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

A commonly activated signaling cascade in many human malignancies,including glioblastoma multiforme,is the Akt pathway. This pathway can be activated via numerous upstream alterations including genomic amplification of epidermal growth factor receptor,PTEN deletion,or PIK3CA mutations. In this study,we screened phosphatidylinositol 3-kinase/Akt small-molecule inhibitors in an isogenic cell culture system with an activated Akt pathway secondary to a PIK3CA mutation. One small molecule,A-443654,showed the greatest selective inhibition of cells with the mutant phenotype. Based on these findings,this inhibitor was screened in vitro against a panel of glioblastoma multiforme cell lines. All cell lines tested were sensitive to A-443654 with a mean IC(50) of approximately 150 nmol/L. An analogue of A-443654,methylated at a region that blocks Akt binding,was on average 36-fold less active. Caspase assays and dual flow cytometric analysis showed an apoptotic mechanism of cell death. A-443654 was further tested in a rat intracranial model of glioblastoma multiforme. Animals treated intracranially with polymers containing A-443654 had significantly extended survival compared with control animals; animals survived 79% and 43% longer than controls when A-443654-containing polymers were implanted simultaneously or in a delayed fashion,respectively. This small molecule also inhibited glioblastoma multiforme stem-like cells with similar efficacy compared with traditionally cultured glioblastoma multiforme cell lines. These results suggest that local delivery of an Akt small-molecule inhibitor is effective against experimental intracranial glioma,with no observed resistance to glioblastoma multiforme cells grown in stem cell conditions. View Publication

Neural progenitor cell (NPC) migration is an essential process for brain development,adult neurogenesis,and neuroregeneration after brain injury. Stromal cell-derived factor-1 (SDF-1,CXCL12) and its traditional receptor CXCR4 are well known to regulate NPC migration. However,the discovery of CXCR7,a newly identified CXCL12 receptor,adds to the dynamics of the existing CXCL12/CXCR4 pair. Antagonists for either CXCR4 or CXCR7 blocked CXCL12-mediated NPC migration in a transwell chemotaxis assay,suggesting that both receptors are required for CXCL12 action. We derived NPC cultures from Cxcr4 knockout (KO) mice and used transwell and stripe assays to determine the cell migration. NPCs derived from Cxcr4 KO mice polarized and migrated in response to CXCL12 gradient,suggesting that CXCR7 could serve as an independent migration receptor. Furthermore,Cxcr4 KO NPCs transplanted into the adult mouse striatum migrated in response to the adjacent injection of CXCL12,an effect that was blocked by a CXCR7 antagonist,suggesting that CXCR7 also mediates NPC migration in vivo. Molecular mechanism studies revealed that CXCR7 interact with Rac1 in the leading edge of the polarized NPCs in the absence of CXCR4. Both CXCR7 and Rac1 are required for extracellular signal-regulated kinases (ERK) 1/2 activation and subsequent NPC migration,indicating that CXCR7 could serve as a functional receptor in CXCL12-mediated NPC migration independent of CXCR4. Together these results reveal an essential role of CXCR7 for CXCL12-mediated NPC migration that will be important to understand neurogenesis during development and in adulthood. View Publication

Although human induced pluripotent stem cells (hiPSCs) hold great potential for the study of human diseases affecting disparate cell types,they have been underutilized in seeking mechanistic insights into the pathogenesis of congenital craniofacial disorders. Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder caused by mutations in EFNB1 and characterized by craniofacial,skeletal,and neurological anomalies. Heterozygous females are more severely affected than hemizygous males,a phenomenon termed cellular interference that involves mosaicism for EPHRIN-B1 function. Although the mechanistic basis for cellular interference in CFNS has been hypothesized to involve Eph/ephrin-mediated cell segregation,no direct evidence for this has been demonstrated. Here,by generating hiPSCs from CFNS patients,we demonstrate that mosaicism for EPHRIN-B1 expression induced by random X inactivation in heterozygous females results in robust cell segregation in human neuroepithelial cells,thus supplying experimental evidence that Eph/ephrin-mediated cell segregation is relevant to pathogenesis in human CFNS patients. View Publication

BIX01294 (Bix) is known to be a euchromatic histone-lysine N-methyltransferase 2 inhibitor and treatment with Bix suppresses cancer cell survival and proliferation. In the present study,it was observed that sequential treatment with low-dose Bix notably increases glioblastoma cell migration and metastasis. It was demonstrated that U251 cells sequentially treated with low-dose Bix exhibited induced characteristic changes in critical epithelial-mesenchymal transition (EMT) markers,including E-cadherin,N-cadherin,β-catenin and zinc finger protein SNAI2. Notably,sequential treatment with Bix also increased the expression of cancer stem cell-associated markers,including sex determining region Y-box 2,octamer-binding transcription factor 4 and cluster of differentiation 133. Neurosphere formation was significantly enhanced in cells sequentially treated with Bix,compared with control cells (control: P=0.011; single treatment of Bix,P=0.045). The results of the present study suggest that accumulation of low-dose Bix enhanced the migration and metastatic potential of glioblastoma cells by regulating EMT-associated gene expression,which may be the cause of the altered properties of glioblastoma stem cells. View Publication

Glycolipids are amphipathic molecules which are highly expressed on cell membranes in skin and brain where they mediate several key cellular processes. Neural stem cells are defined as undifferentiated,proliferative,multipotential cells with extensive self-renewal and are responsive to brain injury. Di-rhamnolipid: α-L-rhamnopyranosyl-(1-2)α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,also referred to as di-rhamnolipid BAC-3,is a glycolipid isolated from the bacteria Pseudomonas aeruginosa. In the previous studies,di-rhamnolipid enhanced dermal tissue healing and regeneration. The present study provides the first assessment of di-rhamnolipid,and glycolipid biosurfactants in general,on the nervous system. Treatment of neural stem cells isolated from the lateral ventricle of adult mice and cultured in defined media containing growth factors at 0.5 and 1 μg/ml of di-rhamnolipid increased the number of neurospheres (2.7- and 2.8-fold,respectively) compared to controls and this effect remained even after passaging in the absence of di-rhamnolipid. In addition,neural stem cells treated with di-rhamnolipid at 50 and 100 μg/ml in defined media supplemented with fetal calf serum and without growth factors exhibited increased cell viability,indicating an interaction between di-rhamnolipid and serum components in the regulation of neural stem cells and neuroprogenitors. Intracerebroventricular administration of di-rhamnolipid at 300 and 120 ng/day increased the number of neurospheres (1.3- and 1.63-fold,respectively) that could be derived from the anterior lateral ventricles of adult mice. These results indicate that di-rhamnolipid stimulates proliferation of neural stem cells and increases their endogenous pools which may have therapeutic potential in managing neurodegenerative or neuropsychiatric disorders and promoting nervous tissue regeneration following injury. View Publication

Activation of neural stem/progenitor cells (NSPCs) is a potential therapeutic strategy of neurological disorders. In this study,NSPCs of subventricular zone were isolated and cultured from platelet-derived growth factor-β-receptor-knockout (PDGFR-β(-/-)) mice of postnatal day 1 (P1) and P28,and the roles of PDGFR-β were examined in these cells. In PDGFR-β-preserving control NSPCs,stem cell activities,such as numbers and diameters of secondary neurospheres,cell proliferation and survival rates,were significantly higher in P1 NSPCs than those in P28 NSPCs. In PDGFR-β(-/-) NSPCs,most of these parameters were decreased as compared with age-matched controls. Among them,the decrease of secondary neurosphere formation was most striking in P1 and P28 PDGFR-β(-/-) NSPCs and in P28 control NSPCs as compared with P1 control NSPCs. PCR-array and following quantitative real-time PCR (qRT-PCR) analyses demonstrated that expressions of fibroblast growth factor-2 (FGF2) and exons IV-IX of brain-derived neurotrophic factor (BDNF) were decreased,and noggin was increased in P1 PDGFR-β(-/-) as compared with P1 controls. Addition of BDNF rescued the number and diameter of secondary neurospheres in P1 PDGFR-β(-/-) NSPCs to similar levels as controls. The expressions of PDGFs and PDGFRs in control NSPCs were increased along with the differentiation-induction,where phosphorylated PDGFR-β was co-localized with neuronal and astrocyte differentiation markers. In controls,the neuronal differentiation was decreased,and the glial differentiation was increased from P1 to P28 NSPCs. Compared with P1 controls,neuronal differentiation was reduced in P1 PDGFR-β(-/-) NSPCs,whereas glial differentiation was comparable between the two genotypes. These results suggest that PDGFR-β signaling is important for the self-renewal and multipotency of NSPCs,particularly in neonatal NSPCs. BDNF,FGF2,and noggin may be involved in the effects of PDGFR-β signaling in these cells. Accordingly,the activation of PDGFR-β in NSPCs may be a novel therapeutic strategy of neurological diseases. View Publication

BACKGROUND: Primitive brain tumors are the leading cause of cancer-related death in children. Tumor cells with stem-like properties (TSCs),thought to account for tumorigenesis and therapeutic resistance,have been isolated from high-grade gliomas in adults. Whether TSCs are a common component of pediatric brain tumors and are of clinical relevance remains to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Tumor cells with self-renewal properties were isolated with cell biology techniques from a majority of 55 pediatric brain tumors samples,regardless of their histopathologies and grades of malignancy (57% of embryonal tumors,57% of low-grade gliomas and neuro-glial tumors,70% of ependymomas,91% of high-grade gliomas). Most high-grade glioma-derived oncospheres (10/12) sustained long-term self-renewal akin to neural stem cells (textgreater7 self-renewals),whereas cells with limited renewing abilities akin to neural progenitors dominated in all other tumors. Regardless of tumor entities,the young age group was associated with self-renewal properties akin to neural stem cells (P = 0.05,chi-square test). Survival analysis of the cohort showed an association between isolation of cells with long-term self-renewal abilities and a higher patient mortality rate (P = 0.013,log-rank test). Sampling of low- and high-grade glioma cultures showed that self-renewing cells forming oncospheres shared a molecular profile comprising embryonic and neural stem cell markers. Further characterization performed on subsets of high-grade gliomas and one low-grade glioma culture showed combination of this profile with mesenchymal markers,the radio-chemoresistance of the cells and the formation of aggressive tumors after intracerebral grafting. CONCLUSIONS/SIGNIFICANCE: In brain tumors affecting adult patients,TSCs have been isolated only from high-grade gliomas. In contrast,our data show that tumor cells with stem cell-like or progenitor-like properties can be isolated from a wide range of histological sub-types and grades of pediatric brain tumors. They suggest that cellular mechanisms fueling tumor development differ between adult and pediatric brain tumors. View Publication

Glioblastoma is the most common form of primary malignant brain tumour. These tumours are highly proliferative and infiltrative resulting in a median patient survival of only 14 months from diagnosis. The current treatment regimens are ineffective against the small population of cancer stem cells residing in the tumourigenic niche; however,a new therapeutic approach could involve the removal of these cells from the microenvironment that maintains the cancer stem cell phenotype. We have isolated multipotent sphere-forming cells from human high grade glioma (glioma sphere-forming cells (GSCs)) to investigate the adhesive and migratory properties of these cells in vitro. We have focused on the role of two closely related metalloproteinases ADAM10 and ADAM17 due to their high expression in glioblastoma and GSCs and their ability to activate cytokines and growth factors. Here,we report that ADAM10 and ADAM17 inhibition selectively increases GSC,but not neural stem cell,migration and that the migrated GSCs exhibit a differentiated phenotype. We also observed a correlation between nestin,a stem/progenitor marker,and fibronectin,an extracellular matrix protein,expression in high grade glioma tissues. GSCs adherence on fibronectin is mediated by α5β1 integrin,where fibronectin further promotes GSC migration and is an effective candidate for in vivo cancer stem cell migration out of the tumourigenic niche. Our results suggest that therapies against ADAM10 and ADAM17 may promote cancer stem cell migration away from the tumourigenic niche resulting in a differentiated phenotype that is more susceptible to treatment. View Publication

BACKGROUND Mechanisms of glioma invasion remain to be fully elucidated. Glioma cells within glioblastoma multiforme (GBM) range from well-differentiated tumor cells to less-differentiated brain tumor-initiating cells (BTICs). The β2-subunit of Na(+)/K(+)-ATPase,called the adhesion molecule on glia (AMOG),is highly expressed in normal glia but is thought to be universally downregulated in GBM. To test our hypothesis that expression of AMOG is heterogeneous in GBM and confers a less invasive phenotype,we compared it between BTICs and differentiated cells from patient-matched GBM and then tested GBM invasion in vitro after AMOG overexpression. METHODS Immunohistochemistry,immunoblotting,and real-time PCR were used to characterize AMOG protein and mRNA expression in tumor samples,BTICs,and differentiated cells. Matrigel invasion assay,scratch assay,and direct cell counting were used for testing in vitro invasion,migration,and proliferation,respectively. RESULTS While AMOG expression is heterogeneous in astrocytomas of grades II-IV,it is lost in most GBM. BTICs express higher levels of AMOG mRNA and protein compared with patient-matched differentiated tumor cells. Overexpression of AMOG decreased GBM cell and BTIC invasion without affecting migration or proliferation. Knockdown of AMOG expression in normal human astrocytes increased invasion. CONCLUSIONS AMOG expression inhibits GBM invasion. Its downregulation increases invasion in glial cells and may also represent an important step in BTIC differentiation. These data provide compelling evidence implicating the role of AMOG in glioma invasion and provide impetus for further investigation. View Publication

Glioblastoma (GBM) is the most aggressive primary brain tumor and is resistant to all therapeutic regimens. Relapse occurs regularly and might be caused by a poorly characterized tumor stem cell (TSC) subpopulation escaping therapy. We suggest aldehyde dehydrogenase 1 (ALDH1) as a novel stem cell marker in human GBM. Using the neurosphere formation assay as a functional method to identify brain TSCs,we show that high protein levels of ALDH1 facilitate neurosphere formation in established GBM cell lines. Even single ALDH1 positive cells give rise to colonies and neurospheres. Consequently,the inhibition of ALDH1 in vitro decreases both the number of neurospheres and their size. Cell lines without expression of ALDH1 do not form tumor spheroids under the same culturing conditions. High levels of ALDH1 seem to keep tumor cells in an undifferentiated,stem cell-like state indicated by the low expression of beta-III-tubulin. In contrast,ALDH1 inhibition induces premature cellular differentiation and reduces clonogenic capacity. Primary cell cultures obtained from fresh tumor samples approve the established GBM cell line results. View Publication

Here we demonstrate,both in vivo and in vitro,that growth hormone (GH) mediates precursor cell activation in the subventricular zone (SVZ) of the aged (12-month-old) brain following exercise,and that GH signaling stimulates precursor activation to a similar extent to exercise. Our results reveal that both addition of GH in culture and direct intracerebroventricular infusion of GH stimulate neural precursor cells in the aged brain. In contrast,no increase in neurosphere numbers was observed in GH receptor null animals following exercise. Continuous infusion of a GH antagonist into the lateral ventricle of wild-type animals completely abolished the exercise-induced increase in neural precursor cell number. Given that the aged brain does not recover well after injury,we investigated the direct effect of exercise and GH on neural precursor cell activation following irradiation. This revealed that physical exercise as well as infusion of GH promoted repopulation of neural precursor cells in irradiated aged animals. Conversely,infusion of a GH antagonist during exercise prevented recovery of precursor cells in the SVZ following irradiation. View Publication