技术资料

Ischemia-induced progenitor cell proliferation is a prominent example of the adult mammalian brain's ability to regenerate injured tissue resulting from pathophysiological processes. In order to better understand and exploit the cell signaling mechanisms that regulate ischemia-induced proliferation,we examined the role of the p42/44 mitogen-activated protein kinase (MAPK) cascade effector ribosomal S6 kinase (RSK) in this process. Here,using the endothelin-1 ischemia model in wild type mice,we show that the activated form of RSK is expressed in the progenitor cells of the subgranular zone (SGZ) after intrahippocampal cerebral ischemia. Further,RSK inhibition significantly reduces ischemia-induced SGZ progenitor cell proliferation. Using the neurosphere assay,we also show that both SGZ- and subventricular zone (SVZ)-derived adult neural stem cells (NSC) exhibit a significant reduction in proliferation in the presence of RSK and MAPK inhibitors. Taken together,these data reveal RSK as a regulator of ischemia-induced progenitor cell proliferation,and as such,suggest potential therapeutic value may be gained by specifically targeting the regulation of RSK in the progenitor cell population of the SGZ. View Publication

OBJECTIVE To analyze the combination therapy of Sinomenine (SIN) and Methotrexate (MTX) in rheumatoid arthritis (RA),we herein demonstrated the combination effect of SIN and MTX on collagen-induced arthritis (CIA) in rats through their modulation on osteoclast-related cytokines. METHODS CIA was induced by the immunization of type II collagen (CII) in SD rats. SIN and MTX were administrated alone or in combination after the onset of arthritis. Arthritis index and histological analysis were used to evaluate the effect of treatments. Effects of SIN and MTX on expression of receptor activator of NF-κB ligand (RANKL) and osteopontin (OPN) in synovial tissues were assayed by immunohistochemistry. RANKL,osteoprotegerin (OPG),IL-6,IL-17 and matrix metalloproteinases (MMPs) in rat serum were measured by ELISA. The expression of osteoclast-related cytokines in fibroblast-like synoviocytes (FLS) from RA patients was assayed by RT-PCR. RESULTS SIN and MTX combination additively reduced the inflammatory symptoms and joint damage in CIA. Combination of SIN and MTX significantly repressed synovial RANKL and OPN production. SIN and MTX exhibited complementary and synergistic effect upon down-regulating RANKL,IL-6,IL-17 and MMPs in rat serum. SIN and MTX also modulated the expression of RANKL and OPG in RA-FLS. CONCLUSION SIN and MTX have additive effects,decreasing inflammation and joint damage in CIA rats by modulating osteoclast-related cytokines. These results are indicative of the combined effect of SIN and MTX for anti-arthritic treatment in RA. View Publication

Summary Human embryonic stem cells (hESCs) regularly acquire nonrandom genomic aberrations during culture,raising concerns about their safe therapeutic application. The International Stem Cell Initiative identified a copy number variant (CNV) amplification of chromosome 20q11.21 in 25% of hESC lines displaying a normal karyotype. By comparing four cell lines paired for the presence or absence of this CNV,we show that those containing this amplicon have higher population doubling rates,attributable to enhanced cell survival through resistance to apoptosis. Of the three genes encoded within the minimal amplicon and expressed in hESCs,only overexpression of BCL2L1 (BCL-XL isoform) provides control cells with growth characteristics similar to those of CNV-containing cells,whereas inhibition of BCL-XL suppresses the growth advantage of CNV cells,establishing BCL2L1 as a driver mutation. Amplification of the 20q11.21 region is also detectable in human embryonal carcinoma cell lines and some teratocarcinomas,linking this mutation with malignant transformation. View Publication

Although transcriptional and posttranscriptional events are detected in RNA-Seq data from second-generation sequencing,full-length mRNA isoforms are not captured. On the other hand,third-generation sequencing,which yields much longer reads,has current limitations of lower raw accuracy and throughput. Here,we combine second-generation sequencing and third-generation sequencing with a custom-designed method for isoform identification and quantification to generate a high-confidence isoform dataset for human embryonic stem cells (hESCs). We report 8,084 RefSeq-annotated isoforms detected as full-length and an additional 5,459 isoforms predicted through statistical inference. Over one-third of these are novel isoforms,including 273 RNAs from gene loci that have not previously been identified. Further characterization of the novel loci indicates that a subset is expressed in pluripotent cells but not in diverse fetal and adult tissues; moreover,their reduced expression perturbs the network of pluripotency-associated genes. Results suggest that gene identification,even in well-characterized human cell lines and tissues,is likely far from complete. View Publication

The cyclooxygenase pathway is strongly implicated in breast cancer progression but the role of this pathway in the biology of breast cancer stem/progenitor cells has not been defined. Recent attention has focused on targeting the cyclooxygenase 2 (COX-2) pathway downstream of the COX-2 enzyme by blocking the activities of individual prostaglandin E (EP) receptors. Prostaglandin E receptor 4 (EP4) is widely expressed in primary invasive ductal carcinomas of the breast and antagonizing this receptor with small molecule inhibitors or shRNA directed to EP4 inhibits metastatic potential in both syngeneic and xenograft models. Breast cancer stem/progenitor cells are defined as a subpopulation of cells that drive tumor growth,metastasis,treatment resistance,and relapse. Mammosphere-forming breast cancer cells of human (MDA-MB-231,SKBR3) or murine (66.1,410.4) origin of basal-type,Her-2 phenotype and/or with heightened metastatic capacity upregulate expression of both EP4 and COX-2 and are more tumorigenic compared to the bulk population. In contrast,luminal-type or non-metastatic counterparts (MCF7,410,67) do not increase COX-2 and EP4 expression in mammosphere culture. Treatment of mammosphere-forming cells with EP4 inhibitors (RQ-15986,AH23848,Frondoside A) or EP4 gene silencing,but not with a COX inhibitor (Indomethacin) reduces both mammosphere-forming capacity and the expression of phenotypic markers (CD44(hi)/CD24(low),aldehyde dehydrogenase) of breast cancer stem cells. Finally,an orally delivered EP4 antagonist (RQ-08) reduces the tumor-initiating capacity and markedly inhibits both the size of tumors arising from transplantation of mammosphere-forming cells and phenotypic markers of stem cells in vivo. These studies support the continued investigation of EP4 as a potential therapeutic target and provide new insight regarding the role of EP4 in supporting a breast cancer stem cell/tumor-initiating phenotype. View Publication

Cancer-initiating cells (CICs) that are responsible for tumor initiation,propagation,and resistance to standard therapies have been isolated from human solid tumors,including colorectal cancer (CRC). The aim of this study was to obtain an immunological profile of CRC-derived CICs and to identify CIC-associated target molecules for T cell immunotherapy. We have isolated cells with CIC properties along with their putative non-CIC autologous counterparts from human primary CRC tissues. These CICs have been shown to display tumor-initiating/stemness" properties� View Publication

Resistance to chemotherapeutic treatment,which is indirectly responsible for many cancer deaths,is normally associated with an aggressive phenotype including increased cell motility and acquisition of invasive properties. Here we describe how breast cancer cells overcome doxorubicin-induced senescence and become drug resistant by overexpression of the microRNA (miR)-106b˜25 cluster. Although all three miRs in the cluster contribute to the generation of doxorubicin resistance,miR-25 is the major contributor to this phenotype. All three miRs in this cluster target EP300,a transcriptional activator of E-cadherin,resulting in cells acquiring a phenotype characteristic of cells undergoing epithelial-to-mesenchymal transition (EMT),including an increase in both cell motility and invasion,as well as the ability to proliferate after treatment with doxorubicin. These findings provide a novel drug resistance/EMT regulatory pathway controlled by the miR-106b˜25 cluster by targeting a transcriptional activator of E-cadherin. View Publication

We previously demonstrated that leukemia cell lines expressing CD44 and hematopoietic progenitor cells (HPC) from umbilical cord blood (CB) showed rolling on hyaluronic acid (HA)-coated surfaces under physiological shear stress. In the present study,we quantitatively assessed the interaction of HPC derived from CB,mobilized peripheral blood (mPB) and bone marrow (BM) from healthy donors,as well as primary leukemia blasts from PB and BM of patients with acute myeloid leukemia (AML) with HA. We have demonstrated that HPC derived from healthy donors showed relative homogeneous rolling and adhesion to HA. In contrast,highly diverse behavioral patterns were found for leukemia blasts under identical conditions. The monoclonal CD44 antibody (clone BU52) abrogated the shear stress-induced rolling of HPC and leukemia blasts,confirming the significance of CD44 in this context. On the other hand,the immobile adhesion of leukemia blasts to the HA-coated surface was,in some cases,not or incompletely inhibited by BU52. The latter property was associated with non-responsiveness to induction chemotherapy and subsequently poor clinical outcome. View Publication

A human invitro cardiac tissue model would be a significant advancement for understanding,studying,and developing new strategies for treating cardiac arrhythmias and related cardiovascular diseases. We developed an invitro model of three-dimensional (3D) human cardiac tissue by populating synthetic filamentous matrices with cardiomyocytes derived from healthy wild-type volunteer (WT) and patient-specific long QT syndrome type 3 (LQT3) induced pluripotent stem cells (iPS-CMs) to mimic the condensed and aligned human ventricular myocardium. Using such a highly controllable cardiac model,we studied the contractility malfunctions associated with the electrophysiological consequences of LQT3 and their response to a panel of drugs. By varying the stiffness of filamentous matrices,LQT3 iPS-CMs exhibited different level of contractility abnormality and susceptibility to drug-induced cardiotoxicity. textcopyright 2013 Elsevier Ltd. View Publication

In February 2013,zoonotic transmission of a novel influenza A virus of the H7N9 subtype was reported in China. Although at present no sustained human-to-human transmission has been reported,a pandemic outbreak of this H7N9 virus is feared. Since neutralizing antibodies to the hemagglutinin (HA) globular head domain of the virus are virtually absent in the human population,there is interest in identifying other correlates of protection,such as cross-reactive CD8(+) T cells (cytotoxic T lymphocytes [CTLs]) elicited during seasonal influenza A virus infections. These virus-specific CD8(+) T cells are known to recognize conserved internal proteins of influenza A viruses predominantly,but it is unknown to what extent they cross-react with the newly emerging H7N9 virus. Here,we assessed the cross-reactivity of seasonal H3N2 and H1N1 and pandemic H1N1 influenza A virus-specific polyclonal CD8(+) T cells,obtained from HLA-typed study subjects,with the novel H7N9 virus. The cross-reactivity of CD8(+) T cells to H7N9 variants of known influenza A virus epitopes and H7N9 virus-infected cells was determined by their gamma interferon (IFN-γ) response and lytic activity. It was concluded that,apart from recognition of individual H7N9 variant epitopes,CD8(+) T cells to seasonal influenza viruses display considerable cross-reactivity with the novel H7N9 virus. The presence of these cross-reactive CD8(+) T cells may afford some protection against infection with the new virus. View Publication

Human embryonic stem cells (hESCs) were used as a model system of human pancreas development to study characteristics of the polyhormonal cells that arise during fetal pancreas development. HESCs were differentiated into fetal-like pancreatic cells in vitro using a 33-day,7-stage protocol. Cultures were ˜90-95% PDX1-positive by day (d) 11 and 70-75% NKX6.1-positive by d17. Polyhormonal cells were scattered at d17,but developed into islet-like clusters that expressed key transcription factors by d33. Human C-peptide and glucagon secretion were first detected at d17 and increased thereafter in parallel with INS and GCG transcript levels. HESC-derived cells were responsive to KCl and arginine,but not glucose in perifusion studies. Compared to adult human islets,hESC-derived cells expressed ˜10-fold higher levels of glucose transporter 1 (GLUT1) mRNA,but similar levels of glucokinase (GCK). In situ hybridization confirmed the presence of GLUT1 transcript within endocrine cells. However,GLUT1 protein was excluded from this population and was instead observed predominantly in non-endocrine cells,whereas GCK was co-expressed in insulin-positive cells. In rubidium efflux assays,hESC-derived cells displayed mild potassium channel activity,but no responsiveness to glucose,metabolic inhibitors or glibenclamide. Western blotting experiments revealed that the higher molecular weight SUR1 band was absent in hESC-derived cells,suggesting a lack of functional KATP channels at the cell surface. In addition,KATP channel subunit transcript levels were not at a 1:1 ratio,as would be expected (SUR1 levels were ˜5-fold lower than KIR6.2). Various ratios of SUR1:KIR6.2 plasmids were transfected into COSM6 cells and rubidium efflux was found to be particularly sensitive to a reduction in SUR1. These data suggest that an impaired ratio of SUR1:KIR6.2 may contribute to the observed KATP channel defects in hESC-derived islet endocrine cells,and along with lack of GLUT1,may explain the absence of glucose-stimulated insulin secretion.?? 2013 Elsevier B.V. View Publication

PURPOSE: Retinal pigmented epithelium derived from human induced pluripotent stem (iPS) cells (iPS-RPE) may be a source of cells for transplantation. For this reason,it is essential to determine the functional competence of iPS-RPE. One key role of the RPE is uptake and processing of retinoids via the visual cycle. The purpose of this study is to investigate the expression of visual cycle proteins and the functional ability of the visual cycle in iPS-RPE.$$n$$nMETHODS: iPS-RPE was derived from human iPS cells. Immunocytochemistry,RT-PCR,and Western blot analysis were used to detect expression of RPE genes lecithin-retinol acyl transferase (LRAT),RPE65,cellular retinaldehyde-binding protein (CRALBP),and pigment epithelium-derived factor (PEDF). All-trans retinol was delivered to cultured cells or whole cell homogenate to assess the ability of the iPS-RPE to process retinoids.$$n$$nRESULTS: Cultured iPS-RPE expresses visual cycle genes LRAT,CRALBP,and RPE65. After incubation with all-trans retinol,iPS-RPE synthesized up to 2942 ± 551 pmol/mg protein all-trans retinyl esters. Inhibition of LRAT with N-ethylmaleimide (NEM) prevented retinyl ester synthesis. Significantly,after incubation with all-trans retinol,iPS-RPE released 188 ± 88 pmol/mg protein 11-cis retinaldehyde into the culture media.$$n$$nCONCLUSIONS: iPS-RPE develops classic RPE characteristics and maintains expression of visual cycle proteins. The results of this study confirm that iPS-RPE possesses the machinery to process retinoids for support of visual pigment regeneration. Inhibition of all-trans retinyl ester accumulation by NEM confirms LRAT is active in iPS-RPE. Finally,the detection of 11-cis retinaldehyde in the culture medium demonstrates the cells' ability to process retinoids through the visual cycle. This study demonstrates expression of key visual cycle machinery and complete visual cycle activity in iPS-RPE. View Publication