技术资料

Although gene-gene interaction,or epistasis,plays a large role in complex traits in model organisms,genome-wide by genome-wide searches for two-way interaction have limited power in human studies. We thus used knowledge of a biological pathway in order to identify a contribution of epistasis to autism spectrum disorders (ASDs) in humans,a reverse-pathway genetic approach. Based on previous observation of increased ASD symptoms in Mendelian disorders of the Ras/MAPK pathway (RASopathies),we showed that common SNPs in RASopathy genes show enrichment for association signal in GWAS (P = 0.02). We then screened genome-wide for interactors with RASopathy gene SNPs and showed strong enrichment in ASD-affected individuals (P < 2.2 x 10-16),with a number of pairwise interactions meeting genome-wide criteria for significance. Finally,we utilized quantitative measures of ASD symptoms in RASopathy-affected individuals to perform modifier mapping via GWAS. One top region overlapped between these independent approaches,and we showed dysregulation of a gene in this region,GPR141,in a RASopathy neural cell line. We thus used orthogonal approaches to provide strong evidence for a contribution of epistasis to ASDs,confirm a role for the Ras/MAPK pathway in idiopathic ASDs,and to identify a convergent candidate gene that may interact with the Ras/MAPK pathway. View Publication

Human apolipoprotein E (ApoE) apolipoprotein is primarily expressed in three isoforms (ApoE2,ApoE3,and ApoE4) that differ only by two residues. ApoE4 constitutes the most important genetic risk factor for Alzheimer's disease (AD),ApoE3 is neutral,and ApoE2 is protective. How ApoE isoforms influence AD pathogenesis,however,remains unclear. Using ES-cell-derived human neurons,we show that ApoE secreted by glia stimulates neuronal Aβ production with an ApoE4< ApoE3< ApoE2 potency rank order. We demonstrate that ApoE binding to ApoE receptors activates dual leucine-zipper kinase (DLK),a MAP-kinase kinase kinase that then activates MKK7 and ERK1/2 MAP kinases. Activated ERK1/2 induces cFos phosphorylation,stimulating the transcription factor AP-1,which in turn enhances transcription of amyloid-β precursor protein (APP) and thereby increases amyloid-β levels. This molecular mechanism also regulates APP transcription in mice in vivo. Our data describe a novel signal transduction pathway in neurons whereby ApoE activates a non-canonical MAP kinase cascade that enhances APP transcription and amyloid-β synthesis. View Publication

BACKGROUND: Induced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming,which uses a defined set of transcription factors,iPS cells represent important sources of patient-specific cells for clinical applications. However,before these cells can be used in therapeutic designs,it is essential to understand their genetic stability. METHODOLOGY/PRINCIPAL FINDINGS: Here,we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation,iPS cells activate checkpoint signaling,evidenced by phosphorylation of ATM,NBS1,CHEK2,and TP53,localization of ATM to the double strand breaks (DSB),and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2) phase of the cell cycle,displaying a lack of the G(1)/S cell cycle arrest similar to human embryonic stem (ES) cells. Furthermore,both cell types remove DSB within six hours of γ-irradiation,form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally,we report elevated expression of genes involved in DNA damage signaling,checkpoint function,and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts. CONCLUSIONS/SIGNIFICANCE: High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage,dramatic changes in cell cycle structure,including a high percentage of cells in the S phase,increased radiosensitivity and loss of DNA damage-induced G(1)/S cell cycle arrest,were observed in stem cells generated by induced pluripotency. View Publication

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the blastocyst. Because of their ability to differentiate into a variety of cell types,human embryonic stem cells (hESCs) provide an unlimited source of cells for clinical medicine and have begun to be used in clinical trials. Presently,although several hundred hESC lines are available in the word,only few have been widely used in basic and applied research. More and more hESC lines with differing genetic backgrounds are required for establishing a bank of hESCs. Here,we report the first Canadian hESC lines to be generated from cryopreserved embryos and we discuss how we navigated through the Canadian regulatory process. The cryopreserved human zygotes used in this study were cultured to the blastocyst stage,and used to isolate ICM via microsurgery. Unlike previous microsurgery methods,which use specialized glass or steel needles,our method conveniently uses syringe needles for the isolation of ICM and subsequent hESC lines. ICM were cultured on MEF feeders in medium containing FBS or serum replacer (SR). Resulting outgrowths were isolated,cut into several cell clumps,and transferred onto fresh feeders. After more than 30 passages,the two hESC lines established using this method exhibited normal morphology,karyotype,and growth rate. Moreover,they stained positively for a variety of pluripotency markers and could be differentiated both in vitro and in vivo. Both cell lines could be maintained under a variety of culture conditions,including xeno-free conditions we have previously described. We suggest that this microsurgical approach may be conducive to deriving xeno-free hESC lines when outgrown on xeno-free human foreskin fibroblast feeders. View Publication

Human embryonic stem (hES) cells have enormous potential for clinical applications. However,one major challenge is to achieve high cell recovery rate after cryopreservation. Understanding how the conventional cryopreservation protocol fails to protect the cells is a prerequisite for developing efficient and successful cryopreservation methods for hES cell lines and banks. We investigated how the stimuli from cryopreservation result in apoptosis,which causes the low cell recovery rate after cryopreservation. The level of reactive oxygen species (ROS) is significantly increased,F-actin content and distribution is altered,and caspase-8 and caspase-9 are activated after cryopreservation. p53 is also activated and translocated into nucleus. During cryopreservation apoptosis is induced by activation of both caspase-8 through the extrinsic pathway and caspase-9 through the intrinsic pathway. However,exactly how the extrinsic pathway is activated is still unclear and deserves further investigation. View Publication

The use of human pluripotent stem cells,including embryonic and induced pluripotent stem cells,in therapeutic applications will require the development of robust,scalable culture technologies for undifferentiated cells. Advances made in large-scale cultures of other mammalian cells will facilitate expansion of undifferentiated human embryonic stem cells (hESCs),but challenges specific to hESCs will also have to be addressed,including development of defined,humanized culture media and substrates,monitoring spontaneous differentiation and heterogeneity in the cultures,and maintaining karyotypic integrity in the cells. This review will describe our current understanding of environmental factors that regulate hESC self-renewal and efforts to provide these cues in various scalable bioreactor culture systems. View Publication

Since the first report of derivation of human embryonic stem cell (hESC) lines in 1998,many progresses have been achieved to reliably and efficiently derive,maintain,and differentiate this therapeutically promising cell type. This chapter introduces some basic and widely recognized methods that we use in our hESC core laboratory. Specifically,it includes methods for (1) deriving hESC lines without using enzyme and antibody to isolate the inner cell mass; (2) sustaining hESC self-renewal under feeder-dependent,feeder-conditioned,and defined conditions as well as pluripotency validation and quality control assays; and (3) inducing hESC differentiation to trophoblast with BMP4. View Publication

Unlocking the clinical potential of stem cell based therapies requires firstly elucidation of the biological mechanisms which direct stem cell fate decisions and thereafter,technical advances which allow these processes to be driven in a fully defined culture environment. Strategies for the generation of defined surfaces for human embryonic stem cell (hESC) and mesenchymal stem cell (MSC) culture remain in their infancy. In this paper we outline a simple,effective and efficient method for presenting proteins or peptides on an otherwise non-fouling Layer-by-Layer (LbL) self-assembled surface of hyaluronic acid (HA) and chitosan (CHI). We are able to generate a surface that has both good temporal stability and the ability to direct biological outcomes based on its defined surface composition. Surface functionalization is achieved through suspending the selected extracellular matrix (ECM) protein domain or extracted full-length protein in buffer containing a cross-linking agent (N-hydroxysulfosuccinimide/N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) over the LbL HA-CHI surface and then allowing the solvent to evaporate overnight. This simple,but important step results in remarkable protein deposition efficiencies often exceeding 50%,whereas traditional cross-linking methods result in such poor deposition of non-collagenous proteins that a.) quantification of bound amounts of protein is outside the resolution of commonly utilized protein assays,and b.) these surfaces are both unable to support cell attachment and growth. The utility of the protein-modified HA-CHI surfaces is demonstrated through the identification of specific hESC attachment efficiencies and through directing MSC osteogenic outcomes on these fully defined surfaces. This simple and scalable method is shown to enable the development of defined stem cell culture conditions,as well as the elucidation of the fundamental biological processes necessary for the realization of stem cell based therapies. View Publication

Microfluidic image cytometry (MIC) has been developed to study phenotypes of various hPSC lines by screening several chemically defined serum/feeder-free conditions. A chemically defined hPSC culture was established using 20 ng mL(-1) of bFGF on 20 microg mL(-1) of Matrigel to grow hPSCs over a week in an undifferentiated state. Following hPSC culture,we conducted quantitative MIC to perform a single cell profiling of simultaneously detected protein expression (OCT4 and SSEA1). Using clustering analysis,we were able to systematically compare the characteristics of various hPSC lines in different conditions. View Publication

The recent development of induced pluripotent stem cells (iPSCs) by ectopic expression of defined reprogramming factors offers enormous therapeutic opportunity. To deliver these factors,murine leukemia virus (MLV)-based vectors have been broadly used in the setting of hematopoietic stem cell transplantation. However,MLV vectors have been implicated in malignancy induced by insertional mutagenesis,whereas lentiviral vectors have not. Furthermore,the infectivity of MLV vectors is limited to dividing cells,whereas lentiviral vectors can also transduce nondividing cells. One important characteristic of MLV vectors is a self-silencing property of the promoter element in pluripotent stem cells,allowing temporal transgene expression in a nonpluripotent state before iPSC derivation. Here we test iPSC generation using a novel chimeric vector carrying a mutant MLV promoter internal to a lentiviral vector backbone,thereby containing the useful properties of both types of vectors. Transgene expression of this chimeric vector was highly efficient compared with that of MLV vectors and was silenced specifically in human embryonic stem cells. Human fetal fibroblasts transduced with the vector encoding each factor were efficiently reprogrammed into a pluripotent state,and these iPSCs had potential to differentiate into a variety of cell types. To explore the possibility of iPSCs for gene therapy,we established iPSC clones expressing a short hairpin RNA (shRNA) targeting chemokine receptor 5 (CCR5),the main coreceptor for HIV-1. Using a reporter construct for CCR5 expression,we confirmed that CCR5 shRNA was expressed and specifically knocked down the reporter expression in iPSCs. These data indicate that our chimeric lentiviral vector is a valuable tool for generation of iPSCs and the combination with vectors encoding transgenes allows for rapid establishment of desired genetically engineered iPSC lines. View Publication

BACKGROUND: To maintain pluripotency of human embryonic stem (huES) cells in feeder-free culture it has been necessary to provide a Matrigel substratum,which is a complex of poorly defined extracellular matrices and growth factors derived from mouse Engelbreth-Holm-Swarm sarcoma cells. Culture of stem cells under ill-defined conditions can inhibit the effectiveness of maintaining cells in a pluripotent state and reduce reproducibility of differentiation protocols. Moreover recent batches of Matrigel have been found to be contaminated with the single stranded RNA virus,Lactate Dehydrogenase Elevating Virus (LDEV),raising concerns regarding the safety of using stem cells that have been cultured on Matrigel in a therapeutic setting. To circumvent such concerns,we attempted to identify a recombinant matrix that could be used as an alternative to Matrigel for the culture of human pluripotent stem cells. huES and human induced pluripotent stem (hiPS) cells were grown on plates coated with a fusion protein consisting of E-cadherin and the IgG Fc domain using mTeSR1 medium.backslashnbackslashnRESULTS: Cells grown under these conditions maintained similar morphology and growth rate to those grown on Matrigel and retained all pluripotent stem cell features,including an ability to differentiate into multiple cell lineages in teratoma assays. We,therefore,present a culture system that maintains the pluripotency of huES and hiPS cells under completely defined conditions.backslashnbackslashnCONCLUSIONS: We propose that this system should facilitate growth of stem cells using good manufacturing practices (GMP),which will be necessary for the clinical use of pluripotent stem cells and their derivatives. View Publication

Pluripotent human embryonic stem (hES) cells are an important experimental tool for basic and applied research,and a potential source of different tissues for transplantation. However,one important challenge for the clinical use of these cells is the issue of immunocompatibility,which may be dealt with by the establishment of hES cell banks to attend different populations. Here we describe the derivation and characterization of a line of hES cells from the Brazilian population,named BR-1,in commercial defined medium. In contrast to the other hES cell lines established in defined medium,BR-1 maintained a stable normal karyotype as determined by genomic array analysis after 6 months in continuous culture (passage 29). To our knowledge,this is the first reported line of hES cells derived in South America. We have determined its genomic ancestry and compared the HLA-profile of BR-1 and another 22 hES cell lines established elsewhere with those of the Brazilian population,finding they would match only 0.011% of those individuals. Our results highlight the challenges involved in hES cell banking for populations with a high degree of ethnic admixture. View Publication