技术资料

Monomeric clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) nucleases have been widely adopted for simple and robust targeted genome editing but also have the potential to induce high-frequency off-target mutations. In principle,two orthogonal strategies for reducing off-target cleavage,truncated guide RNAs (tru-gRNAs) and dimerization-dependent RNA-guided FokI-dCas9 nucleases (RFNs),could be combined as tru-RFNs to further improve genome editing specificity. Here we identify a robust tru-RFN architecture that shows high activity in human cancer cell lines and embryonic stem cells. Additionally,we demonstrate that tru-gRNAs reduce the undesirable mutagenic effects of monomeric FokI-dCas9. Tru-RFNs combine the advantages of two orthogonal strategies for improving the specificity of CRISPR-Cas nucleases and therefore provide a highly specific platform for performing genome editing. View Publication

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) assert a great future for the cardiovascular diseases,both to study them and to explore therapies. However,a comprehensive assessment of the viral vectors used to modify these cells is lacking. In this study,we aimed to compare the transduction efficiency of recombinant adeno-associated vectors (AAV),adenoviruses and lentiviral vectors in hESC,hiPSC,and the derived cardiomyocytes. In undifferentiated cells,adenoviral and lentiviral vectors were superior,whereas in differentiated cells AAV surpassed at least lentiviral vectors. We also tested four AAV serotypes,1,2,6,and 9,of which 2 and 6 were superior in their transduction efficiency. Interestingly,we observed that AAVs severely diminished the viability of undifferentiated cells,an effect mediated by induction of cell cycle arrest genes and apoptosis. Furthermore,we show that the transduction efficiency of the different viral vectors correlates with the abundance of their respective receptors. Finally,adenoviral delivery of the calcium-transporting ATPase SERCA2a to hESC and hiPSC-derived cardiomyocytes successfully resulted in faster calcium reuptake. In conclusion,adenoviral vectors prove to be efficient for both differentiated and undifferentiated lines,whereas lentiviral vectors are more applicable to undifferentiated cells and AAVs to differentiated cells. View Publication

Human pluripotent stem cells (hPSCs),such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),provide a powerful model system for studies of cellular identity and early mammalian development,which hold great promise for regenerative medicine. It is necessary to develop a convenient method to discriminate hPSCs from other cells in clinics and basic research. Herein,a simple and reliable biosensor for stem cell detection was established. In this biosensor system,stage-specific embryonic antigen-3 (SSEA-3) and stage-specific embryonic antigen-4 (SSEA-4) were used to mark human pluripotent stem cells (hPSCs). Antibody specific for SSEA-3 was coated onto magnetic beads for hPSCs enrichment,and antibody specific for SSEA-4 was conjugated with carboxyl-modified tDNA sequence which was used as template for strand displacement amplification (SDA). The amplified single strand DNA (ssDNA) was detected with a lateral flow biosensor (LFB). This biosensor is capable of detecting a minimum of 19 human embryonic stem cells by a strip reader and 100 human embryonic stem cells by the naked eye within 80min. This approach has also shown excellent specificity to distinguish hPSCs from other types of cells,showing that it is promising for specific and handy detection of human pluripotent stem cells. View Publication

Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes,cells from the corneal stroma,may have the potential for restoration of vision in cell therapy and biomedical engineering applications,but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells,maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype. View Publication

Non-viral gene delivery into human embryonic stem cells (hESCs)is an important tool for controlling cell fate. However,the delivery efficiency remains low due in part to the tight colony structure of the cells which prevents effective exposure towards delivery vectors. We herein report a novel approach to enhance non-viral gene delivery to hESCs by transiently altering the cell and colony structure. (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide (Y-27632),a small molecule that inhibits the rho-associated protein kinase pathway,is utilized to induce transient colony spreading which leads to increased transfection efficiency by 1.5 to 2 folds in a spectrum of non-viral transfection reagents including Lipofectamine 2000 and Fugene HD. After removal of Y-27632 post-transfection,cells can revert back to its normal state and do not show alteration of pluripotency. This approach provides a simple,effective tool to enhance non-viral gene delivery into adherent hESCs for genetic manipulation. View Publication

Here we introduce the representative method to culture HESCs under the feeder and feeder-free conditions,the former of which is used to maintain or expand undifferentiated HESCs,and the latter can be used for the preparation of pure HESCs RNA samples,or for screening factors influential on self-renewal of HESCs. We also describe a protocol and tips for conducting gene chip analysis focusing on widely used Affymetrix Microarrays. These techniques will provide us unprecedented scale of biological information that would illuminate a key to decipher complex signaling networks controlling pluripotency. View Publication

Dysbindin is a schizophrenia susceptibility factor and subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) required for lysosome-related organelle biogenesis,and in neurons,synaptic vesicle assembly,neurotransmission,and plasticity. Protein networks,or interactomes,downstream of dysbindin/BLOC-1 remain partially explored despite their potential to illuminate neurodevelopmental disorder mechanisms. Here,we conducted a proteome-wide search for polypeptides whose cellular content is sensitive to dysbindin/BLOC-1 loss of function. We identified components of the vesicle fusion machinery as factors downregulated in dysbindin/BLOC-1 deficiency in neuroectodermal cells and iPSC-derived human neurons,among them the N-ethylmaleimide-sensitive factor (NSF). Human dysbindin/BLOC-1 coprecipitates with NSF and vice versa,and both proteins colocalized in a Drosophila model synapse. To test the hypothesis that NSF and dysbindin/BLOC-1 participate in a pathway-regulating synaptic function,we examined the role for NSF in dysbindin/BLOC-1-dependent synaptic homeostatic plasticity in Drosophila. As previously described,we found that mutations in dysbindin precluded homeostatic synaptic plasticity elicited by acute blockage of postsynaptic receptors. This dysbindin mutant phenotype is fully rescued by presynaptic expression of either dysbindin or Drosophila NSF. However,neither reduction of NSF alone or in combination with dysbindin haploinsufficiency impaired homeostatic synaptic plasticity. Our results demonstrate that dysbindin/BLOC-1 expression defects result in altered cellular content of proteins of the vesicle fusion apparatus and therefore influence synaptic plasticity. View Publication

Many acute and chronic anaemias,including haemolysis,sepsis and genetic bone marrow failure diseases such as Diamond-Blackfan anaemia,are not treatable with erythropoietin (Epo),because the colony-forming unit erythroid progenitors (CFU-Es) that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain sufficient red blood cell production. Treatment of these anaemias requires a drug that acts at an earlier stage of red cell formation and enhances the formation of Epo-sensitive CFU-E progenitors. Recently,we showed that glucocorticoids specifically stimulate self-renewal of an early erythroid progenitor,burst-forming unit erythroid (BFU-E),and increase the production of terminally differentiated erythroid cells. Here we show that activation of the peroxisome proliferator-activated receptor α (PPAR-α) by the PPAR-α agonists GW7647 and fenofibrate synergizes with the glucocorticoid receptor (GR) to promote BFU-E self-renewal. Over time these agonists greatly increase production of mature red blood cells in cultures of both mouse fetal liver BFU-Es and mobilized human adult CD34(+) peripheral blood progenitors,with a new and effective culture system being used for the human cells that generates normal enucleated reticulocytes. Although Ppara(-/-) mice show no haematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis,PPAR-α agonists facilitate recovery of wild-type but not Ppara(-/-) mice from PHZ-induced acute haemolytic anaemia. We also show that PPAR-α alleviates anaemia in a mouse model of chronic anaemia. Finally,both in control and corticosteroid-treated BFU-E cells,PPAR-α co-occupies many chromatin sites with GR; when activated by PPAR-α agonists,additional PPAR-α is recruited to GR-adjacent sites and presumably facilitates GR-dependent BFU-E self-renewal. Our discovery of the role of PPAR-α agonists in stimulating self-renewal of early erythroid progenitor cells suggests that the clinically tested PPAR-α agonists we used may improve the efficacy of corticosteroids in treating Epo-resistant anaemias. View Publication

Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T,$\$-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate,the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%,whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular,atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified,functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling,drug discovery,and regenerative medicine. View Publication

Unintended exposure to teratogenic compounds can lead to various birth defects; however current animal-based testing is limited by time,cost and high inter-species variability. Here,we developed a human-relevant in vitro model,which recapitulated two cellular events characteristic of embryogenesis,to identify potentially teratogenic compounds. We spatially directed mesoendoderm differentiation,epithelial-mesenchymal transition and the ensuing cell migration in micropatterned human pluripotent stem cell (hPSC) colonies to collectively form an annular mesoendoderm pattern. Teratogens could disrupt the two cellular processes to alter the morphology of the mesoendoderm pattern. Image processing and statistical algorithms were developed to quantify and classify the compounds' teratogenic potential. We not only could measure dose-dependent effects but also correctly classify species-specific drug (Thalidomide) and false negative drug (D-penicillamine) in the conventional mouse embryonic stem cell test. This model offers a scalable screening platform to mitigate the risks of teratogen exposures in human. View Publication

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Regeneration of human cartilage is inherently inefficient; an abundant autologous source,such as human induced pluripotent stem cells (hiPSCs),is therefore attractive for engineering cartilage. We report a growth factor-based protocol for differentiating hiPSCs into articular-like chondrocytes (hiChondrocytes) within 2 weeks,with an overall efficiency textgreater90%. The hiChondrocytes are stable and comparable to adult articular chondrocytes in global gene expression,extracellular matrix production,and ability to generate cartilage tissue in vitro and in immune-deficient mice. Molecular characterization identified an early SRY (sex-determining region Y) box (Sox)9(low) cluster of differentiation (CD)44(low)CD140(low) prechondrogenic population during hiPSC differentiation. In addition,2 distinct Sox9-regulated gene networks were identified in the Sox9(low) and Sox9(high) populations providing novel molecular insights into chondrogenic fate commitment and differentiation. Our findings present a favorable method for generating hiPSC-derived articular-like chondrocytes. The hiChondrocytes are an attractive cell source for cartilage engineering because of their abundance,autologous nature,and potential to generate articular-like cartilage rather than fibrocartilage. In addition,hiChondrocytes can be excellent tools for modeling human musculoskeletal diseases in a dish and for rapid drug screening. View Publication