Abstracts / Placenta 34 (2013) A1–A99A6

majority of FGR cases remain idiopathic. These idiopathic FGR pregnanciesare frequently associatedwith placental insufficiency, possibly as a result ofplacental maldevelopment. Understanding the molecular mechanisms ofabnormal placental development in idiopathic FGR is therefore ofincreasing importance. The focus of our studywas to understand the role ofhomeobox gene transcription factors in abnormal placental developmentassociatedwith human idiopathic FGR. The strategy we have employed hasresulted in the identification of homeobox genes, which are expressed innormal placental development and that showed altered expression in FGR.Functional assays following target gene inactivation or over-expression tomimic FGR in cultured cells demonstrated that homeobox genes controlimportant functions inplacental cells. The discoveryof targets of homeoboxgenes has revealed genes, and pathways, not previously implicated in FGR.These target genes and pathways will be further assessed for their thera-peutic anddiagnostic potential for clinicalmanagementof idiopathic FGR infuture.

http://dx.doi.org/10.1016/j.placenta.2013.06.022

PL5.4.OMICS EXPLORATION OF PLACENTAL PATHOLOGIES AND DEVELOP-MENT: TISSUES, CELLS AND GENES

Brian Cox

University of Toronto, Toronto, Canada

Preeclampsia is a dangerous placental and maternal pathology with poorlyunderstood etiology and no cure aside from delivery. Several large scaleclinical trials of proposed therapieshave failed to showsignificantefficacy. Toaddress these challenges many groups have used high-throughput tech-nologies, such as microarrays, proteomics and next-generation sequencingto identify the affectedmolecular pathways and biomarkers of preeclampsia.The assessmentofplacenta tissue samples using these techniqueshas not ledto advancements in diagnosis and treatment as seen in other fields(oncology). We propose a novel methodological approach to overcomeconfounding issues with previous analyses. First, placental biopsy samplescontain multiple cell types that may vary by pathology and cause signalinterference leading to an underestimation of gene expression differences.Second, the systematic belief that the clinical pathology of preeclampsia iscomposed of a single molecular pathology leading to the observation ofstudies being underpowered. We address the first by using a membraneproteomics approach to create a set of signature genes that are enriched to asingle cell type, the syncytiotrophoblast. We address the second by usingdata modelling of the signature gene set to identify multiple molecularsubclusters in the data. Applying thismethod to amicroarray data set of only16 pre-eclamptic patients we identified three distinct molecular pathologygroups consistingof chronic hypertensionwith superimposed preeclampsia,defective angiogenesis and defective hormone homoeostasis. Our novelmethodology is applied to other published data sets and to a large scale setwe generated from 165 samples from Research Centre for Women's andInfants Health (Mt. Sinai, Toronto, Canada). We are now testing if similar orother novelmolecular subtypes can be found in different geographic regions.

http://dx.doi.org/10.1016/j.placenta.2013.06.023

TRAPREGNANCY-RELATED MIRNAS IN TROPHOBLASTIC CELLS

Diana Maria Morales-Prieto, Stephanie Ospina-Prieto,Wittaya Chaiwangyen, Ekkehard Schleussner, Udo R. Markert

Placenta-Lab, University Hospital Jena, Jena, Germany

Objectives: Recent studies revealed a group of miRNAs specificallyexpressed during pregnancy and whose expression correlates with the

gestational age. Since miRNAs regulate cell processes including prolifera-tion and invasion, it is to hypothesize an important role of miRNAs in thecontrol of trophoblast behaviour. Study of miRNome in trophoblast cells isrestricted by the short lifespan and lack of proliferation in vitro. Therefore,the use of trophoblastic cell lines is of outmost importance. We analyzedthe miRNome of isolated trophoblast cells and compared themwith thoseof trophoblastic cell lines. Further, function of some pregnancy-specificmiRNAs belonging to C19MC and C14MC was investigated.Methods: Total RNA was isolated from cytotrophoblast cells from healthyfirst and term placentae and the cell lines HTR-8/SVneo, JEG-3, ACH-3P andAC1-M59. Expression level of 762 miRNAs was quantitatively analyzed byTaqMan HumanMicroRNA Arrays. Silencing of either miR-519d ormiR-134by miRNA-inhibitors was performed in JEG-3 and HTR-8/SVneo cells,respectively. After silencing, cell proliferation and invasion were assessedby BrdU and Matrigel analysis, respectively.Results: In first trimester trophoblast cells, C14MC members are highexpressed while C19MC members are low expressed. This profile changesin third trimester in which C19MC species are high while C14MC speciesare low expressed. Unsupervised clustering analysis revealed that profileof primary cells were more similar to those in JEG-3, ACH-3P and AC1-M59cells than to HTR-8/SVneo cells. Silencing of miR-519d increased JEG-3proliferation but reduced their invasion ability. MiR-134 inhibition resul-ted in an increased in HTR-8/svneo invasion while no changes in prolif-eration were observed.Conclusion: Primary trophoblast cells and cell lines differ in the miRNAprofile which confirms that results obtained from trophoblast cell linesmay require further validation. Our results suggest a regulatory role ofmiR-519d and miR-134 in the proliferation and invasion of trophoblasticcells.

http://dx.doi.org/10.1016/j.placenta.2013.06.024

NI.1.THE YOLK SAC OF NECROMYS LASIURUS (RODENTIA, CRICETIDAE) AS APROMISING SOURCE OF MESENCHYMAL STEM CELLS

Phelipe Favaron 1, Sonia Will 1, Andrea Mess 1,Moacir Franco de Oliveira 2, Maria Angelica Miglino 1

1University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil; 2Department of AnimalScience, Universidade Federal Rural do Semi-Árido, Mossoró, Rio Grande doNorte, Brazil

Recently, the fetal membranes are regarded to be abundant, ethicallyacceptable and easily accessible sources of stem cells. One of thesemembranes, the yolk sac (YS) is structurally diverse andmay be a source ofprogenitor mesenchymal stem cells (MSCs) that cause only minor immu-nogenic troubles. This had been shown for hematopoietic stem cells inlaboratory rodents such as the mouse.Objectives: Herein, we verified the potential of the YS from a rodentspecies native to South America, Necromys lasiurus, to obtain MSCs.Methods: Samples were obtained from a breeding group at UniversidadeFederal Rural do Semi-Árido, Mossoró, Brazil. The explants of 10 YSmembranes from mid gestation were maintained in DMEM-High glucosemedium with 10% fetal bovine serum. The cells were characterised bymorphological analysis, freezing assay, immunophenotyping by flow cit-ometry and immunofluorescence, cell differentiation and tumorigenicityassay.Results: The first adherent cells were observed 7 days after the explantswere plated and included fibroblast-like (92.13%) and epithelial-like cells(6.5%). Fibroblast-like cells were small, elongated and had reduced cyto-plasm, whereas the epithelial cells were large and rounded in shape withsparse cytoplasm and centrally nucleus. During the passages, the epithelialcells died. In contrast, the fibroblast-like cells reacted positively to MSC(Stro1, CD90, CD105, and CD73), pluripotency (Oct4 and Nanog), and he-matopoietic precursor cells (CD117) markers. They showed successfulosteogenic, adipogenic, and chondrogenic differentiation and did notdevelop tumors.