技术资料

Mechanism-based sepsis treatments are unavailable,and their incidence is rising worldwide. Deaths occur during the early acute phase of hyperinflammation or subsequent postacute hypoinflammatory phase with sustained organ failure. The acute sepsis phase shifts rapidly,and multiple attempts to treat early excessive inflammation have uniformly failed. We reported in a sepsis cell model and human sepsis blood leukocytes that nuclear NAD+ sensor SIRT1 deacetylase remodels chromatin at specific gene sets to switch the acute-phase proinflammatory response to hypoinflammatory. Importantly,SIRT1 chromatin reprogramming is reversible,suggesting that inhibition of SIRT1 might reverse postacute-phase hypoinflammation. We tested this concept in septic mice,using the highly specific SIRT1 inhibitor EX-527,a small molecule that closes the NAD+ binding site of SIRT1. Strikingly,when administered 24 h after sepsis,all treated animals survived,whereas only 40% of untreated mice survived. EX-527 treatment reversed the inability of leukocytes to adhere at the small intestine MVI,reversed in vivo endotoxin tolerance,increased leukocyte accumulation in peritoneum,and improved peritoneal bacterial clearance. Mechanistically,the SIRT1 inhibitor restored repressed endothelial E-selectin and ICAM-1 expression and PSGL-1 expression on the neutrophils. Systemic benefits of EX-527 treatment included stabilized blood pressure,improved microvascular blood flow,and a shift toward proimmune macrophages in spleen and bone marrow. Our findings reveal that modifying the SIRT1 NAD+ axis may provide a novel way to treat sepsis in its hypoinflammatory phase. View Publication

Little is known on the embryonic origin and related heterogeneity of adult neural stem cells (aNSCs). We use conditional genetic tracing,activated in a global or mosaic fashion by cell type-specific promoters or focal laser uncaging,coupled with gene expression analyses and Notch invalidations,to address this issue in the zebrafish adult telencephalon. We report that the germinal zone of the adult pallium originates from two distinct subtypes of embryonic progenitors and integrates two modes of aNSC formation. Dorsomedial aNSCs derive from the amplification of actively neurogenic radial glia of the embryonic telencephalon. On the contrary,the lateral aNSC population is formed by stepwise addition at the pallial edge from a discrete neuroepithelial progenitor pool of the posterior telencephalic roof,activated at postembryonic stages and persisting lifelong. This dual origin of the pallial germinal zone allows the temporally organized building of pallial territories as a patchwork of juxtaposed compartments. View Publication

DNA repair plays a crucial role in embryonic and somatic stem cell biology and cell reprogramming. The Fanconi anemia (FA) pathway,which promotes error-free repair of DNA double-strand breaks,is required for somatic cell reprogramming to induced pluripotent stem cells (iPSC). Thus,cells from Fanconi anemia patients,which lack this critical pathway,fail to be reprogrammed to iPSC under standard conditions unless the defective FA gene is complemented. In this study,we utilized the oncogenes of high-risk human papillomavirus 16 (HPV16) to overcome the resistance of FA patient cells to reprogramming. We found that E6,but not E7,recovers FA iPSC colony formation and,furthermore,that p53 inhibition is necessary and sufficient for this activity. The iPSC colonies resulting from each of these approaches stained positive for alkaline phosphatase,NANOG,and Tra-1-60,indicating that they were fully reprogrammed into pluripotent cells. However,FA iPSC were incapable of outgrowth into stable iPSC lines regardless of p53 suppression,whereas their FA-complemented counterparts grew efficiently. Thus,we conclude that the FA pathway is required for the growth of iPSC beyond reprogramming and that p53-independent mechanisms are involved. IMPORTANCE A novel approach is described whereby HPV oncogenes are used as tools to uncover DNA repair-related molecular mechanisms affecting somatic cell reprogramming. The findings indicate that p53-dependent mechanisms block FA cells from reprogramming but also uncover a previously unrecognized defect in FA iPSC proliferation independent of p53. View Publication

Reported herein are two imidazole-based small molecules,termed neurodazine (Nz) and neurodazole (Nzl),which induce neuronal differentiation of pluripotent P19 cells. Their ability to induce neurogenesis of P19 cells is comparable to that of retinoic acid. However,Nz and Nzl were found to be more selective neurogenesis inducers than retinoic acid owing to their unique ability to suppress astrocyte differentiation of P19 cells. Our results also show that Nz and Nzl promote production of physiologically active neurons because P19-cell-derived neurons induced by these substances have functional glutamate responsiveness. The present study suggests that Nz and Nzl could serve as important chemical tools to induce formation of specific populations of neuronal cell types from pluripotent cells. View Publication

Constant deregulation of Id1 and Id3 has been implicated in a wide range of carcinomas. However,underlying molecular evidence for the joint role of Id1 and Id3 in the tumorigenicity of small cell lung cancer (SCLC) is sparse. Investigating the biological significance of elevated expression in SCLC cells,we found that Id1 and Id3 co-suppression resulted in significant reduction of proliferation rate,invasiveness and anchorage-independent growth. Suppressing both Id1 and Id3 expression also greatly reduced the average size of tumors produced by transfectant cells when inoculated subcutaneously into nude mice. Further investigation revealed that suppressed expression of Id1 and Id3 was accompanied by decreased angiogenesis and increased apoptosis. Therefore,the SCLC tumorigenicity suppression effect of double knockdown of Id1 and Id3 may be regulated through pathways of apoptosis and angiogenesis. View Publication

Human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs),a multipotent cell population that is capable of near indefinite expansion and subsequent differentiation into the various cell types that comprise the central nervous system (CNS),could provide an unlimited source of cells for neural-related cell-based therapies and disease modeling. However,the use of NPCs for the study and treatment of a variety of debilitating neurological diseases requires the development of scalable and reproducible protocols for their generation,expansion,characterization,and neuronal differentiation. Here,we describe a serum-free method for the stepwise generation of NPCs from hPSCs through the sequential formation of embryoid bodies (EBs) and neuro-epithelial-like rosettes. NPCs isolated from neural rosette cultures can be homogenously expanded while maintaining high expression of pan-neural markers such as SOX1,SOX2,and Nestin. Finally,this protocol allows for the robust differentiation of NPCs into microtubule-associated protein 2 (MAP2) and β-Tubulin-III (β3T) positive neurons. View Publication

The balance of self-renewal and differentiation in long-term repopulating hematopoietic stem cells (LT-HSC) must be strictly controlled to maintain blood homeostasis and to prevent leukemogenesis. Hematopoietic cytokines can induce differentiation in LT-HSCs; however,the molecular mechanism orchestrating this delicate balance requires further elucidation. We identified the tumor suppressor GADD45G as an instructor of LT-HSC differentiation under the control of differentiation-promoting cytokine receptor signaling. GADD45G immediately induces and accelerates differentiation in LT-HSCs and overrides the self-renewal program by specifically activating MAP3K4-mediated MAPK p38. Conversely,the absence of GADD45G enhances the self-renewal potential of LT-HSCs. Videomicroscopy-based tracking of single LT-HSCs revealed that,once GADD45G is expressed,the development of LT-HSCs into lineage-committed progeny occurred within 36 hr and uncovered a selective lineage choice with a severe reduction in megakaryocytic-erythroid cells. Here,we report an unrecognized role of GADD45G as a central molecular linker of extrinsic cytokine differentiation and lineage choice control in hematopoiesis. View Publication

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function,little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet,understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore,we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs,from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties,including resting membrane potential,action potential,sodium and potassium channel currents,somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons,the resting membrane potential became more negative,the expression of voltage-gated sodium channels increased,the membrane became capable of generating action potentials following adequate depolarization and,at day 48-55,50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step,of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca2+ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology,as electrophysiological properties of iPSC-derived neurons mature over time. View Publication

THAP1 encodes a transcription factor but its regulation is largely elusive. TOR1A was shown to be repressed by THAP1 in vitro. Notably,mutations in both of these genes lead to dystonia (DYT6 or DYT1). Surprisingly,expressional changes of TOR1A in THAP1 mutation carriers have not been detected indicating additional levels of regulation. Here,we investigated whether THAP1 is able to autoregulate its own expression. Using in-silico prediction,luciferase reporter gene assays,and (quantitative) chromatin immunoprecipitation (ChIP),we defined the THAP1 minimal promoter to a 480. bp-fragment and demonstrated specific binding of THAP1 to this region which resulted in repression of the THAP1 promoter. This autoregulation was disturbed by different DYT6-causing mutations. Two mutants (Ser6Phe,Arg13His) were shown to be less stable than wildtype THAP1 adding to the effect of reduced binding to the THAP1 promoter. Overexpressed THAP1 is preferably degraded through the proteasome. Notably,endogenous THAP1 expression was significantly reduced in cells overexpressing wildtype THAP1 as demonstrated by quantitative PCR. In contrast,higher THAP1 levels were detected in induced pluripotent stem cell (iPS)-derived neurons from THAP1 mutation carriers. Thus,we identified a feedback-loop in the regulation of THAP1 expression and demonstrated that mutant THAP1 leads to higher THAP1 expression levels. This compensatory autoregulation may contribute to the mean age at onset in the late teen years or even reduced penetrance in some THAP1 mutation carriers. View Publication

Ligation of erythropoietin (EPO) receptor (EPOR) JAK2 kinase complexes propagates signals within erythroid progenitor cells (EPCs) that are essential for red blood cell production. To reveal hypothesized novel EPOR/JAK2 targets,a phosphotyrosine (PY) phosphoproteomics approach was applied. Beyond known signal transduction factors,32 new targets of EPO-modulated tyrosine phosphorylation were defined. Molecular adaptors comprised one major set including growth factor receptor-bound protein 2 (GRB2)-associated binding proteins 1-3 (GAB1-3),insulin receptor substrate 2 (IRS2),docking protein 1 (DOK1),Src homology 2 domain containing transforming protein 1 (SHC1),and sprouty homologue 1 (SPRY1) as validating targets,and SPRY2,SH2 domain containing 2A (SH2D2A),and signal transducing adaptor molecule 2 (STAM2) as novel candidate adaptors together with an ORF factor designated as regulator of human erythroid cell expansion (RHEX). RHEX is well conserved in Homo sapiens and primates but absent from mouse,rat,and lower vertebrate genomes. Among tissues and lineages,RHEX was elevated in EPCs,occurred as a plasma membrane protein,was rapidly PY-phosphorylated textgreater20-fold upon EPO exposure,and coimmunoprecipitated with the EPOR. In UT7epo cells,knockdown of RHEX inhibited EPO-dependent growth. This was associated with extracellular signal-regulated kinase 1,2 (ERK1,2) modulation,and RHEX coupling to GRB2. In primary human EPCs,shRNA knockdown studies confirmed RHEX regulation of erythroid progenitor expansion and further revealed roles in promoting the formation of hemoglobinizing erythroblasts. RHEX therefore comprises a new EPO/EPOR target and regulator of human erythroid cell expansion that additionally acts to support late-stage erythroblast development. View Publication

Mesenchymal stem cells (MSCs) may be used as powerful tools for the repair and regeneration of damaged tissues. However,isolating tissue specific-derived MSCs may cause pain and increased infection rates in patients,and repetitive isolations may be required. To overcome these difficulties,we have examined alternative methods for MSC production. Here,we show that induced pluripotent stem cells (iPSCs) may be differentiated into mesenchymal stem cells (iMSCs) following exposure to SB431542. Purified iMSCs were administered to mdx mice to study skeletal muscle regeneration in a murine model of muscular dystrophy. Purified iMSCs displayed fibroblast-like morphology,formed three-dimensional spheroid structures,and expressed characteristic mesenchymal stem cell surface markers such as CD29,CD33,CD73,CD90,and CD105. Moreover,iMSCs were capable of differentiating into adipogenic,osteogenic,and chondrogenic lineages. Transplanting iMSC cells to tibialis anterior skeletal muscle tissue in mdx mice lowered oxidative damage as evidenced by a reduction in nitrotyrosine levels,and normal dystrophin expression levels were restored. This study demonstrates the therapeutic potential of purified iMSCs in skeletal muscle regeneration in mdx mice,and suggests that iPSCs are a viable alternate source for deriving MSCs as needed. textcopyright 2014 Elsevier Inc. View Publication

Optimization of pluripotent stem cell expansion and differentiation is facilitated by biological tools that permit non-invasive and dynamic monitoring of pluripotency,and the ability to select for an undifferentiated input cell population. Here we report on the generation and characterisation of clonal human embryonic stem (HES3,H9) and human induced pluripotent stem cell lines (UQEW01i-epifibC11) that have been stably modified with an artificial EOS(C3+) promoter driving expression of EGFP and puromycin resistance-conferring proteins. We show that EGFP expression faithfully reports on the pluripotency status of the cells in these lines and that antibiotic selection allows for an efficient elimination of differentiated cells from the cultures. We demonstrate that the extinction of the expression of the pluripotency reporter during differentiation closely correlates with the decrease in expression of conventional pluripotency markers,such as OCT4 (POU5F1),TRA-1-60 and SSEA4 when screening across conditions with various levels of pluripotency-maintaining or differentiation-inducing signals. We further illustrate the utility of these lines for real-time monitoring of pluripotency in embryoid bodies and microfluidic bioreactors. View Publication