搜索结果: 'NeuroCult Proliferation'

The processes involved in malignant gliomas damage were quantitatively evaluated by microscopy. The near-infrared fluorescent dye IR700 that is conjugated to an anti-CD133 antibody (IR700-CD133) specifically targets malignant gliomas (U87MG) and stem cells (BT142) and is endocytosed into the cells. The gliomas are then photodamaged by the release of reactive oxygen species (ROS) and the heat induced by illumination of IR700 by a red laser,and the motility of the vesicles within these cells is altered as a result of cellular damage. To investigate these changes in motility,we developed a new method that measures fluctuations in the intensity of phase-contrast images obtained from small areas within cells. The intensity fluctuation in U87MG cells gradually decreased as cell damage progressed,whereas the fluctuation in BT142 cells increased. The endocytosed IR700 dye was co-localized in acidic organelles such as endosomes and lysosomes. The pH in U87MG cells,as monitored by a pH indicator,was decreased and then gradually increased by the illumination of IR700,while the pH in BT142 cells increased monotonically. In these experiments,the processes of cell damage were quantitatively evaluated according to the motility of vesicles and changes in pH. View Publication

FBW7 is a crucial component of an SCF-type E3 ubiquitin ligase,which mediates degradation of an array of different target proteins. The Fbw7 locus comprises three different isoforms,each with its own promoter and each suspected to have a distinct set of substrates. Most FBW7 targets have important functions in developmental processes and oncogenesis,including Notch proteins,which are functionally important substrates of SCF(Fbw7). Notch signalling controls a plethora of cell differentiation decisions in a wide range of species. A prominent role of this signalling pathway is that of mediating lateral inhibition,a process where exchange of signals that repress Notch ligand production amplifies initial differences in Notch activation levels between neighbouring cells,resulting in unequal cell differentiation decisions. Here we show that the downstream Notch signalling effector HES5 directly represses transcription of the E3 ligase Fbw7β,thereby directly bearing on the process of lateral inhibition. Fbw7(Δ/+) heterozygous mice showed haploinsufficiency for Notch degradation causing impaired intestinal progenitor cell and neural stem cell differentiation. Notably,concomitant inactivation of Hes5 rescued both phenotypes and restored normal stem cell differentiation potential. In silico modelling suggests that the NICD/HES5/FBW7β positive feedback loop underlies Fbw7 haploinsufficiency. Thus repression of Fbw7β transcription by Notch signalling is an essential mechanism that is coupled to and required for the correct specification of cell fates induced by lateral inhibition. 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

Specific neuronal types derived from embryonic stem cells (ESCs) can facilitate mechanistic studies and potentially aid in regenerative medicine. Existing induction methods,however,mostly rely on the effects of the combined action of multiple added growth factors,which generally tend to result in mixed populations of neurons. Here,we report that overexpression of specific transcription factors (TFs) in ESCs can rather guide the differentiation of ESCs towards specific neuron lineages. Analysis of data on gene expression changes 2 d after induction of each of 185 TFs implicated candidate TFs for further ESC differentiation studies. Induction of 23 TFs (out of 49 TFs tested) for 6 d facilitated neural differentiation of ESCs as inferred from increased proportion of cells with neural progenitor marker PSA-NCAM. We identified early activation of the Notch signaling pathway as a common feature of most potent inducers of neural differentiation. The majority of neuron-like cells generated by induction of Ascl1,Smad7,Nr2f1,Dlx2,Dlx4,Nr2f2,Barhl2,and Lhx1 were GABA-positive and expressed other markers of GABAergic neurons. In the same way,we identified Lmx1a and Nr4a2 as inducers for neurons bearing dopaminergic markers and Isl1,Fezf2,and St18 for cholinergic motor neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific messenger RNA (mRNA) and microRNAs (miRNAs). Sets of Ascl1-induced mRNAs and miRNAs were enriched in Ascl1 targets. In further studies,enrichment of cells obtained with the induction of Ascl1,Smad7,and Nr2f1 using microbeads resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and expressed markers of GABAergic neurons. In summary,this study indicates that induction of transcription factors is a promising approach to generate cultures that show the transcription profiles characteristic of specific neural cell types. View Publication

BACKGROUND Epigenetic modifications,particularly DNA methylation and posttranslational histone modifications regulate heritable changes in transcription without changes in the DNA sequence. Despite a number of studies showing clear links between environmental factors and DNA methylation,little is known about the effect of environmental factors on the recently identified histone lysine methylation. Since their identification numerous studies have establish critical role played by these enzymes in mammalian development. OBJECTIVES Identification of the Jumonji (Jmj) domain containing histone lysine demethylase have added a new dimension to epigenetic control of gene expression by dynamic regulation of histone methylation marks. The objective of our study was to evaluate the effect of prohexadione and trinexapac,widely used plant growth regulators of the acylcyclohexanediones class,on the enzymatic activity of histone lysine demethylases and histone modifications during the neural stem/progenitor cell differentiation. METHODS Here we show that prohexadione,but not trinexapac,directly inhibits non-heme iron (II),2-oxoglutarate-dependent histone lysine demethylase such as Jmjd2a. We used molecular modeling to show binding of prohexadione to Jmjd2a. We also performed in vitro demethylation assays to show the inhibitory effect of prohexadione on Jmjd2a. Further we tested this molecule in cell culture model of mouse hippocampal neural stem/progenitor cells to demonstrate its effect toward neuronal proliferation and differentiation. RESULTS Molecular modeling studies suggest that prohexadione binds to the 2-oxoglutarate binding site of Jmjd2a demethylase. Treatment of primary neural stem/progenitor cells with prohexadione showed a concentration dependent reduction in their proliferation. Further,the prohexadione treated neurospheres were induced toward neurogenic lineage upon differentiation. CONCLUSIONS Our results describe an important chemico-biological interaction of prohexadione,in light of critical roles played by histone lysine demethylases in human health and diseases. View Publication

Glioblastoma (GBM) is the most common and deadly malignant brain cancer,with a median survival of <2 years. GBM displays a cellular complexity that includes brain tumour-initiating cells (BTICs),which are considered as potential key targets for GBM therapies. Here we show that the transcription factors FOXG1 and Groucho/TLE are expressed in poorly differentiated astroglial cells in human GBM specimens and in primary cultures of GBM-derived BTICs,where they form a complex. FOXG1 knockdown in BTICs causes downregulation of neural stem/progenitor and proliferation markers,increased replicative senescence,upregulation of astroglial differentiation genes and decreased BTIC-initiated tumour growth after intracranial transplantation into host mice. These effects are phenocopied by Groucho/TLE knockdown or dominant inhibition of the FOXG1:Groucho/TLE complex. These results provide evidence that transcriptional programmes regulated by FOXG1 and Groucho/TLE are important for BTIC-initiated brain tumour growth,implicating FOXG1 and Groucho/TLE in GBM tumourigenesis. View Publication

Neurogenesis occurs continuously in two brain regions of adult mammals,underpinned by a pool of resident neural stem cells (NSCs) that can differentiate into all neural cell types. To advance our understanding of NSC function and to develop therapeutic and diagnostic approaches,it is important to accurately identify and enrich for NSCs. There are no definitive markers for the identification and enrichment of NSCs present in the mouse brain. Recently,a fluorescent rosamine dye,CDy1,has been identified as a label for pluripotency in cultured human embryonic and induced pluripotent stem cells. As similar cellular characteristics may enable the uptake and retention of CDy1 by other stem cell populations,we hypothesized that this dye may also enrich for primary NSCs from the mouse brain. Because the subventricular zone (SVZ) and the hippocampus represent brain regions that are highly enriched for NSCs in adult mammals,we sampled cells from these areas to test this hypothesis. These experiments revealed that CDy1 staining indeed allows for enrichment and selection of all neurosphere-forming cells from both the SVZ and the hippocampus. We next examined the effectiveness of CDy1 to select for NSCs derived from the SVZ of aged animals,where the total pool of NSCs present is significantly lower than in young animals. We found that CDy1 effectively labels the NSCs in adult and aged animals as assessed by the neurosphere assay and reflects the numbers of NSCs present in aged animals. CDy1,therefore,appears to be a novel marker for enrichment of NSCs in primary brain tissue preparations. View Publication

The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here,we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal,proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive),neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells,as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs,an effect blocked by Notch pathway inhibition. Moreover,R-m-AEA treatment for 48 h,increased proliferation as assessed by BrdU incorporation assay,an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly,stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation),at 7 days,as shown by counting the number of NeuN-positive neurons in the cultures. Moreover,by monitoring intracellular calcium concentrations ([Ca(2+)]i) in single cells following KCl and histamine stimuli,a method that allows the functional evaluation of neuronal differentiation,we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251,for 7 days,thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation,R-m-AEA also increased neurite growth,as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together,these results demonstrate that CB1R activation induces proliferation,self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures. View Publication

The blood-brain barrier (BBB),comprised of brain endothelial cells with tight junctions (TJ) between them,regulates the extravasation of molecules and cells into and out of the central nervous system (CNS). Overcoming the difficulty of delivering therapeutic agents to specific regions of the brain presents a major challenge to treatment of a broad range of brain disorders. Current strategies for BBB opening are invasive,not specific,and lack precise control over the site and timing of BBB opening,which may limit their clinical translation. In the present report,we describe a novel approach based on a combination of stem cell delivery,heat-inducible gene expression and mild heating with high-intensity focused ultrasound (HIFU) under MRI guidance to remotely permeabilize BBB. The permeabilization of the BBB will be controlled with,and limited to where selected pro-inflammatory factors will be secreted secondary to HIFU activation,which is in the vicinity of the engineered stem cells and consequently both the primary and secondary disease foci. This therapeutic platform thus represents a non-invasive way for BBB opening with unprecedented spatiotemporal precision,and if properly and specifically modified,can be clinically translated to facilitate delivery of different diagnostic and therapeutic agents which can have great impact in treatment of various disease processes in the central nervous system. View Publication

Pituitary somatotroph adenomas result in dysregulated growth hormone (GH) hypersecretion and acromegaly; however,regulatory mechanisms that promote GH hypersecretion remain elusive. Here,we provide evidence that STAT3 directly induces somatotroph tumor cell GH. Evaluation of pituitary tumors revealed that STAT3 expression was enhanced in human GH-secreting adenomas compared with that in nonsecreting pituitary tumors. Moreover,STAT3 and GH expression were concordant in a somatotroph adenoma tissue array. Promoter and expression analysis in a GH-secreting rat cell line (GH3) revealed that STAT3 specifically binds the Gh promoter and induces transcription. Stable expression of STAT3 in GH3 cells induced expression of endogenous GH,and expression of a constitutively active STAT3 further enhanced GH production. Conversely,expression of dominant-negative STAT3 abrogated GH expression. In primary human somatotroph adenoma-derived cell cultures,STAT3 suppression with the specific inhibitor S3I-201 attenuated GH transcription and reduced GH secretion in the majority of derivative cultures. In addition,S3I-201 attenuated somatotroph tumor growth and GH secretion in a rat xenograft model. GH induced STAT3 phosphorylation and nuclear translocation,indicating a positive feedback loop between STAT3 and GH in somatotroph tumor cells. Together,these results indicate that adenoma GH hypersecretion is the result of STAT3-dependent GH induction,which in turn promotes STAT3 expression,and suggest STAT3 as a potential therapeutic target for pituitary somatotroph adenomas. View Publication

Malignant gliomas are the most common and deadly primary malignant brain tumors. In vivo orthotopic models could doubtless represent an appropriate tool to test novel treatment for gliomas. However,methods commonly used to monitor the growth of glioma inside the mouse brain are time consuming and invasive. We tested the reliability of a minimally invasive procedure,based on a secreted luciferase (Gaussia luciferase),to frequently monitor the changes of glioma size. Gluc activity was evaluated from blood samples collected from the tail tip of mice twice a week,allowing to make a growth curve for the tumors. We validated the correlation between Gluc activity and tumor size by analysing the tumor after brain dissection. We found that this method is reliable for monitoring human glioma transplanted in immunodeficient mice,but it has strong limitation in immunocompetent models,where an immune response against the luciferase is developed during the first weeks after transplant. View Publication