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Haploid human pluripotent stem cells (PSCs) integrate haploidy and pluripotency,providing a novel system for functional genomics and developmental research in humans. We have recently derived haploid human embryonic stem cells (ESCs) by parthenogenesis and demonstrated their wide differentiation potential and applicability for genetic screening. Because haploid cells can spontaneously become diploid,their enrichment at an early passage is key for successful derivation. In this protocol,we describe two methodologies,namely metaphase spread analysis and cell sorting,for the identification of haploid human cells within parthenogenetic ESC lines. The cell sorting approach also enables the isolation of haploid cells at low percentages,as well as the maintenance of highly enriched haploid ESC lines throughout passaging. The isolation of essentially pure populations of haploid human ESCs by this protocol requires basic PSC culture expertise and can be achieved within 4-6 weeks. View Publication

OBJECTIVE To elucidate the genetic background of a patient with neonatal-onset multisystem inflammatory disease (NOMID) with no NLRP3 mutation. METHODS A Japanese male child diagnosed as having NOMID was studied. The patient did not have any NLRP3 mutation,even as low-frequency mosaicism. We performed whole-exome sequencing on the patient and his parents. Induced pluripotent stem cells (iPSCs) were established from the patient's fibroblasts. The iPSCs were then differentiated into monocyte lineage to evaluate the cytokine profile. RESULTS We established multiple iPSC clones from a patient with NOMID and incidentally found that the phenotypes of monocytes from iPSC clones were heterogeneous and could be grouped into disease and normal phenotypes. Because each iPSC clone was derived from a single somatic cell,we hypothesized that the patient had somatic mosaicism of an interleukin-1β-related gene. Whole-exome sequencing of both representative iPSC clones and the patient's blood revealed a novel heterozygous NLRC4 mutation,p.T177A (c.529AtextgreaterG),as a specific mutation in diseased iPSC clones. Knockout of the NLRC4 gene using the clustered regularly interspaced short palindromic repeat/Cas9 system in a mutant iPSC clone abrogated the pathogenic phenotype. CONCLUSION Our findings indicate that the patient has somatic mosaicism of a novel NLRC4 mutation. To our knowledge,this is the first case showing that somatic mutation of NLRC4 causes autoinflammatory symptoms compatible with NOMID. The present study demonstrates the significance of prospective genetic screening combined with iPSC-based phenotype dissection for individualized diagnoses. View Publication

Surface marker expression forms the basis for characterization and isolation of human embryonic stem cells (hESCs). Currently,there are few well-defined protein epitopes that definitively mark hESCs. Here we combine immunotranscriptional profiling of hESC lines with membrane-polysome translation state array analysis (TSAA) to determine the full set of genes encoding potential hESC surface marker proteins. Three independently isolated hESC lines (HES2,H9,and MEL1) grown under feeder and feeder-free conditions were sorted into subpopulations by fluorescence-activated cell sorting based on coimmunoreactivity to the hESC surface markers GCTM-2 and CD9. Colony-forming assays confirmed that cells displaying high coimmunoreactivity to GCTM-2 and CD9 constitute an enriched subpopulation displaying multiple stem cell properties. Following microarray profiling,820 genes were identified that were common to the GCTM-2(high)/CD9(high) stem cell-like subpopulation. Membrane-polysome TSAA analysis of hESCs identified 1,492 mRNAs encoding actively translated plasma membrane and secreted proteins. Combining these data sets,88 genes encode proteins that mark the pluripotent subpopulation,of which only four had been previously reported. Cell surface immunoreactivity was confirmed for two of these markers: TACSTD1/EPCAM and CDH3/P-Cadherin,with antibodies for EPCAM able to enrich for pluripotent hESCs. This comprehensive listing of both hESCs and spontaneous differentiation-associated transcripts and survey of translated membrane-bound and secreted proteins provides a valuable resource for future study into the role of the extracellular environment in both the maintenance of pluripotency and directed differentiation. View Publication

BACKGROUND: Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators with important functions in development and disease. Here,we sought to identify and functionally characterize novel lncRNAs critical for vertebrate development. METHODS AND RESULTS: By relying on human pluripotent stem cell differentiation models,we investigated lncRNAs differentially regulated at key steps during human cardiovascular development with a special focus on vascular endothelial cells. RNA sequencing led to the generation of large data sets that serve as a gene expression roadmap highlighting gene expression changes during human pluripotent cell differentiation. Stage-specific analyses led to the identification of 3 previously uncharacterized lncRNAs,TERMINATOR,ALIEN,and PUNISHER,specifically expressed in undifferentiated pluripotent stem cells,cardiovascular progenitors,and differentiated endothelial cells,respectively. Functional characterization,including localization studies,dynamic expression analyses,epigenetic modification monitoring,and knockdown experiments in lower vertebrates,as well as murine embryos and human cells,confirmed a critical role for each lncRNA specific for each analyzed developmental stage. CONCLUSIONS: We have identified and functionally characterized 3 novel lncRNAs involved in vertebrate and human cardiovascular development,and we provide a comprehensive transcriptomic roadmap that sheds new light on the molecular mechanisms underlying human embryonic development,mesodermal commitment,and cardiovascular specification. View Publication

Huntington's disease is caused by expanded CAG repeats in HTT,conferring toxic gain of function on mutant HTT (mHTT) protein. Reducing mHTT amounts is postulated as a strategy for therapeutic intervention. We conducted genome-wide RNA interference screens for genes modifying mHTT abundance and identified 13 hits. We tested 10 in vivo in a Drosophila melanogaster Huntington's disease model,and 6 exhibited activity consistent with the in vitro screening results. Among these,negative regulator of ubiquitin-like protein 1 (NUB1) overexpression lowered mHTT in neuronal models and rescued mHTT-induced death. NUB1 reduces mHTT amounts by enhancing polyubiquitination and proteasomal degradation of mHTT protein. The process requires CUL3 and the ubiquitin-like protein NEDD8 necessary for CUL3 activation. As a potential approach to modulating NUB1 for treatment,interferon-β lowered mHTT and rescued neuronal toxicity through induction of NUB1. Thus,we have identified genes modifying endogenous mHTT using high-throughput screening and demonstrate NUB1 as an exemplar entry point for therapeutic intervention of Huntington's disease. View Publication

Human embryonic stem cells (hESCs) and induced pluripotent cells have the potential to provide an unlimited source of tissues for regenerative medicine. For this purpose,development of defined/xeno-free culture systems under feeder-free conditions is essential for the expansion of hESCs. Most defined/xeno-free media for the culture of hESCs contain basic fibroblast growth factor (bFGF). Therefore,bFGF is thought to have an almost essential role for the expansion of hESCs in an undifferentiated state. Here,we report identification of small molecules,some of which were neurotransmitter antagonists (trimipramine and ethopropazine),which promote long-term hESC self-renewal without bFGF in the medium. The hESCs maintained high expression levels of pluripotency markers,had a normal karyotype after 20 passages,and could differentiate into all three germ layers. ?? 2013 Elsevier Inc. View Publication

A potential application of embryonic and inducible pluripotent stem cells for the therapy of degenerative diseases involves pure somatic cells,free of tumorigenic undifferentiated embryonic and inducible pluripotent stem cells. In complex collections of chemicals with pharmacological potential we expect to find molecules able to induce specific pluripotent stem cell death,which could be used in some cell therapy settings to eliminate undifferentiated cells. Therefore,we have screened a chemical library of 1120 small chemicals to identify compounds that induce specifically apoptotic cell death in undifferentiated mouse embryonic stem cells (ESCs). Interestingly,three compounds currently used as clinically approved drugs,nortriptyline,benzethonium chloride and methylbenzethonium chloride,induced differential effects in cell viability in ESCs versus mouse embryonic fibroblasts (MEFs). Nortriptyline induced apoptotic cell death in MEFs but not in ESCs,whereas benzethonium and methylbenzethonium chloride showed the opposite effect. Nortriptyline,a tricyclic antidepressant,has also been described as a potent inhibitor of mitochondrial permeability transition,one of two major mechanisms involved in mitochondrial membrane permeabilization during apoptosis. Benzethonium chloride and methylbenzethonium chloride are quaternary ammonium salts used as antimicrobial agents with broad spectrum and have also been described as anticancer agents. A similar effect of benzethonium chloride was observed in human induced pluripotent stem cells (hiPSCs) when compared to both primary human skin fibroblasts and an established human fibroblast cell line. Human fibroblasts and hiPSCs were similarly resistant to nortriptyline,although with a different behavior. Our results indicate differential sensitivity of ESCs,hiPSCs and fibroblasts to certain chemical compounds,which might have important applications in the stem cell-based therapy by eliminating undifferentiated pluripotent stem cells from stem cell-derived somatic cells to prevent tumor formation after transplantation for therapy of degenerative diseases. View Publication

Recent technological advances in cell reprogramming by generation of induced pluripotent stem cells (iPSC) offer major perspectives in disease modelling and future hopes for providing novel stem cells sources in regenerative medicine. However,research on iPSC still requires refining the criteria of the pluripotency stage of these cells and exploration of their equivalent functionality to human embryonic stem cells (ESC). We report here on the use of infrared microspectroscopy to follow the spectral modification of somatic cells during the reprogramming process. We show that induced pluripotent stem cells (iPSC) adopt a chemical composition leading to a spectral signature indistinguishable from that of embryonic stem cells (ESC) and entirely different from that of the original somatic cells. Similarly,this technique allows a distinction to be made between partially and fully reprogrammed cells. We conclude that infrared microspectroscopy signature is a novel methodology to evaluate induced pluripotency and can be added to the tests currently used for this purpose. View Publication

Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value,thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity,only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2,PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb,heart and embryonic development related transcription factors and biological processes. Moreover,this study uncovered novel possible mechanisms,such as the inhibition of RANBP1,that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1,GSTA2),that protect the cell from secondary oxidative stress. As a proof of principle,we demonstrated that a combination of transcriptomics and proteomics,along with consistent differentiation of hESCs,enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide. View Publication

In a prior study on electronic cigarette (EC) refill fluids,Cinnamon Ceylon was the most cytotoxic of 36 products tested. The purpose of the current study was to determine if high cytotoxicity is a general feature of cinnamon-flavored EC refill fluids and to identify the toxicant(s) in Cinnamon Ceylon. Eight cinnamon-flavored refill fluids,which were screened using the MTT assay,varied in their cytotoxicity with most being cytotoxic. Human embryonic stem cells were generally more sensitive than human adult pulmonary fibroblasts. Most products were highly volatile and produced vapors that impaired survival of cells in adjacent wells. Cinnamaldehyde (CAD),2-methoxycinnamaldehyde (2MOCA),dipropylene glycol,and vanillin were identified in the cinnamon-flavored refill fluids using gas chromatography-mass spectrometry and high-pressure liquid chromatography (HPLC). When authentic standards of each chemical were tested using the MTT assay,only CAD and 2MOCA were highly cytotoxic. The amount of each chemical in the refill fluids was quantified using HPLC,and cytotoxicity correlated with the amount of CAD/product. Duplicate bottles of the same product were similar,but varied in their concentrations of 2MOCA. These data show that the cinnamon flavorings in refill fluids are linked to cytotoxicity,which could adversely affect EC users. ?? 2013 Elsevier Ltd. View Publication

Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast™ chemical screening and prioritization research project. Metabolites from hES cultures were evaluated for known and novel signatures that may be indicative of developmental toxicity. Significant fold changes in endogenous metabolites were detected for 83 putatively annotated mass features in response to the subset of ToxCast chemicals. The annotations were mapped to specific human metabolic pathways. This revealed strong effects on pathways for nicotinate and nicotinamide metabolism,pantothenate and CoA biosynthesis,glutathione metabolism,and arginine and proline metabolism pathways. Predictivity for adverse outcomes in mammalian prenatal developmental toxicity studies used ToxRefDB and other sources of information,including Stemina Biomarker Discovery's predictive DevTox® model trained on 23 pharmaceutical agents of known developmental toxicity and differing potency. The model initially predicted developmental toxicity from the blinded ToxCast compounds in concordance with animal data with 73% accuracy. Retraining the model with data from the unblinded test compounds at one concentration level increased the predictive accuracy for the remaining concentrations to 83%. These preliminary results on a 11-chemical subset of the ToxCast chemical library indicate that metabolomics analysis of the hES secretome provides information valuable for predictive modeling and mechanistic understanding of mammalian developmental toxicity. View Publication