技术资料

BACKGROUND: Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body. Ets variant 2 (ETV2) is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification. Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells. Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal.backslashnbackslashnFINDINGS: We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells (ESCs) to determine when the peak of ETV2 expression occurs. We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression.backslashnbackslashnCONCLUSIONS: Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification. This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic. View Publication

There is a foundational need for quality control tools in stem cell laboratories engaged in basic research,regenerative therapies,and toxicological studies. These tools require automated methods for evaluating cell processes and quality during in vitro passaging,expansion,maintenance,and differentiation. In this paper,an unbiased,automated high-content profiling toolkit,StemCellQC,is presented that non-invasively extracts information on cell quality and cellular processes from time-lapse phase-contrast videos. Twenty four (24) morphological and dynamic features were analyzed in healthy,unhealthy,and dying human embryonic stem cell (hESC) colonies to identify those features that were affected in each group. Multiple features differed in the healthy versus unhealthy/dying groups,and these features were linked to growth,motility,and death. Biomarkers were discovered that predicted cell processes before they were detectable by manual observation. StemCellQC distinguished healthy and unhealthy/dying hESC colonies with 96% accuracy by non-invasively measuring and tracking dynamic and morphological features over 48 hours. Changes in cellular processes can be monitored by StemCellQC and predictions can be made about the quality of pluripotent stem cell colonies. This toolkit reduced the time and resources required to track multiple pluripotent stem cell colonies and eliminated handling errors and false classifications due to human bias. StemCellQC provided both user-specified and classifier-determined analysis in cases where the affected features are not intuitive or anticipated. Video analysis algorithms allowed assessment of biological phenomena using automatic detection analysis,which can aid facilities where maintaining stem cell quality and/or monitoring changes in cellular processes are essential. In the future StemCellQC can be expanded to include other features,cell types,treatments,and differentiating cells. View Publication

Copy number variation (CNV) is a common chromosomal alteration that can occur during in vitro cultivation of human cells and can be accompanied by the accumulation of mutations in coding region sequences. We describe here a systematic application of current molecular technologies to provide a detailed understanding of genomic and sequence profiles of human embryonic stem cell (hESC) lines that were derived under GMP-compliant conditions. We first examined the overall chromosomal integrity using cytogenetic techniques to determine chromosome count,and to detect the presence of cytogenetically aberrant cells in the culture (mosaicism). Assays of copy number variation,using both microarray and sequence-based analyses,provide a detailed view genomic variation in these lines and shows that in early passage cultures of these lines,the size range and distribution of CNVs are entirely consistent with those seen in the genomes of normal individuals. Similarly,genome sequencing shows variation within these lines that is completely within the range seen in normal genomes. Important gene classes,such as tumor suppressors and genetic disease genes,do not display overtly disruptive mutations that could affect the overall safety of cell-based therapeutics. Complete sequence also allows the analysis of important transplantation antigens,such as ABO and HLA types. The combined application of cytogenetic and molecular technologies provides a detailed understanding of genomic and sequence profiles of GMP produced ES lines for potential use as therapeutic agents. View Publication

BACKGROUND: Gene delivery can potentially be used as a therapeutic for treating genetic diseases,including neurodegenerative diseases,as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts.backslashnbackslashnMETHODS: A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling.backslashnbackslashnRESULTS: 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents,including Lipofectamine® 2000,FuGENE® HD,and 25 kDa branched polyethylenimine,for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa,and enabled coexpression of exogenously delivered genes,as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation,but not by poly(beta-amino ester) reprogramming,could be differentiated toward the neuronal lineage,specifically pseudostratified optic cups.backslashnbackslashnCONCLUSION: This study shows that certain nonviral reprogramming methods may not necessarily be safer than viral approaches and that maximizing exogenous gene expression of reprogramming factors is not sufficient to ensure successful reprogramming. View Publication

Human induced pluripotent stem cells (hiPSCs) are considered as a powerful tool for drug and chemical screening and development of new in vitro testing strategies in the field of toxicology,including neurotoxicity evaluation. These cells are able to expand and efficiently differentiate into different types of neuronal and glial cells as well as peripheral neurons. These human cells-based neuronal models serve as test systems for mechanistic studies on different pathways involved in neurotoxicity. One of the well-known mechanisms that are activated by chemically-induced oxidative stress is the Nrf2 signaling pathway. Therefore,in the current study,we evaluated whether Nrf2 signaling machinery is expressed in human induced pluripotent stem cells (hiPSCs)-derived mixed neuronal/glial culture and if so whether it becomes activated by rotenone-induced oxidative stress mediated by complex I inhibition of mitochondrial respiration. Rotenone was found to induce the activation of Nrf2 signaling particularly at the highest tested concentration (100 nM),as shown by Nrf2 nuclear translocation and the up-regulation of the Nrf2-downstream antioxidant enzymes,NQO1 and SRXN1. Interestingly,exposure to rotenone also increased the number of astroglial cells in which Nrf2 activation may play an important role in neuroprotection. Moreover,rotenone caused cell death of dopaminergic neurons since a decreased percentage of tyrosine hydroxylase (TH(+)) cells was observed. The obtained results suggest that hiPSC-derived mixed neuronal/glial culture could be a valuable in vitro human model for the establishment of neuronal specific assays in order to link Nrf2 pathway activation (biomarker of oxidative stress) with additional neuronal specific readouts that could be applied to in vitro neurotoxicity evaluation. View Publication

Familial amyloid polyneuropathy (FAP) is caused by mutations of the transthyretin (TTR) gene,predominantly expressed in the liver. Two compounds that knockdown TTR,comprising a small interfering RNA (siRNA; ALN-TTR-02) and an antisense oligonucleotide (ASO; IONIS-TTRRx),are currently being evaluated in clinical trials. Since primary hepatocytes from FAP patients are rarely available for molecular analysis and commercial tissue culture cells or animal models lack the patient-specific genetic background,this study uses primary cells derived from urine of FAP patients. Urine-derived cells were reprogrammed to induced pluripotent stem cells (iPSCs) with high efficiency. Hepatocyte-like cells (HLCs) showing typical hepatic marker expression were obtained from iPSCs of the FAP patients. TTR mRNA expression of FAP HLCs almost reached levels measured in human hepatocytes. To assess TTR knockdown,siTTR1 and TTR-ASO were introduced to HLCs. A significant downregulation (textgreater80%) of TTR mRNA was induced in the HLCs by both oligonucleotides. TTR protein present in the cell culture supernatant of HLCs was similarly downregulated. Gene expression of other hepatic markers was not affected by the therapeutic oligonucleotides. Our data indicate that urine cells (UCs) after reprogramming and hepatic differentiation represent excellent primary human target cells to assess the efficacy and specificity of novel compounds. View Publication

Evi1 (ecotropic viral integration site 1) is essential for proliferation of hematopoietic stem cells and implicated in the development of myeloid disorders. Particularly,high Evi1 expression defines one of the largest clusters in acute myeloid leukemia and is significantly associated with extremely poor prognosis. However,mechanistic basis of Evi1-mediated leukemogenesis has not been fully elucidated. Here,we show that Evi1 directly represses phosphatase and tensin homologue deleted on chromosome 10 (PTEN) transcription in the murine bone marrow,which leads to activation of AKT/mammalian target of rapamycin (mTOR) signaling. In a murine bone marrow transplantation model,Evi1 leukemia showed modestly increased sensitivity to an mTOR inhibitor rapamycin. Furthermore,we found that Evi1 binds to several polycomb group proteins and recruits polycomb repressive complexes for PTEN down-regulation,which shows a novel epigenetic mechanism of AKT/mTOR activation in leukemia. Expression analyses and ChIPassays with human samples indicate that our findings in mice models are recapitulated in human leukemic cells. Dependence of Evi1-expressing leukemic cells on AKT/mTOR signaling provides the first example of targeted therapeutic modalities that suppress the leukemogenic activity of Evi1. The PTEN/AKT/mTOR signaling pathway and the Evi1-polycomb interaction can be promising therapeutic targets for leukemia with activated Evi1. View Publication

Xenopus in vitro studies have implicated both transforming growth factor beta (TGF-beta) and fibroblast growth factor (FGF) families in mesoderm induction. Although members of both families are present during mouse mesoderm formation,there is little evidence for their functional role in mesoderm induction. We show that mouse embryonic stem cells,which resemble primitive ectoderm,can differentiate to mesoderm in vitro in a chemically defined medium (CDM) in the absence of fetal bovine serum. In CDM,this differentiation is responsive to TGF-beta family members in a concentration-dependent manner,with activin A mediating the formation of dorsoanterior-like mesoderm and bone morphogenetic protein 4 mediating the formation of ventral mesoderm,including hematopoietic precursors. These effects are not observed in CDM alone or when TGF-beta 1,-beta 2,or -beta 3,acid FGF,or basic FGF is added individually to CDM. In vivo,at day 6.5 of mouse development,activin beta A RNA is detectable in the decidua and bone morphogenetic protein 4 RNA is detectable in the egg cylinder. Together,our data strongly implicate the TGF-beta family in mammalian mesoderm development and hematopoietic cell formation. View Publication

Cockayne syndrome (CS) is a human premature aging disorder associated with neurological and developmental abnormalities,caused by mutations mainly in the CS group B gene (ERCC6). At the molecular level,CS is characterized by a deficiency in the transcription-couple DNA repair pathway. To understand the role of this molecular pathway in a pluripotent cell and the impact of CSB mutation during human cellular development,we generated induced pluripotent stem cells (iPSCs) from CSB skin fibroblasts (CSB-iPSC). Here,we showed that the lack of functional CSB does not represent a barrier to genetic reprogramming. However,iPSCs derived from CSB patient's fibroblasts exhibited elevated cell death rate and higher reactive oxygen species (ROS) production. Moreover,these cellular phenotypes were accompanied by an up-regulation of TXNIP and TP53 transcriptional expression. Our findings suggest that CSB modulates cell viability in pluripotent stem cells,regulating the expression of TP53 and TXNIP and ROS production. View Publication

Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state via the introduction of defined transcription factors. Although iPS is a potentially valuable resource for regenerative medicine and drug development,several issues regarding their pluripotency,differentiation propensity and potential for tumorigenesis remain to be elucidated. Analysis of cell surface glycans has arisen as an interesting tool for the characterization of iPS. An appropriate characterization of glycan surface molecules of human embryonic stem (hES) cells and iPS cells might generate crucial data to highlight their role in the acquisition and maintenance of pluripotency. In this study,we characterized the surface glycans of iPS generated from menstrual blood-derived mesenchymal cells (iPS-MBMC). We demonstrated that,upon spontaneous differentiation,iPS-MBMC present high amounts of terminal $\$-galactopyranoside residues,pointing to an important role of terminal-linked sialic acids in pluripotency maintenance. The removal of sialic acids by neuraminidase induces iPS-MBMC and hES cells differentiation,prompting an ectoderm commitment. Exposed $\$-galactopyranose residues might be recognized by carbohydrate-binding molecules found on the cell surface,which could modulate intercellular or intracellular interactions. Together,our results point for the first time to the involvement of the presence of terminal sialic acid in the maintenance of embryonic stem cell pluripotency and,therefore,the modulation of sialic acid biosynthesis emerges as a mechanism that may govern stem cell differentiation. View Publication

Sensory stimuli drive the maturation and function of the mammalian nervous system in part through the activation of gene expression networks that regulate synapse development and plasticity. These networks have primarily been studied in mice,and it is not known whether there are species- or clade-specific activity-regulated genes that control features of brain development and function. Here we use transcriptional profiling of human fetal brain cultures to identify an activity-dependent secreted factor,Osteocrin (OSTN),that is induced by membrane depolarization of human but not mouse neurons. We find that OSTN has been repurposed in primates through the evolutionary acquisition of DNA regulatory elements that bind the activity-regulated transcription factor MEF2. In addition,we demonstrate that OSTN is expressed in primate neocortex and restricts activity-dependent dendritic growth in human neurons. These findings suggest that,in response to sensory input,OSTN regulates features of neuronal structure and function that are unique to primates. View Publication