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BACKGROUND: Due to the inherent sensitivity of human embryonic stem cells (hESCs) to manipulations,the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be low. Additionally,a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK) inhibitor,Y-27632,previously has been identified as enhancing survival of hESCs upon single-cell dissociation,as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions. METHODOLOGY/PRINCIPAL FINDINGS: HESCs were sorted using markers for SSEA-3,TRA-1-81,and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions,cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically,treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632,hESCs were further analyzed. Specifically,hESCs sorted with and without the addition of Y-27632 retained normal morphology,expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry,and maintained a stable karyotype. In addition,the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions. CONCLUSIONS/SIGNIFICANCE: The application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency,and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types,identification and isolation of stem cell subpopulations,and generation of single cell clones. Finally,these results demonstrate an additional application of ROCK inhibition to hESC research. View Publication

Almost 25 centuries ago,Hippocrates,the father of medicine,proclaimed Let food be thy medicine and medicine be thy food." Exploring the association between diet and health continues today. For example� View Publication

It is increasingly evident that neutrophils are able to cross-talk with other leukocytes to shape ongoing inflammatory and immune responses. In this study,we analyzed whether human NK cells may influence the survival and activation of neutrophils under co-culture conditions. We report that NK cells exposed to either IL-15 or IL-18 alone strongly protect the survival of neutrophils via the release of IFNγ and granulocyte macrophage colony-stimulating factor (GM-CSF) plus IFNγ,respectively,and cause a slight up-regulation of neutrophil CD64 and CD11b expression. In comparison,NK cells exposed to both IL-15 and IL-18 show a lesser ability to increase the survival of neutrophils but can more potently up-regulate CD64 and CD11b expression,as well as induce the de novo surface expression of CD69,in neutrophils. Analysis of the events occurring in neutrophil/NK co-cultures exposed to IL-15 plus IL-18 revealed that (i) neutrophil survival is positively affected by NK-derived GM-CSF but negatively influenced by a CD18-dependent neutrophil/NK contact,(ii) NK-derived IFNγ is almost entirely responsible for the induction of CD64,(iii) both soluble factors (primarily GM-CSF) and direct cell-cell contact up-regulate CD11b and CD69 and (iv) NK-derived GM-CSF induces the expression of biologically active heparin-binding EGF-like growth factor (HB-EGF) in neutrophils. Finally,we demonstrate that NK cells can also express HB-EGF when stimulated with either IL-2 or IL-15,yet independently of endogenous GM-CSF. Altogether,our results define a novel interaction within the innate immune system whereby NK cells,by directly modulating neutrophil functions,might contribute to the pathogenesis of inflammatory diseases. View Publication

The development of acquired resistance to trastuzumab remains a prevalent challenge in the treatment of patients whose tumors express human epidermal growth factor 2 (HER2). We previously reported that HER2 overexpressing breast cancers are dependent on Y-box binding protein-1 (YB-1) for growth and survival. As YB-1 is also linked to drug resistance in other types of cancer,we address its possible role in trastuzumab insensitivity. Employing an in vivo model of acquired resistance,we demonstrate that resistant cell lines have elevated levels of P-YB-1(S102) and its activating kinase P-RSK and these levels are sustained following trastuzumab treatment. Further,to demonstrate the importance of YB-1 in mediating drug resistance,the expression of the active mutant YB-1(S102D) rendered the BT474 cell line insensitive to trastuzumab. Questioning the role of tumor-initiating cells (TIC) and their ability to escape cancer therapies,we investigate YB-1's role in inducing the cancer stem cell marker CD44. Notably,the resistant cells express more CD44 mRNA and protein compared with BT474 cells,which correlated with increased mammosphere formation. Expression of YB-1(S102D) in the BT474 cells increase CD44 protein levels,resulting in enhanced mammosphere formation. Further,exposing BT474 cells to trastuzumab selected for a resistant sub-population enriched for CD44. Conversely,small intefering RNA inhibition of CD44 restored trastuzumab sensitivity in the resistant cell lines. Our findings provide insight on a novel mechanism employed by tumor cells to acquire the ability to escape the effects of trastuzumab and suggest that targeting YB-1 may overcome resistance by eliminating the unresponsive TIC population,rendering the cancer sensitive to therapy. View Publication

Tumor-initiating cells (TICs) are defined by their ability to form tumors after xenotransplantation in immunodeficient mice and appear to be relatively rare in most human cancers. Recent data in melanoma indicate that the frequency of TICs increases dramatically via more permissive xenotransplantation conditions,raising the possibility that the true frequency of TICs has been greatly underestimated in most human tumors. We compared the growth of human pancreatic,non-small cell lung,and head and neck carcinomas in NOD/SCID and NSG mice. Although TIC frequency was detected up to 10-fold higher in NSG mice,it remained low (textless1 in 2500 cells) in all cases. Moreover,aldehyde dehydrogenase-positive (ALDH(+)) and CD44(+)CD24(+) cells,phenotypically distinct cells enriched in TICs,were equally tumorigenic in NOD/SCID and NSG mice. Our findings demonstrate that TICs are rare in these cancers and that the identification of TICs and their frequency in other human malignancies should be validated via primary tumors and highly permissive xenotransplantation conditions. View Publication

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CRKL (CRK-like) is an adapter protein predominantly phosphorylated in cells that express the tyrosine kinase p210(BCR-ABL),the fusion product of a (9;22) chromosomal translocation causative for chronic myeloid leukemia. It has been unclear,however,whether CRKL plays a functional role in p210(BCR-ABL) transformation. Here,we show that CRKL is required for p210(BCR-ABL) to support interleukin-3-independent growth of myeloid progenitor cells and long-term outgrowth of B-lymphoid cells from fetal liver-derived hematopoietic progenitor cells. Furthermore,a synthetic phosphotyrosyl peptide that binds to the CRKL SH2 domain with high affinity blocks association of endogenous CRKL with the p210(BCR-ABL) complex and reduces c-MYC levels in K562 human leukemic cells as well as in mouse hematopoietic cells transformed by p210(BCR-ABL) or the imatinib-resistant mutant T315I. These results indicate that the function of CRKL as an adapter protein is essential for p210(BCR-ABL)-induced transformation. View Publication

Tumor angiogenesis is essential for malignant growth and metastasis. Bone marrow (BM)-derived endothelial progenitor cells (EPC) contribute to angiogenesis-mediated tumor growth. EPC ablation can reduce tumor growth; however,the lack of a marker that can track EPCs from the BM to tumor neovasculature has impeded progress in understanding the molecular mechanisms underlying EPC biology. Here,we report the use of transgenic mouse and lentiviral models to monitor the BM-derived compartment of the tumor stroma; this approach exploits the selectivity of the transcription factor inhibitor of DNA binding 1 (Id1) for EPCs to track EPCs in the BM,blood,and tumor stroma,as well as mature EPCs. Acute ablation of BM-derived EPCs using Id1-directed delivery of a suicide gene reduced circulating EPCs and yielded significant defects in angiogenesis-mediated tumor growth. Additionally,use of the Id1 proximal promoter to express microRNA-30-based short hairpin RNA inhibited the expression of critical EPC-intrinsic factors,confirming that signaling through vascular endothelial growth factor receptor 2 is required for EPC-mediated tumor biology. By exploiting the selectivity of Id1 gene expression in EPCs,our results establish a strategy to track and target EPCs in vivo,clarifying the significant role that EPCs play in BM-mediated tumor angiogenesis. View Publication

In mammals DNA methylation occurs at position 5 of cytosine in a CpG context and regulates gene expression. It plays an important role in diseases and inhibitors of DNA methyltransferases (DNMTs)--the enzymes responsible for DNA methylation--are used in clinics for cancer therapy. The most potent inhibitors are 5-azacytidine and 5-azadeoxycytidine. Zebularine (1-(beta-D-ribofuranosyl)-2(1H)- pyrimidinone) is another cytidine analog described as a potent inhibitor that acts by forming a covalent complex with DNMT when incorporated into DNA. Here we bring additional experiments to explain its mechanism of action. First,we observe an increase in the DNA binding when zebularine is incorporated into the DNA,compared to deoxycytidine and 5-fluorodeoxycytidine,together with a strong decrease in the dissociation rate. Second,we show by denaturing gel analysis that the intermediate covalent complex between the enzyme and the DNA is reversible,differing thus from 5-fluorodeoxycytidine. Third,no methylation reaction occurs when zebularine is present in the DNA. We confirm that zebularine exerts its demethylation activity by stabilizing the binding of DNMTs to DNA,hindering the methylation and decreasing the dissociation,thereby trapping the enzyme and preventing turnover even at other sites. View Publication

It has recently been shown that nucleosome distribution,histone modifications and RNA polymerase II (Pol II) occupancy show preferential association with exons (exon-intron marking")� View Publication

BACKGROUND The development of novel influenza vaccines inducing a broad immune response is an important objective. The aim of this study was to evaluate live vaccines which induce both strong humoral and cell-mediated immune responses against the novel human pandemic H1N1 influenza virus,and to show protection in a lethal animal challenge model. METHODOLOGY/PRINCIPAL FINDINGS For this purpose,the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A/California/07/2009 (H1N1) strain (CA/07) were inserted into the replication-deficient modified vaccinia Ankara (MVA) virus--a safe poxviral live vector--resulting in MVA-H1-Ca and MVA-N1-Ca vectors. These live vaccines,together with an inactivated whole virus vaccine,were assessed in a lung infection model using immune competent Balb/c mice,and in a lethal challenge model using severe combined immunodeficient (SCID) mice after passive serum transfer from immunized mice. Balb/c mice vaccinated with the MVA-H1-Ca virus or the inactivated vaccine were fully protected from lung infection after challenge with the influenza H1N1 wild-type strain,while the neuraminidase virus MVA-N1-Ca induced only partial protection. The live vaccines were already protective after a single dose and induced substantial amounts of neutralizing antibodies and of interferon-gamma-secreting (IFN-gamma) CD4- and CD8 T-cells in lungs and spleens. In the lungs,a rapid increase of HA-specific CD4- and CD8 T cells was observed in vaccinated mice shortly after challenge with influenza swine flu virus,which probably contributes to the strong inhibition of pulmonary viral replication observed. In addition,passive transfer of antisera raised in MVA-H1-Ca vaccinated immune-competent mice protected SCID mice from lethal challenge with the CA/07 wild-type virus. CONCLUSIONS/SIGNIFICANCE The non-replicating MVA-based H1N1 live vaccines induce a broad protective immune response and are promising vaccine candidates for pandemic influenza. View Publication

The ability of human embryonic stem cells (hESCs) to differentiate into essentially all somatic cell types has made them a valuable tool for studying human development and has positioned them for broad applications in toxicology,regenerative medicine,and drug discovery. This unit describes a protocol for the large-scale expansion and maintenance of hESCs in vitro. hESC cultures must maintain a balance between the cellular states of pluripotency and differentiation; thus,researchers must use care when growing these technically demanding cells. The culture system is based largely on the use of a proprietary serum-replacement product and basic fibroblast growth factor (bFGF),with mouse embryonic fibroblasts as a feeder layer. These conditions provide the basis for relatively inexpensive maintenance and expansion of hESCs,as well as their engineered counterparts,human induced pluripotent stem cells (hiPSCs). View Publication