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Astrocytes are the most abundant glial cells in the brain and are responsible for diverse functions, from modulating synapse function to regulating the blood-brain barrier. In vivo, these cells exhibit a star-shaped morphology with multiple radial processes that contact synapses and completely surround brain capillaries. In response to trauma or CNS disease, astrocytes become activated, a state associated with profound changes in gene expression, including upregulation of intermediate filament proteins, such as glial fibrillary acidic protein (GFAP). The inability to recapitulate the complex structure of astrocytes and maintain their quiescent state in vitro is a major roadblock to further developments in tissue engineering and regenerative medicine. Here, we characterize astrocyte morphology and activation in various hydrogels to assess the feasibility of developing a matrix that mimics key aspects of the native microenvironment. We show that astrocytes seeded in optimized matrix composed of collagen, hyaluronic acid, and matrigel exhibit a star-shaped morphology with radial processes and do not upregulate GFAP expression, hallmarks of quiescent astrocytes in the brain. In these optimized gels, collagen I provides structural support, HA mimics the brain extracellular matrix, and matrigel provides endothelial cell compatibility and was found to minimize GFAP upregulation. This defined 3D microenvironment for maintaining human astrocytes in vitro provides new opportunities for developing improved models of the blood-brain barrier and studying their response to stress signals., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The subventricular zone of the lateral ventricles (SVZ) is the largest source of neural stem cells (NSCs) in the adult mammalian brain. Newly generated neuroblasts from the SVZ form cellular chains that migrate through the rostral migratory stream (RMS) into the olfactory bulb (OB), where they become mature neurons. Migration through the RMS is a highly regulated process of intrinsic and extrinsic factors, orchestrated to achieve direction and integration of neuroblasts into OB circuitry. These factors include internal cytoskeletal and volume regulators, extracellular matrix proteins, and chemoattractant and chemorepellent proteins. All these molecules direct the cells away from the SVZ, through the RMS, and into the OB guaranteeing their correct integration. Following brain injury, some neuroblasts escape the RMS and migrate into the lesion site to participate in regeneration, a phenomenon that is also observed with brain tumors. This review focuses on factors that regulate the migration of SVZ precursor cells in the healthy and pathologic brain. A better understanding of the factors that control the movement of newly generated cells may be crucial for improving the use of NSC-replacement therapy for specific neurological diseases.

It is increasingly important to understand the molecular basis for the plasticity of neoplastic cells and their capacity to transition between differentiated and stemlike phenotypes. Kruppel-like factor-9 (KLF9), a member of the large KLF transcription factor family, has emerged as a regulator of oncogenesis, cell differentiation, and neural development; however, the molecular basis for the diverse contextual functions of KLF9 remains unclear. This study focused on the functions of KLF9 in human glioblastoma stemlike cells. We established for the first time a genome-wide map of KLF9-regulated targets in human glioblastoma stemlike cells and show that KLF9 functions as a transcriptional repressor and thereby regulates multiple signaling pathways involved in oncogenesis and stem cell regulation. A detailed analysis of one such pathway, integrin signaling, showed that the capacity of KLF9 to inhibit glioblastoma cell stemness and tumorigenicity requires ITGA6 repression. These findings enhance our understanding of the transcriptional networks underlying cancer cell stemness and differentiation and identify KLF9-regulated molecular targets applicable to cancer therapeutics., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Unlabelled: Glioblastoma (GBM) remains the most aggressive primary brain cancer in adults. Similar to other cancers, GBM cells undergo metabolic reprogramming to promote proliferation and survival. Glycolytic inhibition is widely used to target such reprogramming. However, the stability of glycolytic inhibition in GBM remains unclear especially in a hypoxic tumor microenvironment. In this study, it was determined that glucose-6-phosphatase (G6PC/G6Pase) expression is elevated in GBM when compared with normal brain. Human-derived brain tumor-initiating cells (BTIC) use this enzyme to counteract glycolytic inhibition induced by 2-deoxy-d-glucose (2DG) and sustain malignant progression. Downregulation of G6PC renders the majority of these cells unable to survive glycolytic inhibition, and promotes glycogen accumulation through the activation of glycogen synthase (GYS1) and inhibition of glycogen phosphorylase (PYGL). Moreover, BTICs that survive G6PC knockdown are less aggressive (reduced migration, invasion, proliferation, and increased astrocytic differentiation). Collectively, these findings establish G6PC as a key enzyme with promalignant functional consequences that has not been previously reported in GBM and identify it as a potential therapeutic target., Implications: This study is the first to demonstrate a functional relationship between the critical gluconeogenic and glycogenolytic enzyme G6PC with the metabolic adaptations during GBM invasion., (©2014 American Association for Cancer Research.)

Glioblastoma (GBM) stem cells (GSC) are a subpopulation of tumor cells that display stem-like characteristics (stemness) and play unique roles in tumor propagation, therapeutic resistance, and tumor recurrence. Therapeutic targets in GSCs are a focus of increasing interest to improve GBM therapy. Here we report that the hyaluronan-mediated motility receptor (HMMR) is highly expressed in GBM tumors, where it supports the self-renewal and tumorigenic potential of GSCs. HMMR silencing impairs GSC self-renewal and inhibits the expression of GSC markers and regulators. Furthermore, HMMR silencing suppresses GSC-derived tumor growth and extends the survival of mice bearing GSC xenografts. Conversely, HMMR overexpression promotes GSC self-renewal and intracranial tumor propagation. In human GBM tumor specimens, HMMR expression is correlated positively with the expression of stemness-associated markers and regulators. Our findings identify HMMR as a candidate therapeutic target to GSCs as a GBM treatment strategy., (©2014 American Association for Cancer Research.)

Purpose: Glioblastoma is the most common adult primary malignant intracranial cancer. It is associated with poor outcomes because of its invasiveness and resistance to multimodal therapies. Human adipose-derived mesenchymal stem cells (hAMSC) are a potential treatment because of their tumor tropism, ease of isolation, and ability to be engineered. In addition, bone morphogenetic protein 4 (BMP4) has tumor-suppressive effects on glioblastoma and glioblastoma brain tumor-initiating cells (BTIC), but is difficult to deliver to brain tumors. We sought to engineer BMP4-secreting hAMSCs (hAMSCs-BMP4) and evaluate their therapeutic potential on glioblastoma., Experimental Design: The reciprocal effects of hAMSCs on primary human BTIC proliferation, differentiation, and migration were evaluated in vitro. The safety of hAMSC use was evaluated in vivo by intracranial coinjections of hAMSCs and BTICs in nude mice. The therapeutic effects of hAMSCs and hAMSCs-BMP4 on the proliferation and migration of glioblastoma cells as well as the differentiation of BTICs, and survival of glioblastoma-bearing mice were evaluated by intracardiac injection of these cells into an in vivo intracranial glioblastoma murine model., Results: hAMSCs-BMP4 targeted both the glioblastoma tumor bulk and migratory glioblastoma cells, as well as induced differentiation of BTICs, decreased proliferation, and reduced the migratory capacity of glioblastomas in vitro and in vivo. In addition, hAMSCs-BMP4 significantly prolonged survival in a murine model of glioblastoma. We also demonstrate that the use of hAMSCs in vivo is safe., Conclusions: Both unmodified and engineered hAMSCs are nononcogenic and effective against glioblastoma, and hAMSCs-BMP4 are a promising cell-based treatment option for glioblastoma., (©2014 AACR.)

Neurogenesis persists in the adult subventricular zone (SVZ) of the mammalian brain. During aging, the SVZ neurogenic capacity undergoes a progressive decline, which is attributed to a decrease in the population of neural stem cells (NSCs). However, the behavior of the NSCs that remain in the aged brain is not fully understood. Here we performed a comparative ultrastructural study of the SVZ niche of 2-month-old and 24-month-old male C57BL/6 mice, focusing on the NSC population. Using thymidine-labeling, we showed that residual NSCs in the aged SVZ divide less frequently than those in young mice. We also provided evidence that ependymal cells are not newly generated during senescence, as others studies suggest. Remarkably, both astrocytes and ependymal cells accumulated a high number of intermediate filaments and dense bodies during aging, resembling reactive cells. A better understanding of the changes occurring in the neurogenic niche during aging will allow us to develop new strategies for fighting neurological disorders linked to senescence.

Astrocytes are the most prevalent type of glial cell in the brain, participating in a variety of diverse functions from regulating cerebral blood flow to controlling synapse formation. Astrocytes and astrocyte-conditioned media are widely used in models of the blood-brain barrier (BBB), however, very little is known about astrocyte culture in 2D. To test the hypothesis that surface coating and soluble factors influence astrocyte morphology in 2D, we quantitatively analyzed the morphology of human fetal derived astrocytes on glass, matrigel, fibronectin, collagen IV, and collagen I, and after the addition soluble factors including platelet-derived growth factor (PDGF), laminin, basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF). Matrigel surface coatings, as well as addition of leukemia inhibitory factor (LIF) to the media, were found to have the strongest effects on 2D astrocyte morphology, and may be important in improving existing BBB models. In addition, the novel set of quantitative parameters proposed in this paper provide a test for determining the influence of compounds on astrocyte morphology, both to screen for new endothelial cell-secreted factors that influence astrocytes, and to determine in a high-throughput way which factors are important for translation to more complex, 3D BBB models.

Radiation is a common tool in the treatment of brain tumors that induces neurological deficits as a side effect. Some of these deficits appear to be related to the impact of radiation on the neurogenic niches, producing a drastic decrease in the proliferative capacity of these regions. In the adult mammalian brain, the subventricular zone (SVZ) of the lateral ventricles is the main neurogenic niche. Neural stem/precursor cells (NSCs) within the SVZ play an important role in brain repair following injuries. However, the irradiated NSCs' ability to respond to damage has not been previously elucidated. In this study, we evaluated the effects of localized radiation on the SVZ ability to respond to a lysolecithin-induced demyelination of the striatum. We demonstrated that the proliferation rate of the irradiated SVZ was increased after brain damage and that residual NSCs were reactivated. The irradiated SVZ had an expansion of doublecortin positive cells that appeared to migrate from the lateral ventricles toward the demyelinated striatum, where newly generated oligodendrocytes were found. In addition, in the absence of demyelinating damage, remaining cells in the irradiated SVZ appeared to repopulate the neurogenic niche a year post-radiation. These findings support the hypothesis that NSCs are radioresistant and can respond to a brain injury, recovering the neurogenic niche. A more complete understanding of the effects that localized radiation has on the SVZ may lead to improvement of the current protocols used in the radiotherapy of cancer., (Copyright © 2013 AlphaMed Press.)

The subventricular zone (SVZ) is the largest source of newly generated cells in the adult mammalian brain. SVZ-derived neuroblasts migrate via the rostral migratory stream (RMS) to the olfactory bulb (OB), where they differentiate into mature neurons. Additionally, a small proportion of SVZ-derived cells contribute to the generation of myelinating oligodendrocytes. The production of new cells in the SVZ decreases during aging, affecting the incorporation of new neurons into the OB. However, the age-related changes that occur across the RMS are not fully understood. In this study we evaluate how aging affects the cellular organization of migrating neuroblast chains, the proliferation, and the fate of the newly generated cells in the SVZ-OB system. By using electron microscopy and immunostaining, we found that the RMS path becomes discontinuous and its cytoarchitecture is disorganized in aged mice (24-month-old mice). Subsequently, OB neurogenesis was impaired in the aged brain while the production of oligodendrocytes was not compromised. These findings provide new insight into oligodendrocyte preservation throughout life. Further exploration of this matter could help the development of new strategies to prevent neurological disorders associated with senescence.

Purpose: Neural progenitor cells in the subventricular zone (SVZ) have a controversial role in glioblastoma multiforme (GBM) as potential tumor-initiating cells. The purpose of this study was to examine the relationship between radiation dose to the SVZ and survival in GBM patients., Methods and Materials: The study included 116 patients with primary GBM treated at the Johns Hopkins Hospital between 2006 and 2009. All patients underwent surgical resection followed by adjuvant radiation therapy with intensity modulated radiation therapy (60 Gy/30 fractions) and concomitant temozolomide. Ipsilateral, contralateral, and bilateral SVZs were contoured on treatment plans by use of coregistered magnetic resonance imaging and computed tomography. Multivariate Cox regression was used to examine the relationship between mean SVZ dose and progression-free survival (PFS), as well as overall survival (OS). Age, Karnofsky Performance Status score, and extent of resection were used as covariates. The median age was 58 years (range, 29-80 years)., Results: Of the patients, 12% underwent biopsy, 53% had subtotal resection (STR), and 35% had gross total resection (GTR). The Karnofsky Performance Status score was less than 90 in 54 patients and was 90 or greater in 62 patients. The median ipsilateral, contralateral, and bilateral mean SVZ doses were 48.7 Gy, 34.4 Gy, and 41.5 Gy, respectively. Among patients who underwent GTR, a mean ipsilateral SVZ dose of 40 Gy or greater was associated with a significantly improved PFS compared with patients who received less than 40 Gy (15.1 months vs 10.3 months; P=.028; hazard ratio, 0.385 [95% confidence interval, 0.165-0.901]) but not in patients undergoing STR or biopsy. The subgroup of GTR patients who received an ipsilateral dose of 40 Gy or greater also had a significantly improved OS (17.5 months vs 15.6 months; P=.027; hazard ratio, 0.385 [95% confidence interval, 0.165-0.895]). No association was found between SVZ radiation dose and PFS and OS among patients who underwent STR or biopsy., Conclusion: A mean radiation dose of 40 Gy or greater to the ipsilateral SVZ was associated with a significantly improved PFS and OS in patients with GBM after GTR., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Introduction: Glioblastoma is the most common primary malignant brain tumor, and is refractory to surgical resection, radiation, and chemotherapy. Human mesenchymal stem cells (hMSC) may be harvested from bone marrow (BMSC) and adipose (AMSC) tissue. These cells are a promising avenue of investigation for the delivery of adjuvant therapies. Despite extensive research into putative mechanisms for the tumor tropism of MSCs, there remains no direct comparison of the efficacy and specificity of AMSC and BMSC tropism towards glioma., Methods: Under an IRB-approved protocol, intraoperative human Adipose MSCs (hAMSCs) were established and characterized for cell surface markers of mesenchymal stem cell origin in conjunction with the potential for tri-lineage differentiation (adipogenic, chondrogenic, and osteogenic). Validated experimental hAMSCs were compared to commercially derived hBMSCs (Lonza) and hAMSCs (Invitrogen) for growth responsiveness and glioma tropism in response to glioma conditioned media obtained from primary glioma neurosphere cultures., Results: Commercial and primary culture AMSCs and commercial BMSCs demonstrated no statistically significant difference in their migration towards glioma conditioned media in vitro. There was statistically significant difference in the proliferation rate of both commercial AMSCs and BMSCs as compared to primary culture AMSCs, suggesting primary cultures have a slower growth rate than commercially available cell lines., Conclusions: Adipose- and bone marrow-derived mesenchymal stem cells have similar in vitro glioma tropism. Given the well-documented ability to harvest larger numbers of AMSCs under local anesthesia, adipose tissue may provide a more efficient source of MSCs for research and clinical applications, while minimizing patient morbidity during cell harvesting.

Glioblastoma (GB) is a highly invasive and lethal brain tumor due to its universal recurrence. Although it has been suggested that the electroneutral Na(+)-K(+)-Cl(-) cotransporter 1 (NKCC1) can play a role in glioma cell migration, the precise mechanism by which this ion transporter contributes to GB aggressiveness remains poorly understood. Here, we focused on the role of NKCC1 in the invasion of human primary glioma cells in vitro and in vivo. NKCC1 expression levels were significantly higher in GB and anaplastic astrocytoma tissues than in grade II glioma and normal cortex. Pharmacological inhibition and shRNA-mediated knockdown of NKCC1 expression led to decreased cell migration and invasion in vitro and in vivo. Surprisingly, knockdown of NKCC1 in glioma cells resulted in the formation of significantly larger focal adhesions and cell traction forces that were approximately 40% lower than control cells. Epidermal growth factor (EGF), which promotes migration of glioma cells, increased the phosphorylation of NKCC1 through a PI3K-dependant mechanism. This finding is potentially related to WNK kinases. Taken together, our findings suggest that NKCC1 modulates migration of glioma cells by two distinct mechanisms: (1) through the regulation of focal adhesion dynamics and cell contractility and (2) through regulation of cell volume through ion transport. Due to the ubiquitous expression of NKCC1 in mammalian tissues, its regulation by WNK kinases may serve as new therapeutic targets for GB aggressiveness and can be exploited by other highly invasive neoplasms., Competing Interests: The authors have declared that no competing interests exist.

Blast-Induced Traumatic Brain Injury (bTBI) describes a spectrum of injuries caused by an explosive force that results in changes in brain function. The mechanism responsible for primary bTBI following a blast shockwave remains unknown. We have developed a pneumatic device that delivers shockwaves, similar to those known to induce bTBI, within a chamber optimal for fluorescence microscopy. Abrupt changes in pressure can be created with and without the presence of shear forces at the surface of cells. In primary cultures of human central nervous system cells, the cellular calcium response to shockwaves alone was negligible. Even when the applied pressure reached 15 atm, there was no damage or excitation, unless concomitant shear forces, peaking between 0.3 to 0.7 Pa, were present at the cell surface. The probability of cellular injury in response to a shockwave was low and cell survival was unaffected 20 hours after shockwave exposure.

Tumor-initiating stem cells (alternatively called cancer stem cells, CSCs) are a subpopulation of tumor cells that plays unique roles in tumor propagation, therapeutic resistance, and tumor recurrence. It is becoming increasingly important to understand the molecular signaling that regulates the self-renewal and differentiation of CSCs. Transcription factors are critical for the regulation of normal and neopolastic stem cells. Here, we examined the expression and function of the Krüppel-like family of transcription factors (KLFs) in human glioblastoma (GBM)-derived neurosphere lines and low-passage primary GBM-derived neurospheres that are enriched for tumor-initiating stem cells. We identify KLF9 as a relatively unique differentiation-induced transcription factor in GBM-derived neurospheres. KLF9 is shown to induce neurosphere cell differentiation, inhibit neurosphere formation, and inhibit neurosphere-derived xenograft growth in vivo. We also show that KLF9 regulates GBM neurosphere cells by binding to the Notch1 promoter and suppressing Notch1 expression and downstream signaling. Our results show for the first time that KLF9 has differentiating and tumor-suppressing functions in tumor-initiating stem cells.

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants. The brain lesion that an injecting needle produces during its trajectory is a major concern especially in the mouse brain for not only the brain is small but also sometimes multiple injections are needed. We show here a method to produce glass capillary needles with a 50-μm lumen which significantly reduces the brain damage and allows a precise targeting into the rodent brain. This method allows a delivery of small volumes (from 20 to 100 nl), reduces bleeding risks, and minimizes passive diffusion of drugs into the brain parenchyma. By using different size of capillary glass tubes, or changing the needle lumen, several types of substances and cells can be injected. Microinjections with a glass capillary tube represent a significant improvement in injection techniques and deep brain targeting with minimal collateral damage in small rodents.

The discoveries of neural (NSCs) and brain tumor stem cells (BTSCs) in the adult human brain and in brain tumors, respectively, have led to a new era in neuroscience research. These cells represent novel approaches to studying normal phenomena such as memory and learning, as well as pathological conditions such as Parkinson's disease, stroke, and brain tumors. This new paradigm stresses the importance of understanding how these cells behave in vitro and in vivo. It also stresses the need to use human-derived tissue to study human disease because animal models may not necessarily accurately replicate the processes that occur in humans. An important, but often underused, source of human tissue and, consequently, both NSCs and BTSCs, is the operating room. This study describes in detail both current and newly developed laboratory techniques, which in our experience are used to process and study human NSCs and BTSCs from tissue obtained directly from the operating room.

Objective: There has been an increased focus on the region adjacent to the lateral ventricles (LV) as a potential source of malignant tumors and/or more aggressive disease. We set out to determine if glioblastoma multiforme (GBM) bordering the LV was associated with decreased survival as compared to non-LV GBM., Methods: We reviewed the clinical records of 69 consecutive patients undergoing craniotomy for GBM at a single academic institution. Twenty-six patients were identified with contrast-enhancing lesions (CEL) bordering the LV (LV CEL). These 26 patients were matched with 26 patients with CEL not bordering the LV (non-LV CEL). These cohorts were matched for factors consistently shown to be associated with survival, which were age, tumor size, Karnofsky performance score, extent of resection, Gliadel implantation, and Temodar chemotherapy. Overall survival was compared between the cohorts via Log-rank analysis., Results: Despite similarities in pre-operative clinical status, tumor size, peri-operative outcome, and treatment regimens, the median survival for patients with LV CEL was significantly decreased as compared to patients with non-LV CEL (8 months vs. 11 months), P = 0.02. Additionally, survival analysis in patients stratified by primary and secondary resection also demonstrated a strong trend towards decreased survival after resection of LV CEL. After primary and secondary resection, patients with LV CEL versus non-LV CEL had a median survival of 11 months vs. 14 months (P = 0.10) and 7 months vs. 10 months (P = 0.11), respectively., Conclusion: While the causal factors underlying this observation are not provided with this observational study, GBM bordering the LV may carry a prognostic significance.

Aims: The article highlights the general structural characteristics, functional properties and distribution of glutamate transporters, as well as the role they play in epilepsy and oxidative stress., Development: Transporters of amino acids such as glutamate are considered to be proteins that are extremely important in the central nervous system because they participate in the capture of the neurotransmitter following its release in the synaptic cleft, thus putting an end to its effect and limiting glutamate-mediated excitability. These proteins belong to the family of Na+/K+ dependent transporters. A growing body of evidence has been gathered to show that these transporters are involved in several neuronal disorders, such as epilepsy and cerebral ischaemia. In this regard, it is considered that some defect in the structure of the transporters could affect their functioning and, therefore, favour the hyperexcitability produced by glutamate; this in turn would lead to the pathological disorders that are found in epilepsy., Conclusions: A detailed study of the structure and functioning of these transporters, as well as the role they play in the more common neurological diseases, such as epilepsy, would afford us a clearer view of new therapeutic alternatives with which to fight this kind of neuronal disorder in the future.

Pig neural cells express glycoproteins with sialylated N-linked oligosaccharide chains (SNOC) which are used by the porcine rubulavirus (PoRv) as receptors. Pig neuronal or glial cell cultures were employed to investigate (a) whether PoRv infects such cells using a molecule expressing SNOC, and (b) the role of viral envelope glycoproteins in establishing the infection. Enriched neuronal or glial cell cultures were exposed to PoRv and infection was detected immunocytochemically. Neuronal cultures prepared from neonatal pigs were treated enzymatically to eliminate sialic acid or N-linked oligosaccharide chains. Primary neural cultures were exposed to anti-HN or anti-F preincubated with PoRv to study the role of the viral glycoproteins. In enriched cultures, PoRv infected neurons and glial cells, and sialic acid expressed in N-linked oligosaccharide chains appeared to play a central role in infection. It was concluded that HN and F viral glycoproteins are required to infect neurons and glial cells.