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Seven years ago,Aurora Biomed Inc. (Vancouver,BC) recognized the need to create a forum for scientific discourse spanning the spectrum of ion channel disciplines. Since then,researchers from both academia and industry have been invited each year to share their knowledge on the advancement of ion channel research and technology,drug discovery,and safety pharmacology. Aurora Biomed's 2009 Retreat continued this tradition by covering a variety of topics including Ion Channels as Disease and Pain Targets,TRP Ion Channels,Ion Channel Screening Technologies,Ion Channels in Safety Pharmacology,Structure & Function of Ion Channels,Ion Channels in Disease Pathology,and New Horizons in Life Sciences. View Publication

Reprogramming of a limited number of human cell types has been achieved through ectopic expression of four transcription factors to yield induced pluripotent stem (iPS) cells that closely resemble human embryonic stem cells (ESCs). Here,we determined functional and epigenetic properties of iPS cells generated from human umbilical vein endothelial cells (HUVEC) by conventional method of direct reprogramming. Retroviral overexpression of four transcription factors resets HUVEC to the pluripotency. Human endothelial cell-derived iPS (endo-iPS) cells were similar to human ESCs in morphology,gene expression,in vitro and in vivo differentiation capacity. Endo-iPS cells were efficiently differentiated in vitro into endothelial cells. Using genome-wide methylation profiling we show that promoter elements of endothelial specific genes were methylated following reprogramming whereas pluripotency-related gene promoters were hypomethylated similar to levels observed in ESCs. Genome-wide methylation analysis of CpG sites located in the functional regions of over than 14,000 genes indicated that human endo-iPS cells were highly similar to human ES cells,although differences in methylation levels of 46 genes were found. Overall CpG methylation of promoter regions in the pluripotent cells was higher than in somatic. We also show that during reprogramming female human endo-iPS cells exhibited reactivation of the somatically silenced X chromosome. Our findings demonstrate that iPS cells can be generated from human endothelial cells and reprogramming resets epigenetic status of endothelial cells to pluripotency. View Publication

BACKGROUND: Specific populations of highly tumorigenic cells are thought to exist in many human tumors,including pancreatic adenocarcinoma. However,the clinical significance of these tumor-initiating (ie,cancer stem) cells remains unclear. Aldehyde dehydrogenase (ALDH) activity can identify tumor-initiating cells and normal stem cells from several human tissues. We examined the prognostic significance and functional features of ALDH expression in pancreatic adenocarcinoma. METHODS: ALDH expression was analyzed by immunohistochemistry in 269 primary surgical specimens of pancreatic adenocarcinoma and examined for association with clinical outcomes and in paired primary tumors and metastatic lesions from eight pancreatic cancer patients who had participated in a rapid autopsy program. The clonogenic growth potential of ALDH-positive pancreatic adenocarcinoma cells was assessed in vitro by a colony formation assay and by tumor growth in immunodeficient mice (10-14 mice per group). Mesenchymal features of ALDH-positive pancreatic tumor cells were examined by using quantitative reverse transcription-polymerase chain reaction and an in vitro cell invasion assay. Gene expression levels and the invasive potential of ADLH-positive pancreatic cancer cells relative to the bulk cell population were examined by reverse transcription-polymerase chain reaction and an in vitro invasion assays,respectively. All statistical tests were two-sided. RESULTS: ALDH-positive tumor cells were detected in 90 of the 269 primary surgical specimens,and their presence was associated with worse survival (median survival for patients with ALDH-positive vs ALDH-negative tumors: 14 vs 18 months,hazard ratio of death = 1.28,95% confidence interval = 1.02 to 1.68,P = .05). Six (75%) of the eight patients with matched primary and metastatic tumor samples had ALDH-negative primary tumors,and in four (67%) of these six patients,the matched metastatic lesions (located in liver and lung) contained ALDH-positive cells. ALDH-positive cells were approximately five- to 11-fold more clonogenic in vitro and in vivo compared with unsorted or ALHD-negative cells,expressed genes consistent with a mesenchymal state,and had in vitro migratory and invasive potentials that were threefold greater than those of unsorted cells. CONCLUSIONS: ALDH expression marks pancreatic cancer cells that have stem cell and mesenchymal features. The enhanced clonogenic growth and migratory properties of ALDH-positive pancreatic cancer cells suggest that they play a key role in the development of metastatic disease that negatively affects the overall survival of patients with pancreatic adenocarcinoma. View Publication

PURPOSE: In vitro sensitivity to the proapoptotic receptor agonists dulanermin (rhApo2L/TRAIL) and drozitumab (DR5-agonist antibody) is strongly predicted by the expression of the O-glycosylation enzymes GALNT14 in non-small cell lung cancer (NSCLC) cell lines (among others) and of FUT3/6 in colorectal cancer (CRC) cell lines. We developed immunohistochemistry (IHC) assays that measure GALNT14 and FUT3/6 levels in archival formalin-fixed,paraffin-embedded human tumor tissue to determine marker prevalence in NSCLC and CRC tissue and to enable the future examination of these markers in clinical trials. EXPERIMENTAL DESIGN: GALNT14 or FUT3/6 ELISA-positive hybridoma clones were screened through IHC on cell pellets with known mRNA levels. The specificity of staining was examined in cell lines,normal tissue,and tumor tissue. RESULTS: GALNT14 and FUT3/6 IHC exhibited a golgi staining pattern and correlated with GALNT14 and FUT3/6 (but not GALNT2 and FUT4) mRNA expression levels in cell lines and normal tissues,suggesting specificity. GALNT14 and FUT3/6 H-scores were significantly higher in cell lines sensitive to dulanermin (P = 0.01 and P = 0.0004,respectively) and drozitumab (P = 0.03 and P textless 0.0001,respectively) versus resistant cell lines. GALNT14 and FUT3/6 H-scores varied widely,with approximately 45% of NSCLC samples exhibiting weak to moderate GALNT14 staining (H-score of at least 25) and 70% of CRC samples exhibiting moderate to strong FUT3/6 staining (H-score of at least 125). CONCLUSIONS: GALNT14 and FUT3/6 expression can be assessed in human tumors using sensitive and specific IHC assays. Both assays are being deployed in ongoing clinical trials of dulanermin and drozitumab to assess potential utility for patient selection. View Publication

Breast cancer stem cells are a group of undifferentiated cells with self-renewal and multidifferentiation potential. Chemotherapeutic and radiotherapeutic resistance,hypoxic resistance,high tumorigenicity,high cell invasion,and metastatic abilities are characteristics of these cells,which are responsible for breast cancer recurrence. Therefore,the correct sorting and identification of breast cancer stem cells is a primary step for research in this field. This article briefly describes the recent progress on sorting and identification technologies for breast cancer stem cells. Sorting technologies include the side population technique,technologies that depend on cell surface markers,ALDEFLUOR assays,and in situ detection. Identification technologies include mammosphere cultures,limited dilution in vitro,and in-vivo animal models. This review provides an important reference for breast cancer stem cell research,which will explore new methods for the treatment of patients with breast cancer. View Publication

Mesenchymal stem cells (MSCs) have multilineage differentiation potential which includes cell lineages of the central nervous system; hence MSCs might be useful in the treatment of neurodegenerative diseases such as Parkinson's disease. Although mesenchymal stem cells have been shown to differentiate into the neural lineage,there is still little knowledge about the underlying mechanisms of differentiation particularly towards specialized neurons such as dopaminergic neurons. Here,we show that MSCs derived from human umbilical cord blood (MSC(hUCBs)) are capable of expressing tyrosine hydroxylase (TH) and Nurr1,markers typically associated with DA neurons. We also found differential phosphorylation of TH isoforms indicating the presence of post-translational mechanisms possibly activating and modifying TH in MSC(hUCB). Furthermore,functional dissection of components in the differentiation medium revealed that dibutyryl-cAMP (db-cAMP),3-isobutyl-1-methylxanthine (IBMX) and retinoic acid (RA) are involved in the regulation of Nurr1 and Neurofilament-L expression as well as in the differential phosphorylation of TH. We also demonstrate a possible inhibitory role of the protein kinase A signaling pathway in the phosphorylation of specific TH isoforms. View Publication

We have derived 30 human embryonic stem cell lines from supernumerary blastocysts in our laboratory. During the derivation process,we have studied new and safe method to establish good quality lines. All our human embryonic stem cell lines have been derived using human foreskin fibroblasts as feeder cells. The 26 more recent lines were derived in a medium containing serum replacement instead of fetal calf serum. Mechanical isolation of the inner cell mass using flexible metal needles was used in deriving the 10 latest lines. The lines are karyotypically normal,but culture adaptation in two lines has been observed. Our human embryonic stem cell lines are banked,and they are available for researchers. View Publication

Rearrangement of the actin cytoskeleton is essential for dynamic cellular processes. Decreased actin turnover and rigidity of cytoskeletal structures have been associated with aging and cell death. Gelsolin is a Ca(2+)-activated actin-severing protein that is widely expressed throughout the adult mammalian brain. Here,we used gelsolin-deficient (Gsn(-/-)) mice as a model system for actin filament stabilization. In Gsn(-/-) mice,emigration of newly generated cells from the subventricular zone into the olfactory bulb was slowed. In vitro,gelsolin deficiency did not affect proliferation or neuronal differentiation of adult neural progenitors cells (NPCs) but resulted in retarded migration. Surprisingly,hippocampal neurogenesis was robustly induced by gelsolin deficiency. The ability of NPCs to intrinsically sense excitatory activity and thereby implement coupling between network activity and neurogenesis has recently been established. Depolarization-induced [Ca(2+)](i) increases and exocytotic neurotransmitter release were enhanced in Gsn(-/-) synaptosomes. Importantly,treatment of Gsn(-/-) synaptosomes with mycotoxin cytochalasin D,which,like gelsolin,produces actin disassembly,decreased enhanced Ca(2+) influx and subsequent exocytotic norepinephrine release to wild-type levels. Similarly,depolarization-induced glutamate release from Gsn(-/-) brain slices was increased. Furthermore,increased hippocampal neurogenesis in Gsn(-/-) mice was associated with a special microenvironment characterized by enhanced density of perfused vessels,increased regional cerebral blood flow,and increased endothelial nitric oxide synthase (NOS-III) expression in hippocampus. Together,reduced filamentous actin turnover in presynaptic terminals causes increased Ca(2+) influx and,subsequently,elevated exocytotic neurotransmitter release acting on neural progenitors. Increased neurogenesis in Gsn(-/-) hippocampus is associated with a special vascular niche for neurogenesis. View Publication

We investigated functional relationships between microRNA 221/222 (miR-221/222) cluster and p27,a key regulator of cell cycle,in chronic lymphocytic leukemia (CLL). The enforced expression of miR-221/222 in the CLL cell line MEC1 induced a significant down-regulation of p27 protein and conferred a proliferative advantage to the transduced cells that exhibited faster progression into the S phase of the cell cycle. Accordingly,expression of miR-221/miR-222 and p27 was found to be inversely related in leukemic cells obtained from peripheral blood (PB) of 38 patients with CLL. Interestingly,when miR-221/222 and p27 protein were evaluated in different anatomic compartments (lymph nodes or bone marrow) of the same patients,increased expression of the 2 miRNAs became apparent compared with PB. This finding was paralleled by a low expression of p27. In addition,when CLL cells were induced in vitro to enter cell cycle (eg,with cytosine phosphate guanine oligodeoxynucleotide),a significant increase of miR-221/222 expression and a marked down-regulation of p27 protein were evident. These data indicate that the miR-221/222 cluster modulates the expression of p27 protein in CLL cells and lead to suggest that miR-221/222 and p27 may represent a regulatory loop that helps maintaining CLL cells in a resting condition. View Publication

The type-III interferon (IFN) family is composed of 3 molecules in humans: IFN-lambda1 (interleukin-29 [IL-29]),IFN-lambda2 (IL-28A),and IFN-lambda3 (IL-28B),each of which signals through the same receptor complex. Plasmacytoid dendritic cells (pDCs) are major IFN-lambda producers among peripheral lymphocytes. Recently,it has been shown that IFN-lambda1 exerts a powerful inhibitory effect over the T-helper 2 (Th2) response by antagonizing the effect of IL-4 on CD4(+) T cells and inhibiting the production of Th2-associated cytokines. Here,we asked whether Th2 cytokines exert reciprocal control over IFN-lambda production. IL-4 treatment during stimulation of human peripheral lymphocytes significantly elevated IFN-lambda1 transcription and secretion. However,pDCs were not directly responsive to IL-4. Using depletion and reconstitution experiments,we showed that IL-4-responsive monocytes are an intermediary cell,responding to IL-4 by elevating their secretion of IL-1 receptor antagonist (IL-Ra); this IL-1Ra acts on pDCs to elevate their IFN-lambda1 output. Thus,our experiments revealed a novel mechanism for regulation of both IFN-lambda1 production and pDC function,and suggests an expanded immunomodulatory role for Th2-associated cytokines. View Publication

The major goal of researchers and oncologists is to develop promising ground for novel therapeutic strategies to prevent recurrence or relapse of cancer. Recent evidences suggest that a subset of cells called cancer stem cells (CSCs) are present within the tumor mass which possess tumorigenic capacity and may be responsible for propagation,relapse,and metastatic dissemination. These cells have certain stem cell-like properties,e.g. quiescence,selfrenewal,asymmetric division,and multidrug resistance which allow them to drive tumor growth and evade conventional therapies. A number of markers and assays have been designed to isolate and characterize the CSC population from the bulk tumor. The objective now is to selectively target the CSCs in order to eliminate the tumor from root,overcoming the emergence of clones capable of evading traditional therapy. This approach may help in increasing the overall disease-free survival in some cancers. View Publication

The NKG2D receptor activates natural killer (NK) cell cytotoxicity and cytokine production on recognition of self-molecules induced by cellular stress under different conditions such as viral infections. The importance of NKG2D in the immune response to human cytomegalovirus (HCMV) is supported by the identification of several viral molecules that prevent the expression of NKG2D ligands by infected cells. In this study we report that,paradoxically,a significant,selective,and transient reduction of NKG2D expression on NK cells is detected during HCMV infection of peripheral blood mononuclear cells if needed. Antagonizing type I interferon (IFN),interleukin-12 (IL-12),and IFNgamma prevented HCMV-induced down-regulation of surface NKG2D. Moreover,treatment of purified NK cells with recombinant IFNbeta1 and IL-12 mimicked the effect,supporting a direct role of these cytokines in regulating NKG2D surface expression in NK cells. The loss of NKG2D expression selectively impaired NK-cell cytotoxicity against cells expressing NKG2D ligands but preserved the response triggered through other activating receptors. These results support that down-regulation of NKG2D expression on NK cells by cytokines with a key role in antiviral immune response may constitute a physiologic mechanism to control NK-cell reactivity against normal cells expressing NKG2D ligands in the context of inflammatory responses to viral infections. View Publication