[Show abstract][Hide abstract]ABSTRACT: Glioblastoma multiforme (GBM) is the most malignant World Health Organization grade IV brain tumor. GBM patients have a poor prognosis because of its resistance to standard therapies, such as chemotherapy and radiation. Since stem-like cells have been associated with the treatment resistance of GBM, novel therapies targeting the cancer stem cell (CSC) population is critically required. However, GBM CSCs share molecular and functional characteristics with normal neural stem cells (NSCs). To elucidate differential therapeutic targets of GBM CSCs, we compared surface markers of GBM CSCs with adult human NSCs and found that GD2 and CD90 were specifically overexpressed in GBM CSCs. We further tested whether the GBM CSC specific markers are associated with the cancer stemness using primarily cultured patient-derived GBM cells. However, results consistently indicated that GBM cells with or without GD2 and CD90 had similar in vitro sphere formation capacity, a functional characteristics of CSCs. Therefore, GD2 and CD90, GBM specific surface markers, might not be used as specific therapeutic targets for GBM CSCs, although they could have other clinical utilities.
Full-text Article · Mar 2015 · Anatomy & cell biology
[Show abstract][Hide abstract]ABSTRACT: The present invention relates to stem cells in which a gene that activates signaling is introduced and to a method for proliferating the stem cells. More specifically, the invention relates to a method of significantly increasing the ability of stem cells to proliferate, either by transfecting stem cells with the Notch intracellular domain (NICD) to activate the Notch signaling pathway, or by transfecting stem cells with the c-MET gene and treating the transfected stem cells with the HGF ligand protein to activate the c-MET/HGF signaling pathway. According to the present invention, as a result of activating the Notch signaling pathway or the c-MET/HGF signaling pathway, stem cells having an excellent ability to proliferate can be produced in large amounts. Particularly, since neural stem cells which have been difficult to culture in vitro can be proliferated in large amounts, thus the neural stem cells will be more useful for the preparation of cell therapeutic agents for treating cranial nerve diseases.
[Show abstract][Hide abstract]ABSTRACT: An unfortunate consequence of improvements in the treatments of advanced primary cancers is the concurrent increase of metastatic brain tumors. Despite of unfavorable clinical prognosis, radiation therapy is still the only viable treatment option for brain metastases. Expression of c-Met induces cell migration and invasion in many cancers, which are indispensable steps for metastasis. Accordingly, we examined the effects of gene silencing of c-Met on brain metastasis to evaluate the possibility of c-Met as a potential target. MDA-MB-435 cells were transfected with c-Met targeting short hairpin RNAs (shRNAs). Effects of c-Met shRNAs on the expression of epithelial mesenchymal transition (EMT) related proteins, in vitro migration, and in vivo brain metastasis were examined. Expression of mesenchymal markers and in vitro migration of MDA-MB-435 cells were significantly inhibited by introduction of c-Met shRNAs. When c-Met-silenced MDA-MB-435 cells were stereotactically implanted into the brains of immune-compromised mice or injected into the right internal carotid arteries, c-Met-silenced MDA-MB-435 cells produced significantly smaller tumor masses or survival time was significantly prolonged, respectively, compared with MDA-MB-435 cells transfected with control shRNA. The data reveal the novel function of c-Met in the process of brain metastasis and its potential as a preventive and/or therapeutic target in this disease.
Article · Apr 2013 · Clinical and Experimental Metastasis
[Show abstract][Hide abstract]ABSTRACT: Autologous adult human neural stem cells may be used for regenerative cell therapies bypass potential ethical problems. However, stable in vitro expansion protocols and experimental/clinical factors influencing primary cultures need to be further elucidated for clinically applicable techniques. To address these issues, we obtained biopsy specimens from 23 temporal lobe epilepsy patients and adult human multipotent neural cells (ahMNCs) were primarily cultured in a defined attachment culture condition. When the success of primary cultures was defined as stable expansion of cells (>ten in vitro passages) and expression of NSC markers, success rate of the primary culture was 39% (nine of 23 temporal lobes). During the long-term expansion, expressions of NSC markers and differentiation potentials into astrocytes and neurons were maintained. After the 18th sub-culture, spontaneous senescence and differentiation were observed, and the cultivated ahMNCs ceased their proliferation. The culture results were not affected by seizure characteristics, however, an older age (>40 years) and a smaller sample volume (<2ml) were found to exert negative influences on the primary culture results. Furthermore therapeutic effects of ahMNCs against stroke were analyzed in an animal model. Transplantation of ahMNCs cells reduced infarction volumes and enhanced motor activity, significantly. The results here would provide promising experimental and clinical strategy of using patient-specific autologous ahMNCs in regenerative medicine in the future.
Full-text Article · Nov 2012 · Experimental Neurology
[Show abstract][Hide abstract]ABSTRACT: Stem cell therapy is a promising approach for stroke. However, low survival rates and potential tumorigenicity of implanted cells could undermine the efficacy of the cell-based treatment. The use of stem cell-conditioned medium (CM) may be a feasible approach to overcome these limitations. Especially, specific stem cell culture condition and continuous infusion of CM into ischemic brains would have better therapeutic results. The CM was prepared by culturing human adipose-derived stem cells in a three-dimensional spheroid form to increase the secretion of angiogenic/neuroprotective factors. Ischemic stroke was induced by standard middle cerebral artery occlusion methods in the brain of 8-week-old Sprague-Dawley rats. Continuous infusion of CM or αMEM media (0.5 μl/hr) into the lateral ventricle was initiated 8 days after the surgery and maintained for 7 days. Alteration in the motor function was monitored by the rotarod test. Infarction volume and the number of microvessels or TUNEL-positive neural cells were analyzed 15 days after the surgery. Compared with αMEM, continuous CM infusion reduced the infarction volume and maintained motor function. The number of CD31-positive microvessels and TUNEL-positive neural cells significantly increased and decreased, respectively, in the penumbra regions. Although the apoptosis of all neural cell types decreased, reduction in the microglial apoptosis and astrogliosis was prominent and significant. In this study, the therapeutic effects of the CM against stroke were confirmed in an animal model. Increased endothelial cell proliferation, reduced neural cell apoptosis, and milder astrogliosis may play important roles in the treatment effects of CM.
Full-text Article · Sep 2012 · Journal of Neuroscience Research
[Show abstract][Hide abstract]ABSTRACT: Glioblastomas multiforme (GBM) contain highly tumorigenic, self-renewing populations of stem/initiating cells [glioblastoma stem cells (GSC)] that contribute to tumor propagation and treatment resistance. However, our knowledge of the specific signaling pathways that regulate GSCs is limited. The MET tyrosine kinase is known to stimulate the survival, proliferation, and invasion of various cancers including GBM. Here, we identified a distinct fraction of cells expressing a high level of MET in human primary GBM specimens that were preferentially localized in perivascular regions of human GBM biopsy tissues and were found to be highly clonogenic, tumorigenic, and resistant to radiation. Inhibition of MET signaling in GSCs disrupted tumor growth and invasiveness both in vitro and in vivo, suggesting that MET activation is required for GSCs. Together, our findings indicate that MET activation in GBM is a functional requisite for the cancer stem cell phenotype and a promising therapeutic target.
[Show abstract][Hide abstract]ABSTRACT: Fetal mouse NSCs expressing GFP were primarily cultured from brains of 13.5 day old GFP transgenic C57BL/6 mouse embryos. Expression of NSC markers (Nestin, Musashi, Sox2, and CD133) and differentiated neural cell markers (Tuj1 for the neuron; GFAP for the astrocyte; Olig2 for the oligodendrocyte) was examined by immunocytochemistry (A, C, D, E) or flow cytometry (B). NSCs forming neurospheres in the NSC culture condition without serum were utilized (A, B). NSCs maintained in 10% FBS/DMEM on PLO-coated slides for overnight (C), 4 days (D), and 2 weeks (E) were analyzed.
[Show abstract][Hide abstract]ABSTRACT: The gamma knife surgery device can concentrate 50% of the maximal irradiation dose (50% isodose) within 1 mm. The concentration capacity was tested using radiosensitive films. Blue spots represent the intensity of the irradiation and the intensity is presented as graphs.
[Show abstract][Hide abstract]ABSTRACT: The chronic inflammatory response of microglia after the brain irradiation was observed in the irradiated mice by anti-CD68 immunohistochemistry at seven weeks after the last irradiation. There were few CD68-positive cells in the brains of the control mice. CD68-positive cells (arrowheads) were magnified in the insets.
[Show abstract][Hide abstract]ABSTRACT: GFP-negative human umbilical venous endothelial cells (HUVECs) made numerous tubes in matrigels when given differentiation conditions, while GFP-positive NSCs remained as spheres.
[Show abstract][Hide abstract]ABSTRACT: Expression of endothelial or endothelial progenitor cell markers (CD31, CD34 and Sca-1) of primarily cultured GFP+ NSCs was analyzed by flow cytometry and compared with those of endothelial cells (bEND.3). Few primarily cultured GFP+ NSCs expressed the endothelial or endothelial progenitor cell markers.
[Show abstract][Hide abstract]ABSTRACT: Radiation therapy is an indispensable therapeutic modality for various brain diseases. Though endogenous neural stem cells (NSCs) would provide regenerative potential, many patients nevertheless suffer from radiation-induced brain damage. Accordingly, we tested beneficial effects of exogenous NSC supplementation using in vivo mouse models that received whole brain irradiation. Systemic supplementation of primarily cultured mouse fetal NSCs inhibited radiation-induced brain atrophy and thereby preserved brain functions such as short-term memory. Transplanted NSCs migrated to the irradiated brain and differentiated into neurons, astrocytes, or oligodendrocytes. In addition, neurotrophic factors such as NGF were significantly increased in the brain by NSCs, indicating that both paracrine and replacement effects could be the therapeutic mechanisms of NSCs. Interestingly, NSCs also differentiated into brain endothelial cells, which was accompanied by the restoration the cerebral blood flow that was reduced from the irradiation. Inhibition of the VEGF signaling reduced the migration and trans-differentiation of NSCs. Therefore, trans-differentiation of NSCs into brain endothelial cells by the VEGF signaling and the consequential restoration of the cerebral blood flow would also be one of the therapeutic mechanisms of NSCs. In summary, our data demonstrate that exogenous NSC supplementation could prevent radiation-induced functional loss of the brain. Therefore, successful combination of brain radiation therapy and NSC supplementation would provide a highly promising therapeutic option for patients with various brain diseases.
[Show abstract][Hide abstract]ABSTRACT: Expression of VEGF receptors in NSCs was analyzed by semi-quantitative RT-PCR (A), immunocytochemistry (B), and flow cytometry (C). The VEGF receptor 2 (Flk-1) was predominantly expressed in NSCs while all VEGF receptors were highly expressed in endothelial cells. GAPDH = internal control. VEGF concentration in the culture medium (D) and VEGF expression of NSCs (E) were analyzed by ELISA and Real-Time PCR, respectively, at 24 hours after 0, 2, 4, or 8 Gy in vitro irradiation. n = 3 for each group. * P<0.05. (F) Changes in VEGF concentration of the brain were examined by ELISA at 24 hours after 0, 5, or 10 Gy whole brain irradiation (n = 5 for each group). * P<0.05.
[Show abstract][Hide abstract]ABSTRACT: Nitric oxide (NO) modulates the activities of various channels and receptors to participate in the regulation of neuronal intracellular Ca(2+) levels. Ca(2+) binding protein (CaBP) expression may also be altered by NO. Accordingly, we examined expression changes in calbindin-D28k, calretinin, and parvalbumin in the cerebral cortex and hippocampal region of neuronal NO synthase knockout(-/-) (nNOS(-/-)) mice using immunohistochemistry. For the first time, we demonstrate that the expression of CaBPs is specifically altered in the cerebral cortex and hippocampal region of nNOS(-/-) mice and that their expression changed according to neuronal type. As changes in CaBP expression can influence temporal and spatial intracellular Ca(2+) levels, it appears that NO may be involved in various functions, such as modulating neuronal Ca(2+) homeostasis, regulating synaptic transmission, and neuroprotection, by influencing the expression of CaBPs. Therefore, these results suggest another mechanism by which NO participates in the regulation of neuronal Ca(2+) homeostasis. However, the exact mechanisms of this regulation and its functional significance require further investigation.
Full-text Article · Jun 2011 · Anatomy & cell biology
[Show abstract][Hide abstract]ABSTRACT: One of the most detrimental hallmarks of glioblastoma multiforme (GBM) is cellular invasiveness, which is considered a potential cause of tumor recurrence. Infiltrated GBM cells are difficult to completely eradicate surgically and with local therapeutic modalities. Although much effort has focused on understanding the various mechanisms controlling GBM invasiveness, its nature remains poorly understood. In this study, we established highly serial intracranial transplantation. U87R4 cells were highly invasive and displayed stem cell-like properties, as compared to noninvasive but proliferative U87L4 cells. Microarray analysis during serial transplantation revealed that apoptosis-inducing genes (caspase3 and PDCD4) were downregulated whereas several cancer stem cell-relevant genes [Frizzled 4 (FZD4) and CD44] were upregulated in more invasive cells. U87R4 cells were resistant to anticancer drug-induced cell death, partly due to downregulation of caspase3 and PDCD4, and they retained activated Wnt/β-catenin signaling due to upregulation of Frizzled 4, which was sufficient to control neurosphere formation. We also found that FZD4 promoted expression of the epithelial to mesenchymal transition regulator SNAI1, along with acquisition of a mesenchymal phenotype. Taken together, our results argue that Frizzled 4 is a member of the Wnt signaling family that governs both stemness and invasiveness of glioma stem cells, and that it may be a major cause of GBM recurrence and poor prognosis.
[Show abstract][Hide abstract]ABSTRACT: The aim of present study is to investigate more functional neural stem cells (NSCs) could be isolated from brains with amyotrophic lateral sclerosis (ALS) and expanded in vitro, based on previous reports demonstrating de novo neurogenesis is enhanced to replace degenerating neural tissue.
Thirteen- or eighteen-week-old mutant human Cu/Zn superoxide dismutase (SOD1(G93A)) transgenic ALS and wild-type SOD1 transgenic control mice were utilized. Changes in numbers of NSCs in the dentate gyrus were analyzed by immunohistochemistry against nestin and CD133. NSCs were primarily cultured from hippocampus of ALS or control mice. Expression of NSC markers, in vitro expansion capacity, and differentiating potential were compared.
Hippocampus of 13-week-old pre-symptomatic ALS mice harbor more cells that can be propagated for more than 12 passages in vitro, compared with same age control mice. Primarily-cultured cells formed neurospheres in the NSC culture medium, expressed NSC markers, and differentiated into cells with differentiated neural cell characteristics in the differentiation condition confirming that they are NSCs. In contrast, long-term expansible NSCs could not be derived from brains of 18-week-old symptomatic ALS mice with the same experimental techniques, although they had comparable nestin-immunoreactive cells in the dentate gyrus.
These results would suggest that increased neuroregeneration in early phase of ALS could be translated to regenerative approaches; however, long-term exposure to ALS microenvironments could abolish functional capacities of NSCs.
Full-text Article · Jan 2011 · Neurological Research
[Show abstract][Hide abstract]ABSTRACT: Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) have been implicated in a large array of physiological and patho-physiological processes through their receptors (VPAC(1), VPAC(2), and PAC(1) receptor) in the central nervous system. Previously, we demonstrated age-related decreases in VPAC(1) receptor expression in the rat brain providing a possible basis of several age-induced functional changes in the aged brain. In the current study, we also examined age-related changes in PAC(1) and VPAC(2) receptors in aged rat brains using an immunohistochemical approach. We found that PAC1 immunoreactivity was significantly increased in the hippocampal formation, hypothalamus, thalamus, midbrain septal nuclei, and white matter of aged rats compared with young control rats although its distribution pattern was not altered. In contrast, both distribution pattern and immunoreactivity of VPAC(2) receptor remained unchanged in aged rat brains. These results suggest that the PACAP/VIP receptors exhibit specific expressional changes in the aged brain and that these specific changes could underlie age-associated memory and cognitive functional declines as well as several other age-induced functional changes in the brain. However, the exact regulatory mechanism and its functional significance require further elucidation.