技术资料

The fabrication of high-density polymer microarray is described,allowing the simultaneous and efficient evaluation of more than 7000 different polymers in a single-cellular-based screen. These high-density polymer arrays are applied in the search for synthetic substrates for hESCs culture. Up-scaling of the identified hit polymers enables long-term cellular cultivation and promoted successful stem-cell maintenance. View Publication

The airway epithelium constitutes an essential immunological and cytoprotective barrier to inhaled insults,such as cigarette smoke,environmental particles,or viruses. Although bronchial epithelial integrity is crucial for airway homeostasis,defective epithelial barrier function contributes to chronic obstructive pulmonary disease (COPD). Tight junctions at the apical side of epithelial cell-cell contacts determine epithelial permeability. Cigarette smoke exposure,the major risk factor for COPD,is suggested to impair tight junction integrity; however,detailed mechanisms thereof remain elusive. We investigated whether cigarette smoke extract (CSE) and transforming growth factor (TGF)-$$1 affected tight junction integrity. Exposure of human bronchial epithelial cells (16HBE14o(-)) and differentiated primary human bronchial epithelial cells (pHBECs) to CSE significantly disrupted tight junction integrity and barrier function. Specifically,CSE decreased transepithelial electrical resistance (TEER) and tight junction-associated protein levels. Zonula occludens (ZO)-1 and ZO-2 protein levels were significantly reduced and dislocated from the cell membrane,as observed by fractionation and immunofluorescence analysis. These findings were reproduced in isolated bronchi exposed to CSE ex vivo,as detected by real-time quantitative reverse-transcriptase PCR and immunohistochemistry. Combined treatment of 16HBE14o(-) cells or pHBECs with CSE and TGF-$$1 restored ZO-1 and ZO-2 levels. TGF-$$1 cotreatment restored membrane localization of ZO-1 and ZO-2 protein and prevented CSE-mediated TEER decrease. In conclusion,CSE led to the disruption of tight junctions of human bronchial epithelial cells,and TGF-$$1 counteracted this CSE-induced effect. Thus,TGF-$$1 may serve as a protective factor for bronchial epithelial cell homeostasis in diseases such as COPD. View Publication

Many studies have compared the genetic and epigenetic profiles of human induced pluripotent stem cells (hiPSCs) to human embryonic stem cells (hESCs) and yet the picture remains unclear. To address this,we derived a population of neural precursor cells (NPCs) from the H1 (WA01) hESC line and generated isogenic iPSC lines by reprogramming. The gene expression and methylation profile of three lines were compared to the parental line and intermediate NPC population. We found no gene probe with expression that differed significantly between hESC and iPSC samples under undifferentiated or differentiated conditions. Analysis of the global methylation pattern also showed no significant difference between the two PSC populations. Both undifferentiated populations were distinctly different from the intermediate NPC population in both gene expression and methylation profiles. One point to note is that H1 is a male line and so extrapolation to female lines should be cautioned. However,these data confirm our previous findings that there are no significant differences between hESCs and hiPSCs at the gene expression or methylation level. View Publication

Recurrent chromosomal alterations have been repeatedly reported in cultured human embryonic stem cells (hESCs). The effects of these alterations on the capability of pluripotent cells to differentiate and on growth potential of their specific differentiated derivatives remain unclear. Here,we report that the hESC lines HUES-7 and -9 carrying multiple chromosomal alterations produce in vitro mesenchymal stem cells (MSCs) that show progressive growth arrest and enter senescence after 15 and 16 passages,respectively. There was no difference in their proliferative potential when compared with bone marrow-derived MSCs. Array comparative genomic hybridization analysis (aCGH) of hESCs and their mesenchymal derivatives revealed no significant differences in chromosomal alterations,suggesting that genetically altered hESCs are not selected out during differentiation. Our findings indicate that genetically unstable hESCs maintain their capacity to differentiate in vitro into MSCs,which exhibit an in vitro growth pattern of normal MSCs and not that of transformed cells. View Publication

BACKGROUND Granulocyte colony-stimulating factor (G-CSF) is a pro-inflammatory cytokine that stimulates myeloid stem cell maturation,proliferation,and migration into circulation. Despite being a known growth factor,the impact of G-CSF on solid tumours has not been well examined. G-CSF receptor (G-CSFR) is expressed by some tumours,and thus the aim of this study was to examine the expression and impact of G-CSF and G-CSFR on gastrointestinal tumours. METHODS In this study,G-CSF expression was examined in human gastric and colon tumours and by tumour-derived stromal myofibroblasts and carcinoma cells. G-CSFR expression was examined on carcinoma cells isolated from human tissues. The effects of G-CSF on gastric and colon carcinoma cell proliferation,migration,and signalling were examined. RESULTS G-CSFR was highly expressed in 90% of human gastric and colon carcinomas. G-CSF was also found to be highly produced by stromal myofibroblasts and carcinoma cells. Exposure of carcinoma cells to G-CSF led to increased proliferation and migration,and expansion of a sub-population of carcinoma cells expressing stem-like markers. These processes were dependent on ERK1/2 and RSK1 phosphorylation. CONCLUSIONS These data suggest that the G-CSF/R axis promotes gastric and colorectal cancer development and suggest they are potential tumour targets. View Publication

INTRODUCTION: Upregulation of PI3K/Akt/mTOR signalling in endocrine-resistant breast cancer (BC) has identified mTOR as an attractive target alongside anti-hormones to control resistance. RAD001 (everolimus/Afinitor®),an allosteric mTOR inhibitor,is proving valuable in this setting; however,some patients are inherently refractory or relapse during treatment requiring alternative strategies. Here we evaluate the potential for novel dual mTORC1/2 mTOR kinase inhibitors,exemplified by AZD8055,by comparison with RAD001 in ER + endocrine resistant BC cells. METHODS: In vitro models of tamoxifen (TamR) or oestrogen deprivation resistance (MCF7-X) were treated with RAD001 or AZD8055 alone or combined with anti-hormone fulvestrant. Endpoints included growth,cell proliferation (Ki67),viability and migration,with PI3K/AKT/mTOR signalling impact monitored by Western blotting. Potential ER cross-talk was investigated by immunocytochemistry and RT-PCR. RESULTS: RAD001 was a poor growth inhibitor of MCF7-derived TamR and MCF7-X cells (IC50 ≥1 μM),rapidly inhibiting mTORC1 but not mTORC2/AKT signalling. In contrast AZD8055,which rapidly inhibited both mTORC1 and mTORC2/AKT activity,was a highly effective (P textless0.001) growth inhibitor of TamR (IC50 18 nM) and MCF7-X (IC50 24 nM),and of a further T47D-derived tamoxifen resistant model T47D-tamR (IC50 19 nM). AZD8055 significantly (P textless0.05) inhibited resistant cell proliferation,increased cell death and reduced migration. Furthermore,dual treatment of TamR or MCF7-X cells with AZD8055 plus fulvestrant provided superior control of resistant growth versus either agent alone (P textless0.05). Co-treating with AZD8055 alongside tamoxifen (P textless0.01) or oestrogen deprivation (P textless0.05) also effectively inhibited endocrine responsive MCF-7 cells. Although AZD8055 inhibited oestrogen receptor (ER) ser167 phosphorylation in TamR and MCF7-X,it had no effect on ER ser118 activity or expression of several ER-regulated genes,suggesting the mTOR kinase inhibitor impact was largely ER-independent. The capacity of AZD8055 for ER-independent activity was further evidenced by growth inhibition (IC5018 and 20 nM) of two acquired fulvestrant resistant models lacking ER. CONCLUSIONS: This is the first report demonstrating dual mTORC1/2 mTOR kinase inhibitors have potential to control acquired endocrine resistant BC,even under conditions where everolimus fails. Such inhibitors may prove of particular benefit when used alongside anti-hormonal treatment as second-line therapy in endocrine resistant disease,and also potentially alongside anti-hormones during the earlier endocrine responsive phase to hinder development of resistance. View Publication

Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion,differentiation,migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin,which both localize to stem cell niches in vivo. This matrix allows clonal derivation,clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes. View Publication

Tunneling nanotubes (TnTs) are long,non-adherent,actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study,we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24-48. h; and this effect was most prominent in media conditions (low-serum,hyperglycemic medium) that support TnT formation (1.3-1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs,in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs,which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation,and also lipid raft formation as a potential biomarker for TnT-forming cells. textcopyright 2014 Elsevier Inc. View Publication

The generation of functional retinal pigment epithelium (RPE) is of great therapeutic interest to the field of regenerative medicine and may provide possible cures for retinal degenerative diseases,including age-related macular degeneration (AMD). Although RPE cells can be produced from either embryonic stem cells or induced pluripotent stem cells,direct cell reprogramming driven by lineagedetermining transcription factors provides an immediate route to their generation. By monitoring a human RPE specific Best1::GFP reporter,we report the conversion of human fibroblasts into RPE lineage using defined sets of transcription factors. We found that Best1::GFP positive cells formed colonies and exhibited morphological and molecular features of early stage RPE cells. Moreover,they were able to obtain pigmentation upon activation of Retinoic acid (RA) and Sonic Hedgehog (SHH) signaling pathways. Our study not only established an ideal platform to investigate the transcriptional network regulating the RPE cell fate determination,but also provided an alternative strategy to generate functional RPE cells that complement the use of pluripotent stem cells for disease modeling,drug screening,and cell therapy of retinal degeneration. View Publication

Our previous studies showed that anti-β2M monoclonal antibodies (mAbs) at high doses have direct apoptotic effects on myeloma cells,suggesting that anti-β2M mAbs might be developed as a novel therapeutic agent. In this study,we investigated the ability of the mAbs at much lower concentrations to indirectly kill myeloma cells by utilizing immune effector cells or molecules. Our results showed that anti-β2M mAbs effectively lysed MM cells via antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC),which were correlated with and dependent on the surface expression of β2M on MM cells. The presence of MM bone marrow stromal cells or addition of IL-6 did not attenuate anti-β2M mAb-induced ADCC and CDC activities against MM cells. Furthermore,anti-β2M mAbs only showed limited cytotoxicity toward normal B cells and nontumorous mesenchymal stem cells,indicating that the ADCC and CDC activities of the anti-β2M mAbs were more prone to the tumor cells. Lenalidomide potentiated in vitro ADCC activity against MM cells and in vivo tumor inhibition capacity induced by the anti-β2M mAbs by enhancing the activity of NK cells. These results support clinical development of anti-β2M mAbs,both as a monotherapy and in combination with lenalidomide,to improve MM patient outcome. View Publication

Despite high remission rates after therapy,60% to 70% of patients with acute myeloid leukemia (AML) do not survive 5 years after their initial diagnosis. The main cause of treatment failures may be insufficient eradication of a subpopulation of leukemic stem-like cells (LSC),which are thought to be responsible for relapse by giving rise to more differentiated leukemic progenitors (LP). To address the need for therapeutic targets in LSCs,we compared microRNA (miRNA) expression patterns in highly enriched healthy CD34(+)CD38(-) hematopoietic stem cells (HSC),CD34(+)CD38(-) LSCs,and CD34(+)CD38(+) LPs,all derived from the same patients' bone marrow (BM) specimens. In this manner,we identified multiple differentially expressed miRNAs,in particular miR-126,which was highly expressed in HSCs and increased in LSCs compared with LPs,consistent with a stem-like cell function. High miR-126 expression in AML was associated with poor survival,higher chance of relapse,and expression of genes present in LSC/HSC signatures. Notably,attenuating miR-126 expression in AML cells reduced in vitro cell growth by inducing apoptosis,but did not affect the survival of normal BM in which it instead enhanced expansion of HSCs. Furthermore,targeting miR-126 in LSCs and LPs reduced their clonogenic capacity and eliminated leukemic cells,again in the absence of similar inhibitory effects on normal BM cells. Our results define miR-126 as a therapeutic focus to specifically eradicate LSCs and improve AML outcome. View Publication

Human pluripotent stem cells (PSCs) are critical in vitro tools forbackslashnunderstanding mechanisms that regulate lineage differentiation inbackslashnthe human embryo as well as a potentially unlimited supply of stembackslashncells for regenerative medicine. Pluripotent human and mouse embryonicbackslashnstem cells (ESCs) derived from the inner cell mass of blastocystsbackslashnshare a similar transcription factor network to maintain pluripotencybackslashnand self-renewal,yet there are considerable molecular differencesbackslashnreflecting the diverse environments in which mouse and human ESCsbackslashnare derived. In the current study we evaluated the role of Proteinbackslashnarginine methyltransferase 5 (PRMT5) in human ESC (hESC) self-renewalbackslashnand pluripotency given its critical role in safeguarding mouse ESCbackslashnpluripotency. Unlike the mouse,we discovered that PRMT5 has no rolebackslashnin hESC pluripotency. Using microarray analysis we discovered thatbackslashna significant depletion in PRMT5 RNA and protein from hESCs changedbackslashnthe expression of only 78 genes,with the majority being repressed.backslashnFunctionally,we discovered that depletion of PRMT5 had no effectbackslashnon expression of OCT4,NANOG or SOX2,and did not prevent teratomabackslashnformation. Instead,we show that PRMT5 functions in hESCs to regulatebackslashnproliferation in the self-renewing state by regulating the fractionbackslashnof cells in Gap 1 (G1) of the cell cycle and increasing expressionbackslashnof the G1 cell cycle inhibitor P57. Taken together our data unveilsbackslashna distinct role for PRMT5 in hESCs and identifies P57 as new target. View Publication