搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Large scale purification of endothelial cells is of great interest as it could improve tissue transplantation,reperfusion of ischemic tissues and treatment of pathologies in which an endothelial cell dysfunction exists. In this study,we describe a novel genetic approach that selects for endothelial cells from differentiating embryonic stem (ES) cells. Our strategy is based on the establishment of ES-cell clones that carry an integrated puromycin resistance gene under the control of a vascular endothelium-specific promoter,tie-1. Using EGFP as a reporter gene,we first confirmed the endothelial specificity of the tie-1 promoter in the embryoid body model and in cells differentiated in 2D cultures. Subsequently,tie-1-EGFP ES cells were used as recipients for the tie-1-driven puror transgene. The resulting stable clones were expanded and differentiated for seven days in the presence of VEGF before puromycin selection. As expected,puromycin-resistant cells were positive for EGFP and also expressed several endothelial markers,including CD31,CD34,VEGFR-1,VEGFR-2,Tie-1,VE-cadherin and ICAM-2. Release from the puromycin selection resulted in the appearance of alpha-smooth muscle actin-positive cells. Such cells became more numerous when the population was cultured on laminin-1 or in the presence of TGF-beta1,two known inducers of smooth muscle cell differentiation. The hypothesis that endothelial cells or their progenitors may differentiate towards a smooth muscle cell phenotype was further supported by the presence of cells expressing both CD31 and alpha-smooth muscle actin markers. Finally,we show that purified endothelial cells can incorporate into the neovasculature of transplanted tumors in nude mice. Taken together,these results suggest that application of endothelial lineage selection to differentiating ES cells may become a useful approach for future pro-angiogenic and endothelial cell replacement therapies. View Publication

BACKGROUND There has been increasing interest recently in the plasticity of mesenchymal stem cells (MSCs) and their potential to differentiate into neural lineages. To unravel the roles and effects of different growth factors in the differentiation of MSCs into neural lineages,we have differentiated MSCs into neural lineages using different combinations of growth factors. Based on previous studies of the roles of insulin-like growth factor 1 (IGF-1) in neural stem cell isolation in the laboratory,we hypothesized that IGF-1 can enhance proliferation and reduce apoptosis in neural progenitor-like cells (NPCs) during differentiation of MSCs into NCPs.We induced MSCs differentiation under four different combinations of growth factors: (A) EGF%+%bFGF,(B) EGF%+%bFGF%+%IGF-1,(C) EGF%+%bFGF%+%LIF,(D) EGF%+%bFGF%+%BDNF,and (E) without growth factors,as a negative control. The neurospheres formed were characterized by immunofluorescence staining against nestin,and the expression was measured by flow cytometry. Cell proliferation and apoptosis were also studied by MTS and Annexin V assay,respectively,at three different time intervals (24 hr,3 days,and 5 days). The neurospheres formed in the four groups were then terminally differentiated into neuron and glial cells. RESULTS The four derived NPCs showed a significantly higher expression of nestin than was shown by the negative control. Among the groups treated with growth factors,NPCs treated with IGF-1 showed the highest expression of nestin. Furthermore,NPCs derived using IGF-1 exhibited the highest cell proliferation and cell survival among the treated groups. The NPCs derived from IGF-1 treatment also resulted in a better yield after the terminal differentiation into neurons and glial cells than that of the other treated groups. CONCLUSIONS Our results suggested that IGF-1 has a crucial role in the differentiation of MSCs into neuronal lineage by enhancing the proliferation and reducing the apoptosis in the NPCs. This information will be beneficial in the long run for improving both cell-based and cell-free therapy for neurodegenerative diseases. View Publication

First developed for hematologic disorders,the concept of cancer stem cells (CSCs) was expanded to solid tumors,including colorectal cancer (CRC). The traditional model of colon carcinogenesis includes several steps that occur via mutational activation of oncogenes and inactivation of tumor suppressor genes. Intestinal epithelial cells exist for a shorter amount of time than that required to accumulate tumor-inducing genetic changes,so researchers have investigated the concept that CRC arises from the long-lived stem cells,rather than from the differentiated epithelial cells. Colon CSCs were originally identified through the expression of the CD133 glycoprotein using an antibody directed to its epitope AC133. It is not clear if CD133 is a marker of colon CSCs-other cell surface markers,such as epithelial-specific antigen,CD44,CD166,Musashi-1,CD29,CD24,leucine-rich repeat-containing G-protein-coupled receptor 5,and aldehyde dehydrogenase 1,have been proposed. In addition to initiating and sustaining tumor growth,CSCs are believed to mediate cancer relapse after chemotherapy. How can we identify and analyze colon CSCs and what agents are being designed to kill this chemotherapy-refractory population? View Publication

BACKGROUND Transplantation of endothelial progenitor cells (EPCs) improves neovascularization after ischemia,but patients with coronary artery disease (CAD) or diabetes mellitus show a reduced number of EPCs and impaired functional activity. Therefore,we investigated the effects of risk factors,such as glucose and TNF-alpha,on the number of EPCs in vitro to elucidate the underlying mechanisms. METHODS AND RESULTS EPCs of patients or healthy subjects were isolated from peripheral blood. Incubation with glucose or TNF-alpha dose-dependently reduced the number of EPCs (79.9+/-1.3% and 74.3+/-8.1% of control; Ptextless0.05,respectively). This reduction was not caused by apoptosis. TNF-alpha and glucose induced a dose- and time-dependent activation of the p38 MAP kinase,the downstream kinase mitogen- and stress-activated kinase 1,and the transcription factor cAMP-responsive element-binding protein (CREB),in EPCs. Moreover,EPCs from CAD patients had significantly higher basal p38-phosphorylation levels (1.83+/-0.2-fold increase; Ptextless0.05) compared with healthy subjects. The inhibition of the p38-kinase by SB203580 or infection with a dominant negative p38 kinase adenovirus significantly increased basal number of EPCs (136.7+/-6.3% and 142.9+/-18% versus control,respectively). Likewise,ex vivo cultivation of EPCs from patients with CAD with SB203580 significantly increased the number of EPCs and partially reversed the impaired capacity for neovascularization of EPCs in vivo (relative blood flow: 0.40+/-0.03 versus 0.64+/-0.08,Ptextless0.05). The increased numbers of EPCs by SB203580 were associated with an augmentation of EPC proliferation and a reduction of cells expressing the monocytic marker proteins CD14 and CD64,suggesting that p38 regulates proliferation and differentiation events. CONCLUSIONS These results demonstrate that p38 MAP kinase plays a pivotal role in the signal transduction pathways regulating the number of EPCs ex vivo. SB203580 can prevent the negative effects of TNF-alpha and glucose on the number of EPCs and may be useful to improve the number of EPCs for potential cell therapy. View Publication

Development of resistance to therapy continues to be a serious clinical problem in breast cancer management. Cancer stem/progenitor cells have been shown to play roles in resistance to chemo�? and radiotherapy. Here,we examined their role in the development of resistance to the oestrogen receptor antagonist tamoxifen. Tamoxifen�?resistant cells were enriched for stem/progenitors and expressed high levels of the stem cell marker Sox2. Silencing of the SOX2 gene reduced the size of the stem/progenitor cell population and restored sensitivity to tamoxifen. Conversely,ectopic expression of Sox2 reduced tamoxifen sensitivity in vitro and in vivo. Gene expression profiling revealed activation of the Wnt signalling pathway in Sox2�?expressing cells,and inhibition of Wnt signalling sensitized resistant cells to tamoxifen. Examination of patient tumours indicated that Sox2 levels are higher in patients after endocrine therapy failure,and also in the primary tumours of these patients,compared to those of responders. Together,these results suggest that development of tamoxifen resistance is driven by Sox2�?dependent activation of Wnt signalling in cancer stem/progenitor cells. View Publication

The site-directed recombinase Cre can be employed to delete or express genes in cell lines or animals. Clearly,the ability to control remotely the activity of this enzyme would be highly desirable. To this end we have constructed expression vectors for fusion proteins consisting of the Cre recombinase and a mutated hormone-binding domain of the murine oestrogen receptor. The latter still binds the anti-oestrogen drug tamoxifen but no longer 17 beta-oestradiol. We show here that in embryonic stem cells expressing such fusion proteins,tamoxifen can efficiently induce Cre-mediated recombination,thereby activating a stably integrated LacZ reporter gene. In the presence of either 10 microM tamoxifen or 800 nM 4-hydroxy-tamoxifen,recombination of the LacZ gene is complete within 3-4 days. By placing a tamoxifen-binding domain on both ends of the Cre protein,the enzymatic activity of Cre can be even more tightly controlled. Transgenic mice expressing such an tamoxifen-inducible Cre enzyme may thus provide a new and useful genetic tool to mutate or delete genes at specific times during development or in adult animals. View Publication

The decreased proportion of antigen-inexperienced,na{\{i}}ve T cells is a hallmark of aging in both humans and mice and contributes to reduced immune responses particularly against novel and re-emerging pathogens. Na{\"{i}}ve T cells depend on survival signals received during their circulation among the lymph nodes by direct contacts with stroma in particular fibroblastic reticular cells. Macroscopic changes to the architecture of the lymph nodes have been described but it is unclear how lymph node stroma are altered with age and whether these changes contribute to reduced na{\"{i}}ve T cell maintenance. Here using 2-photon microscopy we determined that the aged lymph node displayed increased fibrosis and correspondingly that na{\"{i}}ve T-cell motility was impaired in the aged lymph node especially in proximity to fibrotic deposition. Functionally adoptively transferred young na{\"{i}}ve T-cells exhibited reduced homeostatic turnover in aged hosts supporting the role of T cell-extrinsic mechanisms that regulate their survival. Further we determined that early development of resident fibroblastic reticular cells was impaired which may correlate to the declining levels of na{\"{i}}ve T-cell homeostatic factors observed in aged lymph nodes. Thus our study addresses the controversy as to whether aging impacts the composition lymph node stroma and supports a model in which impaired differentiation of lymph node fibroblasts and increased fibrosis inhibits the interactions necessary for na{\"{i}}ve T cell homeostasis." View Publication

Recurrent/metastatic head and neck cancer remains a devastating disease with insufficient treatment options. We investigated the MET receptor tyrosine kinase as a novel target for the treatment of head and neck squamous cell carcinoma (HNSCC). MET/phosphorylated MET and HGF expression was analyzed in 121 tissues (HNSCC/normal) by immunohistochemistry,and in 20 HNSCC cell lines by immunoblotting. The effects of MET inhibition using small interfering RNA/two small-molecule inhibitors (SU11274/PF-2341066) on signaling,migration,viability,and angiogenesis were determined. The complete MET gene was sequenced in 66 head and neck cancer tissue samples and eight cell lines. MET gene copy number was determined in 14 cell lines and 23 tumor tissues. Drug combinations of SU11274 with cisplatin or erlotinib were tested in SCC35/HN5 cell lines. Eighty-four percent of the HNSCC samples showed MET overexpression,whereas 18 of 20 HNSCC cell lines (90%) expressed MET. HGF overexpression was present in 45% of HNSCC. MET inhibition with SU11274/PF-2341066 abrogated MET signaling,cell viability,motility/migration in vitro,and tumor angiogenesis in vivo. Mutational analysis of 66 tumor tissues and 8 cell lines identified novel mutations in the semaphorin (T230M/E168D/N375S),juxtamembrane (T1010I/R988C),and tyrosine kinase (T1275I/V1333I) domains (incidence: 13.5%). Increased MET gene copy number was present with textgreater10 copies in 3 of 23 (13%) tumor tissues. A greater-than-additive inhibition of cell growth was observed when combining a MET inhibitor with cisplatin or erlotinib and synergy may be mediated via erbB3/AKT signaling. MET is functionally important in HNSCC with prominent overexpression,increased gene copy number,and mutations. MET inhibition abrogated MET functions,including proliferation,migration/motility,and angiogenesis. MET is a promising,novel target for HNSCC and combination approaches with cisplatin or EGFR inhibitors should be explored. View Publication

We demonstrate an essential role for the proteasome complex in two proteolytic processes required for activation of the transcription factor NF-kappa B. The p105 precursor of the p50 subunit of NF-kappa B is processed in vitro by an ATP-dependent process that requires proteasomes and ubiquitin conjugation. The C-terminal region of p105 is rapidly degraded,leaving the N-terminal p50 domain. p105 processing can be blocked in intact cells with inhibitors of the proteasome or in yeast with proteasome mutants. These inhibitors also block the activation of NF-kappa B and the rapid degradation of I kappa B alpha induced by tumor necrosis factor alpha. Thus,the ubiquitin-proteasome pathway functions not only in the complete degradation of polypeptides,but also in the regulated processing of precursors into active proteins. View Publication