技术资料

OBJECTIVE The aim of this study was to evaluate the distribution,concentration and toxicity of cinnamaldehyde in electronic cigarette (e-cigarette) refill fluids and aerosols. METHODS The distribution and concentration of cinnamaldehyde were determined in 39 e-cigarette refill fluids plus 6 duplicates using gas chromatography and mass spectrometry (GC/MS). A cinnamaldehyde toxicity profile was established for embryonic and adult cells using a live cell imaging assay,immunocytochemistry,the comet assay and a recovery assay. RESULTS Twenty of the 39 refill fluids contained cinnamaldehyde at concentrations that are cytotoxic to human embryonic and lung cells in the MTT assay. Cinnamon Ceylon aerosol produced in a cartomizer-style e-cigarette was cytotoxic. Cinnamon Ceylon aerosols and refill fluid aerosols (80% propylene glycol or cinnamaldehyde/propylene glycol) made using a tank/boxmod e-cigarette were more cytotoxic at 5 V than 3 V. Using GC/MS,aerosols produced at 5 V contained 10 additional peaks not present in aerosol generated at 3 V. One of these,2,3-butandione (diacetyl),was confirmed with an authentic standard. Cinnamaldehyde depolymerised microtubules in human pulmonary fibroblasts. At concentrations that produced no effect in the MTT assay,cinnamaldehyde decreased growth,attachment and spreading; altered cell morphology and motility; increased DNA strand breaks; and increased cell death. At the MTT IC50 concentration,lung cells were unable to recover from cinnamaldehyde after 2 hours of treatment,whereas embryonic cells recovered after 8 hours. CONCLUSIONS Cinnamaldehyde-containing refill fluids and aerosols are cytotoxic,genotoxic and low concentrations adversely affect cell processes and survival. These data indicate that cinnamaldehyde in e-cigarette refill fluids/aerosols may impair homeostasis in the respiratory system. View Publication

Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyte trafficking and also contain resident myeloid cells with features of dendritic cells. However,the pathophysiological roles of these cells are poorly understood. Here we found that intimal myeloid cells underwent reverse transendothelial migration (RTM) into the arterial circulation after systemic stimulation of pattern-recognition receptors (PRRs). This process was dependent on expression of the chemokine receptor CCR7 and its ligand CCL19 by intimal myeloid cells. In mice infected with the intracellular pathogen Chlamydia muridarum,blood monocytes disseminated infection to the intima. Subsequent CCL19-CCR7-dependent RTM was critical for the clearance of intimal C. muridarum. This process was inhibited by hypercholesterolemia. Thus,RTM protects the normal arterial intima,and compromised RTM during atherogenesis might contribute to the intracellular retention of pathogens in atherosclerotic lesions. View Publication

Redirecting T cells to attack cancer using engineered chimeric receptors provides powerful new therapeutic capabilities. However,the effectiveness of therapeutic T cells is constrained by the endogenous T cell response: certain facets of natural response programs can be toxic,whereas other responses,such as the ability to overcome tumor immunosuppression,are absent. Thus,the efficacy and safety of therapeutic cells could be improved if we could custom sculpt immune cell responses. Synthetic Notch (synNotch) receptors induce transcriptional activation in response to recognition of user-specified antigens. We show that synNotch receptors can be used to sculpt custom response programs in primary T cells: they can drive a la carte cytokine secretion profiles,biased T cell differentiation,and local delivery of non-native therapeutic payloads,such as antibodies,in response to antigen. SynNotch T cells can thus be used as a general platform to recognize and remodel local microenvironments associated with diverse diseases. View Publication

In several clinical contexts,the measurement of ATP concentration in T lymphocytes has been proposed as a biomarker of immune status,predictive of secondary infections. However,the use of such biomarker in lymphopenic patients requires some adaptations in the ATP dosage protocol. We used blood from healthy volunteers to determine the optimal experimental settings. We investigated technical aspects such as the type of anticoagulant for blood sampling,the effect of freeze and thaw cycles,the reagent and sample mixing sequence,and the optimal dilution buffer. We also shortened the incubation time to 8h,and even showed that a 30min incubation may be sufficient. To evaluate the ATP rise upon lymphocyte activation,the optimal dose of stimulant was defined to be 4μg/mL of phytohaemagglutinin. Lastly,we determined that the number of T cells needed for this measurement was as low as 50,000,which is compatible with the existing lymphopenia in clinical settings. This optimized protocol appears ready to be assessed in lymphopenic patients to further investigate the interconnection between T lymphocyte metabolism and impaired phenotype and functions. View Publication

Spatial organization of the genome plays a central role in gene expression,DNA replication,and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see),a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ,cell sorting,and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion,termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility,and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control. View Publication

Purpose The purpose of this study was to characterize the secretion profile of induced pluripotent stem cell-derived retinal pigment epithelium (iPS-RPE) during wound healing. iPS-RPE was used to develop an in vitro wound healing model. We hypothesized that iPS-RPE secretes cytokines and growth factors which act in an autocrine manner to promote migration and proliferation of cells during wound healing. Methods iPS-RPE was grown in transwells until fully confluent and pigmented. The monolayers were scratched to induce a wound. Levels of Ki-67,$$-catenin,e-cadherin,n-cadherin,and S100A4 expression were analyzed by immunofluorescent labeling. Cell culture medium samples were collected from both the apical and basolateral sides of the transwells every 72 hours for 21 days. The medium samples were analyzed using multiplex ELISA to detect secreted growth factors and cytokines. The effects of conditioned medium on collagen gel contraction,cell proliferation,and migration were measured. Results iPS-RPE underwent epithelial-mesenchymal transition (EMT) during wound healing as indicated by the translocation of $$-catenin to the nucleus,cadherin switch,and expression of S100A4. GRO,GM-CSF,MCP-1,IL-6,and IL-8 were secreted by both the control and the wounded cell cultures. VEGF,FGF-2,and TGF$$ expression were detected at higher levels after wounding than those in control. The proteins were found to be secreted in a polarized manner. The conditioned medium from wounded monolayers promoted collagen gel contraction,as well as proliferation and migration of ARPE 19 cells. Conclusions These results indicate that after the monolayer is wounded,iPS-RPE secretes proteins into the culture medium that promote increased proliferation,contraction,and migration. View Publication

Haploid human pluripotent stem cells (PSCs) integrate haploidy and pluripotency,providing a novel system for functional genomics and developmental research in humans. We have recently derived haploid human embryonic stem cells (ESCs) by parthenogenesis and demonstrated their wide differentiation potential and applicability for genetic screening. Because haploid cells can spontaneously become diploid,their enrichment at an early passage is key for successful derivation. In this protocol,we describe two methodologies,namely metaphase spread analysis and cell sorting,for the identification of haploid human cells within parthenogenetic ESC lines. The cell sorting approach also enables the isolation of haploid cells at low percentages,as well as the maintenance of highly enriched haploid ESC lines throughout passaging. The isolation of essentially pure populations of haploid human ESCs by this protocol requires basic PSC culture expertise and can be achieved within 4-6 weeks. View Publication

Tissue-specific control of gene expression is an invaluable tool for studying various biological processes and medical applications. Efficient regulatory systems have been utilized to control transgene expression in various types of DNA viral or integrating viral vectors. However,existing regulatory systems are difficult to transfer into negative-strand RNA virus vector platforms because of significant differences in their transcriptional machineries. In this study,we developed a novel strategy for regulating transgene expression mediated by a cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp). Because of the capacity of Sendai virus (SeV) nonstructural C proteins to specifically inhibit viral RNA synthesis,overexpression of C protein significantly reduced transgene expression mediated by SeVdp vectors. We found that SeV C overexpression concomitantly reduced SeVdp mRNA levels and genomic RNA synthesis. To control C expression,target sequences for an endogenous microRNA were incorporated into the 3' untranslated region of the C genes. Incorporation of target sequences for miR-21 into the SeVdp vector restored transgene expression in HeLa cells by decreasing C expression. Furthermore,the SeVdp vector containing target sequences for let-7a enabled cell-specific control of transgene expression in human fibroblasts and induced pluripotent stem cells. Our findings demonstrate that SeV C can be used as an effective regulator for controlling transgene expression. This strategy will contribute to efficient and less toxic SeVdp-mediated gene transfer in various biological applications. View Publication

Fragile X syndrome (FXS) is a common cause of intellectual disability that is most often due to a CGG-repeat expansion mutation in the FMR1 gene that triggers epigenetic gene silencing. Epigenetic modifying drugs can only transiently and modestly induce FMR1 reactivation in the presence of the elongated CGG repeat. As a proof-of-principle,we excised the expanded CGG-repeat in both somatic cell hybrids containing the human fragile X chromosome and human FXS iPS cells using the CRISPR/Cas9 genome editing. We observed transcriptional reactivation in approximately 67% of the CRISPR cut hybrid colonies and in 20% of isolated human FXS iPSC colonies. The reactivated cells produced FMRP and exhibited a decline in DNA methylation at the FMR1 locus. These data demonstrate the excision of the expanded CGG-repeat from the fragile X chromosome can result in FMR1 reactivation. View Publication

Cell division is characterized by a sequence of events by which a cell gives rise to two daughter cells. Quantitative measurements of cell-cycle dynamics in single cells showed that despite variability in G1-,S-,and G2 phases,duration of mitosis is short and remarkably constant. Surprisingly,there is no correlation between cell-cycle length and mitotic duration,suggesting that mitosis is temporally insulated from variability in earlier cell-cycle phases. By combining live cell imaging and computational modeling,we showed that positive feedback is the molecular mechanism underlying the temporal insulation of mitosis. Perturbing positive feedback gave rise to a sluggish,variable entry and progression through mitosis and uncoupled duration of mitosis from variability in cell cycle length. We show that positive feedback is important to keep mitosis short,constant,and temporally insulated and anticipate it might be a commonly used regulatory strategy to create modularity in other biological systems. View Publication

Human pluripotent stem cells hold great promise for applications in drug discovery and regenerative medicine. Microfluidic technology is a promising approach for creating artificial microenvironments; however,although a proper 3D microenvironment is required to achieve robust control of cellular phenotypes,most current microfluidic devices provide only 2D cell culture and do not allow tuning of physical and chemical environmental cues simultaneously. Here,the authors report a 3D cellular microenvironment plate (3D-CEP),which consists of a microfluidic device filled with thermoresponsive poly(N-isopropylacrylamide)-β-poly(ethylene glycol) hydrogel (HG),which enables systematic tuning of both chemical and physical environmental cues as well as in situ cell monitoring. The authors show that H9 human embryonic stem cells (hESCs) and 253G1 human induced pluripotent stem cells in the HG/3D-CEP system maintain their pluripotent marker expression under HG/3D-CEP self-renewing conditions. Additionally,global gene expression analyses are used to elucidate small variations among different test environments. Interestingly,the authors find that treatment of H9 hESCs under HG/3D-CEP self-renewing conditions results in initiation of entry into the neural differentiation process by induction of PAX3 and OTX1 expression. The authors believe that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines and facilitate advances in drug screening and regenerative medicine. View Publication

MicroRNAs (miRNA) are central regulators of diverse biological processes and are important in the regulation of stem cell self-renewal. One of the widely studied miRNA-protein regulators is the Lin28-Let-7 pair. In this study,we demonstrate that contrary to the well-established models of mouse ES cells (mESC) and transformed human cancer cells,the pluripotent state of human ES cells (hESC) involves expression of mature Let-7 family miRNAs with concurrent expression of all LIN28 proteins. We show that mature Let-7 miRNAs are regulated during hESC differentiation and have opposite expression profile with LIN28B. Moreover,mature Let-7 miRNAs fine tune the expression levels of LIN28B protein in pluripotent hESCs,whereas silencing of LIN28 proteins have no effect on mature Let-7 levels. These results bring novel information to the highly complex network of human pluripotency and suggest that maintenance of hESC pluripotency differs greatly from the mESCs in regard to LIN28-Let-7 regulation. View Publication