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Developmental Study of Fragile X Syndrome Using Human Embryonic Stem Cells Derived from Preimplantation Genetically Diagnosed Embryos

Department of Genetics, Silberman Institute of Life Science, The Hebrew University, Jerusalem 91904, Israel.
Cell Stem Cell (Impact Factor: 22.15). 12/2007; 1(5):568-77. DOI: 10.1016/j.stem.2007.09.001
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ABSTRACT We report on the establishment of a human embryonic stem cell (HESC) line from a preimplantation fragile X-affected embryo and demonstrate its value as an appropriate model to study developmentally regulated events that are involved in the pathogenesis of this disorder. Fragile X syndrome results from FMR1 gene inactivation due to a CGG expansion at the 5'UTR region of the gene. Early events in FMR1 silencing have not been fully characterized due to the lack of appropriate animal or cellular models. Here we show that, despite the presence of a full mutation, affected undifferentiated HESCs express FMR1 and are DNA unmethylated. However, epigenetic silencing by DNA methylation and histone modification occurs upon differentiation. Our unique cell system allows the dissection of the sequence by which these epigenetic changes are acquired and illustrates the importance of HESCs in unraveling developmentally regulated mechanisms associated with human genetic disorders.

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Available from: Amir Eden, Jul 29, 2015
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    • "For example , the most widely used mouse model was created via targeted exon disruption (1994). Although patients with FXS might express some FMRP during early embryonic development when the gene is thought to be active [Eiges et al., 2007; Colak et al., 2014], the Fmr1 KO mice do not express FMRP at all. Furthermore, many FXS patients are mosaics either due to the presence of partially silenced alleles or premutation (PM) length (CGG repeats between 55 and 200) alleles that are not silenced and thus express variable levels of FMRP [Wohrle et al., 1998]. "
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    ABSTRACT: Fragile X Syndrome (FXS) is the most frequent cause of inherited intellectual disability and autism. It is caused by the absence of the fragile X mental retardation 1 (FMR1) gene product, FMRP, an RNA-binding protein involved in the regulation of translation of a subset of brain mRNAs. In Fmr1 knockout (KO) mice, the absence of FMRP results in elevated protein synthesis in the brain as well as increased signaling of many translational regulators. Whether protein synthesis is also dysregulated in FXS patients is not firmly established. Here, we demonstrate that fibroblasts from FXS patients have significantly elevated rates of basal protein synthesis along with increased levels of phosphorylated mechanistic target of rapamycin (p-mTOR), phosphorylated extracellular signal regulated kinase 1/2 (p-ERK 1/2) and phosphorylated p70 ribosomal S6 kinase 1 (p-S6K1). Treatment with small molecules that inhibit S6K1, and a known FMRP target, phosphoinositide 3-kinase (P13K) catalytic subunit p110β, lowered the rates of protein synthesis in both control and patient fibroblasts. Our data thus demonstrate that fibroblasts from FXS patients may be a useful in vitro model to test the efficacy and toxicity of potential therapeutics prior to clinical trials, as well as for drug screening and designing personalized treatment approaches.This article is protected by copyright. All rights reserved
    Human Mutation 12/2014; 35(12). DOI:10.1002/humu.22699 · 5.05 Impact Factor
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    • "On the other hand they can be differentiated into virtually any cell type in the body, including extra-embryonic lineages (cyto-and syncytiotrophoblasts) (Drukker et al., 2012; Sudheer et al., 2012; Giakoumopoulos and Golos, 2013), and can therefore serve as a highly effective tool for studying early stages of implantation. hESCs that carry chromosomal aberrations can be derived directly from affected embryos following preimplantation genetic diagnosis (PGD) (Eiges et al., 2007; Ben-Yosef et al., 2008; Deleu et al., 2009; Frumkin et al., 2010; Seriola et al., 2011) or from karyotypically normal hESCs by inducing DNA double-strand breaks at specified loci using zinc finger nucleases (Brunet et al., 2009). These translocated hESCs can then serve as a human in vitro model for studying genetic disorders. "
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    ABSTRACT: Carriers of the balanced translocation t(11;22), the most common reciprocal translocation in humans, are at high risk of creating gametes with unbalanced translocation, leading to repeated miscarriages. Current research models for studying translocated embryos and the biological basis for their implantation failure are limited. The aim of this study was to elucidate whether human embryonic stem cells (hESCs) carrying the unbalanced chromosome translocation t(11;22) can provide an explanation for repeated miscarriages of unbalanced translocated embryos. Fluorescent in-situ hybridization and karyotype analysis were performed to analyze the translocation t(11;22) in embryos during PGD and in the derived hESC line. The hESC line was characterized by RT-PCR and FACS analysis for pluripotent markers. Directed differentiation to trophoblasts was carried out by bone morphogenetic protein 4 (BMP4). Trophoblast development was analyzed by measuring β-hCG secretion, by β-hCG immunostaining and by gene expression of trophoblastic markers. We derived the first hESC line carrying unbalanced t(11;22), which showed the typical morphological and molecular characteristics of a hESC line. Control hESCs differentiated into trophoblasts secreted increasing levels of β-hCG and concomitantely expressed the trophoblast genes, CDX2, TP63, KRT7, ERVW1, CGA, GCM1, KLF4 and PPARG. In contrast, differentiated translocated hESCs displayed reduced and delayed secretion of β-hCG concomitant with impaired expression of the trophoblastic genes. The reduced activation of trophoblastic genes may be responsible for the impaired trophoblastic differentiation in translocation t(11;22)-hESCs, associated with implantation failure in unbalanced translocation t(11;22) embryos. Our t(11;22) hESCs are presented as a valuable human model for studying the mechanisms underlying implantation failure.
    Molecular Human Reproduction 11/2014; 21(3). DOI:10.1093/molehr/gau104 · 3.48 Impact Factor
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    • "All cell lines were established in Shaare Zedek Medical Center (Institutional Review Board [IRB] 87/07) apart from LS-FX9, which was kindly provided by the Racine IVF Unit, Tel-Aviv Sourasky Medical Center (IRB 7/04-043). Cell line derivation and characterization were carried out as previously described (Eiges et al., 2007). "
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    ABSTRACT: Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It results from epigenetic silencing of the X-linked FMR1 gene by a CGG expansion in its 5′-untranslated region. Taking advantage of a large set of FXS-affected human embryonic stem cell (HESC) lines and isogenic subclones derived from them, we show that FMR1 hypermethylation commonly occurs in the undifferentiated state (six of nine lines, ranging from 24% to 65%). In addition, we demonstrate that hypermethylation is tightly linked with FMR1 transcriptional inactivation in undifferentiated cells, coincides with loss of H3K4me2 and gain of H3K9me3, and is unrelated to CTCF binding. Taken together, these results demonstrate that FMR1 epigenetic gene silencing takes place in FXS HESCs and clearly highlights the importance of examining multiple cell lines when investigating FXS and most likely other epigenetically regulated diseases.
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