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Protein kinase CK2 (formerly casein kinase II) is a serine/threonine kinase overexpressed in many human tumors,transformed cell lines,and rapidly proliferating tissues. Recent data have shown that many cancers involve inappropriate reactivation of Wnt signaling through ectopic expression of Wnts themselves,as has been seen in a number of human breast cancers,or through mutation of intermediates in the Wnt pathway,such as adenomatous polyposis coli or beta-catenin,as described in colon and other cancers. Wnts are secreted factors that are important in embryonic development,but overexpression of certain Wnts,such as Wnt-1,leads to proliferation and transformation of cells. We report that upon stable transfection of Wnt-1 into the mouse mammary epithelial cell line C57MG,morphological changes and increased proliferation are accompanied by increased levels of CK2,as well as of beta-catenin. CK2 and beta-catenin co-precipitate with the Dvl proteins,which are Wnt signaling intermediates. A major phosphoprotein of the size of beta-catenin appears in in vitro kinase reactions performed on the Dvl immunoprecipitates. In vitro translated beta-catenin,Dvl-2,and Dvl-3 are phosphorylated by CK2. The selective CK2 inhibitor apigenin blocks proliferation of Wnt-1-transfected cells,abrogates phosphorylation of beta-catenin,and reduces beta-catenin and Dvl protein levels. These results demonstrate that endogenous CK2 is a positive regulator of Wnt signaling and growth of mammary epithelial cells. View Publication

Embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of the developing blastocyst that can proliferate extensively in vitro and are capable of adopting all the cell fates in a developing embryo. Clinical interest in the use of ES cells has been stimulated by studies showing that isolated human cells with ES properties from the inner cell mass or developing germ cells can provide a source of somatic precursors. Previous studies have defined in vitro conditions for promoting the development of specific somatic fates,specifically,hematopoietic,mesodermal,and neurectodermal. In this study,we present a method for obtaining dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells in vitro. Furthermore,we demonstrate that the ES cells can be obtained in unlimited numbers and that these neuron types are generated efficiently. We generated CNS progenitor populations from ES cells,expanded these cells and promoted their differentiation into dopaminergic and serotonergic neurons in the presence of mitogen and specific signaling molecules. The differentiation and maturation of neuronal cells was completed after mitogen withdrawal from the growth medium. This experimental system provides a powerful tool for analyzing the molecular mechanisms controlling the functions of these neurons in vitro and in vivo,and potentially for understanding and treating neurodegenerative and psychiatric diseases. View Publication

BACKGROUND: Airway remodeling is a proposed mechanism that underlies the persistent loss of lung function associated with childhood asthma. Previous studies have demonstrated that human lung fibroblasts (HLFs) co-cultured with primary human bronchial epithelial cells (BECs) from asthmatic children exhibit greater expression of extracellular matrix (ECM) components compared to co-culture with BECs derived from healthy children. Myofibroblasts represent a population of differentiated fibroblasts that have greater synthetic activity. We hypothesized co-culture with asthmatic BECs would lead to greater fibroblast to myofibroblast transition (FMT) compared to co-culture with healthy BECs. METHODS: BECs were obtained from well-characterized asthmatic and healthy children and were proliferated and differentiated at an air-liquid interface (ALI). BEC-ALI cultures were co-cultured with HLFs for 96 hours. RT-PCR was performed in HLFs for alpha smooth muscle actin ($$-SMA) and flow cytometry was used to assay for $$-SMA antibody labeling of HLFs. RT-PCR was also preformed for the expression of tropomyosin-I as an additional marker of myofibroblast phenotype. In separate experiments,we investigated the role of TGF$$2 in BEC-HLF co-cultures using monoclonal antibody inhibition. RESULTS: Expression of $$-SMA by HLFs alone was greater than by HLFs co-cultured with healthy BECs,but not different than $$-SMA expression by HLFs co-cultured with asthmatic BECs. Flow cytometry also revealed significantly less $$-SMA expression by healthy co-co-cultures compared to asthmatic co-cultures or HLF alone. Monoclonal antibody inhibition of TGF$$2 led to similar expression of $$-SMA between healthy and asthmatic BEC-HLF co-cultures. Expression of topomyosin-I was also significantly increased in HLF co-cultured with asthmatic BECs compared to healthy BEC-HLF co-cultures or HLF cultured alone. CONCLUSION: These findings suggest dysregulation of FMT in HLF co-cultured with asthmatic as compared to healthy BECs. Our results suggest TGF$$2 may be involved in the differential regulation of FMT by asthmatic BECs. These findings further illustrate the importance of BEC-HLF cross-talk in asthmatic airway remodeling. View Publication

CD8(+) T cells have a central role in antitumour immunity,but their activity is suppressed in the tumour microenvironment. Reactivating the cytotoxicity of CD8(+) T cells is of great clinical interest in cancer immunotherapy. Here we report a new mechanism by which the antitumour response of mouse CD8(+) T cells can be potentiated by modulating cholesterol metabolism. Inhibiting cholesterol esterification in T cells by genetic ablation or pharmacological inhibition of ACAT1,a key cholesterol esterification enzyme,led to potentiated effector function and enhanced proliferation of CD8(+) but not CD4(+) T cells. This is due to the increase in the plasma membrane cholesterol level of CD8(+) T cells,which causes enhanced T-cell receptor clustering and signalling as well as more efficient formation of the immunological synapse. ACAT1-deficient CD8(+) T cells were better than wild-type CD8(+) T cells at controlling melanoma growth and metastasis in mice. We used the ACAT inhibitor avasimibe,which was previously tested in clinical trials for treating atherosclerosis and showed a good human safety profile,to treat melanoma in mice and observed a good antitumour effect. A combined therapy of avasimibe plus an anti-PD-1 antibody showed better efficacy than monotherapies in controlling tumour progression. ACAT1,an established target for atherosclerosis,is therefore also a potential target for cancer immunotherapy. View Publication

Evidence is presented that bone marrow (BM) in addition to CD45(positive) hematopoietic stem cells contains a rare population of heterogenous CD45(negative) nonhematopoietic tissue committed stem cells (TCSC). These nonhematopoietic TCSC (i) are enriched in population of CXCR4(+) CD34(+) AC133(+) lin(-) CD45(-) and CXCR4(+) Sca-1(+) lin(-) CD45(-) in humans and mice,respectively,(ii) display several markers of pluripotent stem cells (PSC) and (iii) as we envision are deposited in BM early in development. Thus,since BM contains versatile nonhematopoietic stem cells,previous studies on plasticity trans-dedifferentiation of BM-derived hematopoietic stem cells (HSC) that did not include proper controls to exclude this possibility could lead to wrong interpretations. Therefore,in this spotlight review we present this alternative explanation of 'plasticity' of BM-derived stem cells based on the assumption that BM stem cells are heterogenous. We also discuss a potential relationship of TCSC/PSC identified by us with other BM-derived CD45(negative) nonhematopoietic stem cells that were recently identified by other investigators (eg MSC,MAPC,USSC and MIAMI cells). Finally,we discuss perspectives and pitfalls in potential application of these cells in regenerative medicine. View Publication

In the central nervous system,neural stem cells proliferate in the ventricular zone (VZ) and sequentially give rise to both neurons and glial cells in a temporally and spatially regulated manner,suggesting that stem cells may differ from one another in different brain regions and at different developmental stages. For the purpose of marking and purifying neural stem cells to ascertain whether such differences exist,we generated transgenic mice using promoters from Hes genes (pHes1 or pHes5) to drive expression of destabilized enhanced green fluorescent protein. In the developing brains of these transgenic mice,GFP expression was restricted to undifferentiated cells in the VZ,which could asymmetrically produce a Numb-positive neuronal daughter and a GFP-positive progenitor cell in clonal culture,indicating that they retain the capacity to self-renew. Our results suggest that pHes-EGFP transgenic mice can be used to explore similarities and differences among neural stem cells during development. View Publication

Glioblastoma multiforme is the most malignant type of primary brain tumor with a poor prognosis. These tumors consist of a heterogeneous population of malignant cells,including well-differentiated tumor cells and less differentiated cells with stem cell properties. These cancer stem cells,known as brain tumor initiating cells,likely contribute to glioma recurrence,as they are highly invasive,mobile,resistant to radiation and chemotherapy,and have the capacity to self-renew. Glioblastoma tumor cells release excitotoxic levels of glutamate,which may be a key process in the death of peritumoral neurons,formation of necrosis,local inflammation,and glioma-related seizures. Moreover,elevated glutamate levels in the tumor may act in paracrine and autocrine manner to activate glutamate receptors on glioblastoma tumor cells,resulting in proliferation and invasion. Using a previously described culturing condition that selectively promotes the growth of brain tumor initiating cells,which express the stem cell markers nestin and SOX-2,we characterize the expression of α-amino-3-hydroxy-5-methyl-4-isozolepropionic acid (AMPA)-type glutamate receptor subunits in brain tumor initiating cells derived from glioblastomas. Here we show for the first time that glioblastoma brain tumor initiating cells express high concentrations of functional calcium-permeable AMPA receptors,compared to the differentiated tumor cultures consisting of non-stem cells. Up-regulated calcium-permeable AMPA receptor expression was confirmed by immunoblotting,immunocytochemistry,and intracellular calcium imaging in response to specific agonists. Our findings raise the possibility that glutamate secretion in the GBM tumor microenvironment may stimulate brain tumor derived cancer stem cells. View Publication

SOX2 (Sex-determining region Y (SRY)-Box2) has important functions during embryonic development and is involved in cancer stem cell (CSC) maintenance,in which it impairs cell growth and tumorigenicity. However,the function of SOX2 in pancreatic cancer cells is unclear. The objective of this study was to analyze SOX2 expression in human pancreatic tumors and determine the role of SOX2 in pancreatic cancer cells regulating CSC properties. In this report,we show that SOX2 is not expressed in normal pancreatic acinar or ductal cells. However,ectopic expression of SOX2 is observed in 19.3% of human pancreatic tumors. SOX2 knockdown in pancreatic cancer cells results in cell growth inhibition via cell cycle arrest associated with p21(Cip1) and p27(Kip1) induction,whereas SOX2 overexpression promotes S-phase entry and cell proliferation associated with cyclin D3 induction. SOX2 expression is associated with increased levels of the pancreatic CSC markers ALDH1,ESA and CD44. Importantly,we show that SOX2 is enriched in the ESA(+)/CD44(+) CSC population from two different patient samples. Moreover,we show that SOX2 directly binds to the Snail,Slug and Twist promoters,leading to a loss of E-Cadherin and ZO-1 expression. Taken together,our findings show that SOX2 is aberrantly expressed in pancreatic cancer and contributes to cell proliferation and stemness/dedifferentiation through the regulation of a set of genes controlling G1/S transition and epithelial-to-mesenchymal transition (EMT) phenotype,suggesting that targeting SOX2-positive cancer cells could be a promising therapeutic strategy. View Publication

BACKGROUND: Niemann-Pick type C1 disease (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. In this lysosomal storage disorder the intracellular transport and sequestration of several lipids like cholesterol is severely impaired,resulting in an accumulation of lipids in late endosomes and lysosomes. The neurological manifestation of the disease is caused by dysfunction and cell death in the central nervous system. Several animal models were used to analyze the impaired pathways. However,the underlying pathogenic mechanisms are still not completely understood and the genetic variability in humans cannot be reflected in these models. Therefore,a human model using patient-specific induced pluripotent stem cells provides a promising approach. METHODS: We reprogrammed human fibroblasts from a NPC1 patient and a healthy control by retroviral transduction with Oct4,Klf4,Sox2 and c-Myc. The obtained human induced pluripotent stem cells (hiPSCs) were characterized by immunocytochemical analyses. Neural progenitor cells were generated and patch clamp recordings were performed for a functional analysis of derived neuronal cells. Filipin stainings and the Amplex Red assay were used to demonstrate and quantify cholesterol accumulation. RESULTS: The hiPSCs expressed different stem cell markers,e.g. Nanog,Tra-1-81 and SSEA4. Using the embryoid body assay,the cells were differentiated in cells of all three germ layers and induced teratoma in immunodeficient mice,demonstrating their pluripotency. In addition,neural progenitor cells were derived and differentiated into functional neuronal cells. Patch clamp recordings revealed voltage dependent channels,spontaneous action potentials and postsynaptic currents. The accumulation of cholesterol in different tissues is the main hallmark of NPC1. In this study we found an accumulation of cholesterol in fibroblasts of a NPC1 patient,derived hiPSCs,and neural progenitor cells,but not in cells derived from fibroblasts of a healthy individual. These findings were quantified by the Amplex Red assay,demonstrating a significantly elevated cholesterol level in cells derived from fibroblasts of a NPC1 patient. CONCLUSIONS: We generated a neuronal model based on induced pluripotent stem cells derived from patient fibroblasts,providing a human in vitro model to study the pathogenic mechanisms of NPC1 disease. View Publication

Bone marrow-derived mesenchymal stem cells (BM-MSCs) can be induced to differentiate into myogenic cells. Despite their potential,previous studies have not been successful in producing a high percentage of cardiac-like cells with a muscle phenotype. We hypothesized that cardiac lineage development in BM-MSC is related to cell passage,culture milieu,and enrichment for specific cell subtypes before and during differentiation. Our study demonstrated that Lin(-) BM-MSC at an intermediate passage (IP; P8-P12) expressed cardiac troponin T (cTnT) after 21 days in culture. Cardiac TnT expression was similar whether IP cells were differentiated in media containing 5-azacytidine+2% FBS (AZA; 14%) or 2% FBS alone (LS; 12%) and both were significantly higher than AZA+5% FBS. This expression was potentiated by first enriching for CD117/Sca-1 cells followed by differentiation (AZA,39% and LS,28%). A second sequential enrichment for the dihydropyridine receptor subunit alpha2delta1 (DHPR-alpha2) resulted in cardiac TnT expressed in 54% of cultured cells compared to 28% of cells after CD117/Sca-1(+) enrichment. Cells enriched for CD117/Sca-1 and subjected to differentiation displayed spontaneous intracellular Ca(2+) transients with an increase in transient frequency and a 60% decrease in the transient duration amplitude between days 14 and 29. In conclusion,IP CD117/Sca-1(+) murine BM-MSCs display robust cardiac muscle lineage development that can be induced independent of AZA but is diminished under higher serum concentrations. Furthermore,temporal changes in calcium kinetics commensurate with increased cTnT expression suggest progressive maturation of a cardiac muscle lineage. Enrichment with CD117/Sca-1 to establish lineage commitment followed by DHPR-alpha2 in lineage developing cells may enhance the therapeutic potential of these cells for transplantation. View Publication

The detection of growth hormone (GH) and its receptor in germinal regions of the mammalian brain prompted our investigation of GH and its role in the regulation of endogenous neural precursor cell activity. Here we report that the addition of exogenous GH significantly increased the expansion rate in long-term neurosphere cultures derived from wild-type mice,while neurospheres derived from GH null mice exhibited a reduced expansion rate. We also detected a doubling in the frequency of large (i.e. stem cell-derived) colonies for up to 120 days following a 7-day intracerebroventricular infusion of GH suggesting the activation of endogenous stem cells. Moreover,gamma irradiation induced the ablation of normally quiescent stem cells in GH-infused mice,resulting in a decline in olfactory bulb neurogenesis. These results suggest that GH activates populations of resident stem and progenitor cells,and therefore may represent a novel therapeutic target for age-related neurodegeneration and associated cognitive decline. View Publication

BACKGROUND: Recurrence is a common problem in bladder cancer; this has been attributed to cancer stem cells. In this study,we characterized potential cancer stem cell populations isolated from three cell lines that demonstrate different responses to cisplatin. MATERIALS AND METHODS: The ALDEFLUOR® assay was used to isolate cells from TCCSUP,T24,and 5637 cell lines,and these cells were evaluated for their ability to form colonies,differentiate,migrate and invade. RESULTS: The cell lines demonstrate a spectrum of aldehyde dehydrogenase high (ALDH(High)) populations that correlate with resistance to cisplatin. In the two resistant cell lines,T24 and 5637,the ALDH(High) cells demonstrate increased colony formation,migration,invasion,and ability to differentiate. The resistant T24 and 5637 cell lines may serve as models to investigate alternative therapies for bladder cancer. View Publication