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In fragile X syndrome (FXS),CGG repeat expansion greater than 200 triplets is believed to trigger FMR1 gene silencing and disease etiology. However,FXS siblings have been identified with more than 200 CGGs,termed unmethylated full mutation (UFM) carriers,without gene silencing and disease symptoms. Here,we show that hypomethylation of the FMR1 promoter is maintained in induced pluripotent stem cells (iPSCs) derived from two UFM individuals. However,a subset of iPSC clones with large CGG expansions carries silenced FMR1. Furthermore,we demonstrate de novo silencing upon expansion of the CGG repeat size. FMR1 does not undergo silencing during neuronal differentiation of UFM iPSCs,and expression of large unmethylated CGG repeats has phenotypic consequences resulting in neurodegenerative features. Our data suggest that UFM individuals do not lack the cell-intrinsic ability to silence FMR1 and that inter-individual variability in the CGG repeat size required for silencing exists in the FXS population. View Publication

Background Proteoglycans are found on the cell surface and in the extracellular matrix,and serve as prime sites for interaction with signaling molecules. Proteoglycans help regulate pathways that control stem cell fate,and therefore represent an excellent tool to manipulate these pathways. Despite their importance,there is a dearth of data linking glycosaminoglycan structure within proteoglycans with stem cell differentiation. Methods Human embryonic stem cell line WA09 (H9) was differentiated into early mesoderm and endoderm lineages,and the glycosaminoglycanomic changes accompanying these transitions were studied using transcript analysis,immunoblotting,immunofluorescence and disaccharide analysis. Results Pluripotent H9 cell lumican had no glycosaminoglycan chains whereas in splanchnic mesoderm lumican was glycosaminoglycanated. H9 cells have primarily non-sulfated heparan sulfate chains. On differentiation towards splanchnic mesoderm and hepatic lineages N-sulfo group content increases. Differences in transcript expression of NDST1,HS6ST2 and HS6ST3,three heparan sulfate biosynthetic enzymes,within splanchnic mesoderm cells compared to H9 cells correlate to changes in glycosaminoglycan structure. Conclusions Differentiation of embryonic stem cells markedly changes the proteoglycanome. General significance The glycosaminoglycan biosynthetic pathway is complex and highly regulated,and therefore,understanding the details of this pathway should enable better control with the aim of directing stem cell differentiation. ?? 2014 Elsevier B.V. All rights reserved. View Publication

Pluripotent stem cells,including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),have the potential to treat type 1 diabetes through cell replacement therapy. However,the protocols used to generate insulin-expressing cells in vitro frequently result in cells which have an immature phenotype and are functionally restricted. MicroRNAs (miRNAs) are now known to be important in cell fate specification,and a unique miRNA signature characterises pancreatic development at the definitive endoderm stage. Several studies have described differences in miRNA expression between ESCs and iPSCs. Here we have used microarray analysis both to identify miRNAs up- or down-regulated upon endoderm formation,and also miRNAs differentially expressed between ESCs and iPSCs. Several miRNAs fulfilling both these criteria were identified,suggesting that differences in the expression of these miRNAs may affect the ability of pluripotent stem cells to differentiate into definitive endoderm. The expression of these miRNAs was validated by qRT-PCR,and the relationship between one of these miRNAs,miR-151a-5p,and its predicted target gene,SOX17,was investigated by luciferase assay,and suggested an interaction between miR-151a-5p and this key transcription factor. In conclusion,these findings demonstrate a unique miRNA expression pattern for definitive endoderm derived from both embryonic and induced pluripotent stem cells. View Publication

Human embryonic stem cells (hESCs) provide great opportunities for regenerative medicine,pharmacological and toxicological investigation,and the study of human embryonic development. These applications require proper derivation,maintenance,and extensive characterization of undifferentiated cells before being used for differentiation into cells of interest. Undifferentiated hESCs possess several unique features,including their extensive proliferation capacity in the undifferentiated state,ability to maintain a normal karyotype after long-term culture,expression of markers characteristic of stem cells,high constitutive telomerase activity,and capacity to differentiate into essentially all somatic cell types. This chapter will summarize the current development in culture conditions and provide technical details for the evaluation and characterization of hESCs. View Publication

PAIRED (PRD)-like homeobox genes belong to a class of predicted transcription factor genes. Several of these PRD-like homeobox genes have been predicted in silico from genomic sequence but until recently had no evidence of transcript expression. We found recently that nine PRD-like homeobox genes,ARGFX,CPHX1,CPHX2,DPRX,DUXA,DUXB,NOBOX,TPRX1 and TPRX2,were expressed in human preimplantation embryos. In the current study we characterized these PRD-like homeobox genes in depth and studied their functions as transcription factors. We cloned multiple transcript variants from human embryos and showed that the expression of these genes is specific to embryos and pluripotent stem cells. Overexpression of the genes in human embryonic stem cells confirmed their roles as transcription factors as either activators (CPHX1,CPHX2,ARGFX) or repressors (DPRX,DUXA,TPRX2) with distinct targets that could be explained by the amino acid sequence in homeodomain. Some PRD-like homeodomain transcription factors had high concordance of target genes and showed enrichment for both developmentally important gene sets and a 36 bp DNA recognition motif implicated in Embryo Genome Activation (EGA). Our data implicate a role for these previously uncharacterized PRD-like homeodomain proteins in the regulation of human embryo genome activation and preimplantation embryo development. View Publication

The enteric nervous system (ENS) is recognized as a second brain because of its complexity and its largely autonomic control of bowel function. Recent progress in studying the interactions between the ENS and the central nervous system (CNS) has implicated alterations of the gut/brain axis as a possible mechanism in the pathophysiology of autism spectrum disorders (ASDs),Parkinson's disease (PD) and other human CNS disorders,whereas the underlying mechanisms are largely unknown because of the lack of good model systems. Human induced pluripotent stem cells (hiPSCs) have the ability to proliferate indefinitely and differentiate into cells of all three germ layers,thus making iPSCs an ideal source of cells for disease modelling and cell therapy. Here,hiPSCs were induced to differentiate into neural crest stem cells (NCSCs) efficiently. When co-cultured with smooth muscle layers of ganglionic gut tissue,the NCSCs differentiated into different subtypes of mature enteric-like neurons expressing nitric oxide synthase (nNOS),vasoactive intestinal polypeptide (VIP),choline acetyltransferase (ChAT) or calretinin with typical electrophysiological characteristics of functional neurons. Furthermore,when they were transplanted into aneural or aganglionic chick,mouse or human gut tissues in ovo,in vitro or in vivo,hiPSC-derived NCSCs showed extensive migration and neural differentiation capacity,generating neurons and glial cells that expressed phenotypic markers characteristic of the enteric nervous system. Our results indicate that enteric NCSCs derived from hiPSCs supply a powerful tool for studying the pathogenesis of gastrointestinal disorders and brain/gut dysfunction and represent a potentially ideal cell source for enteric neural transplantation treatments.Molecular Psychiatry advance online publication,25 October 2016; doi:10.1038/mp.2016.191. View Publication

Bipolar disorder (BD) is a common neuropsychiatric disorder characterized by chronic recurrent episodes of depression and mania. Despite evidence for high heritability of BD,little is known about its underlying pathophysiology. To develop new tools for investigating the molecular and cellular basis of BD,we applied a family-based paradigm to derive and characterize a set of 12 induced pluripotent stem cell (iPSC) lines from a quartet consisting of two BD-affected brothers and their two unaffected parents. Initially,no significant phenotypic differences were observed between iPSCs derived from the different family members. However,upon directed neural differentiation,we observed that CXCR4 (CXC chemokine receptor-4) expressing central nervous system (CNS) neural progenitor cells (NPCs) from both BD patients compared with their unaffected parents exhibited multiple phenotypic differences at the level of neurogenesis and expression of genes critical for neuroplasticity,including WNT pathway components and ion channel subunits. Treatment of the CXCR4(+) NPCs with a pharmacological inhibitor of glycogen synthase kinase 3,a known regulator of WNT signaling,was found to rescue a progenitor proliferation deficit in the BD patient NPCs. Taken together,these studies provide new cellular tools for dissecting the pathophysiology of BD and evidence for dysregulation of key pathways involved in neurodevelopment and neuroplasticity. Future generation of additional iPSCs following a family-based paradigm for modeling complex neuropsychiatric disorders in conjunction with in-depth phenotyping holds promise for providing insights into the pathophysiological substrates of BD and is likely to inform the development of targeted therapeutics for its treatment and ideally prevention. View Publication

The purpose of the present study was to characterize primitive epithelial progenitor populations present in adult normal human mammary tissue using a combination of flow cytometry and in vitro colony assay procedures. Three types of human breast epithelial cell (HBEC) progenitors were identified: luminal-restricted,myoepithelial-restricted and bipotent progenitors. The first type expressed epithelial cell adhesion molecule (EpCAM),alpha6 integrin and MUC1 and generated colonies composed exclusively of cells positive for the luminal-associated markers keratin 8/18,keratin 19,EpCAM and MUC1. Bipotent progenitors produced colonies containing a central core of cells expressing luminal markers surrounded by keratin 14+ myoepithelial-like cells. Single cell cultures confirmed the bipotentiality of these progenitors. Their high expression of alpha6 integrin and low expression of MUC1 suggests a basal position of these cells in the mammary epithelium in vivo. Serial passage in vitro of an enriched population of bipotent progenitors demonstrated that only myoepithelial-restricted progenitors could be readily generated under the culture conditions used. These results support a hierarchical branching model of HBEC progenitor differentiation from a primitive uncommitted cell to luminal- and myoepithelial-restricted progenitors. View Publication

BACKGROUND: The presence of chromosomal abnormalities could have a negative impact for human embryonic stem cell (hESC) applications both in regenerative medicine and in research. A biomarker that allows the identification of chromosomal abnormalities induced in hESC in culture before they take over the culture would represent an important tool for defining optimal culture conditions for hESC. Here we investigate the expression of CD30,reported to be a biomarker of hESCs with abnormal karyotype,in undifferentiated and spontaneously differentiated hESC.backslashnbackslashnMETHODS AND RESULTS: hESC were derived and cultured on mouse fibroblasts in KO-SR containing medium (serum free media) and passaged mechanically. Our results based on analysis at mRNA (RT-PCR) and protein (fluorescence-activated cell sorting and immunocytochemistry) level show that CD30 is expressed in undifferentiated hESC,even at very early passages,without any correlation with the presence of chromosomal anomalies. We also show that the expression of CD30 is rapidly lost during early spontaneous differentiation of hESC.backslashnbackslashnCONCLUSION: We conclude that CD30 expression in hESC cultures is probably a consequence of culture conditions,and that KO-SR may play a role. In addition,the expression of so-called 'stemness' markers does not change in undifferentiated hESC during long-term culture or when cells acquire chromosomal abnormalities. View Publication

Tissue engineering strategies,based on solid/porous scaffolds,suffer from several limitations,such as ineffective vascularization,poor cell distribution and organization within scaffold,in addition to low final cell density,among others. Therefore,the search for other tissue engineering approaches constitutes an active area of investigation. Decellularized matrices (DM) present major advantages compared to solid scaffolds,such as ideal chemical composition,the preservation of vascularization structure and perfect three-dimensional structure. In the present study,we aimed to characterize and investigate murine heart decellularized matrices as biomaterials for regular and whole organ tissue engineering. Heart decellularized matrices were characterized according to: 1. DNA content,through DNA quantificationo and PCR of isolated genomic DNA; 2. Histological structure,assessed after Hematoxylin and Eosin,as well as Masson's Trichrome stainings; 3. Surface nanostructure analysis,performed,using SEM. Those essays allowed us to conclude that DM was indeed decellularized,with preserved extracellular matrix structure. Following characterization,decellularized heart slices were seeded with induced Pluripotent Stem cells (iPS). As expected,but - to the best of our knowledge - never shown before,decellularization of murine heart matrices maintained matrix biocompatibility,as iPS cells rapidly attached to the surface of the material and proliferated. Strikingly though,heart DM presented a differentiation induction effect over those cells,which lost their pluripotency markers after 7 days of culture in the DM. Such loss of differentiation markers was observed,even though bFGF containing media mTSR was used during such period. Gene expression of iPS cells cultured on DM will be further analyzed,in order to assess the effects of culturing pluripotent stem cells in decellularized heart matrices. View Publication

We performed gene expression microarray analysis coupled with spherical self-organizing map (sSOM) for artificially developed cancer stem cells (CSCs). The CSCs were developed from human induced pluripotent stem cells (hiPSCs) with the conditioned media of cancer cell lines,whereas the CSCs were induced from primary cell culture of human cancer tissues with defined factors (OCT3/4,SOX2,and KLF4). These cells commonly expressed human embryonic stem cell (hESC)/hiPSC-specific genes (POU5F1,SOX2,NANOG,LIN28,and SALL4) at a level equivalent to those of control hiPSC 201B7. The sSOM with unsupervised method demonstrated that the CSCs could be divided into three groups based on their culture conditions and original cancer tissues. Furthermore,with supervised method,sSOM nominated TMED9,RNASE1,NGFR,ST3GAL1,TNS4,BTG2,SLC16A3,CD177,CES1,GDF15,STMN2,FAM20A,NPPB,CD99,MYL7,PRSS23,AHNAK,and LOC152573 genes commonly upregulating among the CSCs compared to hiPSC,suggesting the gene signature of the CSCs. View Publication

Recent evidence suggests that protease release by neutrophils in the bone marrow may contribute to hematopoietic progenitor cell (HPC) mobilization. Matrix metalloproteinase-9 (MMP-9),neutrophil elastase (NE),and cathepsin G (CG) accumulate in the bone marrow during granulocyte colony-stimulating factor (G-CSF) treatment,where they are thought to degrade key substrates including vascular cell adhesion molecule-1 (VCAM-1) and CXCL12. To test this hypothesis,HPC mobilization was characterized in transgenic mice deficient in one or more hematopoietic proteases. Surprisingly,HPC mobilization by G-CSF was normal in MMP-9-deficient mice,NE x CG-deficient mice,or mice lacking dipeptidyl peptidase I,an enzyme required for the functional activation of many hematopoietic serine proteases. Moreover,combined inhibition of neutrophil serine proteases and metalloproteinases had no significant effect on HPC mobilization. VCAM-1 expression on bone marrow stromal cells decreased during G-CSF treatment of wild-type mice but not NE x CG-deficient mice,indicating that VCAM-1 cleavage is not required for efficient HPC mobilization. G-CSF induced a significant decrease in CXCL12 alpha protein expression in the bone marrow of Ne x CG-deficient mice,indicating that these proteases are not required to down-regulate CXCL12 expression. Collectively,these data suggest a complex model in which both protease-dependent and -independent pathways may contribute to HPC mobilization. View Publication