技术资料

Long non-coding RNAs (lncRNAs) are known players in the regulatory circuitry of the self-renewal in human embryonic stem cells (hESCs). However,most hESC-specific lncRNAs remain uncharacterized. Here we demonstrate that growth-arrest-specific transcript 5 (GAS5),a known tumour suppressor and growth arrest-related lncRNA,is highly expressed and directly regulated by pluripotency factors OCT4 and SOX2 in hESCs. Phenotypic analysis shows that GAS5 knockdown significantly impairs hESC self-renewal,but its overexpression significantly promotes hESC self-renewal. Using RNA sequencing and functional analysis,we demonstrate that GAS5 maintains NODAL signalling by protecting NODAL expression from miRNA-mediated degradation. Therefore,we propose that the above pluripotency factors,GAS5 and NODAL form a feed-forward signalling loop that maintains hESC self-renewal. As this regulatory function of GAS5 is stem cell specific,our findings also indicate that the functions of lncRNAs may vary in different cell types due to competing endogenous mechanisms. View Publication

IL-25,a new member of the IL-17 cytokine family,is involved in type 2 immunity initiation and has been associated with the pathogenesis of rheumatoid arthritis (RA). However,its exact role remains unclear. Here,we aimed to analyse IL-25 expression in the serum and synovial fluid of RA patients and evaluated the correlations between serum IL-25 levels,clinical and laboratory values and inflammation cytokines. Additionally,we investigated whether IL-25 can suppress Th1/Th17 responses involved in RA pathogenesis. We further determined whether IL-25 can alleviate collagen-induced arthritis (CIA) development in mice and the underlying mechanisms using in vitro and in vivo experiments. Our results showed that IL-25 was upregulated in the serum and synovial fluid of RA patients. Increased serum IL-25 levels were associated with disease severity and inflammatory response in RA patients. Furthermore,IL-25 inhibited CD4(+) T-cell activation and differentiation into Th17 cells,without affecting Th1 cells in human RA and CIA models. Administration of IL-25 could attenuate CIA development by Th17 suppression in an IL-13-dependent manner. Our findings indicate that IL-25 plays a potent immunosuppressive role in the pathogenesis of RA and CIA by downregulating Th17 cell response,and thus,may be a potential therapeutic agent for RA. View Publication

Imprime PGG (Imprime),an intravenously-administered,soluble $\beta$-glucan,has shown compelling efficacy in multiple phase 2 clinical trials with tumor targeting or anti-angiogenic antibodies. Mechanistically,Imprime acts as pathogen-associated molecular pattern (PAMP) directly activating innate immune effector cells,triggering a coordinated anti-cancer immune response. Herein,using whole blood from healthy human subjects,we show that Imprime-induced anti-cancer functionality is dependent on immune complex formation with naturally-occurring,anti-$\beta$ glucan antibodies (ABA). The formation of Imprime-ABA complexes activates complement,primarily via the classical complement pathway,and is opsonized by iC3b. Immune complex binding depends upon Complement Receptor 3 and Fcg Receptor IIa,eliciting phenotypic activation of,and enhanced chemokine production by,neutrophils and monocytes,enabling these effector cells to kill antibody-opsonized tumor cells via the generation of reactive oxygen species and antibody-dependent cellular phagocytosis. Importantly,these innate immune cell changes were not evident in subjects with low ABA levels but could be rescued with exogenous ABA supplementation. Together,these data indicate that pre-existing ABA are essential for Imprime-mediated anti-cancer immune activation and suggest that pre-treatment ABA levels may provide a plausible patient selection biomarker to delineate patients most likely to benefit from Imprime-based therapy. View Publication

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are strongly associated with familial Parkinson's disease (PD). High expression levels in immune cells suggest a role of LRRK2 in regulating the immune system. In this study,we investigated the effect of the LRRK2 (G2019S) mutation in monocytes,using a human stem cell-derived model expressing LRRK2 at endogenous levels. We discovered alterations in the differentiation pattern of LRRK2 mutant,compared to non-mutant isogenic controls,leading to accelerated monocyte production and a reduction in the non-classical CD14+CD16+ monocyte subpopulation in the LRRK2 mutant cells. LPS-treatment of the iPSC-derived monocytes significantly increased the release of pro-inflammatory cytokines,demonstrating a functional response without revealing any significant differences between the genotypes. Assessment of the migrational capacity of the differentiated monocytes revealed moderate deficits in LRRK2 mutant cells,compared to their respective controls. Our findings indicate a pivotal role of LRRK2 in hematopoietic fate decision,endorsing the involvement of the immune system in the development of PD. View Publication

Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease,including congenital birth defects during pregnancy(1). To develop candidate therapeutic agents against ZIKV,we isolated a panel of human monoclonal antibodies (mAbs) from subjects with prior ZIKV infection. A subset of mAbs recognized diverse epitopes on the envelope (E) protein and exhibited potently neutralizing activity. One of the most inhibitory mAbs,ZIKV-117,broadly neutralized infection of ZIKV strains corresponding to African,Asian,and American lineages. Epitope mapping studies revealed that ZIKV-117 recognized a unique quaternary epitope on the E protein dimer-dimer interface. We evaluated the therapeutic efficacy of ZIKV-117 in pregnant and non-pregnant mice. mAb treatment markedly reduced tissue pathology,placental and fetal infection,and mortality in mice. Thus,neutralizing human mAbs can protect against maternal-fetal transmission,infection and disease,and reveal important determinants for structure-based rational vaccine design efforts. View Publication

The $\beta$-haemoglobinopathies,such as sickle cell disease and $\beta$-thalassaemia,are caused by mutations in the $\beta$-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure $\beta$-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably,we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90{\%} targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that,after differentiation into erythrocytes,express adult $\beta$-globin (HbA) messenger RNA,which confirms intact transcriptional regulation of edited HBB alleles. Collectively,these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for $\beta$-haemoglobinopathies. View Publication

The influence of GABA receptor agonists on the terminal differentiation in vitro of dopaminergic (DA) neurons derived from IPS cells was investigated. GABA-A agonist muscimol induced transient elevation of intracellular Ca(2+) level ([Ca(2+)] i ) in the investigated cells at days 5 to 21 of differentiation. Differentiation of cells in the presence of muscimol reduced tyrosine hydroxylase expression. Thus,the presence of active GABA-A receptors,associated with phenotype determination via Ca(2+)-signalling was demonstrated in differentiating human DA neurons. View Publication

Genetic changes in human pluripotent stem cells (hPSCs) gained during culture can confound experimental results and potentially jeopardize the outcome of clinical therapies. Particularly common changes in hPSCs are trisomies of chromosomes 1,12,17,and 20. Thus,hPSCs should be regularly screened for such aberrations. Although a number of methods are used to assess hPSC genotypes,there has been no systematic evaluation of the sensitivity of the commonly used techniques in detecting low-level mosaicism in hPSC cultures. We have performed mixing experiments to mimic the naturally occurring mosaicism and have assessed the sensitivity of chromosome banding,qPCR,fluorescence in situ hybridization,and digital droplet PCR in detecting variants. Our analysis highlights the limits of mosaicism detection by the commonly employed methods,a pivotal requirement for interpreting the genetic status of hPSCs and for setting standards for safe applications of hPSCs in regenerative medicine. View Publication

Sensory stimuli drive the maturation and function of the mammalian nervous system in part through the activation of gene expression networks that regulate synapse development and plasticity. These networks have primarily been studied in mice,and it is not known whether there are species- or clade-specific activity-regulated genes that control features of brain development and function. Here we use transcriptional profiling of human fetal brain cultures to identify an activity-dependent secreted factor,Osteocrin (OSTN),that is induced by membrane depolarization of human but not mouse neurons. We find that OSTN has been repurposed in primates through the evolutionary acquisition of DNA regulatory elements that bind the activity-regulated transcription factor MEF2. In addition,we demonstrate that OSTN is expressed in primate neocortex and restricts activity-dependent dendritic growth in human neurons. These findings suggest that,in response to sensory input,OSTN regulates features of neuronal structure and function that are unique to primates. View Publication

In fragile X syndrome (FXS),CGG repeat expansion greater than 200 triplets is believed to trigger FMR1 gene silencing and disease etiology. However,FXS siblings have been identified with more than 200 CGGs,termed unmethylated full mutation (UFM) carriers,without gene silencing and disease symptoms. Here,we show that hypomethylation of the FMR1 promoter is maintained in induced pluripotent stem cells (iPSCs) derived from two UFM individuals. However,a subset of iPSC clones with large CGG expansions carries silenced FMR1. Furthermore,we demonstrate de novo silencing upon expansion of the CGG repeat size. FMR1 does not undergo silencing during neuronal differentiation of UFM iPSCs,and expression of large unmethylated CGG repeats has phenotypic consequences resulting in neurodegenerative features. Our data suggest that UFM individuals do not lack the cell-intrinsic ability to silence FMR1 and that inter-individual variability in the CGG repeat size required for silencing exists in the FXS population. View Publication