技术资料

GTF2IRD1 is one of the three members of the GTF2I gene family,clustered on chromosome 7 within a 1.8 Mb region that is prone to duplications and deletions in humans. Hemizygous deletions cause Williams-Beuren syndrome (WBS) and duplications cause WBS duplication syndrome. These copy number variations disturb a variety of developmental systems and neurological functions. Human mapping data and analyses of knockout mice show that GTF2IRD1 and GTF2I underpin the craniofacial abnormalities,mental retardation,visuospatial deficits and hypersociability of WBS. However,the cellular role of the GTF2IRD1 protein is poorly understood due to its very low abundance and a paucity of reagents. Here,for the first time,we show that endogenous GTF2IRD1 has a punctate pattern in the nuclei of cultured human cell lines and neurons. To probe the functional relationships of GTF2IRD1 in an unbiased manner,yeast two-hybrid libraries were screened,isolating 38 novel interaction partners,which were validated in mammalian cell lines. These relationships illustrate GTF2IRD1 function,as the isolated partners are mostly involved in chromatin modification and transcriptional regulation,whilst others indicate an unexpected role in connection with the primary cilium. Mapping of the sites of protein interaction also indicates key features regarding the evolution of the GTF2IRD1 protein. These data provide a visual and molecular basis for GTF2IRD1 nuclear function that will lead to an understanding of its role in brain,behaviour and human disease. View Publication

Cardiomyocytes (CMs) generated from human pluripotent stem cells (hPSCs) are a potential cell source for regenerative therapies,drug discovery and disease modeling. All these applications require a routine supply of relatively large quantities of in vitro-generated CMs. This protocol describes a suspension culture-based strategy for the generation of hPSC-CMs as cell-only aggregates,which facilitates process development and scale-up. Aggregates are formed for 4 d in hPSC culture medium followed by 10 d of directed differentiation by applying chemical Wnt pathway modulators. The protocol is applicable to static multiwell formats supporting fast adaptation to specific hPSC line requirements. We also demonstrate how to apply the protocol using stirred tank bioreactors at a 100-ml scale,providing a well-controlled upscaling platform for CM production. In bioreactors,the generation of 40-50 million CMs per differentiation batch at textgreater80% purity without further lineage enrichment can been achieved within 24 d. View Publication

Induced pluripotent stem cells (iPSC) and their differentiated derivatives offer a unique source of human primary cells for toxicity screens. Here,we report on the comparative cytotoxicity of 80 compounds (neurotoxicants,developmental neurotoxicants,and environmental compounds) in iPSC as well as isogenic iPSC-derived neural stem cells (NSC),neurons,and astrocytes. All compounds were tested over a 24-h period at 10 and 100$\$,in duplicate,with cytotoxicity measured using the MTT assay. Of the 80 compounds tested,50 induced significant cytotoxicity in at least one cell type; per cell type,32,38,46,and 41 induced significant cytotoxicity in iPSC,NSC,neurons,and astrocytes,respectively. Four compounds (valinomycin,3,3',5,5'-tetrabromobisphenol,deltamethrin,and triphenyl phosphate) were cytotoxic in all four cell types. Retesting these compounds at 1,10,and 100$\$ using the same exposure protocol yielded consistent results as compared with the primary screen. Using rotenone,we extended the testing to seven additional iPSC lines of both genders; no substantial difference in the extent of cytotoxicity was detected among the cell lines. Finally,the cytotoxicity assay was simplified by measuring luciferase activity using lineage-specific luciferase reporter iPSC lines which were generated from the parental iPSC line. This article is part of a Special Issue entitled SI: PSC and the brain. View Publication

Recently,direct reprogramming between divergent lineages has been achieved by the introduction of regulatory transcription factors. This approach may provide alternative cell resources for drug discovery and regenerative medicine,but applications could be limited by the genetic manipulation involved. Here,we show that mouse fibroblasts can be directly converted into neuronal cells using only a cocktail of small molecules,with a yield of up to textgreater90% being TUJ1-positive after 16 days of induction. After a further maturation stage,these chemically induced neurons (CiNs) possessed neuron-specific expression patterns,generated action potentials,and formed functional synapses. Mechanistically,we found that a BET family bromodomain inhibitor,I-BET151,disrupted the fibroblast-specific program,while the neurogenesis inducer ISX9 was necessary to activate neuron-specific genes. Overall,our findings provide a proof of principle" for chemically induced direct reprogramming of somatic cell fates across germ layers without genetic manipulation� View Publication

In an attempt to bring pluripotent stem cell biology closer to reaching its full potential,many groups have focused on improving reprogramming protocols over the past several years. The episomal modified Sendai virus-based vector has emerged as one of the most practical ones. Here we describe reprogramming of mesenchymal stromal/stem cells (MSC) derived from umbilical cord Wharton's Jelly into induced pluripotent stem cells (iPSC) using genome non-integrating Sendai virus-based vectors. The detailed protocols of iPSC colony cryopreservation (vitrification) and adaption to feeder-free culture conditions are also included. View Publication

Substrate composition significantly impacts human pluripotent stem cell (hPSC) self-renewal and differentiation,but relatively little is known about the role of endogenously produced extracellular matrix (ECM) components in regulating hPSC fates. Here we identify $\$-5 laminin as a signature ECM component endogenously synthesized by undifferentiated hPSCs cultured on defined substrates. Inducible shRNA knockdown and Cas9-mediated disruption of the LAMA5 gene dramatically reduced hPSC self-renewal and increased apoptosis without affecting the expression of pluripotency markers. Increased self-renewal and survival was restored to wild-type levels by culturing the LAMA5-deficient cells on exogenous laminin-521. Furthermore,treatment of LAMA5-deficient cells with blebbistatin or a ROCK inhibitor partially restored self-renewal and diminished apoptosis. These results demonstrate that endogenous $\$-5 laminin promotes hPSC self-renewal in an autocrine and paracrine manner. This finding has implications for understanding how stem cells dynamically regulate their microenvironment to promote self-renewal and provides guidance for efforts to design substrates for stem cell bioprocessing. View Publication

During differentiation,human embryonic stem cells (hESCs) shut down the regulatory network conferring pluripotency in a process we designated pluripotent state dissolution (PSD). In a high-throughput RNAi screen using an inclusive set of differentiation conditions,we identify centrally important and context-dependent processes regulating PSD in hESCs,including histone acetylation,chromatin remodeling,RNA splicing,and signaling pathways. Strikingly,we detected a strong and specific enrichment of cell-cycle genes involved in DNA replication and G2 phase progression. Genetic and chemical perturbation studies demonstrate that the S and G2 phases attenuate PSD because they possess an intrinsic propensity toward the pluripotent state that is independent of G1 phase. Our data therefore functionally establish that pluripotency control is hardwired to the cell-cycle machinery,where S and G2 phase-specific pathways deterministically restrict PSD,whereas the absence of such pathways in G1 phase potentially permits the initiation of differentiation. View Publication

UNLABELLED: Human bocavirus 1 (HBoV1) is a single-stranded DNA parvovirus that causes lower respiratory tract infections in young children worldwide. In this study,we identified novel splice acceptor and donor sites,namely,A1' and D1',in the large nonstructural protein (NS1)-encoding region of the HBoV1 precursor mRNA. The novel small NS proteins (NS2,NS3,and NS4) were confirmed to be expressed following transfection of an HBoV1 infectious proviral plasmid and viral infection of polarized human airway epithelium cultured at an air-liquid interface (HAE-ALI). We constructed mutant pIHBoV1 infectious plasmids which harbor silent mutations (sm) smA1' and smD1' at the A1' and D1' splice sites,respectively. The mutant infectious plasmids maintained production of HBoV1 progeny virions at levels less than five times lower than that of the wild-type plasmid. Importantly,the smA1' mutant virus that does not express NS3 and NS4 replicated in HAE-ALI as effectively as the wild-type virus; however,the smD1' mutant virus that does not express NS2 and NS4 underwent an abortive infection in HAE-ALI. Thus,our study identified three novel NS proteins,NS2,NS3,and NS4,and suggests an important function of the NS2 protein in HBoV1 replication in HAE-ALI. IMPORTANCE: Human bocavirus 1 infection causes respiratory diseases,including acute wheezing in infants,of which life-threatening cases have been reported. In vitro,human bocavirus 1 infects polarized human bronchial airway epithelium cultured at an air-liquid interface that mimics the environment of human lower respiratory airways. Viral nonstructural proteins are often important for virus replication and pathogenesis in infected tissues or cells. In this report,we identified three new nonstructural proteins of human bocavirus 1 that are expressed during infection of polarized human bronchial airway epithelium. Among them,we proved that one nonstructural protein is critical to the replication of the virus in polarized human bronchial airway epithelium. The creation of nonreplicating infectious HBoV1 mutants may have particular utility in vaccine development for this virus. View Publication

T-cell genome engineering holds great promise for cell-based therapies for cancer,HIV,primary immune deficiencies,and autoimmune diseases,but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types� View Publication

The transforming growth factor-$\$(TGF-$\$) family member activin A exerts multiple neurotrophic and protective effects in the brain. Activin also modulates cognitive functions and affective behavior and is a presumed target of antidepressant therapy. Despite its important role in the injured and intact brain,the mechanisms underlying activin effects in the CNS are still largely unknown. Our goal was to identify the first target genes of activin signaling in the hippocampus in vivo. Electroconvulsive seizures,a rodent model of electroconvulsive therapy in humans,were applied to C57BL/6J mice to elicit a strong increase in activin A signaling. Chromatin immunoprecipitation experiments with hippocampal lysates subsequently revealed that binding of SMAD2/3,the intracellular effectors of activin signaling,was significantly enriched at the Pmepa1 gene,which encodes a negative feedback regulator of TGF-$\$ in cancer cells,and at the Kdm6b gene,which encodes an epigenetic regulator promoting transcriptional plasticity. Underlining the significance of these findings,activin treatment also induced PMEPA1 and KDM6B expression in human forebrain neurons generated from embryonic stem cells suggesting interspecies conservation of activin effects in mammalian neurons. Importantly,physiological stimuli such as provided by environmental enrichment proved already sufficient to engender a rapid and significant induction of activin signaling concomitant with an upregulation of Pmepa1 and Kdm6b expression. Taken together,our study identified the first target genes of activin signaling in the brain. With the induction of Kdm6b expression,activin is likely to gain impact on a presumed epigenetic regulator of activity-dependent neuronal plasticity. View Publication

Type 2 diabetes mellitus (T2D) is a chronic metabolic disorder resulting from defects in both insulin secretion and insulin activity. The deficit and dysfunction of insulin secreting $\$-cells are signature symptoms of T2D. Additionally,in pancreatic $\$-cells,a small group of genes that are abundantly expressed in most other tissues is highly selectively repressed. Monocarboxylate transporter 1 (MCT1) is one of these genes. In this study,we identified an MCT1-suppressing microRNA (hsa-miR-495) and used this microRNA together with human embryonic stem cell (hESC) derived pancreatic endoderm (PE) cells transplanted into a high-fat diet induced T2D mouse model. Glucose metabolism significantly improved and other symptoms of T2D were attenuated after the procedure. Our findings support the potential for T2D treatment using the combination of microRNA and hESC differentiated PE cells. View Publication

Decabromodiphenyl ether (BDE-209) has been detected in human serum,semen,placenta,cord blood and milk worldwide. However,little is known regarding the potential effects on the early human embryonic development of BDE-209. In this study,human embryonic stem cell lines FY-hES-10 and FY-hES-26 were used to evaluate the potential effects and explore the toxification mechanisms using low-level BDE-209 exposure. Our data showed that BDE-209 exposure (1,10 and 100 nM) reduced the expression of pluripotent genes such as OCT4,SOX2 and NANOG and induced human embryonic stem cells (hESCs) apoptosis. The downregulation of BIRC5/BCL2 and upregulation of BAX were related to apoptosis of hESCs induced by BDE-209 exposure. A mechanism study showed that OCT4 down-regulation accompanied by OCT4 promoter hypermethylation and increasing miR-145/miR-335 levels,OCT4 inhibitors. Moreover,BDE-209 could increase the generation of intracellular reactive oxygen species (ROS) and decrease SOD2 expression. The ROS increase and OCT4 downregulation after BDE-209 exposure could be reversed partly by antioxidant N-acetylcysteine supplement. These findings showed that BDE-209 exposure could decrease pluripotent genes expression via epigenetic regulation and induce apoptosis through ROS generation in human embryonic stem cells in vitro. View Publication