技术资料

BACKGROUND Evaluation of cystic fibrosis transmembrane conductance regulator (CFTR) functional activity to assess new therapies and define diagnosis of cystic fibrosis (CF) is cumbersome. It is known that leukocytes express detectable levels of CFTR but the molecule has not been characterized in these cells. In this study we aim at setting up and validating a blood test to evaluate CFTR expression and function in leukocytes. DESCRIPTION Western blot,PCR,immunofluorescence and cell membrane depolarization analysis by single-cell fluorescence imaging,using the potential-sensitive DiSBAC(2)(3) probe were utilized. Expression of PKA phosphorylated,cell membrane-localized CFTR was detected in non-CF monocytes,being undetectable or present in truncated form in monocytes derived from CF patients presenting with nonsense mutations. CFTR agonist administration induced membrane depolarization in monocytes isolated from non-CF donors (31 subjects) and,to a lesser extent,obligate CFTR heterozygous carriers (HTZ: 15 subjects),but it failed in monocytes from CF patients (44 subjects). We propose an index,which values in CF patients are significantly (ptextless0.001) lower than in the other two groups. Nasal Potential Difference,measured in selected subjects had concordant results with monocytes assay (Kappa statistic 0.93,95%CI: 0.80-1.00). RESULTS AND SIGNIFICANCE CFTR is detectable and is functional in human monocytes. We also showed that CFTR-associated activity can be evaluated in 5 ml of peripheral blood and devise an index potentially applicable for diagnostic purposes and both basic and translational research: from drug development to evaluation of functional outcomes in clinical trials. View Publication

Pluripotent stem cells exist in naive and primed states,epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs; ref. 1). In the naive state of ESCs,the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks. In contrast,EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types. The transition from naive to primed pluripotency therefore represents a pivotal event in cellular differentiation. But the signals that control this fundamental differentiation step remain unclear. We show here that paracrine and autocrine Wnt signals are essential self-renewal factors for ESCs,and are required to inhibit their differentiation into EpiSCs. Moreover,we find that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors,and support the derivation of new ESC lines,including ones from non-permissive mouse strains. Our results not only demonstrate that Wnt signals regulate the naive-to-primed pluripotency transition,but also identify Wnt as an essential and limiting ESC self-renewal factor. View Publication

Cellular decisions of self-renewal or differentiation arise from integration and reciprocal titration of numerous regulatory networks. Notch and Wnt/β-catenin signalling often intersect in stem and progenitor cells and regulate each other transcriptionally. The biological outcome of signalling through each pathway often depends on the context and timing as cells progress through stages of differentiation. Here,we show that membrane-bound Notch physically associates with unphosphorylated (active) β-catenin in stem and colon cancer cells and negatively regulates post-translational accumulation of active β-catenin protein. Notch-dependent regulation of β-catenin protein did not require ligand-dependent membrane cleavage of Notch or the glycogen synthase kinase-3β-dependent activity of the β-catenin destruction complex. It did,however,require the endocytic adaptor protein Numb and lysosomal activity. This study reveals a previously unrecognized function of Notch in negatively titrating active β-catenin protein levels in stem and progenitor cells. View Publication

Microbial growth in damp indoor environments has been correlated with risks to human health. This study was aimed to determine the cytotoxicity of 1-octen-3-ol (mushroom alcohol")� View Publication

APOBEC3G (A3G) is an intrinsic antiviral factor that inhibits the replication of human immunodeficiency virus (HIV) by deaminating cytidine residues to uridine. This causes guanosine-to-adenosine hypermutation in the opposite strand and results in inactivation of the virus. HIV counteracts A3G through the activity of viral infectivity factor (Vif),which promotes degradation of A3G. We report that viral protein R (Vpr),which interacts with a uracil glycosylase,also counteracted A3G by diminishing the incorporation of uridine. However,this process resulted in activation of the DNA-damage–response pathway and the expression of natural killer (NK) cell–activating ligands. Our results show that pathogen-induced deamination of cytidine and the DNA-damage response to virus-mediated repair of the incorporation of uridine enhance the recognition of HIV-infected cells by NK cells. View Publication

UNLABELLED The current protocols for generation of induced pluripotent stem (iPS) cells involve genome integrating viral vectors which may induce tumorgenesis. The aim of this study was to develop and optimize a non-viral method without genetic manipulation for reprogramming of skeletal myoblasts (SMs) using small molecules. METHODS AND RESULTS SMs from young male Oct3/4-GFP(+) transgenic mouse were treated with DNA methyltransferase (DNMT) inhibitor,RG108. Two weeks later,GFP(+) colonies of SM derived iPS cells (SiPS) expressing GFP and with morphological similarity of mouse embryonic stem (ESCs) were formed and propagated in vitro. SiPS were positive for alkaline phosphatase activity,expressed SSEA1,displayed ES cell specific pluripotency markers and formed teratoma in nude mice. Optimization of culture conditions for embryoid body (EBs) formation yielded spontaneously contracting EBs having morphological,molecular,and ultra-structural similarities with cardiomyocytes and expressed early and late cardiac markers. miR profiling showed abrogation of let-7 family and upregulation of ESCs specific miR-290-295 cluster thus indicating that SiPS were similar to ESCs in miR profile. Four weeks after transplantation into the immunocompetent mice model of acute myocardial infarction (n = 12 per group),extensive myogenesis was observed in SiPS transplanted hearts as compared to DMEM controls (n = 6 per group). A significant reduction in fibrosis and improvement in global heart function in the hearts transplanted with SiPS derived cardiac progenitor cells were observed. CONCLUSIONS Reprogramming of SMs by DNMT inhibitor is a simple,reproducible and efficient technique more likely to generate transgene integration-free iPS cells. Cardiac progenitors derived from iPS cells propagated extensively in the infarcted myocardium without tumorgenesis and improved cardiac function. View Publication

ETV6-RUNX1 gene fusion is usually an early,prenatal event in childhood acute lymphoblastic leukemia (ALL). Transformation results in the generation of a persistent (> 14 years) preleukemic clone,which postnatally converts to ALL after the acquisition of necessary secondary genetic alterations. Many cancer cells show some expression of the erythropoietin receptor (EPOR) gene,although the functionality" of any EPOR complexes and their relevant signaling pathways in nonerythroid cells has not been validated. EPOR mRNA is selectively and ectopically expressed in ETV6-RUNX1(+) ALL but the presence of a functional EPOR on the cell surface and its role in leukemogenesis driven by ETV6-RUNX1 remains to be identified. Here we show that ETV6-RUNX1 directly binds the EPOR promoter and that expression of ETV6-RUNX1 alone in normal pre-B cells is sufficient to activate EPOR transcription. We further reveal that murine and human ETV6-RUNX1(+) cells expressing EPOR mRNA have EPO ligand binding activity that correlates with an increased cell survival through activation of the JAK2-STAT5 pathway and up-regulation of antiapoptotic BCL-XL. These data support the contention that ETV6-RUNX1 directly activates ectopic expression of a functional EPOR and provides cell survival signals that may contribute critically to persistence of covert premalignant clones in children. View Publication

This unit describes the generation of tetracycline-inducible short hairpin RNA interference (shRNAi) human embryonic stem cell (hESC) lines. Using this vector-based approach enables stable and long-term expression of target hairpins under the control of doxycycline/tetracycline. Target degradation can be controlled in both a dose- and time-dependent manner that can even be switched off,depending upon the particular requirements of the study. View Publication

Sorting (or isolation) and manipulation of rare cells with high recovery rate and purity are of critical importance to a wide range of physiological applications. In the current paper,we report on a generic single cell manipulation tool that integrates optical tweezers and microfluidic chip technologies for handling small cell population sorting with high accuracy. The laminar flow nature of microfluidics enables the targeted cells to be focused on a desired area for cell isolation. To recognize the target cells,we develop an image processing methodology with a recognition capability of multiple features,e.g.,cell size and fluorescence label. The target cells can be moved precisely by optical tweezers to the desired destination in a noninvasive manner. The unique advantages of this sorter are its high recovery rate and purity in small cell population sorting. The design is based on dynamic fluid and dynamic light pattern,in which single as well as multiple laser traps are employed for cell transportation,and a recognition capability of multiple cell features. Experiments of sorting yeast cells and human embryonic stem cells are performed to demonstrate the effectiveness of the proposed cell sorting approach. View Publication

Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast™ chemical screening and prioritization research project. Metabolites from hES cultures were evaluated for known and novel signatures that may be indicative of developmental toxicity. Significant fold changes in endogenous metabolites were detected for 83 putatively annotated mass features in response to the subset of ToxCast chemicals. The annotations were mapped to specific human metabolic pathways. This revealed strong effects on pathways for nicotinate and nicotinamide metabolism,pantothenate and CoA biosynthesis,glutathione metabolism,and arginine and proline metabolism pathways. Predictivity for adverse outcomes in mammalian prenatal developmental toxicity studies used ToxRefDB and other sources of information,including Stemina Biomarker Discovery's predictive DevTox® model trained on 23 pharmaceutical agents of known developmental toxicity and differing potency. The model initially predicted developmental toxicity from the blinded ToxCast compounds in concordance with animal data with 73% accuracy. Retraining the model with data from the unblinded test compounds at one concentration level increased the predictive accuracy for the remaining concentrations to 83%. These preliminary results on a 11-chemical subset of the ToxCast chemical library indicate that metabolomics analysis of the hES secretome provides information valuable for predictive modeling and mechanistic understanding of mammalian developmental toxicity. View Publication

Activation of P2X(7) receptor (P2X(7)R) and pannexin have been implicated in membrane permeabilization associated with ischemic cell death and many other inflammatory processes. P2X(7)R has a unique property of forming large pore upon repeated or prolonged application of agonist like ATP or 2',3'-(4-benzoyl) benzoyl ATP. It has been proposed that pannexin 1 (panx1) hemichannel associates with P2X(7)R to form large pore,though the actual mechanism is not yet understood. Calcium concentration in extracellular milieu drops in many patho-physiological conditions,e.g. ischemia,when P2X(7)R/pannexin is also known to be activated. Therefore,we hypothesize that extracellular calcium ([Ca(2+)](o)) plays an important role in the coupling of P2X(7)R-panx1 and subsequent membrane permeabilization. In this study we show that membrane permeability of the P2X(7)R and panx1 expressing N2A cell increases in ([Ca(2+)](o))-free solution. In [Ca(2+)](o)-free solution,fluorescent dye calcein trapped cells exhibited time-dependent dye leakage resulting in about 50% decrease of fluorescence intensity in 30 min. Control cells in 2 mM [Ca(2+)](o) did not show such leakage. Like N2A cells,mixed culture of neuron and glia,derived from hippocampal progenitor cells showed similar dye leakage. Dye leakage was blocked either by pannexin-specific blocker,carbenoxolone or P2X(7)R antagonists,Brilliant Blue G,and oxidized ATP. Furthermore P2X(7)R and panx1 were co-immunoprecipitated. The amount of P2X(7)R protein pulled-down with panx1,increased by twofold when cells were incubated 30 min in [Ca(2+)](o)-free buffer. Taken together,the results of this study demonstrate the activation and association of P2X(7)R-panx1,triggered by the removal of [Ca(2+)](o). View Publication

Pluripotent stem cell-derived cardiomyocytes are currently being investigated for in vitro human heart models and as potential therapeutics for heart failure. In this study,we have developed a differentiation protocol that minimizes the need for specific human embryonic stem cell (hESC) line optimization. We first reduced the heterogeneity that exists within the starting population of bulk cultured hESCs by using cells adapted to single-cell passaging in a 2-dimensional (2D) culture format. Compared with bulk cultures,single-cell cultures comprised larger fractions of TG30(hi)/OCT4(hi) cells,corresponding to an increased expression of pluripotency markers OCT4 and NANOG,and reduced expression of early lineage-specific markers. A 2D temporal differentiation protocol was then developed,aimed at reducing the inherent heterogeneity and variability of embryoid body-based protocols,with induction of primitive streak cells using bone morphogenetic protein 4 and activin A,followed by cardiogenesis via inhibition of Wnt signaling using the small molecules IWP-4 or IWR-1. IWP-4 treatment resulted in a large percentage of cells expressing low amounts of cardiac myosin heavy chain and expression of early cardiac progenitor markers ISL1 and NKX2-5,thus indicating the production of large numbers of immature cardiomyocytes (˜65,000/cm(2) or ˜1.5 per input hESC). This protocol was shown to be effective in HES3,H9,and,to a lesser,extent,MEL1 hESC lines. In addition,we observed that IWR-1 induced predominantly atrial myosin light chain (MLC2a) expression,whereas IWP-4 induced expression of both atrial (MLC2a) and ventricular (MLC2v) forms. The intrinsic flexibility and scalability of this 2D protocol mean that the output population of primitive cardiomyocytes will be particularly accessible and useful for the investigation of molecular mechanisms driving terminal cardiomyocyte differentiation,and potentially for the future treatment of heart failure. View Publication