技术资料

Neural tissue formation is induced by growth factors that activate networks of signal transduction cascades that ultimately lead to the expression of early neural genes,including transcription factors of the SoxB family. Here,we report that fibroblast growth factor (FGF)-induced Erk1/2 (Mapk3 and Mapk1,respectively) mitogen-activated protein kinase (MAPK),but not phosphatidylinositol 3'-OH kinase (PI3K,Pik3r1),signalling is required for neural specification in mouse embryonic stem (ES) cells and in the chick embryo. Further,blocking Erk1/2 inhibits the onset of key SoxB genes in both mouse ES cells (Sox1) and chick embryos (Sox2 and Sox3) and,in both contexts,Erk1/2 signalling is required during only a narrow time window,as neural specification takes place. In the absence of Erk1/2 signalling,differentiation of ES cells stalls following Fgf5 upregulation. Using differentiating ES cells as a model for neural specification,we demonstrate that sustained Erk1/2 activation controls the transition from an Fgf5-positive,primitive ectoderm-like cell state to a neural progenitor cell state without attenuating bone morphogenetic protein (BMP) signalling and we also define the minimum period of Erk1/2 activity required to mediate this key developmental step. Together,these findings identify a conserved,specific and stage-dependent requirement for Erk1/2 signalling downstream of FGF-induced neural specification in higher vertebrates and provide insight into the signalling dynamics governing this process. View Publication

We determined the embryonic origins of adult forebrain subventricular zone (SVZ) stem cells by Cre-lox fate mapping in transgenic mice. We found that all parts of the telencephalic neuroepithelium,including the medial ganglionic eminence and lateral ganglionic eminence (LGE) and the cerebral cortex,contribute multipotent,self-renewing stem cells to the adult SVZ. Descendants of the embryonic LGE and cortex settle in ventral and dorsal aspects of the dorsolateral SVZ,respectively. Both populations contribute new (5-bromo-2'-deoxyuridine-labeled) tyrosine hydroxylase- and calretinin-positive interneurons to the adult olfactory bulb. However,calbindin-positive interneurons in the olfactory glomeruli were generated exclusively by LGE-derived stem cells. Thus,different SVZ stem cells have different embryonic origins,colonize different parts of the SVZ,and generate different neuronal progeny,suggesting that some aspects of embryonic patterning are preserved in the adult SVZ. This could have important implications for the design of endogenous stem cell-based therapies in the future. View Publication

In the present study,we investigated the in vitro effect of resveratrol (RSVL),a polyphenolic phytoestrogen,on cell proliferation and osteoblastic maturation in human bone marrow-derived mesenchymal stem cell (HBMSC) cultures. RSVL (10(-8)-10(-5) M) increased cell growth dose-dependently,as measured by [(3)H]-thymidine incorporation,and stimulated osteoblastic maturation as assessed by alkaline phosphatase (ALP) activity,calcium deposition into the extracellular matrix,and the expression of osteoblastic markers such as RUNX2/CBFA1,Osterix and Osteocalcin in HBMSCs cell cultures. Further studies found that RSVL (10(-6)M) resulted in a rapid activation of both extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) signaling in HBMSCs cultures. The effects of RSVL were mimicked by 17beta-estrodial (10(-8) M) and were abolished by estrogen receptor (ER) antagonist ICI182780. An ERK1/2 pathway inhibitor,PD98059,significantly attenuated RSVL-induced ERK1/2 phosphorylation,consistent with the reduction of cell proliferation and osteoblastic differentiation as well as expression of osteoblastic markers. In contrast,SB203580,a p38 MAPK pathway blocker,blocked RSVL-induced p38 phosphorylation,but resulted in an increase of cell proliferation and a more osteoblastic maturation. These data suggest that RSVL stimulates HBMSCs proliferation and osteoblastic differentiation through an ER-dependent mechanism and coupling to ERK1/2 activation. View Publication

BACKGROUND: The testing of cord blood (CB) progenitor and stem cell units for transplantation suitability involves enumeration of total nucleated cells before freezing. CD34+ cell counts may also be a means of determining suitability. Studies have been conducted to evaluate how specific storage conditions influence cell counts. STUDY DESIGN AND METHODS: CB units were processed by hydroxyethyl starch volume reduction. Cryopreserved-thawed samples were diluted 1:3 without washing. CD34+ cells were measured with three commercially available assay methods. In specific studies,apoptosis-indicating reagents were included. CB units were analyzed for nucleated cells,aldehyde dehydrogenase-containing cells,and progenitor colonies. RESULTS: CD34+ cell levels and nucleated cells were retained during storage in test tubes at 1 to 6 degrees C for 3 days. Cryopreserved-thawed samples showed a reduction in CD34+ cells relative to prefreeze levels with the largest decrease with the Stem-Kit (Beckman Coulter) restricted gating procedure. Prefreeze samples contained minimal numbers of presumed apoptotic cells detected with 7-aminoactinomycin D or SYTO16,but after cryopreservation-thawing there was an increase. Nucleated cell levels determined with a hematology analyzer or flow cytometry were reduced after thawing. Cryopreservation-thawing reduced the percentage of CD34+ cells positive for the presence of aldehyde dehydrogenase and the number of progenitor colonies. These differences were significant. CONCLUSION: These studies indicate that CD34+ cell counts were maintained when CB samples were stored at 1 to 6 degrees C in test tubes for 3 days. Cryopreservation-thawing resulted in changes in a number of parameters including the percentage of CD34+ cells that were aldehyde dehydrogenase(+) and the number of 7-aminoactinomycin D(+) cells and SYTO16(low) cells. View Publication

The specificities of 65 compounds reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70-80 protein kinases. On the basis of this information,the effects of compounds that we have studied in cells and other data in the literature,we recommend the use of the following small-molecule inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated protein kinase) isoforms,PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases,PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5,Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt),rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)-raptor (regulatory associated protein of mTOR) complex],CT 99021 to inhibit GSK3 (glycogen synthase kinase 3),BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase),D4476 to inhibit CK1 (casein kinase 1),VX680 to inhibit Aurora kinases,and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule inhibitors of protein kinases to assess the physiological roles of these enzymes. Despite being used widely,many of the compounds that we analysed were too non-specific for useful conclusions to be made,other than to exclude the involvement of particular protein kinases in cellular processes. View Publication

OBJECTIVE: Studies in pulmonary arteries (PA) of patients with chronic obstructive pulmonary disease (COPD) suggest that bone marrow-derived endothelial progenitor cells (CD133(+)) may infiltrate the intima and differentiate into smooth muscle cells (SMC). This study aimed to evaluate the plasticity of CD133(+) cells to differentiate into SMC and endothelial cells (EC) in both cell culture and human isolated PA. METHODS: Plasticity of granulocyte-colony stimulator factor (G-CSF)-mobilized peripheral blood CD133(+) cells was assessed in co-cultures with primary lines of human PA endothelial cells (PAEC) or SMC (PASMC) and in isolated human PA. We also evaluated if the phenotype of differentiated progenitor cells was acquired by fusion or differentiation. RESULTS: The in vitro studies demonstrated CD133(+) cells may acquire the morphology and phenotype of the cells they were co-cultured with. CD133(+) cells co-incubated with human isolated PA were able to migrate into the intima and differentiate into SMC. Progenitor cell differentiation was produced without fusion with mature cells. CONCLUSIONS: We provide evidence of plasticity of CD133(+) cells to differentiate into both endothelial cells and SMC,reinforcing the idea of their potential role in the remodeling process of PA in COPD. This process was conducted by transdifferentiation and not by cell fusion. View Publication

Mesenchymal stem cells (MSC) establish close interactions with bone marrow sinusoids in a putative perivascular niche. These vessels contain a large storage pool of mature nonproliferating neutrophils. Here,we have investigated the effects of human bone marrow MSC on neutrophil survival and effector functions. MSC from healthy donors,at very low MSC:neutrophil ratios (up to 1:500),significantly inhibited apoptosis of resting and interleukin (IL)-8-activated neutrophils and dampened N-formyl-l-methionin-l-leucyl-l-phenylalanine (f-MLP)-induced respiratory burst. The antiapoptotic activity of MSC did not require cell-to-cell contact,as shown by transwell experiments. Antibody neutralization experiments demonstrated that the key MSC-derived soluble factor responsible for neutrophil protection from apoptosis was IL-6,which signaled by activating STAT-3 transcription factor. Furthermore,IL-6 expression was detected in MSC by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Finally,recombinant IL-6 was found to protect neutrophils from apoptosis in a dose-dependent manner. MSC had no effect on neutrophil phagocytosis,expression of adhesion molecules,and chemotaxis in response to IL-8,f-MLP,or C5a. These results support the following conclusions: (a) in the bone marrow niche,MSC likely protect neutrophils of the storage pool from apoptosis,preserving their effector functions and preventing the excessive or inappropriate activation of the oxidative metabolism,and (b) a novel mechanism whereby the inflammatory potential of activated neutrophils is harnessed by inhibition of apoptosis and reactive oxygen species production without impairing phagocytosis and chemotaxis has been identified. View Publication

Increasing evidence suggests that the deposition of amyloid plaques,composed primarily of the amyloid-beta protein (Abeta),within the cerebrovasculature is a frequent occurrence in Alzheimer's disease and may play a significant role in disease progression. Accordingly,the pathogenic mechanisms by which Abeta can alter vascular function may have therapeutic implications. Despite observations that Abeta elicits a number of physiological responses in endothelial cells,ranging from alteration of protein expression to cell death,the Abeta species accountable for these responses remains unexplored. In the current study,we show that isolated soluble Abeta aggregation intermediates activate human brain microvascular endothelial cells for both adhesion and subsequent transmigration of monocyte cells in the absence of endothelial cell death and monolayer disruption. In contrast,unaggregated Abeta monomer and mature Abeta fibril fail to induce any change in endothelial adhesion or transmigration. Correlations between average Abeta aggregate size and observed increases in adhesion illustrate that smaller soluble aggregates are more potent activators of endothelium. These results support previous studies demonstrating heightened neuronal activity of soluble Abeta aggregates,including Abeta-derived diffusible ligands,oligomers,and protofibrils,and further show that soluble aggregates also selectively exhibit activity in a vascular cell model. View Publication

The amyloid-beta precursor protein (AbetaPP) is a ubiquitously expressed adhesion and neuritogenic protein whose processing has previously been shown to be regulated by reproductive hormones including the gonadotropin luteinizing hormone (LH) in human neuroblastoma cells. We report for the first time the expression of AbetaPP in human embryonic stem (hES) cells at the mRNA and protein levels. Using N- and C-terminal antibodies against AbetaPP,we detected both the mature and immature forms of AbetaPP as well as truncated variants ( approximately 53kDa,47kDa,and 29kDa) by immunoblot analyses. Expression of AbetaPP is regulated by both the stemness of the cells and pregnancy-associated hormones. Addition of human chorionic gonadotropin,the fetal equivalent of LH that is dramatically elevated during pregnancy,markedly increased the expression of all AbetaPP forms. These results indicate a critical molecular signaling link between the hormonal environment of pregnancy and the expression of AbetaPP in hES cells that is suggestive of an important function for this protein during early human embryogenesis prior to the formation of neural precursor cells. View Publication

BACKGROUND: Ewing's sarcoma cell lines may represent a good in vitro model for the understanding of tumor biology in this heterogeneous group of diseases. In the present study,we report the establishment and characterization of a primary Ewing's sarcoma cell line (LDS-Falck 01). MATERIALS AND METHODS: LDS-Falck 01 was generated from a malignant pleural effusion of a patient with metastatic peripheral primitive neuroectodermal tumor arising from the chest wall. Extensive characterization of the cells was accomplished using immunocytochemical,RT-PCR and cytogenetic studies. RESULTS: In vitro LDS-Falck 01 cells had both anchorage-dependent and -independent growth patterns. Immunocytochemical studies showed that cells were PAS-,vimentin-,CD99- and NSE-positive,EGFR- and CD117-negative. Cytogenetic analysis revealed a complex hyperdiploid karyotype with multiple chromosomal aberrations including an unbalanced translocation t(11;22)(q24;q12). The EWS/FLI1 chimeric transcript type 1 was detected. CONCLUSION: This cell line may represent a valid tool for investigating the biomolecular characteristics of this group of neoplasms and their sensitivity to therapeutic agents. View Publication

We have shown that epigallocatechin-3-gallate (EGCG),a polyphenolic compound from green tea,inhibits growth and induces apoptosis in human pancreatic cancer cells. However,the preclinical potential of EGCG in a suitable mouse model has not been examined. In this study,we examined the molecular mechanisms by which EGCG inhibited growth,invasion,metastasis and angiogenesis of human pancreatic cancer cells in a xenograft model system. EGCG inhibited viability,capillary tube formation and migration of HUVEC,and these effects were further enhanced in the presence of an ERK inhibitor. In vivo,AsPC-1 xenografted tumors treated with EGCG showed significant reduction in volume,proliferation (Ki-67 and PCNA staining),angiogenesis (vWF,VEGF and CD31) and metastasis (MMP-2,MMP-7,MMP-9 and MMP-12) and induction in apoptosis (TUNEL),caspase-3 activity and growth arrest (p21/WAF1). EGCG also inhibited circulating endothelial growth factor receptor 2 (VEGF-R2) positive endothelial cells derived from xenografted mice. Tumor samples from EGCG treated mice showed significantly reduced ERK activity,and enhanced p38 and JNK activities. Overall,our data suggest that EGCG inhibits pancreatic cancer growth,invasion,metastasis and angiogenesis,and thus could be used for the management of pancreatic cancer prevention and treatment. View Publication

Over the past 5 years,the Raf kinase family has emerged as a promising target for protein-directed cancer therapy development. The goal of this review is to first provide a concise summary of the data validating Raf proteins as high-interest therapeutic targets. The authors then outline the mode of action of Raf kinases,emphasizing how Raf activities and protein interactions suggest specific approaches to inhibiting Raf. The authors then summarize the set of drugs,antisense reagents and antibodies available or in development for therapeutically targeting Raf or Raf-related proteins,as well as existing strategies combining these and other therapeutic agents. Finally,the authors discuss recent results from systems biology analyses that have the potential to increasingly guide the intelligent selection of combination therapies involving Raf-targeting agents and other therapeutics. View Publication