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The origin and function of human double negative (DN) TCR-alphabeta+ T cells is unknown. They are thought to contribute to the pathogenesis of systemic lupus erythematosus because they expand and accumulate in inflamed organs. In this study,we provide evidence that human TCR-alphabeta+ CD4- CD8- DN T cells can derive from activated CD8+ T cells. Freshly isolated TCR-alphabeta+ DN T cells display a distinct gene expression and cytokine production profile. DN cells isolated from peripheral blood as well as DN cells derived in vitro from CD8+ T cells produce a defined array of proinflammatory mediators that includes IL-1beta,IL-17,IFN-gamma,CXCL3,and CXCL2. These results indicate that,upon activation,CD8+ T cells have the capacity to acquire a distinct phenotype that grants them inflammatory capacity. View Publication

Transcripts expressed in cytotoxic T lymphocytes (CTL) have mechanistic and diagnostic importance in transplantation. We used microarrays to select CTL-associated transcripts (CATs) expressed in human CD4(+) CTL,CD8(+) CTL and NK cells,excluding transcripts expressed in B cells,monocytes and kidney. This generated three transcript sets: CD4(+)-associated,CD8(+)-associated and NK-associated. Surprisingly,many CATs were expressed in effector memory cells e.g. granzyme B/GZMB,interferon-gamma/IFNG. Transcript expression was very similar between CD4(+) and CD8(+) CTL. There were no transcripts highly selective for CD4(+) CTL or CD8(+) CTL: for example,cytotoxic molecule transcripts (perforin,granzymes,granulysin) were shared between CD8(+) CTL and CD4(+) CTL although expression remained higher in CD8(+) CTL. Transcripts that differentiated between CD8(+) CTL and CD4(+) CTL were primarily those shared between CD8(+) CTL and NK cells (e.g. NK receptors KLRC1,KLRC3,KLRD1,KLRK1). No transcripts could differentiate CD4(+) CTL from CD8(+) CTL but NK cell-associated transcripts could differentiate NK cells from CTL. This study serves as a foundation for the interpretation of CATs in rejecting allografts and highlights the extensive sharing of CATs among CD4(+) CTL,CD8(+) CTL and effector memory T cells. View Publication

The optimization of a purely negative depletion,enrichment process for circulating tumor cells (CTCs) in the peripheral blood of head and neck cancer patients is presented. The enrichment process uses a red cell lysis step followed by immunomagnetic labeling,and subsequent depletion,of CD45 positive cells. A number of relevant variables are quantified,or attempted to be quantified,which control the performance of the enrichment process. Six different immunomagnetic labeling combinations were evaluated as well as the significant difference in performance with respect to the blood source: buffy coats purchased from the Red Cross,fresh,peripheral blood from normal donors,and fresh peripheral blood from human cancer patients. After optimization,the process is able to reduce the number of normal blood cells in a cancer patient's blood from 4.05 x 10(9) to 8.04 x 10(3) cells/mL and still recover,on average,2.32 CTC per mL of blood. For all of the cancer patient blood samples tested in which CTC were detected (20 out of 26 patients) the average recovery of CTCs was 21.7 per mL of blood,with a range of 282 to 0.53 CTC. Since the initial number of CTC in a patient's blood is unknown,and most probably varies from patient to patient,the recovery of the CTC is unknown. However,spiking studies of a cancer cell line into normal blood,and subsequent enrichment using the optimized protocol indicated an average recovery of approximately 83%. Unlike a majority of other published studies,this study focused on quantifying as many factors as possible to facilitate both the optimization of the process as well as provide information for current and future performance comparisons. The authors are not aware any other reported study which has achieved the performance reported here (a 5.66 log(10)) in a purely negative enrichment mode of operation. Such a mode of operation of an enrichment process provides significant flexibility in that it has no bias with respect to what attributes define a CTC; thereby allowing the researcher or clinician to use any maker they choose to define whether the final,enrich product contains CTCs or other cell type relevant to the specific question (i.e.,does the CTC have predominantly epithelial or mesenchymal characteristics?). View Publication

Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs),but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing,we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel,we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally,to demonstrate the scalable potential of these procedures,we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging,we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis. View Publication

Booster of pluripotency: RSC133,a new synthetic derivative of indoleacrylic acid/indolepropionic acid,exhibits dual activity by inhibiting histone deacetylase and DNA methyltransferase. Furthermore it potently improves the reprogramming of human somatic cells into a pluripotent state and aids the growth and maintenance of human pluripotent stem cells (hPSCs). View Publication

Inhibition of cytosolic DNA sensing represents a strategy that tumor cells use for immune evasion,but the underlying mechanisms are unclear. Here we have shown that CD47-signal regulatory protein α (SIRPα) axis dictates the fate of ingested DNA in DCs for immune evasion. Although macrophages were more potent in uptaking tumor DNA,increase of DNA sensing by blocking the interaction of SIRPα with CD47 preferentially occurred in dendritic cells (DCs) but not in macrophages. Mechanistically,CD47 blockade enabled the activation of NADPH oxidase NOX2 in DCs,which in turn inhibited phagosomal acidification and reduced the degradation of tumor mitochondrial DNA (mtDNA) in DCs. mtDNA was recognized by cyclic-GMP-AMP synthase (cGAS) in the DC cytosol,contributing to type I interferon (IFN) production and antitumor adaptive immunity. Thus,our findings have demonstrated how tumor cells inhibit innate sensing in DCs and suggested that the CD47-SIRPα axis is critical for DC-driven antitumor immunity. View Publication

This study investigated the effect of mechanical load on human mesenchymal stem cell (hMSC) differentiation under different exogenous transforming growth factor-beta1 (TGF-beta(1)) concentrations (0,1 or 10 ng/ml).The role of the TGF-beta signalling pathway in this process was also studied. Human MSCs were seeded into fibrin-biodegradable polyurethane scaffolds at a cell density of 5 x 10(6) cells per scaffold and stimulated using our bioreactor. One hour of surface motion superimposed on cyclic compression was applied once a day over seven consecutive days. Scaffolds were analysed for gene expression,DNA content and glycosaminoglycan amount. Addition of TGF-beta(1) in the culture medium was sufficient to induce chondrogenesis of hMSCs. Depending on the TGF-beta(1) concentration of the culture medium,mechanical load stimulated chondrogenesis of hMSCs compared to the unloaded scaffolds,with a much stronger effect on gene expression at lower TGF-beta(1) concentrations. With TGF-beta(1) absent in the culture medium,mechanical load stimulated gene transcripts and protein synthesis of TGF-beta(1) and TGF-beta(3). TGF-beta type I receptor inhibitor LY364947 blocked the up-regulation on TGF-beta(1) and TGF-beta(3) production stimulated by mechanical load,and also blocked the chondrogenesis of hMSCs. Taken together,these findings suggest that mechanical load promotes chondrogenesis of hMSCs through TGF-beta pathway by up-regulating TGF-beta gene expression and protein synthesis. View Publication

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors. However,the low efficiency and slow kinetics of the reprogramming process have hampered progress with this technology. Here we report that a natural compound,vitamin C (Vc),enhances iPSC generation from both mouse and human somatic cells. Vc acts at least in part by alleviating cell senescence,a recently identified roadblock for reprogramming. In addition,Vc accelerates gene expression changes and promotes the transition of pre-iPSC colonies to a fully reprogrammed state. Our results therefore highlight a straightforward method for improving the speed and efficiency of iPSC generation and provide additional insights into the mechanistic basis of the reprogramming process. View Publication

Human embryonic stem cells (hESC) present a novel platform for in vitro investigation of the early embryonic cellular response to ionizing radiation. Thus far,no study has analyzed the genome-wide transcriptional response to ionizing radiation in hESCs,nor has any study assessed their ability to form teratomas,the definitive test of pluripotency. In this study,we use microarrays to analyze the global gene expression changes in hESCs after low-dose (0.4 Gy),medium-dose (2 Gy),and high-dose (4 Gy) irradiation. We identify genes and pathways at each radiation dose that are involved in cell death,p53 signaling,cell cycling,cancer,embryonic and organ development,and others. Using Gene Set Enrichment Analysis,we also show that the expression of a comprehensive set of core embryonic transcription factors is not altered by radiation at any dose. Transplantation of irradiated hESCs to immune-deficient mice results in teratoma formation from hESCs irradiated at all doses,definitive proof of pluripotency. Further,using a bioluminescence imaging technique,we have found that irradiation causes hESCs to initially die after transplantation,but the surviving cells quickly recover by 2 weeks to levels similar to control. To conclude,we show that similar to somatic cells,irradiated hESCs suffer significant death and apoptosis after irradiation. However,they continue to remain pluripotent and are able to form all three embryonic germ layers. Studies such as this will help define the limits for radiation exposure for pregnant women and also radiotracer reporter probes for tracking cellular regenerative therapies. View Publication

Pluripotency and self-renewal of human embryonic stem cells (hESCs) is mediated by a complex interplay between extra- and intracellular signaling pathways,which regulate the expression of pluripotency-specific transcription factors. The homeodomain transcription factor NANOG plays a central role in maintaining hESC pluripotency,but the precise role and regulation of NANOG are not well defined. View Publication