited Cldn3 and Cldn7, whereas Cldn10 was only detectable in glandular epithelium. At 4.5 dpc, Cldn3 protein shifted to an apical localization, whereas Cldn7 vanished in the epithelium of the implantation chamber. At this stage, there was no stromal signal for Cldn3 and Cldn7, but a strong induction of Cldn10 in the primary decidual zone. Cldn3 proteins emerged at 5.5 dpc spreading considerably from 6.5 dpc onward in the endothelial cells of the decidual blood sinusoids and in the decidual cells of the compact antimesometrial region. In addition to Cldn3, Cldn10 was identified in human endometrial epithelia. Both proteins were not detected in human first trimester decidual cells. Cldn3 was shown in murine trophoblast giant cells as well as in human extravillous trophoblast cells and thus may have an impact on trophoblast invasion in both species. We here showed a specific claudin signature during early decidualization pointing to a role in decidual angiogenesis and regulation of trophoblast invasion. PMID:26340953

  19. Tube formation in the first trimester placental trophoblast cells: Differential effects of angiogenic growth factors and fatty acids.

    PubMed

    Pandya, Abhilash D; Das, Mrinal K; Sarkar, Arnab; Vilasagaram, Srinivas; Basak, Sanjay; Duttaroy, Asim K

    2016-06-01

    The study aims to investigate whether cytosolic fatty acid-binding protein-4 (FABP4) is involved in angiogenic growth factors- and fatty acid-induced tube formation in first trimester placental trophoblast cells, HTR8/SVneo. We determined the tube formation both at basal as well as stimulated levels in the absence and presence of inhibitors of FABP4 and VEGF signaling pathways. Basal level of tube formation was maximally reduced in the presence of 50 µM of FABP4 inhibitor compared with those by VEGF signaling pathway inhibitors (rapamycin, L-NAME, and p38 MAP kinase inhibitor). Whereas docosahexaenoic acid, 22:6n-3 (DHA)-, and VEGF-induced tube formation was maximally inhibited by p38 MAP kinase inhibitor (63.7 and 34.5%, respectively), however, leptin-induced tube formation was inhibited maximally by FABP4 inhibitor (50.7%). ANGPTL4 and oleic acid (OA)-induced tube formation was not blocked by any of these inhibitors. The FABP4 inhibitor inhibited cell growth stimulated by DHA, leptin, VEGF, and OA (P < 0.05) but was not affected by ANGPTL4. VEGF, leptin, and OA also increased FABP4 protein level in these cells, though the uptake of fatty acids by these cells was not affected by the presence of FABP4 inhibitor. Our data demonstrate that FABP4 may be involved in part in the basal level, and stimulated tube formation by VEGF, DHA, and leptin, whereas it has little or no effect in ANGPTL4- and OA-induced tube formation in these cells. Thus, FABP4 may play a differential role in fatty acids and angiogenic growth factors-mediated tube formation in the first trimester trophoblast cells in vitro.

  20. Peritoneal trophoblastic implant.

    PubMed

    Rachagan, S P; Kutty, K; Govindan, K S

    1997-09-01

    A case of persistent trophoblastic tissue on the pelvic peritoneum is presented. While most cases are secondary to conservative surgery for tubal ectopic pregnancy, primary implantation can also occur as highlighted by this case. A brief pathophysiology of the condition is presented. The importance of monitoring the serum for beta subunit human chorionic gonadotrophin (HCG) is emphasised.

  1. Oleic acid stimulates system A amino acid transport in primary human trophoblast cells mediated by toll-like receptor 4.

    PubMed

    Lager, Susanne; Gaccioli, Francesca; Ramirez, Vanessa I; Jones, Helen N; Jansson, Thomas; Powell, Theresa L

    2013-03-01

    Obese women have an increased risk to deliver large babies. However, the mechanisms underlying fetal overgrowth in these pregnancies are not well understood. Obese pregnant women typically have elevated circulating lipid levels. We tested the hypothesis that fatty acids stimulate placental amino acid transport, mediated via toll-like receptor 4 (TLR4) and mammalian target of rapamycin (mTOR) signaling pathways. Circulating NEFA levels and placental TLR4 expression were assessed in women with varying prepregnancy body mass index (BMI). The effects of oleic acid on system A and system L amino acid transport, and on the activation of the mTOR (4EBP1, S6K1, rpS6), TLR4 (IĸB, JNK, p38 MAPK), and STAT3 signaling pathways were determined in cultured primary human trophoblast cells. Maternal circulating NEFAs (n = 33), but not placental TLR4 mRNA expression (n = 16), correlated positively with BMI (P < 0.05). Oleic acid increased trophoblast JNK and STAT3 phosphorylation (P < 0.05), whereas mTOR activity was unaffected. Furthermore, oleic acid doubled trophoblast system A activity (P < 0.05), without affecting system L activity. siRNA-mediated silencing of TLR4 expression prevented the stimulatory effect of oleic acid on system A activity. Our data suggest that maternal fatty acids can increase placental nutrient transport via TLR4, thereby potentially affecting fetal growth.

  2. Effects of maternal diabetes on trophoblast cells.

    PubMed

    Aires, Marlúcia Bastos; Dos Santos, Anne Carolline Veríssimo

    2015-03-15

    Diabetes mellitus (DM) is a health condition characterized by hyperglycemia over a prolonged period. There are three main types of DM: DM type 1 (DM1), DM2 and gestational DM (GDM). Maternal diabetes, which includes the occurrence of DM1 and DM2 during pregnancy or GDM, increases the occurrence of gesttional complications and adverse fetal outcomes. The hyperglycemic intrauterine environment affects not only the fetus but also the placental development and function in humans and experimental rodents. The underlying mechanisms are still unclear, but some evidence indicates alterations in trophoblast proliferation, apoptosis and cell cycle control in diabetes. A proper coordination of trophoblast proliferation, differentiation and invasion is required for placental development. Initially, increased expression of proliferative markers in junctional and labyrinth zones of rat placentas and villous cytotrophoblast, syncytiotrophoblast, stromal cells and fetal endothelial cells in human placentas is reported among diabetics. Moreover, reduced apoptotic index and expression of some apoptotic genes are described in placentas of GDM women. In addition, cell cycle regulators including cyclins and cyclin-dependent kinase inhibitors seem to be affected by the hyperglycemic environment. More studies are necessary to check the balance between proliferation, apoptosis and differentiation in trophoblast cells during maternal diabetes. PMID:25789116

  3. Up-regulation of lymphocyte antigen 6 complex expression in side-population cells derived from a human trophoblast cell line HTR-8/SVneo.

    PubMed

    Inagaki, Tetsunori; Kusunoki, Soshi; Tabu, Kouichi; Okabe, Hitomi; Yamada, Izumi; Taga, Tetsuya; Matsumoto, Akemi; Makino, Shintaro; Takeda, Satoru; Kato, Kiyoko

    2016-01-01

    The continual proliferation and differentiation of trophoblasts are critical for the maintenance of pregnancy. It is well known that the tissue stem cells are associated with the development of tissues and pathologies. It has been demonstrated that side-population (SP) cells identified by fluorescence-activated cell sorting (FACS) are enriched with stem cells. The SP cells in HTR-8/SVneo cells derived from human primary trophoblast cells were isolated by FACS. HTR-8/SVneo-SP cell cultures generated both SP and non-SP (NSP) subpopulations. In contrast, NSP cell cultures produced NSP cells and failed to produce SP cells. These SP cells showed self-renewal capability by serial colony-forming assay. Microarray expression analysis using a set of HTR-8/SVneo-SP and -NSP cells revealed that SP cells overexpressed several stemness genes including caudal type homeobox2 (CDX2) and bone morphogenic proteins (BMPs), and lymphocyte antigen 6 complex locus D (LY6D) gene was the most highly up-regulated in HTR-8/SVneo-SP cells. LY6D gene reduced its expression in the course of a 7-day cultivation in differentiation medium. SP cells tended to reduce its fraction by treatment of LY6D siRNA indicating that LY6D had potential to maintain cell proliferation of HTR-8/SVneo-SP cells. On ontology analysis, epithelial-mesenchymal transition (EMT) pathway was involved in the up-regulated genes on microarray analysis. HTR-SVneo-SP cells showed enhanced migration. This is the first report that LY6D was important for the maintenance of HTR-8/SVneo-SP cells. EMT was associated with the phenotype of these SP cells.

  4. The Hand1, Stra13 and Gcm1 transcription factors override FGF signaling to promote terminal differentiation of trophoblast stem cells.

    PubMed

    Hughes, Martha; Dobric, Nikolina; Scott, Ian C; Su, Lin; Starovic, Maja; St-Pierre, Benoit; Egan, Sean E; Kingdom, John C P; Cross, James C

    2004-07-01

    The trophoblast cell lineage is an interesting model system because it is composed of a limited number of cell types that are spatially patterned. Trophoblast stem (TS) cells reside within a layer called the chorion and either remain as stem cells or differentiate into spongiotrophoblast (SpT), trophoblast giant (TG) cells or syncytiotrophoblast cells (SynT) of the labyrinth. Maintenance of the TS phenotype is dependent on stimulation by FGF4, whereas differentiation and/or maintenance of the differentiated derivatives are dependent on key transcription factors: Mash2 for SpT, Hand1 for TG cells and Gcm1 for SynT cells. TS cells proliferate and retain their stem cell phenotype in culture in response to FGF4 and an additional factor(s) that can be provided by conditioned medium from embryonic fibroblast feeder cells (CM). To understand the functions of Hand1, Mash2 and Gcm1 at a cellular level, we tested the effects of their ectopic and over-expression on the ability of TS cells to either continue to proliferate or differentiate into their alternative fates. Expression of Mash2 alone had no effects on TS cell differentiation. However, Mash2-transfected cells continued to divide longer after withdrawal of FGF/CM. Hand1 promoted TGC differentiation, even in the continued presence of FGF4/CM. Stra13, another bHLH factor gene that is expressed in TG cells, also induced TG differentiation. Gcm1 induced a rapid arrest of TS proliferation but, in contrast to Hand1 and Stra13, blocked TG cell differentiation. Although Gcm1 was not sufficient to promote SynT formation, expression of an antisense Gcm1 transcript blocked SynT differentiation. These data suggest that Mash2 functions to promote transient FGF4-independent amplification of trophoblast cells that are progressing towards the SpT and TG cell phenotype. By contrast, Hand1 and Stra13 promote cell cycle exit and restrict cells towards the TG fate, whereas Gcm1 promotes cell cycle exit and restriction towards the Syn

  5. Dynamic change of Adamalysin 19 (ADAM19) in human placentas and its effects on cell invasion and adhesion in human trophoblastic cells.

    PubMed

    Zhao, MeiRong; Qiu, Wei; Li, YuXia; Sang, QingXiang Amy; Wang, YanLing

    2009-08-01

    Human ADAM19 is a recently identified member of the ADAM family. It is highly expressed in human placentas, but its dynamic change and function at the human feto-maternal interface during placentation remain to be elucidated. In this present study, the spatial and temporal expression and cellular localization of ADAM19 in normal human placentas were first demonstrated, and the effects of ADAM19 on trophoblast cell adhesion and invasion were further investigated by using a human choriocarcinoma cell line (JEG-3) as an in vitro model. The data demonstrated that ADAM19 was widely distributed in villous cytotrophoblast cells, syncytiotrophoblast cells, column trophoblasts, and villous capillary endothelial cells during early pregnancy. The mRNA and protein level of ADAM19 in placentas was high at gestational weeks 8-9, but diminished significantly at mid- and term pregnancy. In JEG-3 cells, the overexpression of ADAM19 led to diminished cell invasion, as well as increases in cell adhesiveness and the expression of E-cadherin, with no changes in beta-catenin expression observed. These data indicate that ADAM19 may participate in the coordinated regulation of human trophoblast cell behaviors during the process of placentation.

  6. Epithelioid Trophoblastic Tumor in a Postmenopausal Woman: A Case Report

    PubMed Central

    Park, Jung-Woo

    2016-01-01

    Epithelioid trophoblastic tumor (ETT) is a rare gestational trophoblastic neoplasm composed of intermediate trophoblasts. Most cases of ETT are reported in women of reproductive age following a prior gestation within 2 weeks to 30 years. ETT is extremely rare in postmenopausal women. It is commonly misdiagnosed as a poorly differentiated carcinoma or another type of gestational trophoblastic tumor. We report a case of ETT in a 56-year-old woman that developed 23 years after the patient's last pregnancy. PMID:27152314

  7. Estrogen regulates progesterone production by human placental trophoblast cells in culture

    SciTech Connect

    Grimes, R.W.

    1990-01-01

    We have suggested that estrogen regulates placental low-density lipoprotein (LDL) uptake and thus progesterone (P{sub 4}) production during primate pregnancy based on results obtained in antiestrogen-treated baboons. The objectives of the present study, were to determine whether estrogen is also important to regulation of P{sub 4} formation by the human placenta, and whether effects of estrogen were mediated by availability of cholesterol substrate via the LDL, de novo, or deesterification pathways. Term human placenta were dispersed in 0.125% trypsin, cytotrophoblasts were purified via a 70-5% Percoll gradient, incubated 72 h in DMEM with 10% FBS to stimulate formation of syncytia, then incubated an additional 48 h with estradiol (E2). In Experiment 1, 1 {mu}g/ml E{sub 2} and 500 {mu}g/MI LDL-protein, stimulated P{sub 4} (P < .05) two-fold above that with LDL alone, suggesting that E{sub 2} increased LDL uptake. Scatchard analysis indicated that trophoblast uptake of ({sup 125}I)LDL (ng/mg cell protein) was 50% greater (P < .05) with E{sub 2} (mean {plus minus} SE, 638 +/- 23; n = 6) than DMEM in the presence of antiestrogen MER-25. Moreover, uptake and degradation of LDL, and cellular content of free and esterified cholesterol, increased in a dose-dependent manner with 0.1 to 1000 ng/ml E{sub 2}. These results suggest that estrogen regulates placental cell uptake of LDL and thus availability of cholesterol for P{sub 4} biosynthesis during human pregnancy. In Experiment 2, E{sub 2} Stimulated P{sub 4} formation (ng/mg cell protein/48 h) from a control level of 194 {plus minus} 25 to 357 {plus minus} 62, in the absence of LDL. Under these conditions, cholesterol for P{sub 4} biosynthesis must have been derived from de novo synthesis and/or deesterification of cholesteryl ester stores.

  8. RhoGDI2 is expressed in human trophoblasts and involved in their migration by inhibiting the activation of RAC1.

    PubMed

    Liu, Sishi; Cui, Hong; Li, Qiuling; Zhang, Lijuan; Na, Quan; Liu, Caixia

    2014-04-01

    The invasion of placental trophoblast cells into the maternal decidua is an essential aspect of placental embedment. The process of placentation bears several striking similarities to tumor cell metastasis. However, trophoblastic migration during implantation and placentation is stringently controlled both in space and time. RhoGDI2 belongs to a family of Rho guanosine diphosphate dissociation inhibitors (RhoGDIs), and RhoGDI2 is a metastasis suppressor gene and a marker of aggressive human cancer. We evaluated whether RhoGDI2 has a physiological role in embryo implantation, particularly trophoblast migration. The mRNA and protein expression levels of RhoGDI2 were higher in term placentas compared with first-trimester placentas as detected by real-time PCR and Western blot. Immunohistochemical studies indicated that RhoGDI2 localized to the cytotrophoblast layer and extravillous trophoblast in first-trimester placentas and was distributed in the syncytiotrophoblast layers of term placentas. Overexpression of RhoGDI2 in HTR-8/SVneo cells was associated with reduced RAC1-guanosine triphosphate (GTP) levels and inhibited cell migration. Conversely, small interfering RNA-mediated downregulation of RhoGDI2 resulted in significantly increased RAC1-GTP levels. Altered RhoGDI2 expression had no significant effects on cell proliferation. In conclusion, RhoGDI2 inhibits trophoblast cell migration, and this function may involve suppression of RAC1 activation. PMID:24554735

  9. Human trophoblast cells induced MDSCs from peripheral blood CD14+ myelomonocytic cells via elevated levels of CCL2

    PubMed Central

    Zhang, Yun; Qu, Daiwei; Sun, Jintang; Zhao, Lei; Wang, Qingjie; Shao, Qianqian; Kong, Beihua; Zhang, Yun; Qu, Xun

    2016-01-01

    Successful human pregnancy requires the maternal immune system to recognize and tolerate the semi-allogeneic fetus. Myeloid-derived suppressor cells (MDSCs), which are capable of inhibiting T-cell responses, are highly increased in the early stages of pregnancy. Although recent reports indicate a role for MDSCs in fetal–maternal tolerance, little is known about the expansion of MDSCs during pregnancy. In the present study, we demonstrated that the trophoblast cell line HTR8/SVneo could instruct peripheral CD14+ myelomonocytic cells toward a novel subpopulation of MDSCs, denoted as CD14+HLA-DR−/low cells, with suppressive activity and increased expression of IDO1, ARG-1, and COX2. After interaction with HTR8/SVneo cells, CD14+ myelomonocytic cells secrete high levels of CCL2, promoting the expression of signal transducer and activator of transcription 3. We utilized a neutralizing monoclonal antibody to reveal the prominent role of CCL2 in the induction of CD14+HLA-DR−/low MDSCs. In combination, the results of the present study support a novel role for the cross-talk between the trophoblast cell line HTR8/SVneo and maternal CD14+ myelomonocytic cells in initiating MDSCs induction, prompting a tolerogenic immune response to ensure a successful pregnancy. PMID:26027727

  10. Maternal DNA Methylation Regulates Early Trophoblast Development

    PubMed Central

    Branco, Miguel R.; King, Michelle; Perez-Garcia, Vicente; Bogutz, Aaron B.; Caley, Matthew; Fineberg, Elena; Lefebvre, Louis; Cook, Simon J.; Dean, Wendy; Hemberger, Myriam; Reik, Wolf

    2016-01-01

    Summary Critical roles for DNA methylation in embryonic development are well established, but less is known about its roles during trophoblast development, the extraembryonic lineage that gives rise to the placenta. We dissected the role of DNA methylation in trophoblast development by performing mRNA and DNA methylation profiling of Dnmt3a/3b mutants. We find that oocyte-derived methylation plays a major role in regulating trophoblast development but that imprinting of the key placental regulator Ascl2 is only partially responsible for these effects. We have identified several methylation-regulated genes associated with trophoblast differentiation that are involved in cell adhesion and migration, potentially affecting trophoblast invasion. Specifically, trophoblast-specific DNA methylation is linked to the silencing of Scml2, a Polycomb Repressive Complex 1 protein that drives loss of cell adhesion in methylation-deficient trophoblast. Our results reveal that maternal DNA methylation controls multiple differentiation-related and physiological processes in trophoblast via both imprinting-dependent and -independent mechanisms. PMID:26812015

  11. Involvement of Reactive Oxygen Species in Brominated Diphenyl Ether-47-induced Inflammatory Cytokine Release from Human Extravillous Trophoblasts in vitro

    PubMed Central

    Park, Hae-Ryung; Kamau, Patricia W.; Loch-Caruso, Rita

    2014-01-01

    Polybrominated diphenyl ethers (PBDEs) are widely used flame retardant compounds. Brominated diphenyl ether (BDE)-47 is one of the most prevalent PBDE congeners found in human breast milk, serum and placenta. Despite the presence of PBDEs in human placenta, effects of PBDEs on placental cell function are poorly understood. The present study investigated BDE-47-induced reactive oxygen species (ROS) formation and its role in BDE-47-stimulated proinflammatory cytokine release in a first trimester human extravillous trophoblast cell line, HTR-8/SVneo. Exposure of HTR-8/SVneo cells for 4 h to 20 μM BDE-47 increased ROS generation 1.7 fold as measured by the dichlorofluorescein (DCF) assay. Likewise, superoxide anion production increased approximately 5 fold at 10 and 15 μM and 9 fold at 20 μM BDE-47 with a 1-h exposure, as measured by cytochrome c reduction. BDE-47 (10, 15 and 20 μM) decreased the mitochondrial membrane potential by 47–64.5% at 4, 8 and 24 h as assessed with the fluorescent probe Rh123. Treatment with 15 and 20 μM BDE-47 stimulated cellular release and mRNA expression of IL-6 and IL-8 after 12 and 24 h exposures: the greatest increases were a 35-fold increased mRNA expression at 12 h and a 12-fold increased protein concentration at 24 h for IL-6. Antioxidant treatments (deferoxamine mesylate, (±)α-tocopherol, or tempol) suppressed BDE-47-stimulated IL-6 release by 54.1%, 56.3% and 37.7%, respectively, implicating a role for ROS in regulation of inflammatory pathways in HTR-8/SVneo cells. Solvent (DMSO) controls exhibited statistically significantly decreased responses compared with non-treated controls for IL-6 release and IL-8 mRNA expression, but these responses were not consistent across experiments and times. Nonetheless, it is possible that DMSO (used to dissolve BDE-47) may have attenuated the stimulatory actions of BDE-47 on cytokine responses. Because abnormal activation of proinflammatory responses can disrupt trophoblast functions

  12. Involvement of reactive oxygen species in brominated diphenyl ether-47-induced inflammatory cytokine release from human extravillous trophoblasts in vitro

    SciTech Connect

    Park, Hae-Ryung Kamau, Patricia W.; Loch-Caruso, Rita

    2014-01-15

    Polybrominated diphenyl ethers (PBDEs) are widely used flame retardant compounds. Brominated diphenyl ether (BDE)-47 is one of the most prevalent PBDE congeners found in human breast milk, serum and placenta. Despite the presence of PBDEs in human placenta, effects of PBDEs on placental cell function are poorly understood. The present study investigated BDE-47-induced reactive oxygen species (ROS) formation and its role in BDE-47-stimulated proinflammatory cytokine release in a first trimester human extravillous trophoblast cell line, HTR-8/SVneo. Exposure of HTR-8/SVneo cells for 4 h to 20 μM BDE-47 increased ROS generation 1.7 fold as measured by the dichlorofluorescein (DCF) assay. Likewise, superoxide anion production increased approximately 5 fold at 10 and 15 μM and 9 fold at 20 μM BDE-47 with a 1-h exposure, as measured by cytochrome c reduction. BDE-47 (10, 15 and 20 μM) decreased the mitochondrial membrane potential by 47–64.5% at 4, 8 and 24 h as assessed with the fluorescent probe Rh123. Treatment with 15 and 20 μM BDE-47 stimulated cellular release and mRNA expression of IL-6 and IL-8 after 12 and 24-h exposures: the greatest increases were a 35-fold increased mRNA expression at 12 h and a 12-fold increased protein concentration at 24 h for IL-6. Antioxidant treatments (deferoxamine mesylate, (±)α-tocopherol, or tempol) suppressed BDE-47-stimulated IL-6 release by 54.1%, 56.3% and 37.7%, respectively, implicating a role for ROS in the regulation of inflammatory pathways in HTR-8/SVneo cells. Solvent (DMSO) controls exhibited statistically significantly decreased responses compared with non-treated controls for IL-6 release and IL-8 mRNA expression, but these responses were not consistent across experiments and times. Nonetheless, it is possible that DMSO (used to dissolve BDE-47) may have attenuated the stimulatory actions of BDE-47 on cytokine responses. Because abnormal activation of proinflammatory responses can disrupt trophoblast functions

  13. Mammalian target of rapamycin signalling modulates amino acid uptake by regulating transporter cell surface abundance in primary human trophoblast cells.

    PubMed

    Rosario, Fredrick J; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

    2013-02-01

    Abnormal fetal growth increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Emerging evidence suggests that changes in placental amino acid transport directly contribute to altered fetal growth. However, the molecular mechanisms regulating placental amino acid transport are largely unknown. Here we combined small interfering (si) RNA-mediated silencing approaches with protein expression/localization and functional studies in cultured primary human trophoblast cells to test the hypothesis that mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) regulate amino acid transporters by post-translational mechanisms. Silencing raptor (inhibits mTORC1) or rictor (inhibits mTORC2) markedly decreased basal System A and System L amino acid transport activity but had no effect on growth factor-stimulated amino acid uptake. Simultaneous inhibition of mTORC1 and 2 completely inhibited both basal and growth factor-stimulated amino acid transport activity. In contrast, mTOR inhibition had no effect on serotonin transport. mTORC1 or mTORC2 silencing markedly decreased the plasma membrane expression of specific System A (SNAT2, SLC38A2) and System L (LAT1, SLC7A5) transporter isoforms without affecting global protein expression. In conclusion, mTORC1 and mTORC2 regulate human trophoblast amino acid transporters by modulating the cell surface abundance of specific transporter isoforms. This is the first report showing regulation of amino acid transport by mTORC2. Because placental mTOR activity and amino acid transport are decreased in human intrauterine growth restriction our data are consistent with the possibility that dysregulation of placental mTOR plays an important role in the development of abnormal fetal growth.

  14. Leptin stimulates protein synthesis-activating translation machinery in human trophoblastic cells.

    PubMed

    Pérez-Pérez, Antonio; Maymó, Julieta; Gambino, Yésica; Dueñas, José L; Goberna, Raimundo; Varone, Cecilia; Sánchez-Margalet, Víctor

    2009-11-01

    Leptin was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in placenta, where it may work as an autocrine hormone, mediating angiogenesis, growth, and immunomodulation. Leptin receptor (LEPR, also known as Ob-R) shows sequence homology to members of the class I cytokine receptor (gp130) superfamily. In fact, leptin may function as a proinflammatory cytokine. We have previously found that leptin is a trophic and mitogenic factor for trophoblastic cells. In order to further investigate the mechanism by which leptin stimulates cell growth in JEG-3 cells and trophoblastic cells, we studied the phosphorylation state of different proteins of the initiation stage of translation and the total protein synthesis by [(3)H]leucine incorporation in JEG-3 cells. We have found that leptin dose-dependently stimulates the phosphorylation and activation of the translation initiation factor EIF4E as well as the phosphorylation of the EIF4E binding protein EIF4EBP1 (PHAS-I), which releases EIF4E to form active complexes. Moreover, leptin dose-dependently stimulates protein synthesis, and this effect can be partially prevented by blocking mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PIK3) pathways. In conclusion, leptin stimulates protein synthesis, at least in part activating the translation machinery, via the activation of MAPK and PIK3 pathways.

  15. Antisperm antibody-mediated alterations in the cellular activity of human trophoblast cells in culture.

    PubMed

    Sinha, D; Chattopadhyay, S

    1994-04-01

    Immune recognition of the fetus is well documented, yet the immunological basis of pregnancy loss awaits elucidation. Identification of trophoblast membrane epitopes as non-self either by preformed immunoglobulins or by circulating immunocompetent cells would lead to immunological rejection of the tissue. Such an event may occur in cases of cross-reacting antibodies developed as a consequence of exposure of sperm surface antigens. This hypothesis was tested by developing specific antibodies in rabbits against intact sperm surface antigens. These were subjected to different schedules of IgG purification and characterization. By means of nuclide precursor incorporation, the effect of antisperm antibody on DNA, RNA and protein synthesis of trophoblast cells in culture were studied. The results showed that the antibody inhibits incorporation into cells but after a delay of 72 hours some cells gradually recover. The interaction also led to a reduced rate of hCG production. Lysosomal enzyme activity was inhibited in the spent medium of antibody-treated cells but lysosome rich fractions showed no effect. This indicated that the major effect of the antibody was growth inhibitory rather than cytolytic. PMID:7520885

  16. Caspase-8 activity is part of the BeWo trophoblast cell defense mechanisms against Trypanosoma cruzi infection.

    PubMed

    Carrillo, Ileana; Droguett, Daniel; Castillo, Christian; Liempi, Ana; Muñoz, Lorena; Maya, Juan Diego; Galanti, Norbel; Kemmerling, Ulrike

    2016-09-01

    Congenital Chagas disease is caused by the protozoan parasite Trypanosoma cruzi that must cross the placental barrier during transmission. The trophoblast constitutes the first tissue in contact with the maternal-blood circulating parasite. Importantly, the congenital transmission rates are low, suggesting the presence of local placental defense mechanisms. Cellular proliferation and differentiation as well as apoptotic cell death are induced by the parasite and constitute part of the epithelial turnover of the trophoblast, which has been suggested to be part of those placental defenses. On the other hand, caspase-8 is an essential molecule in the modulation of trophoblast turnover by apoptosis and by epithelial differentiation. As an approach to study whether T. cruzi induced trophoblast turnover and infection is mediated by caspase-8, we infected BeWo cells (a trophoblastic cell line) with the parasite and determined in the infected cells the expression and enzymatic activity of caspase-8, DNA synthesis (as proliferation marker), β-human chorionic gonadotropin (β-hCG) (as differentiation marker) and activity of Caspase-3 (as apoptotic death marker). Parasite load in BeWo cells was measured by DNA quantification using qPCR and cell counting. Our results show that T. cruzi induces caspase-8 activity and that its inhibition increases trophoblast cells infection while decreases parasite induced cellular differentiation and apoptotic cell death, but not cellular proliferation. Thus, caspase-8 activity is part of the BeWo trophoblast cell defense mechanisms against T. cruzi infection. Together with our previous results, we suggest that the trophoblast turnover is part of local placental anti-parasite mechanisms.

  17. mTOR mediates human trophoblast invasion through regulation of matrix-remodeling enzymes and is associated with serine phosphorylation of STAT3

    SciTech Connect

    Busch, Susann; Renaud, Stephen J.; Schleussner, Ekkehard; Graham, Charles H.; Markert, Udo R.

    2009-06-10

    The intracellular signaling molecule mammalian target of rapamycin (mTOR) is essential for cell growth and proliferation. It is involved in mouse embryogenesis, murine trophoblast outgrowth and linked to tumor cell invasiveness. In order to assess the role of mTOR in human trophoblast invasion we analyzed the in vitro invasiveness of HTR-8/SVneo immortalized first-trimester trophoblast cells in conjunction with enzyme secretion upon mTOR inhibition and knockdown of mTOR protein expression. Additionally, we also tested the capability of mTOR to trigger signal transducer and activator of transcription (STAT)-3 by its phosphorylation status. Rapamycin inhibited mTOR kinase activity as demonstrated with a lower phosphorylation level of the mTOR substrate p70 S6 kinase (S6K). With the use of rapamycin and siRNA-mediated mTOR knockdown we could show that cell proliferation, invasion and secretion of matrix-metalloproteinases (MMP)-2 and -9, urokinase-like plasminogen activator (uPA) and its major physiological uPA inhibitor (PAI)-1 were inhibited. While tyrosine phosphorylation of STAT3 was unaffected by mTOR inhibition and knockdown, serine phosphorylation was diminished. We conclude that mTOR signaling is one major mechanism in a tightly regulated network of intracellular signal pathways including the JAK/STAT system to regulate invasion in human trophoblast cells by secretion of enzymes that remodel the extra-cellular matrix (ECM) such as MMP-2, -9, uPA and PAI-1. Dysregulation of mTOR may contribute to pregnancy-related pathologies caused through impaired trophoblast invasion.

  18. Interleukin-1β inhibits insulin signaling and prevents insulin-stimulated system A amino acid transport in primary human trophoblasts.

    PubMed

    Aye, Irving L M H; Jansson, Thomas; Powell, Theresa L

    2013-12-01

    Interleukin-1β (IL-1β) promotes insulin resistance in tissues such as liver and skeletal muscle; however the influence of IL-1β on placental insulin signaling is unknown. We recently reported increased IL-1β protein expression in placentas of obese mothers, which could contribute to insulin resistance. In this study, we tested the hypothesis that IL-1β inhibits insulin signaling and prevents insulin-stimulated amino acid transport in cultured primary human trophoblast (PHT) cells. Cultured trophoblasts isolated from term placentas were treated with physiological concentrations of IL-1β (10pg/ml) for 24h. IL-1β increased the phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser307 (inhibitory) and decreased total IRS-1 protein abundance but did not affect insulin receptor β expression. Furthermore, IL-1β inhibited insulin-stimulated phosphorylation of IRS-1 (Tyr612, activation site) and Akt (Thr308) and prevented insulin-stimulated increase in PI3K/p85 and Grb2 protein expression. IL-1β alone stimulated cRaf (Ser338), MEK (Ser221) and Erk1/2 (Thr202/Tyr204) phosphorylation. The inflammatory pathways nuclear factor kappa B and c-Jun N-terminal kinase, which are involved in insulin resistance, were also activated by IL-1β treatment. Moreover, IL-1β inhibited insulin-stimulated System A, but not System L amino acid uptake, indicating functional impairment of insulin signaling. In conclusion, IL-1β inhibited the insulin signaling pathway by inhibiting IRS-1 signaling and prevented insulin-stimulated System A transport, thereby promoting insulin resistance in cultured PHT cells. These findings indicate that conditions which lead to increased systemic maternal or placental IL-1β levels may attenuate the effects of maternal insulin on placental function and consequently fetal growth.

  19. The effects of progesterone on apoptosis in the human trophoblast-derived HTR-8/SV neo cells.

    PubMed

    Liu, Jin; Matsuo, Hiroya; Laoag-Fernandez, Jovelle B; Xu, Qin; Maruo, Takeshi

    2007-12-01

    Progesterone (P4) is frequently used in the treatment of threatened abortion, prevention of recurrent miscarriage and threatened preterm labor. However, little is known about the molecular mechanism of P4 in the regulation of extravillous trophoblasts' (EVTs) function. This study was designed to examine the presence of progesterone receptor (PR) in the human trophoblast-derived HTR-8/SV neo cell line, which is a possible model of EVTs, and the effects of P4 on apoptosis in those cells. The HTR-8/SV neo cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 microg/ml streptomycin. When the cell the population reached 50% confluency, the cells were stepped down to serum-free conditions in the presence or absence of graded concentrations of P4 (1, 10 and 100 ng/ml) for 48 h. The cultured cells were used for RT-PCR, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) assay, immunocytochemistry and western blot analyses. Immunocytochemistry and western blot analyses revealed that PR was evident in HTR-8/SV neo cells. Compared with untreated cultures, treatment with P4 (10 and 100 ng/ml) resulted in significant decreases in the TUNEL-positive rate, Fas, Fas ligand (Fas-L), caspase-8, caspase-3 and poly (ADP-ribose) polymerase (PARP) expression in HTR-8/SV neo cells, and a significant increase in Bcl-2 expression in those cells. Consistently, Fas mRNA expression in those cells was significantly inhibited by the treatment with 10 ng/ml P4 compared with untreated cultures. This study suggests that PR exists in HTR-8/SV neo cells and that P4 inhibits apoptosis by down-regulating Fas, Fas-L, caspase-8, caspase-3 and PARP expression as well as up-regulating Bcl-2 expression in HTR-8/SV neo cells. PMID:17962376

  20. Calcitriol inhibits TNF-alpha-induced inflammatory cytokines in human trophoblasts.

    PubMed

    Díaz, Lorenza; Noyola-Martínez, Nancy; Barrera, David; Hernández, Guillermo; Avila, Euclides; Halhali, Ali; Larrea, Fernando

    2009-07-01

    Elevated placental proinflammatory cytokine release is associated with miscarriage, preterm labor and preeclampsia. Specifically, tumor necrosis factor-alpha (TNF-alpha)-induced cytokines may threaten pregnancy outcome. Since trophoblasts produce calcitriol, a hormone with strong immunosuppressive properties, we assessed the effects of this secosteroid on inflammatory cytokines induced in trophoblasts by challenge with TNF-alpha. The effects of calcitriol on synthesis of mRNAs encoding interleukin-6 (IL-6), interferon-gamma (IFN-gamma), and TNF-alpha were measured by real time RT-PCR. Secreted cytokines were quantified by ELISA. The effects of TNF-alpha on CYP24A1, chorionic gonadotropin (hCG), 3beta-hydroxysteroid dehydrogenase (HSD3B1) and P(450)-aromatase (CYP19) mRNA expression were also studied. TNF-alpha stimulated IL-6, IFN-gamma and its own expression more than 3-fold over controls (P<0.05). Calcitriol inhibited the expression profile of inflammatory cytokine genes in a dose-response manner (P<0.05). This effect was prevented by addition of the vitamin D receptor antagonist TEI-9647. TNF-alpha also significantly inhibited expression of hCG, HSD3B1 and CYP19 genes, and stimulated CYP24A1 gene expression. These data show that calcitriol prevents TNF-alpha induction of inflammatory cytokines through a process likely to be mediated by the vitamin D receptor. We conclude that TNF-alpha inhibits placental hormone synthesis and stimulates calcitriol catabolism by regulating enzymes involved in these processes.

  1. Elsevier Trophoblast Research Award lecture: Molecular mechanisms underlying estrogen functions in trophoblastic cells--focus on leptin expression.

    PubMed

    Gambino, Y P; Maymó, J L; Pérez Pérez, A; Calvo, J C; Sánchez-Margalet, V; Varone, C L

    2012-02-01

    The steroid hormone 17β-estradiol is an estrogen that influences multiple aspects of placental function and fetal development in humans. During early pregnancy it plays a role in the regulation of blastocyst implantation, trophoblast differentiation and invasiveness, remodeling of uterine arteries, immunology and trophoblast production of hormones such as leptin. Estradiol exerts some effects through the action of classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors and regulate gene expression. In addition, estradiol can elicit rapid responses from membrane-associated receptors, like activation of protein-kinase pathways. Thus, the cellular effects of estradiol will depend on the specific receptors expressed and the integration of their signaling events. Leptin, the 16,000MW protein product of the obese gene, was originally considered an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy. The leptin gene is expressed in placenta, where leptin promotes proliferation and survival of trophoblastic cells. Expression of leptin in placenta is highly regulated by key pregnancy molecules as hCG and estradiol. The aim of this paper is to review the molecular mechanisms underlying estrogen functions in trophoblastic cells; focusing on mechanisms involved in estradiol regulation of placental leptin expression.

  2. Oxcarbazepine-loaded polymeric nanoparticles: development and permeability studies across in vitro models of the blood–brain barrier and human placental trophoblast

    PubMed Central

    Lopalco, Antonio; Ali, Hazem; Denora, Nunzio; Rytting, Erik

    2015-01-01

    Encapsulation of antiepileptic drugs (AEDs) into nanoparticles may offer promise for treating pregnant women with epilepsy by improving brain delivery and limiting the transplacental permeability of AEDs to avoid fetal exposure and its consequent undesirable adverse effects. Oxcarbazepine-loaded nanoparticles were prepared by a modified solvent displacement method from biocompatible polymers (poly(lactic-co-glycolic acid) [PLGA] with or without surfactant and PEGylated PLGA [Resomer® RGPd5055]). The physical properties of the developed nanoparticles were determined with subsequent evaluation of their permeability across in vitro models of the blood–brain barrier (hCMEC/D3 cells) and human placental trophoblast cells (BeWo b30 cells). Oxcarbazepine-loaded nanoparticles with encapsulation efficiency above 69% were prepared with sizes ranging from 140–170 nm, polydispersity indices below 0.3, and zeta potential values below -34 mV. Differential scanning calorimetry and X-ray diffraction studies confirmed the amorphous state of the nanoencapsulated drug. The apparent permeability (Pe) values of the free and nanoencapsulated oxcarbazepine were comparable across both cell types, likely due to rapid drug release kinetics. Transport studies using fluorescently-labeled nanoparticles (loaded with coumarin-6) demonstrated increased permeability of surfactant-coated nanoparticles. Future developments in enzyme-prodrug therapy and targeted delivery are expected to provide improved options for pregnant patients with epilepsy. PMID:25792832

  3. NK Cells and Trophoblasts

    PubMed Central

    Parham, Peter

    2004-01-01

    In placental mammals, viviparity—the production of living young within the mother's body—evolved under the auspices of the immune system. Elements of immunity were incorporated, giving pregnancy a mildly inflammatory character. Formation of the placenta, the organ that feeds the fetus, involves a cooperation between maternal natural killer (NK) cells and fetal trophoblast cells that remodels the blood supply. Recent research reveals that this process and human reproductive success are influenced by polymorphic HLA-C ligands and their killer cell immunoglobulin-like receptors (KIR). PMID:15492121

  4. Oxygen-Sensitive K+ Channels Modulate Human Chorionic Gonadotropin Secretion from Human Placental Trophoblast.

    PubMed

    Díaz, Paula; Sibley, Colin P; Greenwood, Susan L

    2016-01-01

    Human chorionic gonadotropin (hCG) is a key autocrine/paracrine regulator of placental syncytiotrophoblast, the transport epithelium of the human placenta. Syncytiotrophoblast hCG secretion is modulated by the partial pressure of oxygen (pO2), reactive oxygen species (ROS) and potassium (K+) channels. Here we test the hypothesis that K+ channels mediate the effects of pO2 and ROS on hCG secretion. Placental villous explants from normal term pregnancies were cultured for 6 days at 6% (normoxia), 21% (hyperoxia) or 1% (hypoxia) pO2. On days 3-5, explants were treated with 5mM 4-aminopyridine (4-AP) or tetraethylammonium (TEA), blockers of pO2-sensitive voltage-gated K+ (KV) channels, or ROS (10-1000μM H2O2). hCG secretion and lactate dehydrogenase (LDH) release, a marker of necrosis, were determined daily. At day 6, hCG and LDH were measured in tissue lysate and 86Rb (K+) efflux assessed to estimate syncytiotrophoblast K+ permeability. hCG secretion and 86Rb efflux were significantly greater in explants maintained in 21% pO2 than normoxia. 4-AP/TEA inhibited hCG secretion to a greater extent at 21% than 6% and 1% pO2, and reduced 86Rb efflux at 21% but not 6% pO2. LDH release and tissue LDH/hCG were similar in 6%, 21% and 1% pO2 and unaffected by 4-AP/TEA. H2O2 stimulated 86Rb efflux and hCG secretion at normoxia but decreased 86Rb efflux, without affecting hCG secretion, at 21% pO2. 4-AP/TEA-sensitive K+ channels participate in pO2-sensitive hCG secretion from syncytiotrophoblast. ROS effects on both hCG secretion and 86Rb efflux are pO2-dependent but causal links between the two remain to be established. PMID:26863525

  5. Oxygen-Sensitive K+ Channels Modulate Human Chorionic Gonadotropin Secretion from Human Placental Trophoblast

    PubMed Central

    Díaz, Paula; Sibley, Colin P.; Greenwood, Susan L.

    2016-01-01

    Human chorionic gonadotropin (hCG) is a key autocrine/paracrine regulator of placental syncytiotrophoblast, the transport epithelium of the human placenta. Syncytiotrophoblast hCG secretion is modulated by the partial pressure of oxygen (pO2), reactive oxygen species (ROS) and potassium (K+) channels. Here we test the hypothesis that K+ channels mediate the effects of pO2 and ROS on hCG secretion. Placental villous explants from normal term pregnancies were cultured for 6 days at 6% (normoxia), 21% (hyperoxia) or 1% (hypoxia) pO2. On days 3–5, explants were treated with 5mM 4-aminopyridine (4-AP) or tetraethylammonium (TEA), blockers of pO2-sensitive voltage-gated K+ (KV) channels, or ROS (10–1000μM H2O2). hCG secretion and lactate dehydrogenase (LDH) release, a marker of necrosis, were determined daily. At day 6, hCG and LDH were measured in tissue lysate and 86Rb (K+) efflux assessed to estimate syncytiotrophoblast K+ permeability. hCG secretion and 86Rb efflux were significantly greater in explants maintained in 21% pO2 than normoxia. 4-AP/TEA inhibited hCG secretion to a greater extent at 21% than 6% and 1% pO2, and reduced 86Rb efflux at 21% but not 6% pO2. LDH release and tissue LDH/hCG were similar in 6%, 21% and 1% pO2 and unaffected by 4-AP/TEA. H2O2 stimulated 86Rb efflux and hCG secretion at normoxia but decreased 86Rb efflux, without affecting hCG secretion, at 21% pO2. 4-AP/TEA-sensitive K+ channels participate in pO2-sensitive hCG secretion from syncytiotrophoblast. ROS effects on both hCG secretion and 86Rb efflux are pO2-dependent but causal links between the two remain to be established. PMID:26863525

  6. Serum Amyloid A in the Placenta and Its Role in Trophoblast Invasion

    PubMed Central

    Sandri, Silvana; Urban Borbely, Alexandre; Fernandes, Isabella; Mendes de Oliveira, Edson; Knebel, Franciele Hinterholz; Ruano, Rodrigo; Zugaib, Marcelo; Filippin-Monteiro, Fabiola; Bevilacqua, Estela; Campa, Ana

    2014-01-01

    The serum amyloid A (SAA) protein is known to function in the acute phase response and immunoregulation. Recently, SAA has been shown to be involved in cell proliferation, differentiation and migratory behavior in different cell types. Here, we evaluated whether exogenous SAA could influence trophoblast invasion and differentiation using both the trophoblast-like BeWo cell line and fully differentiated human extravillous trophoblast cells (EVT) isolated from term placentae. SAA stimulated BeWo cell invasion, as measured in Matrigel invasion assays, and induced metalloprotease mRNA expression and activity. Given that BeWo cells express Toll-like receptor 4 (TLR4), a known receptor for SAA, we examined the role of TLR4 in SAA-induced invasion using a TLR4 neutralizing antibody. We also tested whether SAA could affect markers of trophoblast syncytialization in BeWo cells. We observed that SAA decreased βhCG secretion and did not influence trophoblast syncytialization. Using EVT cells isolated from human term basal plates, we confirmed that SAA at 1 and 10 µg/mL doubled EVT invasion in a TLR4-dependent manner, but at 20 µg/mL inhibited EVT cells invasiveness. In addition, we observed that SAA was expressed in both BeWo cells and human term placentae, specifically in the syncytiotrophoblast, decidual cells and EVT. In conclusion, SAA was identified as a molecule that functions in the placental microenvironment to regulate metalloprotease activity and trophoblast invasion, which are key processes in placentation and placental homeostasis. PMID:24614130

  7. Protective effect of (±)α-tocopherol on brominated diphenyl ether-47-stimulated prostaglandin pathways in human extravillous trophoblasts in vitro.

    PubMed

    Park, Hae-Ryung; Loch-Caruso, Rita

    2015-10-01

    Brominated diphenyl ether (BDE)-47 is a prevalent flame retardant chemical found in human tissues and is linked to adverse pregnancy outcomes in humans. Because dysregulation of the prostaglandin pathway is implicated in adverse pregnancy outcomes, the present study investigates BDE-47 induction of prostaglandin synthesis in a human extravillous trophoblast cell line, HTR-8/SVneo, examining the hypothesis that BDE-47 increases generation of reactive oxygen species (ROS) to stimulate the prostaglandin response. Treatment with 20 μM BDE-47 significantly increased mRNA expression of prostaglandin-endoperoxide synthase 2 (PTGS2) at 4, 12 and 24 h, and 24-h treatment significantly increased cyclooxygenase (COX)-2 cellular protein expression and prostaglandin E2 (PGE2) concentration in culture medium. The BDE-47-stimulated PGE2 release was inhibited by the COX inhibitors indomethacin and NS398, implicating COX activity. Exposure to 20 μM BDE-47 significantly increased ROS generation as measured by carboxydichlorofluorescein fluorescence, and this response was blocked by cotreatment with the peroxyl radical scavenger (±)-α-tocopherol. (±)-α-Tocopherol cotreatment suppressed BDE-47-stimulated increases of PGE2 release without significant effects on COX-2 mRNA and protein expression, implicating a role for ROS in post-translational regulation of COX activity. Because prostaglandins regulate trophoblast functions necessary for placentation and pregnancy, further investigation is warranted of BDE-47 impacts on trophoblast responses. PMID:26026498

  8. Protective Effect of (±)α-Tocopherol on Brominated Diphenyl Ether-47-Stimulated Prostaglandin Pathways in Human Extravillous Trophoblasts In Vitro

    PubMed Central

    Park, Hae-Ryung; Loch-Caruso, Rita

    2015-01-01

    Brominated diphenyl ether (BDE)-47 is a prevalent flame retardant chemical found in human tissues and is linked to adverse pregnancy outcomes in humans. Because dysregulation of the prostaglandin pathway is implicated in adverse pregnancy outcomes, the present study investigates BDE-47 induction of prostaglandin synthesis in a human extravillous trophoblast cell line, HTR-8/SVneo, examining the hypothesis that BDE-47 increases generation of reactive oxygen species (ROS) to stimulate the prostaglandin response. Treatment with 20 μM BDE-47 significantly increased mRNA expression of prostaglandin-endoperoxide synthase 2 (PTGS2) at 4, 12 and 24 h, and 24-h treatment significantly increased cyclooxygenase (COX)-2 cellular protein expression and prostaglandin E2 (PGE2) concentration in culture medium. The BDE-47-stimulated PGE2 release was inhibited by the COX inhibitors indomethacin and NS398, implicating COX activity. Exposure to 20 μM BDE-47 significantly increased ROS generation as measured by carboxydichlorofluorescein fluorescence, and this response was blocked by cotreatment with the peroxyl radical scavenger (±)-α-tocopherol. (±)-α-Tocopherol cotreatment suppressed BDE-47-stimulated increases of PGE2 release without significant effects on COX-2 mRNA and protein expression, implicating a role for ROS in post-translational regulation of COX activity. Because prostaglandins regulate trophoblast functions necessary for placentation and pregnancy, further investigation is warranted of BDE-47 impacts on trophoblast responses. PMID:26026498

  9. Novel expression of EGFL7 in placental trophoblast and endothelial cells and its implication in preeclampsia

    PubMed Central

    Lacko, Lauretta A.; Massimiani, Micol; Sones, Jenny L.; Hurtado, Romulo; Salvi, Silvia; Ferrazzani, Sergio; Davisson, Robin L.; Campagnolo, Luisa; Stuhlmann, Heidi

    2014-01-01

    The mammalian placenta is the site of nutrient and gas exchange between the mother and fetus, and is comprised of two principal cell types, trophoblasts and endothelial cells. Proper placental development requires invasion and differentiation of trophoblast cells, together with coordinated fetal vasculogenesis and maternal vascular remodeling. Disruption in these processes can result in placental pathologies such as preeclampsia (PE), a disease characterized by late gestational hypertension and proteinuria. Epidermal Growth Factor Like Domain 7 (EGFL7) is a largely endothelial-restricted secreted factor that is critical for embryonic vascular development, and functions by modulating the Notch signaling pathway. However, the role of EGFL7 in placental development remains unknown. In this study, we use mouse models and human placentas to begin to understand the role of EGFL7 during normal and pathological placentation. We show that Egfl7 is expressed by the endothelium of both the maternal and fetal vasculature throughout placental development. Importantly, we uncovered a previously unknown site of EGFL7 expression in the trophoblast cell lineage, including the trophectoderm, trophoblast stem cells, and placental trophoblasts. Our results demonstrate significantly reduced Egfl7 expression in human PE placentas, concurrent with a downregulation of Notch target genes. Moreover, using the BPH/5 mouse model of PE, we show that the downregulation of Egfl7 in compromised placentas occurs prior to the onset of characteristic maternal signs of PE. Together, our results implicate Egfl7 as a possible factor in normal placental development and in the etiology of PE. PMID:24751645

  10. The role of invasive trophoblast in implantation and placentation of primates.

    PubMed

    Carter, Anthony M; Enders, Allen C; Pijnenborg, Robert

    2015-03-01

    We here review the evolution of invasive placentation in primates towards the deep penetration of the endometrium and its arteries in hominoids. The strepsirrhine primates (lemurs and lorises) have non-invasive, epitheliochorial placentation, although this is thought to be derived from a more invasive type. In haplorhine primates, there is differentiation of trophoblast at the blastocyst stage into syncytial and cellular trophoblast. Implantation involves syncytiotrophoblast that first removes the uterine epithelium then consolidates at the basal lamina before continuing into the stroma. In later stages of pregnancy, especially in Old World monkeys and apes, cytotrophoblast plays a greater role in the invasive process. Columns of trophoblast cells advance to the base of the implantation site where they spread out to form a cytotrophoblastic shell. In addition, cytotrophoblasts advance into the lumen of the spiral arteries. They are responsible for remodelling these vessels to form wide, low-resistance conduits. In human and great apes, there is additional invasion of the endometrium and its vessels by trophoblasts originating from the base of the anchoring villi. Deep trophoblast invasion that extends remodelling of the spiral arteries to segments in the inner myometrium evolved in the common ancestor of gorilla, chimp and human. PMID:25602074

  11. The role of invasive trophoblast in implantation and placentation of primates

    PubMed Central

    Carter, Anthony M.; Enders, Allen C.; Pijnenborg, Robert

    2015-01-01

    We here review the evolution of invasive placentation in primates towards the deep penetration of the endometrium and its arteries in hominoids. The strepsirrhine primates (lemurs and lorises) have non-invasive, epitheliochorial placentation, although this is thought to be derived from a more invasive type. In haplorhine primates, there is differentiation of trophoblast at the blastocyst stage into syncytial and cellular trophoblast. Implantation involves syncytiotrophoblast that first removes the uterine epithelium then consolidates at the basal lamina before continuing into the stroma. In later stages of pregnancy, especially in Old World monkeys and apes, cytotrophoblast plays a greater role in the invasive process. Columns of trophoblast cells advance to the base of the implantation site where they spread out to form a cytotrophoblastic shell. In addition, cytotrophoblasts advance into the lumen of the spiral arteries. They are responsible for remodelling these vessels to form wide, low-resistance conduits. In human and great apes, there is additional invasion of the endometrium and its vessels by trophoblasts originating from the base of the anchoring villi. Deep trophoblast invasion that extends remodelling of the spiral arteries to segments in the inner myometrium evolved in the common ancestor of gorilla, chimp and human. PMID:25602074

  12. The human leukocyte antigen G promotes trophoblast fusion and β-hCG production through the Erk1/2 pathway in human choriocarcinoma cell lines

    SciTech Connect

    Wang, Ji-meng; Zhao, Hong-xi; Wang, Li; Gao, Zhi-ying; Yao, Yuan-qing

    2013-05-10

    Highlights: •HLA-G expression promotes BeWo cells fusion and fusogenic gene expression. •HLA-G is capable of inducing β-hCG production in human choriocarcinoma cell lines. •Up-regulation of β-hCG production by HLA-G is mediated via the Erk1/2 pathway. -- Abstract: The human leukocyte antigen G (HLA-G) is expressed on the fetal–maternal interface and plays a role in protecting fetal-derived trophoblasts from the maternal immune response, allowing trophoblasts to invade the uterus. However, HLA-G also possesses immune suppressing-independent functions. We found that HLA-G expressing BeWo choriocarcinoma cells increased cell–cell fusion compared to control BeWo cells under forskolin treatment. Regardless of forskolin treatment, the expression of fusogenic gene mRNAs, including syncytin-1, the transcription factor glial cell missing 1 (Gcm1), and beta human chorionic gonadotropin (β-hCG) were elevated. HLA-G up-regulates β-hCG production in human choriocarcinoma cells because HLA-G knockdown in JEG-3 cells induces a dramatic decrease in β-hCG compared with control cells. The defect in β-hCG production in HLA-G knocked-down cells could not be completely overcome by stimulating hCG production through increasing intracellular cAMP levels. HLA-G expressing cells have increased phosphorylation levels for extracellular signal-regulated kinase1/2 (Erk1/2) in BeWo cells. The Erk1/2 pathway is inactivated after the inhibition of HLA-G expression in JEG-3 cells. Finally, Erk1/2 inhibition was able to suppress the increased hCG production induced by HLA-G expression. Together, these data suggest novel roles for HLA-G in regulating β-hCG production via the modulation of the Erk1/2 pathway and by inducing trophoblast cell fusion.

  13. Intermediate Conductance Ca2+-Activated K+ Channels Modulate Human Placental Trophoblast Syncytialization

    PubMed Central

    Díaz, Paula; Wood, Amber M.; Sibley, Colin P.; Greenwood, Susan L.

    2014-01-01

    Regulation of human placental syncytiotrophoblast renewal by cytotrophoblast migration, aggregation/fusion and differentiation is essential for successful pregnancy. In several tissues, these events are regulated by intermediate conductance Ca2+-activated K+ channels (IKCa), in part through their ability to regulate cell volume. We used cytotrophoblasts in primary culture to test the hypotheses that IKCa participate in the formation of multinucleated syncytiotrophoblast and in syncytiotrophoblast volume homeostasis. Cytotrophoblasts were isolated from normal term placentas and cultured for 66 h. This preparation recreates syncytiotrophoblast formation in vivo, as mononucleate cells (15 h) fuse into multinucleate syncytia (66 h) concomitant with elevated secretion of human chorionic gonadotropin (hCG). Cells were treated with the IKCa inhibitor TRAM-34 (10 µM) or activator DCEBIO (100 µM). Culture medium was collected to measure hCG secretion and cells fixed for immunofluorescence with anti-IKCa and anti-desmoplakin antibodies to assess IKCa expression and multinucleation respectively. K+ channel activity was assessed by measuring 86Rb efflux at 66 h. IKCa immunostaining was evident in nucleus, cytoplasm and surface of mono- and multinucleate cells. DCEBIO increased 86Rb efflux 8.3-fold above control and this was inhibited by TRAM-34 (85%; p<0.0001). Cytotrophoblast multinucleation increased 12-fold (p<0.05) and hCG secretion 20-fold (p<0.05), between 15 and 66 h. Compared to controls, DCEBIO reduced multinucleation by 42% (p<0.05) and hCG secretion by 80% (p<0.05). TRAM-34 alone did not affect cytotrophoblast multinucleation or hCG secretion. Hyposmotic solution increased 86Rb efflux 3.8-fold (p<0.0001). This effect was dependent on extracellular Ca2+, inhibited by TRAM-34 and 100 nM charybdotoxin (85% (p<0.0001) and 43% respectively) but unaffected by 100 nM apamin. In conclusion, IKCa are expressed in cytotrophoblasts and their activation inhibits the formation

  14. IFPA Meeting 2012 Workshop Report II: epigenetics and imprinting in the placenta, growth factors and villous trophoblast differentiation, role of the placenta in regulating fetal exposure to xenobiotics during pregnancy, infection and the placenta.

    PubMed

    Ahmed, M S; Aleksunes, L M; Boeuf, P; Chung, M K; Daoud, G; Desoye, G; Díaz, P; Golos, T G; Illsley, N P; Kikuchi, K; Komatsu, R; Lao, T; Morales-Prieto, D M; Nanovskaya, T; Nobuzane, T; Roberts, C T; Saffery, R; Tamura, I; Tamura, K; Than, N G; Tomi, M; Umbers, A; Wang, B; Weedon-Fekjaer, M S; Yamada, S; Yamazaki, K; Yoshie, M; Lash, G E

    2013-03-01

    Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2012 there were twelve themed workshops, four of which are summarized in this report. These workshops related to various aspects of placental biology: 1) epigenetics and imprinting in the placenta; 2) growth factors and villous trophoblast differentiation; 3) role of the placenta in regulating fetal exposure to xenobiotics during pregnancy; 4) infection and the placenta.

  15. In vitro approaches to evaluate placental drug transport by using differentiating JEG-3 human choriocarcinoma cells.

    PubMed

    Ikeda, Kenji; Utoguchi, Naoki; Tsutsui, Hidenobu; Yamaue, Satoko; Homemoto, Manami; Nakao, Erina; Hukunaga, Yumi; Yamasaki, Kyohei; Myotoku, Michiaki; Hirotani, Yoshihiko

    2011-02-01

    Human choriocarcinoma cells have been used as models for studying transcellular drug transport through placental trophoblasts. However, these models allow the transport of low-molecular-weight drugs through intercellular gap junctions. This study aimed at investigating the differentiation patterns of JEG-3 choriocarcinoma cells under different culture conditions and establishing the appropriate model of in vitro syncytiotrophoblast drug transport. Paracellular permeability was estimated by measuring the transepithelial electrical resistance (TEER) across JEG-3 cell layers. The mRNA expression levels of non-expressed in choriocarcinoma clone 1 (NECC1) and breast cancer resistance protein (BCRP), and those of E-cadherin (ECAD) and cadherin-11 (CDH11), which are adherens junction-associated proteins related to fusogenic ability of syncytiotrophoblasts differentiated from cytotrophoblasts, protein expression levels were considered as the differentiation signals. The highest TEER values were obtained in the JEG-3 cells cultured in the Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 (1:1) mixed medium (CS-C(®) ; Dainippon Sumitomo Pharma Co. Ltd., Osaka, Japan). By comparing the TEER values and the differentiation signals, the authors identified at least five JEG-3 cell-differentiation patterns. The differentiation pattern of JEG-3 cultured in CS-C resembled the syncytiotrophoblast-like differentiation signal characterizations in vivo. In conclusion, the syncytiotrophoblast-like models of differentiating JEG-3 cells cultured in CS-C might be appropriate for evaluating drug transport across the placental trophoblast.

  16. TNF-α stimulates System A amino acid transport in primary human trophoblast cells mediated by p38 MAPK signaling.

    PubMed

    Aye, Irving L M H; Jansson, Thomas; Powell, Theresa L

    2015-10-01

    Maternal obesity and gestational diabetes mellitus (GDM) increase the risk of delivering infants that are large for gestational age with greater adiposity, who are prone to the development of metabolic disease in childhood and beyond. These maternal conditions are also associated with increased levels of the proinflammatory cytokine TNF-α in maternal tissues and the placenta. Recent evidence suggests that changes in placental amino acid transport contribute to altered fetal growth. TNF-α was previously shown to stimulate System A amino acid transport in primary human trophoblasts (PHTs), however the molecular mechanisms remain unknown. In this study, we tested the hypothesis that TNF-α regulates amino acid uptake in cultured PHTs by a mitogen-activated protein kinase (MAPK)-dependent mechanism. Treatment of PHTs with TNF-α significantly increased System A amino acid transport, as well as Erk and p38 MAPK signaling. Pharmacological antagonism of p38, but not Erk MAPK activity, inhibited TNF-α stimulated System A activity. Silencing of p38 MAPK using siRNA transfections prevented TNF-α stimulated System A transport in PHTs. TNF-α significantly increased the protein expression of System A transporters SNAT1 and SNAT2, but did not affect their mRNA expression. The effects of TNF-α on SNAT1 and SNAT2 protein expression were reversed by p38 MAPK siRNA silencing. In conclusion, TNF-α regulates System A activity through increased SNAT1 and SNAT2 transporter protein expression in PHTs. These findings suggest that p38 MAPK may represent a critical mechanistic link between elevated proinflammatory cytokines and increased placental amino acid transport in obese and GDM pregnancies associated with fetal overgrowth.

  17. TNF-α stimulates System A amino acid transport in primary human trophoblast cells mediated by p38 MAPK signaling.

    PubMed

    Aye, Irving L M H; Jansson, Thomas; Powell, Theresa L

    2015-10-01

    Maternal obesity and gestational diabetes mellitus (GDM) increase the risk of delivering infants that are large for gestational age with greater adiposity, who are prone to the development of metabolic disease in childhood and beyond. These maternal conditions are also associated with increased levels of the proinflammatory cytokine TNF-α in maternal tissues and the placenta. Recent evidence suggests that changes in placental amino acid transport contribute to altered fetal growth. TNF-α was previously shown to stimulate System A amino acid transport in primary human trophoblasts (PHTs), however the molecular mechanisms remain unknown. In this study, we tested the hypothesis that TNF-α regulates amino acid uptake in cultured PHTs by a mitogen-activated protein kinase (MAPK)-dependent mechanism. Treatment of PHTs with TNF-α significantly increased System A amino acid transport, as well as Erk and p38 MAPK signaling. Pharmacological antagonism of p38, but not Erk MAPK activity, inhibited TNF-α stimulated System A activity. Silencing of p38 MAPK using siRNA transfections prevented TNF-α stimulated System A transport in PHTs. TNF-α significantly increased the protein expression of System A transporters SNAT1 and SNAT2, but did not affect their mRNA expression. The effects of TNF-α on SNAT1 and SNAT2 protein expression were reversed by p38 MAPK siRNA silencing. In conclusion, TNF-α regulates System A activity through increased SNAT1 and SNAT2 transporter protein expression in PHTs. These findings suggest that p38 MAPK may represent a critical mechanistic link between elevated proinflammatory cytokines and increased placental amino acid transport in obese and GDM pregnancies associated with fetal overgrowth. PMID:26508738

  18. Effect of oxygen on the expression of renin-angiotensin system components in a human trophoblast cell line.

    PubMed

    Delforce, Sarah J; Wang, Yu; Van-Aalst, Meg E; Corbisier de Meaultsart, Celine; Morris, Brian J; Broughton-Pipkin, Fiona; Roberts, Claire T; Lumbers, Eugenie R; Pringle, Kirsty G

    2016-01-01

    During the first trimester, normal placental development occurs in a low oxygen environment that is known to stimulate angiogenesis via upregulation of vascular endothelial growth factor (VEGF). Expression of the placental renin-angiotensin system (RAS) is highest in early pregnancy. While the RAS and oxygen both stimulate angiogenesis, how they interact within the placenta is unknown. We postulated that low oxygen increases expression of the proangiogenic RAS pathway and that this is associated with increased VEGF in a first trimester human trophoblast cell line (HTR-8/SVneo). HTR-8/SVneo cells were cultured in one of three oxygen tensions (1%, 5% and 20%). RAS and VEGF mRNA expression were determined by qPCR. Prorenin, angiotensin converting enzyme (ACE) and VEGF protein levels in the supernatant, as well as prorenin and ACE in cell lysates, were measured using ELISAs. Low oxygen significantly increased the expression of both angiotensin II type 1 receptor (AGTR1) and VEGF (both P < 0.05). There was a positive correlation between AGTR1 and VEGF expression at low oxygen (r = 0.64, P < 0.005). Corresponding increases in VEGF protein were observed with low oxygen (P < 0.05). Despite no change in ACE1 mRNA expression, ACE levels in the supernatant increased with low oxygen (1% and 5%, P < 0.05). Expression of other RAS components did not change. Low oxygen increased AGTR1 and VEGF expression, as well as ACE and VEGF protein levels, suggesting that the proangiogenic RAS pathway is activated. This highlights a potential role for the placental RAS in mediating the proangiogenic effects of low oxygen in placental development.

  19. Imaging and Clinical Data of Placental Site Trophoblastic Tumor: A Case Report

    PubMed Central

    Niknejadi, Maryam; Ahmadi, Firoozeh; Akhbari, Farnaz

    2016-01-01

    Placental site trophoblastic tumor (PSTT) is a very rare variant of gestational trophoblastic tumor. It can occur after normal termination of pregnancy or spontaneous abortion and ectopic or molar pregnancy. There is a wide range of clinical manifestations from a benign condition to an aggressive disease with fatal outcome. One of the most important characteristics of PSTT, unlike other forms of gestational trophoblastic diseases (GTD) is the presence of low beta-subunit of human chorionic gonadotropin (β-hCG) levels because it is a neoplastic proliferation of intermediate trophoblastic cells. However, human placental lactogen (hPL) is increased on histologic section and in the serum of patients too. We present a case of PSTT and discuss the differential diagnosis in order to further familiarize physicians with the diagnosis and treatment of this disease. It has a varied clinical spectrum and usually presents with irregular vaginal bleeding or amenorrhea. Diagnosis is confirmed by dilatation and curettage (D and C) and hysterectomy. Because chemotherapy is not effective, surgery is the cornerstone of treatment. This case is presented because it is a rare neoplasm with different treatments and it should be differentiated from molar pregnancy.

  20. Imaging and Clinical Data of Placental Site Trophoblastic Tumor: A Case Report.

    PubMed

    Niknejadi, Maryam; Ahmadi, Firoozeh; Akhbari, Farnaz

    2016-04-01

    Placental site trophoblastic tumor (PSTT) is a very rare variant of gestational trophoblastic tumor. It can occur after normal termination of pregnancy or spontaneous abortion and ectopic or molar pregnancy. There is a wide range of clinical manifestations from a benign condition to an aggressive disease with fatal outcome. One of the most important characteristics of PSTT, unlike other forms of gestational trophoblastic diseases (GTD) is the presence of low beta-subunit of human chorionic gonadotropin (β-hCG) levels because it is a neoplastic proliferation of intermediate trophoblastic cells. However, human placental lactogen (hPL) is increased on histologic section and in the serum of patients too. We present a case of PSTT and discuss the differential diagnosis in order to further familiarize physicians with the diagnosis and treatment of this disease. It has a varied clinical spectrum and usually presents with irregular vaginal bleeding or amenorrhea. Diagnosis is confirmed by dilatation and curettage (D and C) and hysterectomy. Because chemotherapy is not effective, surgery is the cornerstone of treatment. This case is presented because it is a rare neoplasm with different treatments and it should be differentiated from molar pregnancy. PMID:27679689

  1. Pulmonary disease in gestational trophoblastic neoplasms.

    PubMed Central

    McNair, O. M.; Polk, O. D.

    1992-01-01

    Gestational trophoblastic neoplasms can present as pulmonary nodules without significant disease of the reproductive organs. This article describes a case of metastatic gestational trophoblastic disease to the lungs. This entity must be considered in the differential diagnosis in any female of reproductive age who presents with multiple pulmonary nodules. Thoracotomy has a limited role in the initial evaluation of patients with this disease. However, it may be needed in patients who have evidence of persistent pulmonary disease, despite appropriate therapy. Images Figure 1 Figure 2 Figure 3 PMID:1324326

  2. [Gestational trophoblastic disease].

    PubMed

    Allias, Fabienne; Bolze, Pierre-Adrien; Gaillot-Durand, Lucie; Devouassoux-Shisheboran, Mojgan

    2014-12-01

    Gestational trophoblastic disease encompresses a group of interrelated diseases, following a pregnancy after a variable period of time. Hydatiform mole corresponds to premalignant disorders composed of villi with excess of paternal genetic material, with a malignant potential more important for complete mole than partial mole. Gestational trophoblastic neoplasia includes invasive mole, choriocarcinoma, placental site trophoblatic tumor and epithelioid trophoblastic tumor. Their histological diagnosis may be problematic on curettage material and needs to be correlated to serum hCG level and radiological findings. The use of chemotherapy has dramatically improved the prognosis of these lesions. All patients with this rare disease need to be registered in the national service for gestational trophoblastic disease (http://www.mole-chorio.com), which coordinates their management at the national level.

  3. Trophoblast expression dynamics of the tumor suppressor gene gastrokine 2.

    PubMed

    Fahlbusch, Fabian B; Ruebner, Matthias; Huebner, Hanna; Volkert, Gudrun; Bartunik, Hannah; Winterfeld, Ilona; Hartner, Andrea; Menendez-Castro, Carlos; Noegel, Stephanie C; Marek, Ines; Wachter, David; Schneider-Stock, Regine; Beckmann, Matthias W; Kehl, Sven; Rascher, Wolfgang

    2015-09-01

    Gastrokines (GKNs) were originally described as stomach-specific tumor suppressor genes. Recently, we identified GKN1 in extravillous trophoblasts (EVT) of human placenta. GKN1 treatment reduced the migration of the trophoblast cell line JEG-3. GKN2 is known to inhibit the proliferation, migration and invasion of gastric cancer cells and may interact with GKN1. Recently, GKN2 was detected in the placental yolk sac of mice. We therefore aimed to further characterize placental GKN2 expression. By immunohistochemistry, healthy first-trimester placenta showed ubiquitous staining for GKN2 at its early gestational stage. At later gestational stages, a more differentiated expression pattern in EVT and villous cytotrophoblasts became evident. In healthy third-trimester placenta, only EVT retained strong GKN2 immunoreactivity. In contrast, HELLP placentas showed a tendency of increased levels of GKN2 expression with a more prominent GKN2 staining in their syncytiotrophoblast. Choriocarcinoma cell lines did not express GKN2. Besides its trophoblastic expression, we found human GKN2 in fibrotic villi, in amniotic membrane and umbilical cord. GKN2 co-localized with smooth muscle actin in villous myofibroblasts and with HLA-G and GKN1 in EVT. In the rodent placenta, GKN2 was specifically located in the spongiotrophoblast layer. Thus, the gestational age-dependent and compartment-specific expression pattern of GKN2 points to a role for placental development. The syncytial expression of GKN2 in HELLP placentas might represent a reduced state of functional differentiation of the syncytiotrophoblast. Moreover, the specific GKN2 expression in the rodent spongiotrophoblast layer (equivalent to human EVT) might suggest an important role in EVT physiology. PMID:26070363

  4. Invasive Trophoblasts Stimulate Vascular Smooth Muscle Cell Apoptosis by a Fas Ligand-Dependent Mechanism

    PubMed Central

    Harris, Lynda K.; Keogh, Rosemary J.; Wareing, Mark; Baker, Philip N.; Cartwright, Judith E.; Aplin, John D.; Whitley, Guy St J.

    2006-01-01

    During pregnancy, trophoblasts migrate from the placenta into uterine spiral arteries, transforming them into wide channels that lack vasoconstrictive properties. In pathological pregnancies, this process is incomplete. To define the fundamental events involved in spiral artery remodeling, we have studied the effect of trophoblasts on vascular smooth muscle cells (SMCs). Here we demonstrate for the first time that apoptosis of SMCs can be initiated by invading trophoblasts. When trophoblasts isolated from normal placenta (primary trophoblasts) or conditioned medium was perfused into spiral or umbilical artery segments, apoptosis of SMCs resulted. Culture of human aortic SMCs (HASMCs) with primary trophoblasts, primary trophoblast-conditioned medium, or a trophoblast-derived cell line (SGHPL-4) also significantly increased SMC apoptosis. Fas is expressed by spiral artery SMCs, and a Fas-activating antibody triggered HASMC apoptosis. Furthermore, a Fas ligand (FasL)-blocking antibody significantly inhibited HASMC apoptosis induced by primary trophoblasts, SGHPL-4, or trophoblast-conditioned medium. Depleting primary trophoblast-conditioned medium of FasL also abrogated SMC apoptosis in vessels in situ. These results suggest that apoptosis triggered by the release of soluble FasL from invading trophoblasts contributes to the loss of smooth muscle from the walls of spiral arteries during pregnancy. PMID:17071607

  5. Differentiation of Trophoblast Giant Cells and Their Metabolic Functions Are Dependent on Peroxisome Proliferator-Activated Receptor β/δ

    PubMed Central

    Nadra, Karim; Anghel, Silvia I.; Joye, Elisabeth; Tan, Nguan Soon; Basu-Modak, Sharmila; Trono, Didier; Wahli, Walter; Desvergne, Béatrice

    2006-01-01

    Mutation of the nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) severely affects placenta development, leading to embryonic death at embryonic day 9.5 (E9.5) to E10.5 of most, but not all, PPARβ/δ-null mutant embryos. While very little is known at present about the pathway governed by PPARβ/δ in the developing placenta, this paper demonstrates that the main alteration of the placenta of PPARβ/δ-null embryos is found in the giant cell layer. PPARβ/δ activity is in fact essential for the differentiation of the Rcho-1 cells in giant cells, as shown by the severe inhibition of differentiation once PPARβ/δ is silenced. Conversely, exposure of Rcho-1 cells to a PPARβ/δ agonist triggers a massive differentiation via increased expression of 3-phosphoinositide-dependent kinase 1 and integrin-linked kinase and subsequent phosphorylation of Akt. The links between PPARβ/δ activity in giant cells and its role on Akt activity are further strengthened by the remarkable pattern of phospho-Akt expression in vivo at E9.5, specifically in the nucleus of the giant cells. In addition to this phosphatidylinositol 3-kinase/Akt main pathway, PPARβ/δ also induced giant cell differentiation via increased expression of I-mfa, an inhibitor of Mash-2 activity. Finally, giant cell differentiation at E9.5 is accompanied by a PPARβ/δ-dependent accumulation of lipid droplets and an increased expression of the adipose differentiation-related protein (also called adipophilin), which may participate to lipid metabolism and/or steroidogenesis. Altogether, this important role of PPARβ/δ in placenta development and giant cell differentiation should be considered when contemplating the potency of PPARβ/δ agonist as therapeutic agents of broad application. PMID:16581799

  6. Uterine Spiral Artery Remodeling: The Role of Uterine Natural Killer Cells and Extravillous Trophoblasts in Normal and High-Risk Human Pregnancies.

    PubMed

    Tessier, Daniel R; Yockell-Lelièvre, Julien; Gruslin, Andrée

    2015-07-01

    The process of uterine spiral artery remodeling in the first trimester of human pregnancy is an essential part of establishing adequate blood perfusion of the placenta that will allow optimal nutrient/waste exchange to meet fetal demands during later development. Key regulators of spiral artery remodeling are the uterine natural killer cells and the invasive extravillous trophoblasts. The functions of these cells as well as regulation of their activation states and temporal regulation of their localization within the uterine tissue are beginning to be known. In this review, we discuss the roles of these two cell lineages in arterial remodeling events, their interaction/influence on one another and the outcomes of altered temporal, and spatial regulation of these cells in pregnancy complications.

  7. Regulation of amino acid transporter trafficking by mTORC1 in primary human trophoblast cells is mediated by the ubiquitin ligase Nedd4-2.

    PubMed

    Rosario, Fredrick J; Dimasuay, Kris Genelyn; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

    2016-04-01

    Changes in placental amino acid transfer directly contribute to altered fetal growth, which increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Placental amino acid transfer is critically dependent on the expression of specific transporters in the plasma membrane of the trophoblast, the transporting epithelium of the human placenta. However, the molecular mechanisms regulating this process are largely unknown. Nedd4-2 is an ubiquitin ligase that catalyses the ubiquitination of proteins, resulting in proteasomal degradation. We hypothesized that inhibition of mechanistic target of rapamycin complex 1 (mTORC1) decreases amino acid uptake in primary human trophoblast (PHT) cells by activation of Nedd4-2, which increases transporter ubiquitination resulting in decreased transporter expression in the plasma membrane. mTORC 1 inhibition increased the expression of Nedd4-2, promoted ubiquitination and decreased the plasma membrane expression of SNAT2 (an isoform of the System A amino acid transporter) and LAT1 (a System L amino acid transporter isoform), resulting in decreased cellular amino acid uptake. Nedd4-2 silencing markedly increased the trafficking of SNAT2 and LAT1 to the plasma membrane, which stimulated cellular amino acid uptake. mTORC1 inhibition by silencing of raptor failed to decrease amino acid transport following Nedd4-2 silencing. In conclusion, we have identified a novel link between mTORC1 signalling and ubiquitination, a common posttranslational modification. Because placental mTORC1 is inhibited in fetal growth restriction and activated in fetal overgrowth, we propose that regulation of placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth.

  8. Biological markers during early pregnancy: trophoblastic signals of the peri-implantation period.

    PubMed Central

    Glasser, S R; Julian, J; Munir, M I; Soares, M J

    1987-01-01

    The peri-implantation period extends from the time the blastocyst is free in the uterus, through the processes of recognition and attachment, to the beginning of trophoblast differentiation and the interactions between the embryo and the uterine endometrium which initiate establishment of the hemochorial placenta. It is during the peri-implantation period that the embryo and hormonally regulated endometrial cells appear to be most sensitive to factors which introduce risk into the intrauterine environment. There are no markers which can be used practically to assess pregnancy risk during the peri-implantation period of either human or laboratory rodents. Experimental studies, using in vitro laboratory models of differentiating trophoblast cells, have identified peptide hormone markers of pivotal developmental processes. Exposure of trophoblast during the expression of these processes could have severe and far-reaching effects individually and societally. While these trophoblast signals are limited in their utility with respect to health monitoring extrapolation of these findings to human pregnancy, the signals could serve to identify more practical and sensitive markers to assess risk in early gestation. Human chorionic gonadotropin (hCG) has been used extensively as a marker to assess risk during the early stages of pregnancy. Extrapolation of experimental data indicates how hCG could be used more effectively in analyses of possible cause and effect relationships. The limitations of hCG as a marker for risk during the human peri-implantation period are discussed. Peptide hormones which could serve to assess risk during this critical period of extraordinary sensitivity to toxic factors are introduced. PMID:3319548

  9. Trophoblast Deportation to the Lungs of Cotton Rats (Sigmodon hispidus)

    PubMed Central

    Perle, Krista M D La; Green, M Gia; Niewiesk, Stefan

    2014-01-01

    Cotton rats (Sigmodon hispidus) have been used to study a variety of infectious agents, particularly human respiratory viral pathogens. During the course of comprehensive pathologic evaluations of aging breeders from our breeding colony, 6 of 22 (27%) female cotton rats had histologic evidence, limited to the lungs, of embolized cells that were confirmed to be trophoblastic in origin by HSD3B1 immunoreactivity. When pulmonary trophoblast emboli were numerous, they usually were associated with additional histologic findings in the lungs, including pulmonary edema and hemorrhage, endothelial hypertrophy, fibrinoid vascular necrosis, and abundant alveolar macrophages containing fresh fibrin and hemolyzing erythrocytes. Of the 6 cotton rats with pulmonary trophoblast emboli, 5 (83%) were at 8 to 18 d of the 27-d gestation period, with the greatest number of emboli per lung present between days 10 through 14. The remaining cotton rat had a focal pulmonary trophoblast embolus and was not pregnant but had delivered a litter 3 mo previously. Three other cotton rats in either the early or late stages of gestation showed no histologic evidence of pulmonary trophoblast deportation. This report is the first to document pulmonary trophoblast emboli in cotton rats. This finding suggests that cotton rats may be an alternative animal model for the study of normal and aberrant trophoblast deportation in routine pregnancies and gestational pathologic conditions in women. PMID:25527025

  10. Effect of Microcystin-LR on human trophoblast differentiation in vitro

    EPA Science Inventory

    Background: Microcystin LR is a potent protein phosphatase 2a (PP2a) inhibitor and generates reactive oxygen species (ROS) believed to be an essential component of a toxic effect. Toxicological studies have demonstrated microcystin (MCYST) disruption of cytoskeletal function and...

  11. Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells.

    PubMed

    Kubaczka, Caroline; Schorle, Hubert

    2016-01-01

    Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo. PMID:27500445

  12. Protective Effect of Nuclear Factor E2-Related Factor 2 on Inflammatory Cytokine Response to Brominated Diphenyl Ether-47 in the HTR-8/SVneo Human First Trimester Extravillous Trophoblast Cell Line

    PubMed Central

    Park, Hae-Ryung; Loch-Caruso, Rita

    2014-01-01

    Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants, and BDE-47 is a prevalent PBDE congener detected in human tissues. Exposure to PBDEs has been linked to adverse pregnancy outcomes in humans. Although the underlying mechanisms of adverse birth outcomes are poorly understood, critical roles for oxidative stress and inflammation are implicated. The present study investigated antioxidant responses in a human extravillous trophoblast cell line, HTR-8/SVneo, and examined the role of nuclear factor E2-related factor 2 (Nrf2), an antioxidative transcription factor, in BDE-47-induced inflammatory responses in the cells. Treatment of HTR-8/SVneo cells with 5, 10, 15, and 20 μM BDE-47 for 24 h increased intracellular glutathione (GSH) levels compared to solvent control. Treatment of HTR-8/SVneo cells with 20 μM BDE-47 for 24 h induced the antioxidant response element (ARE) activity, indicating Nrf2 transactivation by BDE-47 treatment, and resulted in differential expression of redox-sensitive genes compared to solvent control. Pretreatment with tert-butyl hydroquinone (tBHQ) or sulforaphane, known Nrf2 inducers, reduced BDE-47-stimulated IL-6 release with increased ARE reporter activity, reduced nuclear factor kappa B (NF-κB) reporter activity, increased GSH production, and stimulated expression of antioxidant genes compared to non-Nrf2 inducer pretreated groups, suggesting that Nrf2 may play a protective role against BDE-47-mediated inflammatory responses in HTR-8/SVneo cells. These results suggest that Nrf2 activation significantly attenuated BDE-47-induced IL-6 release by augmentation of cellular antioxidative system via upregulation of Nrf2 signaling pathways, and that Nrf2 induction may be a potential therapeutic target to reduce adverse pregnancy outcomes associated with toxicant-induced oxidative stress and inflammation. PMID:25305463

  13. Phosphoinositide 3-Kinase (PI3K) Subunit p110δ Is Essential for Trophoblast Cell Differentiation and Placental Development in Mouse

    PubMed Central

    Hu, Xiwen; Li, Jiangchao; Zhang, Qianqian; Zheng, Lingyun; Wang, Guang; Zhang, Xiaohan; Zhang, Jingli; Gu, Quliang; Ye, Yuxiang; Guo, Sun-Wei; Yang, Xuesong; Wang, Lijing

    2016-01-01

    Maternal PI3K p110δ has been implicated in smaller litter sizes in mice, but its underlying mechanism remains unclear. The placenta is an indispensable chimeric organ that supports mammalian embryonic development. Using a mouse model of genetic inactivation of PI3K p110δ (p110δD910A/D910A), we show that fetuses carried by p110δD910A/D910A females were growth retarded and showed increased mortality in utero mainly during placentation. The placentas in p110δD910A/D910A females were anomalously anemic, exhibited thinner spongiotrophoblast layer and looser labyrinth zone, which indicate defective placental vasculogenesis. In addition, p110δ was detected in primary trophoblast giant cells (P-TGC) at early placentation. Maternal PI3K p110δ inactivation affected normal TGCs generation and expansion, impeded the branching of chorioallantoic placenta but enhanced the expression of matrix metalloproteinases (MMP-2, MMP-12). Poor vasculature support for the developing fetoplacental unit resulted in fetal death or gross growth retardation. These data, taken together, provide the first in vivo evidence that p110δ may play an important role in placental vascularization through manipulating trophoblast giant cell. PMID:27306493

  14. The trophoblast model of cancer.

    PubMed

    Ross, Colin A

    2015-01-01

    John Beard, the British embryologist and histologist, first proposed his trophoblast model of cancer in 1902. The model has subsequently been expanded by Kelley, and in current times, Gonzalez and Isaacs. The trophoblast model of cancer can be stated as a specified, scientifically testable model, including its core predictions that 1) adult stem cells are ectopic trophoblasts that have migrated to other tissues early in embryogenesis; 2) pancreatic enzymes are the key signal that converts the trophoblast into the stable placenta; 3) cancer arises from trophoblasts that have escaped regulatory control; and 4) pancreatic enzymes can be used to treat cancer. The author reviewed the literature on the trophoblast model of cancer and the use of pancreatic enzymes for the treatment of cancer and organized its key tenets into a set of specified scientific hypotheses. The trophoblast model of cancer can be stated as a set of 11 core predictions and 3 adjunctive or nonessential components. The trophoblast model of cancer is a detailed, testable model that should be investigated within an overlapping set of fields including oncology, histology, physiology, molecular biology, and embryology.

  15. The Elsevier Trophoblast Research Award Lecture: Importance of metzincin proteases in trophoblast biology and placental development: a focus on ADAM12.

    PubMed

    Aghababaei, Mahroo; Beristain, Alexander G

    2015-04-01

    Placental development is a highly regulated process requiring signals from both fetal and maternal uterine compartments. Within this complex system, trophoblasts, placental cells of epithelial lineage, form the maternal-fetal interface controlling nutrient, gas and waste exchange. The commitment of progenitor villous cytotrophoblasts to differentiate into diverse trophoblast subsets is a fundamental process in placental development. Differentiation of trophoblasts into invasive stromal- and vascular-remodeling subtypes is essential for uterine arterial remodeling and placental function. Inadequate placentation, characterized by defects in trophoblast differentiation, may underlie the earliest cellular events driving pregnancy disorders such as preeclampsia and fetal growth restriction. Molecularly, invasive trophoblasts acquire characteristics defined by profound alterations in cell-cell and cell-matrix adhesion, cytoskeletal reorganization and production of proteolytic factors. To date, most studies have investigated the importance of the matrix metalloproteinases (MMPs) and their ability to efficiently remodel components of the extracellular matrix (ECM). However, it is now becoming clear that besides MMPs, other related proteases regulate trophoblast invasion via mechanisms other than ECM turnover. In this review, we will summarize the current knowledge on the regulation of trophoblast invasion by members of the metzincin family of metalloproteinases. Specifically, we will discuss the emerging roles that A Disintegrin and Metalloproteinases (ADAMs) play in placental development, with a particular focus on the ADAM subtype, ADAM12.

  16. The Elsevier Trophoblast Research Award Lecture: Importance of metzincin proteases in trophoblast biology and placental development: a focus on ADAM12.

    PubMed

    Aghababaei, Mahroo; Beristain, Alexander G

    2015-04-01

    Placental development is a highly regulated process requiring signals from both fetal and maternal uterine compartments. Within this complex system, trophoblasts, placental cells of epithelial lineage, form the maternal-fetal interface controlling nutrient, gas and waste exchange. The commitment of progenitor villous cytotrophoblasts to differentiate into diverse trophoblast subsets is a fundamental process in placental development. Differentiation of trophoblasts into invasive stromal- and vascular-remodeling subtypes is essential for uterine arterial remodeling and placental function. Inadequate placentation, characterized by defects in trophoblast differentiation, may underlie the earliest cellular events driving pregnancy disorders such as preeclampsia and fetal growth restriction. Molecularly, invasive trophoblasts acquire characteristics defined by profound alterations in cell-cell and cell-matrix adhesion, cytoskeletal reorganization and production of proteolytic factors. To date, most studies have investigated the importance of the matrix metalloproteinases (MMPs) and their ability to efficiently remodel components of the extracellular matrix (ECM). However, it is now becoming clear that besides MMPs, other related proteases regulate trophoblast invasion via mechanisms other than ECM turnover. In this review, we will summarize the current knowledge on the regulation of trophoblast invasion by members of the metzincin family of metalloproteinases. Specifically, we will discuss the emerging roles that A Disintegrin and Metalloproteinases (ADAMs) play in placental development, with a particular focus on the ADAM subtype, ADAM12. PMID:25589360

  17. Protective effect of nuclear factor E2-related factor 2 on inflammatory cytokine response to brominated diphenyl ether-47 in the HTR-8/SVneo human first trimester extravillous trophoblast cell line

    SciTech Connect

    Park, Hae-Ryung Loch-Caruso, Rita

    2014-11-15

    Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants, and BDE-47 is a prevalent PBDE congener detected in human tissues. Exposure to PBDEs has been linked to adverse pregnancy outcomes in humans. Although the underlying mechanisms of adverse birth outcomes are poorly understood, critical roles for oxidative stress and inflammation are implicated. The present study investigated antioxidant responses in a human extravillous trophoblast cell line, HTR-8/SVneo, and examined the role of nuclear factor E2-related factor 2 (Nrf2), an antioxidative transcription factor, in BDE-47-induced inflammatory responses in the cells. Treatment of HTR-8/SVneo cells with 5, 10, 15, and 20 μM BDE-47 for 24 h increased intracellular glutathione (GSH) levels compared to solvent control. Treatment of HTR-8/SVneo cells with 20 μM BDE-47 for 24 h induced the antioxidant response element (ARE) activity, indicating Nrf2 transactivation by BDE-47 treatment, and resulted in differential expression of redox-sensitive genes compared to solvent control. Pretreatment with tert-butyl hydroquinone (tBHQ) or sulforaphane, known Nrf2 inducers, reduced BDE-47-stimulated IL-6 release with increased ARE reporter activity, reduced nuclear factor kappa B (NF-κB) reporter activity, increased GSH production, and stimulated expression of antioxidant genes compared to non-Nrf2 inducer pretreated groups, suggesting that Nrf2 may play a protective role against BDE-47-mediated inflammatory responses in HTR-8/SVneo cells. These results suggest that Nrf2 activation significantly attenuated BDE-47-induced IL-6 release by augmentation of cellular antioxidative system via upregulation of Nrf2 signaling pathways, and that Nrf2 induction may be a potential therapeutic target to reduce adverse pregnancy outcomes associated with toxicant-induced oxidative stress and inflammation. - Highlights: • BDE-47 stimulated ARE reporter activity and GSH production. • BDE-47 resulted in differential

  18. Krüppel-Like Factor 6 Expression Changes during Trophoblast Syncytialization and Transactivates ßhCG and PSG Placental Genes

    PubMed Central

    Racca, Ana C.; Camolotto, Soledad A.; Ridano, Magali E.; Bocco, José L.; Genti-Raimondi, Susana; Panzetta-Dutari, Graciela M.

    2011-01-01

    Background Krüppel-like factor-6 (KLF6) is a widely expressed member of the Sp1/KLF family of transcriptional regulators involved in differentiation, cell cycle control and proliferation in several cell systems. Even though the highest expression level of KLF6 has been detected in human and mice placenta, its function in trophoblast physiology is still unknown. Methodology/Principal Findings Herein, we explored KLF6 expression and sub-cellular distribution in human trophoblast cells differentiating into the syncytial pathway, and its role in the regulation of genes associated with placental development and pregnancy maintenance. Confocal immunofluorescence microscopy demonstrated that KLF6 is expressed throughout human cytotrophoblast differentiation showing no evident modifications in its nuclear and cytoplasmic localization pattern. KLF6 transcript and protein peaked early during the syncytialization process as determined by qRT-PCR and western blot assays. Overexpression of KLF6 in trophoblast-derived JEG-3 cells showed a preferential nuclear signal correlating with enhanced expression of human β-chorionic gonadotropin (βhCG) and pregnancy-specific glycoprotein (PSG) genes. Moreover, KLF6 transactivated βhCG5, PSG5 and PSG3 gene promoters. Deletion of KLF6 Zn-finger DNA binding domain or mutation of the consensus KLF6 binding site abolished transactivation of the PSG5 promoter. Conclusions/Significance Results are consistent with KLF6 playing a role as transcriptional regulator of relevant genes for placental differentiation and physiology such as βhCG and PSG, in agreement with an early and transient increase of KLF6 expression during trophoblast syncytialization. PMID:21799854

  19. Biological markers during early pregnancy: trophoblastic signals of the peri-implantation period

    SciTech Connect

    Glasser, S.R.; Julian, J.; Munir, M.I.; Soares, M.J.

    1987-10-01

    The peri-implantation period extends from the time the blastocyst is free in the uterus, through the processes of recognition and attachment, to the beginning of trophoblast differentiation and the interactions between the embryo and the uterine endometrium which initiate establishment of the hemochorial placenta. It is during the peri-implantation period that the embryo and hormonally regulated endometrial cells appear to be most sensitive to factors which introduce risk into the intrauterine environment. There are no markers which can be used practically to assess pregnancy risk during the peri-implantation period of either human or laboratory rodents. Experimental studies, using in vitro laboratory models of differentiating trophoblasst cells, have identified peptide hormone markers of pivotal developmental processes. Exposure of trophoblast during the expression of these processes could have severe and far-reaching effects individually and societally. Human chorionic gonadotropin (hCG) has been used extensively as a marker to assess risk during the early stages of pregnancy. Extrapolation of experimental data indicates how hCG could be used more effectively in analyses of possible cause and effect relationships. The limitations of hCG as a marker for risk during the human peri-implantation period are discussed. Peptide hormones which could serve to assess risk during this critical period of extraordinary sensitivity to toxic factors are introduced.

  20. Interaction of extravillous trophoblast galectin-1 and mucin(s)-Is there a functional relevance?

    PubMed

    Bojić-Trbojević, Žanka; Jovanović Krivokuća, Milica; Kolundžić, Nikola; Kadoya, Toshihiko; Radojčić, Ljiljana; Vićovac, Ljiljana

    2016-03-01

    In the course of embryo implantation extensive interaction of the trophoblast with uterine tissue is crucial for adequate trophoblast invasion. This interaction is highly controlled, and it has been pointed out that a specific glycocode and changes in glycosylation may be important for successful implantation and maintenance of pregnancy. Both uterine and trophoblast cells have been shown to express cell surface glycoconjugates and sugar binding proteins, such as mucins (MUC) and galectins (gals). An increasing number of studies have investigated potential candidates interacting in this process. However, knowledge about the biochemical nature of the interactions and their importance for trophoblast cell function, and, consequently, for pregnancy outcome are still lacking. This review is aimed at deliberating the possibility that mucins, as heavily glycosylated proteins, might be among the functionally relevant galectin ligands in human trophoblast, based on both published data and our original research. PMID:26418067

  1. The YY1/MMP2 axis promotes trophoblast invasion at the maternal-fetal interface.

    PubMed

    Tian, Fu-Ju; Cheng, Yan-Xiang; Li, Xiao-Cui; Wang, Fa; Qin, Chuan-Mei; Ma, Xiao-Ling; Yang, Jing; Lin, Yi

    2016-05-01

    YY1 is a sequence-specific DNA-binding transcription factor that has many important biological roles. However, its function in trophoblasts at the maternal-fetal interface remains to be elucidated. In this study, we used an mRNA microarray and reverse transcription qPCR and compared the YY1 mRNA expression level in trophoblasts between patients with recurrent miscarriage (RM) and healthy control subjects. Our results revealed that YY1 mRNA expression was significantly lower in the trophoblasts of the RM group compared with the healthy control group. Furthermore, immunofluorescence and immunohistochemical data showed that YY1 was highly expressed in human placental villi during early pregnancy, especially in cytotrophoblast cells and invasive extravillous trophoblasts, and it was expressed at a much lower level in the placental villi of term pregnancy. YY1 overexpression enhanced, and knockdown repressed, the invasion and proliferation of trophoblasts. Antibody array screening revealed that YY1 significantly promoted MMP2 expression in trophoblasts. Bioinformatics analysis identified three YY1-binding sites in the MMP2 promoter region, and chromatin immunoprecipitation analysis verified that YY1 binds directly to its promoter region. Importantly, inhibition of YY1 by siRNA clearly decreased trophoblast invasion in an ex vivo explant culture model. Overall, our findings revealed a new regulatory pathway of YY1/MMP2 in trophoblast cell invasion during early pregnancy and indicated that YY1 may be involved in the pathogenesis of RM. PMID:27071480

  2. Formation of atypical podosomes in extravillous trophoblasts regulates extracellular matrix degradation

    PubMed Central

    Patel, Anand; Dash, Philip R.

    2012-01-01

    Throughout pregnancy the cytotrophoblast, the stem cell of the placenta, gives rise to the differentiated forms of trophoblasts. The two main cell lineages are the syncytiotrophoblast and the invading extravillous trophoblast. A successful pregnancy requires extravillous trophoblasts to migrate and invade through the decidua and then remodel the maternal spiral arteries. Many invasive cells use specialised cellular structures called invadopodia or podosomes in order to degrade extracellular matrix. Despite being highly invasive cells, the presence of invadapodia or podosomes has not previously been investigated in trophoblasts. In this study these structures have been identified and characterised in extravillous trophoblasts. The role of specialised invasive structures in trophoblasts in the degradation of the extracellular matrix was compared with well characterised podosomes and invadopodia in other invasive cells and the trophoblast specific structures were characterised by using a sensitive matrix degradation assay which enabled visualisation of the structures and their dynamics. We show trophoblasts form actin rich protrusive structures which have the ability to degrade the extracellular matrix during invasion. The degradation ability and dynamics of the structures closely resemble podosomes, but have unique characteristics that have not previously been described in other cell types. The composition of these structures does not conform to the classic podosome structure, with no distinct ring of plaque proteins such as paxillin or vinculin. In addition, trophoblast podosomes protrude more deeply into the extracellular matrix than established podosomes, resembling invadopodia in this regard. We also show several significant pathways such as Src kinase, MAPK kinase and PKC along with MMP-2 and 9 as key regulators of extracellular matrix degradation activity in trophoblasts, while podosome activity was regulated by the rigidity of the extracellular matrix. PMID

  3. Metastasis associated lung adenocarcinoma transcript 1 is up-regulated in placenta previa increta/percreta and strongly associated with trophoblast-like cell invasion in vitro.

    PubMed

    Tseng, Jenn-Jhy; Hsieh, Yeun-Ting; Hsu, Shih-Lan; Chou, Min-Min

    2009-11-01

    Placenta previa increta/percreta (I/P) is a severe form of invasive placentation associated with massive peripartum hemorrhage, which often requires Cesarean hysterectomy. The pathogenesis of invasive placentation is multidimensional, involving decidual deficiency, endomyometrial damage and excessively deep trophoblast invasion into the uterus. In this study, annealing control primer-polymerase chain reaction (ACP-PCR) was used to identify differentially expressed genes, which may impair placentation resulting in placenta previa I/P. Placental tissues from I/P and non-increta/percreta (non-I/P) sites were concomitantly collected from patients undergoing Cesarean hysterectomy. After ACP-PCR experiments (three patients), the differentially expressed bands, consistently showing up- or down-regulated trends between each of the I/P and non-I/P tissue pairs, were cloned and sequenced. Human non-protein coding metastasis associated lung adenocarcinoma transcript 1 (MALAT-1) gene was identified. Real-time quantitative PCR (10 patients) confirmed significant overexpression of MALAT-1 in I/P samples (P = 0.005). To investigate the role of MALAT-1 gene in the regulation of trophoblast cell invasion, targeting of MALAT-1 mRNA expression with short interfering RNA (siRNA) in trophoblast-like BeWo, JAR and JEG-3 choriocarcinoma cells was performed. The invasion ability of these cells was significantly suppressed after siRNA silencing (P < 0.001), and this was not correlated with abnormal MMP-2 and MMP-9 enzyme activities. Our results suggest that MALAT-1 expression in placenta previa I/P is increased and its down-regulation inhibits trophoblast-like cell invasion in vitro. MALAT-1 might be involved in regulating trophoblast invasion during the development of advanced invasive placentation.

  4. Angiogenin and gestational trophoblastic tumors, a promising prognostic marker.

    PubMed

    Shaarawy, Mohamed; El-Mallah, Samira Y; Sheiba, Mamdouh

    2003-03-01

    The aim of this study was to evaluate the diagnostic and prognostic values of serum angiogenin concentration in cases with gestational trophoblastic diseases (GTDs). Seventy-two patients with GTDs and 20 first trimester healthy pregnant women (controls) participated in this study. According to the WHO scoring system, GTDs were subgrouped into 24 hydatiform mole spontaneous regression (HMSR), 18 postmolar gestational trophoblastic tumors of high risk (PMHR), 16 low-risk choriocarcinoma, and 14 high-risk choriocarcinoma. Before treatment, a blood sample from each case was assayed for human chorionic gonadotrophin , subunit (hCGb) by radioimmunoassay and angiogenin by enzyme immunoassay. Follow-up hCGb and angiogenin assays were carried out for 1 year after treatment. Pretreatment of abnormal values of serum angiogenin (> 711 ng/ml, upper 95% confidence interval of controls) was encountered in 100% of PMHR cases compared to no single case of HMSR. Serum angiogenin levels in low- and high-risk cases with choriocarcinoma were significantly higher than in controls. Abnormal high values were encountered in 25% and 86% of cases, respectively. None of the low-risk cases exceeded 920 ng/ml, while 72% of high-risk cases exceeded this value. Serial angiogenin assays were correlated with disease progression and were positively correlated with serum hCGb (r = 0.75, p < 0.01). In conclusion, serum angiogenin may be a valuable marker of differential diagnosis of GTDs and its serial measurements are suggestive of remission and effective therapeutic intervention or disease progression.

  5. New insights for Ets2 function in trophoblast using lentivirus-mediated gene knockdown in trophoblast stem cells.

    PubMed

    Odiatis, C; Georgiades, P

    2010-07-01

    Mouse trophoblast stem (TS) cells represent a unique in vitro system that provides an unlimited supply of TS cells for the study of trophoblast differentiation and TS cell self-renewal. Although the mouse transcription factor Ets2 is required for TS cell self-renewal, its role in this and in TS cell differentiation has not been explored fully, partly due to the early lethality of Ets2 null mice. To address this, we developed a novel lentivirus-based system that resulted in efficient Ets2 knockdown in the overwhelming majority of TS cells. This system enables functional studies in TS cells, especially for genes required for TS cell self-renewal because TS cell derivation using gene-knockout embryos for such genes depends on TS cell self-renewal. Using morphological/morphometric criteria and gene expression analysis, we show that the requirement for Ets2 in self-renewal of TS cells cultured in 'stem cell medium' (SCM) involves maintenance of the expression of genes that inhibit TS cell differentiation in SCM, such as Cdx2 and Esrrb, and preservation of the undifferentiated TS cell morphology. During TS cell differentiation caused by Cdx2/Esrrb downregulation, due to either Ets2 knockdown in SCM or culture in differentiation medium (DM), Ets2 is also required for the promotion of trophoblast giant cell (TGC) and junctional zone trophoblast (JZT) differentiation. This TGC differentiation involves Ets2-dependent expression of Hand1, a gene required for the differentiation of all TGC types. This study uncovers new roles for Ets2 in TS cell self-renewal and differentiation and demonstrates the usefulness of this lentivirus system for gene function studies in TS cells.

  6. Xanthohumol impairs glucose uptake by a human first-trimester extravillous trophoblast cell line (HTR-8/SVneo cells) and impacts the process of placentation.

    PubMed

    Correia-Branco, Ana; Azevedo, Cláudia F; Araújo, João R; Guimarães, João T; Faria, Ana; Keating, Elisa; Martel, Fátima

    2015-10-01

    In this study, we aimed to investigate modulation of glucose uptake by the HTR-8/SVneo human first-trimester extravillous trophoblast cell line by a series of compounds and to study its consequences upon cell proliferation, viability and migration. We observed that uptake of (3)H-deoxy-d-glucose ((3)H-DG; 10 nM) was time-dependent, saturable, inhibited by cytochalasin B (50 and 100 µM), phloretin (0.5 mM) and phloridzin (1 mM), insulin-insensitive and sodium-independent. In the short term (30 min), neither 5-HT (100-1000 µM), melatonin (10 nM) nor the drugs of abuse ethanol (100 mM), nicotine (100 µM), cocaine (25 µM), amphetamine (10-25 µM) and 3,4-methylenedioxy-N-methamphetamine (10 µM) affected (3)H-DG uptake, while dexamethasone (100-1000 µM), fluoxetine (100-300 µM), quercetin, epigallocatechin-3-gallate (30-1000 µM), xanthohumol (XH) and resveratrol (1-500 µM) decreased it. XH was the most potent inhibitor [IC50 = 3.55 (1.37-9.20) µM] of (3)H-DG uptake, behaving as a non-competitive inhibitor of (3)H-DG uptake, both after short- and long-term (24 h) treatment. The effect of XH (5 µM; 24 h) upon (3)H-DG uptake involved mammalian target of rapamycin, tyrosine kinases and c-Jun N-terminal kinases intracellular pathways. Moreover, XH appeared to decrease cellular uptake of lactate due to inhibition of the monocarboxylate transporter 1. Additionally, XH (24 h; 5 µM) decreased cell viability, proliferation, culture growth and migration. The effects of XH upon cell viability and culture growth, but not the antimigratory effect, were mimicked by low extracellular glucose conditions and reversed by high extracellular glucose conditions. We thus suggest that XH, by inhibiting glucose cellular uptake and impairing HTR-8/SVneo cell viability and proliferation, may have a deleterious impact in the process of placentation. PMID:26194608

  7. Stem cell differentiation and human liver disease

    PubMed Central

    Zhou, Wen-Li; Medine, Claire N; Zhu, Liang; Hay, David C

    2012-01-01

    Human stem cells are scalable cell populations capable of cellular differentiation. This makes them a very attractive in vitro cellular resource and in theory provides unlimited amounts of primary cells. Such an approach has the potential to improve our understanding of human biology and treating disease. In the future it may be possible to deploy novel stem cell-based approaches to treat human liver diseases. In recent years, efficient hepatic differentiation from human stem cells has been achieved by several research groups including our own. In this review we provide an overview of the field and discuss the future potential and limitations of stem cell technology. PMID:22563188

  8. Detection of the alpha-subunit of inhibin in trophoblastic neoplasia.

    PubMed

    Pelkey, T J; Frierson, H F; Mills, S E; Stoler, M H

    1999-01-01

    Placental trophoblasts are the primary source of serum inhibin during pregnancy. We sought to characterize inhibin immunolabeling in trophoblastic neoplasms and compare the results with those for established markers of trophoblastic differentiation. Formalin-fixed, paraffin-embedded tissues from three normal term placentas, 13 hydatidiform moles (HM), five choriocarcinomas (CC) (three gestational, one testicular, one mediastinal), and five placental site trophoblastic tumors (PSTT) were immunolabeled with antibodies to the alpha-subunit of inhibin, hPL, and beta-hCG. Additionally, six first-trimester placentas were immunolabeled with antibody to inhibin alone. Trophoblastic subpopulations were assessed for the number of positive cells (1% to 24% = 1+,25% to 49% = 2+,50% to 74% = 3+, 75% to 100% = 4+). Immunolabeling in term placenta and HM was similar, because beta-hCG was the most sensitive marker (4+) of syncytiotrophoblasts followed by inhibin (3-4+) and hPL (2-3+). Immunolabeling of syncytiotrophoblasts in CC was similar to that in term placenta and HM, except for negative hPL staining in two cases. In HM, inhibin labeling of intermediate trophoblasts (2-3+) was less than that for hPL (3-4+). In CC, inhibin and hPL labeling of intermediate trophoblasts was more variable with negative hPL reactivity in two cases. Inhibin and hPL did not stain cytotrophoblasts. In PSTT, inhibin immunolabeling was a better marker than hPL in two cases, inferior in two, and similar in one. Inhibin labeling of syncytiotrophoblasts was less than that for beta-hCG, but did not have the often marked background staining that was present with beta-hCG. Immunolabeling of trophoblast subpopulations for inhibin was similar between first-trimester placenta, term placenta, and HM. We conclude that inhibin is a useful immunohistochemical marker of trophoblastic neoplasia and should be included in the antibody panel with beta-hCG and hPL.

  9. ADAM12-directed ectodomain shedding of E-cadherin potentiates trophoblast fusion.

    PubMed

    Aghababaei, M; Hogg, K; Perdu, S; Robinson, W P; Beristain, A G

    2015-12-01

    Trophoblasts, placental cells of epithelial lineage, undergo extensive differentiation to form the cellular components of the placenta. Trophoblast progenitor cell differentiation into the multinucleated syncytiotrophoblast is a key developmental process required for placental function, where defects in syncytiotrophoblast formation and turnover associate with placental pathologies and link to poor pregnancy outcomes. The cellular and molecular processes governing syncytiotrophoblast formation are poorly understood, but require the activation of pathways that direct cell fusion. The protease, A Disintegrin and Metalloproteinase 12 (ADAM12), controls cell fusion in myoblasts and is highly expressed in the placenta localizing to multiple trophoblast populations. However, the importance of ADAM12 in regulating trophoblast fusion is unknown. Here, we describe a function for ADAM12 in regulating trophoblast fusion. Using two distinct trophoblast models of cell fusion, we show that ADAM12 is dynamically upregulated and is under the transcriptional control of protein kinase A. siRNA-directed loss of ADAM12 impedes spontaneous fusion of primary cytotrophoblasts, whereas overexpression of the secreted variant, ADAM12S, potentiates cell fusion in the Bewo trophoblast cell line. Mechanistically, both ectopic and endogenous levels of ADAM12 were shown to control trophoblast fusion through E-cadherin ectodomain shedding and remodeling of intercellular boundaries. This study describes a novel role for ADAM12 in placental development, specifically highlighting its importance in controlling the differentiation of villous cytotrophoblasts into multinucleated cellular structures. Moreover, this work identifies E-cadherin as a novel ADAM12 substrate, and highlights the significance that cell adhesion molecule ectodomain shedding has in normal development.

  10. Role of hypoxia-inducible transcription factors 1alpha and 2alpha in the regulation of plasminogen activator inhibitor-1 expression in a human trophoblast cell line.

    PubMed

    Meade, E S; Ma, Y Y; Guller, S

    2007-10-01

    The plasminogen activator inhibitors (PAIs) play critical roles in regulating hemostatic and invasive functions of trophoblasts through suppression of plasmin-dependent fibrinolysis and extracellular matrix degradation. The expression of PAI-1 is increased under hypoxic conditions, although the mechanism remains incompletely understood. In the current study we used HTR-8/SVneo cells, a first trimester extravillous trophoblast cell line, and siRNA technology to examine the role of hypoxia-inducible transcription factors (HIFs)-1alpha and -2alpha in the regulation of PAI-1 expression. Using serum-containing and serum-free media culture media it was initially noted that levels of PAI-1, but not PAI-2 protein, were markedly induced by hypoxic (2-3% oxygen) treatment. Under hypoxic conditions, Western blotting revealed that the presence of siRNAs to HIF-1alpha and HIF-2alpha suppressed expression of their respective proteins, whereas treatment with non-targeting and cyclophilin B siRNAs did not. Importantly, incubation with siRNA to HIF-1alpha or HIF-2alpha alone reduced PAI-1 protein levels to a similar extent, with the combined treatment inducing a more profound effect. The presence of HIF siRNAs reduced levels of PAI-1 mRNA as measured by quantitative real-time PCR, indicating that HIF-1alpha and HIF-2 alpha regulate PAI-1 expression at a transcriptional level. These results indicate that both HIF-1alpha and HIF-2alpha play important and similar roles in hypoxia-mediated stimulation of PAI-1 expression in HTR-8/SVneo cells. Our findings provide insight into the physiological regulation of trophoblast PAI-1 expression in early pregnancy when placental oxygen levels are low, as well as a mechanism for over-expression of placental PAI-1 noted in pregnancies with preeclampsia.

  11. Ethanol cytotoxic effect on trophoblast cells.

    PubMed

    Clave, S; Joya, X; Salat-Batlle, J; Garcia-Algar, O; Vall, O

    2014-03-01

    Prenatal ethanol exposure may cause both, altered fetal neurodevelopment and impaired placental function. These disturbances can lead to growth retardation, which is one of the most prevalent features in Fetal Alcohol Syndrome (FAS). It is not known whether there is a specific pattern of cytotoxicity caused by ethanol that can be extrapolated to other cell types. The aim of this study was to determine the cytotoxic effects caused by sustained exposure of trophoblast cells to ethanol. The cytotoxic effect of sustained exposure to standard doses of ethanol on an in vitro human trophoblast cell line, JEG3, was examined. Viable cell count by exclusion method, total protein concentration, lactate dehydrogenase (LDH) activity and activation of apoptotic markers (P-H2AX, caspase-3 and PARP-1) were determined. Sustained exposure to ethanol decreased viable cell count and total protein concentration. LDH activity did not increased in exposed cells but apoptotic markers were detected. In addition, there was a dose-dependent relationship between ethanol concentration and apoptotic pathways activation. Sustained ethanol exposure causes cellular cytotoxicity by apoptotic pathways induction as a result of DNA damage. This apoptotic induction may partially explain the altered function of placental cells and the damage previously detected in other tissues.

  12. Minor Histocompatibility Antigens Are Expressed in Syncytiotrophoblast and Trophoblast Debris

    PubMed Central

    Holland, Olivia J.; Linscheid, Caitlin; Hodes, Herbert C.; Nauser, Traci L.; Gilliam, Melissa; Stone, Peter; Chamley, Larry W.; Petroff, Margaret G.

    2012-01-01

    The fetal semi-allograft can induce expansion and tolerance of antigen-specific maternal T and B cells through paternally inherited major histocompatibility complex and minor histocompatibility antigens (mHAgs). The effects of these antigens have important consequences on the maternal immune system both during and long after pregnancy. Herein, we investigate the possibility that the placental syncytiotrophoblast and deported trophoblastic debris serve as sources of fetal mHAgs. We mapped the expression of four mHAgs (human mHAg 1, pumilio domain-containing protein KIAA0020, B-cell lymphoma 2–related protein A1, and ribosomal protein S4, Y linked) in the placenta. Each of these proteins was expressed in several placental cell types, including the syncytiotrophoblast. These antigens and two additional Y chromosome–encoded antigens [DEAD box polypeptide 3, Y linked (DDX3Y), and lysine demethylase5D] were also identified by RT-PCR in the placenta, purified trophoblast cells, and cord blood cells. Finally, we used a proteomic approach to investigate the presence of mHAgs in the syncytiotrophoblast and trophoblast debris shed from first-trimester placenta. By this method, four antigens (DDX3Y; ribosomal protein S4, Y linked; solute carrier 1A5; and signal sequence receptor 1) were found in the syncytiotrophoblast, and one antigen (DDX3Y) was found in shed trophoblast debris. The finding of mHAgs in the placenta and in trophoblast debris provides the first direct evidence that fetal antigens are present in debris shed from the human placenta. The data, thus, suggest a mechanism by which the maternal immune system is exposed to fetal alloantigens, possibly explaining the relationship between parity and graft-versus-host disease. PMID:22079431

  13. The impact of ionizing radiation on placental trophoblasts

    PubMed Central

    Kanter, D.J.; O'Brien, M.B.; Shi, X.-H.; Chu, T.; Mishima, T.; Beriwal, S.; Epperly, M.W.; Wipf, P.; Greenberger, J.S.; Sadovsky, Y.

    2014-01-01

    Introduction Exposure to low-dose radiation is widespread and attributable to natural sources. However, occupational, medical, accidental, and terrorist-related exposures remain a significant threat. Information on radiation injury to the feto-placental unit is scant and largely observational. We hypothesized that radiation causes trophoblast injury, and alters the expression of injury-related transcripts in vitro or in vivo, thus affecting fetal growth. Methods Primary human trophoblasts (PHTs), BeWo or NCCIT cells were irradiated in vitro, and cell number and viability were determined. Pregnant C57Bl/6HNsd mice were externally irradiated on E13.5, and placentas examined on E17.5. RNA expression was analyzed using microarrays and RT-qPCR. The experiments were repeated in the presence of the gramicidin S (GS)-derived nitroxide JP4-039, used to mitigate radiation-induced cell injury. Results We found that survival of in vitro–irradiated PHT cell was better than that of irradiated BeWo trophoblast cell line or the radiosensitive NCCIT mixed germ cell tumor line. Radiation altered the expression of several trophoblast genes, with a most dramatic effect on CDKN1A (p21, CIP1). Mice exposed to radiation at E13.5 exhibited a 25% reduction in mean weight by E17.5, and a 9% reduction in placental weight, which was associated with relatively small changes in placental gene expression. JP4-039 had a minimal effect on feto-placental growth or on gene expression in irradiated PHT cells or mouse placenta. Discussion and conclusion While radiation affects placental trophoblasts, the established placenta is fairly resistant to radiation, and changes in this tissue may not fully account for fetal growth restriction induced by ionizing radiation. PMID:24418702

  14. Expression of glypican 3 in placental site trophoblastic tumor

    PubMed Central

    2010-01-01

    Background Glypican-3 (GPC3) is a membrane-bound heparan sulfate proteoglycan that functions in embryonic cell growth and differentiation and is highly expressed in the placenta. GPC3 is mutated in Simpson-Golabi-Behmel syndrome, which is characterized by tissue overgrowth and an increased risk of embryonal malignancies. GPC3 has also been implicated in sporadic cancer, particularly hepatocellular carcinoma, for which it has been shown to be a useful diagnostic marker. Although GPC3 expression has been studied in non-neoplastic placental tissue, its presence in gestational trophoblastic diseases has not been previously explored. The purpose of this study was to investigate the immunohistochemical expression of GPC3 in placental site trophoblastic tumor (PSTT), a very rare gestational trophoblastic neoplasm which may be morphologically confused with non-trophoblastic tumors, and to assess its possible utility as a diagnostic marker. Methods Fifteen cases of PSTT, as well as samples from placental site nodule (PSN) (n = 2), leiomyosarcoma (n = 1), leiomyoma (n = 1), invasive cervical squamous cell carcinoma (n = 7) and endometrial adenocarcinoma (n = 11) were examined. Immunoreactivity was semi-quantitatively evaluated as negative (0, < 5% of cells stained), focally positive (1+, 5-10% of cells stained), positive (2+, 11-50% of cells stained) or diffusely positive (3+, > 50% of cells stained). Staining intensity for each subtype was graded from 0 to 3 and a mean intensity was calculated. Results Eighty percent of PSTT (12/15) were immunoreactive for GPC3 (0, 20; 1+, 20%; 2+, 40%; 3+, 20%) with a mean intensity of 1.3. Stronger, predominately cytoplasmic staining was seen in larger multi- and mononucleated cells with smaller mononucleate cells showing weak muddy cytoplasmic staining. Both PSN cases were positive (1+, 50%; 2+, 50%) and two of nine invasive cervical squamous cell carcinomas showed staining (0, 57%; 1+, 29%; 2+, 14%), predominately in a basal

  15. Ovine trophoblasts express cathelicidin host defence peptide in response to infection.

    PubMed

    Coyle, Christopher; Wheelhouse, Nick; Jacques, Maxime; Longbottom, David; Svoboda, Pavel; Pohl, Jan; Duncan, W Colin; Rae, Michael T; Barlow, Peter G

    2016-09-01

    Cationic host defence peptides (CHDP; also known as antimicrobial peptides) are key components of the immune response in the female reproductive tract. The role of the placental trophoblast in ovine host defence remains poorly understood. This study characterises expression of genes for cathelicidin and defensin peptides in primary ovine placental tissues, the ovine trophoblast cell line (AH-1) and in response to the TLR-4 ligand LPS, the abortifacient organism Waddlia chondrophila and 1α,25-dihydroxyvitamin D3. Using RT-PCR, expression of the CHDP SMAP-29, sBD-1 and sBD-2 was assessed in the AH-1 cell line in response to LPS, 1α,25-dihydroxyvitamin D3 exposure (a known stimulator of cathelicidin gene expression), or W. chondrophila infection. Expression of cathelicidin in the trophoblast compartment of the ovine placenta and in the ovine trophoblast cell line (AH-1) was also established. AH-1 cells did not upregulate expression of CHDP in response to LPS, but sBD-1 and sBD-2 expression was significantly increased in response to W. chondrophila infection. SMAP-29 expression was not altered by in vitro exposure to 1α,25-dihydroxyvitamin D3. This study demonstrates that the ovine trophoblast expresses cathelicidins, but does not upregulate expression of CHDP in response to LPS. Ovine trophoblasts are shown to differentially regulate expression of CHDP and lack a demonstrable vitamin D-mediated cathelicidin response. PMID:27348190

  16. Establishment and Characterization of a Telomerase-Immortalized Sheep Trophoblast Cell Line

    PubMed Central

    Zhang, Yufei; Liu, Shuying

    2016-01-01

    The primary sheep trophoblast cells (STCs) have a finite lifespan in culture. This feature limits the scope for long-term in vitro studies with STCs. This study was an attempt to establish and characterize a telomerase-immortalized sheep trophoblast cell line. STCs were isolated and purified by using Percoll and specific immunoaffinity purification, respectively. The purified STCs were transfected with a plasmid carrying sequences of human telomerase reverse transcriptase (hTERT) to create immortalized sheep trophoblast cell line (hTERT-STCs). hTERT-STCs showed a stable expression of hTERT gene, serially passaged for a year, and showed active proliferation without signs of senescence. Cytokeratin 7 (CK-7), secreted human chorionic gonadotrophin subunit β (CG-β), placental lactogen (PL), and endogenous jaagsiekte sheep retrovirus (enJSRV) envelope genes were expressed in hTERT-STCs. Transwell cell invasion assay indicated that hTERT-STCs still possessed the same invasive characteristics as normal primary sheep trophoblast cells. hTERT-STCs could not grow in soft agar and did not develop into tumors in nude mice. In this study, we established a strain of immortalized sheep trophoblast cell line which could be gainfully employed in the future as an experimental model to study trophoblast cells with secretory function, invasive features, and probable biological function of enJSRV envelope genes. PMID:26998488

  17. Fatal Cases of Gestational Trophoblastic Neoplasia in a National Trophoblastic Disease Reference Center in Dakar Senegal

    PubMed Central

    Gueye, Mamour; Ndiaye-Gueye, Mame Diarra; Kane Gueye, Serigne Modou; Moreau, Jean Charles

    2016-01-01

    Objectives: The objectives of this study were to analyze deaths after gestational trophoblastic neoplasia and to determine the factors of treatment failure. Methods: This is a retrospective study in Aristide Le Dantec teaching Hospital in Dakar, Senegal, between 1 January 2006 and 31 December 2014. We took into account socio-epidemiological characteristics of patients, initial diagnosis, time between uterine evacuation and admission, time to onset of gestational trophoblastic neoplasia (GTN), treatment received (deadlines, protocols), difficulties experienced in the diagnosis and the initiation of treatment and survival. Results: In total, 1044 patients were admitted during the study period; 164 cases of GTN were diagnosed (15.7%); and 21 deaths occurred leading to a specific lethality of 12.8%. The average age was 30 years. Almost all patients (n = 18; 85.7%) had low income or no income. Eight out of 21 patients (38.1%) were seen in our department after GTN onset. The mean time to onset of GTN of all patients was 22.1 weeks. For 66.6%, histology was not available; the diagnosis of hydatidiform mole was made on the clinical history and sonographic features and GTN on human chorionic gonadotrophin (hCG) evolution and ultrasound findings. None of the patients had regular chemotherapy due to financial reasons. Patients who died within 3 months after diagnosis had metastatic tumors (7 of 21). All these women had resistance to treatment or progressed after three courses of chemotherapy. Ten of the 12 women with high-risk GTN were not treated with multi-agent chemotherapy (EMA-CO) for purely financial reasons. Conclusion and Global Health Implications: The high incidence and mortality require a profound reorganization of our health system and a high awareness of practitioners to refer to time or to declare all suspected cases of hydatidiform mole or gestational trophoblastic neoplasia.

  18. Fatal Cases of Gestational Trophoblastic Neoplasia in a National Trophoblastic Disease Reference Center in Dakar Senegal

    PubMed Central

    Gueye, Mamour; Ndiaye-Gueye, Mame Diarra; Kane Gueye, Serigne Modou; Moreau, Jean Charles

    2016-01-01

    Objectives: The objectives of this study were to analyze deaths after gestational trophoblastic neoplasia and to determine the factors of treatment failure. Methods: This is a retrospective study in Aristide Le Dantec teaching Hospital in Dakar, Senegal, between 1 January 2006 and 31 December 2014. We took into account socio-epidemiological characteristics of patients, initial diagnosis, time between uterine evacuation and admission, time to onset of gestational trophoblastic neoplasia (GTN), treatment received (deadlines, protocols), difficulties experienced in the diagnosis and the initiation of treatment and survival. Results: In total, 1044 patients were admitted during the study period; 164 cases of GTN were diagnosed (15.7%); and 21 deaths occurred leading to a specific lethality of 12.8%. The average age was 30 years. Almost all patients (n = 18; 85.7%) had low income or no income. Eight out of 21 patients (38.1%) were seen in our department after GTN onset. The mean time to onset of GTN of all patients was 22.1 weeks. For 66.6%, histology was not available; the diagnosis of hydatidiform mole was made on the clinical history and sonographic features and GTN on human chorionic gonadotrophin (hCG) evolution and ultrasound findings. None of the patients had regular chemotherapy due to financial reasons. Patients who died within 3 months after diagnosis had metastatic tumors (7 of 21). All these women had resistance to treatment or progressed after three courses of chemotherapy. Ten of the 12 women with high-risk GTN were not treated with multi-agent chemotherapy (EMA-CO) for purely financial reasons. Conclusion and Global Health Implications: The high incidence and mortality require a profound reorganization of our health system and a high awareness of practitioners to refer to time or to declare all suspected cases of hydatidiform mole or gestational trophoblastic neoplasia. PMID:27622010

  19. Opioids and differentiation in human cancer cells.

    PubMed

    Zagon, Ian S; McLaughlin, Patricia J

    2005-10-01

    This study was designed to examine the role of opioids on cell differentiation, with an emphasis on the mechanism of opioid growth factor (OGF, [Met5]-enkephalin)-dependent growth inhibition. Three human cancer cell lines (SK-N-SH neuroblastoma and SCC-1 and CAL-27 squamous cell carcinoma of the head and neck), along with OGF and the opioid antagonist naltrexone (NTX) at a dosage (10(-6) M) known to repress or increase, respectively, cell replication, were utilized. The effects on differentiation (neurite formation, process lengths, betaIII-tubulin, involucrin) were investigated in cells exposed to OGF or NTX for up to 6 days. In addition, the influence of a variety of other natural and synthetic opioids on differentiation was examined. OGF, NTX, naloxone, [D-Pen2,5]-enkephalin, dynorphin A1-8, beta-endorphin, endomorphin-1 and -2, [D-Ala2, MePhe4, Glycol5]-enkephalin (DAMGO), morphine, and U69,593 at concentrations of 10(-6) M did not alter cell differentiation of any cancer cell line. In NTX-treated SK-N-SH cells, cellular area was increased 23%, and nuclear area was decreased 17%, from control levels; no changes in cell or nuclear area were recorded in OGF-exposed cells. F-actin concentration was increased 40% from control values in SK-N-SH cells subjected to NTX, whereas alpha-tubulin was decreased 53% in OGF-treated cells. These results indicate that the inhibitory or stimulatory actions of OGF and NTX, respectively, on cell growth in tissue culture are not due to alterations in differentiation pathways. However, exposure to OGF and NTX modified some aspects of cell structure, but this was independent of differentiation. The absence of effects on cancer cell differentiation by a variety of other opioids supports the previously reported lack of growth effects of these compounds.

  20. The YY1/MMP2 axis promotes trophoblast invasion at the maternal–fetal interface

    PubMed Central

    Tian, Fu‐Ju; Cheng, Yan‐Xiang; Li, Xiao‐Cui; Wang, Fa; Qin, Chuan‐Mei; Ma, Xiao‐Ling

    2016-01-01

    Abstract YY1 is a sequence‐specific DNA‐binding transcription factor that has many important biological roles. However, its function in trophoblasts at the maternal–fetal interface remains to be elucidated. In this study, we used an mRNA microarray and reverse transcription qPCR and compared the YY1 mRNA expression level in trophoblasts between patients with recurrent miscarriage (RM) and healthy control subjects. Our results revealed that YY1 mRNA expression was significantly lower in the trophoblasts of the RM group compared with the healthy control group. Furthermore, immunofluorescence and immunohistochemical data showed that YY1 was highly expressed in human placental villi during early pregnancy, especially in cytotrophoblast cells and invasive extravillous trophoblasts, and it was expressed at a much lower level in the placental villi of term pregnancy. YY1 overexpression enhanced, and knockdown repressed, the invasion and proliferation of trophoblasts. Antibody array screening revealed that YY1 significantly promoted MMP2 expression in trophoblasts. Bioinformatics analysis identified three YY1‐binding sites in the MMP2 promoter region, and chromatin immunoprecipitation analysis verified that YY1 binds directly to its promoter region. Importantly, inhibition of YY1 by siRNA clearly decreased trophoblast invasion in an ex vivo explant culture model. Overall, our findings revealed a new regulatory pathway of YY1/MMP2 in trophoblast cell invasion during early pregnancy and indicated that YY1 may be involved in the pathogenesis of RM. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. PMID:27071480

  1. Who let the dogs in: a canine trophoblast invasion model for pre-eclampsia.

    PubMed

    Kutzler, M; Sahlfeld, L; Fellows, E

    2012-12-01

    Pre-eclampsia affects 2-8% of pregnant women worldwide and is the third leading cause of maternal mortality in the United States, accounting for 20% of maternal deaths, for which the only known cure is delivery of the placenta. It is known that pre-eclampsia results from defects within the trophoblast invasion of the endometrium and myometrium. At a morphological level within the pre-eclamptic human placenta, trophoblast invasion is shallow, and this results in hypoperfusion, which is a life-threatening condition for both the mother and the foetus. Pre-eclampsia has been intensively investigated for over 50 years, and yet the causes are largely unknown. Despite a large body of data, it is still unknown exactly which mechanisms regulate trophoblast invasion. An effective animal model may be crucial to understanding the underlying causes of pre-eclampsia. A canine model is a proposed improvement on the current efforts to investigate disorders of shallow trophoblast invasion throughout gestation and to improve understanding of the factors that regulate trophoblast invasion. The objectives of this research were to elucidate and compare cellular and molecular similarities between normal canine trophoblasts with those from recently published reports on pre-eclampsia in women.

  2. Silencing of Paternally Expressed Gene 10 Inhibits Trophoblast Proliferation and Invasion

    PubMed Central

    Chen, Haiying; Sun, Manni; Liu, Jing; Tong, Chunxiao; Meng, Tao

    2015-01-01

    Paternally expressed gene 10 (PEG10) is an imprinted and monoallelic expressed gene. Previous study using a knockout mouse model revealed a crucial role of PEG10 in placental development, yet the exact function of PEG10 during placentation remains to be elucidated. In this study, denuded chorionic villi were prepared from first trimester human placentas, and transduced with PEG10 small interference RNA (siRNA) or non-targeting control sequence by lentiviral infection. Immunohistochemical staining revealed that silencing of PEG10 in the chorionic villous explants resulted in reduced immune-reactivity to CK7, Ki67 and integrin α5, implying that silencing of PEG10 impaired the proliferation of villous trophoblasts and may interfere with the activity of extravillous trophoblasts. We further investigated the role of PEG10 in the proliferation, migration and invasion of JEG-3 trophoblast cell line and the primary chorionic villous cells. PEG10-silenced JEG-3 cells and primary chorionic villous cells displayed a reduced proliferation rate and impaired invasiveness in vitro. Silencing of PEG10 in trophoblast cells led to upregulated expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) as well as downregulated expression of matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, knockdown of TIMP-1 reversed the suppressed invasiveness of PEG10 siRNA-transduced JEG-3 cells. In conclusion, our study demonstrates that PEG10 plays an important role in trophoblast proliferation and promotes trophoblast invasion through TIMP-1. PMID:26680220

  3. Unusual Presentation of Hypothyroidism in a Pregnant Woman, Mimicking Gestational Trophoblastic Neoplasm

    PubMed Central

    Aminimoghaddam, Soheila; Mazloomi, Maryam; Rahimi, Maryam

    2016-01-01

    Hypothyroidism is a common health issue worldwide with varying clinical manifestations. We report a woman who experienced an incomplete abortion and undiagnosed hypothyroidism who was referred to the oncologist with the suspicion of metastatic gestational trophoblastic neoplasm (GTN). A 29-year-old woman with incomplete abortion was referred to an oncologist for possible GTN due to persistent active vaginal bleeding, an elevated beta human chorionic gonadotropin (hCG), abnormal cervical inspection exam, abnormal liver function tests, ovarian enlargement, ascites, and a pleural effusion. She was found to have hypothyroidism in further work-up. She was managed with thyroid hormone replacement therapy and her condition improved after 6 weeks. Complete resolution of the ovarian mass and pericardial and pleural effusion was achieved. This case describes an important experience; hypothyroidism should be considered in the differential diagnosis of any woman with an incomplete abortion presenting with an ovarian mass. Evaluation and correct diagnosis are important to prevent mismanagement. PMID:27034864

  4. MTA3 regulates CGB5 and Snail genes in trophoblast

    SciTech Connect

    Chen, Ying; Miyazaki, Jun; Nishizawa, Haruki; Kurahashi, Hiroki; Leach, Richard; Wang, Kai

    2013-04-19

    Highlights: •Impaired MTA3, raised CGB5 and Snail expression are associated with preeclampsia. •Knock-down of MTA3 causes up-regulation of CGB5 and Snail genes in BeWo cells. •MTA3 occupies CGB5 and Snail gene promoters in BeWo cells. -- Abstract: Secreted by the placental trophoblast, human chorionic gonadotropin (hCG) is an important hormone during pregnancy and is required for the maintenance of pregnancy. Previous studies have shown that dys-regulation of hCG expression is associated with preeclampsia. However, the exact relationship between altered hCG levels and development of preeclampsia is unknown. Metastasis associated protein 3 (MTA3), a chromatin remodeling protein, is abundantly expressed in the placental trophoblasts, but its function is unknown. In breast cancer, MTA3 has been shown to repress the expression of Snail and cell migration. However, whether MTA3 acts similarly in the trophoblast has not been investigated. In the present study, we examined the role of MTA3 in regulating the hCG β-subunit gene (gene name: CGB5) and Snail expression in the trophoblast cell line, BeWo, as well as its relevance to the high hCG expression levels seen in preeclampsia. First, we investigated MTA3 expression in preeclamptic placenta as compared to normal control placenta via gene expression microarray and qRT-PCR and found that MTA3 was significantly down-regulated, whereas both CGB5 and Snail were up-regulated in preeclamptic placenta. Secondly, we knocked down MTA3 gene in trophoblast cell line BeWo and found Snail and hCG were both up-regulated, suggesting that MTA3 represses Snail and hCG gene expression in trophoblasts. Next, we cloned the CGB5 and Snail promoters into the pGL3-basic vector individually and found that silencing of MTA3 by siRNA resulted in an increase of both CGB5 and Snail promoter activities. To confirm that this MTA3 inhibition is a direct effect, we performed a chromatin immune-precipitation (ChIP) assay and found that MTA3

  5. MTA3 regulates CGB5 and Snail genes in trophoblast.

    PubMed

    Chen, Ying; Miyazaki, Jun; Nishizawa, Haruki; Kurahashi, Hiroki; Leach, Richard; Wang, Kai

    2013-04-19

    Secreted by the placental trophoblast, human chorionic gonadotropin (hCG) is an important hormone during pregnancy and is required for the maintenance of pregnancy. Previous studies have shown that dys-regulation of hCG expression is associated with preeclampsia. However, the exact relationship between altered hCG levels and development of preeclampsia is unknown. Metastasis associated protein 3 (MTA3), a chromatin remodeling protein, is abundantly expressed in the placental trophoblasts, but its function is unknown. In breast cancer, MTA3 has been shown to repress the expression of Snail and cell migration. However, whether MTA3 acts similarly in the trophoblast has not been investigated. In the present study, we examined the role of MTA3 in regulating the hCG β-subunit gene (gene name: CGB5) and Snail expression in the trophoblast cell line, BeWo, as well as its relevance to the high hCG expression levels seen in preeclampsia. First, we investigated MTA3 expression in preeclamptic placenta as compared to normal control placenta via gene expression microarray and qRT-PCR and found that MTA3 was significantly down-regulated, whereas both CGB5 and Snail were up-regulated in preeclamptic placenta. Secondly, we knocked down MTA3 gene in trophoblast cell line BeWo and found Snail and hCG were both up-regulated, suggesting that MTA3 represses Snail and hCG gene expression in trophoblasts. Next, we cloned the CGB5 and Snail promoters into the pGL3-basic vector individually and found that silencing of MTA3 by siRNA resulted in an increase of both CGB5 and Snail promoter activities. To confirm that this MTA3 inhibition is a direct effect, we performed a chromatin immune-precipitation (ChIP) assay and found that MTA3 occupied the proximal promoter regions of both Snail and hCG within BeWo cells. Furthermore, we examined MTA3 expression in placental trophoblast by immunohistochemistry and found that MTA3 expression was higher in villous cytotrophoblasts versus

  6. A rare case of combined placental site trophoblastic tumour with mature cystic teratoma and mixed germ cell tumour in the testis.

    PubMed

    Leow, Wei Qiang; Loh, Hwai Liang Alwin; Lee, Lui Shiong; Goh, Chin Hong Ronald

    2015-08-01

    A 20-year-old male presented with persistent right testicular pain. Following ultrasound detection of testicular nodules and biopsy for intraoperative consultation which yielded germ cell tumour, he underwent radical orchidectomy. A predominantly whitish cyst and a lobulated, variegated nodule were identified. Histology showed a mature cystic teratoma with a focus of infiltrative epithelioid cells containing eosinophilic cytoplasm and pleomorphic nuclei, invading ectatic vessel wall associated with fibrinoid change. These cells were positive for cytokeratin, human placental lactogen and inhibin, while negative for Melan-A, p63 and alpha-fetoprotein, consistent with placental site trophoblastic tumor (PSTT). The variegated nodule was a mixed germ cell tumour composed of embryonal carcinoma and immature teratoma. Aside from choriocarcinoma, primary trophoblastic tumors such as PSTT, which are derived from intermediate trophoblasts, are extremely rare in the testis. Aside from a case of pure testicular PSTT, 2 other cases have been described in association with germ cell tumour, of which one is a mature teratoma with PSTT that demonstrated gain of chromosome 12p. The other presented with PSTT in retroperitoneal recurrence of a testicular mixed germ cell tumour. We discussed the features of this tumour in the testis and important differentials in its diagnosis.

  7. Average Gait Differential Image Based Human Recognition

    PubMed Central

    Chen, Jinyan; Liu, Jiansheng

    2014-01-01

    The difference between adjacent frames of human walking contains useful information for human gait identification. Based on the previous idea a silhouettes difference based human gait recognition method named as average gait differential image (AGDI) is proposed in this paper. The AGDI is generated by the accumulation of the silhouettes difference between adjacent frames. The advantage of this method lies in that as a feature image it can preserve both the kinetic and static information of walking. Comparing to gait energy image (GEI), AGDI is more fit to representation the variation of silhouettes during walking. Two-dimensional principal component analysis (2DPCA) is used to extract features from the AGDI. Experiments on CASIA dataset show that AGDI has better identification and verification performance than GEI. Comparing to PCA, 2DPCA is a more efficient and less memory storage consumption feature extraction method in gait based recognition. PMID:24895648

  8. Vasoactive Intestinal Peptide modulates trophoblast-derived cell line function and interaction with phagocytic cells through autocrine pathways

    PubMed Central

    Vota, Daiana; Paparini, Daniel; Hauk, Vanesa; Toro, Ayelén; Merech, Fatima; Varone, Cecilia; Ramhorst, Rosanna; Pérez Leirós, Claudia

    2016-01-01

    Trophoblast cells migrate and invade the decidual stroma in a tightly regulated process to maintain immune homeostasis at the maternal-placental interface during the first weeks of pregnancy. Locally synthesized factors modulate trophoblast cell function and their interaction with maternal leukocytes to promote the silent clearance of apoptotic cells. The vasoactive intestinal peptide (VIP) is a pleiotropic polypeptide with trophic and anti-inflammatory effects in murine pregnancy models. We explored the effect of VIP on two human first trimester trophoblast cell lines, particularly on their migration, invasiveness and interaction with phagocytic cells, and the signalling and regulatory pathways involved. We found that VIP enhanced trophoblast cell migration and invasion through the activation of high affinity VPAC receptors and PKA-CRE signalling pathways. VIP knocked-down trophoblast cells showed reduced migration in basal and leukemic inhibitor factor (LIF)-elicited conditions. In parallel, VIP-silenced trophoblast cells failed to induce the phagocytosis of apoptotic bodies and the expression of immunosuppressant markers by human monocytes. Our results suggest that VIP-mediated autocrine pathways regulate trophoblast cell function and contribute to immune homeostasis maintenance at placentation and may provide new clues for therapeutic intervention in pregnancies complicated by defective deep placentation. PMID:27212399

  9. Vasoactive Intestinal Peptide modulates trophoblast-derived cell line function and interaction with phagocytic cells through autocrine pathways.

    PubMed

    Vota, Daiana; Paparini, Daniel; Hauk, Vanesa; Toro, Ayelén; Merech, Fatima; Varone, Cecilia; Ramhorst, Rosanna; Pérez Leirós, Claudia

    2016-01-01

    Trophoblast cells migrate and invade the decidual stroma in a tightly regulated process to maintain immune homeostasis at the maternal-placental interface during the first weeks of pregnancy. Locally synthesized factors modulate trophoblast cell function and their interaction with maternal leukocytes to promote the silent clearance of apoptotic cells. The vasoactive intestinal peptide (VIP) is a pleiotropic polypeptide with trophic and anti-inflammatory effects in murine pregnancy models. We explored the effect of VIP on two human first trimester trophoblast cell lines, particularly on their migration, invasiveness and interaction with phagocytic cells, and the signalling and regulatory pathways involved. We found that VIP enhanced trophoblast cell migration and invasion through the activation of high affinity VPAC receptors and PKA-CRE signalling pathways. VIP knocked-down trophoblast cells showed reduced migration in basal and leukemic inhibitor factor (LIF)-elicited conditions. In parallel, VIP-silenced trophoblast cells failed to induce the phagocytosis of apoptotic bodies and the expression of immunosuppressant markers by human monocytes. Our results suggest that VIP-mediated autocrine pathways regulate trophoblast cell function and contribute to immune homeostasis maintenance at placentation and may provide new clues for therapeutic intervention in pregnancies complicated by defective deep placentation. PMID:27212399

  10. Human myostatin negatively regulates human myoblast growth and differentiation.

    PubMed

    McFarlane, Craig; Hui, Gu Zi; Amanda, Wong Zhi Wei; Lau, Hiu Yeung; Lokireddy, Sudarsanareddy; Xiaojia, Ge; Mouly, Vincent; Butler-Browne, Gillian; Gluckman, Peter D; Sharma, Mridula; Kambadur, Ravi

    2011-07-01

    Myostatin, a member of the transforming growth factor-β superfamily, has been implicated in the potent negative regulation of myogenesis in murine models. However, little is known about the mechanism(s) through which human myostatin negatively regulates human skeletal muscle growth. Using human primary myoblasts and recombinant human myostatin protein, we show here that myostatin blocks human myoblast proliferation by regulating cell cycle progression through targeted upregulation of p21. We further show that myostatin regulates myogenic differentiation through the inhibition of key myogenic regulatory factors including MyoD, via canonical Smad signaling. In addition, we have for the first time demonstrated the capability of myostatin to regulate the Notch signaling pathway during inhibition of human myoblast differentiation. Treatment with myostatin results in the upregulation of Hes1, Hes5, and Hey1 expression during differentiation; moreover, when we interfere with Notch signaling, through treatment with the γ-secretase inhibitor L-685,458, we find enhanced myotube formation despite the presence of excess myostatin. Therefore, blockade of the Notch pathway relieves myostatin repression of differentiation, and myostatin upregulates Notch downstream target genes. Immunoprecipitation studies demonstrate that myostatin treatment of myoblasts results in enhanced association of Notch1-intracellular domain with Smad3, providing an additional mechanism through which myostatin targets and represses the activity of the myogenic regulatory factor MyoD. On the basis of these results, we suggest that myostatin function and mechanism of action are very well conserved between species, and that myostatin regulation of postnatal myogenesis involves interactions with numerous downstream signaling mediators, including the Notch pathway. PMID:21508334

  11. Differential network analysis in human cancer research.

    PubMed

    Gill, Ryan; Datta, Somnath; Datta, Susmita

    2014-01-01

    A complex disease like cancer is hardly caused by one gene or one protein singly. It is usually caused by the perturbation of the network formed by several genes or proteins. In the last decade several research teams have attempted to construct interaction maps of genes and proteins either experimentally or reverse engineer interaction maps using computational techniques. These networks were usually created under a certain condition such as an environmental condition, a particular disease, or a specific tissue type. Lately, however, there has been greater emphasis on finding the differential structure of the existing network topology under a novel condition or disease status to elucidate the perturbation in a biological system. In this review/tutorial article we briefly mention some of the research done in this area; we mainly illustrate the computational/statistical methods developed by our team in recent years for differential network analysis using publicly available gene expression data collected from a well known cancer study. This data includes a group of patients with acute lymphoblastic leukemia and a group with acute myeloid leukemia. In particular, we describe the statistical tests to detect the change in the network topology based on connectivity scores which measure the association or interaction between pairs of genes. The tests under various scores are applied to this data set to perform a differential network analysis on gene expression for human leukemia. We believe that, in the future, differential network analysis will be a standard way to view the changes in gene expression and protein expression data globally and these types of tests could be useful in analyzing the complex differential signatures.

  12. Chemoresistant gestational trophoblastic neoplasia: a case report.

    PubMed

    Cp, Sudha; M, Sahana

    2014-07-01

    Gestational trophoblastic neoplasia (GTN) is a disease of women in reproductive age. It is one of the most chemotherapy responsive and highly curable cancer. It is diagnosed when there is clinical, radiologic, pathologic, and/or hormonal evidence of persistent or relapsed gestational trophoblastic disease. In most instances, it is cured by surgical evacuation of the uterus. If persistent, it is treated with chemotherapy which provides response in >90% of the cases. In the unresponsive persistent cases and if the women has completed her child bearing, hysterectomy is generally recommended. Here, we report a rare case of chemoresistant GTN which was confirmed to be placental-site trophoblastic tumour (PSTT) on biopsy.

  13. Use of sonar in diagnosis and management of invasive gestational trophoblastic tumors.

    PubMed

    Tsai, W S

    1974-01-01

    Use of sonar in differential diagnosis between normal and molar pregnancies is well established. In this study, invasive trophoblastic tumors were investigated. Intrauterine masses were demonstrated sonographically in all 4 cases, As well as H.C.G. assay and chest X-ray, sonographic follow-up also seems to be of value in evaluating the effectiveness of the chemotherapy.

  14. Drugs Approved for Gestational Trophoblastic Disease

    Cancer.gov

    This page lists cancer drugs approved by the Food and Drug Administration (FDA) for gestational trophoblastic disease. The list includes generic names and brand names. The drug names link to NCI's Cancer Drug Information summaries.

  15. NADPH-diaphorase activity and nitric oxide synthase isoforms in the trophoblast of Calomys callosus

    PubMed Central

    MORAES, NECI; ZAGO, DOUGLAS; GAGIOTI, SONIA; HOSHIDA, MARA SANDRA; BEVILACQUA, ESTELA

    2001-01-01

    The pattern of expression of a variety of placental nitric oxide synthase isoforms has contributed to elucidating the regulatory mechanisms of nitric oxide (NO) synthesis during gestation. The maintenance of vascular tone, attenuation of vasoconstriction, prevention of platelet and leukocyte adhesion to the trophoblast surface, and possible participation in uterine blood flow seem to be the main functions of NO generated at the fetal-maternal interface in humans and mice. Extending this knowledge to other rodent species commonly used as laboratory animals, in this study we focus on NADPH-diaphorase activity and the distribution of nitric oxide synthase isoforms (NOS) in the trophoblast cells of Calomys callosus during different phases of pregnancy. NADPH-diaphorase activity was evaluated cytochemically and the presence of NOS isoforms detected by immunohistochemistry. These techniques were performed on pre- and postimplantation embryos in situ and in vitro, as well as in placentae on d 14 and 18 of pregnancy. Neither NADPH-diaphorase activity nor inducible or endothelial NOS isoforms were found in pre-implanting embryos except after culturing for at least 48 h, when some of the embryonic cells were positive for the diaphorase reaction. On d 6·5 of pregnancy, trophoblast cells showed intense diaphorase activity both in situ and under in vitro conditions. A positive reaction was also found in the different placental trophoblast cells on d 14 and 18 of pregnancy. The inducible NOS (iNOS) isoform, but not the endothelial isoform, was immunodetected in trophoblast cells from the placenta and from postimplantation embryos in situ and under in vitro conditions. These results strongly suggest the production of NO by the iNOS isoform in the trophoblast of Calomys callosus after embryo implantation. The data also emphasise a possible role for the trophoblast in producing and releasing cytotoxic molecules at the fetal-maternal interface. PMID:11327206

  16. Deep trophoblast invasion and spiral artery remodelling in the placental bed of the chimpanzee.

    PubMed

    Pijnenborg, R; Vercruysse, L; Carter, A M

    2011-05-01

    Deep trophoblast invasion is usually considered to be a unique feature of human placentation as compared to other primates. Because of the occasional occurrence of preeclampsia in great apes, which in the human is associated with impaired deep invasion, this uniqueness may be questioned. The availability of two well-documented pregnant chimpanzee uteri in the Hubrecht Collection (Museum für Naturkunde, Berlin) allowed us to evaluate the extent of trophoblast invasion in this species. By adjusting currently used protocols, we obtained successful immunohistochemical staining for cytokeratin and α-actin, as well as Ulex europaeus agglutinin 1 (UEA1) lectin staining, in this archival material. In both specimens interstitial trophoblast invasion had occurred in both decidua and myometrium. Because of a lack of published data on fetal growth for this species, fetal sizes (7cm and 13cm) could not be strictly related to gestational ages and thus be compared with the time-course of human trophoblast invasion. However, since the earlier specimen did not show any endovascular trophoblast invasion in spiral arteries - in contrast to pregnant human uteri with equivalent fetal sizes - endovascular migration seems to begin at a different gestational age in the chimpanzee. In the later specimen endovascular trophoblast was associated with spiral artery remodelling in the inner myometrium, and this invasion was extended to include a radial artery, which at that stage still showed relatively intact vascular smooth muscle and elastic lamina. We conclude that invasion depth and spiral artery remodelling are basically similar in chimpanzees and humans, although the seemingly different time of onset may have implications for uteroplacental oxygen supply and fetal development.

  17. Glioma-associated Oncogene 2 Is Essential for Trophoblastic Fusion by Forming a Transcriptional Complex with Glial Cell Missing-a.

    PubMed

    Tang, Chao; Tang, Lanfang; Wu, Xiaokai; Xiong, Wenyi; Ruan, Hongfeng; Hussain, Musaddique; Wu, Junsong; Zou, Chaochun; Wu, Ximei

    2016-03-11

    Cell-cell fusion of human villous trophoblasts, referred to as a process of syncytialization, acts as a prerequisite for the proper development and functional maintenance of the human placenta. Given the fact that the main components of the Hedgehog signaling pathway are expressed predominantly in the syncytial layer of human placental villi, in this study, we investigated the potential roles and underlying mechanisms of Hedgehog signaling in trophoblastic fusion. Activation of Hedgehog signaling by a variety of approaches robustly induced cell fusion and the expression of syncytial markers, whereas suppression of Hedgehog signaling significantly attenuated cell fusion and the expression of syncytial markers in both human primary cytotrophoblasts and trophoblast-like BeWo cells. Moreover, among glioma-associated oncogene (GLI) family transcriptional factors in Hedgehog signaling, knockdown of GLI2 but not GLI1 and GLI3 significantly attenuated Hedgehog-induced cell fusion, whereas overexpression of the GLI2 activator alone was sufficient to induce cell fusion. Finally, GLI2 not only stabilized glial cell missing-a, a pivotal transcriptional factor for trophoblastic syncytialization, but also formed a transcriptional heterodimer with glial cell missing-a to transactivate syncytin-1, a trophoblastic fusogen, and promote trophoblastic syncytialization. Taken together, this study uncovered a so far uncharacterized role of Hedgehog/GLI2 signaling in trophoblastic fusion, implicating that Hedgehog signaling, through GLI2, could be required for human placental development and pregnancy maintenance.

  18. Studying the HPV life cycle in 3A trophoblasts and resulting pathophysiology.

    PubMed

    Liu, Yong; You, Hong; Hermonat, Paul L

    2005-01-01

    Human papillomavirus (HPV) productive infection has been long considered to be restricted to the squamous epithelium. However, we have demonstrated that both HPV type 16 (HPV-16) and type 31b (HPV-31b) productively replicate in a trophoblast cell line, 3A. Trophoblasts are an important cell type, as these are the cells of the placenta that directly contact maternal tissues. There are a number of studies that suggest that HPV infection may be found commonly in placental material and might be linked with spontaneous abortions. This chapter describes the preparation and transfection of HPV genomes into 3A cells, suggests ways to analyze viral replication and transcription, and describes how to assay the effect of HPV infection on the ability of the trophoblasts to bind endometrial cells.

  19. [Human pluripotent stem cell and neural differentiation].

    PubMed

    Wataya, Takafumi; Muguruma, Keiko; Sasai, Yoshiki

    2008-10-01

    Recovery of lost brain function is an important issue in medical studies because neurons of the central nervous system (CNS) have poor potential for regeneration. Since few CNS diseases can be treated completely by medicines, regenerative therapy by using stem cells should be studied as a new type of therapeutic intervention. The efficacy of cell replacement therapy in Parkinson's disease has been well investigated. Several studies on fetal tissue transplantation have revealed that quantity and purity of transplanted cells are necessary for recovery of symptoms. SFEB (Serum-free floating culture of embryoid body-like aggregates) method is capable of inducing multi-potential CNS progenitors that can be steered to differentiate into region-specific tissues. On the basis of the existing knowledge of embryology, we have succeeded in the generating of various types of neurons such as telencephalic, cerebeller (Purkinje and granule cells), retinal (photoreceptor cells) and hypothalamic neurons. Application of this culture method to human ES (hES) cells is necessary for clinical purpose: however, poor survival of hES cells in SFEB culture might limit the possibility of using these cells for future medical applications. We found that a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, markedly diminished the dissociation-induced apoptosis of hES cells and enabled the cells to form aggregates in SFEB culture. For both mouse and human ES cells, SFEB culture is a favorable method that can generate large amounts of region-specific neurons. However, stem cell-based therapy continues to face several obstacles. It is important that researchers in the basic sciences and clinical medicine should discuss these problems together to overcome both scientific and ethical issues related to stem cells.

  20. Differentiated regions of human placental cell surface associated with attachment of chorionic villi, phagocytosis of maternal erythrocytes and syncytiotrophoblast repair.

    PubMed

    Clint, J M; Wakely, J; Ockleford, C D

    1979-05-23

    Scanning electron micrographs of human placental cell surface show: (1) Differentiated zones of trophoblast which may be covered by fewer 'microvilli' than the adjacent syncytial cell surface and which extend as a narrow, usually distal protrusion of the chorionic villus. This narrow outgrowth terminates as a fractured end. Presumably since preparations were obtained from therapeutic terminations of pregnancy or Caesarian deliveries these broken ends represent the yield point in the anchoring 'villi' ruptured as a result of surgery. Similar anchoring 'villi' with fractured ends were observed in unfixed material with the use of Nomarski interference contrast microscopy. (2) It appears that, during apparent phagocytic uptake of maternal erythrocytes by syncytiotrophoblast, cell surface lining the forming vacuole still retains an irregular microvillous surface. This observation indicates the potential location of phagocytosis receptors for red blood cells in the placental cell surface. (3) Areas of human placenta which appears to have been damaged and may be undergoing repair exhibit masses of cells with conspicuous microvillar cell surfaces. The origin of these cells is discussed in relation to the usual processes of syncytiotrophoblast formation.

  1. Dysregulated expression of IDO may cause unexplained recurrent spontaneous abortion through suppression of trophoblast cell proliferation and migration.

    PubMed

    Zong, Shanshan; Li, Chunqing; Luo, Chengfeng; Zhao, Xin; Liu, Chunhong; Wang, Kai; Jia, Wenwen; Bai, Mingliang; Yin, Minghong; Bao, Shihua; Guo, Jie; Kang, Jiuhong; Duan, Tao; Zhou, Qian

    2016-01-27

    In pregnancy, trophoblast proliferation, migration and invasion are important for the establishment and maintenance of a successful pregnancy. Impaired trophoblast function has been implicated in recurrent spontaneous abortion (RSA), a major complication of pregnancy, but the underlying mechanisms remain unclear. Indoleamine 2,3-dioxygenase (IDO), an enzyme that catabolizes tryptophan along the kynurenine pathway, is highly expressed in the placenta and serum during pregnancy. Here, we identified a novel function of IDO in regulating trophoblast cell proliferation and migration. We showed that IDO expression and activity were decreased in unexplained recurrent spontaneous abortion (URSA) compared to normal pregnancy. Furthermore, blocking IDO in human trophoblast cells led to reduced proliferation and migration, along with decreased STAT3 phosphorylation and MMP9 expression. Increased STAT3 phosphorylation reversed the IDO knockdown-suppressed trophoblast cell proliferation and migration. In addition, the overexpression of IDO promoted cell proliferation and migration, which could be abolished by the STAT3 signaling inhibitor (AG490). Finally, we observed similar reductions of STAT3 phosphorylation and MMP9 expression in URSA patients. These results indicate that the level of IDO expression may be associated with pregnancy-related complications, such as URSA, by affecting trophoblast cell proliferation and migration via the STAT3 signaling pathway.

  2. Chemoresistant Gestational Trophoblastic Neoplasia: A Case Report

    PubMed Central

    M, Sahana

    2014-01-01

    Gestational trophoblastic neoplasia (GTN) is a disease of women in reproductive age. It is one of the most chemotherapy responsive and highly curable cancer. It is diagnosed when there is clinical, radiologic, pathologic, and/or hormonal evidence of persistent or relapsed gestational trophoblastic disease. In most instances, it is cured by surgical evacuation of the uterus. If persistent, it is treated with chemotherapy which provides response in >90% of the cases. In the unresponsive persistent cases and if the women has completed her child bearing, hysterectomy is generally recommended. Here, we report a rare case of chemoresistant GTN which was confirmed to be placental-site trophoblastic tumour (PSTT) on biopsy. PMID:25177610

  3. Split Immunological Tolerance to Trophoblast

    PubMed Central

    de Mestre, Amanda; Noronha, Leela; Wagner, Bettina; Antczak, Douglas F.

    2010-01-01

    Split immunological tolerance refers to states in which an individual is capable of mounting certain types of immune responses to a particular antigenic challenge, but is tolerant of the same antigen in other compartments of the immune system. This concept is applicable to the immunological relationship between mother and fetus, and particularly relevant in equine pregnancy. In pregnant mares, antibody responses to paternal foreign Major Histocompatibility Complex class I antigens are robust, while anti-paternal cytotoxic T cell responses are diminished compared to those mounted by non-pregnant mares. Here we compared the distribution of the major lymphocyte subsets, the percentage of lymphocytes expressing Interferon Gamma (IFNG) and Interleukin 4 (IL4) and the level of expression of the immunoregulatory transcription factor FOXP3 between pregnant and non-pregnant mares, and between peripheral blood and the endometrium during pregnancy. In a cohort of mares in which peripheral blood lymphocytes were tested during early pregnancy and in the non-pregnant state, there were only slight changes observed during pregnancy. In contrast, comparison of peripheral blood lymphocytes with lymphocytes isolated from the endometrial cups of pregnant mares revealed striking differences in lymphocyte sub-populations. The endometrial cups contained higher numbers of IFNG+ lymphocytes, and lower numbers of lymphocytes expressing IL4. The endometrial cup lymphocytes also had higher numbers of FOXP3+ cells compared to peripheral blood lymphocytes. Taken together, these results strengthen the evidence for a state of split tolerance to trophoblast, and furthermore define sharp differences in immune reactivity during equine pregnancy between peripheral blood lymphocytes and lymphocytes at the maternal-fetal interface. PMID:19876828

  4. Restraint of Trophoblast Invasion of the Uterus by Decorin: Role in Pre-eclampsia.

    PubMed

    Nandi, Pinki; Siddiqui, Mohammad Fyyaz; Lala, Peeyush K

    2016-03-01

    Decorin (DCN) is a leucine-rich, TGF-β binding proteoglycan produced by mesenchymal cells including chondrocytes, dermal fibroblasts, and uterine decidual cells. It exerts multiple physiological functions including collagen fibrillogenesis, myogenesis, angiostasis, and restraining placental invasiveness. We discovered that decidua-derived DCN restrains proliferation, migration, and invasion of extravillous trophoblast (EVT) cells of the human placenta in a TGF-β-independent manner. These functions were differentially mediated by binding of DCN to multiple tyrosine kinase receptors (TKR) including EGFR, IGFR1, and VEGFR2. DCN blocked VEGFR-2 dependent EVT cell migration and endovascular differentiation by inhibiting P38MAPK and ERK1/2 pathways.We identified the avid VEGFR2 binding site in DCN protein as a 12 amino acids (LGTNPLKSSGIE) span in the Leucine-rich-repeat (LRR) 5 region of domain III. A single amino acid mutation (substitution of K to A) of DCN at this site abrogated VEGFR-2- dependent DCN actions. Also, DCN mRNA expression, measured with in situ hybridization, was selectively upregulated in decidual cells in placentas from mothers suffering from pre-eclampsia (PE), whereas the expression levels remained unchanged in chorionic villus mesenchymal cells. This difference between PE and control placentas was present at all gestational ages, indicating the pathogenic role of DCN in PE. We hypothesize that increased blood DCN levels could be a candidate biomarker for PE. PMID:26554635

  5. GABA A receptor π subunit promotes apoptosis of HTR-8/SVneo trophoblastic cells: Implications in preeclampsia

    PubMed Central

    LU, JUNJIE; ZHANG, QIAN; TAN, DONGMEI; LUO, WENPING; ZHAO, HAI; MA, JING; LIANG, HAO; TAN, YI

    2016-01-01

    Gamma-aminobutyric acid (GABA) functions primarily as an inhibitory neurotransmitter through its receptors in the mature central nervous system. The GABA type A receptor π subunit (GABRP) has been identified in the tissues of the reproductive system, particularly in the uterus. In addition, we have previously detected GABRP expression in both human and mouse placentas. To examine the role of GABRP in trophoblastic cell invasion, we constructed a pIRES2-GABRP-EGFP plasmid which was used for the transfection of a human placental cell line derived from first trimester extravillous trophoblasts (HTR-8/SVneo). The number of invaded cells was decreased by GABRP overexpression. Notably, the decrease in the invasive cell number may be due to the increased apoptosis of the HTR-8/SVneo cells following GABRP transfection, which was further confirmed by flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Based on the increased apoptosis of trophoblastic cells in pregnancies complicated by preeclampsia (PE) and the fact that GABRP promotes the apoptosis of trophoblastic cells, we hypothesized that GABRP expression is increased in the placental tissues from patients with PE compared with that in the normal groups and this hypothesis was confirmed by RT-qPCR and immunohistochemical analysis. Taken together, these findings imply that GABRP plays an important role in placentation and this pathway may be a promising molecular target for the development of novel therapeutic strategies for PE. PMID:27221053

  6. Folate ameliorates dexamethasone-induced fetal and placental growth restriction potentially via improvement of trophoblast migration.

    PubMed

    Zhou, Linfang; Zhang, Ai; Wang, Kai; Zhou, Qian; Duan, Tao

    2015-01-01

    Overexposure to prenatal dexamethasone (Dex) leads to small placental and fetal size and the alteration of fetal programming. Folate plays important roles in processes associated with successful pregnancy, including angiogenesis and trophoblast invasion. Placental folate transport is altered with prenatal Dex administration. The purpose of this study was to investigate the protective role of maternal folate administration in placentas exposed to Dex. In vitro, four groups of C57BL/6J pregnant mice were utilized: 1) normal drinking water+Saline injection group (NN); 2) normal drinking water+Dex injection group (ND); 3) drinking water with folate+Saline injection group (FN); and 4) drinking water with folate+Dex injection group (FD). In vivo, four treatment groups of the human extravillous trophoblast HTR-8/SVneo cells were classified: 1) control (NN); 2) Dex treatment (ND); 3) folate treatment (FN); and 4) folate and Dex treatment (FD). The results showed the maternal folate increases the placental size, birth weight, and expression of matrix metalloproteinases 9 (MMP9) in a mice model of Dex overexposure. In human extravillous trophoblast HTR8/SVneo, folate ameliorated the Dex-induced supress of cell migration and improved the expression/activity of MMP2 and MMP9. In conclusion, folate might be a potential therapy intervention to reduce the adverse effects of prenatal Dex exposure partially via improved trophoblast migration.

  7. Development of Non-Viral, Trophoblast-Specific Gene Delivery for Placental Therapy.

    PubMed

    Abd Ellah, Noura; Taylor, Leeanne; Troja, Weston; Owens, Kathryn; Ayres, Neil; Pauletti, Giovanni; Jones, Helen

    2015-01-01

    Low birth weight is associated with both short term problems and the fetal programming of adult onset diseases, including an increased risk of obesity, diabetes and cardiovascular disease. Placental insufficiency leading to intrauterine growth restriction (IUGR) contributes to the prevalence of diseases with developmental origins. Currently there are no therapies for IUGR or placental insufficiency. To address this and move towards development of an in utero therapy, we employ a nanostructure delivery system complexed with the IGF-1 gene to treat the placenta. IGF-1 is a growth factor critical to achieving appropriate placental and fetal growth. Delivery of genes to a model of human trophoblast and mouse placenta was achieved using a diblock copolymer (pHPMA-b-pDMAEMA) complexed to hIGF-1 plasmid DNA under the control of trophoblast-specific promoters (Cyp19a or PLAC1). Transfection efficiency of pEGFP-C1-containing nanocarriers in BeWo cells and non-trophoblast cells was visually assessed via fluorescence microscopy. In vivo transfection and functionality was assessed by direct placental-injection into a mouse model of IUGR. Complexes formed using pHPMA-b-pDMAEMA and CYP19a-923 or PLAC1-modified plasmids induce trophoblast-selective transgene expression in vitro, and placental injection of PLAC1-hIGF-1 produces measurable RNA expression and alleviates IUGR in our mouse model, consequently representing innovative building blocks towards human placental gene therapies.

  8. SETD7 Regulates the Differentiation of Human Embryonic Stem Cells.

    PubMed

    Castaño, Julio; Morera, Cristina; Sesé, Borja; Boue, Stephanie; Bonet-Costa, Carles; Martí, Merce; Roque, Alicia; Jordan, Albert; Barrero, Maria J

    2016-01-01

    The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knock down that might affect the proper silencing of these genes during differentiation.

  9. SETD7 Regulates the Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Castaño, Julio; Morera, Cristina; Sesé, Borja; Boue, Stephanie; Bonet-Costa, Carles; Martí, Merce; Roque, Alicia; Jordan, Albert; Barrero, Maria J.

    2016-01-01

    The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knock down that might affect the proper silencing of these genes during differentiation. PMID:26890252

  10. Mitochondrial respiration regulates adipogenic differentiation of human mesenchymal stem cells.

    PubMed

    Zhang, Yanmin; Marsboom, Glenn; Toth, Peter T; Rehman, Jalees

    2013-01-01

    Human mesenchymal stem cells (MSCs) are adult multipotent stem cells which can be isolated from bone marrow, adipose tissue as well as other tissues and have the capacity to differentiate into a variety of mesenchymal cell types such as adipocytes, osteoblasts and chondrocytes. Differentiation of stem cells into mature cell types is guided by growth factors and hormones, but recent studies suggest that metabolic shifts occur during differentiation and can modulate the differentiation process. We therefore investigated mitochondrial biogenesis, mitochondrial respiration and the mitochondrial membrane potential during adipogenic differentiation of human MSCs. In addition, we inhibited mitochondrial function to assess its effects on adipogenic differentiation. Our data show that mitochondrial biogenesis and oxygen consumption increase markedly during adipogenic differentiation, and that reducing mitochondrial respiration by hypoxia or by inhibition of the mitochondrial electron transport chain significantly suppresses adipogenic differentiation. Furthermore, we used a novel approach to suppress mitochondrial activity using a specific siRNA-based knockdown of the mitochondrial transcription factor A (TFAM), which also resulted in an inhibition of adipogenic differentiation. Taken together, our data demonstrates that increased mitochondrial activity is a prerequisite for MSC differentiation into adipocytes. These findings suggest that metabolic modulation of adult stem cells can maintain stem cell pluripotency or direct adult stem cell differentiation.

  11. Apolipoprotein E promotes lipid accumulation and differentiation in human adipocytes

    SciTech Connect

    Lasrich, Dorothee; Bartelt, Alexander; Grewal, Thomas; Heeren, Joerg

    2015-09-10

    Several studies in mice indicate a role for apolipoprotein E (APOE) in lipid accumulation and adipogenic differentiation in adipose tissue. However, little is yet known if APOE functions in a similar manner in human adipocytes. This prompted us to compare lipid loading and expression of adipocyte differentiation markers in APOE-deficient and control adipocytes using the differentiated human mesenchymal stem cell line hMSC-Tert as well as primary human and mouse adipocytes as model systems. Differentiated hMSC-Tert were stably transduced with or without siRNA targeting APOE while murine adipocytes were isolated from wild type and Apoe knockout mice. Human APOE knockdown hMSC-Tert adipocytes accumulated markedly less triglycerides compared to control cells. This correlated with strongly decreased gene expression levels of adipocyte markers such as adiponectin (ADIPOQ) and fatty acid binding protein 4 (FABP4) as well as the key transcription factor driving adipocyte differentiation, peroxisome proliferator activator receptor gamma (PPARG), in particular the PPARG2 isoform. Similarly, differentiation of murine Apoe-deficient adipocytes was characterized by reduced gene expression of Adipoq, Fabp4 and Pparg. Interestingly, incubation of APOE-deficient hMSC-Tert adipocytes with conditioned media from APOE3-overexpressing adipocytes or APOE-containing Very Low Density Lipoprotein (VLDL) partially restored triglyceride accumulation, but were unable to induce adipocyte differentiation, as judged by expression of adipocyte markers. Taken together, depletion of endogenous APOE in human adipocytes severely impairs lipid accumulation, which is associated with an inability to initiate differentiation. - Highlights: • Immortalized human mesenchymal stem cells were used to study adipocyte development. • Knockdown of endogenous APOE lead to impaired lipid accumulation and adipogenesis. • APOE supplementation partially restored lipid accumulation but not differentiation.

  12. Directed Myogenic Differentiation of Human Induced Pluripotent Stem Cells.

    PubMed

    Shoji, Emi; Woltjen, Knut; Sakurai, Hidetoshi

    2016-01-01

    Patient-derived induced pluripotent stem cells (iPSCs) have opened the door to recreating pathological conditions in vitro using differentiation into diseased cells corresponding to each target tissue. Yet for muscular diseases, a method for reproducible and efficient myogenic differentiation from human iPSCs is required for in vitro modeling. Here, we introduce a myogenic differentiation protocol mediated by inducible transcription factor expression that reproducibly and efficiently drives human iPSCs into myocytes. Delivering a tetracycline-inducible, myogenic differentiation 1 (MYOD1) piggyBac (PB) vector to human iPSCs enables the derivation of iPSCs that undergo uniform myogenic differentiation in a short period of time. This differentiation protocol yields a homogenous skeletal muscle cell population, reproducibly reaching efficiencies as high as 70-90 %. MYOD1-induced myocytes demonstrate characteristics of mature myocytes such as cell fusion and cell twitching in response to electric stimulation within 14 days of differentiation. This differentiation protocol can be applied widely in various types of patient-derived human iPSCs and has great prospects in disease modeling particularly with inherited diseases that require studies of early pathogenesis and drug screening. PMID:25971915

  13. Directed Myogenic Differentiation of Human Induced Pluripotent Stem Cells.

    PubMed

    Shoji, Emi; Woltjen, Knut; Sakurai, Hidetoshi

    2016-01-01

    Patient-derived induced pluripotent stem cells (iPSCs) have opened the door to recreating pathological conditions in vitro using differentiation into diseased cells corresponding to each target tissue. Yet for muscular diseases, a method for reproducible and efficient myogenic differentiation from human iPSCs is required for in vitro modeling. Here, we introduce a myogenic differentiation protocol mediated by inducible transcription factor expression that reproducibly and efficiently drives human iPSCs into myocytes. Delivering a tetracycline-inducible, myogenic differentiation 1 (MYOD1) piggyBac (PB) vector to human iPSCs enables the derivation of iPSCs that undergo uniform myogenic differentiation in a short period of time. This differentiation protocol yields a homogenous skeletal muscle cell population, reproducibly reaching efficiencies as high as 70-90 %. MYOD1-induced myocytes demonstrate characteristics of mature myocytes such as cell fusion and cell twitching in response to electric stimulation within 14 days of differentiation. This differentiation protocol can be applied widely in various types of patient-derived human iPSCs and has great prospects in disease modeling particularly with inherited diseases that require studies of early pathogenesis and drug screening.

  14. Dynamic Transcriptional and Epigenetic Regulation of Human Epidermal Keratinocyte Differentiation

    PubMed Central

    Cavazza, Alessia; Miccio, Annarita; Romano, Oriana; Petiti, Luca; Malagoli Tagliazucchi, Guidantonio; Peano, Clelia; Severgnini, Marco; Rizzi, Ermanno; De Bellis, Gianluca; Bicciato, Silvio; Mavilio, Fulvio

    2016-01-01

    Summary Human skin is maintained by the differentiation and maturation of interfollicular stem and progenitors cells. We used DeepCAGE, genome-wide profiling of histone modifications and retroviral integration analysis, to map transcripts, promoters, enhancers, and super-enhancers (SEs) in prospectively isolated keratinocytes and transit-amplifying progenitors, and retrospectively defined keratinocyte stem cells. We show that >95% of the active promoters are in common and differentially regulated in progenitors and differentiated keratinocytes, while approximately half of the enhancers and SEs are stage specific and account for most of the epigenetic changes occurring during differentiation. Transcription factor (TF) motif identification and correlation with TF binding site maps allowed the identification of TF circuitries acting on enhancers and SEs during differentiation. Overall, our study provides a broad, genome-wide description of chromatin dynamics and differential enhancer and promoter usage during epithelial differentiation, and describes a novel approach to identify active regulatory elements in rare stem cell populations. PMID:27050947

  15. Circulating Cell Free DNA in the Diagnosis of Trophoblastic Tumors

    PubMed Central

    Openshaw, Mark R.; Harvey, Richard A.; Sebire, Neil J.; Kaur, Baljeet; Sarwar, Naveed; Seckl, Michael J.; Fisher, Rosemary A.

    2015-01-01

    Gestational trophoblastic neoplasia (GTN) represents a group of diseases characterized by production of human chorionic gonadotropin (hCG). Since non-gestational tumors may occasionally secrete hCG, histopathological diagnosis is important for appropriate clinical management. However, a histopathological diagnosis is not always available. We therefore investigated the feasibility of extracting cell free DNA (cfDNA) from the plasma of women with GTN for use as a “liquid biopsy” in patients without histopathological diagnosis. cfDNA was prepared from the plasma of 20 women with a diagnosis of GTN and five with hCG-secreting tumors of unknown origin. Genotyping of cfDNA from the patient, genomic DNA from her and her partner and DNA from the tumor tissue identified circulating tumor DNA (ctDNA) (from 9% to 53% of total cfDNA) in 12 of 20 patients with GTN. In one case without a tissue diagnosis, ctDNA enabled a diagnosis of GTN originating in a non-molar conception and in another a diagnosis of non-gestational tumor, based on the high degree of allelic instability and loss of heterozygosity in the ctDNA. In summary ctDNA can be detected in the plasma of women with GTN and can facilitate the diagnosis of both gestational and non-gestational trophoblastic tumors in cases without histopathological diagnosis. PMID:26981554

  16. StarD7 Knockdown Modulates ABCG2 Expression, Cell Migration, Proliferation, and Differentiation of Human Choriocarcinoma JEG-3 Cells

    PubMed Central

    Flores-Martín, Jésica; Rena, Viviana; Márquez, Sebastián; Panzetta-Dutari, Graciela M.; Genti-Raimondi, Susana

    2012-01-01

    Background StAR-related lipid transfer domain containing 7 (StarD7) is a member of the START-domain protein family whose function still remains unclear. Our data from an explorative microarray assay performed with mRNAs from StarD7 siRNA-transfected JEG-3 cells indicated that ABCG2 (ATP-binding cassette sub-family G member 2) was one of the most abundantly downregulated mRNAs. Methodology/Principal Findings Here, we have confirmed that knocking down StarD7 mRNA lead to a decrease in the xenobiotic/lipid transporter ABCG2 at both the mRNA and protein levels (−26.4% and −41%, p<0.05, at 48 h of culture, respectively). Also a concomitant reduction in phospholipid synthesis, bromodeoxyuridine (BrdU) uptake and 3H-thymidine incorporation was detected. Wound healing and transwell assays revealed that JEG-3 cell migration was significantly diminished (p<0.05). Conversely, biochemical differentiation markers such as human chorionic gonadotrophin β-subunit (βhCG) protein synthesis and secretion as well as βhCG and syncytin-1 mRNAs were increased approximately 2-fold. In addition, desmoplakin immunostaining suggested that there was a reduction of intercellular desmosomes between adjacent JEG-3 cells after knocking down StarD7. Conclusions/Significance Altogether these findings provide evidence for a role of StarD7 in cell physiology indicating that StarD7 modulates ABCG2 multidrug transporter level, cell migration, proliferation, and biochemical and morphological differentiation marker expression in a human trophoblast cell model. PMID:22952907

  17. Hepatic Differentiation from Human Ips Cells Using M15 Cells.

    PubMed

    Umeda, Kahoko; Shiraki, Nobuaki; Kume, Shoen

    2016-01-01

    Here, we describe a procedure of human iPS cells differentiation into the definitive endoderm, further into albumin-expressing and albumin-secreting hepatocyte, using M15, a mesonephros- derived cell line. Approximately 90 % of human iPS cells differentiated into SOX17-positive definitive endoderm then approximately 50 % of cells became albumin-positive cells, and secreted ALB protein. This M15 feeder system for endoderm and hepatic differentiation is a simple and efficient method, and useful for elucidating molecular mechanisms for hepatic fate decision, and could represent an attractive approach for a surrogate cell source for pharmaceutical studies.

  18. Directed Differentiation of Human Embryonic Stem Cells into Neural Progenitors.

    PubMed

    Banda, Erin; Grabel, Laura

    2016-01-01

    A variety of protocols have been used to produce neural progenitors from human embryonic stem cells. We have focused on a monolayer culture approach that generates neural rosettes. To initiate differentiation, cells are plated in a serum-free nutrient-poor medium in the presence of a BMP inhibitor. Depending on the cell line used, additional growth factor inhibitors may be required to promote neural differentiation. Long-term culture and addition of the Notch inhibitor DAPT can promote terminal neuronal differentiation. Extent of differentiation is monitored using immunocytochemistry for cell type-specific markers.

  19. Soft matrix supports osteogenic differentiation of human dental follicle cells

    SciTech Connect

    Viale-Bouroncle, Sandra; Voellner, Florian; Moehl, Christoph; Kuepper, Kevin; Brockhoff, Gero; Reichert, Torsten E.; Schmalz, Gottfried; Morsczeck, Christian

    2011-07-08

    Highlights: {yields} Rigid stiffness supports osteogenic differentiation in mesenchymal stem cells (MSCs). {yields} Our study examined stiffness and differentiation of dental follicle cells (DFCs). {yields} Soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs. {yields} DFCs and MSCs react contrarily to soft and rigid surface stiffness. -- Abstract: The differentiation of stem cells can be directed by the grade of stiffness of the developed tissue cells. For example a rigid extracellular matrix supports the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs). However, less is known about the relation of extracellular matrix stiffness and cell differentiation of ectomesenchymal dental precursor cells. Our study examined for the first time the influence of the surface stiffness on the proliferation and osteogenic differentiation of human dental follicle cells (DFCs). Cell proliferation of DFCs was only slightly decreased on cell culture surfaces with a bone-like stiffness. The osteogenic differentiation in DFCs could only be initiated with a dexamethasone based differentiation medium after using varying stiffness. Here, the softest surface improved the induction of osteogenic differentiation in comparison to that with the highest stiffness. In conclusion, different to bone marrow derived MSCs, soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs.

  20. Trophoblast phagocytic program: roles in different placental systems.

    PubMed

    Bevilacqua, Estela; Hoshida, Mara-Sandra; Amarante-Paffaro, Andrea; Albieri-Borges, Andrea; Zago Gomes, Sara

    2010-01-01

    Although not belonging to the class of professional phagocytes, in many species trophoblast cells exhibit intense phagocytic activity. The complete range of physiological functions of trophoblast phagocytosis has not yet been fully characterized. Close association between the trophoblast and nutrition was determined many years ago. Hubrecht (1889) when proposing for the first time the name trophoblast to the external layer of the blastocyst, directly established the nutritive significance of this embryonic layer. Indeed, histotrophic phagocytosis, i.e. the internalization of maternal cells and secreted materials, is considered an important function of the trophoblast before the completion of the placenta. Recently, however, unexpected characteristics of the trophoblast have significantly enhanced our understanding of this process. Roles in acquisition of space for embryo development, in tissue remodeling during implantation and placentation and in defense mechanisms are highlighting how this cellular activity may be relevant for the maternal-fetal relationship beyond its nutritional function.

  1. Metropolitan Professional Sexual Differentiation: Toward a Human Ecological Theory.

    ERIC Educational Resources Information Center

    Johnson, Gordon C., Jr.

    A human ecology theoretical framework for the study of sexual differentiation in metropolitan area professional occupations is presented in this paper. Human ecology theory attempts to describe characteristics of organizations for a population as a whole. Differences in the characteristics of organizations among populations represent the…

  2. Serum-Induced Differentiation of Human Meibomian Gland Epithelial Cells

    PubMed Central

    Sullivan, David A.; Liu, Yang; Kam, Wendy R.; Ding, Juan; Green, Karin M.; Shaffer, Scott A.; Hatton, Mark P.; Liu, Shaohui

    2014-01-01

    Purpose. We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. Methods. Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression. Results. Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells. Conclusions. Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland. PMID:24867579

  3. Expression of heparin-binding EGF-like growth factor in term chorionic villous explants and its role in trophoblast survival

    PubMed Central

    Imudia, Anthony N.; Kilburn, Brian A.; Petkova, Anelia; Edwin, Samuel S.; Romero, Roberto; Armant, D. Randall

    2008-01-01

    Heparin-binding EGF-like growth factor (HBEGF) induces trophoblast extravillous differentiation and prevents apoptosis. These functions are compromised in preeclampsia. Because HBEGF is downregulated in placentas delivered by women with preeclampsia, we have examined its expression and cytoprotective activity in term villous explants. Chorionic villous explants prepared from non-pathological placentas collected by cesarean section at term were cultured at either 20% or 2% O2 and treated with the HBEGF antagonist CRM197 or recombinant HBEGF. Paraffin sections were assayed for trophoblast death, proliferation and HBEGF expression using the TUNEL method, immunohistochemistry for nuclear Ki67 expression and semi-quantitative immunohistochemistry with image analysis, respectively. Trophoblast cell death was increased significantly after 8 h of culture with CRM197 or by culture for 2 h at 2% O2. Exogenous HBEGF prevented cell death due to hypoxia. Proliferative capacity was not affected by culture at either 20% or 2% O2. Contrary to first trimester placenta, term trophoblasts do not elevate HBEGF expression in response to hypoxia. However, low endogenous levels of HBEGF are required to maintain survival. Therefore, HBEGF-mediated signaling significantly reduces trophoblast cell death at term and its deficiency in preeclampsia could negatively impact trophoblast survival. PMID:18691754

  4. The Efficient Derivation of Trophoblast Cells from Porcine In Vitro Fertilized and Parthenogenetic Blastocysts and Culture with ROCK Inhibitor Y-27632

    PubMed Central

    Hou, Dongxia; Su, Min; Li, Xiawei; Li, Zhiying; Yun, Ting; Zhao, Yuhang; Zhang, Manling; Zhao, Lihua; Li, Rongfeng; Yu, Haiquan; Li, Xueling

    2015-01-01

    Trophoblasts (TR) are specialized cells of the placenta and play an important role in embryo implantation. The in vitro culture of trophoblasts provided an important tool to investigate the mechanisms of implantation. In the present study, porcine trophoblast cells were derived from pig in vitro fertilized (IVF) and parthenogenetically activated (PA) blastocysts via culturing in medium supplemented with KnockOut serum replacement (KOSR) and basic fibroblast growth factor (bFGF) on STO feeder layers, and the effect of ROCK (Rho-associated coiled-coil protein kinases) inhibiter Y-27632 on the cell lines culture was tested. 5 PA blastocyst derived cell lines and 2 IVF blastocyst derived cell lines have been cultured more than 20 passages; one PA cell lines reached 110 passages without obvious morphological alteration. The derived trophoblast cells exhibited epithelium-like morphology, rich in lipid droplets, and had obvious defined boundaries with the feeder cells. The cells were histochemically stained positive for alkaline phosphatase. The expression of TR lineage markers, such as CDX2, KRT7, KRT18, TEAD4, ELF5 and HAND1, imprinted genes such as IGF2, PEG1 and PEG10, and telomerase activity related genes TERC and TERF2 were detected by immunofluorescence staining, reverse transcription PCR and quantitative real-time PCR analyses. Both PA and IVF blastocysts derived trophoblast cells possessed the ability to differentiate into mature trophoblast cells in vitro. The addition of Y-27632 improved the growth of both PA and IVF blastocyst derived cell lines and increased the expression of trophoblast genes. This study has provided an alternative highly efficient method to establish trophoblast for research focused on peri-implantation and placenta development in IVF and PA embryos. PMID:26555939

  5. The Efficient Derivation of Trophoblast Cells from Porcine In Vitro Fertilized and Parthenogenetic Blastocysts and Culture with ROCK Inhibitor Y-27632.

    PubMed

    Hou, Dongxia; Su, Min; Li, Xiawei; Li, Zhiying; Yun, Ting; Zhao, Yuhang; Zhang, Manling; Zhao, Lihua; Li, Rongfeng; Yu, Haiquan; Li, Xueling

    2015-01-01

    Trophoblasts (TR) are specialized cells of the placenta and play an important role in embryo implantation. The in vitro culture of trophoblasts provided an important tool to investigate the mechanisms of implantation. In the present study, porcine trophoblast cells were derived from pig in vitro fertilized (IVF) and parthenogenetically activated (PA) blastocysts via culturing in medium supplemented with KnockOut serum replacement (KOSR) and basic fibroblast growth factor (bFGF) on STO feeder layers, and the effect of ROCK (Rho-associated coiled-coil protein kinases) inhibiter Y-27632 on the cell lines culture was tested. 5 PA blastocyst derived cell lines and 2 IVF blastocyst derived cell lines have been cultured more than 20 passages; one PA cell lines reached 110 passages without obvious morphological alteration. The derived trophoblast cells exhibited epithelium-like morphology, rich in lipid droplets, and had obvious defined boundaries with the feeder cells. The cells were histochemically stained positive for alkaline phosphatase. The expression of TR lineage markers, such as CDX2, KRT7, KRT18, TEAD4, ELF5 and HAND1, imprinted genes such as IGF2, PEG1 and PEG10, and telomerase activity related genes TERC and TERF2 were detected by immunofluorescence staining, reverse transcription PCR and quantitative real-time PCR analyses. Both PA and IVF blastocysts derived trophoblast cells possessed the ability to differentiate into mature trophoblast cells in vitro. The addition of Y-27632 improved the growth of both PA and IVF blastocyst derived cell lines and increased the expression of trophoblast genes. This study has provided an alternative highly efficient method to establish trophoblast for research focused on peri-implantation and placenta development in IVF and PA embryos.

  6. Matrix metalloproteinase expression and activity in trophoblast-decidual tissues at organogenesis in CF-1 mouse.

    PubMed

    Fontana, Vanina; Coll, Tamara A; Sobarzo, Cristian M A; Tito, Leticia Perez; Calvo, Juan Carlos; Cebral, Elisa

    2012-10-01

    During early placentation, matrix metalloproteinases (MMPs) play important roles in decidualization, trophoblast migration, invasion, angiogenesis, vascularization and extracellular matrix (ECM) remodeling of the endometrium. The aim of our study was to analyze the localization, distribution and differential expression of MMP-2 and -9 in the organogenic implantation site and to evaluate in vivo and in vitro decidual MMP-2 and -9 activities on day 10 of gestation in CF-1 mouse. Whole extracts for Western blotting of organogenic E10-decidua expressed MMP-2 and -9 isoforms. MMP-2 immunoreactivity was found in a granular and discrete pattern in ECM of mesometrial decidua (MD) near maternal blood vessels and slightly in non-decidualized endometrium (NDE). Immunoexpression of MMP-9 was also detected in NDE, in cytoplasm of decidual cells and ECM of vascular MD, in trophoblastic area and in growing antimesometrial deciduum. Gelatin zymography showed that MMP-9 activity was significantly lower in CM compared to the active form of direct (not cultured) and cultured decidua. The decidual active MMP-9 was significantly higher than the active MMP-2. These results show differential localization, protein expression and enzymatic activation of MMPs, suggesting specific roles for MMP-2 and MMP-9 in decidual and trophoblast tissues related to organogenic ECM remodeling and vascularization during early establishment of mouse placentation. PMID:22714107

  7. Knockdown of TACC3 inhibits trophoblast cell migration and invasion through the PI3K/Akt signaling pathway.

    PubMed

    Zhu, Xiaojun; Cao, Qianqian; Li, Xia; Wang, Zhengping

    2016-10-01

    The insufficient invasion of trophoblasts is known to be correlated with the development of preeclampsia. Transforming acidic coiled‑coil protein 3 (TACC3), a member of the TACC domain family, is important in the regulation of cell differentiation, migration and invasion. However, the role of TACC3 in trophoblast function during placental development remains to be fully elucidated. The present study aimed to determine the expression and function of TACC3 in human placenta and to examine the underlying mechanisms. TACC3 expression was analyzed in preeclamptic placentas using reverse transcription‑quantitative polymerase chain reaction and western blotting. Cell proliferation was determined by the MTT assay, and cell migration and invasion were measured using Transwell assays. The expression levels of TACC3, matrix metalloproteinase (MMP)‑2, MMP‑9, tissue inhibitor of metalloproteinase (TIMP)‑1, TIMP‑2, phosphoinositide 3‑kinase (PI3K), phosphorylated (p)‑PI3K, AKT and p‑AKT were detected by western blotting. The results showed that the expression of TACC3 was downregulated in preeclamptic placentas. The knockdown of TACC3 significantly inhibited HTR8/SVneo cell proliferation, migration and invasion, and inhibited the expression of matrix metalloproteinases. In addition, the knockdown of TACC3 significantly reduced the levels of p‑PI3K and Akt in the HTR8/SVneo cells. Taken together, the results of the present study demonstrated that the knockdown of TACC3 inhibited the migration and invasion of HTR8/SVneo cells through suppression of the PI3K/Akt signaling pathway. Therefore, TACC3 may serve as a novel potential target for treating preeclampsia. PMID:27572091

  8. Transcriptome dynamics during human erythroid differentiation and development.

    PubMed

    Yang, Yadong; Wang, Hai; Chang, Kai-Hsin; Qu, Hongzhu; Zhang, Zhaojun; Xiong, Qian; Qi, Heyuan; Cui, Peng; Lin, Qiang; Ruan, Xiuyan; Yang, Yaran; Li, Yajuan; Shu, Chang; Li, Quanzhen; Wakeland, Edward K; Yan, Jiangwei; Hu, Songnian; Fang, Xiangdong

    2013-01-01

    To explore the mechanisms controlling erythroid differentiation and development, we analyzed the genome-wide transcription dynamics occurring during the differentiation of human embryonic stem cells (HESCs) into the erythroid lineage and development of embryonic to adult erythropoiesis using high throughput sequencing technology. HESCs and erythroid cells at three developmental stages: ESER (embryonic), FLER (fetal), and PBER (adult) were analyzed. Our findings revealed that the number of expressed genes decreased during differentiation, whereas the total expression intensity increased. At each of the three transitions (HESCs-ESERs, ESERs-FLERs, and FLERs-PBERs), many differentially expressed genes were observed, which were involved in maintaining pluripotency, early erythroid specification, rapid cell growth, and cell-cell adhesion and interaction. We also discovered dynamic networks and their central nodes in each transition. Our study provides a fundamental basis for further investigation of erythroid differentiation and development, and has implications in using ESERs for transfusion product in clinical settings.

  9. Gestational and Non-gestational Trophoblastic Disease. Guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF Registry No. 032/049, December 2015)

    PubMed Central

    Tempfer, C.; Horn, L.-C.; Ackermann, S.; Beckmann, M. W.; Dittrich, R.; Einenkel, J.; Günthert, A.; Haase, H.; Kratzsch, J.; Kreissl, M. C.; Polterauer, S.; Ebert, A. D.; Schneider, K. T. M.; Strauss, H. G.; Thiel, F.

    2016-01-01

    Purpose: The aim was to establish an official interdisciplinary guideline, published and coordinated by the German Society of Gynecology and Obstetrics (DGGG). The guideline was developed for use in German-speaking countries. In addition to the Germany Society of Gynecology and Obstetrics, the guideline has also been approved by the Swiss Society of Gynecology and Obstetrics (SGGG) and the Austrian Society of Gynecology and Obstetrics (OEGGG). The aim was to standardize diagnostic procedures and the management of gestational and non-gestational trophoblastic disease in accordance with the principles of evidence-based medicine, drawing on the current literature and the experience of the colleagues involved in compiling the guideline. Methods: This s2k guideline represents the consensus of a representative panel of experts with a range of different professional backgrounds commissioned by the DGGG. Following a review of the international literature and international guidelines on trophoblastic tumors, a structural consensus was achieved in a formalized, multi-step procedure. This was done using uniform definitions, objective assessments, and standardized management protocols. Recommendations: The recommendations of the guideline cover the epidemiology, classification and staging of trophoblastic tumors; the measurement of human chorionic gonadotropin (hCG) levels in serum, and the diagnosis, management, and follow-up of villous trophoblastic tumors (e.g., partial mole, hydatidiform mole, invasive mole) and non-villous trophoblastic tumors (placental site nodule, exaggerated placental site, placental site tumor, epitheloid trophoblastic tumor, and choriocarcinoma). PMID:26941444

  10. Distinct differentiation characteristics of individual human embryonic stem cell lines

    PubMed Central

    Mikkola, Milla; Olsson, Cia; Palgi, Jaan; Ustinov, Jarkko; Palomaki, Tiina; Horelli-Kuitunen, Nina; Knuutila, Sakari; Lundin, Karolina; Otonkoski, Timo; Tuuri, Timo

    2006-01-01

    Background Individual differences between human embryonic stem cell (hESC) lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare their individual differentiation characteristic. Results The cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could be passaged enzymatically only on mouse feeders. However, this was found to lead to chromosomal instability after prolonged culture. All hESC lines expressed the established markers of pluripotent cells as well as several primordial germ cell (PGC) marker genes in a uniform manner. However, the cell lines showed distinct features in their spontaneous differentiation patterns. The embryoid body (EB) formation frequency of FES 30 cell line was significantly lower than that of other lines and cells within the EBs differentiated less readily. Likewise, teratomas derived from FES 30 cells were constantly cystic and showed only minor solid tissue formation with a monotonous differentiation pattern as compared with the other lines. Conclusion hESC lines may differ substantially in their differentiation properties although they appear similar in the undifferentiated state. PMID:16895598

  11. Isolation, identification and differentiation of human embryonic cartilage stem cells.

    PubMed

    Fu, Changhao; Yan, Zi; Xu, Hao; Zhang, Chen; Zhang, Qi; Wei, Anhui; Yang, Xi; Wang, Yi

    2015-07-01

    We isolated human embryonic cartilage stem cells (hECSCs), a novel stem cell population, from the articular cartilage of eight-week-old human embryos. These stem cells demonstrated a marker expression pattern and differentiation potential intermediate to those of human embryonic stem cells (hESCs) and human adult stem cells (hASCs). hECSCs expressed markers associated with both hESCs (OCT4, NANOG, SOX2, SSEA-3 and SSEA-4) and human adult stem cells (hASCs) (CD29, CD44, CD90, CD73 and CD10). These cells also differentiated into adipocytes, osteoblasts, chondrocytes, neurons and islet-like cells under specific inducing conditions. We identified N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) as an inducer of chondrogenic differentiation in hECSCs. Similar results using N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) were obtained for two other types of human embryonic tissue-derived stem cells, human embryonic hepatic stem cells (hEHSCs) and human embryonic amniotic fluid stem cells (hEASCs), both of which exhibited a marker expression pattern similar to that of hECSCs. The isolation of hECSCs and the discovery that N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) induces chondrogenic differentiation in different stem cell populations might aid the development of strategies in tissue engineering and cartilage repair.

  12. DIFFERENTIATING HUMAN FROM ANIMAL ISOLATES OF CRYPTOSPORIDIUM PARVUM

    EPA Science Inventory

    We analyzed 9s Cryptosporidium parvum isolates from humans and animals by a polymerase chain reaction/restriction fragment length polymorphism method based on the thrombospondin-related anonymous protein 2 gene sequence. Used as a molecular marker, this method can differentiate ...

  13. Gossypol-Induced Differentiation in Human Leukemia HL-60 Cells

    PubMed Central

    Wang, Wen-Qing; Li, Rong; Bai, Qing-Xian; Liu, Yu-Hong; Zhang, Wei-Ping; Wang, Juan-Hong; Wang, Zhe; Li, Yuan-Fei; Chen, Xie-Qun; Huang, Gao-Sheng

    2006-01-01

    The main treatment of leukemia is traditional radiochemotherapy, which is associated with serious side effects. In the past twenty years, differentiation was found as an important effective measure to treat leukemia with fewer side effects. Gossypol, a natural compound which has been used as an effective contraceptive drug, has been proposed to be a potent drug to treat leukemia, but the differentiation effect has not been studied. In the present study, we investigated the pro-differentiated effects, in vitro, of gossypol on the classic human myeloid leukemia HL-60 cell line. The effects of gossypol were investigated by using morphological changes, nitroblue tetrazolium (NBT) reduction, surface markers, cell-cycle analysis and Western blot analysis, etc. When HL-60 cells were incubated with low concentrations of gossypol (2-5μM) for 48hr, a prominent G0/G1 arrest was observed. At 96 hr of treatment, 90% of HL-60 cells differentiated, as evidenced by morphological changes, NBT reduction, and increase in cell surface expression of some molecules were detected. This study is the first to identify gossypol’s pro-differentiated effects on the leukemia cell line, and it induced differentiation through the PBK (PDZ-binding kinase)/TOPK (T-LAKcell-originated protein kinase) (PBK/TOPK) pathway. It is concluded that gossypol could induce differentiation in the leukemia HL-60 cells, and it may be a potential therapeutic agent, chemoprevention or chemotherapeutic adjuvant especially in combination drug therapy for leukemia. PMID:23675007

  14. ApoE Receptor 2 mediates trophoblast dysfunction and pregnancy complications induced by antiphospholipid antibodies in mice

    PubMed Central

    Ulrich, Victoria; Gelber, Shari E.; Vukelic, Milena; Sacharidou, Anastasia; Herz, Joachim; Urbanus, Rolf T.; de Groot, Philip G.; Natale, David R.; Harihara, Anirudha; Redecha, Patricia; Abrahams, Vikki M.; Shaul, Philip W.

    2015-01-01

    Objective Pregnancies in women with the antiphospholipid syndrome (APS) are frequently complicated by fetal loss and intrauterine growth restriction (IUGR). How circulating antiphospholipid antibodies (aPL) cause pregnancy complications in APS is poorly understood. We sought to determine if the LDL receptor family member apoE receptor 2 (apoER2) mediates trophoblast dysfunction and pregnancy complications induced by aPL. Methods Placental and trophoblast apoER2 expression was evaluated by immunohistochemistry and immunoblotting. Normal human IgG (NHIgG) and aPL were purified from healthy individuals and APS patients, respectively. The role of apoER2 in aPL-induced changes in trophoblast proliferation, migration and kinase activation was assessed using RNA interference in HTR-8/SVneo cells. The participation of apoER2 in aPL-induced pregnancy loss and IUGR was evaluated in pregnant apoER2+/+ and apoER2−/− mice injected with aPL or NHIgG. Results We found that apoER2 is abundant in human and mouse placental trophoblasts, and in multiple trophoblast-derived cell lines including HTR-8/SVneo cells. ApoER2 and its interaction with the cell surface protein β2-glycoprotein I were required for aPL-induced inhibition of cultured trophoblast proliferation and migration. In parallel, aPL antagonism of Akt kinase activation by EGF in trophoblasts was mediated by apoER2. Furthermore, in a murine passive transfer model of pregnancy complications of APS, apoER2−/− mice were protected from both aPL-induced fetal loss and aPL-induced IUGR. Conclusion ApoER2 plays a major role in the attenuation of trophoblast function by aPL, and the receptor mediates aPL-induced pregnancy complications in vivo in mice. ApoER2-directed interventions can now potentially be developed to combat the pregnancy complications associated with APS. PMID:26474194

  15. Derivation and differentiation of haploid human embryonic stem cells.

    PubMed

    Sagi, Ido; Chia, Gloryn; Golan-Lev, Tamar; Peretz, Mordecai; Weissbein, Uri; Sui, Lina; Sauer, Mark V; Yanuka, Ofra; Egli, Dieter; Benvenisty, Nissim

    2016-04-01

    Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development.

  16. Derivation and differentiation of haploid human embryonic stem cells.

    PubMed

    Sagi, Ido; Chia, Gloryn; Golan-Lev, Tamar; Peretz, Mordecai; Weissbein, Uri; Sui, Lina; Sauer, Mark V; Yanuka, Ofra; Egli, Dieter; Benvenisty, Nissim

    2016-04-01

    Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development. PMID:26982723

  17. Leptin promotes cell proliferation and survival of trophoblastic cells.

    PubMed

    Magariños, María Paula; Sánchez-Margalet, Víctor; Kotler, Mónica; Calvo, Juan Carlos; Varone, Cecilia L

    2007-02-01

    Leptin, the 16-kDa protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta. In the present work, we studied a possible effect of leptin on trophoblastic cell proliferation, survival, and apoptosis. Recombinant human leptin added to JEG-3 and BeWo choriocarcinoma cell lines showed a stimulatory effect on cell proliferation up to 3 and 2.4 times, respectively, measured by (3)H-thymidine incorporation and cell counting. These effects were time and dose dependent. Maximal effect was achieved at 250 ng leptin/ml for JEG-3 cells and 50 ng leptin/ml for BeWo cells. Moreover, by inhibiting endogenous leptin expression with 2 microM of an antisense oligonucleotide (AS), cell proliferation was diminished. We analyzed cell population distribution during the different stages of cell cycle by fluorescence-activated cell sorting, and we found that leptin treatment displaced the cells towards a G2/M phase. We also found that leptin upregulated cyclin D1 expression, one of the key cell cycle-signaling proteins. Since proliferation and death processes are intimately related, the effect of leptin on cell apoptosis was investigated. Treatment with 2 microM leptin AS increased the number of apoptotic cells 60 times, as assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and the caspase-3 activity was increased more than 2 fold. This effect was prevented by the addition of 100 ng leptin/ml. In conclusion, we provide evidence that suggests that leptin is a trophic and mitogenic factor for trophoblastic cells by virtue of its inhibiting apoptosis and promoting proliferation. PMID:17021346

  18. Variable DNA methylation changes during differentiation of human melanoma cells.

    PubMed

    Steigerwald, S D; Pfeifer, G P

    1988-09-01

    The DNA 5-methylcytosine content has been analyzed in the human melanoma cell line M21 at several time points after induction of differentiation by a variety of inducers. 5-Aza-2'-deoxycytidine reduces DNA methylation to about 50% of the control level and this demethylation occurs prior to the establishment of the differentiated phenotype. The DNA synthesis inhibitors cytosine arabinoside, aphidicolin, and hydroxyurea exert different effects on DNA methylation in these cells. Cytosine arabinoside induces an early DNA hypermethylation, which is however reversible and drops to the original level after 24 h. Hydroxyurea induces DNA hypermethylation after a lag period of more than 48 h and the DNA polymerase alpha inhibitor aphidicolin has no effect on the DNA methylation level. Treatment of cells with phorbol 12-myristate 13-acetate, another potent inducer of melanoma cell differentiation, does not result in a change of total DNA methylation over a period of 96 h. These results indicate that differentiation of human melanoma cells can be accompanied by variable changes of the DNA methylation pattern. These changes can be neither generally related to the differentiation process itself nor related to the effects of DNA synthesis inhibition on DNA methylation, but may more likely reflect a direct or indirect particular effect of the inducer on the DNA methylation process.

  19. Integrating human stem cell expansion and neuronal differentiation in bioreactors

    PubMed Central

    Serra, Margarida; Brito, Catarina; Costa, Eunice M; Sousa, Marcos FQ; Alves, Paula M

    2009-01-01

    Background Human stem cells are cellular resources with outstanding potential for cell therapy. However, for the fulfillment of this application, major challenges remain to be met. Of paramount importance is the development of robust systems for in vitro stem cell expansion and differentiation. In this work, we successfully developed an efficient scalable bioprocess for the fast production of human neurons. Results The expansion of undifferentiated human embryonal carcinoma stem cells (NTera2/cl.D1 cell line) as 3D-aggregates was firstly optimized in spinner vessel. The media exchange operation mode with an inoculum concentration of 4 × 105 cell/mL was the most efficient strategy tested, with a 4.6-fold increase in cell concentration achieved in 5 days. These results were validated in a bioreactor where similar profile and metabolic performance were obtained. Furthermore, characterization of the expanded population by immunofluorescence microscopy and flow cytometry showed that NT2 cells maintained their stem cell characteristics along the bioreactor culture time. Finally, the neuronal differentiation step was integrated in the bioreactor process, by addition of retinoic acid when cells were in the middle of the exponential phase. Neurosphere composition was monitored and neuronal differentiation efficiency evaluated along the culture time. The results show that, for bioreactor cultures, we were able to increase significantly the neuronal differentiation efficiency by 10-fold while reducing drastically, by 30%, the time required for the differentiation process. Conclusion The culture systems developed herein are robust and represent one-step-forward towards the development of integrated bioprocesses, bridging stem cell expansion and differentiation in fully controlled bioreactors. PMID:19772662

  20. Dynamic changes in the human methylome during differentiation.

    PubMed

    Laurent, Louise; Wong, Eleanor; Li, Guoliang; Huynh, Tien; Tsirigos, Aristotelis; Ong, Chin Thing; Low, Hwee Meng; Kin Sung, Ken Wing; Rigoutsos, Isidore; Loring, Jeanne; Wei, Chia-Lin

    2010-03-01

    DNA methylation is a critical epigenetic regulator in mammalian development. Here, we present a whole-genome comparative view of DNA methylation using bisulfite sequencing of three cultured cell types representing progressive stages of differentiation: human embryonic stem cells (hESCs), a fibroblastic differentiated derivative of the hESCs, and neonatal fibroblasts. As a reference, we compared our maps with a methylome map of a fully differentiated adult cell type, mature peripheral blood mononuclear cells (monocytes). We observed many notable common and cell-type-specific features among all cell types. Promoter hypomethylation (both CG and CA) and higher levels of gene body methylation were positively correlated with transcription in all cell types. Exons were more highly methylated than introns, and sharp transitions of methylation occurred at exon-intron boundaries, suggesting a role for differential methylation in transcript splicing. Developmental stage was reflected in both the level of global methylation and extent of non-CpG methylation, with hESC highest, fibroblasts intermediate, and monocytes lowest. Differentiation-associated differential methylation profiles were observed for developmentally regulated genes, including the HOX clusters, other homeobox transcription factors, and pluripotence-associated genes such as POU5F1, TCF3, and KLF4. Our results highlight the value of high-resolution methylation maps, in conjunction with other systems-level analyses, for investigation of previously undetectable developmental regulatory mechanisms. PMID:20133333

  1. Trophoblast Major Histocompatibility Complex Class I Expression Is Associated with Immune-Mediated Rejection of Bovine Fetuses Produced by Cloning.

    PubMed

    Rutigliano, Heloisa M; Thomas, Aaron J; Wilhelm, Amanda; Sessions, Benjamin R; Hicks, Brady A; Schlafer, Donald H; White, Kenneth L; Davies, Christopher J

    2016-08-01

    Trophoblast cells from bovine somatic cell nuclear transfer (SCNT) conceptuses express major histocompatibility complex class I (MHC-I) proteins early in gestation, and this may be one cause of the significant first-trimester embryonic mortality observed in these pregnancies. MHC-I homozygous-compatible (n = 9), homozygous-incompatible (n = 8), and heterozygous-incompatible (n = 5) SCNT pregnancies were established. The control group consisted of eight pregnancies produced by artificial insemination. Uterine and placental samples were collected on Day 35 ± 1 of pregnancy, and expression of MHC-I, leukocyte markers, and cytokines were examined by immunohistochemistry. Trophoblast cells from all SCNT pregnancies expressed MHC-I, while trophoblast cells from age-matched control pregnancies were negative for MHC-I expression. Expression of MHC-I antigens by trophoblast cells from SCNT pregnancies was associated with lymphocytic infiltration in the endometrium. Furthermore, MHC-I-incompatible conceptuses, particularly the heterozygous-incompatible ones, induced a more pronounced lymphocytic infiltration than MHC-I-compatible conceptuses. Cells expressing cluster of differentiation (CD) 3, gamma/deltaTCR, and MHC-II were increased in the endometrium of SCNT pregnancies compared to the control group. CD4(+) lymphocytes were increased in MHC-I-incompatible pregnancies compared to MHC-I-compatible and control pregnancies. CD8(+), FOXP3(+), and natural killer cells were increased in MHC-I heterozygous-incompatible SCNT pregnancies compared to homozygous SCNT and control pregnancies.

  2. Cell proliferation potency is independent of FGF4 signaling in trophoblast stem cells derived from androgenetic embryos

    PubMed Central

    OGAWA, Hidehiko; TAKYU, Ryuichi; MORIMOTO, Hiromu; TOEI, Shuntaro; SAKON, Hiroshi; GOTO, Shiori; MORIYA, Shota; KONO, Tomohiro

    2015-01-01

    We previously established trophoblast stem cells from mouse androgenetic embryos (AGTS cells). In this study, to further characterize AGTS cells, we compared cell proliferation activity between trophoblast stem (TS) cells and AGTS cells under fibroblast growth factor 4 (FGF4) signaling. TS cells continued to proliferate and maintained mitotic cell division in the presence of FGF4. After FGF4 deprivation, the cell proliferation stopped, the rate of M-phase cells decreased, and trophoblast giant cells formed. In contrast, some of AGTS cells continued to proliferate, and the rate of M-phase cells did not decrease after FGF4 deprivation, although the other cells differentiated into giant cells. RO3306, an ATP competitor that selectively inhibits CDK1, inhibited the cell proliferation of both TS and AGTS cells. Under RO3306 treatment, cell death was induced in AGTS cells but not in TS cells. These results indicate that RO3306 caused TS cells to shift mitotic cell division to endoreduplication but that some of AGTS cells did not shift to endoreduplication and induced cell death. In conclusion, the paternal genome facilitated the proliferation of trophoblast cells without FGF4 signaling. PMID:26498204

  3. Trophoblast Major Histocompatibility Complex Class I Expression Is Associated with Immune-Mediated Rejection of Bovine Fetuses Produced by Cloning.

    PubMed

    Rutigliano, Heloisa M; Thomas, Aaron J; Wilhelm, Amanda; Sessions, Benjamin R; Hicks, Brady A; Schlafer, Donald H; White, Kenneth L; Davies, Christopher J

    2016-08-01

    Trophoblast cells from bovine somatic cell nuclear transfer (SCNT) conceptuses express major histocompatibility complex class I (MHC-I) proteins early in gestation, and this may be one cause of the significant first-trimester embryonic mortality observed in these pregnancies. MHC-I homozygous-compatible (n = 9), homozygous-incompatible (n = 8), and heterozygous-incompatible (n = 5) SCNT pregnancies were established. The control group consisted of eight pregnancies produced by artificial insemination. Uterine and placental samples were collected on Day 35 ± 1 of pregnancy, and expression of MHC-I, leukocyte markers, and cytokines were examined by immunohistochemistry. Trophoblast cells from all SCNT pregnancies expressed MHC-I, while trophoblast cells from age-matched control pregnancies were negative for MHC-I expression. Expression of MHC-I antigens by trophoblast cells from SCNT pregnancies was associated with lymphocytic infiltration in the endometrium. Furthermore, MHC-I-incompatible conceptuses, particularly the heterozygous-incompatible ones, induced a more pronounced lymphocytic infiltration than MHC-I-compatible conceptuses. Cells expressing cluster of differentiation (CD) 3, gamma/deltaTCR, and MHC-II were increased in the endometrium of SCNT pregnancies compared to the control group. CD4(+) lymphocytes were increased in MHC-I-incompatible pregnancies compared to MHC-I-compatible and control pregnancies. CD8(+), FOXP3(+), and natural killer cells were increased in MHC-I heterozygous-incompatible SCNT pregnancies compared to homozygous SCNT and control pregnancies. PMID:27385783

  4. Polymeric membranes modulate human keratinocyte differentiation in specific epidermal layers.

    PubMed

    Salerno, Simona; Morelli, Sabrina; Giordano, Francesca; Gordano, Amalia; Bartolo, Loredana De

    2016-10-01

    In vitro models of human bioengineered skin substitutes are an alternative to animal experimentation for testing the effects and toxicity of drugs, cosmetics and pollutants. For the first time specific and distinct human epidermal strata were engineered by using membranes and keratinocytes. To this purpose, biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT-PCL were prepared by phase-inversion technique and characterized in order to evaluate their morphological, physico-chemical and mechanical properties. The capability of membranes to modulate keratinocyte differentiation inducing specific interactions in epidermal membrane systems was investigated. The overall results demonstrated that the membrane properties strongly influence the cell morpho-functional behaviour of human keratinocytes, modulating their terminal differentiation, with the creation of specific epidermal strata or a fully proliferative epidermal multilayer system. In particular, human keratinocytes adhered on CHT and CHT-PCL membranes, forming the structure of the epidermal top layers, such as the corneum and granulosum strata, characterized by withdrawal or reduction from the cell cycle and cell proliferation. On the PCL membrane, keratinocytes developed an epidermal basal lamina, with high proliferating cells that stratified and migrated over time to form a complete differentiating epidermal multilayer system. PMID:27371895

  5. Inorganic arsenic impairs differentiation and functions of human dendritic cells

    SciTech Connect

    Macoch, Mélinda; Morzadec, Claudie; Fardel, Olivier; Vernhet, Laurent

    2013-01-15

    Experimental studies have demonstrated that the antileukemic trivalent inorganic arsenic prevents the development of severe pro-inflammatory diseases mediated by excessive Th1 and Th17 cell responses. Differentiation of Th1 and Th17 subsets is mainly regulated by interleukins (ILs) secreted from dendritic cells (DCs) and the ability of inorganic arsenic to impair interferon-γ and IL-17 secretion by interfering with the physiology of DCs is unknown. In the present study, we demonstrate that high concentrations of sodium arsenite (As(III), 1–2 μM) clinically achievable in plasma of arsenic-treated patients, block differentiation of human peripheral blood monocytes into immature DCs (iDCs) by inducing their necrosis. Differentiation of monocytes in the presence of non-cytotoxic concentrations of As(III) (0.1 to 0.5 μM) only slightly impacts endocytotic activity of iDCs or expression of co-stimulatory molecules in cells activated with lipopolysaccharide. However, this differentiation in the presence of As(III) strongly represses secretion of IL-12p70 and IL-23, two major regulators of Th1 and Th17 activities, from iDCs stimulated with different toll-like receptor (TLR) agonists in metalloid-free medium. Such As(III)-exposed DCs also exhibit reduced mRNA levels of IL12A and/or IL12B genes when activated with TLR agonists. Finally, differentiation of monocytes with non-cytotoxic concentrations of As(III) subsequently reduces the ability of activated DCs to stimulate the release of interferon-γ and IL-17 from Th cells. In conclusion, our results demonstrate that clinically relevant concentrations of inorganic arsenic markedly impair in vitro differentiation and functions of DCs, which may contribute to the putative beneficial effects of the metalloid towards inflammatory autoimmune diseases. Highlights: ► Inorganic arsenic impairs differentiation and functions of human dendritic cells (DCs) ► Arsenite (> 1 μM) blocks differentiation of dendritic cells by

  6. Human macrophage differentiation involves an interaction between integrins and fibronectin

    SciTech Connect

    Laouar, A.; Chubb, C.B.H.; Collart, F.; Huberman, E.

    1997-03-14

    The authors have examined the role of integrins and extracellular matrix (ECM) proteins in macrophage differentiation of (1) human HL-60 myeloid leukemia cells induced by phorbol 12-myristate 13-acetate (PMA) and (2) human peripheral blood monocytes induced by either PMA or macrophage-colony stimulating factor (M-CSF). Increased {beta}{sub 1} integrin and fibronectin (FN) gene expression was observed in PMA-treated HL-60 cells and PMA- or M-CSF-treated monocytes, even at a time preceding the manifestation of macrophage markers. Treated HL-60 cells and monocytes also released and deposited FN on the culture dishes. An HL-60 cell variant, HL-525, which is deficient in protein kinase C {beta} (PKC{beta}) and resistant to PMA-induced differentiation, failed to express FN after PMA treatment. Restoration of PKC{beta} resulted in PMA-induced FN gene expression and macrophage differentiation. The macrophage phenotype induced in HL-60 cells or monocytes was attenuated by anti-{beta}{sub 1} integrin or anti-FN MAbs. The authors suggest that macrophage differentiation involves activation of PKC and expression of specific integrins and ECM proteins. The stimulated cells, through their integrins, attach and spread on these substrates by binding to the deposited ECM proteins. This attachment and spreading in turn, through integrin signaling, leads to the macrophage phenotype.

  7. [THE IDENTIFICATION AND DIFFERENTIATION OF BACTERIOPHAGES OF HUMAN PATHOGENIC VIBRIO].

    PubMed

    Gaevskaia, N E; Kudriakova, T A; Makedonova, L D; Kachkina, G V

    2015-04-01

    The issue of identification and differentiation of large group of bacteriophages of human pathogenic vibrio is still unresolved. In research and practical applied purposes it is important to consider characteristics of bacteriophages for establishing similarity and differences between them. The actual study was carried out to analyze specimens of DNA-containing bacteriophages of pathogenic vibrio. The overwhelming majority of them characterized by complicated type of symmetry--phages with double-helical DNA and also phages with mono-helical DNA structure discovered recently in vibrio. For the first time, the general framework of identification and differentiation of bacteriophages of pathogenic vibrio was developed. This achievement increases possibility to establish species assignment of phages and to compare with phages registered in the database. "The collection of bacteriophages and test-strains of human pathogenic vibrio" (No2010620549 of 24.09.210).

  8. Ultrastructural differentiation of the glomerular mesangium from the human metanephros.

    PubMed

    Cotrutz, C; Constantinov, M; Ionescu, C

    1989-01-01

    In section of metanephros tissue obtained from 32 human embryos and fetuses, 8 to 24 weeks old, the mesangial cells differentiate beginning with the 12th week. Some of the cells from the central mass of the metanephros blastema showed endocytosis phenomena characteristic for the adult mesangial cells. In the 13th week the three cellular types from the renal corpuscle individualize, and from the 14th to the 16th week they acquired the adult morphological aspect.

  9. Differentially Expressed Genes and Signature Pathways of Human Prostate Cancer

    PubMed Central

    Myers, Jennifer S.; von Lersner, Ariana K.; Robbins, Charles J.; Sang, Qing-Xiang Amy

    2015-01-01

    Genomic technologies including microarrays and next-generation sequencing have enabled the generation of molecular signatures of prostate cancer. Lists of differentially expressed genes between malignant and non-malignant states are thought to be fertile sources of putative prostate cancer biomarkers. However such lists of differentially expressed genes can be highly variable for multiple reasons. As such, looking at differential expression in the context of gene sets and pathways has been more robust. Using next-generation genome sequencing data from The Cancer Genome Atlas, differential gene expression between age- and stage- matched human prostate tumors and non-malignant samples was assessed and used to craft a pathway signature of prostate cancer. Up- and down-regulated genes were assigned to pathways composed of curated groups of related genes from multiple databases. The significance of these pathways was then evaluated according to the number of differentially expressed genes found in the pathway and their position within the pathway using Gene Set Enrichment Analysis and Signaling Pathway Impact Analysis. The “transforming growth factor-beta signaling” and “Ran regulation of mitotic spindle formation” pathways were strongly associated with prostate cancer. Several other significant pathways confirm reported findings from microarray data that suggest actin cytoskeleton regulation, cell cycle, mitogen-activated protein kinase signaling, and calcium signaling are also altered in prostate cancer. Thus we have demonstrated feasibility of pathway analysis and identified an underexplored area (Ran) for investigation in prostate cancer pathogenesis. PMID:26683658

  10. Epigenetic regulation of human adipose-derived stem cells differentiation.

    PubMed

    Daniunaite, Kristina; Serenaite, Inga; Misgirdaite, Roberta; Gordevicius, Juozas; Unguryte, Ausra; Fleury-Cappellesso, Sandrine; Bernotiene, Eiva; Jarmalaite, Sonata

    2015-12-01

    Adult stem cells have more restricted differentiation potential than embryonic stem cells (ESCs), but upon appropriate stimulation can differentiate into cells of different germ layers. Epigenetic factors, including DNA modifications, take a significant part in regulation of pluripotency and differentiation of ESCs. Less is known about the epigenetic regulation of these processes in adult stem cells. Gene expression profile and location of DNA modifications in adipose-derived stem cells (ADSCs) and their osteogenically differentiated lineages were analyzed using Agilent microarrays. Methylation-specific PCR and restriction-based quantitative PCR were applied for 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) detection in selected loci. The level of DNA modifications in the POU5F1 locus was quantified with deep sequencing. Expression levels of selected genes were assayed by real-time PCR. ADSCs differentiation into osteogenic lineages involved marked changes in both 5mC and 5hmC profiles, but 5hmC changes were more abundant. 5mC losses and 5hmC gains were the main events observed during ADSCs differentiation, and were accompanied by increased expression of TET1 (P = 0.009). In ADSCs, POU5F1 was better expressed than NANOG or SOX2 (P ≤ 0.001). Both 5mC and 5hmC marks were present in the POU5F1 locus, but only hydroxymethylation of specific cytosine showed significant effect on the gene expression. In summary, the data of our study suggest significant involvement of changes in 5hmC profile during the differentiation of human adult stem cells.

  11. Autophagy facilitates organelle clearance during differentiation of human erythroblasts

    PubMed Central

    Betin, Virginie M.S.; Singleton, Belinda K.; Parsons, Stephen F.; Anstee, David J.; Lane, Jon D.

    2013-01-01

    Wholesale depletion of membrane organelles and extrusion of the nucleus are hallmarks of mammalian erythropoiesis. Using quantitative EM and fluorescence imaging we have investigated how autophagy contributes to organelle removal in an ex vivo model of human erythroid differentiation. We found that autophagy is induced at the polychromatic erythroid stage, and that autophagosomes remain abundant until enucleation. This stimulation of autophagy was concomitant with the transcriptional upregulation of many autophagy genes: of note, expression of all ATG8 mammalian paralog family members was stimulated, and increased expression of a subset of ATG4 family members (ATG4A and ATG4D) was also observed. Stable expression of dominant-negative ATG4 cysteine mutants (ATG4BC74A; ATG4DC144A) did not markedly delay or accelerate differentiation of human erythroid cells; however, quantitative EM demonstrated that autophagosomes are assembled less efficiently in ATG4BC74A-expressing progenitor cells, and that cells expressing either mutant accumulate enlarged amphisomes that cannot be degraded. The appearance of these hybrid autophagosome/endosome structures correlated with the contraction of the lysosomal compartment, suggesting that the actions of ATG4 family members (particularly ATG4B) are required for the control of autophagosome fusion with late, degradative compartments in differentiating human erythroblasts. PMID:23508006

  12. Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells

    SciTech Connect

    Atay, Safinur; Gercel-Taylor, Cicek; Kesimer, Mehmet; Taylor, Douglas D.

    2011-05-01

    Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit a density between 1.134 and 1.173 g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165 nm {+-} 0.5 nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of 'normal' early pregnancy.

  13. CDX2 regulates multiple trophoblast genes in bovine trophectoderm CT-1 cells.

    PubMed

    Schiffmacher, Andrew T; Keefer, Carol L

    2013-10-01

    The bovine trophectoderm (TE) undergoes a dramatic morphogenetic transition prior to uterine endometrial attachment. Many studies have documented trophoblast-specific gene expression profiles at various pre-attachment stages, yet genetic interactions within the transitioning TE gene regulatory network are not well characterized. During bovine embryogenesis, transcription factors OCT4 and CDX2 are co-expressed during early trophoblast elongation. In this study, the bovine trophectoderm-derived CT-1 cell line was utilized as a genetic model to examine the roles of CDX2 and OCT4 within the bovine trophoblast gene regulatory network. An RT-PCR screen for TE-lineage transcription factors identified expression of CDX2, ERRB, ID2, SOX15, ELF5, HAND1, and ASCL2. CT-1 cells also express a nuclear-localized, 360 amino acid OCT4 ortholog of the pluripotency-specific human OCT4A. To delineate the roles of CDX2 and OCT4 within the CT-1 gene network, CDX2 and OCT4 levels were manipulated via overexpression and siRNA-mediated knockdown. An increase in CDX2 negatively regulated OCT4 expression, but increased expression of IFNT, HAND1, ASCL2, SOX15, and ELF5. A reduction of CDX2 levels exhibited a reciprocal effect, resulting in decreased expression of IFNT, HAND1, ASCL2, and SOX15. Both overexpression and knockdown of CDX2 increased ETS2 transcription. In contrast to CDX2, manipulation of OCT4 levels only revealed a positive autoregulatory mechanism and upregulation of ASCL2. Together, these results suggest that CDX2 is a core regulator of multiple trophoblast genes within CT-1 cells.

  14. Cystic trophoblastic tumor: a nonaggressive lesion in postchemotherapy resections of patients with testicular germ cell tumors.

    PubMed

    Ulbright, Thomas M; Henley, John D; Cummings, Oscar W; Foster, Richard S; Cheng, Liang

    2004-09-01

    Cystic trophoblastic tumor (CTT) is an uncommon lesion that is usually seen after chemotherapy in patients with testicular germ cell tumors. Its clinical significance has not been well studied. We identified 17 patients with CTT in retroperitoneal lymph node dissections (RPLNDs) after cisplatin-based chemotherapy for testicular germ cell tumors. None had other forms of persistent germ cell tumor except for teratoma, and no patient received additional chemotherapy after RPLND. At the time of RPLND, 7 patients were known to have had normal serum levels of beta-subunit of human chorionic gonadotropin (beta-hCG), whereas 5 had relatively mild elevations (1.6-165 mIU/mL, median, 8.0 mIU/mL). The CTTs consisted of circumscribed, small cysts, usually multifocal, lined by mostly mononucleated trophoblast cells with abundant eosinophilic cytoplasm, often with smudged nuclei and showing only infrequent mitotic figures. Although the epithelial lining was often stratified to several layers in thickness or formed intracystic papillary tufts, solid proliferations of trophoblast cells within the stroma were absent, as were clearly biphasic admixtures of mononucleated and multinucleated trophoblast cells. The cysts were either empty or contained fibrinoid material and were set in a hypocellular, fibrous stroma with adjacent teratoma. Stains for hCG highlighted rare cells. On follow-up of 15 patients, 11 were disease free (mean, 80 months). Three recurred with serum alpha-fetoprotein elevations at 25, 31, and 107 months, respectively, and one with beta-hCG elevation at 2 months. The latter patient, however, also had unresected mediastinal tumor postchemotherapy. We conclude that the finding of CTT in postchemotherapy resections does not warrant additional chemotherapy. Its clinical significance appears similar to that of residual teratoma.

  15. Coexisting epithelioid trophoblastic tumor and placental site trophoblastic tumor of the uterus following a term pregnancy: report of a case and review of literature

    PubMed Central

    Zhang, Xiaofei; zhou, Caiyun; Yu, Minghua; Chen, Xiaoduan

    2015-01-01

    Gestational trophoblastic neoplasms are a group of fetal trophoblastic tumors including choriocarcinomas, epithelioid trophoblastic tumors (ETTs), and placental site trophoblastic tumors (PSTTs). Mixed gestational trophoblastic neoplasms are extremely rare. The existence of mixed gestational trophoblastic neoplasms that were composed of choriocarcinoma and/or PSTT and/or ETT was also reported. Herein, we present a case of uterine mixed gestational trophoblastic neoplasm which is ETT admixed with PSTT, and reviewed 9 cases of mixed gestational trophoblastic neoplasms reported in English literature available. The most common combination was a choriocarcinoma admixed with an ETT and/or PSTT. Mixed gestational trophoblastic neoplasms present in women of reproductive age and rare in postmenopausal, Abnormal vaginal bleeding is the most common presenting symptom, serum β-HCG levels are elevated, mostly below 2500 mIU/ml, the tumor was limited to uterus in 7 cases, the rest of 3 with pulmonary metastases at the time of diagnosis. Mixed gestational trophoblastic neoplasms have more similar clinical features with intermediate trophoblastic tumors (ITTs). Total hysterectomy with lymph node dissection is recommended treatment for mixed gestational trophoblastic neoplasms, and chemotherapy should be used in patients with metastatic disease and with nonmetastatic disease who have adverse prognostic factors. PMID:26261623

  16. Cytoplasmic localization of p21 protects trophoblast giant cells from DNA damage induced apoptosis.

    PubMed

    de Renty, Christelle; DePamphilis, Melvin L; Ullah, Zakir

    2014-01-01

    Proliferating trophoblast stem cells (TSCs) can differentiate into nonproliferating but viable trophoblast giant cells (TGCs) that are resistant to DNA damage induced apoptosis. Differentiation is associated with selective up-regulation of the Cip/Kip cyclin-dependent kinase inhibitors p57 and p21; expression of p27 remains constant. Previous studies showed that p57 localizes to the nucleus in TGCs where it is essential for endoreplication. Here we show that p27 also remains localized to the nucleus during TSC differentiation where it complements the role of p57. Unexpectedly, p21 localized to the cytoplasm where it was maintained throughout both the G- and S-phases of endocycles, and where it prevented DNA damage induced apoptosis. This unusual status for a Cip/Kip protein was dependent on site-specific phosphorylation of p21 by the Akt1 kinase that is also up-regulated in TGCs. Although cytoplasmic p21 is widespread among cancer cells, among normal cells it has been observed only in monocytes. The fact that it also occurs in TGCs reveals that p57 and p21 serve nonredundant functions, and suggests that the role of p21 in suppressing apoptosis is restricted to terminally differentiated cells.

  17. Differentiation and characterization of human facial subcutaneous adipocytes.

    PubMed

    Chon, Su-Hyoun; Pappas, Apostolos

    2015-01-01

    Aging is associated with the loss of facial subcutaneous fat and with increased abdominal subcutaneous fat. Site specific differences in adipocyte phenotype and/or gene expression may play a role in these age-related changes. In this study, we isolated and characterized human facial preadipocytes and investigated distinct metabolic properties such as a differentiation pattern in relation to abdominal preadipocytes. Subcutaneous preadipocytes were isolated from human facial and abdominal skin and cultured in the presence of differentiation factors including rosiglitazone, a known peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist, isobutyl-methyl xanthine (IBMX) and insulin. Differentiation was characterized microscopically and by quantitative real-time PCR. Unexpected superior adipogenic capacity of facial preadipocytes was observed; more facial preadipocytes differentiated in response to rosiglitazone than abdominal preadipocytes and facial preadipocytes retained their ability to differentiate through passage 11 compared with passage 5 for abdominal preadipocytes. Experiments confirmed a reduced lipolysis response in facial versus abdominal adipocytes after exposure to isoproterenol, which was consistent with the reduced β2-adrenergic receptor expression by 60% in the facial cells. The expression of other lipid metabolic gene markers was similar in both facial and abdominal adipocytes with the exception of β3-adrenergic receptor which was only found in abdominal adipose tissue. Gene profiling, by microarray analysis, identified that several HOX genes are robustly reduced in facial adipocytes compared to abdominal adipocytes, suggesting different characteristics between the 2 fat depots. These differences may have implications for development of treatments for facial fat loss during aging.

  18. Differentiation and characterization of human facial subcutaneous adipocytes

    PubMed Central

    Chon, Su-Hyoun; Pappas, Apostolos

    2014-01-01

    Aging is associated with the loss of facial subcutaneous fat and with increased abdominal subcutaneous fat. Site specific differences in adipocyte phenotype and/or gene expression may play a role in these age-related changes. In this study, we isolated and characterized human facial preadipocytes and investigated distinct metabolic properties such as a differentiation pattern in relation to abdominal preadipocytes. Subcutaneous preadipocytes were isolated from human facial and abdominal skin and cultured in the presence of differentiation factors including rosiglitazone, a known peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist, isobutyl-methyl xanthine (IBMX) and insulin. Differentiation was characterized microscopically and by quantitative real-time PCR. Unexpected superior adipogenic capacity of facial preadipocytes was observed; more facial preadipocytes differentiated in response to rosiglitazone than abdominal preadipocytes and facial preadipocytes retained their ability to differentiate through passage 11 compared with passage 5 for abdominal preadipocytes. Experiments confirmed a reduced lipolysis response in facial versus abdominal adipocytes after exposure to isoproterenol, which was consistent with the reduced β2-adrenergic receptor expression by 60% in the facial cells. The expression of other lipid metabolic gene markers was similar in both facial and abdominal adipocytes with the exception of β3-adrenergic receptor which was only found in abdominal adipose tissue. Gene profiling, by microarray analysis, identified that several HOX genes are robustly reduced in facial adipocytes compared to abdominal adipocytes, suggesting different characteristics between the 2 fat depots. These differences may have implications for development of treatments for facial fat loss during aging. PMID:26167398

  19. Differential neural network configuration during human path integration.

    PubMed

    Arnold, Aiden E G F; Burles, Ford; Bray, Signe; Levy, Richard M; Iaria, Giuseppe

    2014-01-01

    Path integration is a fundamental skill for navigation in both humans and animals. Despite recent advances in unraveling the neural basis of path integration in animal models, relatively little is known about how path integration operates at a neural level in humans. Previous attempts to characterize the neural mechanisms used by humans to visually path integrate have suggested a central role of the hippocampus in allowing accurate performance, broadly resembling results from animal data. However, in recent years both the central role of the hippocampus and the perspective that animals and humans share similar neural mechanisms for path integration has come into question. The present study uses a data driven analysis to investigate the neural systems engaged during visual path integration in humans, allowing for an unbiased estimate of neural activity across the entire brain. Our results suggest that humans employ common task control, attention and spatial working memory systems across a frontoparietal network during path integration. However, individuals differed in how these systems are configured into functional networks. High performing individuals were found to more broadly express spatial working memory systems in prefrontal cortex, while low performing individuals engaged an allocentric memory system based primarily in the medial occipito-temporal region. These findings suggest that visual path integration in humans over short distances can operate through a spatial working memory system engaging primarily the prefrontal cortex and that the differential configuration of memory systems recruited by task control networks may help explain individual biases in spatial learning strategies.

  20. Differential neural network configuration during human path integration

    PubMed Central

    Arnold, Aiden E. G. F; Burles, Ford; Bray, Signe; Levy, Richard M.; Iaria, Giuseppe

    2014-01-01

    Path integration is a fundamental skill for navigation in both humans and animals. Despite recent advances in unraveling the neural basis of path integration in animal models, relatively little is known about how path integration operates at a neural level in humans. Previous attempts to characterize the neural mechanisms used by humans to visually path integrate have suggested a central role of the hippocampus in allowing accurate performance, broadly resembling results from animal data. However, in recent years both the central role of the hippocampus and the perspective that animals and humans share similar neural mechanisms for path integration has come into question. The present study uses a data driven analysis to investigate the neural systems engaged during visual path integration in humans, allowing for an unbiased estimate of neural activity across the entire brain. Our results suggest that humans employ common task control, attention and spatial working memory systems across a frontoparietal network during path integration. However, individuals differed in how these systems are configured into functional networks. High performing individuals were found to more broadly express spatial working memory systems in prefrontal cortex, while low performing individuals engaged an allocentric memory system based primarily in the medial occipito-temporal region. These findings suggest that visual path integration in humans over short distances can operate through a spatial working memory system engaging primarily the prefrontal cortex and that the differential configuration of memory systems recruited by task control networks may help explain individual biases in spatial learning strategies. PMID:24808849

  1. Do We Know What Causes Gestational Trophoblastic Disease?

    MedlinePlus

    ... know what causes gestational trophoblastic disease? Normally, the sperm and egg cells each provide a set of ... hydatidiform (HY-duh-TIH-dih-form) moles , a sperm cell fertilizes an abnormal egg cell that has ...

  2. Inconformity of CXCL3 plasma level and placenta expression in preeclampsia and its effect on trophoblast viability and invasion.

    PubMed

    Gui, Shunping; Ni, Shanshan; Jia, Jin; Gong, Yunhui; Gao, Linbo; Zhang, Lin; Zhou, Rong

    2014-01-01

    As a member of the chemokine family, CXCL3 was previously known to participate in many pathophysiological events. However, whether CXCL3 stimulates trophoblast invasion as a key process of preeclampsia pathogenesis remains largely unknown. Therefore, the aim of this study was to investigate this hypothesis and determine the effect of CXCL3 on the first trimester trophoblast. Seventy gravidas were included in this study. ELISA was used to detect CXCL3 plasma levels on preeclampsia and normal pregnant groups. CXCL3 protein and mRNA levels were detected via Western blot and real-time quantitative PCR analysis after immunolocalized in human placenta. Moreover, the CXCL3 function in HTR-8/Svneo was analyzed via WST-1 assay, flow cytometry and invasion test. The plasma CXCL3 level in preeclampsia was significantly higher than that in normal pregnancy. CXCL3 expression was observed in the cytoplasm of placental trophoblasts and vascular endothelium in all groups without significant difference between maternal and fetal sides. In addition, placenta CXCL3 expression in severe preeclampsia was significantly lower than those in normal and mild PE groups. Moreover, exogenous CXCL3 can promote the proliferation and invasion of HTR-8/Svneo; however, its effect on apoptosis remains unclear. In summary, a significant abnormality of plasma CXCL3 level and placental CXCL3 expression was discovered in severe preeclampsia; CXCL3 had a function in trophoblast invasion, which indicated its participation in shallow implantation. Therefore CXCL3 might be involved in severe preeclampsia pathogenesis. PMID:25485631

  3. Pregnancy-associated plasma protein A up-regulated by progesterone promotes adhesion and proliferation of trophoblastic cells.

    PubMed

    Wang, Jiao; Liu, Shuai; Qin, Hua-Min; Zhao, Yue; Wang, Xiao-Qi; Yan, Qiu

    2014-01-01

    Embryo implantation and development is a complex biological process for the establishment of the successful pregnancy. Progesterone is a critical factor in the regulation of embryo adhesion to uterine endometrium and proliferation. Although it has been reported that pregnancy-associated plasma protein A (PAPPA) is increased in pregnant women, the relationship between progesterone and PAPPA, and the effects of PAPPA on embryo adhesion and proliferation are still not clear. The present results showed that the serum level of progesterone and PAPPA was closely correlated by ELISA assay (p<0.01). PAPPA was detected in the villi of early embryo by RT-PCR, Western blot, immunohistochemistry and immunofluorescent staining. Moreover, PAPPA was significantly up-regulated by progesterone in trophoblastic (JAR) cells by Real-time PCR and ELISA assay (p<0.01); while the expression was decreased by the progesterone receptor inhibitor RU486. The down-regulation of PAPPA by siRNA transfection or up-regulation of PAPPA by progesterone treatment significantly decreased or increased the adhesion rate of trophoblastic cells to human uterine epithelial cell lines (RL95-2 and HEC-1A), respectively (p<0.01), as well as the proliferation of trophoblastic cells. In conclusion, PAPPA is up-regulated by progesterone, which promotes the adhesion and proliferation potential of trophoblastic cells. PMID:24817938

  4. Human dermal stem cells differentiate into functional epidermal melanocytes.

    PubMed

    Li, Ling; Fukunaga-Kalabis, Mizuho; Yu, Hong; Xu, Xiaowei; Kong, Jun; Lee, John T; Herlyn, Meenhard

    2010-03-15

    Melanocytes sustain a lifelong proliferative potential, but a stem cell reservoir in glabrous skin has not yet been found. Here, we show that multipotent dermal stem cells isolated from human foreskins lacking hair follicles are able to home to the epidermis to differentiate into melanocytes. These dermal stem cells, grown as three-dimensional spheres, displayed a capacity for self-renewal and expressed NGFRp75, nestin and OCT4, but not melanocyte markers. In addition, cells derived from single-cell clones were able to differentiate into multiple lineages including melanocytes. In a three-dimensional skin equivalent model, sphere-forming cells differentiated into HMB45-positive melanocytes, which migrated from the dermis to the epidermis and aligned singly among the basal layer keratinocytes in a similar fashion to pigmented melanocytes isolated from the epidermis. The dermal stem cells were negative for E-cadherin and N-cadherin, whereas they acquired E-cadherin expression and lost NGFRp75 expression upon contact with epidermal keratinocytes. These results demonstrate that stem cells in the dermis of human skin with neural-crest-like characteristics can become mature epidermal melanocytes. This finding could significantly change our understanding of the etiological factors in melanocyte transformation and pigmentation disorders; specifically, that early epigenetic or genetic alterations leading to transformation may take place in the dermis rather than in the epidermis.

  5. Human brain stem structures respond differentially to noxious heat.

    PubMed

    Ritter, Alexander; Franz, Marcel; Dietrich, Caroline; Miltner, Wolfgang H R; Weiss, Thomas

    2013-01-01

    Concerning the physiological correlates of pain, the brain stem is considered to be one core region that is activated by noxious input. In animal studies, different slopes of skin heating (SSH) with noxious heat led to activation in different columns of the midbrain periaqueductal gray (PAG). The present study aimed at finding a method for differentiating structures in PAG and other brain stem structures, which are associated with different qualities of pain in humans according to the structures that were associated with different behavioral significances to noxious thermal stimulation in animals. Brain activity was studied by functional MRI in healthy subjects in response to steep and shallow SSH with noxious heat. We found differential activation to different SSH in the PAG and the rostral ventromedial medulla (RVM). In a second experiment, we demonstrate that the different SSH were associated with different pain qualities. Our experiments provide evidence that brainstem structures, i.e., the PAG and the RVM, become differentially activated by different SSH. Therefore, different SSH can be utilized when brain stem structures are investigated and when it is aimed to activate these structures differentially. Moreover, percepts of first pain were elicited by shallow SSH whereas percepts of second pain were elicited by steep SSH. The stronger activation of these brain stem structures to SSH, eliciting percepts of second vs. first pain, might be of relevance for activating different coping strategies in response to the noxious input with the two types of SSH.

  6. Regulation of human subcutaneous adipocyte differentiation by EID1.

    PubMed

    Vargas, Diana; Shimokawa, Noriaki; Kaneko, Ryosuke; Rosales, Wendy; Parra, Adriana; Castellanos, Ángela; Koibuchi, Noriyuki; Lizcano, Fernando

    2016-02-01

    Increasing thermogenesis in white adipose tissues can be used to treat individuals at high risk for obesity and cardiovascular disease. The objective of this study was to determine the function of EP300-interacting inhibitor of differentiation (EID1), an inhibitor of muscle differentiation, in the induction of beige adipocytes from adipose mesenchymal stem cells (ADMSCs). Subcutaneous adipose tissue was obtained from healthy women undergoing abdominoplasty. ADMSCs were isolated in vitro, grown, and transfected with EID1 or EID1 siRNA, and differentiation was induced after 48 h by administering rosiglitazone. The effects of EID1 expression under the control of the aP2 promoter (aP2-EID1) were also evaluated in mature adipocytes that were differentiated from ADMSCs. Transfection of EID1 into ADMSCs reduced triglyceride accumulation while increasing levels of thermogenic proteins, such as PGC1α, TFAM, and mitochondrial uncoupling protein 1 (UCP1), all of which are markers of energy expenditure and mitochondrial activity. Furthermore, increased expression of the beige phenotype markers CITED1 and CD137 was observed. Transfection of aP2-EID1 transfection induced the conversion of mature white adipocytes to beige adipocytes, as evidenced by increased expression of PGC1α, UCP1, TFAM, and CITED1. These results indicate that EID1 can modulate ADMSCs, inducing a brown/beige lineage. EID1 may also activate beiging in white adipocytes obtained from subcutaneous human adipose tissue. PMID:26643909

  7. Shear stress induces osteogenic differentiation of human mesenchymal stem cells

    PubMed Central

    Yourek, Gregory; McCormick, Susan M; Mao, Jeremy J; Reilly, Gwendolen C

    2014-01-01

    Aim To determine whether fluid flow-induced shear stress affects the differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) into osteogenic cells. Materials & methods hMSCs cultured with or without osteogenic differentiation medium were exposed to fluid flow-induced shear stress and analyzed for alkaline phosphatase activity and expression of osteogenic genes. Results Immediately following shear stress, alkaline phosphatase activity in osteogenic medium was significantly increased. At days 4 and 8 of culture the mRNA expression of bone morphogenetic protein-2 and osteopontin was significantly higher in hMSCs subjected to shear stress than those cultured in static conditions. However, hMSCs cultured in osteogenic differentiation medium were less responsive in gene expression of alkaline phosphatase and bone morphogenetic protein-2. Conclusion These data demonstrate that shear stress stimulates hMSCs towards an osteoblastic phenotype in the absence of chemical induction, suggesting that certain mechanical stresses may serve as an alternative to chemical stimulation of stem cell differentiation. PMID:20868327

  8. Induction of a trophoblast-like phenotype by hydralazine in the p19 embryonic carcinoma cell line.

    PubMed

    O'Driscoll, Cliona M; Coulter, Jonathan B; Bressler, Joseph P

    2013-03-01

    Chemicals that affect cellular differentiation through epigenetic mechanisms have potential utility in treating a wide range of diseases. Hydralazine decreases DNA methylation in some cell types but its effect on differentiation has not been well explored. After five days of exposure to hydralazine, P19 embryocarcinoma cells displayed a giant cell morphology and were binucleate, indicative of a trophoblast-like morphology. Other trophoblast-like properties included the intermediary filament Troma-1/cytokeratin 8 and the transcription factor Tead4. A decrease in CpG methylation at three sites in the TEAD4 promoter and the B1 repeated sequence was observed. Knocking down expression of Tead4 with siRNA blocked the increase in Troma-1/cytokeratin 8 and over expression of Tead4 induced the expression of Troma-1/cytokeratin 8. Cells treated for 5days with hydralazine were no longer capable of undergoing retinoic acid-mediated neuronal differentiation. An irreversible loss of the pluripotent transcription factor Oct-4 was observed following hydralazine exposure. In summary, hydralazine induces P19 cells to assume a trophoblast-like phenotype by upregulating Tead4 expression through a mechanism involving DNA demethylation.

  9. Transcriptome Sequencing from Diverse Human Populations Reveals Differentiated Regulatory Architecture

    PubMed Central

    Lappalainen, Tuuli; Henn, Brenna M.; Kidd, Jeffrey M.; Yee, Muh-Ching; Grubert, Fabian; Cann, Howard M.; Snyder, Michael; Montgomery, Stephen B.; Bustamante, Carlos D.

    2014-01-01

    Large-scale sequencing efforts have documented extensive genetic variation within the human genome. However, our understanding of the origins, global distribution, and functional consequences of this variation is far from complete. While regulatory variation influencing gene expression has been studied within a handful of populations, the breadth of transcriptome differences across diverse human populations has not been systematically analyzed. To better understand the spectrum of gene expression variation, alternative splicing, and the population genetics of regulatory variation in humans, we have sequenced the genomes, exomes, and transcriptomes of EBV transformed lymphoblastoid cell lines derived from 45 individuals in the Human Genome Diversity Panel (HGDP). The populations sampled span the geographic breadth of human migration history and include Namibian San, Mbuti Pygmies of the Democratic Republic of Congo, Algerian Mozabites, Pathan of Pakistan, Cambodians of East Asia, Yakut of Siberia, and Mayans of Mexico. We discover that approximately 25.0% of the variation in gene expression found amongst individuals can be attributed to population differences. However, we find few genes that are systematically differentially expressed among populations. Of this population-specific variation, 75.5% is due to expression rather than splicing variability, and we find few genes with strong evidence for differential splicing across populations. Allelic expression analyses indicate that previously mapped common regulatory variants identified in eight populations from the International Haplotype Map Phase 3 project have similar effects in our seven sampled HGDP populations, suggesting that the cellular effects of common variants are shared across diverse populations. Together, these results provide a resource for studies analyzing functional differences across populations by estimating the degree of shared gene expression, alternative splicing, and regulatory genetics

  10. Neurosphere Based Differentiation of Human iPSC Improves Astrocyte Differentiation.

    PubMed

    Zhou, Shuling; Szczesna, Karolina; Ochalek, Anna; Kobolák, Julianna; Varga, Eszter; Nemes, Csilla; Chandrasekaran, Abinaya; Rasmussen, Mikkel; Cirera, Susanna; Hyttel, Poul; Dinnyés, András; Freude, Kristine K; Avci, Hasan X

    2016-01-01

    Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers, PAX6 and NESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6 expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers  GFAP and aquaporin 4 (AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC homogeneity and also to increase the differentiation potential of iPSC towards astrocytes. PMID:26798357

  11. Human monocyte differentiation stage affects response to arachidonic acid.

    PubMed

    Escobar-Alvarez, Elizabeth; Pelaez, Carlos A; García, Luis F; Rojas, Mauricio

    2010-01-01

    AA-induced cell death mechanisms acting on human monocytes and monocyte-derived macrophages (MDM), U937 promonocytes and PMA-differentiated U937 cells were studied. Arachidonic acid induced apoptosis and necrosis in monocytes and U937 cells but only apoptosis in MDM and U937D cells. AA increased both types of death in Mycobacterium tuberculosis-infected cells and increased the percentage of TNFalpha+ cells and reduced IL-10+ cells. Experiments blocking these cytokines indicated that AA-mediated death was TNFalpha- and IL-10-independent. The differences in AA-mediated cell death could be explained by high ROS, calpain and sPLA-2 production and activity in monocytes. Blocking sPLA-2 in monocytes and treatment with antioxidants favored M. tuberculosis control whereas AA enhanced M. tuberculosis growth in MDM. Such evidence suggested that AA-modulated effector mechanisms depend on mononuclear phagocytes' differentiation stage.

  12. New insights in the regulation of human B cell differentiation

    PubMed Central

    Schmidlin, Heike; Diehl, Sean A.; Blom, Bianca

    2009-01-01

    B lymphocytes provide the cellular basis of the humoral immune response. All stages of this process, from B cell activation to formation of germinal centers and differentiation into memory B cells or plasma cells, are influenced by extrinsic signals and controlled by transcriptional regulation. Compared to naïve B cells, memory B cells display a distinct expression profile, which allows for their rapid secondary responses. Indisputably, many B cell malignancies result from aberrations in the circuitry controlling B cell function, particularly during the GC reaction. Here we review new insights into memory B cell subtypes, recent literature on transcription factors regulating human B cell differentiation, and further evidence for B cell lymphomagenesis emanating from errors during the GC cell reactions. PMID:19447676

  13. Osteogenic differentiation of human dental papilla mesenchymal cells

    SciTech Connect

    Ikeda, Etsuko; Hirose, Motohiro . E-mail: motohiro-hirose@aist.go.jp; Kotobuki, Noriko; Shimaoka, Hideki; Tadokoro, Mika; Maeda, Masahiko; Hayashi, Yoshiko; Kirita, Tadaaki; Ohgushi, Hajime

    2006-04-21

    We isolated dental papilla from impacted human molar and proliferated adherent fibroblastic cells after collagenase treatment of the papilla. The cells were negative for hematopoietic markers but positive for CD29, CD44, CD90, CD105, and CD166. When the cells were further cultured in the presence of {beta}-glycerophosphate, ascorbic acid, and dexamethasone for 14 days, mineralized areas together with osteogenic differentiation evidenced by high alkaline phosphatase activity and osteocalcin contents were observed. The differentiation was confirmed at both protein and gene expression levels. The cells can also be cryopreserved and, after thawing, could show in vivo bone-forming capability. These results indicate that mesenchymal type cells localize in dental papilla and that the cells can be culture expanded/utilized for bone tissue engineering.

  14. Recombinant human bone morphogenetic protein-9 potently induces osteogenic differentiation of human periodontal ligament fibroblasts.

    PubMed

    Fuchigami, Sawako; Nakamura, Toshiaki; Furue, Kirara; Sena, Kotaro; Shinohara, Yukiya; Noguchi, Kazuyuki

    2016-04-01

    To accomplish effective periodontal regeneration for periodontal defects, several regenerative methods using growth and differentiation factors, including bone morphogenetic proteins (BMPs), have been developed. Bone morphogenetic protein-9 exhibits the most potent osteogenic activity of this growth factor family. However, it is unclear whether exogenous BMP-9 can induce osteogenic differentiation in human periodontal ligament (PDL) fibroblasts. Here, we examined the effects of recombinant human (rh) BMP-9 on osteoblastic differentiation in human PDL fibroblasts in vitro, compared with rhBMP-2. Recombinant human BMP-9 potently induced alkaline phosphatase (ALP) activity, mineralization, and increased expression of runt-related transcription factor-2/core binding factor alpha 1 (RUNX2/CBFA1), osterix, inhibitor of DNA binding/differentiation-1 (ID1), osteopontin, and bone sialoprotein genes, compared with rhBMP-2. The levels of rhBMP-9-induced osterix and ALP mRNA were significantly reduced in activin receptor-like kinase-1 and -2 small interfering RNA (siRNA)-transfected human PDL fibroblasts. Recombinant human BMP-9-induced ALP activity was not inhibited by noggin, in contrast to rhBMP-2 induced ALP activity, which was. Phosphorylation of SMAD1/5/8 in human PDL fibroblasts was induced by addition of rhBMP-9. Recombinant human BMP-9-induced ALP activity was suppressed by SB203580, SP600125, and U0126, which are inhibitors of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2), respectively. Our data suggest that rhBMP-9 is a potent inducer of the differentiation of human PDL fibroblasts into osteoblast-like cells and that this may be mediated by the SMAD and mitogen-activated protein kinase (p38, ERK1/2, and JNK) pathways. PMID:26879145

  15. Human macrophage differentiation involves an interaction between integrins and fibronectin

    SciTech Connect

    Laouar, A.; Chubb, C.B.H.; Collart, F.; Huberman, E.

    1996-11-15

    The authors have examined the role of the {beta}{sub 1} integrin family of adhesion receptors (VLA) and the extracellular matrix protein fibronectin (FN) in macrophage differentiation of (1) human HL-60 myeloid leukemia cells induced by phorbol 12-myristate 13-acetate (PMA) and (2) human peripheral blood monocytes induced by either PMA or macrophage-colony stimulating factor (M=CSF). Increased VLA and FN gene expression was observed as early as 4 h after PMA treatment of HL-60 cells and PMA- or M-CSF-treatment of monocytes, and it preceded the manifestation of macrophage markers. Treated HL-60 cells and monocytes also released and deposited FN on the surface of the tissue culture dishes. An HL-60 cell variant, HL-525, which is deficient in protein kinase C {beta} and resistant to PMA-induced differentiation, exhibited elevated levels of the VLA antigen but failed to express the FN gene. Incubation of HL-525 cells on dishes precoated with exogenous FN resulted in a macrophage differentiation. The macrophage phenotype induced in HL-60 cells, HL-525 cells, or monocytes was attenuated to various degrees by anti-VLA or anti-FN MAbs or by exogenous RGDS, a VLA-binding motif on FN. The authors suggest that macrophage differentiation is initiated by the activation of protein kinase C, which leads to the expression of the integrin, FN and related genes. The integrins mediate cell attachment and spreading on appropriate substrates by binding to deposited extracellular proteins such as FN. This attachment and spreading, in turn, leads to the expression of genes that code for the macrophage functions.

  16. Differential role of ICAM ligands in determination of human memory T cell differentiation

    PubMed Central

    Perez, Omar D; Mitchell, Dennis; Nolan, Garry P

    2007-01-01

    Background Leukocyte Function Antigen-1 (LFA-1) is a primary adhesion molecule that plays important roles in T cell activation, leukocyte recirculation, and trans-endothelial migration. By applying a multivariate intracellular phospho-proteomic analysis, we demonstrate that LFA-1 differentially activates signaling molecules. Results Signal intensity was dependent on both ICAM ligand and LFA-1 concentration. In the presence of CD3 and CD28 stimulation, ICAM-2 and ICAM-3 decreased TGFβ1 production more than ICAM-1. In long-term differentiation experiments, stimulation with ICAM-3, CD3, and CD28 generated IFNγ producing CD4+CD45RO+CD62L-CD11aBrightCD27- cells that had increased expression of intracellular BCL2, displayed distinct chemokine receptor profiles, and exhibited distinct migratory characteristics. Only CD3/CD28 with ICAM-3 generated CD4+CD45RO+CD62L-CD11aBrightCD27- cells that were functionally responsive to chemotaxis and exhibited higher frequencies of cells that signaled to JNK and ERK1/2 upon stimulation with MIP3α. Furthermore, these reports identify that the LFA-1 receptor, when presented with multiple ligands, can result in distinct T cell differentiation states and suggest that the combinatorial integration of ICAM ligand interactions with LFA-1 have functional consequences for T cell biology. Conclusion Thus, the ICAM ligands, differentially modulate LFA-1 signaling in T cells and potentiate the development of memory human T cells in vitro. These findings are of importance in a mechanistic understanding of memory cell differentiation and ex vivo generation of memory cell subsets for therapeutic applications. PMID:17233909

  17. Proteinase expression during differentiation of human osteoclasts in vitro.

    PubMed

    Blair, H C; Sidonio, R F; Friedberg, R C; Khan, N N; Dong, S S

    2000-06-12

    Osteoclasts are macrophage-derived polykaryons that degrade bone in an acidic extracellular space. This differentiation includes expression of proteinases and acid transport proteins, cell fusion, and bone attachment, but the sequence of events is unclear. We studied two proteins expressed at high levels only in the osteoclast, cathepsin K, a thiol proteinase, and tartrate-resistant acid phosphatase (TRAP), and compared this expression with acid transport and bone degradation. Osteoclastic differentiation was studied using human apheresis macrophages cocultured with MG63 osteosarcoma cells, which produce cytokines including RANKL and CSF-1 that mediate efficient osteoclast formation. Immunoreactive cathepsin K appeared at 3-5 days. Cathepsin K activity was seen on bone substrate but not within cells, and cathepsin K increased severalfold during further differentiation and multinucleation from 7 to 14 days. TRAP also appeared at 3-5 d, independently of cell fusion or bone attachment, and TRAP activity reached much higher levels in osteoclasts attached to bone fragments. Two proteinases that occur in the precursor macrophages, cathepsin B, a thiol proteinase related to cathepsin K, and an unrelated lysosomal aspartate proteinase, cathepsin D, were also studied to determine the specificity of the differentiation events. Cathepsin B occurred at all times, but increased two- to threefold in parallel with cathepsin K. Cathepsin D activity did not change with differentiation, and secreted activity was not significant. In situ acid transport measurements showed increased acid accumulation after 7 days either in cells on osteosarcoma matrix or attached to bone, but bone pit activity and maximal acid uptake required 10-14 days. We conclude that TRAP and thiol proteinase expression begin at essentially the same time, and precede cell fusion and bone attachment. However, major increases in acid secretion and proteinases expression continue during cell fusion and bone

  18. FBI-1 Is Overexpressed in Gestational Trophoblastic Disease and Promotes Tumor Growth and Cell Aggressiveness of Choriocarcinoma via PI3K/Akt Signaling.

    PubMed

    Mak, Victor C Y; Wong, Oscar G W; Siu, Michelle K Y; Wong, Esther S Y; Ng, Wai-Yan; Wong, Richard W C; Chan, Ka-Kui; Ngan, Hextan Y S; Cheung, Annie N Y

    2015-07-01

    Human placental trophoblasts can be considered pseudomalignant, with tightly controlled proliferation, apoptosis, and invasiveness. Gestational trophoblastic disease (GTD) represents a family of heterogeneous trophoblastic lesions with aberrant apoptotic and proliferative activities and dysregulation of cell signaling pathways. We characterize the oncogenic effects of factor that binds to the inducer of short transcripts of HIV-1 [FBI-1, alias POZ and Krüppel erythroid myeloid ontogenic factor (POKEMON)/ZBTB7A] in GTD and its role in promoting cell aggressiveness in vitro and tumor growth in vivo. IHC studies showed increased nuclear expression of FBI-1, including hydatidiform moles, choriocarcinoma (CCA), and placental site trophoblastic tumor, in GTD. In JAR and JEG-3 CCA cells, ectopic FBI-1 expression opposed apoptosis through repression of proapoptotic genes (eg, BAK1, FAS, and CASP8). FBI-1 overexpression also promoted Akt activation, as indicated by Akt-pS473 phosphorylation. FBI-1 overexpression promoted mobility and invasiveness of JEG-3 and JAR, but not in the presence of the phosphoinositide 3-kinase inhibitor LY294002. These findings suggest that FBI-1 could promote cell migration and invasion via phosphoinositide 3-kinase/Akt signaling. In vivo, nude mice injected with CCA cells with stable FBI-1 knockdown demonstrated reduced tumor growth compared with that in control groups. These findings suggest that FBI-1 is clinically associated with the progression of, and may be a therapeutic target in, GTD, owing to its diverse oncogenic effects on dysregulated trophoblasts. PMID:26093985

  19. FBI-1 Is Overexpressed in Gestational Trophoblastic Disease and Promotes Tumor Growth and Cell Aggressiveness of Choriocarcinoma via PI3K/Akt Signaling.

    PubMed

    Mak, Victor C Y; Wong, Oscar G W; Siu, Michelle K Y; Wong, Esther S Y; Ng, Wai-Yan; Wong, Richard W C; Chan, Ka-Kui; Ngan, Hextan Y S; Cheung, Annie N Y

    2015-07-01

    Human placental trophoblasts can be considered pseudomalignant, with tightly controlled proliferation, apoptosis, and invasiveness. Gestational trophoblastic disease (GTD) represents a family of heterogeneous trophoblastic lesions with aberrant apoptotic and proliferative activities and dysregulation of cell signaling pathways. We characterize the oncogenic effects of factor that binds to the inducer of short transcripts of HIV-1 [FBI-1, alias POZ and Krüppel erythroid myeloid ontogenic factor (POKEMON)/ZBTB7A] in GTD and its role in promoting cell aggressiveness in vitro and tumor growth in vivo. IHC studies showed increased nuclear expression of FBI-1, including hydatidiform moles, choriocarcinoma (CCA), and placental site trophoblastic tumor, in GTD. In JAR and JEG-3 CCA cells, ectopic FBI-1 expression opposed apoptosis through repression of proapoptotic genes (eg, BAK1, FAS, and CASP8). FBI-1 overexpression also promoted Akt activation, as indicated by Akt-pS473 phosphorylation. FBI-1 overexpression promoted mobility and invasiveness of JEG-3 and JAR, but not in the presence of the phosphoinositide 3-kinase inhibitor LY294002. These findings suggest that FBI-1 could promote cell migration and invasion via phosphoinositide 3-kinase/Akt signaling. In vivo, nude mice injected with CCA cells with stable FBI-1 knockdown demonstrated reduced tumor growth compared with that in control groups. These findings suggest that FBI-1 is clinically associated with the progression of, and may be a therapeutic target in, GTD, owing to its diverse oncogenic effects on dysregulated trophoblasts.

  20. Isolation, characterization, and differentiation of human multipotent dermal stem cells.

    PubMed

    Li, Ling; Fukunaga-Kalabis, Mizuho; Herlyn, Meenhard

    2013-01-01

    Skin, as the body's largest organ, has been extensively used to study adult stem cells. Most previous skin-related studies have focused on stem cells isolated from hair follicles and from keratinocytes. Here we present a protocol to isolate multipotent neural crest stem-like dermis-derived stem cells (termed dermal stem cells or DSCs) from human neonatal foreskins. DSCs grow like neural spheres in human embryonic stem cell medium and gain the ability to self-renew and differentiate into several cell lineages including melanocytes, neuronal cells, Schwann cells, smooth muscle cells, adipocytes, and chondrocytes. These cells express neural crest stem cell markers (NGFRp75 and nestin) as well as an embryonic stem cell marker (OCT4).

  1. Silibinin Regulates Lipid Metabolism and Differentiation in Functional Human Adipocytes

    PubMed Central

    Barbagallo, Ignazio; Vanella, Luca; Cambria, Maria T.; Tibullo, Daniele; Godos, Justyna; Guarnaccia, Laura; Zappalà, Agata; Galvano, Fabio; Li Volti, Giovanni

    2016-01-01

    Silibinin, a natural plant flavonolignan is the main active constituent found in milk thistle (Silybum marianum). It is known to have hepatoprotective, anti-neoplastic effect, and suppresses lipid accumulation in adipocytes. Objective of this study was to investigate the effect of silibinin on adipogenic differentiation and thermogenic capacity of human adipose tissue derived mesenchymal stem cells. Silibinin (10 μM) treatment, either at the beginning or at the end of adipogenic differentiation, resulted in an increase of SIRT-1, PPARα, Pgc-1α, and UCPs gene expression. Moreover, silibinin administration resulted in a decrease of PPARγ, FABP4, FAS, and MEST/PEG1 gene expression during the differentiation, confirming that this compound is able to reduce fatty acid accumulation and adipocyte size. Our data showed that silibinin regulated adipocyte lipid metabolism, inducing thermogenesis and promoting a brown remodeling in adipocyte. Taken together, our findings suggest that silibinin increases UCPs expression by stimulation of SIRT1, PPARα, and Pgc-1α, improved metabolic parameters, decreased lipid mass leading to the formation of functional adipocytes. PMID:26834634

  2. Kinetics of growth and differentiation of cultured human epidermal keratinocytes

    SciTech Connect

    Albers, K.M.

    1985-01-01

    A study was made of the interrelationship between replication and differentiation in cultures of human epidermal keratinocytes. Measures of both parameters were made using newly developed methods to quantify the rate at which keratinocytes replicate and the rate at which they withdraw from the cell cycle. Keratinocyte replication was measured by determining the cell doubling time, labeling index, and cell cycle duration. Cell cycle length was measured using a double label assay that determines the length of time between two successive phases of DNA synthesis. The first DNA synthesis phase was marked by labeling keratinocytes with /sup 14/C-thymidine. At the next round of DNA synthesis, cells were labeled with bromodeoxyuridine, a heavy analog of thymidine. The cell cycle length is given by the time required for the /sup 14/C-labeled DNA to become double labeled. To measure keratinocyte differentiation, the rate at which cells withdraw from the cell cycle was determined. To measure withdrawal, the percentage of cells labeled by a pulse of /sup 14/C-thymidine that failed to undergo a second cycle of DNA synthesis, as measured by bromodeoxyuridine incorporation, was determined. Cells which failed to undergo a second cycle of synthesis were considered to have differentiated and withdrawn from the cell cycle.

  3. Trop-2 overexpression in poorly differentiated endometrial endometrioid adenocarcinoma: Implications for immunotherapy with hRS7, a humanized anti-Trop-2 monoclonal antibody

    PubMed Central

    Bignotti, Eliana; Ravaggi, Antonella; Romani, Chiara; Falchetti, Marcella; Lonardi, Silvia; Facchetti, Fabio; Pecorelli, Sergio; Varughese, Joyce; Cocco, Emiliano; Bellone, Stefania; Schwartz, Peter E.; Rutherford, Thomas J.; Santin, Alessandro D.

    2011-01-01

    Objective We evaluated the expression of human trophoblast cell-surface marker (Trop-2) in endometrial endometrioid carcinoma (EEC) and the potential application of hRS7, a humanized monoclonal anti-Trop-2 antibody, as a therapeutic agent against poorly-differentiated EEC. Methods Trop-2 expression was evaluated by immunohistochemistry in 131 EEC with different degrees of differentiation and 32 normal endometrial controls (NEC). Trop-2 expression was also evaluated by real-time polymerase-chain-reaction (qRT-PCR) and flow cytometry in 3 primary EEC cell lines derived from patients harboring poorly-differentiated EEC. Finally, sensitivity of G3 EEC cell lines to hRS7 antibody-dependent cellular-cytotoxicity (ADCC) was tested in standard 5-hours 51Cr-release assays. Results Trop-2 expression was detected in 126 of 131 (96.2%) EEC samples. Tumor tissues showed markedly increased Trop-2 positivity as compared to NEC (p=0.001). Trop-2 expression was significantly higher in all grades of EEC vs. NEC. G3 tumors displayed significantly stronger Trop-2 immunostaining compared to G1 EEC (p=0.01). High Trop-2 expression by qRT-PCR and flow cytometry was found in one G3 EEC primary cell line (EEC-ARK-1). Unlike Trop-2-negative EEC cell lines, EEC-ARK-1 was found highly sensitive to hRS7-mediated ADCC in vitro (range of killing: 33.9% to 50.6%) (p=0.004). Human serum did not significantly inhibit hRS7-mediated-cytotoxicity against EEC-ARK-1 (p= 0.773). Conclusions Trop-2 is highly expressed in EEC and its expression is significantly higher in poorly-differentiated EEC when compared to well-differentiated EEC. Primary G3 EEC overexpressing Trop-2 are highly sensitive to hRS7-mediated cytotoxicity in vitro. hRS7 may represent a novel therapeutic agent for the treatment of high-grade EEC refractory to standard treatment modalities. PMID:21892093

  4. Characterization of Inhibitor of differentiation (Id) proteins in human cornea.

    PubMed

    Mohan, Rajiv R; Morgan, Brandie R; Anumanthan, Govindaraj; Sharma, Ajay; Chaurasia, Shyam S; Rieger, Frank G

    2016-05-01

    Inhibitor of differentiation (Id) proteins are DNA-binding transcription factors involved in cellular proliferation, migration, inflammation, angiogenesis and fibrosis. However, their expression and role in the cornea is unknown. The present study was undertaken to characterize the expression of Id proteins and their interactions with the pro-fibrotic cytokine Transforming Growth Factor β1 (TGFβ1) and anti-fibrotic cytokine, bone morphogenic protein 7 (BMP7) in human cornea. Human donor corneas procured from Eye Bank were used. Id proteins were localized in human corneal sections using immunofluorescence. Primary cultures of human corneal fibroblasts (HCF) were established and treated with either TGFβ1 (5 ng/ml) or BMP7 (10 ng/ml) for 24 h in serum free medium. Expression of Id's in response to TGFβ1, BMP7 and TGFβ1 + BMP7 was analyzed by quantitative real time PCR (qRT-PCR) and western blot analysis. Id1 and Id2 proteins were ubiquitously expressed in the epithelial cells and stromal keratocytes in human cornea. The Id1 was localized to the basal epithelial cells as seen by immunohistochemistry. HCF expressed all known mammalian Id genes (Id1-Id4). In addition, Id1 and Id2 are selectively expressed in HCF. Treatment of human recombinant TGFβ1 (5 ng/ml) to serum-starved HCF showed a significant increase in Id genes (Id1, Id2 and Id4) at 2 h time point compared to BMP7 treatment, which showed time dependent increase in the expression of Id1-Id3 at 24-48 h. Combined treatment with TGFβ1 + BMP7 to HCF showed a significant increase in Id1 transcript and an increasing trend in Id3 and Id4 expression. The results of this study suggest that Id family of genes (Id1-Id4) are localized in the human cornea and expressed in the corneal fibroblasts. Also, Id's were differentially regulated with TGFβ1 and/or BMP7 in a time dependent manner and might serve as a therapeutic target in corneal fibrosis.

  5. Acrylamide inhibits cellular differentiation of human neuroblastoma and glioblastoma cells.

    PubMed

    Chen, Jong-Hang; Chou, Chin-Cheng

    2015-08-01

    This study explores human neuroblastoma (SH-SY5Y) and human glioblastoma (U-1240 MG) cellular differentiation changes under exposure to acrylamide (ACR). Differentiation of SH-SY5Y and U-1240 MG cells were induced by retinoic acid (RA) and butyric acid (BA), respectively. Morphological observations and MTT assay showed that the induced cellular differentiation and cell proliferation were inhibited by ACR in a time- and dose-dependent manner. ACR co-treatment with RA attenuated SH-SY5Y expressions of neurofilament protein-L (NF-L), microtubule-associated protein 1b (MAP1b; 1.2 to 0.7, p < 0.001), MAP2c (2.2 to 0.8, p < 0.05), and Janus kinase1 (JAK1; 1.9 to 0.6, p < 0.001), while ACR co-treatment with BA attenuated U-1240 MG expressions of glial fibrillary acidic protein (GFAP), MAP1b (1.2 to 0.6, p < 0.001), MAP2c (1.5 to 0.7, p < 0.01), and JAK1 (2.1 to 0.5, p < 0.001), respectively. ACR also decreased the phosphorylation of extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) in U-1240 MG cells, while caffeine reversed this suppression of ERK and JNK phosphorylation caused by ACR treatment. These results showed that RA-induced neurogenesis of SH-SY5Y and BA-induced astrogliogenesis of U-1240 MG cells were attenuated by ACR and were associated with down-regulation of MAPs expression and JAK-STAT signaling.

  6. Genome-wide DNA methylation identifies trophoblast invasion-related genes: Claudin-4 and Fucosyltransferase IV control mobility via altering matrix metalloproteinase activity.

    PubMed

    Hu, Yuxiang; Blair, John D; Yuen, Ryan K C; Robinson, Wendy P; von Dadelszen, Peter

    2015-05-01

    Previously we showed that extravillous cytotrophoblast (EVT) outgrowth and migration on a collagen gel explant model were affected by exposure to decidual natural killer cells (dNK). This study investigates the molecular causes behind this phenomenon. Genome wide DNA methylation of exposed and unexposed EVT was assessed using the Illumina Infinium HumanMethylation450 BeadChip array (450 K array). We identified 444 differentially methylated CpG loci in dNK-treated EVT compared with medium control (P < 0.05). The genes associated with these loci had critical biological roles in cellular development, cellular growth and proliferation, cell signaling, cellular assembly and organization by Ingenuity Pathway Analysis (IPA). Furthermore, 23 mobility-related genes were identified by IPA from dNK-treated EVT. Among these genes, CLDN4 (encoding claudin-4) and FUT4 (encoding fucosyltransferase IV) were chosen for follow-up studies because of their biological relevance from research on tumor cells. The results showed that the mRNA and protein expressions of both CLDN4 and FUT4 in dNK-treated EVT were significantly reduced compared with control (P < 0.01 for both CLDN4 and FUT4 mRNA expression; P < 0.001 for CLDN4 and P < 0.01 for FUT4 protein expression), and were inversely correlated with DNA methylation. Knocking down CLDN4 and FUT4 by small interfering RNA reduced trophoblast invasion, possibly through the altered matrix metalloproteinase (MMP)-2 and/or MMP-9 expression and activity. Taken together, dNK alter EVT mobility at least partially in association with an alteration of DNA methylation profile. Hypermethylation of CLDN4 and FUT4 reduces protein expression. CLDN4 and FUT4 are representative genes that participate in modulating trophoblast mobility. PMID:25697377

  7. Genome-wide DNA methylation identifies trophoblast invasion-related genes: Claudin-4 and Fucosyltransferase IV control mobility via altering matrix metalloproteinase activity

    PubMed Central

    Hu, Yuxiang; Blair, John D.; Yuen, Ryan K.C.; Robinson, Wendy P.; von Dadelszen, Peter

    2015-01-01

    Previously we showed that extravillous cytotrophoblast (EVT) outgrowth and migration on a collagen gel explant model were affected by exposure to decidual natural killer cells (dNK). This study investigates the molecular causes behind this phenomenon. Genome wide DNA methylation of exposed and unexposed EVT was assessed using the Illumina Infinium HumanMethylation450 BeadChip array (450 K array). We identified 444 differentially methylated CpG loci in dNK-treated EVT compared with medium control (P < 0.05). The genes associated with these loci had critical biological roles in cellular development, cellular growth and proliferation, cell signaling, cellular assembly and organization by Ingenuity Pathway Analysis (IPA). Furthermore, 23 mobility-related genes were identified by IPA from dNK-treated EVT. Among these genes, CLDN4 (encoding claudin-4) and FUT4 (encoding fucosyltransferase IV) were chosen for follow-up studies because of their biological relevance from research on tumor cells. The results showed that the mRNA and protein expressions of both CLDN4 and FUT4 in dNK-treated EVT were significantly reduced compared with control (P < 0.01 for both CLDN4 and FUT4 mRNA expression; P < 0.001 for CLDN4 and P < 0.01 for FUT4 protein expression), and were inversely correlated with DNA methylation. Knocking down CLDN4 and FUT4 by small interfering RNA reduced trophoblast invasion, possibly through the altered matrix metalloproteinase (MMP)-2 and/or MMP-9 expression and activity. Taken together, dNK alter EVT mobility at least partially in association with an alteration of DNA methylation profile. Hypermethylation of CLDN4 and FUT4 reduces protein expression. CLDN4 and FUT4 are representative genes that participate in modulating trophoblast mobility. PMID:25697377

  8. Genome-wide DNA methylation identifies trophoblast invasion-related genes: Claudin-4 and Fucosyltransferase IV control mobility via altering matrix metalloproteinase activity.

    PubMed

    Hu, Yuxiang; Blair, John D; Yuen, Ryan K C; Robinson, Wendy P; von Dadelszen, Peter

    2015-05-01

    Previously we showed that extravillous cytotrophoblast (EVT) outgrowth and migration on a collagen gel explant model were affected by exposure to decidual natural killer cells (dNK). This study investigates the molecular causes behind this phenomenon. Genome wide DNA methylation of exposed and unexposed EVT was assessed using the Illumina Infinium HumanMethylation450 BeadChip array (450 K array). We identified 444 differentially methylated CpG loci in dNK-treated EVT compared with medium control (P < 0.05). The genes associated with these loci had critical biological roles in cellular development, cellular growth and proliferation, cell signaling, cellular assembly and organization by Ingenuity Pathway Analysis (IPA). Furthermore, 23 mobility-related genes were identified by IPA from dNK-treated EVT. Among these genes, CLDN4 (encoding claudin-4) and FUT4 (encoding fucosyltransferase IV) were chosen for follow-up studies because of their biological relevance from research on tumor cells. The results showed that the mRNA and protein expressions of both CLDN4 and FUT4 in dNK-treated EVT were significantly reduced compared with control (P < 0.01 for both CLDN4 and FUT4 mRNA expression; P < 0.001 for CLDN4 and P < 0.01 for FUT4 protein expression), and were inversely correlated with DNA methylation. Knocking down CLDN4 and FUT4 by small interfering RNA reduced trophoblast invasion, possibly through the altered matrix metalloproteinase (MMP)-2 and/or MMP-9 expression and activity. Taken together, dNK alter EVT mobility at least partially in association with an alteration of DNA methylation profile. Hypermethylation of CLDN4 and FUT4 reduces protein expression. CLDN4 and FUT4 are representative genes that participate in modulating trophoblast mobility.

  9. Differential cytotoxic effects of arsenic on human and animal cells.

    PubMed

    Lee, T C; Ho, I C

    1994-09-01

    Human fibroblasts (HFW) were 10-fold more susceptible than Chinese hamster ovary (CHO-K1) cells to sodium arsenite. Comparison of cellular antioxidant enzyme activities showed that CHO-K1 cells contained 3- and 8-fold more glutathione-peroxidase and catalase activities, respectively, than HFW cells. Since vitamin E, methylamine, and benzyl alcohol could prevent, in part, the arsenite-induced killing of HFW cells, we suggest that arsenite can induce oxidative damage in HFW cells. We have also established arsenic-resistant cells, SA7 and CL3R, from CHO cells and from a human lung adenocarcinoma cell line (CL3), respectively. The arsenic resistance of SA7 cells was attributed mainly to elevation of glutathione S-transferase pi levels, and that of CL3R cells was possibly due to an increase in heme oxygenase activity. Since induction of heme oxygenase is a general response to oxidative stress, we suspect that the differential toxicity of arsenic to human and animal cells could be due to arsenic's more efficient induction of oxidative damage in human cells.

  10. Primary human monocyte differentiation regulated by Nigella sativa pressed oil

    PubMed Central

    2011-01-01

    Background Oxidized low density lipoprotein plays an important role in development of foam cells in atherosclerosis. The study was focused on regulation of primary human monocyte growth and CD11b expression in presence of Nigella sativa oil. Methods Primary human monocytes were isolated from whole blood and grown at 37°C and 5% CO2 saturation for five days prior to treatment with Nigella sativa oil. The cells were plated and washed before treatment with ox-LDL (10 μg/ml) as positive control and combined treatment of ox-LDL (10 μg/ml) and (140 ng/ml) Nigella sativa oil. The growth progression was monitored every 24 hours for 3 days. Results Macrophages showed reduced growth in comparison to monocytes 24 hours after treatment with Nigella sativa oil. The mean cell diameter was significantly different between untreated and treated condition in monocytes and macrophages (p < 0.001). Similarly, intracellular lipid accumulation was hindered in combined treatment with Nigella sativa oil. This was further supported by cell surface expression analysis, where CD11b was markedly reduced in cells treated with combination oxLDL and Nigella sativa oil compared to oxLDL alone. More cells differentiated into macrophage-like cells when monocytes were supplemented with oxidized LDL alone. Conclusions The finding provides preliminary evidence on regulation of cell growth and differentiation in monocyte and monocyte-derived macrophages by Nigella sativa oil. Further investigations need to be conducted to explain its mechanism in human monocyte. PMID:22104447

  11. Rod Photoreceptor Differentiation in Fetal and Infant Human Retina

    PubMed Central

    Hendrickson, Anita; Bumsted-O'Brien, Keely; Natoli, Riccardo; Ramamurthy, Visvanathan; Possin, Daniel; Provis, Jan

    2014-01-01

    Human rods and cones are arranged in a precise spatial mosaic that is critical for optimal functioning of the visual system. However, the molecular processes that underpin specification of cell types within the mosaic are poorly understood. The progressive differentiation of human rods was tracked from fetal week (Fwk) 9 to postnatal (P) 8 months using immunocytochemical markers of key molecules that represent rod progression from post-mitotic precursors to outer segment-bearing functional photoreceptors. We find two phases associated with rod differentiation. The early phase begins in rods on the foveal edge at Fwk 10.5 when rods are first identified, and the rod-specific proteins NRL and NR2e3 are detected. By Fwk 11-12, these rods label for interphotoreceptor retinoid binding protein, recoverin, and aryl hydrocarbon receptor interacting protein-like 1. The second phase occurs over the next month with the appearance of rod opsin at Fwk 15, closely followed by the outer segment proteins rod GTP-gated sodium channel and peripherin. TULP is expressed relatively late at Fwk 18-20 in rods. Each phase proceeds across the retina in a central-peripheral order, such that rods in far peripheral retina are only entering the early phase at the same time that cells in central retina are entering their late phase. During the second half of gestation rods undergo an intracellular reorganization of these proteins, and cellular and OS elongation which continues into infancy. The progression of rod development shown here provides insight into the possible mechanisms underlying human retinal visual dysfunction when there are mutations affecting key rod-related molecules. PMID:18778702

  12. Early patterning of the chorion leads to the trilaminar trophoblast cell structure in the placental labyrinth.

    PubMed

    Simmons, David G; Natale, David R C; Begay, Valerie; Hughes, Martha; Leutz, Achim; Cross, James C

    2008-06-01

    The labyrinth of the rodent placenta contains villi that are the site of nutrient exchange between mother and fetus. They are covered by three trophoblast cell types that separate the maternal blood sinusoids from fetal capillaries--a single mononuclear cell that is a subtype of trophoblast giant cell (sinusoidal or S-TGC) with endocrine function and two multinucleated syncytiotrophoblast layers, each resulting from cell-cell fusion, that function in nutrient transport. The developmental origins of these cell types have not previously been elucidated. We report here the discovery of cell-layer-restricted genes in the mid-gestation labyrinth (E12.5-14.5) including Ctsq in S-TGCs (also Hand1-positive), Syna in syncytiotrophoblast layer I (SynT-I), and Gcm1, Cebpa and Synb in syncytiotrophoblast layer II (SynT-II). These genes were also expressed in distinct layers in the chorion as early as E8.5, prior to villous formation. Specifically, Hand1 was expressed in apical cells lining maternal blood spaces (Ctsq is not expressed until E12.5), Syna in a layer immediately below, and Gcm1, Cebpa and Synb in basal cells in contact with the allantois. Cebpa and Synb were co-expressed with Gcm1 and were reduced in Gcm1 mutants. By contrast, Hand1 and Syna expression was unaltered in Gcm1 mutants, suggesting that Gcm1-positive cells are not required for the induction of the other chorion layers. These data indicate that the three differentiated trophoblast cell types in the labyrinth arise from distinct and autonomous precursors in the chorion that are patterned before morphogenesis begins.

  13. Differentiation in human amniotic fluid cell cultures: I: Collagen production.

    PubMed Central

    Priest, R E; Priest, J H; Moinuddin, J F; Keyser, A J

    1977-01-01

    The collagen produced by differentiated cells cultured from human amniotic fluid was characterized in two ways. By chain composition and by 4-hydroxyproline:3-hydroxyproline isomer ratio, the collagen synthesized by F-type (fibroblast) cells was indistinguishable from that made by cultured fetal dermal fibroblasts. The predominant cells in young amniotic fluid cultures, termed AF-type, produced collagen with a lower isomer ratio, resembling that of basement membrane collage. The chain composition, as determined by chromatography on carboxymethyl cellulose, varied for different cultures of the AF-type, but the major pattern was consistent with that of basement membrane collagen. On the basis of these characteristics, F cells are of fibroblast origin, whereas most AF cells are of a different origin either endothelial or epithelial. Other evidence (Megaw et al., 1977) suggests an epithelial origin for AF cells. PMID:881704

  14. Differentiation and Genomic Instability in a Human Mammary Cell Model

    NASA Technical Reports Server (NTRS)

    Richmond, R.; Kale, R.; Pettengill, O.; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    Harvest of prophylactic mastectomy specimens from an obligate heterozygote for ataxia-telangiectasia provided autologous fibroblasts as well epithelial cells (HMEC). The routine availability of these autologous cells has provided an opportunity to study cell-cell interactions in coculture and monoculture, and in 3-dimensional cultures grown in the NASA rotating bioreactor. HMEC and stromal fibroblasts grown in 2-dimensional monoculture were both observed to produce extracellular matrix. Similar matrix was encountered in 3-dimensional cultures containing HMEC. Metaphases were analyzed. For stromal fibroblasts, genomic aberrations were found in 18% of metaphase spreads. For HMEC, aberrations were greater such that a majority were found to be abnormal. The level of genomic instability determined for these noncancerous cells in 2-dimensional monoculture should be useful for generating a human cell model that can correlate the effects of differentiation in 3-dimensional coculture on the level of genomic instability.

  15. Blastocyst Elongation, Trophoblastic Differentiation and Embryonic Pattern Formation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular basis behind elongation and concomitant gastrulation in ungulates that occurs during pre-implantation is still poorly understood. In-depth transcriptome analysis of the elongating porcine conceptus at specific stages has demonstrated that protein synthesis, protein trafficking, cell g...

  16. Affine differential geometry analysis of human arm movements.

    PubMed

    Flash, Tamar; Handzel, Amir A

    2007-06-01

    Humans interact with their environment through sensory information and motor actions. These interactions may be understood via the underlying geometry of both perception and action. While the motor space is typically considered by default to be Euclidean, persistent behavioral observations point to a different underlying geometric structure. These observed regularities include the "two-thirds power law", which connects path curvature with velocity, and "local isochrony", which prescribes the relation between movement time and its extent. Starting with these empirical observations, we have developed a mathematical framework based on differential geometry, Lie group theory and Cartan's moving frame method for the analysis of human hand trajectories. We also use this method to identify possible motion primitives, i.e., elementary building blocks from which more complicated movements are constructed. We show that a natural geometric description of continuous repetitive hand trajectories is not Euclidean but equi-affine. Specifically, equi-affine velocity is piecewise constant along movement segments, and movement execution time for a given segment is proportional to its equi-affine arc-length. Using this mathematical framework, we then analyze experimentally recorded drawing movements. To examine movement segmentation and classification, the two fundamental equi-affine differential invariants-equi-affine arc-length and curvature are calculated for the recorded movements. We also discuss the possible role of conic sections, i.e., curves with constant equi-affine curvature, as motor primitives and focus in more detail on parabolas, the equi-affine geodesics. Finally, we explore possible schemes for the internal neural coding of motor commands by showing that the equi-affine framework is compatible with the common model of population coding of the hand velocity vector when combined with a simple assumption on its dynamics. We then discuss several alternative explanations

  17. Differential transfer of dietary flavour compounds into human breast milk.

    PubMed

    Hausner, Helene; Bredie, Wender L P; Mølgaard, Christian; Petersen, Mikael Agerlin; Møller, Per

    2008-09-01

    Transfer of dietary flavour compounds into human milk is believed to constitute the infant's early flavour experiences. This study reports on the time-dependent transfer of flavour compounds from the mother's diet to her breast milk using a within-subject design. Eighteen lactating mothers completed three test days on which they provided a baseline milk sample prior to ingestion of capsules containing 100 mg d-carvone, l-menthol, 3-methylbutyl acetate and trans-anethole. Milk samples were collected 2, 4, 6 and 8 h post-ingestion and analysed by a dynamic headspace method and gas chromatography-mass spectroscopy. The recovery quantities were adjusted for variations in milk fat content. Concentration-time profiles for d-carvone and trans-anethole revealed a maximum around 2 h post-ingestion, whereas the profile for l-menthol showed a plateau pattern. The ester 3-methylbutyl acetate could not be detected in the milk, but a single determination showed traces (<0.4 ppb) in a 1 h milk collection. Flavour compounds appeared to be transmitted differentially from the mother's diet to her milk. The results imply that human milk provides a reservoir for time-dependent chemosensory experiences to the infant; however, volatiles from the diet are transferred selectively and in relatively low amounts.

  18. Scaffold fiber diameter regulates human tendon fibroblast growth and differentiation.

    PubMed

    Erisken, Cevat; Zhang, Xin; Moffat, Kristen L; Levine, William N; Lu, Helen H

    2013-02-01

    The diameter of collagen fibrils in connective tissues, such as tendons and ligaments is known to decrease upon injury or with age, leading to inferior biomechanical properties and poor healing capacity. This study tests the hypotheses that scaffold fiber diameter modulates the response of human tendon fibroblasts, and that diameter-dependent cell responses are analogous to those seen in healthy versus healing tissues. Particularly, the effect of the fiber diameter (320 nm, 680 nm, and 1.80 μm) on scaffold properties and the response of human tendon fibroblasts were determined over 4 weeks of culture. It was observed that scaffold mechanical properties, cell proliferation, matrix production, and differentiation were regulated by changes in the fiber diameter. More specifically, a higher cell number, total collagen, and proteoglycan production were found on the nanofiber scaffolds, while microfibers promoted the expression of phenotypic markers of tendon fibroblasts, such as collagen I, III, V, and tenomodulin. It is possible that the nanofiber scaffolds of this study resemble the matrix in a state of injury, stimulating the cells for matrix deposition as part of the repair process, while microfibers represent the healthy matrix with micron-sized collagen bundles, thereby inducing cells to maintain the fibroblastic phenotype. The results of this study demonstrate that controlling the scaffold fiber diameter is critical in the design of scaffolds for functional and guided connective tissue repair, and provide new insights into the role of matrix parameters in guiding soft tissue healing.

  19. Generation and Use of Trophoblast Stem Cells and Uterine Myocytes to Study the Role of Connexins for Pregnancy and Labor.

    PubMed

    Kibschull, Mark; Lye, Stephen J; Shynlova, Oksana

    2016-01-01

    Transgenic mouse models have demonstrated critical roles for gap junctions in establishing a successful pregnancy. To study the cellular and molecular mechanisms, the use of cell culture systems is essential to discriminate between the effects of different connexin isoforms expressed in individual cells or tissues of the developing conceptus or in maternal reproductive tissues. The generation and analysis of gene-deficient trophoblast stem cell lines from mice clearly revealed the functions of connexins in regulating placental development. This chapter focuses on the use of connexin gene-deficient trophoblast stem cell cultures to reveal the individual role of gap junctions in regulating trophoblast differentiation and proliferation in vitro under controlled conditions. In addition, cultures of primary uterine myocytes, isolated from mice or rats, allow studying the effects of mechanical stretch or ovarian hormones on regulating connexin expression, and thus, to model the molecular mechanisms of uterine growth and development during pregnancy. Here, we describe the derivation of primary uterine myocyte cultures and their use in in vitro stretch experiments to study the mechanisms of myometrial remodeling essential to accommodate the growing fetus throughout gestation. PMID:27207288

  20. Extravillous trophoblast cells-derived exosomes promote vascular smooth muscle cell migration

    PubMed Central

    Salomon, Carlos; Yee, Sarah; Scholz-Romero, Katherin; Kobayashi, Miharu; Vaswani, Kanchan; Kvaskoff, David; Illanes, Sebastian E.; Mitchell, Murray D.; Rice, Gregory E.

    2014-01-01

    Background: Vascular smooth muscle cells (VSMCs) migration is a critical process during human uterine spiral artery (SpA) remodeling and a successful pregnancy. Extravillous trophoblast cells (EVT) interact with VSMC and enhance their migration, however, the mechanisms by which EVT remodel SpA remain to be fully elucidated. We hypothesize that exosomes released from EVT promote VSMC migration. Methods: JEG-3 and HTR-8/SVneo cell lines were used as models for EVT. Cells were cultured at 37°C and humidified under an atmosphere of 5% CO2-balanced N2 to obtain 8% O2. Cell-conditioned media were collected, and exosomes (exo-JEG-3 and exo- HTR-8/SVneo) isolated by differential and buoyant density centrifugation. The effects of exo-EVT on VSMC migration were established using a real-time, live-cell imaging system (Incucyte™). Exosomal proteins where identified by mass spectrometry and submitted to bioinformatic pathway analysis (Ingenuity software). Results: HTR-8/SVneo cells were significantly more (~30%) invasive than JEG-3 cells. HTR-8/SVneo cells released 2.6-fold more exosomes (6.39 × 108 ± 2.5 × 108 particles/106 cells) compared to JEG-3 (2.86 × 108 ± 0.78 × 108 particles/106 cells). VSMC migration was significantly increased in the presence of exo-JEG-3 and exo-HTR-8/SVneo compared to control (−exosomes) (21.83 ± 0.49 h and 15.57 ± 0.32, respectively, vs. control 25.09 ± 0.58 h, p < 0.05). Sonication completely abolished the effect of exosomes on VSMC migration. Finally, mass spectrometry analysis identified unique exosomal proteins for each EVT cell line-derived exosomes. Conclusion: The data obtained in this study are consistent with the hypothesis that the release, content, and bioactivity of exosomes derived from EVT-like cell lines is cell origin-dependent and differentially regulates VSMC migration. Thus, an EVT exosomal signaling pathway may contribute to SpA remodeling by promoting the migration of VSMC out of the vessel walls. PMID:25157233

  1. Persistent trophoblastic tissue following salpingostomy for unruptured ectopic pregnancy

    SciTech Connect

    Rivlin, M.E.; Meeks, G.R.; Cowan, B.D.; Bates, G.W.

    1985-02-01

    Radioimmunoassay of beta-hCG was used to diagnose an ectopic pregnancy in a 30 year old patient and the site of pregnancy was determined by ultrasonography. A salpingostomy was performed; the ectopic pregnancy and the residual trophoblastic tissue were removed. Six weeks later a right salpingectomy was performed to remove persistent trophoblastic tissue. Histologic examination of the surgical specimen demonstrated viable chorionic villi. Serial measurements of beta-hCG are recommended following conservative surgery for ectopic gestation to assure the patient and the surgeon that the tube contains no residual products of conception.

  2. Use of ultrasound in the evaluation of trophoblastic disease and its response to therapy. [Comparison with HCG radioimmunoassay

    SciTech Connect

    Requard, C.K.; Mettler, F.A. Jr.

    1980-05-01

    Thirty-nine patients with trophoblastic disease were studied to determine the usefulness of ultrasound in identifying risk patterns and response to therapy. Serial measurements of serum human chorionic gonadotropin-beta subunit (HCG-BSU) were compared with ultrasonographic uterine and theca lutein cyst volumes. In 16 patients ultrasound demonstrated theca lutein cysts, many of which were not palpable on physical examination. Although there was a significant decrease in uterine volume and a change in the sonographic pattern following evacuation, volume slowly returned to normal over a period of several months. Persistent trophoblastic disease was more accurately detected by HCG-BSU measurements than by ultrasound. Persistent disease developed in 44% of those patients who had theca lutein cysts and in 22% of those without cysts. Patients with theca lutein cysts did not consistently have higher HCG-BSU levels than patients without cysts, and it is concluded that ultrasound is the best method for detecting these cysts.

  3. Comparison of syncytiotrophoblast generated from human embryonic stem cells and from term placentas

    PubMed Central

    Yabe, Shinichiro; Alexenko, Andrei P.; Amita, Mitsuyoshi; Yang, Ying; Schust, Danny J.; Ezashi, Toshihiko; Roberts, R. Michael

    2016-01-01

    Human embryonic stem cells (ESCs) readily commit to the trophoblast lineage after exposure to bone morphogenetic protein-4 (BMP-4) and two small compounds, an activin A signaling inhibitor and a FGF2 signaling inhibitor (BMP4/A83-01/PD173074; BAP treatment). During differentiation, areas emerge within the colonies with the biochemical and morphological features of syncytiotrophoblast (STB). Relatively pure fractions of mononucleated cytotrophoblast (CTB) and larger syncytial sheets displaying the expected markers of STB can be obtained by differential filtration of dispersed colonies through nylon strainers. RNA-seq analysis of these fractions has allowed them to be compared with cytotrophoblasts isolated from term placentas before and after such cells had formed syncytia. Although it is clear from extensive gene marker analysis that both ESC- and placenta-derived syncytial cells are trophoblast, each with the potential to transport a wide range of solutes and synthesize placental hormones, their transcriptome profiles are sufficiently dissimilar to suggest that the two cell types have distinct pedigrees and represent functionally different kinds of STB. We propose that the STB generated from human ESCs represents the primitive syncytium encountered in early pregnancy soon after the human trophoblast invades into the uterine wall. PMID:27051068

  4. Comparison of syncytiotrophoblast generated from human embryonic stem cells and from term placentas.

    PubMed

    Yabe, Shinichiro; Alexenko, Andrei P; Amita, Mitsuyoshi; Yang, Ying; Schust, Danny J; Sadovsky, Yoel; Ezashi, Toshihiko; Roberts, R Michael

    2016-05-10

    Human embryonic stem cells (ESCs) readily commit to the trophoblast lineage after exposure to bone morphogenetic protein-4 (BMP-4) and two small compounds, an activin A signaling inhibitor and a FGF2 signaling inhibitor (BMP4/A83-01/PD173074; BAP treatment). During differentiation, areas emerge within the colonies with the biochemical and morphological features of syncytiotrophoblast (STB). Relatively pure fractions of mononucleated cytotrophoblast (CTB) and larger syncytial sheets displaying the expected markers of STB can be obtained by differential filtration of dispersed colonies through nylon strainers. RNA-seq analysis of these fractions has allowed them to be compared with cytotrophoblasts isolated from term placentas before and after such cells had formed syncytia. Although it is clear from extensive gene marker analysis that both ESC- and placenta-derived syncytial cells are trophoblast, each with the potential to transport a wide range of solutes and synthesize placental hormones, their transcriptome profiles are sufficiently dissimilar to suggest that the two cell types have distinct pedigrees and represent functionally different kinds of STB. We propose that the STB generated from human ESCs represents the primitive syncytium encountered in early pregnancy soon after the human trophoblast invades into the uterine wall. PMID:27051068

  5. Role of HLA-G1 in trophoblast cell proliferation, adhesion and invasion

    SciTech Connect

    Jiang, Feng; Zhao, Hongxi; Wang, Li; Guo, Xinyu; Wang, Xiaohong; Yin, Guowu; Hu, Yunsheng; Li, Yi; Yao, Yuanqing

    2015-02-27

    Trophoblast cells are important in embryo implantation and fetomaternal tolerance. HLA-G is specifically expressed at the maternal–fetal interface and is a regulator in pregnancy. The aim of the present study was to detect the effect of HLA-G1 on trophoblast cell proliferation, adhesion, and invasion. Human trophoblast cell lines (JAR and HTR-8/SVneo cells) were infected with HLA-G1-expressing lentivirus. After infection, HLA-G1 expression of the cells was detected by western blotting. Cell proliferation was detected by the BrdU assay. The cell cycle and apoptosis of JAR and HTR-8/SVneo cells was measured by flow cytometry (FCM). The invasion of the cells under different conditions was detected by the transwell invasion chamber assay. HLA-G1 didn't show any significant influence on the proliferation, apoptosis, adhesion, and invasion of trophocytes in normal culture conditions. However, HLA-G1 inhibited JAR and HTR-8/SVneo cells invasion induced by hepatocyte growth factor (HGF) under normal oxygen conditions. In conditions of hypoxia, HLA-G1 couldn't inhibit the induction of cell invasion by HGF. HLA-G1 is not an independent factor for regulating the trophocytes. It may play an indirect role in embryo implantation and formation of the placenta. - Highlights: • HLA-G1 could not influence trophocytes under normal conditions. • HLA-G1 inhibited cell invasion induced by HGF under normal oxygen condition. • HLA-G1 could not influence cell invasion under hypoxia conditions.

  6. New method to differentiate human peripheral blood monocytes into insulin producing cells: Human hematosphere culture.

    PubMed

    Hur, Jin; Yang, Ji Min; Choi, Jae-Il; Yun, Ji-Yeon; Jang, Jae Hee; Kim, Joonoh; Kim, Ju-Young; Oh, Il-Young; Yoon, Chang-Hwan; Cho, Hyun-Jai; Park, Young-Bae; Kim, Hyo-Soo

    2012-02-24

    Strategy to differentiate stem cells into insulin producing cells (IPCs) in vitro has been a promising one to get cell source of β-cell replacement therapy for diabetes. It has been suggested that islets and neurons share features and nestin-positive cells could differentiate into IPCs. We have recently developed a three-dimensional culture system using human peripheral blood cells named as blood-born hematosphere (BBHS). Here we showed that most of BBHS were composed of nestin-positive cells. Under the four-stage differentiation protocol for IPCs, we plated nestin-positive BBHS onto fibronectin-coated dish. These cells form islet-like clusters and most of them expressed insulin. Pancreatic specific genes were turned on, such as transcription factors (Pdx-1, Ngn3 and Nkx6.1), genes related to endocrine function (Glut-2 and PC2) or β cell function (Kir6.2, SUR1). Furthermore islet differentiation was confirmed by dithizone (DTZ) staining to detect zinc ion which binds insulin protein within the cells. Finally, IPCs derived from BBHS showed capability to secrete insulin in response to glucose stimulation. Taken together, our novel protocol successfully induced islet-like human insulin producing cells out of BBHS. This strategy of ex vivo expansion of IPCs using BBHS provides an autologous therapeutic cell source for the treatment of diabetes. PMID:22310720

  7. Epithelioid trophoblastic tumor of the uterus in a postmenopausal woman: a case report and review of the literature.

    PubMed

    Coulson, L E; Kong, C S; Zaloudek, C

    2000-11-01

    We report an epithelioid trophoblastic tumor (ETT), a recently delineated type of gestational trophoblastic tumor (GTT), discovered in the uterus of a 66-year-old woman. She had been treated for a hydatidiform mole 17 years previously without chemotherapy. The resected uterus contained a solid/cystic tumor located entirely within the myometrium. Microscopically, there was an epithelial-like growth pattern. The tumor was circumscribed, with a pushing border, and the tumor cells grew in cords, nests, and sheets within which were aggregates of hyaline material and necrotic debris. Most tumor cells were mononuclear and had an epithelioid appearance with distinct cell borders, eosinophilic cytoplasm, and nuclei with occasional indistinct nucleoli. Scattered multinucleated cells consistent with syncytiotrophoblastic cells were also present. Immunohistochemical staining revealed strong diffuse reactivity for cytokeratins (CK7, AE1/AE3, CAM 5.2, CK18) and epidermal growth factor receptor, and focal reactivity, mainly in syncytiotrophoblastic cells, for beta-human chorionic gonadotropin, human placental lactogen, and inhibin-alpha. The histologic and immunohistochemical features were characteristic of ETT, and helped to distinguish the tumor from other trophoblastic tumors and squamous cell carcinoma. An unusual observation was a high mitotic count, reflected in a Ki-67 proliferative index of 68.6%. Our findings indicate that ETT, like other types of GTT, can occur in postmenopausal women, even years after a gestational event.

  8. Merosin, a protein specific for basement membranes of Schwann cells, striated muscle, and trophoblast, is expressed late in nerve and muscle development.

    PubMed Central

    Leivo, I; Engvall, E

    1988-01-01

    We have identified a tissue-specific basement membrane-associated protein by using monoclonal antibodies prepared against a protein fraction of human placenta. In immunofluorescence, the monoclonal antibodies stained basement membranes of Schwann cells, striated muscle, and trophoblast, whereas no reaction was seen with any other basement membrane or tissue structure. In antibody-affinity chromatography of proteolytic digests of human placenta, a 65-kDa polypeptide was bound by these monoclonal antibodies. Rabbit antisera and monoclonal antibodies raised against the isolated 65-kDa polypeptide stained human and monkey tissues identically to the original monoclonal antibodies and reacted with an 80-kDa polypeptide in tissue extracts prepared without proteolysis. The 65-kDa and 80-kDa polypeptides were shown to be immunologically distinct from laminin, type IV collagen, fibronectin, and major serum proteins. They presumably represent a novel basement membrane-associated protein, which we have named merosin. No merosin immunoreactivity could be detected in cultures of any of 28 established cell lines. In developing mouse tissues, merosin staining first appeared at the newborn stage. The restricted tissue distribution and late developmental appearance of merosin suggest that the protein has a tissue-specific function associated with a high level of differentiation. Images PMID:3278318

  9. Human cytotrophoblasts acquire aneuploidies as they differentiateto an invasive phenotype

    SciTech Connect

    Weier, Jingly F.; Weier, Heinz-Ulrich G.; Jung, Christine J.; Gormley, Matthew; Zhou, Yuan; Chu, Lisa W.; Genbacev, Olga; Wright, AlexiA.; Fisher, Susan J.

    2004-12-15

    Through an unusual differentiation process, human trophoblast progenitors (cytotrophoblasts) give rise to tumor-like cells that invade the uterus. By an unknown mechanism, invasive cytotrophoblasts exhibit permanent cell cycle withdrawal. Here we report molecular cytogenetic data showing that {approx} 20 to 60 percent of these interphase cells had acquired aneusomies involving chromosomes X, Y, o r16. The incidence positively correlated with gestational age and differentiation to an invasive phenotype. Scoring 12 chromosomes in flow-sorted cytotrophoblasts showed that more than 95 percent of the cells were hyperdiploid. Thus, aneuploidy appears to be an important component of normal placentation, perhaps limiting the proliferative and invasive potential of cytotrophoblasts within the uterus.

  10. Adiponectin inhibits insulin function in primary trophoblasts by PPARα-mediated ceramide synthesis.

    PubMed

    Aye, Irving L M H; Gao, Xiaoli; Weintraub, Susan T; Jansson, Thomas; Powell, Theresa L

    2014-04-01

    Maternal adiponectin (ADN) levels are inversely correlated with birth weight, and ADN infusion in pregnant mice down-regulates placental nutrient transporters and decreases fetal growth. In contrast to the insulin-sensitizing effects in adipose tissue and muscle, ADN inhibits insulin signaling in the placenta. However, the molecular mechanisms involved are unknown. We hypothesized that ADN inhibits insulin signaling and insulin-stimulated amino acid transport in primary human trophoblasts by peroxisome proliferator-activated receptor-α (PPARα)-mediated ceramide synthesis. Primary human term trophoblast cells were treated with ADN and/or insulin. ADN increased the phosphorylation of p38 MAPK and PPARα. ADN inhibited insulin signaling and insulin-stimulated amino acid transport. This effect was dependent on PPARα, because activation of PPARα with an agonist (GW7647) inhibited insulin signaling and function, whereas PPARα-small interfering RNA reversed the effects of ADN on the insulin response. ADN increased ceramide synthase expression and stimulated ceramide production. C2-ceramide inhibited insulin signaling and function, whereas inhibition of ceramide synthase (with Fumonisin B1) reversed the effects of ADN on insulin signaling and amino acid transport. These findings are consistent with the model that maternal ADN limits fetal growth mediated by activation of placental PPARα and ceramide synthesis, which inhibits placental insulin signaling and amino acid transport, resulting in reduced fetal nutrient availability.

  11. Cholinergic Interneurons Are Differentially Distributed in the Human Striatum

    PubMed Central

    Bernácer, Javier; Prensa, Lucía; Giménez-Amaya, José Manuel

    2007-01-01

    Background The striatum (caudate nucleus, CN, and putamen, Put) is a group of subcortical nuclei involved in planning and executing voluntary movements as well as in cognitive processes. Its neuronal composition includes projection neurons, which connect the striatum with other structures, and interneurons, whose main roles are maintaining the striatal organization and the regulation of the projection neurons. The unique electrophysiological and functional properties of the cholinergic interneurons give them a crucial modulating function on the overall striatal response. Methodology/Principle Findings This study was carried out using stereological methods to examine the volume and density (cells/mm3) of these interneurons, as visualized by choline acetyltransferase (ChAT) immunoreactivity, in the following territories of the CN and Put of nine normal human brains: 1) precommissural head; 2) postcommissural head; 3) body; 4) gyrus and 5) tail of the CN; 6) precommissural and 7) postcommissural Put. The distribution of ChAT interneurons was analyzed with respect to the topographical, functional and chemical territories of the dorsal striatum. The CN was more densely populated by cholinergic neurons than the Put, and their density increased along the anteroposterior axis of the striatum with the CN body having the highest neuronal density. The associative territory of the dorsal striatum was by far the most densely populated. The striosomes of the CN precommissural head and the postcommissural Put contained the greatest number of ChAT-ir interneurons. The intrastriosomal ChAT-ir neurons were abundant on the periphery of the striosomes throughout the striatum. Conclusions/Significance All these data reveal that cholinergic interneurons are differentially distributed in the distinct topographical and functional territories of the human dorsal striatum, as well as in its chemical compartments. This heterogeneity may indicate that the posterior aspects of the CN require a

  12. Nerve growth factor promotes human hemopoietic colony growth and differentiation

    SciTech Connect

    Matsuda, H.; Coughlin, M.D.; Bienenstock, J.; Denburg, J.A. )

    1988-09-01

    Nerve growth factor (NGF) is a neurotropic polypeptide necessary for the survival and growth of some central neurons, as well as sensory afferent and sympathetic neurons. Much is now known of the structural and functional characteristics of NGF, whose gene has recently been clones. Since it is synthesized in largest amounts by the male mouse submandibular gland, its role exclusively in nerve growth is questionable. These experiments indicate that NGF causes a significant stimulation of granulocyte colonies grown from human peripheral blood in standard hemopoietic methylcellulose assays. Further, NGF appears to act in a relatively selective fashion to induce the differentiation of eosinophils and basophils/mast cells. Depletion experiments show that the NGF effect may be T-cell dependent and that NGF augments the colony-stimulating effect of supernatants from the leukemic T-cell (Mo) line. The hemopoietic activity of NGF is blocked by {sup 125}I-polyclonal and monoclonal antibodies to NGF. The authors conclude that NGF may indirectly act as a local growth factor in tissues other than those of the nervous system by causing T cells to synthesize or secrete molecules with colony-stimulating activity. In view of the synthesis of NGF in tissue injury, the involvement of basophils/mast cells and eosinophils in allergic and other inflammatory processes, and the association of mast cells with fibrosis and tissue repair, they postulate that NGF plays an important biological role in a variety of repair processes.

  13. Differential effects of planktonic and biofilm MRSA on human fibroblasts.

    PubMed

    Kirker, Kelly R; James, Garth A; Fleckman, Philip; Olerud, John E; Stewart, Philip S

    2012-01-01

    Bacteria colonizing chronic wounds often exist as biofilms, yet their role in chronic wound pathogenesis remains unclear. Staphylococcus aureus biofilms induce apoptosis in dermal keratinocytes, and given that chronic wound biofilms also colonize dermal tissue, it is important to investigate the effects of bacterial biofilms on dermal fibroblasts. The effects of a predominant wound pathogen, methicillin-resistant S. aureus, on normal, human, dermal fibroblasts were examined in vitro. Cell-culture medium was conditioned with equivalent numbers of either planktonic or biofilm methicillin-resistant S. aureus and then fed to fibroblast cultures. Fibroblast response was evaluated using scratch, viability, and apoptosis assays. The results suggested that fibroblasts experience the same fate when exposed to the soluble products of either planktonic or biofilm methicillin-resistant S. aureus, namely limited migration followed by death. Enzyme-linked immunosorbent assays demonstrated that fibroblast production of cytokines, growth factors, and proteases were differentially affected by planktonic and biofilm-conditioned medium. Planktonic-conditioned medium induced more interleukin-6, interleukin-8, vascular endothelial growth factor, transforming growth factor-β1, heparin-bound epidermal growth factor, matrix metalloproteinase-1, and metalloproteinase-3 production in fibroblasts than the biofilm-conditioned medium. Biofilm-conditioned medium induced more tumor necrosis factor-α production in fibroblasts compared with planktonic-conditioned medium, and suppressed metalloproteinase-3 production compared with controls.

  14. Pathogenic role of anti-ß2-glycoprotein I antibodies in antiphospholipid associated fetal loss: characterisation of ß2-glycoprotein I binding to trophoblast cells and functional effects of anti-ß2-glycoprotein I antibodies in vitro

    PubMed Central

    Di, S; Raschi, E; Testoni, C; Castellani, R; D'Asta, M; Shi, T; Krilis, S; Caruso, A; Meroni, P

    2005-01-01

    Background: Antiphospholipid antibodies reacting with ß2-glycoprotein I (ß2GPI) have been associated with recurrent fetal loss and pregnancy complications. Objective: To investigate whether specific mutations in the phospholipid binding site of ß2GPI might affect its binding to trophoblast and in turn the anti-ß2GPI antibody induced functional effects. Methods: ß2GPI adhesion to trophoblast was evaluated as human monoclonal IgM or polyclonal IgG anti-ß2GPI antibody binding to trophoblast monolayers cultured (1) in complete medium; (2) in serum-free medium; (3) after serum starvation in the presence of purified human ß2GPI; or (4) in the presence of ß2GPI with single or multiple mutations in the amino acid loop Cys281-Lys-Asn-Lys-Glu-Lys-Lys-Cys288. The effect of anti-ß2GPI binding to trophoblast was evaluated as chorionic gonadotropin (hCG) mRNA expression, and protein release by RT-PCR and radioimmunoassay, respectively. Results: ß2GPI adhesion to trophoblast and its consequent recognition by the specific antibodies were inversely proportional to the mutation number in the phospholipid binding site. Anti-ß2GPI antibodies reduced gonadotropin release, hormone dependent hCG mRNA expression, and protein synthesis in the presence of ß2GPI, while the addition of the mutants or the absence of ß2GPI had no effect. Conclusions: ß2GPI binds to trophoblast in vitro through its fifth domain, as reported for endothelial cells, and can be recognised by anti-ß2GPI antibodies; the antibody binding downregulates trophoblast hCG synthesis and secretion. Such a mechanism might contribute to defective placentation in women with fetal loss associated with the antiphospholipid syndrome. PMID:15256379

  15. Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation

    NASA Technical Reports Server (NTRS)

    Leong, J.; Hughes-Fulford, M.; Rakhlin, N.; Habib, A.; Maclouf, J.; Goldyne, M. E.

    1996-01-01

    Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

  16. The placental imprintome and imprinted gene function in the trophoblast glycogen cell lineage.

    PubMed

    Lefebvre, Louis

    2012-07-01

    Imprinted genes represent a unique class of autosomal genes expressed from only one of the parental alleles during development. The choice of the expressed allele is not random but rather is determined by the parental origin of the allele. Consequently, the mouse genome contains more than 100 genes expressed preferentially or exclusively from the maternally or the paternally inherited allele. Current research efforts are focused on understanding the molecular mechanism of this epigenetic phenomenon as well as the biological functions of the genes under its regulation. Both theoretical considerations and experimental results support a role for genomic imprinting in the regulation of embryonic growth and placental biology. In this review, recent efforts to establish the complete set of genes showing imprinted expression in the mouse placenta are first discussed. Then, the evidence suggesting that imprinted genes might be implicated in the emergence, maintenance and function of trophoblast glycogen cells is presented. Although the origin and functions of this trophoblast cell lineage are currently unknown, the analysis of mutations in imprinted genes in the mouse are providing new insights into these issues. The implications of this work for placental pathologies in human are also discussed.

  17. A.A.W. Hubrecht and the naming of the trophoblast.

    PubMed

    Pijnenborg, R; Vercruysse, L

    2013-04-01

    The term 'trophoblast' was introduced in 1889 by the Dutch embryologist A.W.W. Hubrecht during his study of early postimplantation stages in the hedgehog. He had selected this species because of its supposed relatedness to ancestral early mammals, which might provide clues to the evolution of viviparity. Although the new term referred in the first place to the nutritive activity of these cells, Hubrecht was well aware of other possible functions, including a protective role by their positioning at the interface between mother and conceptus. He also paid attention to the invasive activity of the trophoblast into the decidua, but was confused about the nature of the trophospongial layer of the placenta. Because of the structural analogy between early postimplantation stages in hedgehogs and humans, he speculated that, in contrast to other primate groups, anthropoids might have been derived from an evolutionary line separate from ancestral insectivore-like mammals. Although the latter idea has obviously become out of date, Hubrecht's writings provide a fascinating insight in the evolutionary thinking at that time, and therefore still merit a close study.

  18. Identification of Pathways Mediating Growth Differentiation Factor5-Induced Tenogenic Differentiation in Human Bone Marrow Stromal Cells

    PubMed Central

    Tan, Sik-Loo; Ahmad, Tunku Sara; Ng, Wuey-Min; Azlina, Amir Abbas; Azhar, Mahmood Merican; Selvaratnam, Lakshmi; Kamarul, Tunku

    2015-01-01

    To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and

  19. Integrin αVβ3 and αVβ5 are required for leukemia inhibitory factor-mediated the adhesion of trophoblast cells to the endometrial cells.

    PubMed

    Chung, Tae-Wook; Park, Mi-Ju; Kim, Hyung Sik; Choi, Hee-Jung; Ha, Ki-Tae

    2016-01-22

    The embryo implantation including adhesion between trophoblast and endometrium is a crucial process for the successful pregnancy. LIF and adhesion molecules including integrins are known as significant factors for embryo implantation. However, the function of LIF on the regulation of adhesion molecule expression and promotion of trophoblast adhesion to endometrial cells has not been fully elucidated. Here we show that LIF significantly induced mRNA expression of ITGAV, ITGB3, and ITGB5 in endometrial cells, as evidenced by RT-PCR and qRT-PCR analysis. Based on the results from treatment of antagonist for LIF receptor (hLA), LIF positively regulates expression of integrin αV, β3, and β5, and adhesion of the human trophectoderm-derived JAr cells to endometrial Ishikawa cells. Furthermore, the adhesion between trophoblastic cells and LIF-stimulated endometrial cells was significantly reduced by neutralization of LIF-mediated integrin β3 and β5 expression on endometrial cell surface with integrin subunit β3 and β5 antibodies. Taken together, we firstly demonstrate that LIF enhances the adhesion of trophoblastic cells to endometrial cells by up-regulating expression of integrin heterodimer αVβ3 and αVβ5, indicating the promotion of endometrial receptivity for embryo implantation. PMID:26723254

  20. Cytomegalovirus Replicates in Differentiated but not in Undifferentiated Human Embryonal Carcinoma Cells

    NASA Astrophysics Data System (ADS)

    Gonczol, Eva; Andrews, Peter W.; Plotkin, Stanley A.

    1984-04-01

    To study the mode of action of human cytomegalovirus, an important teratogenic agent in human populations, the susceptibility of a pluripotent human embryonal carcinoma cell line to the virus was investigated. Viral antigens were not expressed nor was infectious virus produced by human embryonal carcinoma cells after infection, although the virus was able to penetrate these cells. In contrast, retinoic acid-induced differentiated derivatives of embryonal carcinoma cells were permissive for antigen expression and infectious virus production. Replication of human cytomegalovirus in human teratocarcinoma cells may therefore depend on cellular functions associated with differentiation.

  1. Low oxygen tension induces Krüppel-Like Factor 6 expression in trophoblast cells.

    PubMed

    Racca, A C; Ridano, M E; Bandeira, C L; Bevilacqua, E; Avvad Portari, E; Genti-Raimondi, S; Graham, C H; Panzetta-Dutari, G M

    2016-09-01

    The transcription factor Krüppel-Like Factor 6 (KLF6) has important roles in cell differentiation, angiogenesis, apoptosis, and proliferation. Furthermore, there is evidence that KLF6 is required for proper placental development. While oxygen is a critical mediator of trophoblast differentiation and function, the involvement of oxygen in the regulation of KLF6 expression remains unexplored. In the present study we examined the expression of KLF6 in placental tissue from uncomplicated and preeclamptic pregnancies, the latter often characterized by an inadequately perfused placenta. We also determined the effect of hypoxia and the involvement of Hypoxia-Inducible Factor 1α (HIF-1α) on the expression of KLF6 in cultured trophoblast cells and placental tissues. Results revealed that villous, interstitial and endovascular extravillous cytotrophoblasts from placentas from normal and preeclamptic pregnancies express KLF6. In addition, KLF6 immunoreactivity was higher in the placental bed of preeclamptic pregnancies than in those of uncomplicated pregnancies. We demonstrated that hypoxia induced an early and transient increase in KLF6 protein levels in HTR8/SVneo extravillous cytotrophoblast cells and in placental explants. Reoxygenation returned KLF6 protein to basal levels. Moreover, hypoxia-induced up-regulation of KLF6 expression was dependent on HIF-1α as revealed by siRNA knockdown in HTR8/SVneo cells. These results indicate that KLF6 may mediate some of the effects of hypoxia in placental development. The regulation of KLF6 protein levels by oxygen has significant implications for understanding its putative role in diseases affected by tissue hypoxia. PMID:27577710

  2. Low oxygen tension induces Krüppel-Like Factor 6 expression in trophoblast cells.

    PubMed

    Racca, A C; Ridano, M E; Bandeira, C L; Bevilacqua, E; Avvad Portari, E; Genti-Raimondi, S; Graham, C H; Panzetta-Dutari, G M

    2016-09-01

    The transcription factor Krüppel-Like Factor 6 (KLF6) has important roles in cell differentiation, angiogenesis, apoptosis, and proliferation. Furthermore, there is evidence that KLF6 is required for proper placental development. While oxygen is a critical mediator of trophoblast differentiation and function, the involvement of oxygen in the regulation of KLF6 expression remains unexplored. In the present study we examined the expression of KLF6 in placental tissue from uncomplicated and preeclamptic pregnancies, the latter often characterized by an inadequately perfused placenta. We also determined the effect of hypoxia and the involvement of Hypoxia-Inducible Factor 1α (HIF-1α) on the expression of KLF6 in cultured trophoblast cells and placental tissues. Results revealed that villous, interstitial and endovascular extravillous cytotrophoblasts from placentas from normal and preeclamptic pregnancies express KLF6. In addition, KLF6 immunoreactivity was higher in the placental bed of preeclamptic pregnancies than in those of uncomplicated pregnancies. We demonstrated that hypoxia induced an early and transient increase in KLF6 protein levels in HTR8/SVneo extravillous cytotrophoblast cells and in placental explants. Reoxygenation returned KLF6 protein to basal levels. Moreover, hypoxia-induced up-regulation of KLF6 expression was dependent on HIF-1α as revealed by siRNA knockdown in HTR8/SVneo cells. These results indicate that KLF6 may mediate some of the effects of hypoxia in placental development. The regulation of KLF6 protein levels by oxygen has significant implications for understanding its putative role in diseases affected by tissue hypoxia.

  3. Human Olfactory Mucosa Multipotent Mesenchymal Stromal Cells Promote Survival, Proliferation, and Differentiation of Human Hematopoietic Cells

    PubMed Central

    Diaz-Solano, Dylana; Wittig, Olga; Ayala-Grosso, Carlos; Pieruzzini, Rosalinda

    2012-01-01

    Multipotent mesenchymal stromal cells (MSCs) from the human olfactory mucosa (OM) are cells that have been proposed as a niche for neural progenitors. OM-MSCs share phenotypic and functional properties with bone marrow (BM) MSCs, which constitute fundamental components of the hematopoietic niche. In this work, we investigated whether human OM-MSCs may promote the survival, proliferation, and differentiation of human hematopoietic stem cells (HSCs). For this purpose, human bone marrow cells (BMCs) were co-cultured with OM-MSCs in the absence of exogenous cytokines. At different intervals, nonadherent cells (NACs) were harvested from BMC/OM-MSC co-cultures, and examined for the expression of blood cell markers by flow cytometry. OM-MSCs supported the survival (cell viability >90%) and proliferation of BMCs, after 54 days of co-culture. At 20 days of co-culture, flow cytometric and microscopic analyses showed a high percentage (73%) of cells expressing the pan-leukocyte marker CD45, and the presence of cells of myeloid origin, including polymorphonuclear leukocytes, monocytes, basophils, eosinophils, erythroid cells, and megakaryocytes. Likewise, T (CD3), B (CD19), and NK (CD56/CD16) cells were detected in the NAC fraction. Colony-forming unit–granulocyte/macrophage (CFU-GM) progenitors and CD34+ cells were found, at 43 days of co-culture. Reverse transcriptase–polymerase chain reaction (RT-PCR) studies showed that OM-MSCs constitutively express early and late-acting hematopoietic cytokines (i.e., stem cell factor [SCF] and granulocyte- macrophage colony-stimulating factor [GM-CSF]). These results constitute the first evidence that OM-MSCs may provide an in vitro microenvironment for HSCs. The capacity of OM-MSCs to support the survival and differentiation of HSCs may be related with the capacity of OM-MSCs to produce hematopoietic cytokines. PMID:22471939

  4. Human olfactory mucosa multipotent mesenchymal stromal cells promote survival, proliferation, and differentiation of human hematopoietic cells.

    PubMed

    Diaz-Solano, Dylana; Wittig, Olga; Ayala-Grosso, Carlos; Pieruzzini, Rosalinda; Cardier, Jose E

    2012-11-20

    Multipotent mesenchymal stromal cells (MSCs) from the human olfactory mucosa (OM) are cells that have been proposed as a niche for neural progenitors. OM-MSCs share phenotypic and functional properties with bone marrow (BM) MSCs, which constitute fundamental components of the hematopoietic niche. In this work, we investigated whether human OM-MSCs may promote the survival, proliferation, and differentiation of human hematopoietic stem cells (HSCs). For this purpose, human bone marrow cells (BMCs) were co-cultured with OM-MSCs in the absence of exogenous cytokines. At different intervals, nonadherent cells (NACs) were harvested from BMC/OM-MSC co-cultures, and examined for the expression of blood cell markers by flow cytometry. OM-MSCs supported the survival (cell viability >90%) and proliferation of BMCs, after 54 days of co-culture. At 20 days of co-culture, flow cytometric and microscopic analyses showed a high percentage (73%) of cells expressing the pan-leukocyte marker CD45, and the presence of cells of myeloid origin, including polymorphonuclear leukocytes, monocytes, basophils, eosinophils, erythroid cells, and megakaryocytes. Likewise, T (CD3), B (CD19), and NK (CD56/CD16) cells were detected in the NAC fraction. Colony-forming unit-granulocyte/macrophage (CFU-GM) progenitors and CD34(+) cells were found, at 43 days of co-culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) studies showed that OM-MSCs constitutively express early and late-acting hematopoietic cytokines (i.e., stem cell factor [SCF] and granulocyte- macrophage colony-stimulating factor [GM-CSF]). These results constitute the first evidence that OM-MSCs may provide an in vitro microenvironment for HSCs. The capacity of OM-MSCs to support the survival and differentiation of HSCs may be related with the capacity of OM-MSCs to produce hematopoietic cytokines.

  5. Molecular cloning of the human Hand1 gene/cDNA and its tissue-restricted expression in cytotrophoblastic cells and heart.

    PubMed

    Knöfler, M; Meinhardt, G; Vasicek, R; Husslein, P; Egarter, C

    1998-12-11

    The basic helix-loop-helix (bHLH) factor Hand1 plays a role in the developing chicken heart and is required for trophoblast giant cell differentiation and cardiac looping of mouse embryonic development. Here, we report the cloning of the human Hand1 cDNA and gene from a heart-specific cDNA library and a genomic lambda-DNA library, respectively. We present the nucleotide sequence of a 1.75kb cDNA clone, encoding the presumptive 215 amino acid human Hand1 protein, and show homology comparison of the conserved bHLH region between different species. In vitro transcription-translation of Hand1 mRNA and analysis of protein size suggest that the Hand1 polypeptide is (post)translationally modified. By Southern blot analysis we demonstrate that the isolated genomic DNA clone harbours the entire Hand1 gene and describe molecular structure and sequences of the two 799 and 938bp exons and the single 1.56kb intron. The expression pattern of the mRNA in different human tissues revealed that Hand1 transcripts are restricted to the heart, suggesting that the protein could be required for cardiac-specific gene transcription and function in adults. Hand1 transcripts were undetectable in a non-tumorigenic villous trophoblast cell line, immunopurified cytotrophoblasts undergoing in vitro differentiation, and first trimester placental tissue, suggesting that the transcription factor is not involved in the development of villous and extravillous trophoblast cell lineages. Hand1 mRNA, however, was abundantly expressed in cytotrophoblastic Jeg-3 and BeWo cells, suggesting that Hand1 could be required for early trophoblast differentiation.

  6. sFLT1 in preeclampsia: trophoblast defense against a decidual VEGFA barrage?

    PubMed

    Adamson, S Lee

    2014-11-01

    Preeclampsia, a life-threatening complication of human pregnancy, has a spectrum of clinical signs and is likely caused by an array of pathological mechanisms. However, elevated levels of soluble fms-like tyrosine kinase-1 (sFLT1) in the placenta and in the maternal circulation has emerged as a common finding in women with preeclampsia and likely is a causative factor in this disorder. In this issue of the JCI, Fan and colleagues provide experimental evidence from both humans and mice that suggests placental trophoblast cells overexpress sFLT1 in self defense against excessive VEGFA produced by maternal decidual cells. The authors' work thus implicates the decidual cells of the mother as the culprit responsible for increased placental expression of sFLT1, a VEGFA antagonist that enters the maternal circulation and consequently induces the clinical signs of preeclampsia.

  7. sFLT1 in preeclampsia: trophoblast defense against a decidual VEGFA barrage?

    PubMed Central

    Adamson, S. Lee

    2014-01-01

    Preeclampsia, a life-threatening complication of human pregnancy, has a spectrum of clinical signs and is likely caused by an array of pathological mechanisms. However, elevated levels of soluble fms-like tyrosine kinase-1 (sFLT1) in the placenta and in the maternal circulation has emerged as a common finding in women with preeclampsia and likely is a causative factor in this disorder. In this issue of the JCI, Fan and colleagues provide experimental evidence from both humans and mice that suggests placental trophoblast cells overexpress sFLT1 in self defense against excessive VEGFA produced by maternal decidual cells. The authors’ work thus implicates the decidual cells of the mother as the culprit responsible for increased placental expression of sFLT1, a VEGFA antagonist that enters the maternal circulation and consequently induces the clinical signs of preeclampsia. PMID:25329689

  8. Effects of pulsed electromagnetic field frequencies on the osteogenic differentiation of human mesenchymal stem cells.

    PubMed

    Luo, Fei; Hou, Tianyong; Zhang, Zehua; Xie, Zhao; Wu, Xuehui; Xu, Jianzhong

    2012-04-01

    The purpose of this study was to evaluate the effect of different frequencies of pulsed electromagnetic fields on the osteogenic differentiation of human mesenchymal stem cells. Third-generation human mesenchymal stem cells were irradiated with different frequencies of pulsed electromagnetic fields, including 5, 25, 50, 75, 100, and 150 Hz, with a field intensity of 1.1 mT, for 30 minutes per day for 21 days. Changes in human mesenchymal stem cell morphology were observed using phase contrast microscopy. Alkaline phosphatase activity and osteocalcin expression were also determined to evaluate human mesenchymal stem cell osteogenic differentiation.Different effects were observed on human mesenchymal stem cell osteoblast induction following exposure to different pulsed electromagnetic field frequencies. Levels of human mesenchymal stem cell differentiation increased when the pulsed electromagnetic field frequency was increased from 5 hz to 50 hz, but the effect was weaker when the pulsed electromagnetic field frequency was increased from 50 Hz to 150 hz. The most significant effect on human mesenchymal stem cell differentiation was observed at of 50 hz.The results of the current study show that pulsed electromagnetic field frequency is an important factor with regard to the induction of human mesenchymal stem cell differentiation. Furthermore, a pulsed electromagnetic field frequency of 50 Hz was the most effective at inducing human mesenchymal stem cell osteoblast differentiation in vitro.

  9. Mechanical strain applied to human fibroblasts differentially regulates skeletal myoblast differentiation

    PubMed Central

    Hicks, Michael R.; Cao, Thanh V.; Campbell, David H.

    2012-01-01

    Cyclic short-duration stretches (CSDS) such as those resulting from repetitive motion strain increase the risk of musculoskeletal injury. Myofascial release is a common technique used by clinicians that applies an acyclic long-duration stretch (ALDS) to muscle fascia to repair injury. When subjected to mechanical strain, fibroblasts within muscle fascia secrete IL-6, which has been shown to induce myoblast differentiation, essential for muscle repair. We hypothesize that fibroblasts subjected to ALDS following CSDS induce myoblast differentiation through IL-6. Fibroblast conditioned media and fibroblast-myoblast cocultures were used to test fibroblasts' ability to induce myoblast differentiation. The coculture system applies strain to fibroblasts only but still allows for diffusion of potential differentiation mediators to unstrained myoblasts on coverslips. To determine the role of IL-6, we utilized myoblast unicultures ± IL-6 (0–100 ng/ml) and cocultures ± α-IL-6 (0–200 μg/ml). Untreated uniculture myoblasts served as a negative control. After 96 h, coverslips (n = 6–21) were microscopically analyzed and quantified by blinded observer for differentiation endpoints: myotubes per square millimeter (>3 nuclei/cell), nuclei/myotube, and fusion efficiency (%nuclei within myotubes). The presence of fibroblasts and fibroblast conditioned media significantly enhanced myotube number (P < 0.05). However, in coculture, CSDS applied to fibroblasts did not reproduce this effect. ALDS following CSDS increased myotube number by 78% and fusion efficiency by 96% vs. CSDS alone (P < 0.05). Fibroblasts in coculture increase IL-6 secretion; however, IL-6 secretion did not correlate with enhanced differentiation among strain groups. Exogenous IL-6 in myoblast uniculture failed to induce differentiation. However, α-IL-6 attenuated differentiation in all coculture groups (P < 0.05). Fibroblasts secrete soluble mediators that have profound effects on several measures of

  10. Mechanical strain applied to human fibroblasts differentially regulates skeletal myoblast differentiation.

    PubMed

    Hicks, Michael R; Cao, Thanh V; Campbell, David H; Standley, Paul R

    2012-08-01

    Cyclic short-duration stretches (CSDS) such as those resulting from repetitive motion strain increase the risk of musculoskeletal injury. Myofascial release is a common technique used by clinicians that applies an acyclic long-duration stretch (ALDS) to muscle fascia to repair injury. When subjected to mechanical strain, fibroblasts within muscle fascia secrete IL-6, which has been shown to induce myoblast differentiation, essential for muscle repair. We hypothesize that fibroblasts subjected to ALDS following CSDS induce myoblast differentiation through IL-6. Fibroblast conditioned media and fibroblast-myoblast cocultures were used to test fibroblasts' ability to induce myoblast differentiation. The coculture system applies strain to fibroblasts only but still allows for diffusion of potential differentiation mediators to unstrained myoblasts on coverslips. To determine the role of IL-6, we utilized myoblast unicultures ± IL-6 (0-100 ng/ml) and cocultures ± α-IL-6 (0-200 μg/ml). Untreated uniculture myoblasts served as a negative control. After 96 h, coverslips (n = 6-21) were microscopically analyzed and quantified by blinded observer for differentiation endpoints: myotubes per square millimeter (>3 nuclei/cell), nuclei/myotube, and fusion efficiency (%nuclei within myotubes). The presence of fibroblasts and fibroblast conditioned media significantly enhanced myotube number (P < 0.05). However, in coculture, CSDS applied to fibroblasts did not reproduce this effect. ALDS following CSDS increased myotube number by 78% and fusion efficiency by 96% vs. CSDS alone (P < 0.05). Fibroblasts in coculture increase IL-6 secretion; however, IL-6 secretion did not correlate with enhanced differentiation among strain groups. Exogenous IL-6 in myoblast uniculture failed to induce differentiation. However, α-IL-6 attenuated differentiation in all coculture groups (P < 0.05). Fibroblasts secrete soluble mediators that have profound effects on several measures of myoblast

  11. X-radiation-induced differentiation of xenotransplanted human undifferentiated rhabdomyosarcoma

    SciTech Connect

    Takizawa, T.; Matsui, T.; Maeda, Y.; Okabe, S.; Mochizuki, M.; Tanaka, A.; Kawaguchi, K.; Fukayama, M.; Funata, N.; Koike, M.

    1989-01-01

    A serially xenotransplantable strain of undifferentiated embryonal rhabdomyosarcoma originating from the nasal cavity of a 42-year-old woman has been established in our laboratory. After radiotherapy for the tumor donor, distinct rhabdomyoblastic differentiation of the undifferentiated sarcoma cells appeared in the primary lesion, and it is a reasonable assumption that X-irradiation has a certain potentiality to induce morphologic differentiation of tumor cells. To study this possibility, tissue fragments of undifferentiated embryonal rhabdomyosarcoma that had grown to more than 10 mm after being transplanted to nude mice were selectively irradiated in situ. The degree of rhabdomyoblastic differentiation according to radiation dose was evaluated by light and electron microscopy and by immunostainability for myoglobin, creatine phosphokinase-MM, and desmin. Distinct morphologic differentiation of undifferentiated sarcoma cells could be induced by repeated X-irradiations at several-week intervals.

  12. In vitro toxicological effects of estrogenic mycotoxins on human placental cells: Structure activity relationships

    SciTech Connect

    Prouillac, Caroline; Lecoeur, Sylvaine

    2012-03-15

    Zearalenone (ZEN) is a non-steroid estrogen mycotoxin produced by numerous strains of Fusarium which commonly contaminate cereals. After oral administration, ZEN is reduced via intestinal and hepatic metabolism to α- and β-zearalenol (αZEL and βZEL). These reduced metabolites possess estrogenic properties, αZEL showing the highest affinity for ERs. ZEN and reduced metabolites cause hormonal effects in animals, such as abnormalities in the development of the reproductive tract and mammary gland in female offspring, suggesting a fetal exposure to these contaminants. In our previous work, we have suggested the potential impact of ZEN on placental cells considering this organ as a potential target of xenobiotics. In this work, we first compared the in vitro effects of αZEL and βΖΕL on cell differentiation to their parental molecule on human trophoblast (BeWo cells). Secondly, we investigated their molecular mechanisms of action by investigating the expression of main differentiation biomarkers and the implication of nuclear receptor by docking prediction. Conversely to ZEN, reduced metabolites did not induce trophoblast differentiation. They also induced significant changes in ABC transporter expression by potential interaction with nuclear receptors (LXR, PXR, PR) that could modify the transport function of placental cells. Finally, the mechanism of ZEN differentiation induction seemed not to involve nuclear receptor commonly involved in the differentiation process (PPARγ). Our results demonstrated that in spite of structure similarities between ZEN, αZEL and βZEL, toxicological effects and toxicity mechanisms were significantly different for the three molecules. -- Highlights: ► ZEN and metabolites have differential effect on trophoblast differentiation. ► ZEN and metabolites have differential effect on ABC transporter expression. ► ZEN and metabolites effects involved nuclear receptors interaction.

  13. KeyGenes, a Tool to Probe Tissue Differentiation Using a Human Fetal Transcriptional Atlas

    PubMed Central

    Roost, Matthias S.; van Iperen, Liesbeth; Ariyurek, Yavuz; Buermans, Henk P.; Arindrarto, Wibowo; Devalla, Harsha D.; Passier, Robert; Mummery, Christine L.; Carlotti, Françoise; de Koning, Eelco J.P.; van Zwet, Erik W.; Goeman, Jelle J.; Chuva de Sousa Lopes, Susana M.

    2015-01-01

    Summary Differentiated derivatives of human pluripotent stem cells in culture are generally phenotypically immature compared to their adult counterparts. Their identity is often difficult to determine with certainty because little is known about their human fetal equivalents in vivo. Cellular identity and signaling pathways directing differentiation are usually determined by extrapolating information from either human adult tissue or model organisms, assuming conservation with humans. To resolve this, we generated a collection of human fetal transcriptional profiles at different developmental stages. Moreover, we developed an algorithm, KeyGenes, which uses this dataset to quantify the extent to which next-generation sequencing or microarray data resemble specific cell or tissue types in the human fetus. Using KeyGenes combined with the human fetal atlas, we identified multiple cell and tissue samples unambiguously on a limited set of features. We thus provide a flexible and expandable platform to monitor and evaluate the efficiency of differentiation in vitro. PMID:26028532

  14. Immunofluorescence confocal laser scanning microscopy and immuno-electron microscopic identification of keratins in human materno-foetal interaction zone

    PubMed Central

    Ahenkorah, J; Hottor, B; Byrne, S; Bosio, P; Ockleford, C D

    2009-01-01

    Abstract We show here that at least 5 keratin proteins are present in villous trophoblast and the same 5 in extravillous trophoblast. A further 14 tested were undetectable in these tissues. In contrast, 10 of the 19 keratins tested were present in amniotic epithelium. The marking of amniotic epithelium on the one hand, as distinct from villous and extravillous trophoblast on the other, can be achieved using 5 keratins (K4, 6, 13, 14 and 17) with a mixture of positive and negative discrimination that is expected, in combination, to be highly sensitive. All the specific keratins identified in trophoblast were apparently up-regulated on the pathway to extravillous trophoblast. Co-ordinated differentiation at the molecular expression level is indicated by this finding. The relevant keratins are K5, 7, 8, 18 and 19. Specific keratins have been identified that are down-regulated in villous trophoblast in pre-eclamptic pregnancy. This difference between healthy and pre-eclamptic chorionic villous trophoblast keratin expression was statistically significant in 4 out of the 5 keratins. This was not the case for the extravillous trophoblast at the immunofluorescence confocal level but significant differences were obtained using immunogold electron microscopy. We suggest that the villous trophoblast in pre-eclamptic placentae is cytoskeletally weaker with respect to the filaments made from these specific proteins and that this is one reason why, in pre-eclampsia, trophoblast is deported in greater quantity than in healthy placentae. PMID:18466353

  15. Temporal impact of substrate mechanics on differentiation of human embryonic stem cells to cardiomyocytes.

    PubMed

    Hazeltine, Laurie B; Badur, Mehmet G; Lian, Xiaojun; Das, Amritava; Han, Wenqing; Palecek, Sean P

    2014-02-01

    A significant clinical need exists to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes, enabling tissue modeling for in vitro discovery of new drugs or cell-based therapies for heart repair in vivo. Chemical and mechanical microenvironmental factors are known to impact the efficiency of stem cell differentiation, but cardiac differentiation protocols in hPSCs are typically performed on rigid tissue culture polystyrene (TCPS) surfaces, which do not present a physiological mechanical setting. To investigate the temporal effects of mechanics on cardiac differentiation, we cultured human embryonic stem cells (hESCs) and their derivatives on polyacrylamide hydrogel substrates with a physiologically relevant range of stiffnesses. In directed differentiation and embryoid body culture systems, differentiation of hESCs to cardiac troponin T-expressing (cTnT+) cardiomyocytes peaked on hydrogels of intermediate stiffness. Brachyury expression also peaked on intermediate stiffness hydrogels at day 1 of directed differentiation, suggesting that stiffness impacted the initial differentiation trajectory of hESCs to mesendoderm. To investigate the impact of substrate mechanics during cardiac specification of mesodermal progenitors, we initiated directed cardiomyocyte differentiation on TCPS and transferred cells to hydrogels at the Nkx2.5/Isl1+ cardiac progenitor cell stage. No differences in cardiomyocyte purity with stiffness were observed on day 15. These experiments indicate that differentiation of hESCs is sensitive to substrate mechanics at early stages of mesodermal induction, and proper application of substrate mechanics can increase the propensity of hESCs to differentiate to cardiomyocytes.

  16. Signatures of human NK cell development and terminal differentiation.

    PubMed

    Luetke-Eversloh, Merlin; Killig, Monica; Romagnani, Chiara

    2013-01-01

    Natural killer (NK) cells are part of the innate lymphoid cell (ILC) family and represent the main cytotoxic population. NK cells develop from bone marrow common lymphoid progenitors and undergo terminal differentiation in the periphery, where they finally gain their cytotoxic competence as well as the ability to produce IFN-γ in response to engagement of activating receptors. This process has been at least partially elucidated and several markers have been identified to discriminate different NK cell stages and other ILC populations. NK cell terminal differentiation is not only associated with progressive phenotypic changes but also with defined effector signatures. In this essay, we will describe the phenotypic and functional characteristics of the main stages of NK cell development and terminal differentiation and discuss them in light of recent discoveries of novel ILC populations.

  17. Signatures of Human NK Cell Development and Terminal Differentiation

    PubMed Central

    Luetke-Eversloh, Merlin; Killig, Monica; Romagnani, Chiara

    2013-01-01

    Natural killer (NK) cells are part of the innate lymphoid cell (ILC) family and represent the main cytotoxic population. NK cells develop from bone marrow common lymphoid progenitors and undergo terminal differentiation in the periphery, where they finally gain their cytotoxic competence as well as the ability to produce IFN-γ in response to engagement of activating receptors. This process has been at least partially elucidated and several markers have been identified to discriminate different NK cell stages and other ILC populations. NK cell terminal differentiation is not only associated with progressive phenotypic changes but also with defined effector signatures. In this essay, we will describe the phenotypic and functional characteristics of the main stages of NK cell development and terminal differentiation and discuss them in light of recent discoveries of novel ILC populations. PMID:24416035

  18. Edges of human embryonic stem cell colonies display distinct mechanical properties and differentiation potential

    PubMed Central

    Rosowski, Kathryn A.; Mertz, Aaron F.; Norcross, Samuel; Dufresne, Eric R.; Horsley, Valerie

    2015-01-01

    In order to understand the mechanisms that guide cell fate decisions during early human development, we closely examined the differentiation process in adherent colonies of human embryonic stem cells (hESCs). Live imaging of the differentiation process reveals that cells on the outer edge of the undifferentiated colony begin to differentiate first and remain on the perimeter of the colony to eventually form a band of differentiation. Strikingly, this band is of constant width in all colonies, independent of their size. Cells at the edge of undifferentiated colonies show distinct actin organization, greater myosin activity and stronger traction forces compared to cells in the interior of the colony. Increasing the number of cells at the edge of colonies by plating small colonies can increase differentiation efficiency. Our results suggest that human developmental decisions are influenced by cellular environments and can be dictated by colony geometry of hESCs. PMID:26391588

  19. Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

    PubMed Central

    Liévin-Le Moal, Vanessa

    2013-01-01

    SUMMARY Hosts are protected from attack by potentially harmful enteric microorganisms, viruses, and parasites by the polarized fully differentiated epithelial cells that make up the epithelium, providing a physical and functional barrier. Enterovirulent bacteria interact with the epithelial polarized cells lining the intestinal barrier, and some invade the cells. A better understanding of the cross talk between enterovirulent bacteria and the polarized intestinal cells has resulted in the identification of essential enterovirulent bacterial structures and virulence gene products playing pivotal roles in pathogenesis. Cultured animal cell lines and cultured human nonintestinal, undifferentiated epithelial cells have been extensively used for understanding the mechanisms by which some human enterovirulent bacteria induce intestinal disorders. Human colon carcinoma cell lines which are able to express in culture the functional and structural characteristics of mature enterocytes and goblet cells have been established, mimicking structurally and functionally an intestinal epithelial barrier. Moreover, Caco-2-derived M-like cells have been established, mimicking the bacterial capture property of M cells of Peyer's patches. This review intends to analyze the cellular and molecular mechanisms of pathogenesis of human enterovirulent bacteria observed in infected cultured human colon carcinoma enterocyte-like HT-29 subpopulations, enterocyte-like Caco-2 and clone cells, the colonic T84 cell line, HT-29 mucus-secreting cell subpopulations, and Caco-2-derived M-like cells, including cell association, cell entry, intracellular lifestyle, structural lesions at the brush border, functional lesions in enterocytes and goblet cells, functional and structural lesions at the junctional domain, and host cellular defense responses. PMID:24006470

  20. Clinical and radiological correlations in patients with gestational trophoblastic disease*

    PubMed Central

    Lima, Lana de Lourdes Aguiar; Parente, Raphael Câmara Medeiros; Maestá, Izildinha; Amim Junior, Joffre; de Rezende Filho, Jorge Fonte; Montenegro, Carlos Antonio Barbosa; Braga, Antônio

    2016-01-01

    Gestational trophoblastic disease is an abnormality of pregnancy that encompasses a group of diseases that differ from each other in their propensity for regression, invasion, metastasis, and recurrence. In the past, it was common for patients with molar pregnancy to present with marked symptoms: copious bleeding; theca lutein cysts; uterus larger than appropriate for gestational age; early preeclampsia; hyperemesis gravidarum; and hyperthyroidism. Currently, with early diagnosis made by ultrasound, most patients are diagnosed while the disease is still in the asymptomatic phase. In cases of progression to trophoblastic neoplasia, staging-typically with Doppler flow studies of the pelvis and chest X-ray, although occasionally with computed tomography or magnetic resonance imaging-is critical to the choice of an appropriate antineoplastic therapy regimen. Because it is an unusual and serious disease that affects women of reproductive age, as well as because its appropriate treatment results in high cure rates, it is crucial that radiologists be familiar with gestational trophoblastic disease, in order to facilitate its early diagnosis and to ensure appropriate follow-up imaging. PMID:27777478

  1. STELLA Facilitates Differentiation of Germ Cell and Endodermal Lineages of Human Embryonic Stem Cells

    PubMed Central

    Wongtrakoongate, Patompon; Jones, Mark; Gokhale, Paul J.; Andrews, Peter W.

    2013-01-01

    Stella is a developmentally regulated gene highly expressed in mouse embryonic stem (ES) cells and in primordial germ cells (PGCs). In human, the gene encoding the STELLA homologue lies on chromosome 12p, which is frequently amplified in long-term cultured human ES cells. However, the role played by STELLA in human ES cells has not been reported. In the present study, we show that during retinoic acid (RA)-induced differentiation of human ES cells, expression of STELLA follows that of VASA, a marker of germline differentiation. By contrast, human embryonal carcinoma cells express STELLA at a higher level compared with both karyotypically normal and abnormal human ES cell lines. We found that over-expression of STELLA does not interfere with maintenance of the stem cell state of human ES cells, but following retinoic acid induction it leads to up-regulation of germline- and endodermal-associated genes, whereas neural markers PAX6 and NEUROD1 are down-regulated. Further, STELLA over-expression facilitates the differentiation of human ES cells into BE12-positive cells, in which the expression of germline- and endodermal-associated genes is enriched, and suppresses differentiation of the neural lineage. Taken together, this finding suggests a role for STELLA in facilitating germline and endodermal differentiation of human ES cells. PMID:23457636

  2. Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

    PubMed Central

    Di Pasquale, Elisa; Song, Belle; Condorelli, Gianluigi

    2013-01-01

    In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells. PMID:23851455

  3. [Frequency ratio of two forms of amitotic division of trophoblast cell nuclei in the mink blastocysts during the period of delayed implantation].

    PubMed

    Isakova, G K; Shilova, I E

    2003-01-01

    A comparative study of amitotic division activity of trophoblast cells by constriction and by extrusion in blastocysts of American mink during the obligatory period of delayed implantation has been carried out. The frequency of occurrence of amitotic figures was found to be nearly 10% at the onset of renewal of blastocyst growth (the blastocyst size was 0.4 mm in diameter), and nearly 20% at the stage of active growth (0.9 mm), as well as at the stage of expansion prior to blastocyst attachment to the uterine wall (1.7 mm). The ratios between the frequencies of division by extrusion and by constriction were 2:1, 5:1, and 4:1 at the three stages, respectively. We suggest that the cells generated via different amitotic ways play different roles in trophoblast differentiation. PMID:12942744

  4. Differential expression of the fractalkine chemokine receptor (CX3CR1) in human monocytes during differentiation

    PubMed Central

    Panek, Cecilia Analia; Ramos, Maria Victoria; Mejias, Maria Pilar; Abrey-Recalde, Maria Jimena; Fernandez-Brando, Romina Jimena; Gori, Maria Soledad; Salamone, Gabriela Verónica; Palermo, Marina Sandra

    2015-01-01

    Circulating monocytes (Mos) may continuously repopulate macrophage (MAC) or dendritic cell (DC) populations to maintain homeostasis. MACs and DCs are specialized cells that play different and complementary immunological functions. Accordingly, they present distinct migratory properties. Specifically, whereas MACs largely remain in tissues, DCs are capable of migrating from peripheral tissues to lymphoid organs. The aim of this work was to analyze the expression of the fractalkine receptor (CX3CR1) during the monocytic differentiation process. Freshly isolated Mos express high levels of both CX3CR1 mRNA and protein. During the Mo differentiation process, CX3CR1 is downregulated in both DCs and MACs. However, MACs showed significantly higher CX3CR1 expression levels than did DC. We also observed an antagonistic CX3CR1 regulation by interferon (IFN)-γ and interleukin (IL)-4 during MAC activation through the classical and alternative MAC pathways, respectively. IFN-γ inhibited the loss of CX3CR1, but IL-4 induced it. Additionally, we demonstrated an association between CX3CR1 expression and apoptosis prevention by soluble fractalkine (sCX3CL1) in Mos, DCs and MACs. This is the first report demonstrating sequential and differential CX3CR1 modulation during Mo differentiation. Most importantly, we demonstrated a functional link between CX3CR1 expression and cell survival in the presence of sCX3CL1. PMID:25502213

  5. Economic Development, Human Capital, and Gender Earnings Differentials in China

    ERIC Educational Resources Information Center

    Ng, Ying Chu

    2004-01-01

    Gender earnings differentials in China during the course of development in the post-reform period were examined. The analysis showed that the female-male earnings ratio increased over time in all regions. The region with relatively rapid economic reforms had the highest female-male earnings ratio. Decomposition of the gender earnings differential…

  6. Influence of human myasthenia gravis thymus on the differentiation of human cord blood stem cells in SCID mice.

    PubMed

    Li, Qian Ru; Liu, Ping Ping; Xuan, Xiao Yan; Guan, Sha Sha; Du, Ying; Gao, Feng; Zhang, Qing Yong

    2014-02-01

    The normal thymus contributes to T lymphocytes differentiation and induction of tolerance to self-antigens. Myasthenia gravis (MG) is characterized by abnormal thymic hyperplasia. To assess the potential influence of MG-thymus on the differentiation of T lymphocytes differentiation, we used the MG-thymus transplanted severe combined immunodeficiency (SCID) mice model to evaluate the human cord blood stem cells differentiation. Thymus fragments from MG patient and human cord blood stem cells were transplanted into SCID mice successively. SCID mice were observed to develop sustained human T lymphocytes and a functional anti-tumor immune. The levels of various T cell subsets in SCID mice with MG-thymus were different from that of control group. Among that, the frequency of CD4(+)CD25(+) T cells was significant lower in SCID mice with MG-thymus. The deficiency of CD4(+)CD25(+) T cells seens to contribute to the pathogenesis of MG.

  7. Degradation of extracellular matrix by mouse trophoblast outgrowths: a model for implantation

    PubMed Central

    Glass, RH; Aggeler, J; Spindle, A; Pederson, RA; Werb, Z

    1983-01-01

    During implantation the embryo attaches to the endometrial surface and trophoblast traverses the uterine epithelium, anchoring in the uterine connective tissue. To determine whether trophoblast can facilitate invasion of the uterus by degrading components of normal uterine extracellular matrix, mouse blastocysts were cultured on a radio-labeled extracellular matrix that contained glycoproteins, elastin, and collagen. The embryos attached to the matrix, and trophoblast spread over the surface. Starting on day 5 of culture there was a release of labeled peptides into the medium. The radioactive peptides released from the matrix by the embryos had molecular weights ranging from more than 25,000 to more than 200. By day 7 there were areas where individual trophoblast cells had separated from one another, revealing the underlying substratum that was cleared of matrix. When trophoblast cells were lysed with NH(4)OH on day 8, it was apparent that the area underneath the trophoblast outgrowth had been cleared of matrix. Scanning electron microscopy and time-lapse cinemicrography confirmed that the digestion of matrix was highly localized, taking place only underneath the trophoblast, with no evidence of digestion of the matrix beyond the periphery of the trophoblast outgrowth. The sharp boundaries of degredation observed may be due to localized proteinase secretion by trophoblast, to membrane proteinases on the surface of trophoblast, or to endocytosis. Digestion of the matrix was not dependent on plasminogen, thus ruling out a role for plasminogen activator. Digestion was not inhibited by a variety of hormones and inhibitors, including progesterone, 17β-estradiol, leupeptin, EDTA, colchicine, NH(4)Cl, or ε-aminocaproic acid. This system of culturing embryos on extracellular matrix may be useful in determining the processes that regulate trophoblast migration and invasion into the maternal tissues during implantation.0 PMID:6339525

  8. Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm

    PubMed Central

    Rasmussen, Camilla Holzmann; Petersen, Dorthe Roenn; Moeller, Jonas Bech

    2015-01-01

    Human embryonic stem cells have the ability to generate all cell types in the body and can potentially provide an unlimited source of cells for cell replacement therapy to treat degenerative diseases such as diabetes. Current differentiation protocols of human embryonic stem cells towards insulin producing beta cells focus on soluble molecules whereas the impact of cell-matrix interactions has been mainly unattended. In this study almost 500 different extracellular matrix protein combinations were screened to systemically identify extracellular matrix proteins that influence differentiation of human embryonic stem cells to the definitive endoderm lineage. The percentage of definitive endoderm cells after differentiation on collagen I and fibronectin was >85% and 65%, respectively. The cells on collagen I substrates displayed different morphology and gene expression during differentiation as assessed by time lapse studies compared to cells on the other tested substrates. Global gene expression analysis showed that cells differentiated on collagen I were largely similar to cells on fibronectin after completed differentiation. Collectively, the data suggest that collagen I induces a more rapid and consistent differentiation of stem cells to definitive endoderm. The results shed light on the importance of extracellular matrix proteins for differentiation and also points to a cost effective and easy method to improve differentiation. PMID:26713616

  9. Enhancement by retinoid of hemin-induced differentiation of human leukemia K562 cell line.

    PubMed

    Nakajima, O; Hashimoto, Y; Iwasaki, S

    1993-09-01

    The effect of retinoid on human leukemia K562 cell differentiation induced by hemin was examined. Retinoids (retinoic acid and synthetic retinoids [Am80 and Ch55]) dose-dependently enhanced hemin-induced erythroid differentiation of K562 cells, though these retinoids themselves did not induce the differentiation. Under optimal conditions, these retinoids caused a doubling of the population of hemin-induced differentiated cells. In addition, co-treatment of cells with hemin and retinoid led to longer maintenance of the differentiated state after the removal of hemin, which might imply acquisition of irreversibility of hemin-induced differentiation. These results suggest that the combination of retinoids with other differentiation inducers might be useful for leukemia therapy in cases where the leukemic cells are poorly responsive or unresponsive to retinoids, alone.

  10. Toxicity of organic and inorganic mercury species in differentiated human neurons and human astrocytes.

    PubMed

    Lohren, Hanna; Blagojevic, Lara; Fitkau, Romy; Ebert, Franziska; Schildknecht, Stefan; Leist, Marcel; Schwerdtle, Tanja

    2015-10-01

    Organic mercury (Hg) species exert their toxicity primarily in the central nervous system. The food relevant Hg species methylmercury (MeHg) has been frequently studied regarding its neurotoxic effects in vitro and in vivo. Neurotoxicity of thiomersal, which is used as a preservative in medical preparations, is to date less characterised. Due to dealkylation of organic Hg or oxidation of elemental Hg, inorganic Hg is present in the brain albeit these species are not able to readily cross the blood brain barrier. This study compared for the first time toxic effects of organic MeHg chloride (MeHgCl) and thiomersal as well as inorganic mercury chloride (HgCl2) in differentiated human neurons (LUHMES) and human astrocytes (CCF-STTG1). The three Hg species differ in their degree and mechanism of toxicity in those two types of brain cells. Generally, neurons are more susceptible to Hg species induced cytotoxicity as compared to astrocytes. This might be due to the massive cellular mercury uptake in the differentiated neurons. The organic compounds exerted stronger cytotoxic effects as compared to inorganic HgCl2. In contrast to HgCl2 exposure, organic Hg compounds seem to induce the apoptotic cascade in neurons following low-level exposure. No indicators for apoptosis were identified for both inorganic and organic mercury species in astrocytes. Our studies clearly demonstrate species-specific toxic mechanisms. A mixed exposure towards all Hg species in the brain can be assumed. Thus, prospectively coexposure studies as well as cocultures of neurons and astrocytes could provide additional information in the investigation of Hg induced neurotoxicity.

  11. The management of recurrent and drug-resistant gestational trophoblastic neoplasia (GTN).

    PubMed

    Newlands, E S

    2003-12-01

    Gestational trophoblastic neoplasia (GTN) comprises a spectrum of disease from low-risk disease which can be cured with simple relatively non-toxic treatment, to extremely aggressive tumours which require specialized management. The prognostic variables in patients with GTN are different from those in other gynaecological malignancies, and the major adverse prognostic variables include long interval from antecedent pregnancy, high concentrations of the pregnancy hormone, human chorionic gonadotrophin, metastases in brain and liver and failure of prior treatment. Patients who relapse after their prior treatment can also be categorized into different risk groups. Salvage treatment can vary from single agent actinomycin D to combination chemotherapy and, in selected cases, surgery. With appropriate management, the majority of patients can achieve long-term remission and, in most cases, preserve fertility. The late side-effects of more intensive treatment are a small risk of inducing second tumours and also of bringing forward the age of menopause.

  12. Early Differentiation within the Animate Domain: Are Humans Something Special?

    ERIC Educational Resources Information Center

    Pauen, Sabina

    2000-01-01

    Two experiments investigated whether preverbal infants distinguish between humans and mammals. Study 1 found that 7-, 9-, and 11-month-olds distinguished humans from mammals in an object-examination task. Study 2 found that 7-month-olds but not 5-month-olds showed evidence for category discrimination with the 2-dimensional color photos of toy…

  13. Differential sensitivity of Chironomus and human hemoglobin to gamma radiation.

    PubMed

    Gaikwad, Pallavi S; Panicker, Lata; Mohole, Madhura; Sawant, Sangeeta; Mukhopadhyaya, Rita; Nath, Bimalendu B

    2016-08-01

    Chironomus ramosus is known to tolerate high doses of gamma radiation exposure. Larvae of this insect possess more than 95% of hemoglobin (Hb) in its circulatory hemolymph. This is a comparative study to see effect of gamma radiation on Hb of Chironomus and humans, two evolutionarily diverse organisms one having extracellular and the other intracellular Hb respectively. Stability and integrity of Chironomus and human Hb to gamma radiation was compared using biophysical techniques like Dynamic Light Scattering (DLS), UV-visible spectroscopy, fluorescence spectrometry and CD spectroscopy after exposure of whole larvae, larval hemolymph, human peripheral blood, purified Chironomus and human Hb. Sequence- and structure-based bioinformatics methods were used to analyze the sequence and structural similarities or differences in the heme pockets of respective Hbs. Resistivity of Chironomus Hb to gamma radiation is remarkably higher than human Hb. Human Hb exhibited loss of heme iron at a relatively low dose of gamma radiation exposure as compared to Chironomus Hb. Unlike human Hb, the heme pocket of Chironomus Hb is rich in aromatic amino acids. Higher hydophobicity around heme pocket confers stability of Chironomus Hb compared to human Hb. Previously reported gamma radiation tolerance of Chironomus can be largely attributed to its evolutionarily ancient form of extracellular Hb as evident from the present study.

  14. Differential sensitivity of Chironomus and human hemoglobin to gamma radiation.

    PubMed

    Gaikwad, Pallavi S; Panicker, Lata; Mohole, Madhura; Sawant, Sangeeta; Mukhopadhyaya, Rita; Nath, Bimalendu B

    2016-08-01

    Chironomus ramosus is known to tolerate high doses of gamma radiation exposure. Larvae of this insect possess more than 95% of hemoglobin (Hb) in its circulatory hemolymph. This is a comparative study to see effect of gamma radiation on Hb of Chironomus and humans, two evolutionarily diverse organisms one having extracellular and the other intracellular Hb respectively. Stability and integrity of Chironomus and human Hb to gamma radiation was compared using biophysical techniques like Dynamic Light Scattering (DLS), UV-visible spectroscopy, fluorescence spectrometry and CD spectroscopy after exposure of whole larvae, larval hemolymph, human peripheral blood, purified Chironomus and human Hb. Sequence- and structure-based bioinformatics methods were used to analyze the sequence and structural similarities or differences in the heme pockets of respective Hbs. Resistivity of Chironomus Hb to gamma radiation is remarkably higher than human Hb. Human Hb exhibited loss of heme iron at a relatively low dose of gamma radiation exposure as compared to Chironomus Hb. Unlike human Hb, the heme pocket of Chironomus Hb is rich in aromatic amino acids. Higher hydophobicity around heme pocket confers stability of Chironomus Hb compared to human Hb. Previously reported gamma radiation tolerance of Chironomus can be largely attributed to its evolutionarily ancient form of extracellular Hb as evident from the present study. PMID:27237970

  15. Differentiation of human alloreactive CD8+ T cells in vitro

    PubMed Central

    Rentenaar, Rob J; Vosters, Jelle L G; Van Diepen, Frank N J; Remmerswaal, Ester B M; Van Lier, René A W; Ten Berge, Ineke J M

    2002-01-01

    Expansion and differentiation of alloantigen-reactive CD8+ T cells in mixed lymphocyte cultures was followed by measurement of the loss of carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescence of responder cells. Proliferation of CD8+ T cells became detectable on day 4 of culture and, 2 days later, > 60% of the CD8+ T cells in culture were dividing alloreactive lymphocytes. In parallel with expansion, CD8+ T-cell differentiation was initiated, as evidenced by an increase in the number of CD45RA− and CD27− T cells and acquisition of the ability to produce interferon-γ after restimulation with the specific alloantigen. Finally, although short-term stimulation and measurement of intracellular cytokine production allowed visualization of alloreactive CD8+ T cells expanded in vitro, this procedure did not detect circulating alloreactive CD8+ T cells activated in vivo in recipients of allogeneic kidney grafts. PMID:11918689

  16. Vav promotes differentiation of human tumoral myeloid precursors

    SciTech Connect

    Bertagnolo, Valeria; Brugnoli, Federica; Mischiati, Carlo; Sereni, Alessia; Bavelloni, Alberto; Carini, Cinzia; Capitani, Silvano . E-mail: cps@unife.it

    2005-05-15

    Vav is one of the genetic markers that correlate with the differentiation of hematopoietic cells. In T and B cells, it appears crucial for both development and functions, while, in non-lymphoid hematopoietic cells, Vav seems not involved in cell maturation, but rather in the response of mature cells to agonist-dependent proliferation and phagocytosis. We have previously demonstrated that the amount and the tyrosine phosphorylation of Vav are up-regulated in both whole cells and nuclei of tumoral promyelocytes induced to granulocytic maturation by ATRA and that tyrosine-phosphorylated Vav does not display any ATRA-induced GEF activity but contributes to the regulation of PI 3-K activity. In this study, we report that Vav accumulates in nuclei of ATRA-treated APL-derived cells and that the down-modulation of Vav prevents differentiation of tumoral promyelocytes, indicating that it is a key molecule in ATRA-dependent myeloid maturation. On the other hand, the overexpression of Vav induces an increased expression of surface markers of granulocytic differentiation without affecting the maturation-related changes of the nuclear morphology. Consistent with an effect of Vav on the transcriptional machinery, array profiling shows that the inhibition of the Syk-dependent tyrosine phosphorylation of Vav reduces the number of ATRA-induced genes. Our data support the unprecedented notion that Vav plays crucial functions in the maturation process of myeloid cells, and suggest that Vav can be regarded as a potential target for the therapeutic treatment of myeloproliferative disorders.

  17. Glycosaminoglycans enhance osteoblast differentiation of bone marrow derived human mesenchymal stem cells.

    PubMed

    Mathews, Smitha; Mathew, Suja Ann; Gupta, Pawan Kumar; Bhonde, Ramesh; Totey, Satish

    2014-02-01

    Extracellular matrix plays an important role in regulating cell growth and differentiation. The biomimetic approach of cell-based tissue engineering is based on mirroring this in vivo micro environment for developing a functional tissue engineered construct. In this study, we treated normal tissue culture plates with selected extracellular matrix components consisting of glycosaminoglycans such as chondroitin-4-sulphate, dermatan sulphate, chondroitin-6-sulphate, heparin and hyaluronic acid. Mesenchymal stem cells isolated from adult human bone marrow were cultured on the glycosaminoglycan treated culture plates to evaluate their regulatory role in cell growth and osteoblast differentiation. Although no significant improvement on human mesenchymal stem cell adhesion and proliferation was observed on the glycosaminoglycan-treated tissue culture plates, there was selective osteoblast differentiation, indicating its potential role in differentiation rather than proliferation. Osteoblast differentiation studies showed high osteogenic potential for all tested glycosaminoglycans except chondroitin-4-sulphate. Osteoblast differentiation-associated genes such as osterix, osteocalcin, integrin binding sialoprotein, osteonectin and collagen, type 1, alpha 1 showed significant upregulation. We identified osterix as the key transcription factor responsible for the enhanced bone matrix deposition observed on hyaluronic acid, heparin and chondroitin-6-sulphate. Hyaluronic acid provided the most favourable condition for osteoblast differentiation and bone matrix synthesis. Our results confirm and emphasise the significant role of extracellular matrix in regulating cell differentiation. To summarise, glycosaminoglycans of extracellular matrix played a significant role in regulating osteoblast differentiation and could be exploited in the biomimetic approach of fabricating or functionalizing scaffolds for stem cell based bone tissue engineering.

  18. Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance

    PubMed Central

    Fedorov, Oleg; Castex, Josefina; Tallant, Cynthia; Owen, Dafydd R.; Martin, Sarah; Aldeghi, Matteo; Monteiro, Octovia; Filippakopoulos, Panagis; Picaud, Sarah; Trzupek, John D.; Gerstenberger, Brian S.; Bountra, Chas; Willmann, Dominica; Wells, Christopher; Philpott, Martin; Rogers, Catherine; Biggin, Philip C.; Brennan, Paul E.; Bunnage, Mark E.; Schüle, Roland; Günther, Thomas; Knapp, Stefan; Müller, Susanne

    2015-01-01

    Mammalian SWI/SNF [also called Brg/Brahma-associated factors (BAFs)] are evolutionarily conserved chromatin-remodeling complexes regulating gene transcription programs during development and stem cell differentiation. BAF complexes contain an ATP (adenosine 5′-triphosphate)–driven remodeling enzyme (either BRG1 or BRM) and multiple protein interaction domains including bromodomains, an evolutionary conserved acetyl lysine–dependent protein interaction motif that recruits transcriptional regulators to acetylated chromatin. We report a potent and cell active protein interaction inhibitor, PFI-3, that selectively binds to essential BAF bromodomains. The high specificity of PFI-3 was achieved on the basis of a novel binding mode of a salicylic acid head group that led to the replacement of water molecules typically maintained in other bromodomain inhibitor complexes. We show that exposure of embryonic stem cells to PFI-3 led to deprivation of stemness and deregulated lineage specification. Furthermore, differentiation of trophoblast stem cells in the presence of PFI-3 was markedly enhanced. The data present a key function of BAF bromodomains in stem cell maintenance and differentiation, introducing a novel versatile chemical probe for studies on acetylation-dependent cellular processes controlled by BAF remodeling complexes. PMID:26702435

  19. Human embryonic stem cells differentiate into functional renal proximal tubular-like cells.

    PubMed

    Narayanan, Karthikeyan; Schumacher, Karl M; Tasnim, Farah; Kandasamy, Karthikeyan; Schumacher, Annegret; Ni, Ming; Gao, Shujun; Gopalan, Began; Zink, Daniele; Ying, Jackie Y

    2013-04-01

    Renal cells are used in basic research, disease models, tissue engineering, drug screening, and in vitro toxicology. In order to provide a reliable source of human renal cells, we developed a protocol for the differentiation of human embryonic stem cells into renal epithelial cells. The differentiated stem cells expressed markers characteristic of renal proximal tubular cells and their precursors, whereas markers of other renal cell types were not expressed or expressed at low levels. Marker expression patterns of these differentiated stem cells and in vitro cultivated primary human renal proximal tubular cells were comparable. The differentiated stem cells showed morphological and functional characteristics of renal proximal tubular cells, and generated tubular structures in vitro and in vivo. In addition, the differentiated stem cells contributed in organ cultures for the formation of simple epithelia in the kidney cortex. Bioreactor experiments showed that these cells retained their functional characteristics under conditions as applied in bioartificial kidneys. Thus, our results show that human embryonic stem cells can differentiate into renal proximal tubular-like cells. Our approach would provide a source for human renal proximal tubular cells that are not affected by problems associated with immortalized cell lines or primary cells.

  20. MIR146A inhibits JMJD3 expression and osteogenic differentiation in human mesenchymal stem cells

    PubMed Central

    Huszar, Jessica M.; Payne, Christopher J.

    2014-01-01

    Chromatin remodeling is important for cell differentiation. Histone methyltransferase EZH2 and histone demethylase JMJD3 (KDM6B) modulate levels of histone H3 lysine 27 trimethylation (H3K27me3). Interplay between the two modulators influence lineage specification in stem cells. Here, we identified microRNA MIR146A to be a negative regulator of JMJD3. In the osteogenic differentiation of human mesenchymal stem cells (hMSCs), we observed an upregulation of JMJD3 and a downregulation of MIR146A. Blocking JMJD3 activity in differentiating hMSCs reduced transcript levels of osteogenic gene RUNX2. H3K27me3 levels decreased at the RUNX2 promoter during cell differentiation. Modulation of MIR146A levels in hMSCs altered JMJD3 and RUNX2 expression and affected osteogenic differentiation. We conclude that JMJD3 promotes osteogenesis in differentiating hMSCs, with MIR146A regulating JMJD3. PMID:24726732

  1. Differentiation of Cardiomyocytes from Human Pluripotent Stem Cells Using Monolayer Culture

    PubMed Central

    Batalov, Ivan; Feinberg, Adam W

    2015-01-01

    Human pluripotent stem cells (PSCs) are a promising cell source for cardiac tissue engineering and cell-based therapies for heart repair because they can be expanded in vitro and differentiated into most cardiovascular cell types, including cardiomyocytes. During embryonic heart development, this differentiation occurs under the influence of internal and external stimuli that guide cells to go down the cardiac lineage. In order to differentiate PSCs in vitro, these or similar stimuli need to be provided in a controlled manner. However, because it is not possible to completely recapitulate the embryonic environment, the factors essential for cardiac differentiation of PSCs in vitro need to be experimentally determined and validated. Since PSCs were first developed, significant progress has been made in optimizing techniques for their differentiation toward cardiomyocytes. In this review, we will summarize recent advances in these techniques, with particular focus on monolayer-based methods that have improved the efficiency and scalability of cardiomyocyte differentiation. PMID:26052225

  2. Differential distribution of calpain in human lymphoid cells.

    PubMed

    Deshpande, R V; Goust, J M; Banik, N L

    1993-07-01

    Calpain, a calcium-activated neutral proteinase, is ubiquitously present in human tissues. To determine if lymphoid cells implicated in pathogenesis of demyelination may harbor calpain in a functionally active form, we determined both muCalpain and mCalpain activities in human lymphoid cell lines. DEAE-cellulose and phenylsepharose column chromatography were used to isolate the enzyme from the natural inhibitor, calpastatin. Lymphocytic lines (CCRF-CEM, MOLT-3, MOLT-4, M.R.) showed predominance of muCalpain (55-80%) whereas the monocytic line (U-937) showed predominance of mCalpain (77%). Proportion and subcellular distribution of both isoforms varied among cell lines. Calpains isolated from U-937 cells degraded myelin basic protein. These results indicate that human lymphoid cells harbor functionally active calpain that can degrade myelin components in vitro. The study suggests a degradative role for calpain in demyelinating diseases. PMID:7690115

  3. Immune parameters differentiating active from latent tuberculosis infection in humans.

    PubMed

    Lee, Ji Yeon; Jung, Young Won; Jeong, Ina; Joh, Joon-Sung; Sim, Soo Yeon; Choi, Boram; Jee, Hyeon-Gun; Lim, Dong-Gyun

    2015-12-01

    Tuberculosis remains a highly prevalent infectious disease worldwide. Identification of the immune parameters that differentiate active disease from latent infection will facilitate the development of efficient control measures as well as new diagnostic modalities for tuberculosis. Here, we investigated the cytokine production profiles of monocytes and CD4(+) T lymphocytes upon encountering mycobacterial antigens. In addition, cytokines and lipid mediators with immune-modulating activities were examined in plasma samples ex vivo. Comparison of these parameters in active tuberculosis patients and healthy subjects with latent infection revealed that, active tuberculosis was associated with diminished Th1-type cytokine secretion from CD4(+) T cells and less augmented inflammatory cytokine secretion from monocytes induced by IFN-γ than that in latent tuberculosis infection. In addition, a higher plasma concentration of lipoxin A4 and lower ratio of prostaglandin E2 to lipoxin A4 were observed in active cases than in latent infections. These findings have implications for preparing new therapeutic strategies and for differential diagnosis of the two types of tuberculosis infection.

  4. Effects of Wnt3a on proliferation and differentiation of human epidermal stem cells

    SciTech Connect

    Jia Liwei; Zhou Jiaxi; Peng Sha; Li Juxue; Cao Yujing; Duan Enkui

    2008-04-11

    Epidermal stem cells maintain development and homeostasis of mammalian epidermis throughout life. However, the molecular mechanisms involved in the proliferation and differentiation of epidermal stem cells are far from clear. In this study, we investigated the effects of Wnt3a and Wnt/{beta}-catenin signaling on proliferation and differentiation of human fetal epidermal stem cells. We found both Wnt3a and active {beta}-catenin, two key members of the Wnt/{beta}-catenin signaling, were expressed in human fetal epidermis and epidermal stem cells. In addition, Wnt3a protein can promote proliferation and inhibit differentiation of epidermal stem cells in vitro culture. Our results suggest that Wnt/{beta}-catenin signaling plays important roles in human fetal skin development and homeostasis, which also provide new insights on the molecular mechanisms of oncogenesis in human epidermis.

  5. Neural Differentiation in the Third Dimension: Generating a Human Midbrain.

    PubMed

    Marton, Rebecca M; Paşca, Sergiu P

    2016-08-01

    In recent years, technological improvements in three-dimensional (3D) culture systems have enabled the generation of organoids or spheroids representing a variety of tissues, including the brain. In this issue of Cell Stem Cell, Jo et al. (2016) describe a 3D culture model of the human midbrain containing dopaminergic neurons and neuromelanin. PMID:27494668

  6. Transcriptional PROFILING OF MUCOCILIARY DIFFERENTIATION IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    When cultured at an air-liquid interface (ALI) in the appropriate medium, primary human airway epithelial cells form a polarized, pseudostratified epithelium composed of ciliated and mucus-secreting cells. This culture system provides a useful tool for the in vitro study of...

  7. Effect of Age on Regulation of Human Osteoclast Differentiation

    PubMed Central

    Chung, Ping-Lin; Zhou, Shuanhu; Eslami, Behnam; Shen, Longxiang; LeBoff, Meryl S.; Glowacki, Julie

    2014-01-01

    Human skeletal aging is characterized as a gradual loss of bone mass due to an excess of bone resorption not balanced by new bone formation. Using human marrow cells, we tested the hypothesis that there is an age-dependent increase in osteoclastogenesis due to intrinsic changes in regulatory factors [macrophage-colony stimulating factor (M-CSF), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG)] and their receptors [c-fms and RANK]. In bone marrow cells (BMCs), c-fms (r=0.61, p=0.006) and RANK expression (r=0.59, p=0.008) were increased with age (27-82 years, n=19). In vitro generation of osteoclasts was increased with age (r=0.89, p=0.007). In enriched marrow stromal cells (MSCs), constitutive expression of RANKL was increased with age (r=0.41, p=0.049) and expression of OPG was inversely correlated with age (r=-0.43, p=0.039). Accordingly, there was an age-related increase in RANKL/OPG (r=0.56, p=0.005). These data indicate an age-related increase in human osteoclastogenesis that is associated with an intrinsic increase in expression of c-fms and RANK in osteoclast progenitors, and, in the supporting MSCs, an increase in pro-osteoclastogenic RANKL expression and a decrease in anti-osteoclastogenic OPG. These findings support the hypothesis that human marrow cells and their products can contribute to skeletal aging by increasing the generation of bone-resorbing osteoclasts. These findings help to explain underlying molecular mechanisms of progressive bone loss with advancing age in humans. PMID:24700654

  8. Erythropoietic differentiation of a human embryonic stem cell line harbouring the sickle cell anaemia mutation

    PubMed Central

    Pryzhkova, Marina V; Peters, Ann; Zambidis, Elias T

    2012-01-01

    Herein is reported efficient erythropoietic differentiation of a human embryonic stem cell (ESC) line derived from a preimplantation genetic diagnosis (PGD)-screened embryo that harbours the homozygous sickle cell disease (SCD) haemoglobinopathy mutation. This human ESC line possesses typical pluripotency characteristics and forms multilineage teratomas in vivo. SCD-human ESC efficiently differentiated to the haematopoietic lineage under serum-free and stromal co-culture conditions and gave rise to robust primitive and definitive erythrocytes. Expression of embryonic, fetal and adult sickle globin genes in SCD PGD-derived human ESC-derived erythrocytes was confirmed by quantitative real-time PCR, intracytoplasmic fluorescence-activated cell sorting and insitu immunostaining of PGD-derived human ESC teratoma sections. These data introduce important methodologies and paradigms for using patient-specific human ESC to generate normal and haemoglobinopathic erythroid progenitors for biomedical research. PMID:20541472

  9. Estrogen treatment enhances neurogenic differentiation of human adipose derived stem cells in vitro

    PubMed Central

    Razavi, Shahnaz; Razavi, Mohamad Reza; Ahmadi, Nafiseh; Kazemi, Mohammad

    2015-01-01

    Objective(s): Estrogen is a sexual hormone that has prominent effects on reproductive and non-reproductive tissues. The aim of this study is to evaluate the effects of estrogen on the proliferation and neural differentiation of human adipose derived stem cells (ADSCs) during neurogenic differentiation. Materials and Methods: Isolated human ADSCs were trans-differentiated in neural induction medium containing neurobasal medium, N2 and B27 with or without 17β-estradiol (E2) treatment. Proliferation rate and neural differentiation of human ADSCs were assessed using MTT assay, immunostaining and real time RT- PCR analysis, respectively. Results: Analysis of data show that estradiol treatment can significantly increase proliferation rate of differentiated cells (P<0.05). Immunocytochemical and real time RT-PCR analysis revealed that the expression of precursor and mature neuronal markers (nestin and MAP2) was significantly higher in the E2 treated cell cultures when compared to the untreated cell cultures (P<0.05). Conclusion: According to our findings, estrogen can promote proliferation and neuronal differentiation of human ADSCs. PMID:26557969

  10. Glycogen synthase kinase 3β inhibition promotes human iTreg differentiation and suppressive function.

    PubMed

    Xia, Yongxiang; Zhuo, Han; Lu, Yunjie; Deng, Lei; Jiang, Runqiu; Zhang, Long; Zhu, Qin; Pu, Liyong; Wang, Xuehao; Lu, Ling

    2015-05-01

    Induced regulatory T cells (iTregs) are essential to maintain immunological tolerance, immune homeostasis and prevention of autoimmunity. Some studies suggest that glycogen synthase kinase 3β (GSK3β) is involved in the mouse iTreg differentiation; however, whether GSK3β inhibits or enhances iTreg differentiation is still a matter of controversy. To address this issue, we have utilized human naïve CD4(+) T cells and investigated whether GSK3 activity changes during iTreg differentiation and whether altering GSK3 activity influences the development of iTregs and its suppressive function. As a constitutively activated kinase, during iTreg differentiation GSK3β became quickly deactivated (phosphorylated at serine 9), which is dependent on MAPK pathway rather than PI3-kinase/Akt pathway. Our results indicated that inhibition of GSK3β by specific inhibitors, SB216763 or TDZD-8, promoted the differentiation of iTreg and increased their suppressive activity. In contrast, overexpression of GSK3β significantly inhibited iTreg differentiation. Furthermore, GSK3β inhibition enhanced iTreg differentiation through the TGF-β/Smad3 pathway. Taken together, this study demonstrates that inhibition of GSK3β enhances human iTreg differentiation and its suppressive activity, and provides a rationale to target GSK3β as a novel immunotherapeutic strategy.

  11. Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells.

    PubMed

    Kajiwara, Masatoshi; Aoi, Takashi; Okita, Keisuke; Takahashi, Ryosuke; Inoue, Haruhisa; Takayama, Naoya; Endo, Hiroshi; Eto, Koji; Toguchida, Junya; Uemoto, Shinji; Yamanaka, Shinya

    2012-07-31

    Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, the in vitro directed differentiation of human pluripotent stem cells into mature hepatocytes remains challenging. Little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. In the current study, we developed an improved hepatic differentiation protocol and compared 28 hiPSC lines originated from various somatic cells and derived using retroviruses, Sendai viruses, or episomal plasmids. This comparison indicated that the origins, but not the derivation methods, may be a major determinant of variation in hepatic differentiation. The hiPSC clones derived from peripheral blood cells consistently showed good differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts showed poor differentiation. However, when we compared hiPSCs from peripheral blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. These data underscore the importance of donor differences when comparing the differentiation propensities of hiPSC clones.

  12. Enhanced differentiation of human osteoblasts on Ti surfaces pre-treated with human whole blood.

    PubMed

    Kopf, Brigitte S; Schipanski, Angela; Rottmar, Markus; Berner, Simon; Maniura-Weber, Katharina

    2015-06-01

    Early and effective integration of a metal implant into bone tissue is of crucial importance for its long-term stability. While different material properties including surface roughness and wettability but also initial blood-implant surface interaction are known to influence this osseointegration, implications of the latter process are still poorly understood. In this study, early interaction between blood and the implant surface and how this affects the mechanism of osseointegration were investigated. For this, blood coagulation on a micro-roughened hydrophobic titanium (Ti) surface (SLA-H(phob)) and on a hydrophilic micro-roughened Ti surface with nanostructures (SLActive-H(phil)NS), as well as the effects of whole human blood pre-incubation of these two surfaces on the differentiation potential of primary human bone cells (HBC) was assessed. Interestingly, pre-incubation with blood resulted in a dense fibrin network over the entire surface on SLActive-H(phil)NS but only in single patches of fibrin and small isolated fibre complexes on SLA-H(phob). On SLActive-H(phil)NS, the number of HBCs attaching to the fibrin network was greatly increased and the cells displayed enhanced cell contact to the fibrin network. Notably, HBCs displayed increased expression of the osteogenic marker proteins alkaline phosphatase and collagen-I when cultivated on both surfaces upon blood pre-incubation. Additionally, blood pre-treatment promoted an earlier and enhanced mineralization of HBCs cultivated on SLActive-H(phil)NS compared to SLA-H(phob). The results presented in this study therefore suggest that blood pre-incubation of implant surfaces mimics a more physiological situation, eventually providing a more predictive in vitro model for the evaluation of novel bone implant surfaces.

  13. Uterine arteriovenous malformations following gestational trophoblastic neoplasia: a systematic review.

    PubMed

    Touhami, Omar; Gregoire, Jean; Noel, Patricia; Trinh, Xuan Bich; Plante, Marie

    2014-10-01

    Uterine arteriovenous malformation (AVM) following gestational trophoblastic neoplasia (GTN) is a rare condition. It can be associated with chronic vaginal bleeding or life-threatening heavy bleeding, even after complete resolution of the tumor following chemotherapy. This analysis aimed to perform an extensive systematic review highlighting clinical symptoms, imaging, management and prognosis of this rare complication of GTN. We also describe an additional case of uterine AVM following GTN. We conducted a literature search using Medline, Embase and Cochrane library to analyze the clinical data of 49 published cases of uterine AVM following GTN. Median age of the women diagnosed with AVM was 29 years (range 15-49). Median gravidity was 2 (range 1-8) and 50% of women were nulligravida. Complete molar pregnancy was the most common initial gestational trophoblastic diagnosis (48%). Overall, 44 patients (88%) were symptomatic and presented with chronic or acute abnormal vaginal bleeding. Only 3 patients had an undetectable HCG level at the time of uterine AVM diagnosis. Hypo-echoic space in the myometrium is the most relevant finding on ultrasonography but the gold standard for the definitive diagnosis of AVMs is angiographic examination. Uterine artery embolization was the most common treatment option performed in 82% of the patients and was successful in controlling the bleeding in 85% of cases. We identified 20 pregnancies after successful embolization of uterine AVM following a GTN and 90% of them were successful. Because of the risk of life-threatening heavy bleeding, the diagnosis of uterine AVM should always be considered in patients with a history of recurrent unexplained vaginal bleeding after gestational trophoblastic neoplasia. Angiographic embolization is successful in the majority of cases and does not appear to compromise future pregnancy.

  14. Involvement of miRNAs in the Differentiation of Human Glioblastoma Multiforme Stem-Like Cells

    PubMed Central

    Aldaz, Beatriz; Sagardoy, Ainara; Nogueira, Lorena; Guruceaga, Elizabeth; Grande, Lara; Huse, Jason T.; Aznar, Maria A.; Díez-Valle, Ricardo; Tejada-Solís, Sonia; Alonso, Marta M.; Fernandez-Luna, Jose L.

    2013-01-01

    Glioblastoma multiforme (GBM)-initiating cells (GICs) represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process. PMID:24155920

  15. Non-coding RNAs in pluripotency and neural differentiation of human pluripotent stem cells.

    PubMed

    Lukovic, Dunja; Moreno-Manzano, Victoria; Klabusay, Martin; Stojkovic, Miodrag; Bhattacharya, Shomi S; Erceg, Slaven

    2014-01-01

    Several studies have demonstrated the important role of non-coding RNAs as regulators of posttranscriptional processes, including stem cells self-renewal and neural differentiation. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (ihPSCs) show enormous potential in regenerative medicine due to their capacity to differentiate to virtually any type of cells of human body. Deciphering the role of non-coding RNAs in pluripotency, self-renewal and neural differentiation will reveal new molecular mechanisms involved in induction and maintenances of pluripotent state as well as triggering these cells toward clinically relevant cells for transplantation. In this brief review we will summarize recently published studies which reveal the role of non-coding RNAs in pluripotency and neural differentiation of hESCs and ihPSC.

  16. Elsevier Trophoblast Research Award Lecture: origin, evolution and future of placenta miRNAs.

    PubMed

    Morales-Prieto, D M; Ospina-Prieto, S; Schmidt, A; Chaiwangyen, W; Markert, U R

    2014-02-01

    MicroRNAs (miRNAs) regulate the expression of a large number of genes in plants and animals. Placental miRNAs appeared late in evolution and can be found only in mammals. Nevertheless, these miRNAs are constantly under evolutionary pressure. As a consequence, miRNA sequences and their mRNA targets may differ between species, and some miRNAs can only be found in humans. Their expression can be tissue- or cell-specific and can vary time-dependently. Human placenta tissue exhibits a specific miRNA expression pattern that dynamically changes during pregnancy and is reflected in the maternal plasma. Some placental miRNAs are involved in or associated with major pregnancy disorders, such as preeclampsia, intrauterine growth restriction or preterm delivery and, therefore, have a strong potential for usage as sensitive and specific biomarkers. In this review we summarize current knowledge on the origin of placental miRNAs, their expression in humans with special regard to trophoblast cells, interspecies differences, and their future as biomarkers. It can be concluded that animal models for human reproduction have a different panel of miRNAs and targets, and can only partly reflect or predict the situation in humans.

  17. Fibroblast growth factor-2 stimulates adipogenic differentiation of human adipose-derived stem cells

    SciTech Connect

    Kakudo, Natsuko . E-mail: kakudon@takii.kmu.ac.jp; Shimotsuma, Ayuko; Kusumoto, Kenji

    2007-07-27

    Adipose-derived stem cells (ASCs) have demonstrated a capacity for differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli and specific growth and factors. It is acknowledged that fibroblast growth factor-2 (FGF-2) promotes chondrogenic and inhibits osteogenic differentiation of ASCs, but thorough investigations of its effects on adipogenic differentiation are lacking. In this study, we demonstrate at the cellular and molecular levels the effect of FGF-2 on adipogenic differentiation of ASCs, as induced by an adipogenic hormonal cocktail consisting of 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and indomethacin. FGF-2 significantly enhances the adipogenic differentiation of human ASCs. Furthermore, in cultures receiving FGF-2 before adipogenic induction, mRNA expression of peroxisome proliferator-activated receptor {gamma}2 (PPAR{gamma}2), a key transcription factor in adipogenesis, was upregulated. The results of FGF-2 supplementation suggest the potential applications of FGF-2 and ASCs in adipose tissue regeneration.

  18. The Hematopoietic Differentiation and Production of Mature Myeloid Cells from Human Pluripotent Stem Cells

    PubMed Central

    Choi, Kyung-Dal; Vodyanik, Maxim; Slukvin, Igor I.

    2011-01-01

    Here we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin-CD34+CD43+CD45+ multipotent progenitors. The protocol is comprised of three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells, (ii) short-term expansion of multipotent myeloid progenitors with a high dose of GM-CSF, and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells (DCs), Langerhans cells (LCs), macrophages, and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 days of culture, and an additional 2 days are needed to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5–19 days of culture with cytokines, depending on the cell type. PMID:21372811

  19. Differential regional effects of octreotide on human gastrointestinal motor function.

    PubMed Central

    von der Ohe, M R; Camilleri, M; Thomforde, G M; Klee, G G

    1995-01-01

    The effects of octreotide on regional motor function in the human gut are unclear. In a randomised, blinded study the effects of octreotide (50 micrograms, subcutaneously, three times daily) and placebo on gastric, small bowel, and colonic transit, and colonic motility and tone were assessed in 12 healthy volunteers whose colon had been cleansed. Octreotide accelerated initial gastric emptying (p = 0.05), inhibited small bowel transit (p < 0.01), and reduced ileocolonic bolus transfers (p < 0.05). Colonic transit was unaltered by octreotide; the postprandial colonic tonic response was inhibited (p < 0.05 v placebo), whereas colonic phasic pressure activity was increased by octreotide (p < 0.05 v placebo). These data support the use of octreotide in diarrhoeal states but not in diseases that cause small bowel stasis and bacterial overgrowth. Simultaneous measurements of colonic transit, tone, and phasic contractility are valid in studying the effects of pharmacological changes and may be applicable to the study of the human colon in health and disease. PMID:7797125

  20. Intrahaplotypic Variants Differentiate Complex Linkage Disequilibrium within Human MHC Haplotypes.

    PubMed

    Lam, Tze Hau; Tay, Matthew Zirui; Wang, Bei; Xiao, Ziwei; Ren, Ee Chee

    2015-11-23

    Distinct regions of long-range genetic fixation in the human MHC region, known as conserved extended haplotypes (CEHs), possess unique genomic characteristics and are strongly associated with numerous diseases. While CEHs appear to be homogeneous by SNP analysis, the nature of fine variations within their genomic structure is unknown. Using multiple, MHC-homozygous cell lines, we demonstrate extensive sequence conservation in two common Asian MHC haplotypes: A33-B58-DR3 and A2-B46-DR9. However, characterization of phase-resolved MHC haplotypes revealed unique intra-CEH patterns of variation and uncovered 127 single nucleotide variants (SNVs) which are missing from public databases. We further show that the strong linkage disequilibrium structure within the human MHC that typically confounds precise identification of genetic features can be resolved using intra-CEH variants, as evidenced by rs3129063 and rs448489, which affect expression of ZFP57, a gene important in methylation and epigenetic regulation. This study demonstrates an improved strategy that can be used towards genetic dissection of diseases.

  1. Clonal, Self-Renewing and Differentiating Human and Porcine Urothelial Cells, a Novel Stem Cell Population

    PubMed Central

    Larsson, Hans M.; Gorostidi, Francois; Hubbell, Jeffrey A.; Barrandon, Yann; Frey, Peter

    2014-01-01

    Although urothelial progenitor-like cells have been described in the human urinary tract, the existence of stem cells remains to be proven. Using a culture system that favors clonogenic epithelial cell growth, we evaluated and characterized clonal human urothelial cells. We isolated human urothelial cells that were clonogenic, capable of self-renewal and could develop into fully differentiated urothelium once re-implanted into the subcapsular space of nude mice. In addition to final urothelial cell differentiation, spontaneous formation of bladder-like microstructures was observed. By examining an epithelial stem cell signature marker, we found p63 to correlate with the self-renewal capacity of the isolated human urothelial clonal populations. Since a clinically relevant, long-term model for functional reconstitution of human cells does not exist, we sought to establish a culture method for porcine urothelial cells in a clinically relevant porcine model. We isolated cells from porcine ureter, urethra and bladder that were clonogenic and capable of self-renewal and differentiation into fully mature urothelium. In conclusion, we could isolate human and porcine cell populations, behaving as urothelial stem cells and showing clonogenicity, self-renewal and, once re-implanted, morphological differentiation. PMID:24587183

  2. Disruption of the hormonal network and the enantioselectivity of bifenthrin in trophoblast: maternal-fetal health risk of chiral pesticides.

    PubMed

    Zhao, Meirong; Zhang, Ying; Zhuang, Shulin; Zhang, Quan; Lu, Chengsheng; Liu, Weiping

    2014-07-15

    Endocrine-disrupting chemicals (EDCs) can interfere with normal hormone signaling to increase health risks to the maternal-fetal system, yet few studies have been conducted on the currently used chiral EDCs. This work tested the hypothesis that pyrethroids could enantioselectively interfere with trophoblast cells. Cell viability, hormone secretion, and steroidogenesis gene expression of a widely used pyrethroid, bifenthrin (BF), were evaluated in vitro, and the interactions of BF enantiomers with estrogen receptor (ER) were predicted. At low or noncytotoxic concentrations, both progesterone and human chorionic gonadotropin secretion were induced. The expression levels of progesterone receptor and human leukocyte antigen G genes were significantly stimulated. The key regulators of the hormonal cascade, GnRH type-I and its receptor, were both upregulated. The expression levels of selected steroidogenic genes were also significantly altered. Moreover, a consistent enantioselective interference of hormone signaling was observed, and S-BF had greater effects than R-BF. Using molecular docking, the enantioselective endocrine disruption of BF was predicted to be partially due to enantiospecific ER binding affinity. Thus, BF could act through ER to enantioselectively disturb the hormonal network in trophoblast cells. These converging results suggest that the currently used chiral pesticides are of significant concern with respect to maternal-fetal health.

  3. Distinct mechanisms regulate Cdx2 expression in the blastocyst and in trophoblast stem cells.

    PubMed

    Rayon, Teresa; Menchero, Sergio; Rollán, Isabel; Ors, Inmaculada; Helness, Anne; Crespo, Miguel; Nieto, Andres; Azuara, Véronique; Rossant, Janet; Manzanares, Miguel

    2016-01-01

    The first intercellular differences during mammalian embryogenesis arise in the blastocyst, producing the inner cell mass and the trophectoderm. The trophectoderm is the first extraembryonic tissue and does not contribute to the embryo proper, its differentiation instead forming tissues that sustain embryonic development. Crucial roles in extraembryonic differentiation have been identified for certain transcription factors, but a comprehensive picture of the regulation of this early specification is still lacking. Here, we investigated whether the regulatory mechanisms involved in Cdx2 expression in the blastocyst are also utilized in the postimplantation embryo. We analyzed an enhancer that is regulated through Hippo and Notch in the blastocyst trophectoderm, unexpectedly finding that it is inactive in the extraembryonic structures at postimplantation stages. Further analysis identified other Cdx2 regulatory elements including a stem-cell specific regulatory sequence and an element that drives reporter expression in the trophectoderm, a subset of cells in the extraembryonic region of the postimplantation embryo and in trophoblast stem cells. The cross-comparison in this study of cis-regulatory elements employed in the blastocyst, stem cell populations and the postimplantation embryo provides new insights into early mammalian development and suggests a two-step mechanism in Cdx2 regulation. PMID:27256674

  4. Distinct mechanisms regulate Cdx2 expression in the blastocyst and in trophoblast stem cells.

    PubMed

    Rayon, Teresa; Menchero, Sergio; Rollán, Isabel; Ors, Inmaculada; Helness, Anne; Crespo, Miguel; Nieto, Andres; Azuara, Véronique; Rossant, Janet; Manzanares, Miguel

    2016-06-03

    The first intercellular differences during mammalian embryogenesis arise in the blastocyst, producing the inner cell mass and the trophectoderm. The trophectoderm is the first extraembryonic tissue and does not contribute to the embryo proper, its differentiation instead forming tissues that sustain embryonic development. Crucial roles in extraembryonic differentiation have been identified for certain transcription factors, but a comprehensive picture of the regulation of this early specification is still lacking. Here, we investigated whether the regulatory mechanisms involved in Cdx2 expression in the blastocyst are also utilized in the postimplantation embryo. We analyzed an enhancer that is regulated through Hippo and Notch in the blastocyst trophectoderm, unexpectedly finding that it is inactive in the extraembryonic structures at postimplantation stages. Further analysis identified other Cdx2 regulatory elements including a stem-cell specific regulatory sequence and an element that drives reporter expression in the trophectoderm, a subset of cells in the extraembryonic region of the postimplantation embryo and in trophoblast stem cells. The cross-comparison in this study of cis-regulatory elements employed in the blastocyst, stem cell populations and the postimplantation embryo provides new insights into early mammalian development and suggests a two-step mechanism in Cdx2 regulation.

  5. Distinct mechanisms regulate Cdx2 expression in the blastocyst and in trophoblast stem cells

    PubMed Central

    Rayon, Teresa; Menchero, Sergio; Rollán, Isabel; Ors, Inmaculada; Helness, Anne; Crespo, Miguel; Nieto, Andres; Azuara, Véronique; Rossant, Janet; Manzanares, Miguel

    2016-01-01

    The first intercellular differences during mammalian embryogenesis arise in the blastocyst, producing the inner cell mass and the trophectoderm. The trophectoderm is the first extraembryonic tissue and does not contribute to the embryo proper, its differentiation instead forming tissues that sustain embryonic development. Crucial roles in extraembryonic differentiation have been identified for certain transcription factors, but a comprehensive picture of the regulation of this early specification is still lacking. Here, we investigated whether the regulatory mechanisms involved in Cdx2 expression in the blastocyst are also utilized in the postimplantation embryo. We analyzed an enhancer that is regulated through Hippo and Notch in the blastocyst trophectoderm, unexpectedly finding that it is inactive in the extraembryonic structures at postimplantation stages. Further analysis identified other Cdx2 regulatory elements including a stem-cell specific regulatory sequence and an element that drives reporter expression in the trophectoderm, a subset of cells in the extraembryonic region of the postimplantation embryo and in trophoblast stem cells. The cross-comparison in this study of cis-regulatory elements employed in the blastocyst, stem cell populations and the postimplantation embryo provides new insights into early mammalian development and suggests a two-step mechanism in Cdx2 regulation. PMID:27256674

  6. Schisandrae fructus enhances myogenic differentiation and inhibits atrophy through protein synthesis in human myotubes

    PubMed Central

    Kim, Cy Hyun; Shin, Jin-Hong; Hwang, Sung Jun; Choi, Yung Hyun; Kim, Dae-Seong; Kim, Cheol Min

    2016-01-01

    Schisandrae fructus (SF) has recently been reported to increase skeletal muscle mass and inhibit atrophy in mice. We investigated the effect of SF extract on human myotube differentiation and its acting pathway. Various concentrations (0.1–10 μg/mL) of SF extract were applied on human skeletal muscle cells in vitro. Myotube area and fusion index were measured to quantify myotube differentiation. The maximum effect was observed at 0.5 μg/mL of SF extract, enhancing differentiation up to 1.4-fold in fusion index and 1.6-fold in myotube area at 8 days after induction of differentiation compared to control. Phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 and 70 kDa ribosomal protein S6 kinase, which initiate translation as downstream of mammalian target of rapamycin pathway, was upregulated in early phases of differentiation after SF treatment. SF also attenuated dexamethasone-induced atrophy. In conclusion, we show that SF augments myogenic differentiation and attenuates atrophy by increasing protein synthesis through mammalian target of rapamycin/70 kDa ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1 signaling pathway in human myotubes. SF can be a useful natural dietary supplement in increasing skeletal muscle mass, especially in the aged with sarcopenia and the patients with disuse atrophy. PMID:27330287

  7. The forkhead transcription factor FOXP1 represses human plasma cell differentiation.

    PubMed

    van Keimpema, Martine; Grüneberg, Leonie J; Mokry, Michal; van Boxtel, Ruben; van Zelm, Menno C; Coffer, Paul; Pals, Steven T; Spaargaren, Marcel

    2015-10-29

    Expression of the forkhead transcription factor FOXP1 is essential for early B-cell development, whereas downregulation of FOXP1 at the germinal center (GC) stage is required for GC B-cell function. Aberrantly high FOXP1 expression is frequently observed in diffuse large B-cell lymphoma and mucosa-associated lymphoid tissue lymphoma, being associated with poor prognosis. Here, by gene expression analysis upon ectopic overexpression of FOXP1 in primary human memory B cells (MBCs) and B-cell lines, combined with chromatin immunoprecipitation and sequencing, we established that FOXP1 directly represses expression of PRDM1, IRF4, and XBP1, transcriptional master regulators of plasma cell (PC) differentiation. In accordance, FOXP1 is prominently expressed in primary human naive and MBCs, but expression strongly decreases during PC differentiation. Moreover, as compared with immunoglobulin (Ig) M(+) MBCs, IgG(+) MBCs combine lower expression of FOXP1 with an enhanced intrinsic PC differentiation propensity, and constitutive (over)expression of FOXP1 in B-cell lines and primary human MBCs represses their ability to differentiate into PCs. Taken together, our data indicate that proper control of FOXP1 expression plays a critical role in PC differentiation, whereas aberrant expression of FOXP1 might contribute to lymphomagenesis by blocking this terminal B-cell differentiation. PMID:26289642

  8. The forkhead transcription factor FOXP1 represses human plasma cell differentiation

    PubMed Central

    van Keimpema, Martine; Grüneberg, Leonie J.; Mokry, Michal; van Boxtel, Ruben; van Zelm, Menno C.; Coffer, Paul; Pals, Steven T.

    2015-01-01

    Expression of the forkhead transcription factor FOXP1 is essential for early B-cell development, whereas downregulation of FOXP1 at the germinal center (GC) stage is required for GC B-cell function. Aberrantly high FOXP1 expression is frequently observed in diffuse large B-cell lymphoma and mucosa-associated lymphoid tissue lymphoma, being associated with poor prognosis. Here, by gene expression analysis upon ectopic overexpression of FOXP1 in primary human memory B cells (MBCs) and B-cell lines, combined with chromatin immunoprecipitation and sequencing, we established that FOXP1 directly represses expression of PRDM1, IRF4, and XBP1, transcriptional master regulators of plasma cell (PC) differentiation. In accordance, FOXP1 is prominently expressed in primary human naive and MBCs, but expression strongly decreases during PC differentiation. Moreover, as compared with immunoglobulin (Ig) M+ MBCs, IgG+ MBCs combine lower expression of FOXP1 with an enhanced intrinsic PC differentiation propensity, and constitutive (over)expression of FOXP1 in B-cell lines and primary human MBCs represses their ability to differentiate into PCs. Taken together, our data indicate that proper control of FOXP1 expression plays a critical role in PC differentiation, whereas aberrant expression of FOXP1 might contribute to lymphomagenesis by blocking this terminal B-cell differentiation. PMID:26289642

  9. Differentially expressed protein markers in human submandibular and sublingual secretions.

    PubMed

    Hu, Shen; Denny, Patricia; Denny, Paul; Xie, Yongming; Loo, Joseph A; Wolinsky, Lawrence E; Li, Yang; McBride, Jim; Ogorzalek Loo, Rachel R; Navazesh, Mavash; Wong, David T

    2004-11-01

    Proteome analysis of secretions from individual salivary glands is important for understanding the health of the oral cavity and pathogenesis of certain diseases. However, cross-contamination of submandibular (SM) and sublingual (SL) glandular secretions can occur. The close anatomic relationship of the SM and SL ductal orifices can lead to such contamination. Additionally, these glands may share common ducts. To insure the purity of SM/SL secretions for proteomic analysis, it is important to develop unique biomarkers which could be used to verify the integrity of the individual glandular saliva. In this study, a proteomics approach based on mass spectrometry and gel electrophoresis techniques was utilized to identify and verify a set of proteins (cystatin C, calgranulin B and MUC5B mucin), which are differentially expressed in SM/SL secretions. SM/SL fluids were obtained from nine healthy subjects. Cystatin C was found to be an SM-selective protein as it was found in all SM fluids but not detected in two SL fluids. MUC5B mucin and calgranulin B, on the other hand, were found to be SL-selective proteins. All SL samples contained MUC5B mucin, whereas MUC5B mucin was not detected in four SM samples. Eight of the SL samples contained calgranulin B; however, calgranulin B was absent in eight SM samples. This set of protein markers, especially calgranulin B, can be used to determine the purity of SM/SL samples, and therefore identify potential individuals who do not exhibit cross-contaminated SM/SL secretions, an important requirement for subsequent proteome analysis of pure SM and SL secretions.

  10. Effect of F-spondin on cementoblastic differentiation of human periodontal ligament cells

    SciTech Connect

    Kitagawa, Masae; Kudo, Yasusei; Iizuka, Shinji; Ogawa, Ikuko; Abiko, Yoshimitsu; Miyauchi, Mutsumi; Takata, Takashi . E-mail: ttakata@hiroshima-u.ac.jp

    2006-10-27

    Cementum is a mineralized tissue produced by cementoblasts covering the roots of teeth that provides for the attachment of periodontal ligament to roots and surrounding alveolar bone. To study the mechanism of proliferation and differentiation of cementoblasts is important for understanding periodontal physiology and pathology including periodontal tissue regeneration. However, the detailed mechanism of the proliferation and differentiation of human cementoblasts is still unclear. We previously established human cementoblast-like (HCEM) cell lines. We thought that comparing the transcriptional profiles of HCEM cells and human periodontal ligament (HPL) cells derived from the same teeth could be a good approach to identify genes that influence the nature of cementoblasts. We identified F-spondin as the gene demonstrating the high fold change expression in HCEM cells. Interestingly, F-spondin highly expressing HPL cells showed similar phenotype of cementoblasts, such as up-regulation of mineralized-related genes. Overall, we identified F-spondin as a promoting factor for cementoblastic differentiation.

  11. Quantifying trophoblast migration: In vitro approaches to address in vivo situations.

    PubMed

    James, Joanna; Tun, Win; Clark, Alys

    2016-03-01

    When trophoblasts migrate and invade in vivo, they do so by interacting with a range of other cell types, extracellular matrix proteins, chemotactic factors and physical forces such as fluid shear stress. These factors combine to influence overall trophoblast migration and invasion into the decidua, which in turn determines the success of spiral artery remodelling, and pregnancy itself. Our understanding of these important but complex processes is limited by the simplified conditions in which we often study cell migration in vitro, and many discrepancies are observed between different in vitro models in the literature. On top of these experimental considerations, the migration of individual trophoblasts can vary widely. While time-lapse microscopy provides a wealth of information on trophoblast migration, manual tracking of individual cell migration is a time consuming task that ultimately restricts the numbers of cells quantified, and thus the ability to extract meaningful information from the data. However, the development of automated imaging algorithms is likely to aid our ability to accurately interpret trophoblast migration in vitro, and better allow us to relate these observations to in vivo scenarios. This commentary discusses the advantages and disadvantages of techniques commonly used to quantify trophoblast migration and invasion, both from a cell biology and a mathematical perspective, and examines how such techniques could be improved to help us relate trophoblast migration more accurately to in vivo function in the future.

  12. Macro histone variants are critical for the differentiation of human pluripotent cells.

    PubMed

    Barrero, María J; Sese, Borja; Martí, Mercè; Izpisua Belmonte, Juan Carlos

    2013-05-31

    We have previously shown that macro histone variants (macroH2A) are expressed at low levels in stem cells and are up-regulated during differentiation. Here we show that the knockdown of macro histone variants impaired the in vitro and in vivo differentiation of human pluripotent cells, likely through defects in the silencing of pluripotency-related genes. ChIP experiments showed that during differentiation macro histone variants are recruited to the regulatory regions of pluripotency and developmental genes marked with H3K27me3 contributing to the silencing of these genes.

  13. Inhibition of the differentiation of human myeloid cell lines by redox changes induced through glutathione depletion.

    PubMed Central

    Esposito, F; Agosti, V; Morrone, G; Morra, F; Cuomo, C; Russo, T; Venuta, S; Cimino, F

    1994-01-01

    We have investigated the effect of redox changes in vivo on the differentiation of two human myeloid cell lines, HL-60 and KG-1. The glutathione-depleting agent diethyl maleate (DEM) prevented the development of differentiated features in response to phorbol esters, including adherence of the cells to plastic surfaces and repression of the myeloperoxidase and CD34 genes. Moreover, DEM abolished phorbol 12-myristate 13-acetate-induced activation of the transcription factors AP-1 and Egr-1, suggesting that inhibition of differentiation may be due, at least in part, to redox modifications of these proteins. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7519845

  14. Osteogenic Differentiation of Human Amniotic Fluid Mesenchymal Stem Cells Is Determined by Epigenetic Changes

    PubMed Central

    2016-01-01

    Osteogenic differentiation of human amniotic fluid derived mesenchymal stem cells (AF-MSCs) has been widely studied in vitro and in vivo as a potential tool for regenerative medicine and tissue engineering. While most of the studies analyze changes in transcriptional profile during differentiation to date there is not much information regarding epigenetic changes in AF-MSCs during differentiation. The aim of our study was to evaluate epigenetic changes during osteogenic differentiation of AF-MS cells. Isolated AF-MSCs were characterized morphologically and osteogenic differentiation was confirmed by cell staining and determining expression of alkaline phosphatase and osteopontin by RT-qPCR. Variation in gene expression levels of pluripotency markers and specific microRNAs were also evaluated. Analysis of epigenetic changes revealed that levels of chromatin modifying enzymes such as Polycomb repressive complex 2 (PRC2) proteins (EZH2 and SUZ12), DNMT1, HDAC1, and HDAC2 were reduced after osteogenic differentiation of AF-MSCs. We demonstrated that the level of specific histone markers keeping active state of chromatin (H3K4me3, H3K9Ac, and others) increased and markers of repressed state of chromatin (H3K27me3) decreased. Our results show that osteogenic differentiation of AF-MSCs is conducted by various epigenetic alterations resulting in global chromatin remodeling and provide insights for further epigenetic investigations in human AF-MSCs.

  15. Human milk oligosaccharides are differentially metabolised in neonatal rats.

    PubMed

    Jantscher-Krenn, Evelyn; Marx, Carolin; Bode, Lars

    2013-08-01

    Human milk oligosaccharides (HMO) are complex glycans that are highly abundant in human milk, but not in infant formula. Accumulating data, mostly from in vitro and animal studies, indicate that HMO benefit the breast-fed infant in multiple ways and in different target organs. In vitro incubation studies suggest that HMO can resist the low pH in the infant's stomach and enzymatic degradation in the small intestine and reach the colon in the same composition as in the mother's milk. The oligosaccharide composition in faeces of breast-fed infants is, however, very different from that in the mother's milk, raising questions on when, where and how HMO are metabolised between ingestion and excretion. To answer some of these questions, we established a pulse-chase model in neonatal rats and analysed HMO profiles to track their composition over time in five consecutive equal-length intestinal segments as well as in serum and urine. The relative abundance of individual HMO changed significantly within the first 2 h after feeding and already in the segments of the small intestine prior to reaching the colon. Only 3'-sialyllactose, the major oligosaccharide in rat milk, and hardly any other HMO appeared in the serum and the urine of HMO-fed rats, indicating a selective absorption of rat milk-specific oligosaccharides. The present results challenge the paradigm that HMO reach the colon and other target organs in the same composition as originally secreted with the mother's milk. The present results also raise questions on whether rats and other animals represent suitable models to study the effects of HMO.

  16. The effect of scaffold architecture on odontogenic differentiation of human dental pulp stem cells.

    PubMed

    Wang, Jing; Ma, Haiyun; Jin, Xiaobing; Hu, Jiang; Liu, Xiaohua; Ni, Longxing; Ma, Peter X

    2011-11-01

    Previous studies have shown the superiority of nanofibrous (NF) poly(l-lactic acid) (PLLA) scaffolds in supporting the osteogenic differentiation of a few cell types and bone regeneration. The aim of the current study was to investigate whether NF-PLLA scaffolds are advantageous for the odontogenic differentiation and mineralization of human dental pulp stem cells (DPSCs) over solid-walled (SW) PLLA scaffolds. The in vitro studies demonstrated that, compared with SW scaffolds, NF scaffolds enhanced attachment and proliferation as well as odontogenic differentiation of human DPSCs. The alkaline phosphatase (ALP) activity and the expression of odontogenic genes of human DPSCs were increased on NF scaffolds compared with that on SW scaffolds. In addition, more mineral deposition was observed on the NF scaffolds, as demonstrated by von Kossa staining, calcium content measurement and scanning electron microscopy. Consistent with the in vitro studies, NF scaffolds promoted odontogenic differentiation and hard tissue formation compared with SW scaffolds after 8 weeks of ectopic transplantation in nude mice, as confirmed by von Kossa staining, Masson's trichrome staining and immunohistochemical staining for dentin sialoprotein. In conclusion, NF-PLLA scaffolds enhanced the odontogenic differentiation of human DPSCs and mineralization both in vitro and in vivo, and are promising scaffolds for dentin regeneration.

  17. Gold nanorod delivery of LSD1 siRNA induces human mesenchymal stem cell differentiation.

    PubMed

    Zhao, Xiongfei; Huang, Qianying; Jin, Yiqiang

    2015-09-01

    Over the past decade, theranostic nanoparticles with microsize and multifunctional ability have emerged as a new platform in biomedical field, such as cancer therapy, optical imaging and gene therapy. Gene therapy has been recently shown as a promising tool for tissue engineering as safe and effective nanotechnology-based delivery methods are developed. Controlling adhesion and differentiation of stem cells is critical for tissue regeneration. In this study, we have developed poly-sodium 4-styrenesulfonate (PSS) and poly-allylamine hydrochloride (PAH) coated AuNR-based nanocarriers, which are capable of delivering small interfering RNA (siRNA) against LSD1 to induce the differentiation of human mesenchymal stem cells. To further study the mechanism, we tested the stemness and differentiation genes and found that they have been changed with LSD1 down-regulation. In addition, with the hepatocyte growth factor (HGF), LSD1 siRNA delivery by AuNRs could promote the differentiation of the human mesenchymal stem cells (human MSCs) into a hepatocyte lineage in vitro. Our results suggest for the first time use of AuNRs as nanocarriers of delivery LSD1 siRNA to induce the differentiation of human MSCs into a hepatocyte lineage, and envision the potential application of nanotechnology in tissue remodeling (such as liver and bone) in vivo, eventually translating to clinical applications.

  18. Gold nanorod delivery of LSD1 siRNA induces human mesenchymal stem cell differentiation.

    PubMed

    Zhao, Xiongfei; Huang, Qianying; Jin, Yiqiang

    2015-09-01

    Over the past decade, theranostic nanoparticles with microsize and multifunctional ability have emerged as a new platform in biomedical field, such as cancer therapy, optical imaging and gene therapy. Gene therapy has been recently shown as a promising tool for tissue engineering as safe and effective nanotechnology-based delivery methods are developed. Controlling adhesion and differentiation of stem cells is critical for tissue regeneration. In this study, we have developed poly-sodium 4-styrenesulfonate (PSS) and poly-allylamine hydrochloride (PAH) coated AuNR-based nanocarriers, which are capable of delivering small interfering RNA (siRNA) against LSD1 to induce the differentiation of human mesenchymal stem cells. To further study the mechanism, we tested the stemness and differentiation genes and found that they have been changed with LSD1 down-regulation. In addition, with the hepatocyte growth factor (HGF), LSD1 siRNA delivery by AuNRs could promote the differentiation of the human mesenchymal stem cells (human MSCs) into a hepatocyte lineage in vitro. Our results suggest for the first time use of AuNRs as nanocarriers of delivery LSD1 siRNA to induce the differentiation of human MSCs into a hepatocyte lineage, and envision the potential application of nanotechnology in tissue remodeling (such as liver and bone) in vivo, eventually translating to clinical applications. PMID:26046277

  19. Differentiation of the nuclear groups in the posterior horn of the human embryonic spinal cord.

    PubMed

    Pytel, A; Bruska, M; Woźniak, W

    2011-11-01

    The formation of nuclear groups in the posterior horns of the human embryonic spinal cord was traced in serial sections of embryos of developmental stages 13 to 23 (32 to 56 postovulatory days). The following observations, new for the human, are presented: 1. The differentiation of the neural tube into 3 zones (germinal, mantle and marginal) is detected in the middle of the 5(th) week. 2. The primordia of the posterior horns are marked at stage 14 (33 days). 3. In the middle of the 7(th) week the nucleus proprius and substantia gelatinosa are discerned. 4. Differentiation of the nuclei within the posterior horns proceeds in the ventrodorsal and rostrocaudal gradients.

  20. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells

    SciTech Connect

    Gao, Fei; Kishida, Tsunao; Ejima, Akika; Gojo, Satoshi; Mazda, Osam

    2013-02-08

    Highlights: ► iPS-derived cells express myostatin and its receptor upon myoblast differentiation. ► Myostatin inhibits myoblast differentiation by inhibiting MyoD and Myo5a induction. ► Silencing of myostatin promotes differentiation of human iPS cells into myoblasts. -- Abstract: Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases.

  1. Localized decrease of {beta}-catenin contributes to the differentiation of human embryonic stem cells

    SciTech Connect

    Lam, Hayley; Patel, Shyam; Wong, Janelle; Chu, Julia; Li, Adrian; Li, Song

    2008-08-08

    Human embryonic stem cells (hESC) are pluripotent, and can be directed to differentiate into different cell types for therapeutic applications. To expand hESCs, it is desirable to maintain hESC growth without differentiation. As hESC colonies grow, differentiated cells are often found at the periphery of the colonies, but the underlying mechanism is not well understood. Here, we utilized micropatterning techniques to pattern circular islands or strips of matrix proteins, and examined the spatial pattern of hESC renewal and differentiation. We found that micropatterned matrix restricted hESC differentiation at colony periphery but allowed hESC growth into multiple layers in the central region, which decreased hESC proliferation and induced hESC differentiation. In undifferentiated hESCs, {beta}-catenin primarily localized at cell-cell junctions but not in the nucleus. The amount of {beta}-catenin in differentiating hESCs at the periphery of colonies or in multiple layers decreased significantly at cell-cell junctions. Consistently, knocking down {beta}-catenin decreased Oct-4 expression in hESCs. These results indicate that localized decrease of {beta}-catenin contributes to the spatial pattern of differentiation in hESC colonies.

  2. Transient serum exposure regimes to support dual differentiation of human mesenchymal stem cells.

    PubMed

    France, L A; Scotchford, C A; Grant, D M; Rashidi, H; Popov, A A; Sottile, V

    2014-08-01

    Human mesenchymal stem cells (MSCs), which can generate both osteoblasts and chondrocytes, represent an ideal resource for orthopaedic repair using tissue-engineering approaches. One major difficulty for the development of osteochondral constructs using undifferentiated MSCs is that serum is typically used in culture protocols to promote differentiation of the osteogenic component, whereas existing chondrogenic differentiation protocols rely on the use of serum-free conditions. In order to define conditions which could be compatible with both chondrogenic and osteogenic differentiation in a single bioreactor, we have analysed the efficiency of new biphasic differentiation regimes based on transient serum exposure followed by serum-free treatment. MSC differentiation was assessed either in serum-free medium or with a range of transient exposure to serum, and compared to continuous serum-containing treatment. Although osteogenic differentation was not supported in the complete absence of serum, marker expression and extensive mineralization analyses established that 5 days of transient exposure triggered a level of differentiation comparable to that observed when serum was present throughout. This initial phase of serum exposure was further shown to support the successful chondrogenic differentiation of MSCs, comparable to controls maintained in serum-free conditions throughout. This study indicates that a culture based on temporal serum exposure followed by serum-free treatment is compatible with both osteogenic and chondrogenic differentiation of MSCs. These results will allow the development of novel strategies for osteochondral tissue engineering approaches using MSCs for regenerative medicine. PMID:23161724

  3. Differentiation of human colon cancer cells changes the expression of β-tubulin isotypes and MAPs

    PubMed Central

    Carles, G; Braguer, D; Dumontet, C; Bourgarel, V; Gonçalves, A; Sarrazin, M; Rognoni, J B; Briand, C

    1999-01-01

    The human colon adenocarcinoma HT29-D4 cell line is an interesting model for studies on epithelial cell differentiation. Undifferentiated cells are malignant proliferating cells, whereas differentiated cells act like epithelial polarized cells. In the present study, we first characterized the action of taxoids on the microtubular network of HT29-D4 cells according to the state of differentiation. Microtubular bundles were found in undifferentiated cells but not in differentiated cells, even with 500-fold higher taxoid concentrations for 96 h. This finding led us to study changes in microtubules according to the polarity status of the cell. E-MAP-115 was expressed only in differentiated cells; expression of β-tubulin isotypes was altered in them relative to undifferentiated cells. Classes I, II, III, IVa and IVb isotypes were expressed in both phenotypes; however, differentiated epithelial cells displayed a specific increase in class III β-tubulin. Thus, the increase in expression of this β-tubulin isotype in differentiated cells is not restricted to neuronal cells. Moreover, these expression changes may reflect a higher stability of microtubular network in differentiated cells, which may explain the lower activity of anti-microtubule agents, independently of the mitotic process. These results indicate that the composition of microtubules should be considered as one of the criteria involved in the response of tumour cells to chemotherapy with anti-microtubule agents. © 1999 Cancer Research Campaign PMID:10376967

  4. Adenosine Signaling Mediates Osteogenic Differentiation of Human Embryonic Stem Cells on Mineralized Matrices

    PubMed Central

    Rao, Vikram; Shih, Yu-Ru V.; Kang, Heemin; Kabra, Harsha; Varghese, Shyni

    2015-01-01

    Human embryonic stem cells (hESCs) are attractive cell sources for tissue engineering and regenerative medicine due to their self-renewal and differentiation ability. Design of biomaterials with an intrinsic ability that promotes hESC differentiation to the targeted cell type boasts significant advantages for tissue regeneration. We have previously developed biomineralized calcium phosphate (CaP) matrices that inherently direct osteogenic differentiation of hESCs without the need of osteogenic-inducing chemicals or growth factors. Here, we show that CaP matrix-driven osteogenic differentiation of hESCs occurs through A2b adenosine receptor (A2bR). The inhibition of the receptor with an A2bR-specific antagonist attenuated mineralized matrix-mediated osteogenic differentiation of hESCs. In addition, when cultured on matrices in an environment deficient of CaP minerals, exogenous adenosine promoted osteogenic differentiation of hESCs, but was attenuated by the inhibition of A2bR. Such synthetic matrices that intrinsically support osteogenic commitment of hESCs are not only beneficial for bone tissue engineering but can also be used as a platform to study the effect of the physical and chemical cues to the extracellular milieu on stem cell commitment. Insights into the cell signaling during matrix-induced differentiation of stem cells will also help define the key processes and enable discovery of new targets that promote differentiation of pluripotent stem cells for bone tissue engineering. PMID:26618155

  5. Clonogenic multipotent stem cells in human adipose tissue differentiate into functional smooth muscle cells

    PubMed Central

    Rodríguez, Larissa V.; Alfonso, Zeni; Zhang, Rong; Leung, Joanne; Wu, Benjamin; Ignarro, Louis J.

    2006-01-01

    Smooth muscle is a major component of human tissues and is essential for the normal function of a multitude of organs including the intestine, urinary tract and the vascular system. The use of stem cells for cell-based tissue engineering and regeneration strategies represents a promising alternative for smooth muscle repair. For such strategies to succeed, a reliable source of smooth muscle precursor cells must be identified. Adipose tissue provides an abundant source of multipotent cells. In this study, the capacity of processed lipoaspirate (PLA) and adipose-derived stem cells to differentiate into phenotypic and functional smooth muscle cells was evaluated. To induce differentiation, PLA cells were cultured in smooth muscle differentiation medium. Smooth muscle differentiation of PLA cells induced genetic expression of all smooth muscle markers and further confirmed by increased protein expression of smooth muscle cell-specific α actin (ASMA), calponin, caldesmon, SM22, myosin heavy chain (MHC), and smoothelin. Clonal studies of adipose derived multipotent cells demonstrated differentiation of these cells into smooth muscle cells in addition to trilineage differentiation capacity. Importantly, smooth muscle-differentiated cells, but not their precursors, exhibit the functional ability to contract and relax in direct response to pharmacologic agents. In conclusion, adipose-derived cells have the potential to differentiate into functional smooth muscle cells and, thus, adipose tissue can be a useful source of cells for treatment of injured tissues where smooth muscle plays an important role. PMID:16880387

  6. Laminin 411 and 511 promote the cholangiocyte differentiation of human induced pluripotent stem cells.

    PubMed

    Takayama, Kazuo; Mitani, Seiji; Nagamoto, Yasuhito; Sakurai, Fuminori; Tachibana, Masashi; Taniguchi, Yukimasa; Sekiguchi, Kiyotoshi; Mizuguchi, Hiroyuki

    2016-05-20

    The drug discovery research for cholestatic liver diseases has been hampered by the lack of a well-established human cholangiocyte model. Functional cholangiocyte-like cells differentiated from human induced pluripotent stem (iPS) cells are expected to be a promising candidate for such research, but there remains no well-established method for differentiating cholangiocytes from human iPS cells. In this study, we searched for a suitable extracellular matrix to promote cholangiocyte differentiation from human iPS cells, and found that both laminin 411 and laminin 511 were suitable for this purpose. The gene expression levels of the cholangiocyte markers, aquaporin 1 (AQP1), SRY-box 9 (SOX9), cystic fibrosis transmembrane conductance regulator (CFTR), G protein-coupled bile acid receptor 1 (GPBAR1), Jagged 1 (JAG1), secretin receptor (SCTR), and γ-glutamyl transferase (GGT1) were increased by using laminin 411 or laminin 511 as a matrix. In addition, the percentage of AQP1-positive cells was increased from 61.8% to 92.5% by using laminin 411 or laminin 511. Furthermore, the diameter and number of cysts consisted of cholangiocyte-like cells were increased when using either matrix. We believe that the human iPS cell-derived cholangiocyte-like cells, which were generated by using our differentiation technology, would be useful for the drug discovery research of cholestatic liver diseases. PMID:27103433

  7. Human GPM6A is associated with differentiation and neuronal migration of neurons derived from human embryonic stem cells.

    PubMed

    Michibata, Hideo; Okuno, Tsuyoshi; Konishi, Nae; Kyono, Kiyoshi; Wakimoto, Koji; Aoki, Kan; Kondo, Yasushi; Takata, Kazuyuki; Kitamura, Yoshihisa; Taniguchi, Takashi

    2009-05-01

    Glycoprotein M6A (GPM6A) is known as a transmembrane protein and an abundant cell surface protein on neurons in the central nervous system (CNS). However, the function of GPM6A in the differentiation of neurons derived from human embryonic stem (ES) cells is unknown. To investigate the function of GPM6A in neural differentiation, we generated human ES cell lines with overexpressed (B2h-oeM6A) or suppressed (B2h-shM6A) human GPM6A. Real-time polymerase chain reaction (PCR) showed that overexpression of GPM6A markedly increased the expression of neuroectodermal-associated genes (OTX1, Lmx1b, En1, Pax2, Sox2, and Wnt1), and the number of neural stem cells (NSCs) derived from B2h-oeM6A cells compared to control vector transfected human ES cells (B2h-Mock1). Our results show an increase in the number of differentiated neuronal cells (cholinergic, catecholaminergic, and GABAergic neurons) from NSCs derived from B2h-oeM6A cells. On the other hand, suppression of human GPM6A expression using a short hairpin RNA (shRNA) in human ES cells led to a decrease in both the expression of neuroectodermal-associated genes and the number of NSCs derived from B2h-shM6A cells. In addition, our results show a decrease in the number of differentiated neuronal cells from NSCs in B2h-shM6A cells compared to control vector transfected human ES cells (B2h-shNSP1). Moreover, overexpression or suppression of human GPM6A in human ES cells led to an increase or decrease, respectively, of neuronal migration. Hence, our findings suggest that expression level of GPM6A is, directly or indirectly, associated with the differentiation and neuronal migration of neurons derived from undifferentiated human ES cells. PMID:19298174

  8. New discoveries on the biology and detection of human chorionic gonadotropin.

    PubMed

    Cole, Laurence A

    2009-01-26

    Human chorionic gonadotropin (hCG) is a glycoprotein hormone comprising 2 subunits, alpha and beta joined non covalently. While similar in structure to luteinizing hormone (LH), hCG exists in multiple hormonal and non-endocrine agents, rather than as a single molecule like LH and the other glycoprotein hormones. These are regular hCG, hyperglycosylated hCG and the free beta-subunit of hyperglycosylated hCG. For 88 years regular hCG has been known as a promoter of corpus luteal progesterone production, even though this function only explains 3 weeks of a full gestations production of regular hCG. Research in recent years has explained the full gestational production by demonstration of critical functions in trophoblast differentiation and in fetal nutrition through myometrial spiral artery angiogenesis. While regular hCG is made by fused villous syncytiotrophoblast cells, extravillous invasive cytotrophoblast cells make the variant hyperglycosylated hCG. This variant is an autocrine factor, acting on extravillous invasive cytotrophoblast cells to initiate and control invasion as occurs at implantation of pregnancy and the establishment of hemochorial placentation, and malignancy as occurs in invasive hydatidiform mole and choriocarcinoma. Hyperglycosylated hCG inhibits apoptosis in extravillous invasive cytotrophoblast cells promoting cell invasion, growth and malignancy. Other non-trophoblastic malignancies retro-differentiate and produce a hyperglycosylated free beta-subunit of hCG (hCG free beta). This has been shown to be an autocrine factor antagonizing apoptosis furthering cancer cell growth and malignancy. New applications have been demonstrated for total hCG measurements and detection of the 3 hCG variants in pregnancy detection, monitoring pregnancy outcome, determining risk for Down syndrome fetus, predicting preeclampsia, detecting pituitary hCG, detecting and managing gestational trophoblastic diseases, diagnosing quiescent gestational trophoblastic

  9. New discoveries on the biology and detection of human chorionic gonadotropin

    PubMed Central

    Cole, Laurence A

    2009-01-01

    Human chorionic gonadotropin (hCG) is a glycoprotein hormone comprising 2 subunits, alpha and beta joined non covalently. While similar in structure to luteinizing hormone (LH), hCG exists in multiple hormonal and non-endocrine agents, rather than as a single molecule like LH and the other glycoprotein hormones. These are regular hCG, hyperglycosylated hCG and the free beta-subunit of hyperglycosylated hCG. For 88 years regular hCG has been known as a promoter of corpus luteal progesterone production, even though this function only explains 3 weeks of a full gestations production of regular hCG. Research in recent years has explained the full gestational production by demonstration of critical functions in trophoblast differentiation and in fetal nutrition through myometrial spiral artery angiogenesis. While regular hCG is made by fused villous syncytiotrophoblast cells, extravillous invasive cytotrophoblast cells make the variant hyperglycosylated hCG. This variant is an autocrine factor, acting on extravillous invasive cytotrophoblast cells to initiate and control invasion as occurs at implantation of pregnancy and the establishment of hemochorial placentation, and malignancy as occurs in invasive hydatidiform mole and choriocarcinoma. Hyperglycosylated hCG inhibits apoptosis in extravillous invasive cytotrophoblast cells promoting cell invasion, growth and malignancy. Other non-trophoblastic malignancies retro-differentiate and produce a hyperglycosylated free beta-subunit of hCG (hCG free beta). This has been shown to be an autocrine factor antagonizing apoptosis furthering cancer cell growth and malignancy. New applications have been demonstrated for total hCG measurements and detection of the 3 hCG variants in pregnancy detection, monitoring pregnancy outcome, determining risk for Down syndrome fetus, predicting preeclampsia, detecting pituitary hCG, detecting and managing gestational trophoblastic diseases, diagnosing quiescent gestational trophoblastic

  10. Toward a Differential Diagnosis of Hidden Hearing Loss in Humans

    PubMed Central

    Liberman, M. Charles; Epstein, Michael J.; Cleveland, Sandra S.; Wang, Haobing

    2016-01-01

    Recent work suggests that hair cells are not the most vulnerable elements in the inner ear; rather, it is the synapses between hair cells and cochlear nerve terminals that degenerate first in the aging or noise-exposed ear. This primary neural degeneration does not affect hearing thresholds, but likely contributes to problems understanding speech in difficult listening environments, and may be important in the generation of tinnitus and/or hyperacusis. To look for signs of cochlear synaptopathy in humans, we recruited college students and divided them into low-risk and high-risk groups based on self-report of noise exposure and use of hearing protection. Cochlear function was assessed by otoacoustic emissions and click-evoked electrocochleography; hearing was assessed by behavioral audiometry and word recognition with or without noise or time compression and reverberation. Both groups had normal thresholds at standard audiometric frequencies, however, the high-risk group showed significant threshold elevation at high frequencies (10–16 kHz), consistent with early stages of noise damage. Electrocochleography showed a significant difference in the ratio between the waveform peaks generated by hair cells (Summating Potential; SP) vs. cochlear neurons (Action Potential; AP), i.e. the SP/AP ratio, consistent with selective neural loss. The high-risk group also showed significantly poorer performance on word recognition in noise or with time compression and reverberation, and reported heightened reactions to sound consistent with hyperacusis. These results suggest that the SP/AP ratio may be useful in the diagnosis of “hidden hearing loss” and that, as suggested by animal models, the noise-induced loss of cochlear nerve synapses leads to deficits in hearing abilities in difficult listening situations, despite the presence of normal thresholds at standard audiometric frequencies. PMID:27618300

  11. Differential Chemokine Signature between Human Preadipocytes and Adipocytes

    PubMed Central

    Ignacio, Rosa Mistica C.; Gibbs, Carla R.; Lee, Eun-Sook

    2016-01-01

    Obesity is characterized as an accumulation of adipose tissue mass represented by chronic, low-grade inflammation. Obesity-derived inflammation involves chemokines as important regulators contributing to the pathophysiology of obesity-related diseases such as cardiovascular disease, diabetes and some cancers. The obesity-driven chemokine network is poorly understood. Here, we identified the profiles of chemokine signature between human preadipocytes and adipocytes, using PCR arrays and qRT-PCR. Both preadipocytes and adipocytes showed absent or low levels in chemokine receptors in spite of some changes. On the other hand, the chemokine levels of CCL2, CCL7-8, CCL11, CXCL1-3, CXCL6 and CXCL10-11 were dominantly expressed in preadipocytes compared to adipocytes. Interestingly, CXCL14 was the most dominant chemokine expressed in adipocytes compared to preadipocytes. Moreover, there is significantly higher protein level of CXCL14 in conditioned media from adipocytes. In addition, we analyzed the data of the chemokine signatures in adipocytes obtained from healthy lean and obese postmenopausal women based on Gene Expression Omnibus (GEO) dataset. Adipocytes from obese individuals had significantly higher levels in chemokine signature as follows: CCL2, CCL13, CCL18-19, CCL23, CCL26, CXCL1, CXCL3 and CXCL14, as compared to those from lean ones. Also, among the chemokine networks, CXCL14 appeared to be the highest levels in adipocytes from both lean and obese women. Taken together, these results identify CXCL14 as an important chemokine induced during adipogenesis, requiring further research elucidating its potential therapeutic benefits in obesity. PMID:27340388

  12. Toward a Differential Diagnosis of Hidden Hearing Loss in Humans.

    PubMed

    Liberman, M Charles; Epstein, Michael J; Cleveland, Sandra S; Wang, Haobing; Maison, Stéphane F

    2016-01-01

    Recent work suggests that hair cells are not the most vulnerable elements in the inner ear; rather, it is the synapses between hair cells and cochlear nerve terminals that degenerate first in the aging or noise-exposed ear. This primary neural degeneration does not affect hearing thresholds, but likely contributes to problems understanding speech in difficult listening environments, and may be important in the generation of tinnitus and/or hyperacusis. To look for signs of cochlear synaptopathy in humans, we recruited college students and divided them into low-risk and high-risk groups based on self-report of noise exposure and use of hearing protection. Cochlear function was assessed by otoacoustic emissions and click-evoked electrocochleography; hearing was assessed by behavioral audiometry and word recognition with or without noise or time compression and reverberation. Both groups had normal thresholds at standard audiometric frequencies, however, the high-risk group showed significant threshold elevation at high frequencies (10-16 kHz), consistent with early stages of noise damage. Electrocochleography showed a significant difference in the ratio between the waveform peaks generated by hair cells (Summating Potential; SP) vs. cochlear neurons (Action Potential; AP), i.e. the SP/AP ratio, consistent with selective neural loss. The high-risk group also showed significantly poorer performance on word recognition in noise or with time compression and reverberation, and reported heightened reactions to sound consistent with hyperacusis. These results suggest that the SP/AP ratio may be useful in the diagnosis of "hidden hearing loss" and that, as suggested by animal models, the noise-induced loss of cochlear nerve synapses leads to deficits in hearing abilities in difficult listening situations, despite the presence of normal thresholds at standard audiometric frequencies. PMID:27618300

  13. Hepatic differentiation of human pluripotent stem cells on human liver progenitor HepaRG-derived acellular matrix.

    PubMed

    Kanninen, Liisa K; Porola, Pauliina; Niklander, Johanna; Malinen, Melina M; Corlu, Anne; Guguen-Guillouzo, Christiane; Urtti, Arto; Yliperttula, Marjo L; Lou, Yan-Ru

    2016-02-15

    Human hepatocytes are extensively needed in drug discovery and development. Stem cell-derived hepatocytes are expected to be an improved and continuous model of human liver to study drug candidates. Generation of endoderm-derived hepatocytes from human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, is a complex, challenging process requiring specific signals from soluble factors and insoluble matrices at each developmental stage. In this study, we used human liver progenitor HepaRG-derived acellular matrix (ACM) as a hepatic progenitor-specific matrix to induce hepatic commitment of hPSC-derived definitive endoderm (DE) cells. The DE cells showed much better attachment to the HepaRG ACM than other matrices tested and then differentiated towards hepatic cells, which expressed hepatocyte-specific makers. We demonstrate that Matrigel overlay induced hepatocyte phenotype and inhibited biliary epithelial differentiation in two hPSC lines studied. In conclusion, our study demonstrates that the HepaRG ACM, a hepatic progenitor-specific matrix, plays an important role in the hepatic differentiation of hPSCs.

  14. Benzo(a)pyrene inhibits migration and invasion of extravillous trophoblast HTR-8/SVneo cells via activation of the ERK and JNK pathway.

    PubMed

    Liu, Liyuan; Wang, Yingxiong; Shen, Cha; He, Junlin; Liu, Xueqing; Ding, Yubin; Gao, Rufei; Chen, Xuemei

    2016-07-01

    Benzo(a)pyrene (BaP) is a persistent organic pollutant (POP) that is a serious threat to human health. Numerous studies have shown that BaP causes adverse effects in pregnancy, but the mechanism remains unclear. The moderate invasion of trophoblast cells into the endometrium is an important factor during successful embryo implantation. The aim of this study was to investigate the effect and mechanism of BaP on the invasion and migration of trophoblast cells. HTR-8/SVneo cells were treated with different concentrations (1, 5, 10, 25, 50 and 100 μM) of BaP. The invasion and migration of HTR-8/SVneo cells were observed after BaP treatment. The protein levels related to migration and invasion was detected by Western blot. The results confirmed that BaP inhibits the migration and invasion of extravillous trophoblast HTR-8/SVneo cells. Further investigations indicated that the protein levels of MMP-2, MMP-9 and E-cadherin in HTR-8/SVneo cells were changed by BaP treatment. Moreover, the data demonstrated that BaP activated the MAPK signaling pathway. Pretreatment with specific inhibitors of MAPK rescued BaP-induced change in the migration and invasion of HTR-8/SVneo cells. Taken together, our results indicated that BaP inhibits invasion and the migration of HTR-8/SVneo cells, which might cause a failure in early pregnancy. Copyright © 2015 John Wiley & Sons, Ltd.

  15. Trophoblastic cell lines generated from tumour necrosis factor receptor-deficient mice reveal specific functions for the two tumour necrosis factor receptors.

    PubMed

    Rasmussen, C A; Pace, J L; Banerjee, S; Phillips, T A; Hunt, J S

    1999-01-01

    In mice and humans, expression of the tumour necrosis factor receptor-1 (TNF-R1) gene in placental trophoblast cells is constitutive whereas expression of the TNF-R2 gene is developmentally programmed. In order to study the individual functions of TNF-R1 and -R2 in this lineage, cell lines were generated from placental explants of homozygous matings of gestation day 10 outbred mice (Swiss-Webster), TNF-R1-deficient (TNF-R1-/-) and TNF-R2-/- transgenic mice as well as the background strain for the TNF-R2-/- mice (WT, C57BL/6x129). All of the cells exhibited trophoblast markers; they contained cytokeratin intermediate filaments, expressed alkaline phosphatase activity and displayed transferrin receptors, but were negative for vimentin filaments and the macrophage marker, F4/80. Analysis of DNA by polymerase chain reaction demonstrated the expected TNF-R genotype in each line. In experiments testing the effects of recombinant mouse TNF-alpha (rmTNF-alpha) on viability and proliferation of the cell lines, rmTNF-alpha modestly but dose-dependently inhibited the growth of WT and TNF-R2-/- cells while having no effect on TNF-R1-/- cells. Actinomycin D-treated WT and, to a lesser extent, TNF-R2-/- cells, were more sensitive to growth inhibition than untreated cells whereas TNF-R1-/- cell responses remained unchanged. These data indicated that rmTNF-alpha inhibits growth of trophoblastic cells through TNF-R1 and that newly synthesized protein(s) provide partial protection against toxicity. In contrast to the receptor species-specific effects on cell growth exerted by rmTNF-alpha, both TNF-R mediated inhibition of alkaline phosphatase activity. Collectively, the observations support the postulate that receptor expression is the key factor which determines the nature and extent of TNF-alpha effects on trophoblast cell growth and function.

  16. Delineation of Natural Killer Cell Differentiation from Myeloid Progenitors in Human.

    PubMed

    Chen, Qingfeng; Ye, Weijian; Jian Tan, Wei; Mei Yong, Kylie Su; Liu, Min; Qi Tan, Shu; Loh, Eva; Te Chang, Kenneth; Chye Tan, Thiam; Preiser, Peter R; Chen, Jianzhu

    2015-01-01

    Understanding of natural killer (NK) cell development in human is incomplete partly because of limited access to appropriate human tissues. We have developed a cytokine-enhanced humanized mouse model with greatly improved reconstitution and function of human NK cells. Here we report the presence of a cell population in the bone marrow of the cytokine-treated humanized mice that express both NK cell marker CD56 and myeloid markers such as CD36 and CD33. The CD56(+)CD33(+)CD36(+) cells are also found in human cord blood, fetal and adult bone marrow. Although the CD56(+)CD33(+)CD36(+) cells do not express the common NK cell functional receptors and exhibit little cytotoxic and cytokine-producing activities, they readily differentiate into mature NK cells by acquiring expression of NK cell receptors and losing expression of the myeloid markers. Further studies show that CD33(+)CD36(+) myeloid NK precursors are derived from granulo-myelomonocytic progenitors. These results delineate the pathway of human NK cell differentiation from myeloid progenitors in the bone marrow and suggest the utility of humanized mice for studying human hematopoiesis. PMID:26456148

  17. Nitric Oxide Receptor Soluble Guanylyl Cyclase Undergoes Splicing Regulation in Differentiating Human Embryonic Cells

    PubMed Central

    Sharin, Vladislav G.; Mujoo, Kalpana; Kots, Alexander Y.; Martin, Emil; Murad, Ferid

    2011-01-01

    Nitric oxide (NO), an important mediator molecule in mammalian physiology, initiates a number of signaling mechanisms by activating the enzyme soluble guanylyl cyclase (sGC). Recently, a new role for NO/cyclic guanosine monophosphate signaling in embryonic development and cell differentiation has emerged. The changes in expression of NO synthase isoforms and various sGC subunits has been demonstrated during human and mouse embryonic stem (ES) cells differentiation. Previously, our laboratory demonstrated that nascent α1 sGC transcript undergoes alternative splicing and that expression of α1 sGC splice forms directly affects sGC activity. Expression of sGC splice variants in the process of human ES (hES) cells differentiation has not been investigated. In this report, we demonstrate that α1 sGC undergoes alternative splicing during random hES differentiation for the first time. Our results indicate that C-α1 sGC splice form is expressed at high levels in differentiating cells and its intracellular distribution varies from canonical α1 sGC subunit. Together, our data suggest that alternative splicing of sGC subunits is associated with differentiation of hES cells. PMID:20964618

  18. Comparison of different protocols for neural differentiation of human induced pluripotent stem cells.

    PubMed

    Salimi, Ali; Nadri, Samad; Ghollasi, Marzieh; Khajeh, Khosro; Soleimani, Masoud

    2014-03-01

    Although embryonic stem cells (ESCs) have enormous potentials due to their pluripotency, their therapeutic use is limited by ethical, biological and safety issues. Compared to ESCs, induced pluripotent stem cells (iPSCs) can be obtained from mouse or human fibroblasts by reprogramming. Numerous studies have established many protocols for differentiation of human iPSCs (hiPSCs) into neural lineages. However, the low differentiation efficiency of such protocols motivates researchers to design new protocols for high yield differentiation. Herein, we compared neural differentiation potential of three induction media for conversion of hiPSCs into neural lineages. In this study, hiPSCs-derived embryoid bodies were plated on laminin coated dishes and were treated with three induction media including (1) bFGF, EGF (2) RA and (3) forskolin, IBMX. Immunofluorescence staining and quantitative real-time PCR (qPCR) analysis were used to detect the expression of neural genes and proteins. qPCR analysis showed that the expression of neural genes in differentiated hiPSCs in forskolin, IBMX supplemented media was significantly higher than undifferentiated cells and those in induction media containing bFGF, EGF or RA. In conclusion, our results indicated a successful establishment protocol with high efficiency for differentiation of hiPSCs into neural lineages.

  19. Characterization of lipid metabolism in insulin-sensitive adipocytes differentiated from immortalized human mesenchymal stem cells

    SciTech Connect

    Prawitt, Janne; Niemeier, Andreas; Kassem, Moustapha; Beisiegel, Ulrike; Heeren, Joerg

    2008-02-15

    There is a great demand for cell models to study human adipocyte function. Here we describe the adipogenic differentiation of a telomerase-immortalized human mesenchymal stem cell line (hMSC-Tert) that maintains numerous features of terminally differentiated adipocytes even after prolonged withdrawal of the peroxisome proliferator activated receptor {gamma} (PPAR{gamma}) agonist rosiglitazone. Differentiated hMSC-Tert developed the characteristic monolocular phenotype of mature adipocytes. The expression of adipocyte specific markers was highly increased during differentiation. Most importantly, the presence of the PPAR{gamma} agonist rosiglitazone was not required for the stable expression of lipoprotein lipase, adipocyte fatty acid binding protein and perilipin on mRNA and protein levels. Adiponectin expression was post-transcriptionally down-regulated in the absence of rosiglitazone. Insulin sensitivity as measured by insulin-induced phosphorylation of Akt and S6 ribosomal protein was also independent of rosiglitazone. In addition to commonly used adipogenic markers, we investigated further PPAR{gamma}-stimulated proteins with a role in lipid metabolism. We observed an increase of lipoprotein receptor (VLDLR, LRP1) and apolipoprotein E expression during differentiation. Despite this increased expression, the receptor-mediated endocytosis of lipoproteins was decreased in differentiated adipocytes, suggesting that these proteins may have an additional function in adipose tissue beyond lipoprotein uptake.

  20. Human stem cell neuronal differentiation on silk-carbon nanotube composite

    NASA Astrophysics Data System (ADS)

    Chen, Chi-Shuo; Soni, Sushant; Le, Catherine; Biasca, Matthew; Farr, Erik; Chen, Eric Y.-T.; Chin, Wei-Chun

    2012-02-01

    Human embryonic stem cells [hESCs] are able to differentiate into specific lineages corresponding to regulated spatial and temporal signals. This unique attribute holds great promise for regenerative medicine and cell-based therapy for many human diseases such as spinal cord injury [SCI] and multiple sclerosis [MS]. Carbon nanotubes [CNTs] have been successfully used to promote neuronal differentiation, and silk has been widely applied in tissue engineering. This study aims to build silk-CNT composite scaffolds for improved neuron differentiation efficiency from hESCs. Two neuronal markers (β-III tubulin and nestin) were utilized to determine the hESC neuronal lineage differentiation. In addition, axonal lengths were measured to evaluate the progress of neuronal development. The results demonstrated that cells on silk-CNT scaffolds have a higher β-III tubulin and nestin expression, suggesting augmented neuronal differentiation. In addition, longer axons with higher density were found to associate with silk-CNT scaffolds. Our silk-CNT-based composite scaffolds can promote neuronal differentiation of hESCs. The silk-CNT composite scaffolds developed here can serve as efficient supporting matrices for stem cell-derived neuronal transplants, offering a promising opportunity for nerve repair treatments for SCI and MS patients.

  1. In vitro differentiation potential of human haematopoietic CD34(+) cells towards pancreatic β-cells.

    PubMed

    Sunitha, Manne Mudhu; Srikanth, Lokanathan; Santhosh Kumar, Pasupuleti; Chandrasekhar, Chodimella; Sarma, Potukuchi Venkata Gurunadha Krishna

    2016-10-01

    Haematopoietic stem cells (HSCs) possess multipotent ability to differentiate into various types of cells on providing appropriate niche. In the present study, the differentiating potential of human HSCs into β-cells of islets of langerhans was explored. Human HSCs were apheretically isolated from a donor and cultured. Phenotypic characterization of CD34 glycoprotein in the growing monolayer HSCs was confirmed by immunocytochemistry and flow cytometry techniques. HSCs were induced by selection with beta cell differentiating medium (BDM), which consists of epidermal growth factor (EGF), fibroblast growth factor (FGF), transferrin, Triiodo-l-Tyronine, nicotinamide and activin A. Distinct morphological changes of differentiated cells were observed on staining with dithizone (DTZ) and expression of PDX1, insulin and synaptophysin was confirmed by immunocytochemistry. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed distinct expression of specific β-cell markers, pancreatic and duodenal homeobox-1 (PDX1), glucose transporter-2 (GLUT-2), synaptophysin (SYP) and insulin (INS) in these differentiated cells compared to HSCs. Further, these cells exhibited elevated expression of INS gene at 10 mM glucose upon inducing with different glucose concentrations. The prominent feature of the obtained β-cells was the presence of glucose sensors, which was determined by glucokinase activity and high glucokinase activity compared with CD34(+) stem cells. These findings illustrate the differentiation of CD34(+) HSCs into β-cells of islets of langerhans. PMID:27514733

  2. Soft substrates drive optimal differentiation of human healthy and dystrophic myotubes.

    PubMed

    Serena, Elena; Zatti, Susi; Reghelin, Elena; Pasut, Alessandra; Cimetta, Elisa; Elvassore, Nicola

    2010-04-01

    The in vitro development of human myotubes carrying genetic diseases, such as Duchenne Muscular Dystrophy, will open new perspectives in the identification of innovative therapeutic strategies. Through the proper design of the substrate, we guided the differentiation of human healthy and dystrophic myoblasts into myotubes exhibiting marked functional differentiation and highly defined sarcomeric organization. A thin film of photo cross-linkable elastic poly-acrylamide hydrogel with physiological-like and tunable mechanical properties (elastic moduli, E: 12, 15, 18 and 21 kPa) was used as substrate. The functionalization of its surface by micro-patterning in parallel lanes (75 microm wide, 100 microm spaced) of three adhesion proteins (laminin, fibronectin and matrigel) was meant to maximize human myoblasts fusion. Myotubes formed ont