技术资料

The developmental outcomes of preimplantation mammalian embryos are regulated directly by the surrounding microenvironment,and inappropriate concentrations of amino acids,or the loss of amino acid-sensing mechanisms,can be detrimental and impact further development. A specific role for l-proline in the differentiation of embryonic stem (ES) cells,a cell population derived from the blastocyst,has been shown in culture. l-proline acts as a signalling molecule,exerting its effects through cell uptake and subsequent metabolism. Uptake in ES cells occurs predominantly through the sodium-coupled neutral amino acid transporter 2,Slc38a2 (SNAT2). Dynamic expression of amino acid transporters has been shown in the early mammalian embryo,reflecting functional roles for amino acids in embryogenesis. The expression of SNAT2 and family member Slc38a1 (SNAT1) was determined in mouse embryos from the 2-cell stage through to the early post-implantation pre-gastrulation embryo. Key changes in expression were validated in cell culture models of development. Both transporters showed temporal dynamic expression patterns and changes in intracellular localisation as differentiation progressed. Changes in transporter expression likely reflect different amino acid requirements during development. Findings include the differential expression of SNAT1 in the inner and outer cells of the compacted morula and nuclear localisation of SNAT2 in the trophectoderm and placental lineages. Furthermore,SNAT2 expression was up-regulated in the epiblast prior to primitive ectoderm formation,an expression pattern consistent with a role for the transporter in later developmental decisions within the pluripotent lineage. We propose that the differential expression of SNAT2 in the epiblast provides evidence for an l-proline-mediated mechanism contributing to the regulation of embryonic development. View Publication

Human pluripotent stem cells (hPSCs) have gained a solid foothold in basic research and drug industry as they can be used in??vitro to study human development and have potential to offer limitless supply of various somatic cell types needed in drug development. Although the hepatic differentiation of hPSCs has been extensively studied,only a little attention has been paid to the role of the extracellular matrix. In this study we used laminin-511,laminin-521,and fibronectin,found in human liver progenitor cells,as culture matrices for hPSC-derived definitive endoderm cells. We observed that laminin-511 and laminin-521 either alone or in combination support the hepatic specification and that fibronectin is not a vital matrix protein for the hPSC-derived definitive endoderm cells. The expression of the laminin-511/521-specific integrins increased during the definitive endoderm induction and hepatic specification. The hepatic cells differentiated on laminin matrices showed the upregulation of liver-specific markers both at mRNA and protein levels,secreted human albumin,stored glycogen,and exhibited cytochrome P450 enzyme activity and inducibility. Altogether,we found that laminin-511 and laminin-521 can be used as stage-specific matrices to guide the hepatic specification of hPSC-derived definitive endoderm cells. View Publication

Human iPSC line Oex2054SV.4 was generated from fibroblasts of a patient with an optic atrophy 'plus' phenotype associated with a heterozygous mutation in the OPA1 gene. Reprogramming factors OCT3/4,SOX2,CMYC and KLF4 were delivered using a non-integrative methodology that involves the use of Sendai virus. View Publication

Human embryonic stem cell line BJNhem20-TetR was generated using non-viral method. The construct pCAG-TetRnls was transfected using microporation procedure. BJNhem20-TetR can subsequently be transfected with any vector harbouring a TetO (Tet operator) sequence to generate doxycycline based inducible line. For example,in human embryonic stem cells,the pSuperior based TetO system has been transfected into a TetR containing line to generate OCT4 knockdown cell line (Zafarana et al.,2009). Thus BJNhem20-TetR can be used as a tool to perturb gene expression in human embryonic stem cells. View Publication

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are defined as pluripotent in view of their self-renewal ability and potential to differentiate to cells of all three germ layers. Recent studies have indicated that microRNAs (miRNAs) play an important role in the maintenance of pluripotency and cell cycle regulation. We used a microarray based approach to identify miRNAs that were enriched in hESCs when compared to differentiated cells and at the same time showed significant expression changes between different phases of cell cycle. We identified 34 candidate miRNAs and performed functional studies on one of these,miR-1305,which showed the highest expression change during cell cycle transition. Overexpression of miR-1305 induced differentiation of pluripotent stem cells,increased cell apoptosis and sped up G1/S transition,while its downregulation facilitated the maintenance of pluripotency and increased cell survival. Using target prediction software and luciferase based reporter assays we identified POLR3G as a downstream target by which miR-1305 regulates the fine balance between maintenance of pluripotency and onset of differentiation. Overexpression of POLR3G rescued pluripotent stem cell differentiation induced by miR-1305 overexpression. In contrast,knock-down of POLR3G expression abolished the miR-1305-knockdown mediated enhancement of pluripotency,thus validating its role as miR-1305 target in human pluripotent stem cells. Together our data point to an important role for miR-1305 as a novel regulator of pluripotency,cell survival and cell cycle and uncovers new mechanisms and networks by which these processes are intertwined in human pluripotent stem cells. This article is protected by copyright. All rights reserved. View Publication

We describe a system for culturing human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells on a recombinant form of human laminin-511,a component of the natural hES cell niche. The system is devoid of animal products and feeder cells and contains only one undefined component,human albumin. The hES cells self-renewed with normal karyotype for at least 4 months (20 passages),after which the cells could produce teratomas containing cell lineages of all three germ layers. When plated on laminin-511 in small clumps,hES cells spread out in a monolayer,maintaining cellular homogeneity with approximately 97% OCT4-positive cells. Adhesion of hES cells was dependent on alpha6beta1 integrin. The use of homogeneous monolayer hES or iPS cell cultures provides more controllable conditions for the design of differentiation methods. This xeno-free and feeder-free system may be useful for the development of cell lineages for therapeutic purposes. View Publication

Genetic manipulation of human embryonic stem cells (hESC) has been limited by their general resistance to common methods used to introduce exogenous DNA or RNA. Efficient and high throughput transfection of nucleic acids into hESC would be a valuable experimental tool to manipulate these cells for research and clinical applications. We investigated the ability of two commercially available electroporation systems,the Nucleofection® 96-well Shuttle® System from Lonza and the Neon™ Transfection System from Invitrogen to efficiently transfect hESC. Transfection efficiency was measured by flow cytometry for the expression of the green fluorescent protein and the viability of the transfected cells was determined by an ATP catalyzed luciferase reaction. The transfected cells were also analyzed by flow cytometry for common markers of pluripotency. Both systems are capable of transfecting hESC at high efficiencies with little loss of cell viability. However,the reproducibility and the ease of scaling for high throughput applications led us to perform more comprehensive tests on the Nucleofection® 96-well Shuttle® System. We demonstrate that this method yields a large fraction of transiently transfected cells with minimal loss of cell viability and pluripotency,producing protein expression from plasmid vectors in several different hESC lines. The method scales to a 96-well plate with similar transfection efficiencies at the start and end of the plate. We also investigated the efficiency with which stable transfectants can be generated and recovered under antibiotic selection. Finally,we found that this method is effective in the delivery of short synthetic RNA oligonucleotides (siRNA) into hESC for knockdown of translation activity via RNA interference. Our results indicate that these electroporation methods provide a reliable,efficient,and high-throughput approach to the genetic manipulation of hESC. View Publication

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of personalized in vitro disease models,genomic health analyses,and autologous cell therapy. Here we describe the generation of T lymphocyte-derived iPSCs from small,clinically advantageous volumes of non-mobilized peripheral blood. These T-cell derived iPSCs (TiPS") retain a normal karyotype and genetic identity to the donor. They share common characteristics with human embryonic stem cells (hESCs) with respect to morphology� View Publication

Understanding the complex mechanisms that govern the fate decisions of human embryonic stem cells (hESCs) is fundamental to their use in cell replacement therapies. The progress of dissecting these mechanisms will be facilitated by the availability of robust high-throughput screening assays on hESCs. In this study,we report an image-based high-content assay for detecting compounds that affect hESC survival or pluripotency. Our assay was designed to detect changes in the phenotype of hESC colonies by quantifying multiple parameters,including the number of cells in a colony,colony area and shape,intensity of nuclear staining,and the percentage of cells in the colony that express a marker of pluripotency (TRA-1-60),as well as the number of colonies per well. We used this assay to screen 1040 compounds from two commercial compound libraries,and identified 17 that promoted differentiation,as well as 5 that promoted survival of hESCs. Among the novel small compounds we identified with activity on hESC are several steroids that promote hESC differentiation and the antihypertensive drug,pinacidil,which affects hESC survival. The analysis of overlapping targets of pinacidil and the other survival compounds revealed that activity of PRK2,ROCK,MNK1,RSK1,and MSK1 kinases may contribute to the survival of hESCs. View Publication

Reprogramming of somatic cells to pluripotency promises to boost cellular therapy. Most instances of direct reprogramming have been achieved by forced expression of defined exogenous factors using multiple viral vectors. The most used 4 transcription factors,octamer-binding transcription factor 4 (OCT4),(sex determining region Y)-box 2 (SOX2),Kruppel-like factor 4 (KLF4),and v-myc myelocytomatosis viral oncogene homolog (C-MYC),can induce pluripotency in mouse and human fibroblasts. Here,we report that forced expression of a new combination of transcription factors (T-cell leukemia/lymphoma protein 1A [TCL-1A],C-MYC,and SOX2) is sufficient to promote the reprogramming of human fibroblasts into pluripotent cells. These 3-factor pluripotent cells are similar to human embryonic stem cells in morphology,in the ability to differentiate into cells of the 3 embryonic layers,and at the level of global gene expression. Induced pluripotent human cells generated by a combination of other factors will be of great help for the understanding of reprogramming pathways. This,in turn,will allow us to better control cell-fate and apply this knowledge to cell therapy. View Publication

Teratogens,substances that may cause fetal abnormalities during development,are responsible for a significant number of birth defects. Animal models used to predict teratogenicity often do not faithfully correlate to human response. Here,we seek to develop a more predictive developmental toxicity model based on an in vitro method that utilizes both human embryonic stem (hES) cells and metabolomics to discover biomarkers of developmental toxicity. We developed a method where hES cells were dosed with several drugs of known teratogenicity then LC-MS analysis was performed to measure changes in abundance levels of small molecules in response to drug dosing. Statistical analysis was employed to select for specific mass features that can provide a prediction of the developmental toxicity of a substance. These molecules can serve as biomarkers of developmental toxicity,leading to better prediction of teratogenicity. In particular,our work shows a correlation between teratogenicity and changes of greater than 10% in the ratio of arginine to asymmetric dimethylarginine levels. In addition,this study resulted in the establishment of a predictive model based on the most informative mass features. This model was subsequently tested for its predictive accuracy in two blinded studies using eight drugs of known teratogenicity,where it correctly predicted the teratogenicity for seven of the eight drugs. Thus,our initial data shows that this platform is a robust alternative to animal and other in vitro models for the prediction of the developmental toxicity of chemicals that may also provide invaluable information about the underlying biochemical pathways. ?? 2010 Elsevier Inc. View Publication

Motor neuron diseases (MNDs) are a group of neurological disorders that selectively affect motor neurons. There are currently no cures or efficacious treatments for these diseases. In recent years,significant developments in stem cell research have been applied to MNDs,particularly regarding neuroprotection and cell replacement. However,a consistent source of motor neurons for cell replacement is required. Human embryonic stem cells (hESCs) could provide an inexhaustible supply of differentiated cell types,including motor neurons that could be used for MND therapies. Recently,it has been demonstrated that induced pluripotent stem (iPS) cells may serve as an alternative source of motor neurons,since they share ES characteristics,self-renewal,and the potential to differentiate into any somatic cell type. In this review,we discuss several reproducible methods by which hESCs or iPS cells are efficiently isolated and differentiated into functional motor neurons,and possible clinical applications. View Publication