技术资料

In culture conditions,human induced-pluripotent stem cells (hiPSC)-derived neurons form synaptic connections with other cells and establish neuronal networks,which are expected to be an in vitro model system for drug discovery screening and toxicity testing. While early studies demonstrated effects of co-culture of hiPSC-derived neurons with astroglial cells on survival and maturation of hiPSC-derived neurons,the population spiking patterns of such hiPSC-derived neurons have not been fully characterized. In this study,we analyzed temporal spiking patterns of hiPSC-derived neurons recorded by a multi-electrode array system. We discovered that specific sets of hiPSC-derived neurons co-cultured with astrocytes showed more frequent and highly coherent non-random synchronized spike trains and more dynamic changes in overall spike patterns over time. These temporally coordinated spiking patterns are physiological signs of organized circuits of hiPSC-derived neurons and suggest benefits of co-culture of hiPSC-derived neurons with astrocytes. View Publication

The widespread application of human stem-cell-derived neurons for functional studies is impeded by complicated differentiation protocols,immaturity,and deficient optogene expression as stem cells frequently lose transgene expression over time. Here we report a simple but precise Cre-loxP-based strategy for generating conditional,and thereby stable,optogenetic human stem-cell lines. These cells can be easily and efficiently differentiated into functional neurons,and optogene expression can be triggered by administering Cre protein to the cultures. This conditional expression system may be applied to stem-cell-derived neurons whenever timed transgene expression could help to overcome silencing at the stem-cell level. View Publication

Neurotrophins and their receptors are frequently expressed in malignant gliomas,yet their functions are largely unknown. Previously,we have shown that p75 neurotrophin receptor is required for glioma invasion and proliferation. However,the role of Trk receptors has not been examined. In this study,we investigated the importance of TrkB and TrkC in survival of brain tumor-initiating cells (BTICs). Here,we show that human malignant glioma tissues and also tumor-initiating cells isolated from fresh human malignant gliomas express the neurotrophin receptors TrkB and TrkC,not TrkA,and they also express neurotrophins NGF,BDNF,and neurotrophin 3 (NT3). Specific activation of TrkB and TrkC receptors by ligands BDNF and NT3 enhances tumor-initiating cell viability through activation of ERK and Akt pathways. Conversely,TrkB and TrkC knockdown or pharmacologic inhibition of Trk signaling decreases neurotrophin-dependent ERK activation and BTIC growth. Further,pharmacological inhibition of both ERK and Akt pathways blocked BDNF,and NT3 stimulated BTIC survival. Importantly,attenuation of BTIC growth by EGFR inhibitors could be overcome by activation of neurotrophin signaling,and neurotrophin signaling is sufficient for long term BTIC growth as spheres in the absence of EGF and FGF. Our results highlight a novel role for neurotrophin signaling in brain tumor and suggest that Trks could be a target for combinatorial treatment of malignant glioma. View Publication

OBJECTIVE To develop in vitro adenoid cystic carcinoma cell line as a surrogate for functional studies. MATERIALS AND METHODS Cells obtained from a primary ACC of the base of tongue were cultivated in vitro and immortalized with h-TERT. Morphologic,cytogenetic and functional studies were performed. RESULTS Tumor cells were verified by positive reactions to keratin and smooth muscle actin and phenotypic cellular and nuclear features. In-vitro cell growth and colony formation assay supported their tumor nature. CONCLUSION We authenticated an ACC cell line with hybrid epithelial-myoepithelial feature as a resource for functional experimentation. View Publication

UNLABELLED Certain murine leukemia viruses (MLVs) are capable of inducing fatal progressive spongiform motor neuron disease in mice that is largely mediated by viral Env glycoprotein expression within central nervous system (CNS) glia. While the etiologic mechanisms and the glial subtypes involved remain unresolved,infection of NG2 glia was recently observed to correlate spatially and temporally with altered neuronal physiology and spongiogenesis. Since one role of NG2 cells is to serve as oligodendrocyte (OL) progenitor cells (OPCs),we examined here whether their infection by neurovirulent (FrCasE) or nonneurovirulent (Fr57E) ecotropic MLVs influenced their viability and/or differentiation. Here,we demonstrate that OPCs,but not OLs,are major CNS targets of both FrCasE and Fr57E. We also show that MLV infection of neural progenitor cells (NPCs) in culture did not affect survival,proliferation,or OPC progenitor marker expression but suppressed certain glial differentiation markers. Assessment of glial differentiation in vivo using transplanted transgenic NPCs showed that,while MLVs did not affect cellular engraftment or survival,they did inhibit OL differentiation,irrespective of MLV neurovirulence. In addition,in chimeric brains,where FrCasE-infected NPC transplants caused neurodegeneration,the transplanted NPCs proliferated. These results suggest that MLV infection is not directly cytotoxic to OPCs but rather acts to interfere with OL differentiation. Since both FrCasE and Fr57E viruses restrict OL differentiation but only FrCasE induces overt neurodegeneration,restriction of OL maturation alone cannot account for neuropathogenesis. Instead neurodegeneration may involve a two-hit scenario where interference with OPC differentiation combined with glial Env-induced neuronal hyperexcitability precipitates disease. IMPORTANCE A variety of human and animal retroviruses are capable of causing central nervous system (CNS) neurodegeneration manifested as motor and cognitive deficits. These retroviruses infect a variety of CNS cell types; however,the specific role each cell type plays in neuropathogenesis remains to be established. The NG2 glia,whose CNS functions are only now emerging,are a newly appreciated viral target in murine leukemia virus (MLV)-induced neurodegeneration. Since one role of NG2 glia is that of oligodendrocyte progenitor cells (OPCs),we investigated here whether their infection by the neurovirulent MLV FrCasE contributed to neurodegeneration by affecting OPC viability and/or development. Our results show that both neurovirulent and nonneurovirulent MLVs interfere with oligodendrocyte differentiation. Thus,NG2 glial infection could contribute to neurodegeneration by preventing myelin formation and/or repair and by suspending OPCs in a state of persistent susceptibility to excitotoxic insult mediated by neurovirulent virus effects on other glial subtypes. View Publication

BACKGROUND Glioma is the most common form of primary malignant brain tumor in adults,with approximately 4 cases per 100 000 people each year. Gliomas,like many tumors,are thought to primarily metabolize glucose for energy production; however,the reliance upon glycolysis has recently been called into question. In this study,we aimed to identify the metabolic fuel requirements of human glioma cells. METHODS We used database searches and tissue culture resources to evaluate genotype and protein expression,tracked oxygen consumption rates to study metabolic responses to various substrates,performed histochemical techniques and fluorescence-activated cell sorting-based mitotic profiling to study cellular proliferation rates,and employed an animal model of malignant glioma to evaluate a new therapeutic intervention. RESULTS We observed the presence of enzymes required for fatty acid oxidation within human glioma tissues. In addition,we demonstrated that this metabolic pathway is a major contributor to aerobic respiration in primary-cultured cells isolated from human glioma and grown under serum-free conditions. Moreover,inhibiting fatty acid oxidation reduces proliferative activity in these primary-cultured cells and prolongs survival in a syngeneic mouse model of malignant glioma. CONCLUSIONS Fatty acid oxidation enzymes are present and active within glioma tissues. Targeting this metabolic pathway reduces energy production and cellular proliferation in glioma cells. The drug etomoxir may provide therapeutic benefit to patients with malignant glioma. In addition,the expression of fatty acid oxidation enzymes may provide prognostic indicators for clinical practice. View Publication

The endoplasmic reticulum (ER) is involved in Ca2+ signaling and protein folding. ER Ca2+ depletion and accumulation of unfolded proteins activate the molecular chaperone GRP78 (glucose-regulated protein 78) which in turn triggers the ER stress response (ERSR) pathway aimed to restore ER homeostasis. Failure to adapt to stress,however,results in apoptosis. We and others have shown that malignant cells are more susceptible to ERSR-induced apoptosis than their normal counterparts,implicating the ERSR as a potential target for cancer therapeutics. Predicated on these findings,we developed an assay that uses a GRP78 biosensor to identify small molecule activators of ERSR in glioma cells. We performed a quantitative high-throughput screen (qHTS) against a collection of ˜425,000 compounds and a comprehensive panel of orthogonal secondary assays was formulated for stringent compound validation. We identified novel activators of ERSR,including a compound with a 2,9-diazaspiro[5.5]undecane core,which depletes intracellular Ca2+ stores and induces apoptosis-mediated cell death in several cancer cell lines,including patient-derived and 3D cultures of glioma cells. This study demonstrates that our screening platform enables the identification and profiling of ERSR inducers with cytotoxic activity and advocates for characterization of these compound in in vivo models. View Publication

Glioblastoma multiforme (GBM),the most common and lethal tumor of the adult brain,generally shows chemo- and radioresistance. MicroRNAs (miRs) regulate physiological processes,such as resistance of GBM cells to temozolomide (TMZ). Although miRs are attractive targets for cancer therapeutics,the effectiveness of this approach requires targeted delivery. Mesenchymal stem cells (MSCs) can migrate to the sites of cancers,including GBM. We report on an increase in miR-9 in TMZ-resistant GBM cells. miR-9 was involved in the expression of the drug efflux transporter,P-glycoprotein. To block miR-9,methods were developed with Cy5-tagged anti-miR-9. Dye-transfer studies indicated intracellular communication between GBM cells and MSCs. This occurred by gap junctional intercellular communication and the release of microvesicles. In both cases,anti-miR-9 was transferred from MSCs to GBM cells. However,the major form of transfer occurred with the microvesicles. The delivery of anti-miR-9 to the resistant GBM cells reversed the expression of the multidrug transporter and sensitized the GBM cells to TMZ,as shown by increased cell death and caspase activity. The data showed a potential role for MSCs in the functional delivery of synthetic anti-miR-9 to reverse the chemoresistance of GBM cells.Molecular Therapy-Nucleic Acids (2013) 2,e126; doi:10.1038/mtna.2013.60; published online 1 October 2013. View Publication

While no effective therapy is available for the treatment of methamphetamine (METH)-induced neurotoxicity,aerobic exercise is being proposed to improve depressive symptoms and substance abuse outcomes. The present study focuses on the effect of exercise on METH-induced aberrant neurogenesis in the hippocampal dentate gyrus in the context of the blood-brain barrier (BBB) pathology. Mice were administered with METH or saline by i.p. injections for 5 days with an escalating dose regimen. One set of mice was sacrificed 24 h post last injection of METH,and the remaining animals were either subjected to voluntary wheel running (exercised mice) or remained in sedentary housing (sedentary mice). METH administration decreased expression of tight junction (TJ) proteins and increased BBB permeability in the hippocampus. These changes were preserved post METH administration in sedentary mice and were associated with the development of significant aberrations of neural differentiation. Exercise protected against these effects by enhancing the protein expression of TJ proteins,stabilizing the BBB integrity,and enhancing the neural differentiation. In addition,exercise protected against METH-induced systemic increase in inflammatory cytokine levels. These results suggest that exercise can attenuate METH-induced neurotoxicity by protecting against the BBB disruption and related microenvironmental changes in the hippocampus. View Publication

The potential ability to differentiate dedifferentiated adipocytes into a neural lineage is attracting strong interest as an emerging method of producing model cells for the treatment of a variety of neurological diseases. Here,we describe the efficient conversion of dedifferentiated adipocytes into a neural-like cell population. These cells grew in neurosphere-like structures and expressed a high level of the early neuroectodermal marker Nestin. These neurospheres could proliferate and express stemness genes,suggesting that these cells could be committed to the neural lineage. After neural induction,NeuroD1,Sox1,Double Cortin,and Eno2 were not expressed. Patch clamp data did not reveal different electrophysiological properties,indicating the inability of these cells to differentiate into mature neurons. In contrast,the differentiated cells expressed a high level of CLDN11,as demonstrated using molecular method,and stained positively for the glial cell markers CLDN11 and GFAP,as demonstrated using immunocytochemistry. These data were confirmed by quantitative results for glial cell line-derived neurotrophic factor production,which showed a higher secretion level in neurospheres and the differentiated cells compared with the untreated cells. In conclusion,our data demonstrate morphological,molecular,and immunocytochemical evidence of initial neural differentiation of mature adipocytes,committing to a glial lineage. View Publication

Neurogenesis in the subventricular zone (SVZ) is regulated by diffusible factors and cell-cell contacts. In vivo,SVZ stem cells are associated with the abluminal surface of blood vessels and such interactions are thought to regulate their neurogenic capacity. SVZ neural stem cells (NSCs) have been described to contact endothelial-derived laminin via α6β1 integrin. To elucidate whether heterocellular contacts with brain endothelial cells (BEC) regulate SVZ cells neurogenic capacities,cocultures of SVZ neurospheres and primary BEC,both obtained from C57BL/6 mice,were performed. The involvement of laminin-integrin interactions in SVZ homeostasis was tested in three ways. Firstly,SVZ cells were analyzed following incubation of BEC with the protein synthesis inhibitor cycloheximide (CHX) prior to coculture,a treatment expected to decrease membrane proteins. Secondly,SVZ cells were cocultured with BEC in the presence of an anti-α6 integrin neutralizing antibody. Thirdly,BEC were cultured with β1-/- SVZ cells. We showed that contact with BEC supports,at least in part,proliferation and stemness of SVZ cells,as evaluated by the number of BrdU positive (+) and Sox2+ cells in contact with BEC. These effects are dependent on BEC-derived laminin binding to α6β1 integrin and are decreased in cocultures incubated with anti-α6 integrin neutralizing antibody and in cocultures with SVZ β1-/- cells. Moreover,BEC-derived laminin sustains stemness in SVZ cell cultures via activation of the Notch and mTOR signaling pathways. Our results show that BEC/SVZ interactions involving α6β1 integrin binding to laminin,contribute to SVZ cell proliferation and stemness. View Publication

Glioblastoma multiforme (GBM) is the most common and lethal adult brain tumor. Resistance to standard radiation and chemotherapy is thought to involve survival of GBM cancer stem cells (CSCs). To date,no single marker for identifying GBM CSCs has been able to capture the diversity of CSC populations,justifying the needs for additional CSC markers for better characterization. Employing targeted mass spectrometry,here we present five cell-surface markers HMOX1,SLC16A1,CADM1,SCAMP3,and CLCC1 which were found to be elevated in CSCs relative to healthy neural stem cells (NSCs). Transcriptomic analyses of REMBRANDT and TCGA compendiums also indicated elevated expression of these markers in GBM relative to controls and non-GBM diseases. Two markers SLC16A1 and HMOX1 were found to be expressed among pseudopalisading cells that reside in the hypoxic region of GBM,substantiating the histopathological hallmarks of GBM. In a prospective study (N%=%8) we confirmed the surface expression of HMOX1 on freshly isolated primary GBM cells (P0). Employing functional assays that are known to evaluate stemness,we demonstrate that elevated HMOX1 expression is associated with stemness in GBM and can be modulated through TGFβ. siRNA-mediated silencing of HMOX1 impaired GBM invasion-a phenomenon related to poor prognosis. In addition,surgical resection of GBM tumors caused declines (18%%±%5.1SEM) in the level of plasma HMOX1 as measured by ELISA,in 8/10 GBM patients. These findings indicate that HMOX1 is a robust predictor of GBM CSC stemness and pathogenesis. Further understanding of the role of HMOX1 in GBM may uncover novel therapeutic approaches. Stem Cells 2016;34:2276-2289. View Publication