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Long-term in vitro culture of undifferentiated human embryonic stem cells (hESCs) traditionally requires a fibroblast feeder cell layer. Using feeder cells in hESC cultures is highly laborious and limits large-scale hESC production for potential application in regenerative medicine. Replacing feeder cells with defined human extracellular matrix (ECM) components or synthetic biomaterials would be ideal for large-scale production of clinical-grade hESCs. We tested and compared different feeder cell-free hESC culture methods based on different human ECM proteins,human and animal sera matrices,and a Matrigel matrix. Also selected biomaterials were tested for feeder cell-free propagation of undifferentiated hESCs. The matrices were tested together with conventional and modified hESC culture media,human foreskin fibroblast-conditioned culture medium,chemically defined medium,TeSR1,and modified TeSR1 media. The results showed the undefined,xenogeneic Matrigel to be a superior matrix for hESC culture compared with the purified human ECM proteins,serum matrices,and the biomaterials tested. A long-term,feeder cell-free culture system was successful on Matrigel in combination with mTeSR1 culture medium,but a xeno-free,fully defined,and reproducible feeder cell-free hESC culture method still remains to be developed. View Publication

Objective: Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) constitute unique sources of pluripotent cells,although the molecular mechanisms involved in their differentiation into specific lineages are just beginning to be defined. Here we evaluated the ability of MEDII (medium conditioned by HepG2 cells,a human hepatocarcinoma cell line) to selectively enhance generation of mesodermal derivatives,including hematopoietic cells,from hESCs and hiPSCs. Materials and Methods: Test cells were exposed to MEDII prior to being placed in conditions that promote embryoid body (EB) formation. Hematopoietic activity was measured by clonogenic assays,flow cytometry,quantitative real-time polymerase chain reaction of specific transcript complementary DNAs and the ability of cells to repopulate sublethally irradiated nonobese diabetic/severe combined immunodeficient interleukin-2 receptor ??-chain-null mice for almost 1 year. Results: Exposure of both hESCs and hiPSCs to MEDII induced a rapid and preferential differentiation of hESCs into mesodermal elements. Subsequently produced EBs showed a further enhanced expression of transcripts characteristic of multiple mesodermal lineages,and a concurrent decrease in endodermal and ectodermal cell transcripts. Frequency of all types of clonogenic hematopoietic progenitors in subsequently derived EBs was also increased. In vivo assays of MEDII-treated hESC-derived EBs also showed they contained cells able to undertake low-level but longterm multilineage repopulation of primary and secondary nonobese diabetic/severe combined immunodeficient interleukin-2 receptor ??-chain-null mice. Conclusions: MEDII treatment of hESCs and hiPSCs alike selectively enhances their differentiation into mesodermal cells and allows subsequent generation of detectable levels of hematopoietic progenitors with in vitro and in vivo differentiating activity. ?? 2009 ISEH - Society for Hematology and Stem Cells. View Publication

One of the factors that can impact human embryonic stem cell expansion in stirred microcarrier culture reactors is mechanical stress caused by agitation. Therefore,we have investigated the effects of agitation on human embryonic stem cell growth and expression of pluripotent markers. Agitation of HES-2 cell line in microcarrier cultures in stirred spinner and agitated six-well plates did not affect expression of pluripotent markers,cell viability,and cell doubling times even after seven passages. However,HES-3 cell line was found to be shear sensitive,showing downregulation of three pluripotent markers Oct-4,mAb 84,and Tra-1-60,and lower cell densities in agitated as compared with static cultures,even after one passage. Cell viability was unaffected. The HES-3-agitated cultures showed increased expression of genes and proteins of the three germ layers. We were unable to prevent loss of pluripotent markers or restore doubling times in agitated HES-3 microcarrier cultures by addition of five different known cell protective polymers. In addition,the human induced pluripotent cell line IMR90 was also shown to differentiate in agitated conditions. These results indicate that the effect of agitation on cell growth and differentiation is cell line specific. We assume that the changes in the growth and differentiation of the agitation-sensitive (HES-3) cell line do not result from the effect of shear stress directly on cell viability,but rather by signaling effects that influence the cells to differentiate resulting in slower growth. 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

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

Vogt-Koyanagi-Harada (VKH) disease (and sympathetic ophthalmia) is an ocular inflammatory disease that is considered to be a cell-mediated autoimmune disease against melanocytes. The purpose of this study was to determine the Ags specific to VKH disease and to develop an animal model of VKH disease. We found that exposure of lymphocytes from patients with VKH disease to peptides (30-mer) derived from the tyrosinase family proteins led to significant proliferation of the lymphocytes. Immunization of these peptides into pigmented rats induced ocular and extraocular changes that highly resembled human VKH disease,and we suggest that an experimental VKH disease was induced in these rats. We conclude that VKH disease is an autoimmune disease against the tyrosinase family proteins. View Publication

In recent years,human embryonic stem (hES) cells have become a promising cell source for regenerative medicine. Although hES cells have the ability for unlimited self-renewal,potential adverse effects of long-term cell culture upon hES cells must be investigated before therapeutic applications of hES cells can be realized. Here we investigated changes in molecular profiles associated with young (textless60 passages) and old (textgreater120 passages) cells of the H9 hES cell line as well as young (textless85 passages) and old (textgreater120 passages) cells of the PKU1 hES cell line. Our results show that morphology,stem cell markers,and telomerase activity do not differ significantly between young and old passage cells. Cells from both age groups were also shown to differentiate into derivatives of all 3 germ layers upon spontaneous differentiation in vitro. Interestingly,mitochondrial dysfunction was found to occur with prolonged culture. Old passage cells of both the H9 and PKU1 lines were characterized by higher mitochondrial membrane potential,larger mitochondrial morphology,and higher reactive oxygen species content than their younger counterparts. Teratomas derived from higher passage cells were also found to have an uneven preference for differentiation compared with tumors derived from younger cells. These findings suggest that prolonged culture of hES cells may negatively impact mitochondrial function and possibly affect long-term pluripotency. View Publication

The longevity-promoting NAD+-dependent class III histone deacetylase Sirtuin 1 (SIRT1) is involved in stem cell function by controlling cell fate decision and/or by regulating the p53-dependent expression of NANOG. We show that SIRT1 is down-regulated precisely during human embryonic stem cell differentiation at both mRNA and protein levels and that the decrease in Sirt1 mRNA is mediated by a molecular pathway that involves the RNA-binding protein HuR and the arginine methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). SIRT1 down-regulation leads to reactivation of key developmental genes such as the neuroretinal morphogenesis effectors DLL4,TBX3,and PAX6,which are epigenetically repressed by this histone deacetylase in pluripotent human embryonic stem cells. Our results indicate that SIRT1 is regulated during stem cell differentiation in the context of a yet-unknown epigenetic pathway that controls specific developmental genes in embryonic stem cells. View Publication

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