技术资料

Tissue-specific control of gene expression is an invaluable tool for studying various biological processes and medical applications. Efficient regulatory systems have been utilized to control transgene expression in various types of DNA viral or integrating viral vectors. However,existing regulatory systems are difficult to transfer into negative-strand RNA virus vector platforms because of significant differences in their transcriptional machineries. In this study,we developed a novel strategy for regulating transgene expression mediated by a cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp). Because of the capacity of Sendai virus (SeV) nonstructural C proteins to specifically inhibit viral RNA synthesis,overexpression of C protein significantly reduced transgene expression mediated by SeVdp vectors. We found that SeV C overexpression concomitantly reduced SeVdp mRNA levels and genomic RNA synthesis. To control C expression,target sequences for an endogenous microRNA were incorporated into the 3' untranslated region of the C genes. Incorporation of target sequences for miR-21 into the SeVdp vector restored transgene expression in HeLa cells by decreasing C expression. Furthermore,the SeVdp vector containing target sequences for let-7a enabled cell-specific control of transgene expression in human fibroblasts and induced pluripotent stem cells. Our findings demonstrate that SeV C can be used as an effective regulator for controlling transgene expression. This strategy will contribute to efficient and less toxic SeVdp-mediated gene transfer in various biological applications. View Publication

Haploid human pluripotent stem cells (PSCs) integrate haploidy and pluripotency,providing a novel system for functional genomics and developmental research in humans. We have recently derived haploid human embryonic stem cells (ESCs) by parthenogenesis and demonstrated their wide differentiation potential and applicability for genetic screening. Because haploid cells can spontaneously become diploid,their enrichment at an early passage is key for successful derivation. In this protocol,we describe two methodologies,namely metaphase spread analysis and cell sorting,for the identification of haploid human cells within parthenogenetic ESC lines. The cell sorting approach also enables the isolation of haploid cells at low percentages,as well as the maintenance of highly enriched haploid ESC lines throughout passaging. The isolation of essentially pure populations of haploid human ESCs by this protocol requires basic PSC culture expertise and can be achieved within 4-6 weeks. View Publication

Purpose The purpose of this study was to characterize the secretion profile of induced pluripotent stem cell-derived retinal pigment epithelium (iPS-RPE) during wound healing. iPS-RPE was used to develop an in vitro wound healing model. We hypothesized that iPS-RPE secretes cytokines and growth factors which act in an autocrine manner to promote migration and proliferation of cells during wound healing. Methods iPS-RPE was grown in transwells until fully confluent and pigmented. The monolayers were scratched to induce a wound. Levels of Ki-67,$$-catenin,e-cadherin,n-cadherin,and S100A4 expression were analyzed by immunofluorescent labeling. Cell culture medium samples were collected from both the apical and basolateral sides of the transwells every 72 hours for 21 days. The medium samples were analyzed using multiplex ELISA to detect secreted growth factors and cytokines. The effects of conditioned medium on collagen gel contraction,cell proliferation,and migration were measured. Results iPS-RPE underwent epithelial-mesenchymal transition (EMT) during wound healing as indicated by the translocation of $$-catenin to the nucleus,cadherin switch,and expression of S100A4. GRO,GM-CSF,MCP-1,IL-6,and IL-8 were secreted by both the control and the wounded cell cultures. VEGF,FGF-2,and TGF$$ expression were detected at higher levels after wounding than those in control. The proteins were found to be secreted in a polarized manner. The conditioned medium from wounded monolayers promoted collagen gel contraction,as well as proliferation and migration of ARPE 19 cells. Conclusions These results indicate that after the monolayer is wounded,iPS-RPE secretes proteins into the culture medium that promote increased proliferation,contraction,and migration. View Publication

Normalization of human RNA-seq experiments employing chimpanzee RNA as a spike-in standard is reported. Human and chimpanzee RNAs exhibit single nucleotide variations (SNVs) in average 210-bp intervals. Spike-in chimpanzee RNA would behave the same as the human counterparts during the whole NGS procedures owing to the high sequence similarity. After discrimination of species origins of the NGS reads based on SNVs,the chimpanzee reads were used to read-by-read normalize biases and variations of human reads. By this approach,as many as 10,119 transcripts were simultaneously normalized for the entire NGS procedures leading to accurate and reproducible quantification of differential gene expression. In addition,incomparable data sets from different in-process degradations or from different library preparation methods were made well comparable by the normalization. Based on these results,we expect that the normalization approaches using near neighbor genomes as internal standards could be employed as a standard protocol,which will improve both accuracy and comparability of NGS results across different sample batches,laboratories and NGS platforms. View Publication

The essential functions of Polycomb Repressive Complex 1 (PRC1) in development and gene silencing are thought to involve long non-coding RNAs (lncRNAs),but few specific lncRNAs that guide PRC1 activity are known. We screened for lncRNAs which co-precipitate with PRC1 from chromatin and found candidates that impact Polycomb Group protein (PcG)-regulated gene expression in vivo. A novel lncRNA from this screen,CAT7,regulates expression and PcG binding at the MNX1 locus during early neuronal differentiation. CAT7 contains a unique tandem repeat domain which shares high sequence similarity to a non-syntenic zebrafish analog,cat7l. Defects caused by interference of cat7l RNA during zebrafish embryogenesis were rescued by human CAT7 RNA,enhanced by interference of a PRC1 component,and suppressed by interference of a known PRC1 target gene,demonstrating cat7l genetically interacts with a PRC1. We propose a model whereby PRC1 acts in concert with specific lncRNAs,and that CAT7/cat7l represent convergent lncRNAs that independently evolved to tune PRC1 repression at individual loci. View Publication

The vertebrate retina is a highly multilayered nervous tissue with a large diversity of cellular components. With the development of stem cell technologies,human retinas can be generated in three-dimensional (3-D) culture in vitro. However,understanding the factors modulating key productive processes and the way that they influence development are far from clear. Oxygen,as the most essential element participating in metabolism,is a critical factor regulating organic development. In this study,using 3-D culture of human stem cells,we examined the effect of intermittent high oxygen treatment (40% O2) on the formation and cellular behavior of neural retinas (NR) in the embryonic body (EB). The volume of EB and number of proliferating cells increased significantly under 40% O2 on day 38,50,and 62. Additionally,the ratio of PAX6+ cells within NR was significantly increased. The neural rosettes could only develop with correct apical-basal polarity under 40% O2. In addition,the generation,migration and maturation of retinal ganglion cells were enhanced under 40% O2. All of these results illustrated that 40% O2 strengthened the formation of NR in EB with characteristics similar to the in vivo state,suggesting that the hyperoxic state facilitated the retinal development in vitro. View Publication

Functional variability among human clones of induced pluripotent stem cells (hiPSCs) remains a limitation in assembling high-quality biorepositories. Beyond inter-person variability,the root cause of intra-person variability remains unknown. Mitochondria guide the required transition from oxidative to glycolytic metabolism in nuclear reprogramming. Moreover,mitochondria have their own genome (mitochondrial DNA [mtDNA]). Herein,we performed mtDNA next-generation sequencing (NGS) on 84 hiPSC clones derived from a cohort of 19 individuals,including mitochondrial and non-mitochondrial patients. The analysis of mtDNA variants showed that low levels of potentially pathogenic mutations in the original fibroblasts are revealed through nuclear reprogramming,generating mutant hiPSCs with a detrimental effect in their differentiated progeny. Specifically,hiPSC-derived cardiomyocytes with expanded mtDNA mutations non-related with any described human disease,showed impaired mitochondrial respiration,being a potential cause of intra-person hiPSC variability. We propose mtDNA NGS as a new selection criterion to ensure hiPSC quality for drug discovery and regenerative medicine. View Publication

Next-generation mass spectrometric (MS) techniques such as SWATH-MS have substantially increased the throughput and reproducibility of proteomic analysis,but ensuring consistent quantification of thousands of peptide analytes across multiple liquid chromatography-tandem MS (LC-MS/MS) runs remains a challenging and laborious manual process. To produce highly consistent and quantitatively accurate proteomics data matrices in an automated fashion,we developed TRIC (http://proteomics.ethz.ch/tric/),a software tool that utilizes fragment-ion data to perform cross-run alignment,consistent peak-picking and quantification for high-throughput targeted proteomics. TRIC reduced the identification error compared to a state-of-the-art SWATH-MS analysis without alignment by more than threefold at constant recall while correcting for highly nonlinear chromatographic effects. On a pulsed-SILAC experiment performed on human induced pluripotent stem cells,TRIC was able to automatically align and quantify thousands of light and heavy isotopic peak groups. Thus,TRIC fills a gap in the pipeline for automated analysis of massively parallel targeted proteomics data sets. View Publication

PURPOSE Proliferative vitreoretinopathy (PVR) is a blinding disorder that develops after a retinal tear or detachment. Activation of the retinal pigmented epithelium (RPE) is implicated in PVR; however,the mechanisms leading to enhanced RPE proliferation,migration,and contraction remain largely unknown. This study utilized an in vitro model of PVR to investigate the role of acetylation in RPE activation and its contribution to the progression of this disease. METHODS ARPE-19 cells,primary cultures of porcine RPE,and induced pluripotent stem cell-derived RPE (iPS-RPE) were utilized for cellular and molecular analyses. Cells treated with transforming growth factor beta 2 (TGF$$2; 10 ng/mL) alone or in the presence of the broad-spectrum histone deacetylase (HDAC) inhibitor,trichostatin A (TSA; 0.1 $$M),were assessed for contraction and migration through collagen contraction and scratch assays,respectively. Western blotting and immunofluorescence analysis were performed to assess $$-smooth muscle actin ($$-SMA) and $$-catenin expression after TGF$$2 treatment alone or in combination with TSA. RESULTS TGF$$2 significantly increased RPE cell contraction in collagen matrix and this effect was inhibited in the presence of TSA (0.1 $$M). In agreement with these data,immunofluorescence analysis of TSA-treated iPS-RPE wounded monolayers revealed decreased $$-SMA as compared with control. Scratch assays to assess wound healing revealed TSA inhibited TGF$$2-mediated iPS-RPE cell migration. CONCLUSIONS Our findings indicate a role of acetylation in RPE activation. Specifically,the HDAC inhibitor TSA decreased RPE cell proliferation and TGF$$2-mediated cell contraction and migration. Further investigation of pharmacological compounds that modulate acetylation may hold promise as therapeutic agents for PVR. View Publication

The U1 small nuclear (sn)RNA (U1) is a multifunctional ncRNA,known for its pivotal role in pre-mRNA splicing and regulation of RNA 3' end processing events. We recently demonstrated that a new class of human U1-like snRNAs,the variant (v)U1 snRNAs (vU1s),also participate in pre-mRNA processing events. In this study,we show that several human vU1 genes are specifically upregulated in stem cells and participate in the regulation of cell fate decisions. Significantly,ectopic expression of vU1 genes in human skin fibroblasts leads to increases in levels of key pluripotent stem cell mRNA markers,including NANOG and SOX2. These results reveal an important role for vU1s in the control of key regulatory networks orchestrating the transitions between stem cell maintenance and differentiation. Moreover,vU1 expression varies inversely with U1 expression during differentiation and cell re-programming and this pattern of expression is specifically de-regulated in iPSC-derived motor neurons from Spinal Muscular Atrophy (SMA) type 1 patient's. Accordingly,we suggest that an imbalance in the vU1/U1 ratio,rather than an overall reduction in Uridyl-rich (U)-snRNAs,may contribute to the specific neuromuscular disease phenotype associated with SMA. View Publication

BACKGROUND Aside from its importance in reproduction,estrogen (E2) is known to regulate the proliferation and differentiation of hematopoietic stem cells in rodents. However,the regulatory role of E2 in human hematopoietic system has not been investigated. The purpose of this study is to investigate the effect of E2 on hematopoietic differentiation using human pluripotent stem cells (hPSCs). RESULTS E2 improved hematopoietic differentiation of hPSCs via estrogen receptor alpha (ER-$$)-dependent pathway. During hematopoietic differentiation of hPSCs,ER-$$ is persistently maintained and hematopoietic phenotypes (CD34 and CD45) were exclusively detected in ER-$$ positive cells. Interestingly,continuous E2 signaling is required to promote hematopoietic output from hPSCs. Supplementation of E2 or an ER-$$ selective agonist significantly increased the number of hemangioblasts and hematopoietic progenitors,and subsequent erythropoiesis,whereas ER-$$ selective agonist did not. Furthermore,ICI 182,780 (ER antagonist) completely abrogated the E2-induced hematopoietic augmentation. Not only from hPSCs but also from human umbilical cord bloods,does E2 signaling potentiate hematopoietic development,suggesting universal function of E2 on hematopoiesis. CONCLUSIONS Our study identifies E2 as positive regulator of human hematopoiesis and suggests that endocrine factors such as E2 influence the behavior of hematopoietic stem cells in various physiological conditions. View Publication

Engineered heart tissues made from human pluripotent stem cell-derived cardiomyocytes have been used for modeling cardiac pathologies,screening new therapeutics,and providing replacement cardiac tissue. Current methods measure the functional performance of engineered heart tissue by their twitch force and beating frequency,typically obtained by optical measurements. In this article,we describe a novel method for assessing twitch force and beating frequency of engineered heart tissue using magnetic field sensing,which enables multiple tissues to be measured simultaneously. The tissues are formed as thin structures suspended between two silicone posts,where one post is rigid and another is flexible and contains an embedded magnet. When the tissue contracts it causes the flexible post to bend in proportion to its twitch force. We measured the bending of the post using giant magnetoresistive (GMR) sensors located underneath a 24-well plate containing the tissues. We validated the accuracy of the readings from the GMR sensors against optical measurements. We demonstrated the utility and sensitivity of our approach by testing the effects of three concentrations of isoproterenol and verapamil on twitch force and beating frequency in real-time,parallel experiments. This system should be scalable beyond the 24-well format,enabling greater automation in assessing the contractile function of cardiomyocytes in a tissue-engineered environment. View Publication