技术资料

Pediatric high-grade astrocytomas (HGAs) account for 15-20% of all pediatric central nervous system tumors. These neoplasms predominantly involve the supratentorial hemispheres or the pons--diffuse intrinsic pontine gliomas (DIPG). Assumptions that pediatric HGAs are biologically similar to adult HGAs have recently been challenged,and the development of effective therapeutic modalities for DIPG and supratentorial HGA hinges on a better understanding of their biological properties. Here,20 pediatric HGAs (9 DIPGs and 11 supratentorial HGAs) were subject to gene expression profiling following approval by the research ethics board at our institution. Many of these tumors showed expression signatures composed of genes that promote G1/S and G2/M cell cycle progression. In particular,Aurora kinase B (AURKB) was consistently and highly overexpressed in 6/9 DIPGs and 8/11 HGAs. Array data were validated using quantitative real-time PCR and immunohistochemistry,as well as cross-validation of our data set with previously published series. Inhibition of Aurora B activity in DIPG and in pediatric HGA cell lines resulted in growth arrest accompanied by morphological changes,cell cycle aberrations,nuclear fractionation and polyploidy as well as a reduction in colony formation. Our data highlight Aurora B as a potential therapeutic target in DIPG. View Publication

AIM: The generation and characterization of a human embryonic stem cell (hESC) line stably expressing red fluorescent mCherry protein.backslashnbackslashnMETHODS: Lentiviral transduction of a ubiquitously-expressed human EF-1α promoter driven mCherry transgene was performed in MEL2 hESC. Red fluore-scence was assessed by immunofluorescence and flow cytometry. Pluripotency of stably transduced hESC was determined by immunofluorescent pluripotency marker expression,flow cytometry,teratoma assays and embryoid body-based differentiation followed by reverse transcriptase-polymerase chain reaction. Quantification of cell motility and survival was performed with time lapse microscopy.backslashnbackslashnRESULTS: Constitutively fluorescently-labeled hESCs are useful tools for facile in vitro and in vivo tracking of survival,motility and cell spreading on various surfaces before and after differentiation. Here we describe the generation and characterization of a hESC line (MEL2) stably expressing red fluorescent protein,mCherry. This line was generated by random integration of a fluorescent protein-expressing cassette,driven by the ubiquitously-expressed human EF-1α promoter. Stably transfected MEL2-mCherry hESC were shown to express pluripotency markers in the nucleus (POU5F1/OCT4,NANOG and SOX2) and on the cell surface (SSEA4,TRA1-60 and TG30/CD9) and were shown to maintain a normal karyotype in long-term (for at least 35 passages) culture. MEL2-mCherry hESC further readily differentiated into representative cell types of the three germ layers in embryoid body and teratoma based assays and,importantly,maintained robust mCherry expression throughout differentiation. The cell line was next adapted to single-cell passaging,rendering it compatible with numerous bioengineering applications such as measurement of cell motility and cell spreading on various protein modified surfaces,quantification of cell attachment to nanoparticles and rapid estimation of cell survival.backslashnbackslashnCONCLUSION: The MEL2-mCherry hESC line conforms to the criteria of bona fide pluripotent stem cells and maintains red fluorescence throughout differentiation,making it a useful tool for bioengineering and in vivo tracking experiments. View Publication

De novo DNA methylation is an essential aspect of the epigenetic reprogramming that takes place during early development,yet factors responsible for its instatement at particular genomic loci are poorly defined. Here,we demonstrate that the KRAB-ZFP-mediated recruitment of KAP1 to DNA in embryonic stem cells (ESCs) induces cytosine methylation. This process is preceded by H3K9 trimethylation,and genome-wide analyses reveal that it spreads over short distances from KAP1-binding sites so as to involve nearby CpG islands. In sharp contrast,in differentiated cells,KRAB/KAP1-induced heterochromatin formation does not lead to DNA methylation. Correspondingly,the methylation status of CpG islands in the adult mouse liver correlates with their proximity to KAP1-binding sites in ESCs,not in hepatocytes. Therefore,KRAB-ZFPs and their cofactor KAP1 are in part responsible for the establishment during early embryogenesis of site-specific DNA methylation patterns that are maintained through development View Publication

Neural cells derived from induced pluripotent stem cells (iPSCs) have the potential for autologous cell therapies in treating patients with severe neurological disorders or injury. However,further study of efficacy and safety are needed in large animal preclinical models that have similar neural anatomy and physiology to humans such as the pig. The pig model for pluripotent stem cell therapy has been made possible for the first time with the development of pig iPSCs (piPSCs) capable of in vitro and in vivo differentiation into tissues of all three germ layers. Still,the question remains if piPSCs are capable of undergoing robust neural differentiation using a system similar to those being used with human iPSCs. In this study,we generated a new line of piPSCs from fibroblast cells that expressed pluripotency markers and were capable of embryoid body differentiation into all three germ layers. piPSCs demonstrated robust neural differentiation forming βIII-TUB/MAP2+ neurons,GFAP+ astrocytes,and O4+ oligodendrocytes and demonstrated strong upregulation of neural cell genes representative of all three major neural lineages of the central nervous system. In the presence of motor neuron signaling factors,piPSC-derived neurons showed expression of transcription factors associated with motor neuron differentiation (HB9 and ISLET1). Our findings demonstrate that SSEA4 expression is required for piPSCs to differentiate into neurons,astrocytes,and oligodendrocytes and furthermore develop specific neuronal subtypes. This indicates that the pigs can fill the need for a powerful model to study autologous neural iPSC therapies in a system similar to humans. View Publication

Human pluripotent stem cells (hPSCs) are a promising cell source for tissue engineering and regenerative medicine,especially in the field of neurobiology. Neural differentiation protocols have been developed to differentiate hPSCs into specific neural cells,but these predominantly rely on biochemical cues. Recently,differentiation protocols have incorporated topographical cues to increase the total neuronal yield. However,the means by which these topographical cues improve neuronal yield remains unknown. In this study,we explored the effect of topography on the neural differentiation of hPSC by quantitatively studying the changes in marker expression at a transcript and protein level. We found that 2 ??m gratings increase the rate of neural differentiation,and that an additional culture period of 2 ??m gratings in the absence of neurotrophic signals can improve the neural differentiation of hPSCs. We envisage that this work can be incorporated into future differentiation protocols to decrease the differentiation period as well as the biochemical signals added,thus generating hPSC-derived neural cells in a more cost effective and efficient manner. ?? 2012 Elsevier Ltd. View Publication

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression through translational inhibition and RNA decay and have been implicated in the regulation of cellular differentiation,proliferation,angiogenesis,and apoptosis. In this study,we analyzed global miRNA and mRNA microarrays to predict novel miRNA-mRNA interactions in human embryonic stem cells and induced pluripotent stem cells (iPSCs). In particular,we demonstrate a regulatory feedback loop between the miR-302 cluster and two transcription factors,NR2F2 and OCT4. Our data show high expression of miR-302 and OCT4 in pluripotent cells,while NR2F2 is expressed exclusively in differentiated cells. Target analysis predicts that NR2F2 is a direct target of miR-302,which we experimentally confirm by reporter luciferase assays and real-time polymerase chain reaction. We also demonstrate that NR2F2 directly inhibits the activity of the OCT4 promoter and thus diminishes the positive feedback loop between OCT4 and miR-302. Importantly,higher reprogramming efficiencies were obtained when we reprogrammed human adipose-derived stem cells into iPSCs using four factors (KLF4,C-MYC,OCT4,and SOX2) plus miR-302 (this reprogramming cocktail is hereafter referred to as KMOS3") when compared to using four factors ("KMOS"). Furthermore� View Publication

Aim: Reprogramming of somatic cells utilizing viral free methods provide a remarkable method to generate human induced pluripotent stem cells (hiPSCs) for regenerative medicine. In this study,we evaluate developmental ontogeny of cardiomyocytes following induced differentiation of hiPSCs. Main Methods: Fibroblasts were reprogrammed with episomal vectors to generate hiPSC and were subsequently differentiated to cardiomyocytes. Ontogenic development of cardiomyocytes was studied by real-time PCR. Key findings: Human iPSCs derived from episomal based vectors maintain classical pluripotency markers,generate teratomas and spontaneously differentiate into three germ layers in vitro. Cardiomyogenic induction of these hiPSCs efficiently generated cardiomyocytes. Ontogenic gene expression studies demonstrated that differentiation of cardiomyocytes was initiated by increased expression of mesodermal markers,followed by early cardiac committed markers,structural and ion channel genes. Furthermore,our correlation analysis of gene expression studies with human heart demonstrated that pivotal structural genes like cardiac troponin,actinin,myosin light chain maintained a high correlation with ion channel genes indicating coordinated activation of cardiac transcriptional machinery. Finally,microelectrode recordings show that these cardiomyocytes could respond aptly to pharmacologically active drugs. Cardiomyocytes showed a chronotropic response to isoproterenol,reduced Na+ influx with quinidine,prolongation of beating rate corrected field potential duration (cFPD) with E-4031 and reduced beating frequency and shortened cFPD with verapamil. Significance: Our study shows that viral free hiPSCs efficiently differentiate into cardiomyocytes with cardiac-specific molecular,structural,and functional properties that recapitulate developmental ontogeny of cardiogenesis. These results,coupled with the potential to generate patient-specific hiPSC lines hold great promise for the development of in vitro platform for drug pharmacogenomics; disease modeling and regenerative medicine. textcopyright 2012 Elsevier Inc. All rights reserved. View Publication

Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks,making them attractive candidate HSC regulators. We report that miR-126,a miRNA expressed in HSC and early progenitors,plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion,resulting in expansion of mouse and human long-term repopulating HSC. Conversely,enforced miR-126 expression impaired cell-cycle entry,leading to progressively reduced hematopoietic contribution. In HSC/early progenitors,miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway,attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size,demonstrating the importance of miRNA in the control of HSC function. View Publication

Extrinsic signaling cues in the microenvironment of acute myelogenous leukemia (AML) contribute to disease progression and therapy resistance. Yet,it remains unknown how the bone marrow niche in which AML arises is subverted to support leukemic persistence at the expense of homeostatic function. Exosomes are cell membrane-derived vesicles carrying protein and RNA cargoes that have emerged as mediators of cell-cell communication. In this study,we examined the role of exosomes in developing the AML niche of the bone marrow microenvironment,investigating their biogenesis with a focus on RNA trafficking. We found that both primary AML and AML cell lines released exosome-sized vesicles that entered bystander cells. These exosomes were enriched for several coding and noncoding RNAs relevant to AML pathogenesis. Furthermore,their uptake by bone marrow stromal cells altered their secretion of growth factors. Proof-of-concept studies provided additional evidence for the canonical functions of the transferred RNA. Taken together,our findings revealed that AML exosome trafficking alters the proliferative,angiogenic,and migratory responses of cocultured stromal and hematopoietic progenitor cell lines,helping explain how the microenvironmental niche becomes reprogrammed during invasion of the bone marrow by AML. View Publication

The aldehyde dehydrogenase (ALDH) superfamily is composed of nicotinamide adenine dinucleotide (phosphate) (NAD(P)(+))-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. To date,24 ALDH gene families have been identified in the eukaryotic genome. In addition to aldehyde metabolizing capacity,ALDHs have additional catalytic (e.g. esterase and reductase) and non-catalytic activities. The latter include functioning as structural elements in the eye (crystallins) and as binding molecules to endobiotics and xenobiotics. Mutations in human ALDH genes and subsequent inborn errors in aldehyde metabolism are the molecular basis of several diseases. Most recently ALDH polymorphisms have been associated with gout and osteoporosis. Aldehyde dehydrogenase enzymes also play important roles in embryogenesis and development,neurotransmission,oxidative stress and cancer. This article serves as a comprehensive review of the current state of knowledge regarding the ALDH superfamily and the contribution of ALDHs to various physiological and pathophysiological processes. View Publication

Articular cartilage,which is mainly composed of collagen II,enables smooth skeletal movement. Degeneration of collagen II can be caused by various events,such as injury,but degeneration especially increases over the course of normal aging. Unfortunately,the body does not fully repair itself from this type of degeneration,resulting in impaired movement. Microfracture,an articular cartilage repair surgical technique,has been commonly used in the clinic to induce the repair of tissue at damage sites. Mesenchymal stem cells (MSC) have also been used as cell therapy to repair degenerated cartilage. However,the therapeutic outcomes of all these techniques vary in different patients depending on their age,health,lesion size and the extent of damage to the cartilage. The repairing tissues either form fibrocartilage or go into a hypertrophic stage,both of which do not reproduce the equivalent functionality of endogenous hyaline cartilage. One of the reasons for this is inefficient chondrogenesis by endogenous and exogenous MSC. Drugs that promote chondrogenesis could be used to induce self-repair of damaged cartilage as a non-invasive approach alone,or combined with other techniques to greatly assist the therapeutic outcomes. The recent development of human induced pluripotent stem cell (iPSCs),which are able to self-renew and differentiate into multiple cell types,provides a potentially valuable cell resource for drug screening in a more relevant" cell type. Here we report a screening platform using human iPSCs in a multi-well plate format to identify compounds that could promote chondrogenesis." View Publication

The interaction between HER2 and aryl hydrocarbon receptor (AhR) signaling cascades during mammosphere formation of MCF-7 cells was studied. A newly established clonal MCF-7 cell line (HER2-5),stably overexpressing HER2,showed significantly enhanced levels of AhR mRNA and protein compared with MCF-7 cells. AhR was required for the HER2-mediated induction of interleukin-6 mRNA and for mammosphere formation in HER2-5 and MCF-7 cells. Mammosphere forming efficiency was suppressed by an AhR antagonist in a dose-dependent manner,as well as by knockdown of AhR. Taken together,these results indicate that AhR enhances mammosphere formation by human HER2-overexpressing breast cancer cells. View Publication