搜索结果: 'NeuroCult™ SM1'

Since the discovery of neural stem cells (NSC) in the embryonic and adult mammalian central nervous system (CNS),there have been a growing numbers of tissue culture media and protocols to study and functionally characterize NSCs and its progeny in vitro. One of these culture systems introduced in 1992 is referred to as the Neurosphere Assay,and it has been widely used to isolate,expand,differentiate and even quantify NSC populations. Several years later because its application as a quantitative in vitro assay for measuring NSC frequency was limited,a new single-step semisolid based assay,the Neural Colony Forming Cell (NCFC) assay was developed to accurately measure NSC numbers. The NCFC assay allows the discrimination between NSCs and progenitors by the size of colonies they produce (i.e.,their proliferative potential). The evolution and continued improvements made to these tissue culture tools will facilitate further advances in the promising application of NSCs for therapeutic use. View Publication

Neurodevelopmental defects are observed in the hereditary disorder Cockayne syndrome (CS). The gene most frequently mutated in CS,Cockayne Syndrome B (CSB),is required for the repair of bulky DNA adducts in transcribed genes during transcription-coupled nucleotide excision repair. CSB also plays a role in chromatin remodeling and mitochondrial function. The role of CSB in neural development is poorly understood. Here we report that the abundance of neural progenitors is normal in Csb(-/-) mice and the frequency of apoptotic cells in the neurogenic niche of the adult subependymal zone is similar in Csb(-/-) and wild type mice. Both embryonic and adult Csb(-/-) neural precursors exhibited defective self-renewal in the neurosphere assay. In Csb(-/-) neural precursors,self-renewal progressively decreased in serially passaged neurospheres. The data also indicate that Csb and the nucleotide excision repair protein Xpa preserve embryonic neural stem cell self-renewal after UV DNA damage. Although Csb(-/-) neural precursors do not exhibit altered neuronal lineage commitment after low-dose UV (1J/m(2)) in vitro,neurons differentiated in vitro from Csb(-/-) neural precursors that had been irradiated with 1J/m(2) UV exhibited defective neurite outgrowth. These findings identify a function for Csb in neural precursors. View Publication

Skin-derived precursor (SKP) cells are a valuable resource for tissue engineering and regenerative medicine,because they represent multipotent stem cells that differentiate into neural and mesodermal progenies. Previous studies suggest that the stem cell pool decreases with age. Here,we show that human multipotent SKP cells can be efficiently collected from adult cheek/chin skin,even in aged individuals of 70-78years. SKP cells were isolated from 38 skin samples by serum-free sphere culture and examined for the ability to differentiate into neural and mesodermal lineages. The number of spheres obtained from adult facial skin was significantly higher than that of trunk or extremity skin. SKP cells derived from cheek/chin skin exhibited a high ability to differentiate into neural and mesodermal cells relative to those derived from eyelid,trunk,or extremity skin. Furthermore,cheek/chin skin SKP cells were shown to express markers for undifferentiated stem cells,including a high expression level of the Sox9 gene. These results indicate that cheek/chin skin is useful for the recovery of multipotent stem cells for tissue engineering and regenerative therapy. View Publication

Prominin-1 (CD133) is commonly used to isolate stem and progenitor cells from the developing and adult nervous system and to identify cancer stem cells in brain tumors. However,despite extensive characterization of Prominin-1(+) precursor cells from the adult subventricular zone,no information about the expression of Prominin-1 by precursor cells in the subgranular zone (SGZ) of the adult hippocampus has been available. We show here that Prominin-1 is expressed by a significant number of cells in the SGZ of adult mice in vivo and ex vivo,including postmitotic astrocytes. A small subset of Prominin-1(+) cells coexpressed the nonspecific precursor cell marker Nestin as well as GFAP and Sox2. Upon fluorescence-activated cell sorting,only Prominin-1/Nestin double-positive cells fulfilled the defining stem cell criteria of proliferation,self-renewal,and multipotentiality as assessed by a neurosphere assay. In addition,isolated primary Prominin-1(+) cells preferentially migrated to the neurogenic niche in the SGZ upon transplantation in vivo. Finally,despite its expression by various stem and progenitor cells,Prominin-1 turned out to be dispensable for precursor cell proliferation in vitro and in vivo. Nevertheless,a net decrease in hippocampal neurogenesis,by ∼30% was found in Prominin-1 knock-out mice,suggesting other roles in controlling adult hippocampal neurogenesis. Remarkably,an upregulation of Prominin-2 was detected in Prominin-1-deficient mice highlighting a potential compensatory mechanism,which might explain the lack of severe symptoms in individuals carrying mutations in the Prom1 gene. View Publication

Recent advances in the ability to efficiently characterize tumor genomes is enabling targeted drug development,which requires rigorous biomarker-based patient selection to increase effectiveness. Consequently,representative DNA biomarkers become equally important in pre-clinical studies. However,it is still unclear how well these markers are maintained between the primary tumor and the patient-derived tumor models. Here,we report the comprehensive identification of somatic coding mutations and copy number aberrations in four glioblastoma (GBM) primary tumors and their matched pre-clinical models: serum-free neurospheres,adherent cell cultures,and mouse xenografts. We developed innovative methods to improve the data quality and allow a strict comparison of matched tumor samples. Our analysis identifies known GBM mutations altering PTEN and TP53 genes,and new actionable mutations such as the loss of PIK3R1,and reveals clear patient-to-patient differences. In contrast,for each patient,we do not observe any significant remodeling of the mutational profile between primary to model tumors and the few discrepancies can be attributed to stochastic errors or differences in sample purity. Similarly,we observe 96% primary-to-model concordance in copy number calls in the high-cellularity samples. In contrast to previous reports based on gene expression profiles,we do not observe significant differences at the DNA level between in vitro compared to in vivo models. This study suggests,at a remarkable resolution,the genome-wide conservation of a patient's tumor genetics in various pre-clinical models,and therefore supports their use for the development and testing of personalized targeted therapies. View Publication

The mammalian central nervous system (CNS) undergoes significant expansion postnatally,producing astrocytes,oligodendrocytes and inhibitory neurons to modulate the activity of neural circuits. This is coincident in humans with the emergence of pediatric epilepsy,a condition commonly treated with valproate/valproic acid (VPA),a potent inhibitor of histone deacetylases (HDACs). The sequential activity of specific HDACs,however,may be essential for the differentiation of distinct subpopulations of neurons and glia. Here,we show that different subsets of CNS neural stem cells (NSCs) and progenitors switch expression of HDAC1 and HDAC2 as they commit to a neurogenic lineage in the subventricular zone (SVZ) and dentate gyrus (DG). The administration of VPA for only one week from P7-P14,combined with sequential injections of thymidine analogs reveals that VPA stimulates a significant and differential decrease in the production and differentiation of progeny of NSCs in the DG,rostral migratory stream (RMS),and olfactory bulb (OB). Cross-fostering VPA-treated mice revealed,however,that a postnatal failure to thrive induced by VPA treatment had a greater effect on DG neurogenesis than VPA action directly. By one month after VPA,OB interneuron genesis was significantly and differentially reduced in both periglomerular and granule neurons. Using neurosphere assays to test if VPA directly regulates NSC activity,we found that short term treatment with VPA in vivo reduced neurosphere numbers and size,a phenotype that was also obtained in neurospheres from control mice treated with VPA and an alternative HDAC inhibitor,Trichostatin A (TSA) at 0 and 3 days in vitro (DIV). Collectively,these data show that clinically used HDAC inhibitors like VPA and TSA can perturb postnatal neurogenesis; and their use should be carefully considered,especially in individuals whose brains are actively undergoing key postnatal time windows of development. View Publication

The lipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2),synthesised by PIKfyve,regulates a number of intracellular membrane trafficking pathways. Genetic alteration of the PIKfyve complex,leading to even a mild reduction in PtdIns(3,5)P 2,results in marked neurodegeneration via an uncharacterised mechanism. In the present study we have shown that selectively inhibiting PIKfyve activity,using YM-201636,significantly reduces the survival of primary mouse hippocampal neurons in culture. YM-201636 treatment promoted vacuolation of endolysosomal membranes followed by apoptosis-independent cell death. Many vacuoles contained intravacuolar membranes and inclusions reminiscent of autolysosomes. Accordingly,YM-201636 treatment increased the level of the autophagosomal marker protein LC3-II,an effect that was potentiated by inhibition of lysosomal proteases,suggesting that alterations in autophagy could be a contributing factor to neuronal cell death. View Publication

Insulin-like growth factor (IGF) is involved in regulating many processes during neural development,and IGF binding protein-4 (IGFBP4) functions as a modulator of IGF actions or in an IGF-independent manner (e.g.,via inhibiting Wnt/β-catenin signaling). In the present study,neural progenitor cells (NPCs) were isolated from the forebrain of newborn mice to investigate effects of IGFBP4 on the proliferation and differentiation of NPCs. The proliferation of NPCs was evaluated using Cell Counting Kit-8 (CCK-8) after treatment with or without IGFBP4 as well as blockers of IGF-IR and β-catenin. Phosphorylation levels of Akt,Erk1,2 and p38 were analyzed by Western blotting. The differentiation of NPCs was evaluated using immunofluorescence and Western blotting. It was shown that exogenous IGFBP4 significantly inhibited the proliferation of NPCs and it did not induce a more pronounced inhibition of cell proliferation after blockade of IGF-IR but it did after antagonism of β-catenin. Akt phosphorylation was significantly decreased and phosphorylation levels of Erk1,2 and p38 were not significantly changed in IGFBP4-treated NPCs. Excessive IGFBP4 significantly promoted NPCs to differentiate into astrocytes and neurons. These data suggested that exogenous IGFBP4 inhibits proliferation and promotes differentiation of neural progenitor cells mainly through IGF-IR signaling pathway. View Publication

BACKGROUND Neuroblastoma (NB),a tumor of the primitive neural crest,despite aggressive treatment portends a poor long-term survival for patients with advanced high stage NB. New treatment strategies are required. METHODS We investigated coordinated targeting of essential homeostatic regulatory factors involved in cancer progression,histone deacetylases (HDACs) and carbonic anhydrases (CAs). RESULTS We evaluated the antitumor potential of the HDAC inhibitor (HDACi),pyridylmethyl-N-4-[(2-aminophenyl)-carbamoyl]-benzyl-carbamate (MS-275) in combination with a pan CA inhibitor,acetazolamide (AZ) on NB SH-SY5Y,SK-N-SH and SK-N-BE(2) cells. The key observation was that the combination AZ + MS-275 significantly inhibited growth,induced cell cycle arrest and apoptosis,and reduced migration capacity of NB cell line SH-SY5Y. In addition,this combination significantly inhibited tumor growth in vivo,in a pre-clinical xenograft model. Evidence was obtained for a marked reduction in tumorigenicity and in the expression of mitotic,proliferative,HIF-1α and CAIX. NB xenografts of SH-SY5Y showed a significant increase in apoptosis. CONCLUSION MS-275 alone at nanomolar concentrations significantly reduced the putative cancer stem cell (CSC) fraction of NB cell lines,SH-SY5Y and SK-N-BE(2),in reference to NT2/D1,a teratocarcinoma cell line,exhibiting a strong stem cell like phenotype in vitro. Whereas stemness genes (OCT4,SOX2 and Nanog) were found to be significantly downregulated after MS-275 treatment,this was further enhanced by AZ co-treatment. The significant reduction in initial tumorigenicity and subsequent abrogation upon serial xenografting suggests potential elimination of the NB CSC fraction. The significant potentiation of MS-275 by AZ is a promising therapeutic approach and one amenable for administration to patients given their current clinical utility. View Publication

Glioblastoma multiforme (GBM) is a grade IV malignant brain tumor with high mortality and has been well known to involve many molecular pathways,including G-protein coupled receptor (GPCR)-mediated signaling (such as epithelial growth factor receptor [EGFR] and platelet derived growth factor receptor [PDGFR]). G protein-coupled receptor kinases (GRK) directly regulate GPCR activity by phosphorylating activated agonist-bound receptors to desensitize signaling and internalize receptors through beta-arrestins. Recent studies in various cancers,including prostate and breast cancer,have highlighted the role of change in GRK expression to oncogenesis and tumor proliferation. In this study,we evaluated the expression of GRK5 in grade II to grade IV glioma specimens using immunohistochemistry and found that GRK5 expression levels are highly correlated with aggressiveness of glioma. We used culture conditions to selectively promote the growth of either glioblastoma cells with stem cell markers (GSC) or differentiated glioblastoma cells (DGC) from fresh GBM specimens. GSC are known to be highly invasive and mobile,and have the capacity to self-renew and are more resistant to chemotherapy and radiation compared to differentiated populations of GBM. We examined the expression of GRK5 in these two sets of culturing conditions for GBM cells and found that GRK5 expression is upregulated in GSC compared to differentiated GBM cells. To better understand the role of GRK5 in GBM-derived stem cells,we created stable GRK5 knockdown and evaluated the proliferation rate. Using an ATP chemiluminescence assay,we show,for the first time,that knocking down the expression of GRK5 decreased the proliferation rate of GSC in contrast to control. View Publication

Mechanisms that maintain cancer stem cells are crucial to tumour progression. The ID2 protein supports cancer hallmarks including the cancer stem cell state. HIFα transcription factors,most notably HIF2α (also known as EPAS1),are expressed in and required for maintenance of cancer stem cells (CSCs). However,the pathways that are engaged by ID2 or drive HIF2α accumulation in CSCs have remained unclear. Here we report that DYRK1A and DYRK1B kinases phosphorylate ID2 on threonine 27 (Thr27). Hypoxia downregulates this phosphorylation via inactivation of DYRK1A and DYRK1B. The activity of these kinases is stimulated in normoxia by the oxygen-sensing prolyl hydroxylase PHD1 (also known as EGLN2). ID2 binds to the VHL ubiquitin ligase complex,displaces VHL-associated Cullin 2,and impairs HIF2α ubiquitylation and degradation. Phosphorylation of Thr27 of ID2 by DYRK1 blocks ID2-VHL interaction and preserves HIF2α ubiquitylation. In glioblastoma,ID2 positively modulates HIF2α activity. Conversely,elevated expression of DYRK1 phosphorylates Thr27 of ID2,leading to HIF2α destabilization,loss of glioma stemness,inhibition of tumour growth,and a more favourable outcome for patients with glioblastoma. View Publication

Inflammatory activation precedes and correlates with accumulating τ lesions in Alzheimer's disease and tauopathies. However,the relationship between neuroinflammation and etiology of pathologic τ remains elusive. To evaluate whether inflammatory signaling may promote or accelerate neurofibrillary tangle pathology,we explored the effect of recombinant adeno-associated virus (rAAV)-mediated overexpression of a master inflammatory cytokine,IFN-γ,on τ phosphorylation. In initial studies in primary neuroglial cultures,rAAV-mediated expression of IFN-γ did not alter endogenous τ production or paired helical filament τ phosphorylation. Next,we tested the effect of rAAV-mediated expression of IFN-γ in the brains of 2 mouse models of tauopathy: JNPL3 and rTg4510. In both models,IFN-γ increased 1) signal transducer and activator of transcription 1 levels and gliosis,and 2) hyperphosphorylation and conformational alterations of soluble τ compared with control cohorts. However,sarkosyl-insoluble phosphorylated τ levels and ubiquitin staining were unaltered in the IFN-γ cohorts. Notably,IFN-γ-induced τ hyperphosphorylation was associated with release of the inhibitory effect of glycogen synthase kinase 3β function by decreasing Ser9 phosphorylation. Our data suggest that type II IFN signaling can promote τ phosphorylation by modulating cellular kinase activity,though this is insufficient in accelerating neuritic tangle pathology. View Publication