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miR-126 regulates placental development and glucose metabolism
100
Abstracts / Placenta 45 (2016) 63e133
Methods: Differentiation of hESCs was carried out by BMP addition in hESC colonies of varying size to evaluate effect of BMP on endogenous Activin/Nodal signaling. TB differentiation was also evaluated in the presence or absence of BMP inhibition, when Activin/Nodal signaling was inhibited. TB differentiation was assessed using a stringent set of criteria, as previously discussed (Sarkar et al., J. Biol Chem 2015 290(14):8834-48) Results: Initially, BMP signaling inhibits Activin/Nodal signaling during BMP-induced TB differentiation. Endogenous Activin/Nodal signaling can result in variability of TB differentiation; Activin/Nodal inhibition is necessary for TB differentiation. However, BMP is dispensable in this stage of differentiation, i.e. acquisition of TB fate, as long as Activin/Nodal inhibition is maintained. Yet, during later stages of TB differentiation, BMP signaling is necessary for formation of functional syncytiotrophoblasts (STB). Specifically, although multinucleate cells with morphology and syncytin expression consistent with STB are obtained when BMP is inhibited, we observe that secretion of hCG is negligible compared to corresponding controls wherein BMP signaling is not inhibited. On the other hand, extravillous TB (eCTB) can be obtained even in the presence of BMP inhibition. Conclusion: BMP signaling plays a biphasic role in TB differentiation of hESCs. P1.91 A QUIET DEATH; HYPOXIC STRESS FORCES DIMINISHED GROWTH AND POTENCY AND LARGE, IRREVERSIBLE TROPHOBLAST STEM CELL DIFFERENTIATION Dan Rappolee. Wayne State University, Detroit MI, USA Objectives: Stresses slow trophoblast stem cell (TSC) growth, force potency decrease and differentiation increase to functionally compensate for fewer cells. Previous reports suggest a reversibility period in stressed TSC differentiation. Here we test for periods of normal and stress-forced irreversible differentiation. Methods: In a previously optimized model, TSCs were cultured initially for 1-4 days with FGF4 removal, (normal differentiation) or 2-5 days of hypoxic stress with FGF4 at 0.5% O2 (stress-forced differentiation). After each day of initial culture, commitment to differentiation was tested in a additional 3 day culture in normal differentiation media or normal potency media (with FGF4, heparin, 20% O2). At the end of the initial period and the commitment period, cells were tested for trophoblast giant cell (TGC) population% by micrography and flow cytometry, and potency factor (Cdx2, Id2, ErrB) protein and differentiated protein (PL1) expression by immunoblot. Results: Hypoxic stress at 0.5% O2 forces 60-90% potency factor loss and nil growth despite FGF4. Hypoxic stress increases differentiated trophoblast giant cells (TGC)s to ~50% of cells and a 5-6 fold increase in the TGC product antiluteolytic hormone PL1. Normal differentiation with FGF4 removal for 2 days produced cells that increase TGC% and have near-zero Cdx2 whether or not FGF4 is re-added for 3 additional days. These cells are irreversibly differentiated. Hypoxic stress with FGF4 irreversibly differentiates cells in 4 days; TGC% increase and Cdx2 remains at near-zero levels in either normal or differentiation “commitment” culture. Conclusion: Prolonged reversibility should increase survival probability once stress subsides. Runted stem cell growth, forced differentiation of fewer cells, and irreversible differentiation should initially increase antiluteolytic hormone to maintain pregnancy, followed by plateau. Stressforced irreversible differentiation despite FGF4 presence is surprising. If this occurs in vivo it would lead to embryo lethality without cell death as no TSC stemness reserve would remain. P1.92 MIR-126 REGULATES METABOLISM
PLACENTAL
DEVELOPMENT
AND
GLUCOSE
Abhijeet Sharma, Heidi Stuhlmann. Weill Cornell Medical College, New York, USA Background: A functional placenta develops through a delicate interplay of the maternal and fetal vasculature and the supporting trophoblast compartment. Defects during placental development can lead to adverse
outcomes for the mother and the fetus. Placental development is controlled by a host of known genetic factors. In contrast, the role of noncoding RNAs in placental development has not been investigated. The endothelial-specific microRNA miR-126 regulates angiogenesis in mouse embryos and adults. Loss of miR-126 in mice results in perinatal lethality due to vascular defects in the embryos. However, the role of miR-126 in placental development remains unknown. Objectives: To elucidate the role of miR-126 in mouse placental development. Methods: Placental tissue from congenic miR-126 KO was compared to WT tissue using immunohistochemical and biochemical methods to study histopathology and gene expression. Results: miR-126 KO congenic mice die in utero and are haploinsufficient. miR-126 has a novel expression domain in trophoblasts of the placenta. E15.5 miR-126 KO placentas display hypertrophy of the junctional zone and a reduced fetal labyrinth. Preliminary results indicate that the expansion in the junctional zone is due to reduced proliferating glycogen progenitors and increased glycogen cell differentiation. miR-126 can bind to the 3’UTR of IRS1 and miR-126 KO placentas overexpress IRS1 by 2.5 fold when compared to WT placentas. Based on these results, we examined if miR-126 controls glucose metabolism in adults. Our results show that adult male miR-126+/- mice display fasting hyperglycemia and reduced ability to clear glucose from their bloodstream during a glucose tolerance test. Conclusions: Our findings suggest that miR-126 regulates glycogen cell differentiation in the mouse placenta by regulating insulin signaling through direct control of Irs1 transcripts. Dysregulation of the insulinsignaling pathway in miR-126 KO placentas may implicate a role for miR126 in gestational and adult type 2 diabetes. P1.93 DIAMINE OXIDASE IN PREGNANCY e AN OLD ACQUAINTANCE EXCLUSIVELY RELEASED FROM AN UNEXPECTED SOURCE € fler 1, Thomas Philipp Velicky 1, Sophie Pils 1, Bernd Jilma 1, Martin Kno € hm 1, David Cantonwine 3, Thomas Bo Jürgen McElrath 2, 3, Pollheimer 1. 1 Medical University of Vienna, Vienna, Austria; 2 Harvard Medical School, Boston, Massachusetts, USA; 3 Brigham and Women's Hospital, Boston, Massachusetts, USA Objective: The histamine- and polyamine-degrading enzyme diamine oxidase (DAO, AOC1) is produced in large amounts at the fetal-maternal interface. Consequently, the activity of DAO increases more than 100-fold in serum or plasma of pregnant women. For decades maternal decidual cells have been considered as the source of that increase, possibly as a protective mechanism against adverse effects of histamine such as uterine contractions. In contrast, our present data show that it is not maternal uterine tissue but instead the placenta itself synthesizes and secrets DAO. Methods: Immunofluorescence, laser-scanning microscopy, qRT-PCR, RNA-sequencing, western blotting and a novel DAO ELISA were used to investigate DAO expression in human placental tissue and to measure concentrations in maternal plasma and tissue culture supernatants. A DAO activity assay and siRNA-mediated gene knock-down experiments were performed in primary trophoblast cultures to obtain functional data. Results: Contrary to the published literature, DAO could not be detected in various maternal decidual populations, neither in situ nor in primary isolated cells. Instead, a subpopulation of extravillous trophoblasts (EVTs) showed strong DAO mRNA and protein expression, especially when in close proximity to spiral arteries. During in vitro differentiation AOC1 is amongst the highest upregulated genes in EVTs and an activity assay confirmed that supernatants of differentiated EVT cultures contain functionally active DAO. Over the course of pregnancy, elevated DAO concentrations were detectable as early as the 7th week of gestation and increased up to 100-fold before decreasing rapidly after delivery. Our data suggest decreased DAO serum levels at week 10 of gestation in pregnancies that went on to develop preeclampsia compared to healthy controls. Conclusion: We introduce DAO as an EVT-specific factor acting at the fetalmaternal interface and the maternal circulation to protect the fetus from maternal histamine. Early rise and detectability make it an interesting candidate for the investigation of EVT-related diseases.
Abstracts / Placenta 45 (2016) 63e133
Methods: Differentiation of hESCs was carried out by BMP addition in hESC colonies of varying size to evaluate effect of BMP on endogenous Activin/Nodal signaling. TB differentiation was also evaluated in the presence or absence of BMP inhibition, when Activin/Nodal signaling was inhibited. TB differentiation was assessed using a stringent set of criteria, as previously discussed (Sarkar et al., J. Biol Chem 2015 290(14):8834-48) Results: Initially, BMP signaling inhibits Activin/Nodal signaling during BMP-induced TB differentiation. Endogenous Activin/Nodal signaling can result in variability of TB differentiation; Activin/Nodal inhibition is necessary for TB differentiation. However, BMP is dispensable in this stage of differentiation, i.e. acquisition of TB fate, as long as Activin/Nodal inhibition is maintained. Yet, during later stages of TB differentiation, BMP signaling is necessary for formation of functional syncytiotrophoblasts (STB). Specifically, although multinucleate cells with morphology and syncytin expression consistent with STB are obtained when BMP is inhibited, we observe that secretion of hCG is negligible compared to corresponding controls wherein BMP signaling is not inhibited. On the other hand, extravillous TB (eCTB) can be obtained even in the presence of BMP inhibition. Conclusion: BMP signaling plays a biphasic role in TB differentiation of hESCs. P1.91 A QUIET DEATH; HYPOXIC STRESS FORCES DIMINISHED GROWTH AND POTENCY AND LARGE, IRREVERSIBLE TROPHOBLAST STEM CELL DIFFERENTIATION Dan Rappolee. Wayne State University, Detroit MI, USA Objectives: Stresses slow trophoblast stem cell (TSC) growth, force potency decrease and differentiation increase to functionally compensate for fewer cells. Previous reports suggest a reversibility period in stressed TSC differentiation. Here we test for periods of normal and stress-forced irreversible differentiation. Methods: In a previously optimized model, TSCs were cultured initially for 1-4 days with FGF4 removal, (normal differentiation) or 2-5 days of hypoxic stress with FGF4 at 0.5% O2 (stress-forced differentiation). After each day of initial culture, commitment to differentiation was tested in a additional 3 day culture in normal differentiation media or normal potency media (with FGF4, heparin, 20% O2). At the end of the initial period and the commitment period, cells were tested for trophoblast giant cell (TGC) population% by micrography and flow cytometry, and potency factor (Cdx2, Id2, ErrB) protein and differentiated protein (PL1) expression by immunoblot. Results: Hypoxic stress at 0.5% O2 forces 60-90% potency factor loss and nil growth despite FGF4. Hypoxic stress increases differentiated trophoblast giant cells (TGC)s to ~50% of cells and a 5-6 fold increase in the TGC product antiluteolytic hormone PL1. Normal differentiation with FGF4 removal for 2 days produced cells that increase TGC% and have near-zero Cdx2 whether or not FGF4 is re-added for 3 additional days. These cells are irreversibly differentiated. Hypoxic stress with FGF4 irreversibly differentiates cells in 4 days; TGC% increase and Cdx2 remains at near-zero levels in either normal or differentiation “commitment” culture. Conclusion: Prolonged reversibility should increase survival probability once stress subsides. Runted stem cell growth, forced differentiation of fewer cells, and irreversible differentiation should initially increase antiluteolytic hormone to maintain pregnancy, followed by plateau. Stressforced irreversible differentiation despite FGF4 presence is surprising. If this occurs in vivo it would lead to embryo lethality without cell death as no TSC stemness reserve would remain. P1.92 MIR-126 REGULATES METABOLISM
PLACENTAL
DEVELOPMENT
AND
GLUCOSE
Abhijeet Sharma, Heidi Stuhlmann. Weill Cornell Medical College, New York, USA Background: A functional placenta develops through a delicate interplay of the maternal and fetal vasculature and the supporting trophoblast compartment. Defects during placental development can lead to adverse
outcomes for the mother and the fetus. Placental development is controlled by a host of known genetic factors. In contrast, the role of noncoding RNAs in placental development has not been investigated. The endothelial-specific microRNA miR-126 regulates angiogenesis in mouse embryos and adults. Loss of miR-126 in mice results in perinatal lethality due to vascular defects in the embryos. However, the role of miR-126 in placental development remains unknown. Objectives: To elucidate the role of miR-126 in mouse placental development. Methods: Placental tissue from congenic miR-126 KO was compared to WT tissue using immunohistochemical and biochemical methods to study histopathology and gene expression. Results: miR-126 KO congenic mice die in utero and are haploinsufficient. miR-126 has a novel expression domain in trophoblasts of the placenta. E15.5 miR-126 KO placentas display hypertrophy of the junctional zone and a reduced fetal labyrinth. Preliminary results indicate that the expansion in the junctional zone is due to reduced proliferating glycogen progenitors and increased glycogen cell differentiation. miR-126 can bind to the 3’UTR of IRS1 and miR-126 KO placentas overexpress IRS1 by 2.5 fold when compared to WT placentas. Based on these results, we examined if miR-126 controls glucose metabolism in adults. Our results show that adult male miR-126+/- mice display fasting hyperglycemia and reduced ability to clear glucose from their bloodstream during a glucose tolerance test. Conclusions: Our findings suggest that miR-126 regulates glycogen cell differentiation in the mouse placenta by regulating insulin signaling through direct control of Irs1 transcripts. Dysregulation of the insulinsignaling pathway in miR-126 KO placentas may implicate a role for miR126 in gestational and adult type 2 diabetes. P1.93 DIAMINE OXIDASE IN PREGNANCY e AN OLD ACQUAINTANCE EXCLUSIVELY RELEASED FROM AN UNEXPECTED SOURCE € fler 1, Thomas Philipp Velicky 1, Sophie Pils 1, Bernd Jilma 1, Martin Kno € hm 1, David Cantonwine 3, Thomas Bo Jürgen McElrath 2, 3, Pollheimer 1. 1 Medical University of Vienna, Vienna, Austria; 2 Harvard Medical School, Boston, Massachusetts, USA; 3 Brigham and Women's Hospital, Boston, Massachusetts, USA Objective: The histamine- and polyamine-degrading enzyme diamine oxidase (DAO, AOC1) is produced in large amounts at the fetal-maternal interface. Consequently, the activity of DAO increases more than 100-fold in serum or plasma of pregnant women. For decades maternal decidual cells have been considered as the source of that increase, possibly as a protective mechanism against adverse effects of histamine such as uterine contractions. In contrast, our present data show that it is not maternal uterine tissue but instead the placenta itself synthesizes and secrets DAO. Methods: Immunofluorescence, laser-scanning microscopy, qRT-PCR, RNA-sequencing, western blotting and a novel DAO ELISA were used to investigate DAO expression in human placental tissue and to measure concentrations in maternal plasma and tissue culture supernatants. A DAO activity assay and siRNA-mediated gene knock-down experiments were performed in primary trophoblast cultures to obtain functional data. Results: Contrary to the published literature, DAO could not be detected in various maternal decidual populations, neither in situ nor in primary isolated cells. Instead, a subpopulation of extravillous trophoblasts (EVTs) showed strong DAO mRNA and protein expression, especially when in close proximity to spiral arteries. During in vitro differentiation AOC1 is amongst the highest upregulated genes in EVTs and an activity assay confirmed that supernatants of differentiated EVT cultures contain functionally active DAO. Over the course of pregnancy, elevated DAO concentrations were detectable as early as the 7th week of gestation and increased up to 100-fold before decreasing rapidly after delivery. Our data suggest decreased DAO serum levels at week 10 of gestation in pregnancies that went on to develop preeclampsia compared to healthy controls. Conclusion: We introduce DAO as an EVT-specific factor acting at the fetalmaternal interface and the maternal circulation to protect the fetus from maternal histamine. Early rise and detectability make it an interesting candidate for the investigation of EVT-related diseases.