技术资料

Many natural prion diseases of humans and animals are considered to be acquired through oral consumption of contaminated food or pasture. Determining the route by which prions establish host infection will identify the important factors that influence oral prion disease susceptibility and to which intervention strategies can be developed. After exposure,the early accumulation and replication of prions within small intestinal Peyer's patches is essential for the efficient spread of disease to the brain. To replicate within Peyer's patches,the prions must first cross the gut epithelium. M cells are specialised epithelial cells within the epithelia covering Peyer's patches that transcytose particulate antigens and microorganisms. M cell-development is dependent upon RANKL-RANK-signalling,and mice in which RANK is deleted only in the gut epithelium completely lack M cells. In the specific absence of M cells in these mice,the accumulation of prions within Peyer's patches and the spread of disease to the brain was blocked,demonstrating a critical role for M cells in the initial transfer of prions across the gut epithelium in order to establish host infection. Since pathogens,inflammatory stimuli and aging can modify M cell-density in the gut,these factors may also influence oral prion disease susceptibility. Mice were therefore treated with RANKL to enhance M cell density in the gut. We show that prion uptake from the gut lumen was enhanced in RANKL-treated mice,resulting in shortened survival times and increased disease susceptibility,equivalent to a 10-fold higher infectious titre of prions. Together these data demonstrate that M cells are the critical gatekeepers of oral prion infection,whose density in the gut epithelium directly limits or enhances disease susceptibility. Our data suggest that factors which alter M cell-density in the gut epithelium may be important risk factors which influence host susceptibility to orally acquired prion diseases. View Publication

Incorporation of thymidine analogues in replicating DNA,coupled with antibody and fluorophore staining,allows analysis of cell proliferation,but is currently limited to monolayer cultures,fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation,S phase progression over several division cycles,effects of anti-proliferative drugs and other applications. It is based on the prominent (˜ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain,Hoechst 33342 upon the incorporation of 5-bromo-2'-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content,multi-parametric dynamic analyses,far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures,complex 3D tissue models of tumor cell spheroids and intestinal organoids,and in physiological study with metformin treatment. View Publication

The human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However,the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs) still has much room to improve. Here we attempted to promote the tumor tropism of hiPS-NSCs by manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration. We first developed a baculovirus-delivered CRISPR interference (CRISPRi) system that sterically blocked the E-box element in the promoter of the miR-199a/214 cluster with an RNA-guided catalytically dead Cas9 (dCas9). We then applied this CRISPRi system to hiPS-NSCs and successfully suppressed the expression of miR-199a-5p,miR-199a-3p,and miR-214 in the microRNA gene cluster. Meanwhile,the expression levels of their targets related to regulation of hypoxia-stimulated cell migration,such as HIF1A,MET,and MAPK1,were upregulated. Further migration assays demonstrated that the targeted inhibition of the miR-199a/214 cluster significantly enhanced the tumor tropism of hiPS-NSCs both in vitro and in vivo. These findings suggest a novel application of CRISPRi in NSC-based tumor-targeted gene therapy. View Publication

Previous studies in the mouse indicated that ARID3A plays a critical role in the first cell fate decision required for generation of trophectoderm (TE). Here,we demonstrate that ARID3A is widely expressed during mouse and human placentation and essential for early embryonic viability. ARID3A localizes to trophoblast giant cells and other trophoblast-derived cell subtypes in the junctional and labyrinth zones of the placenta. Conventional Arid3a knockout embryos suffer restricted intrauterine growth with severe defects in placental structural organization. Arid3a null placentas show aberrant expression of subtype-specific markers as well as significant alteration in cytokines,chemokines and inflammatory response-related genes,including previously established markers of human placentation disorders. BMP4-mediated induction of trophoblast stem (TS)-like cells from human induced pluripotent stem cells results in ARID3A up-regulation and cytoplasmic to nuclear translocation. Overexpression of ARID3A in BMP4-mediated TS-like cells up-regulates TE markers,whereas pluripotency markers are down-regulated. Our results reveal an essential,conserved function for ARID3A in mammalian placental development through regulation of both intrinsic and extrinsic developmental programs. View Publication

Success in the differentiating human embryonic stem cells (hESCs) into insulin-secreting β cells raises new hopes for diabetes treatment. In this work,we demonstrated the feasibility of developing islet organoids from hESCs within biomimetic 3D scaffolds. We showed that such a 3D microenvironment is critical to the generation of pancreatic endoderm and endocrine from hESCs. The organoids formed consisted of pancreatic α,β,δ,and pancreatic polypeptide (PP) cells. A high-level co-expression of PDX1,NKX6.1,and NGN3 in these cells suggests the characteristics of pancreatic β cells. More importantly,most insulin-secreting cells generated did not express glucagon,somatostatin,or PP. The expression of mature β cell marker genes such as Pdx1,Ngn3,Insulin,MafA,and Glut2 was detected in these 3D-induced cell clusters. A high-level expression of C-peptide confirmed the de novo endogenous insulin production in these 3D induced cells. Insulin-secretory granules,an indication of β cell maturity,were detected in these cells as well. Glucose challenging experiments suggested that these cells are sensitive to glucose levels due to their elevated maturity. Exposing the cells to a high concentration of glucose induced a sharp increase in insulin secretion. View Publication

Reliance on volunteer blood donors can lead to transfusion product shortages,and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time,known as 'the storage lesion'. Thus,there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh,transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However,it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity,viability,deformability,and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo,we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel,chronically anemic,SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data,stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs,the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product. View Publication

NLRs (nucleotide-binding domain and leucine-rich repeats) belong to a large family of cytoplasmic sensors that regulate an extraordinarily diverse range of biological functions. One of these functions is to contribute to immunity against infectious diseases,but dysregulation of their functional activity leads to the development of inflammatory and autoimmune diseases. Cytoplasmic innate immune sensors,including NLRs,are central regulators of intestinal homeostasis. NLRC3 (also known as CLR16.2 or NOD3) is a poorly characterized member of the NLR family and was identified in a genomic screen for genes encoding proteins bearing leucine-rich repeats (LRRs) and nucleotide-binding domains. Expression of NLRC3 is drastically reduced in the tumour tissue of patients with colorectal cancer compared to healthy tissues,highlighting an undefined potential function for this sensor in the development of cancer. Here we show that mice lacking NLRC3 are hyper-susceptible to colitis and colorectal tumorigenesis. The effect of NLRC3 is most dominant in enterocytes,in which it suppresses activation of the mTOR signalling pathways and inhibits cellular proliferation and stem-cell-derived organoid formation. NLRC3 associates with PI3Ks and blocks activation of the PI3K-dependent kinase AKT following binding of growth factor receptors or Toll-like receptor 4. These findings reveal a key role for NLRC3 as an inhibitor of the mTOR pathways,mediating protection against colorectal cancer. View Publication

Pentraxin-2 (PTX-2),also known as serum amyloid P component (SAP/APCS),is a constitutive,antiinflammatory,innate immune plasma protein whose circulating level is decreased in chronic human fibrotic diseases. Here we show that recombinant human PTX-2 (rhPTX-2) retards progression of chronic kidney disease in Col4a3 mutant mice with Alport syndrome,reducing blood markers of kidney failure,enhancing lifespan by 20%,and improving histological signs of disease. Exogenously delivered rhPTX-2 was detected in macrophages but also in tubular epithelial cells,where it counteracted macrophage activation and was cytoprotective for the epithelium. Computational analysis of genes regulated by rhPTX-2 identified the transcriptional regulator c-Jun along with its activator protein-1 (AP-1) binding partners as a central target for the function of rhPTX-2. Accordingly,PTX-2 attenuates c-Jun and AP-1 activity,and reduces expression of AP-1-dependent inflammatory genes in both monocytes and epithelium. Our studies therefore identify rhPTX-2 as a potential therapy for chronic fibrotic disease of the kidney and an important inhibitor of pathological c-Jun signaling in this setting. View Publication

Amniogenesis-the development of amnion-is a critical developmental milestone for early human embryogenesis and successful pregnancy. However,human amniogenesis is poorly understood due to limited accessibility to peri-implantation embryos and a lack of in vitro models. Here we report an efficient biomaterial system to generate human amnion-like tissue in vitro through self-organized development of human pluripotent stem cells (hPSCs) in a bioengineered niche mimicking the in vivo implantation environment. We show that biophysical niche factors act as a switch to toggle hPSC self-renewal versus amniogenesis under self-renewal-permissive biochemical conditions. We identify a unique molecular signature of hPSC-derived amnion-like cells and show that endogenously activated BMP-SMAD signalling is required for the amnion-like tissue development by hPSCs. This study unveils the self-organizing and mechanosensitive nature of human amniogenesis and establishes the first hPSC-based model for investigating peri-implantation human amnion development,thereby helping advance human embryology and reproductive medicine. View Publication

Induced pluripotent stem cells (iPSCs) are being pursued as a source of cells for autologous therapies,many of which will be aimed at aged patients. To explore the impact of age on iPSC quality,we produced iPSCs from blood cells of 16 donors aged 21-100. We find that iPSCs from older donors retain an epigenetic signature of age,which can be reduced through passaging. Clonal expansion via reprogramming also enables the discovery of somatic mutations present in individual donor cells,which are missed by bulk sequencing methods. We show that exomic mutations in iPSCs increase linearly with age,and all iPSC lines analyzed carry at least one gene-disrupting mutation,several of which have been associated with cancer or dysfunction. Unexpectedly,elderly donors (textgreater90 yrs) harbor fewer mutations than predicted,likely due to a contracted blood progenitor pool. These studies establish that donor age is associated with an increased risk of abnormalities in iPSCs and will inform clinical development of reprogramming technology. View Publication

The chromatin landscape and cellular metabolism both contribute to cell fate determination,but their interplay remains poorly understood. Using genome-wide siRNA screening,we have identified prohibitin (PHB) as an essential factor in self-renewal of human embryonic stem cells (hESCs). Mechanistically,PHB forms protein complexes with HIRA,a histone H3.3 chaperone,and stabilizes the protein levels of HIRA complex components. Like PHB,HIRA is required for hESC self-renewal. PHB and HIRA act together to control global deposition of histone H3.3 and gene expression in hESCs. Of particular note,PHB and HIRA regulate the chromatin architecture at the promoters of isocitrate dehydrogenase genes to promote transcription and,thus,production of α-ketoglutarate,a key metabolite in the regulation of ESC fate. Our study shows that PHB has an unexpected nuclear role in hESCs that is required for self-renewal and that it acts with HIRA in chromatin organization to link epigenetic organization to a metabolic circuit. View Publication

BACKGROUND & AIMS Developmental morphogens play an important role in coordinating the ductular reaction and portal fibrosis occurring in the setting of cholangiopathies. However,little is known about how membrane signaling events in ductular reactive cells (DRCs) are transduced into nuclear transcriptional changes to drive cholangiocyte maturation and matrix deposition. Therefore,the aim of this study was to investigate potential mechanistic links between cell signaling events and epigenetic regulators in DRCs. METHODS Using directed differentiation of induced pluripotent stem cells (iPSC),isolated DRCs,and in vivo models,we examine the mechanisms whereby sonic hedgehog (Shh) overcomes an epigenetic barrier in biliary precursors and promotes both cholangiocyte maturation and deposition of fibronectin (FN). RESULTS We demonstrate,for the first time,that Gli1 influences the differentiation state and fibrogenic capacity of iPSC-derived hepatic progenitors and isolated DRCs. We outline a novel pathway wherein Shh-mediated Gli1 binding in key cholangiocyte gene promoters overcomes an epigenetic barrier conferred by the polycomb protein,enhancer of zeste homolog 2 (EZH2) and initiates the transcriptional program of cholangiocyte maturation. We also define previously unknown functional Gli1 binding sites in the promoters of cytokeratin (CK)7,CK19,and FN. Our in vivo results show that EZH2 KO mice fed the choline-deficient,ethanolamine supplemented (CDE) diet have an exaggerated cholangiocyte expansion associated with more robust ductular reaction and increased peri-portal fibrosis. CONCLUSION We conclude that Shh/Gli1 signaling plays an integral role in cholangiocyte maturation in vitro by overcoming an EZH2-dependent epigenetic barrier and this mechanism also promotes biliary expansion in vivo. View Publication