技术资料

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

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

The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs,FUS and TDP-43,we integrate CLIP-seq and RNA Bind-N-Seq technologies,and show that TAF15 binds to ∼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns,are enriched in 3' untranslated regions and alter genes distinct from TDP-43. However,unlike FUS and TDP-43,TAF15 has a minimal role in alternative splicing. In human neural progenitors,TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons,the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G,but contrasts with late-stage sporadic ALS patients. Taken together,our findings reveal convergent and divergent roles for FUS,TAF15 and TDP-43 in RNA metabolism. View Publication

Efficient delivery of therapeutics across the neuroprotective blood-brain barrier (BBB) remains a formidable challenge for central nervous system drug development. High-fidelity in vitro models of the BBB could facilitate effective early screening of drug candidates targeting the brain. In this study,we developed a microfluidic BBB model that is capable of mimicking in vivo BBB characteristics for a prolonged period and allows for reliable in vitro drug permeability studies under recirculating perfusion. We derived brain microvascular endothelial cells (BMECs) from human induced pluripotent stem cells (hiPSCs) and cocultured them with rat primary astrocytes on the two sides of a porous membrane on a pumpless microfluidic platform for up to 10 days. The microfluidic system was designed based on the blood residence time in human brain tissues,allowing for medium recirculation at physiologically relevant perfusion rates with no pumps or external tubing,meanwhile minimizing wall shear stress to test whether shear stress is required for in vivo-like barrier properties in a microfluidic BBB model. This BBB-on-a-chip model achieved significant barrier integrity as evident by continuous tight junction formation and in vivo-like values of trans-endothelial electrical resistance (TEER). The TEER levels peaked above 4000 $$ textperiodcentered cm(2) on day 3 on chip and were sustained above 2000 $$ textperiodcentered cm(2) up to 10 days,which are the highest sustained TEER values reported in a microfluidic model. We evaluated the capacity of our microfluidic BBB model to be used for drug permeability studies using large molecules (FITC-dextrans) and model drugs (caffeine,cimetidine,and doxorubicin). Our analyses demonstrated that the permeability coefficients measured using our model were comparable to in vivo values. Our BBB-on-a-chip model closely mimics physiological BBB barrier functions and will be a valuable tool for screening of drug candidates. The residence time-based design of a microfluidic platform will enable integration with other organ modules to simulate multi-organ interactions on drug response. Biotechnol. Bioeng. 2016;9999: 1-11. textcopyright 2016 Wiley Periodicals,Inc. 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

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

We performed gene expression microarray analysis coupled with spherical self-organizing map (sSOM) for artificially developed cancer stem cells (CSCs). The CSCs were developed from human induced pluripotent stem cells (hiPSCs) with the conditioned media of cancer cell lines,whereas the CSCs were induced from primary cell culture of human cancer tissues with defined factors (OCT3/4,SOX2,and KLF4). These cells commonly expressed human embryonic stem cell (hESC)/hiPSC-specific genes (POU5F1,SOX2,NANOG,LIN28,and SALL4) at a level equivalent to those of control hiPSC 201B7. The sSOM with unsupervised method demonstrated that the CSCs could be divided into three groups based on their culture conditions and original cancer tissues. Furthermore,with supervised method,sSOM nominated TMED9,RNASE1,NGFR,ST3GAL1,TNS4,BTG2,SLC16A3,CD177,CES1,GDF15,STMN2,FAM20A,NPPB,CD99,MYL7,PRSS23,AHNAK,and LOC152573 genes commonly upregulating among the CSCs compared to hiPSC,suggesting the gene signature of the CSCs. View Publication