Neurosphere Cells Research Articles

Alginate Encapsulation as a Novel Strategy for the Cryopreservation of Neurospheres

Primary cultures of brain cell neurospheres are valuable in vitro models for neurotoxicology and brain cell research. Such applications would greatly benefit from the development of efficient cryopreservation protocols that assure the availability of viable and genetically stable stocks of functional neurospheres. In this work we aimed at developing an integrated strategy allowing for long-term culture and cryopreservation of brain cell neurospheres with high viability and reduced recovery time postthawing. Microencapsulation in clinical-grade, ultrahigh viscous, highly purified alginate uniformly cross-linked with Ba(2+) was evaluated as the main strategy to avoid the commonly observed loss of cell-cell and cell-matrix interactions with consequent aggregate's fragmentation and decrease in cell viability that occurs postthawing. Brain cells isolated from 16-day-old fetal rats were cultured in spinner vessels as neurospheres, encapsulated at the 5th day of culture, and cryopreserved at day 19. Culture characterization and assessment of postthawing recovery, concerning cell metabolism, aggregate's cell type composition, and neuron-astrocyte interactions were performed through analysis of membrane integrity, metabolic activity assays, and immunohistochemistry. Our results show that the encapsulation process does not affect cell viability's central metabolism; neither cell differentiation nor cell extensions into cell networks are usually observed between neurons and astrocytes within the neurosphere structure. Neurosphere encapsulation resulted in reduced recovery time postthawing and significantly less fragmentation. Further, the use of serum-free CryoStor™ solution provided further protection for both nonencapsulated and encapsulated aggregates compared with serum-supplemented culture medium as the cryopreservation medium.

Hypoxia Increases the Expression of Stem-Cell Markers and Promotes Clonogenicity in Glioblastoma Neurospheres

Hypoxia promotes the expansion of non-neoplastic stem and precursor cell populations in the normal brain, and is common in malignant brain tumors. We examined the effects of hypoxia on stem-like cells in glioblastoma (GBM). When GBM-derived neurosphere cultures are grown in 1% oxygen, hypoxia-inducible factor 1alpha (HIF1alpha) protein levels increase dramatically, and mRNA encoding other hypoxic response genes, such as those encoding hypoxia-inducible gene-2, lysyl oxidase, and vascular endothelial growth factor, are induced over 10-fold. Hypoxia increases the stem-like side population over fivefold, and the percentage of cells expressing CD133 threefold or more. Notch pathway ligands and targets are also induced. The rise in the stem-like fraction in GBM following hypoxia is paralleled by a twofold increase in clonogenicity. We believe HIF1alpha plays a causal role in these changes, as when oxygen-stable HIF1alpha is expressed in normoxic glioma cells CD133 is induced. We used digoxin, which has been shown to lower HIF protein levels in vitro and in vivo, to inhibit the hypoxic response. Digoxin suppressed HIF1alpha protein expression, HIF1alpha downstream targets, and slowed tumor growth in vivo. In addition, pretreatment with digoxin reduced GBM flank xenograft engraftment of hypoxic GBM cells, and daily intraperitoneal injections of digoxin were able to significantly inhibit the growth of established subcutaneous glioblastoma xenografts, and suppressed expression of vascular endothelial growth factor.

Bone morphogenetic protein 4 stimulates attachment of neurospheres and astrogenesis of neural stem cells in neurospheres via phosphatidylinositol 3 kinase-mediated upregulation of N-cadherin

The neurosphere culture system is useful for expanding neural stem cells (NSCs) without affecting self-renewal potential and multipotency. However, the extrinsic signals that affect the formation or dissociation of neurospheres are poorly understood. Here, we found that bone morphogenetic protein 4 (BMP4) induced the attachment of neurospheres, astrocytic differentiation, and migration of neurosphere NSCs. These outcomes were accompanied by Akt activation and upregulation of the adhesion molecule, N-cadherin. A phosphatidylinositol 3 kinase (PI3 kinase) inhibitor (LY294002) blocked attachment of neurosphere, astrocytic differentiation, migration, and N-cadherin upregulation of neurosphre NSCs. The PI3 kinase-Akt pathway appeared to selectively mediate the effects of BMP4, as neurosphere attachment was unaffected by MEK inhibitors (PD98059 and U0126). Importantly, a neutralizing N-cadherin antibody inhibited BMP4-induced neurosphere attachment, astrocytic differentiation, and migration of neurosphere NSCs. Together, these findings show that BMP4-induced attachment of neurospheres is related to the astrocytic differentiation of these cells and that these effects are attributable, at least in part, to PI3 kinase-Akt pathway-dependent induction of N-cadherin.

Directing Astroglia from the Cerebral Cortex into Subtype Specific Functional Neurons

Author SummaryThe brain consists of two major cell types: neurons, which transmit information, and glial cells, which support and protect neurons. Interestingly, evidence suggests that some glial cells, including astroglia, can be directly converted into neurons by specific proteins, a transformation that may aid in the functional repair of damaged brain tissue. However, in order for the repaired brain areas to function properly, it is important that astroglia be directed into appropriate neuronal subclasses. In this study, we show that non-neurogenic astroglia from the cerebral cortex can be reprogrammed in vitro using just a single transcription factor to yield fully functional excitatory or inhibitory neurons. We achieved this result through forced expression of the same transcription factors that instruct the genesis of these distinct neuronal subtypes during embryonic forebrain development. Moreover we demonstrate that reactive astroglia isolated from the adult cortex after local injury can be reprogrammed into synapse-forming excitatory or inhibitory neurons following a similar strategy. Our findings provide evidence that endogenous glial cells may prove a promising strategy for replacing neurons that have degenerated due to trauma or disease.

Abstract 4285: C-Met signaling supports a stem-like transcriptional program and phenotype in glioblastoma

Abstract Neoplastic stem-like cells that efficiently propagate tumor xenograft formation exist in solid cancers including glioblastoma. These cells are considered to be responsible for therapeutic resistance and tumor recurrence. The nature of these neoplastic stem-like cells and the molecular signaling pathways that support their phenotype are incompletely defined. The receptor tyrosine kinase c-Met and its ligand HGF promote the formation and growth of multiple cancers including the gliomas. We recently found that GBM-derived neurospheres that are enriched in tumor-initiating stem-like cells widely express activated c-Met. Using flow cytometry and quantitative reverse transcription-PCR, we found that neurosphere cell subpopulations expressing c-Met at high levels also express high levels of the neuroprogenitor/stem cell markers SoX2 and Nestin in comparison to low c-Met expressing cells. Also, CD133+ cells were found to express up to 10-fold higher levels of c-Met relative to CD133- cells. We hypothesize that c-Met might function to support GBM-derived stem-like cells. To test this hypothesis, we examined the effects of c-Met activation/inhibition on the growth and gene expression patterns of established human GBM neurosphere cell lines and low passage primary neurospheres derived from GBM xenograft lines. HGF statistically significantly enhanced cell growth rates and neurosphere forming capacity, a biological marker of the stem cell phenotype. Conversely, inhibiting c-Met kinase activity with SU11274 statistically significantly diminished neurosphere formation and depleted cultures of CD133+ cells. C-Met activation by HGF activated MAPK, AKT and Stat3 and induced expression of SoX2, KLF4 and c-Myc, transcription factors that support stem cell phenotypes. Together, these findings establish a link between c-Met expression/function and the neoplastic stem-like molecular fingerprint and phenotype in GBM-derived cells. The findings suggest that c-Met inhibitors may have a role in the development of therapeutic strategies for targeting tumor-initiating stem-like cells in malignant glioma and other solid cancers. Funded by CA129192 (NIH) and the Brain Tumor Funders Collaborative. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4285.

Abstract 5159: Kruppel-like factor 9 modulates the growth and differentiation of glioblastoma-derived neoplastic stem cells

Abstract Glioblastoma (GBM) is the most prevalent brain tumor with high frequency of recurrence. During the past decade, the identification of neoplastic stem-like cells in diverse human cancers including GBM represents an important conceptual advance in cancer biology and suggests new therapeutic strategies. We found that retinoic acid (RA) induces growth arrest and cell differentiation of GBM-derived stem-like cells (GBM-SCs). RA treatment also inhibited the growth of GBM-SC xenografts in mice. We studied gene expression changes during RA-induced GBM-SC differentiation and found that kruppel-like factor 9 (KLF9) expression increased 8-fold after RA treatment. KLF proteins are evolutionarily conserved zinc finger-containing transcription factors that bind to GC-rich DNA regions. KLF9 is known to function as a progesterone receptor cofactor to regulate progesterone-responsive genes but its function in cancer and stem cell biology is essentially unknown. We used lentivirus-based methods to modulate KLF9 expression in GBM-SCs. KLF9 gain-of-function induced GBM-SC differentiation and inhibited GBM-derived neurosphere formation in clonogenic and neurosphere cell growth assays. KLF9 gain-of-function also prevented the growth of GBM-SC subcutaneous xenografts in mice. Knockdown of KLF9 suppressed RA-induced differentiation of GBM-SCs. These data indicate that KLF9 and possibly other KLF family members modulate transcriptional mechanisms that regulate GBM-SC growth, differentiation, and possibly tumor initiation. Currently, we are utilizing gene expression microarray and chromatin immunoprecipitation assays to study the KLF9 target genes. Our findings provide new insights pertaining to the molecular regulation of the GBM-SC phenotype and could lead to innovative therapeutic strategies for targeting the stem-like cell pool in malignant brain tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5159.

Abstract 4352: Using human neural stem cells to model pediatric high grade gliomas: The role of p53, c-myc, and EGFRvIII in neoplastic transformation

Abstract Brain tumors are a heterogeneous group of cancers that can arise at multiple locations in the central nervous system. Some brain tumors are restricted to specific anatomical sites, such as medulloblastoma, which is found only in the cerebellum. In the context of this heterogeneity, there are common molecular pathways which are altered across different types of brain tumors. Some of these molecular lesions include inactivation of tumor suppressors such as p53, overexpression of pro-proliferative factors such as c-myc, and alteration of cytokine pathways involving PDGF and EGF. We have isolated neural stem and progenitor cells from multiple sites in the developing human brain, including spinal cord, brain stem, cerebellum, thalamus, and cerebral cortex. These cells can be grown as neurospheres in long term culture, and can differentiate into neurons, astrocytes and oligodendrocytes. To test the hypothesis that these human neurosphere cells can give rise to brain tumors, we infected them with lentivirus encoding hTERT as well as dominant-negative p53, c-myc, and EGFRvIII. The resultant clones derived from cortex, brain stem and spinal cord grow at an increased rate compared to control GFP-infected cells. Interestingly, thalamus-derived cells in the initial analysis do not proliferate rapidly in response to dominant negative p53 and c-myc overexpression, highlighting the potential differences in neural stem and progenitor cell response to oncogenic stimuli. Dominant negative p53 and c-myc overexpressing cells maintain an ability to differentiate in vitro, similar to that observed in human glioblastoma neurosphere lines. Experiments to test the in vitro clonogenic potential and in vivo engraftment of control and infected cells are pending. Our studies suggest that, in contrast to some previous reports, human neural precursor cells can be transformed. They also form the basis for future experiments aimed at creating genetically and pathologically relevant models for pediatric brain tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4352.

Abstract 5147: Trancriptional activation of the glioma stem cell marker podoplanin by the homeobox transcription factor prox1

Abstract The homeobox transcription factor Prox1 is related to the Drosophila prospero gene, which mediates cell fate decisions of neuroblasts. Prox1 is a regulator of lymphangiogenesis but it is also expressed in the developing central nervous system. Homeobox proteins are known to play essential role in the determination of cell fate and the development of the body plan. It has been shown in Drosophila that prospero-mediated transcriptional repression of stem cell genes and activation of differentiation genes prevents tumorigenic growth, suggesting that prospero is in fact a tumor suppressor. Due to its homology to prospero, we speculated that prox1 may play a role in glioma tumorigensis. We previously identified the transmembrane glycoprotein podoplanin (pdpn) as a prognostic marker in infiltrating gliomas, the most common and lethal of adult brain tumors, by analyses of gene expression profiling data. Subsequent functional studies revealed that pdpn identified a subpopulation of cells from primary neurophere cultures and fresh tumors that have characteristics of glioma cancer stem cells (GSCs), such as radiation resistance and tumor initiation, and that the protein played a functional role in maintaining the stem cell phenotype. Like prox1, pdpn is expressed both during lymphangiogenesis and neural development and mice null for either protein have a similar phenotype, including lymphedema and the lack of terminal alveoli formation in the lung. While other transcription factors have been implicated in cancer stem cells, such as Bmi-1, prox1 has note previously been implicated to have a role in this tumor-initiating, treatment resistant cell population. We therefore hypothesized that prox1 transcriptionally regulates PDPN expression in GSCs. To test this, we examined gene expression profiling data from a large glioma dataset for correlations between PDPN and PROX1 expression. No correlation was seen between expression of the two genes; however, PROX1 expression was predictive of patient survival independent of tumor grade (p=.0333). Next, we screened primary neurosphere cell lines for levels of prox1 and pdpn by immunoblot. Co-expression with pdpn was observed in all cell lines expressing prox1. To examine the role of prox1 in PDPN expression, we compared the levels of PDPN in cells for whom prox1 expression had been silenced. In preliminary results, real-time PCR for PDPN showed significant decreases in expression in the presence of PROX1-targeting siRNA. Ongoing promotor and functional analyses will validate the role of prox1 in the transcriptional activation of PDPN. Comparison of the expression profiles of isogenic clones differing only in their expression of PROX1 identifies additional transcriptional targets. In summary, we have identified prox1 as a candidate transcription factor in the regulation of the glioma stem cell phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5147.

Abstract 14: Targeting Notch in malignant brain tumors: Crosstalk with Hedgehog as a potential mechanism of treatment resistance

Abstract Developmental signaling cascades such as Notch, Hedgehog, and Wnt are increasingly being investigated as novel therapeutic targets in malignant brain tumors. These pathways are thought to play particularly important roles in stem-like cancer cells, which seem resistant to standard chemo- and radiation therapies. Indeed, inhibiting Notch signaling in medulloblastoma or glioblastoma (GBM) cultures decreases the proportion of stem-like cells. However, we thought that other developmental stem cell maintenance pathways might compensate for the pharmacological inhibition of Notch signaling, and therefore investigated Hedgehog and Wnt. Interestingly, we found that levels of the Hedgehog targets Gli1 and Patched1B increased almost two-fold in response to pharmacological inhibition of Notch in neurosphere lines derived from malignant gliomas. The Wnt target Axin2 was also upregulated in some lines, but in a less reproducible fashion. We hypothesized that there might be direct transcriptional regulation of Hedgehog targets by the Notch pathway, and used chromatin immunoprecipitation to evaluate whether the repressive factor Hes1 bound to the first intron of Gli1. We found that Hes1 does bind to multiple N-boxes in this intron, consistent with the concept that it acts as a transcriptional repressor for Gli1. Together, our data suggest a negative feedback loop between Notch and Hedgehog by which GBMs could escape from pharmacological Notch inhibition by up-regulating Hedgehog signaling. We also investigated whether targeting both Notch and Hedgehog signaling simultaneously would kill brain tumor cells more effectively than as monotherapies. As we hypothesized, while pharmacological inhibition of Notch (using gamma-secretase inhibitor) or Hedgehog (using cyclopamine) signaling alone inhibited the growth and clonogenicity of GBM neurosphere cell lines in a dose-dependent fashion, co-treatment resulted in significantly more robust inhibition of cell growth and colony-forming ability. Moreover, when neurosphere lines capable of forming intracranial tumors were pretreated with one or both pathway inhibitors before injection into mice, co-treatment significantly prolonged survival. This is consistent with our model that inhibiting both pathways more effectively kills the xenograft-initiating cells within the culture. We next investigated whether these findings applied to primary human GBMs and found that freshly cultured neurospheres were highly susceptible to co-treatment but more refractory to monotherapy. Our findings suggest that targeting multiple developmental pathways may be necessary to fully ablate the stem-like population in malignant brain tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 14.

Abstract LB-387: MYCN dependence of tumor propagating cells in an orthotopic model of medulloblastoma

Abstract Medulloblastoma (MB) is the most common paediatric brain tumor and is a major cause of cancer death in children. MYCN expression is critical to cerebellar development, regulates the fate of neural progenitor cells (NPCs) and drives the expansion of tumor propagating cells (TPCs) thought to represent the cell-of-origin for this disease. To examine the role of MYCN in MB tumorigenesis, we established neurosphere cell lines enriched for putative TPCs from the cerebella of GTML mice (transgenic for doxycycline-regulable overexpression of MYCN and luciferase). These mice are predisposed to anaplastic/large-cell MB (AACR abstract LB-79). Neurospheres derived from GTML cerebella with positive luciferase imaging signals but no visible tumor tissue exhibited several characteristics consistent with enrichment for TPCs and/or NPCs. Spheroids proliferated efficiently in the absence of exogenous growth factor, exhibited self-renewal in clonal dilution assays, expressed high levels of Mycn protein and the NPC markers nestin and SOX2, and in response to serum, differentiated along neuronal and glial lineages (increased GFAP and III tubulin expression by immunofluorescence). Finally, orthotopic reimplantation at low titer (<250 cells) recapitulated anaplastic/large-cell MB tumor formation with short latency (<14 days) and complete penetrance. Suppression of MYCN with doxycycline (confirmed by loss of luciferase imaging) caused growth arrest via a G1 blockade of neurospheres in vitro. Pretreatment of implanted spheres significantly reduced tumor penetrance (<60%) and increased latency (2-3 months). Efficient regression of established tumors was observed with concomitant loss of nestin expression and increases in differentiated glial and neuronal markers. Taken together, these data suggest that MYCN expression drives MB tumor formation by maintenance or expansion of a population of relatively undifferentiatedTPCs present in normal cerebella. This model represents a significant tool for further investigation of the role of MYCN in MB initiation and progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-387.

Effective Adenovirus-Mediated Gene Transfer Into Neural Stem Cells Derived From Human Embryonic Stem Cells

Human embryonic stem cell-derived neural stem cells (hESC-NSCs) are an attractive cell type for studying aspects of brain development and pathology. To develop the full potential of this model system, it is important to establish a reliable methodology for the manipulation of gene expression in hNSCs. To address this issue, we used an adenoviral vector with a CMV promoter-driven green fluorescent protein (GFP) reporter gene (Ad5-GFP). We optimized conditions for Ad5-GFP infection and assessed the efficiency of infection of whole and dissociated embryonic stem cell (ESC)-derived neurospheres as well as the effect of adenoviral vectors on cell surface marker expression, proliferation, and differentiation potential. Our results demonstrate that most neurosphere cells ( approximately 70%) express the coxsackie and adenovirus receptor and can be infected with Ad5. More specifically, the CD133+ hESC-NSC population could be infected more efficiently than the CD133 population and both populations expressed GFP at high levels. At low multiplicity of infection (MOI < 25), the virus had no significant effect on stem cell marker expression (CD133 and Nestin), cell survival, cell proliferation rate, or differentiation potential. This model system provides a practical new approach to study human NSC function in the context of neurodegenerative and neoplastic disorders.

CARBON NANOTUBE RIBBON AND THREAD SUPPORT ATTACHMENT AND DIFFERENTIATION OF NEURAL STEM CELLS

Carbon nanotubes (CNTs) have properties that promise an exciting role in nervous tissue repair. CNTs are strong, extremely light weight, biocompatible and electrically conductive. A relatively novel form of CNT material, multiple strands of CNTs spun into thread, accentuates the linear geometry of CNTs while retaining electrical conductivity. We propose that CNT thread, which is strong, pliable and readily manipulated, has the potential to support longitudinal growth of nerves after injury, in nervous system prostheses. Towards understanding how CNT materials support nervous tissue regeneration, we examined the in vitro interactions between CNT materials and neural stem cell-containing neurospheres, prepared from newborn mouse cortices. Intact neurospheres attached to pieces of as-grown arrays of CNTs, CNT "ribbon" material pulled from the arrays, and CNT thread. Stem cells differentiated into both neurons and glia under these conditions. Processes and cells aligned with the longitudinal axis of the CNT materials, both with and without coatings. For comparisons, dissociated neurosphere cells were plated on CNT thread, polypropylene surgical suture thread and commercial carbon fibers. Initial cell attachment (within seconds) and attachment at 24 h was greater on CNT thread than on the other fiber types. Stem cells on CNT threads differentiated into neurons and astrocytes over several days, on all fibers. The presence of serum greatly aided the health and spreading of both neurons and astrocytes. These findings demonstrate that CNT materials, in particular the thread form, are viable preparations for neural cell attachment, outgrowth and differentiation.

Selective chromosome segregation in mouse neurosphere cells

Selective chromosome segregation in mouse neurosphere cells

Genetic instability and diminished differentiation capacity in long-term cultured mouse neurosphere cells

The potential use of neural stem cells in basic research, drug testing and for development of therapeutic strategies requires large scale in vitro amplification, increasing the probability of genetic instability and transformation. Little is known, however, about potential correlations between long-term culture of neural stem and progenitor cells (NSPCs), changed differentiation and self-renewal capacities, and the occurrence of chromosomal instability. This study investigates the effect of extended culture time on self-renewal, differentiation capacity, cell cycle phase distribution, telomere length, telomerase activity and chromosomal stability on fetal brain-derived cells that form floating sphere colonies (neurospheres). We observed that increased sphere-forming capacity indicative of increased proliferation was accompanied by a decreased ability to differentiate into neural lineages. The high mobility group A (Hmga2) gene positively regulates self-renewal via repression of p16(Ink4a) and p19(ARF) gene expression. This study discerned an upregulation of Hmga2 gene and protein expression and decreased p16(Ink4a) and p19(ARF) gene expression, suggesting that Hmga2 might promote the proliferation of neurosphere cells in long-term culture. Further, our analyses revealed a significant decrease in telomere length after 4 weeks of culturing that is paralleled by a moderate upregulation of telomerase activity. Importantly, regular gain of chromosome 1 with random structural chromosomal aberrations was observed within 16 weeks of neurosphere cell culture. Genetic instability and diminished differentiation capacity seem to be a consequence of long-term culture of neurosphere cells. These data indicate the necessity to analyze self-renewal, differentiation capacity, telomere length, tumor suppressor genes and chromosomal stability in neurosphere cultures prior to their usage in basic research, drug testing or the development of therapeutic strategies.

Multipotency of cultured olfactory epithelium neural stem cells

Objective:To investigate the culture method for adult olfactory epithelium neural stem cells and their multipotency during in vitro differentiation.Methods:Olfactory epithelium was sequentially digested with neutral protease and collagenase ⅠA,then the olfactory epithelium neural stem cells were enriched and amplified.Neurospheres were formed by culturing the cells with serum free medium containing bFGF and EGF.Differentiation of neurospheres was induced with medium containing serum;the differentiated cells were identified by immunocytochemistry.Results:Primary cultured olfactory epithelium neural stem cells proliferated and formed colonies.Cells in the colonies expressed the in vivo markers GBC2 and cytokeratin 5.Passaged cells proliferated and formed neurospheres when cultured with serum free medium containing bFGF and EGF.Medium containing serum induced the differentiation of neurospheres.TUJ1 positive neurons,P75NGFR positive olfactory ensheathing cells,GFAP positive astrocytes and NG2 positive oligodendrocyte progenitors were detected in the highly heterogeneous differentiated cells;besides,the cells also contained nestin positive neural stem cells.Conclusion:Cultured adult olfactory epithelium neural stem cells can proliferate and form neurospheres.The neurosphere cells have the multipotency to differentiate into neurons,astrocytes and oligodendrocyte progenitors.

Unconditioned adult-derived neurosphere cells mainly differentiate towards astrocytes upon transplantation in sclerotic rat hippocampus

Cell transplantation is being investigated as an alternative treatment for medically refractory temporal lobe epilepsy (TLE). In this study the fate of adult-derived neurosphere cells was evaluated after transplantation in the lesioned hippocampus of the intrahippocampal kainic acid (KA) model for TLE. Neurosphere-forming cells were derived from the subventricular zone (SVZ) of transgenic green fluorescent protein (GFP) reporter mice and expanded in culture. After 10 passages in vitro neurosphere-derived cells were transplanted in the hippocampus three days (KA3d group) and three weeks (KA3w group) after intrahippocampal KA injection. Survival and differentiation of neurosphere cells were evaluated three and six weeks after transplantation. A fraction (about 1%) of GFP-expressing neurosphere cells survived for at least six weeks after transplantation with a higher and more robust survival rate in the KA3d compared to the KA3w group. Although a small fraction of the cells expressed the neuronal marker NeuN, neurosphere cells mainly differentiated towards astrocytes. Our results indicate that adult-derived neurosphere cells are able to survive upon transplantation in the sclerotic hippocampus. The transplanted cells do not or hardly contribute to neuronal replacement and mainly adopt an astrogliotic fate.

Vascular endothelial growth factor enhances migration of astroglial cells in subventricular zone neurosphere cultures

Vascular endothelial growth factor (VEGF) is an endothelial and neuronal survival factor and a mitogen for endothelial cells and astrocytes in both explant and in vivo injury models. In the CNS, interplay between the vasculature and neural stem progenitor (NSP) cells is required for the maintenance of angiogenic/neurogenic coordination in the germinal niche in the subventricular zone (SVZ) of the lateral ventricle. Using an in vitro SVZ neurosphere (NS) model, this study aimed to understand the direct effects of VEGF and its receptor signaling on neonatal NSP cell growth and migration. Our data indicate that VEGF administration, compared with untreated or brain-derived neurotrophic factor-treated NS, significantly increased growth and migratory capacity of glial fibrillary acidic protein (GFAP)(+) and nestin(+) NSP cells and in secondary cultures induced a stellate astrocyte morphology. Blockade of both VEGF, which is normally expressed in some NS cells, and its flt-1 receptor signaling by neutralizing antibodies caused morphological changes specifically in GFAP(+) cells and disrupted sphere formation and outward migration. These cells did not appear as conventional polygonal astrocytes; their process growth was severely restricted, and overall migration was reduced by up to 76% of control cultures. Blockade of VEGF's flk-1 receptor reduced VEGF expression and caused a lesser, though significant, decrease (29%) in NSP (GFAP(+)) cell migration. The results show that both VEGF and, in particular, flt-1 receptor signaling are critical to the proper configuration of the NS and its subsequent development. VEGF is also an important growth and migratory factor particularly for GFAP(+) cells developing in SVZ-derived NS in culture.

A brain slice culture model for studies of endogenous and exogenous precursor cell migration in the rostral migratory stream

The rostral migratory stream (RMS) is the main pathway by which newly born subventricular zone (SVZ) cells reach the olfactory bulb (OB) in rodents. This migration has been well studied in vivo, but an organotypic in vitro model would facilitate more experimental investigations. Here we introduce a slice culture preparation of the rat forebrain including en suite the rostral part of the lateral ventricle, the RMS and the OB. The preparation was validated with regard to endogenous cell proliferation and migration by tracking bromodeoxyuridine (BrdU)-labelled cells in newly established and 3 and 6 week old cultures. For testing the migratory abilities of exogenous precursor cells, rat SVZ neurospheres and human neural (HNS1 cells) and mesenchymal (hMSC-TERT) stem cell lines were micrografted to the rostral SVZ of 1 and 7 day old cultures. Two weeks later graft derivatives were identified by immunohistochemical staining for human nuclei (HNS1/hMSC-TERT cells) and BrdU (HNS1 cells/neurospheres). Numerous HNS1 cells and BrdU-positive neurosphere cells were found in the RMS. Having reached the OB, subpopulations of the cells expressed the astroglial markers glial fibrillary acidic protein/hAM and the neuronal markers NeuN/tyrosine hydroxylase. Interestingly, the hMSC-TERT cells remained at the implantation site, demonstrating a diversity in migratory capability of different precursor cells. In conclusion, the RMS in rat forebrain slice cultures retains its ability to support migration of endogenous and exogenous neural precursors, making the cultures highly feasible for studies of conditions and factors regulating cell migration.

4 Tubulin Identifies a Primitive Cell Source for Oligodendrocytes in the Mammalian Brain

We have identified a novel population of cells in the subventricular zone (SVZ) of the mammalian brain that expresses beta4 tubulin (betaT4) and has properties of primitive neuroectodermal cells. betaT4 cells are scattered throughout the SVZ of the lateral ventricles in adult human brain and are significantly increased in the SVZs bordering demyelinated white matter in multiple sclerosis brains. In human fetal brain, betaT4 cell densities peak during the latter stages of gliogenesis, which occurs in the SVZ of the lateral ventricles. betaT4 cells represent <2% of the cells present in neurospheres generated from postnatal rat brain but >95% of cells in neurospheres treated with the anti-mitotic agent Ara C. betaT4 cells produce oligodendrocytes, neurons, and astrocytes in vitro. We compared the myelinating potential of betaT4-positive cells with A2B5-positive oligodendrocyte progenitor cells after transplantation (25,000 cells) into postnatal day 3 (P3) myelin-deficient rat brains. At P20, the progeny of betaT4 cells myelinated up to 4 mm of the external capsule, which significantly exceeded that of transplanted A2B5-positive progenitor cells. Such extensive and rapid mature CNS cell generation by a relatively small number of transplanted cells provides in vivo support for the therapeutic potential of betaT4 cells. We propose that betaT4 cells are an endogenous cell source that can be recruited to promote neural repair in the adult telencephalon.

Loss of ATM Impairs Proliferation of Neural Stem Cells Through Oxidative Stress-Mediated p38 MAPK Signaling

Ataxia-telangiectasia (A-T) is a genetic disorder caused by a mutation of the Atm gene, which controls DNA repair, cell cycling, and redox homeostasis. Even though oxidative stress has been implicated in the neurological anomalies in A-T, the effects of ATM loss on neural stem cell (NSC) survival has remained elusive. In this study, we investigated the effects of oxidative stress on NSC proliferation in an animal model for A-T neurodegeneration. We found that cultured subventricular zone neurosphere cells from Atm(-/-) mice show impaired proliferation, as well as intrinsic elevation of reactive oxygen species (ROS) levels, compared with those from Atm(+/+) mice. We also show that increasing the levels of ROS by H(2)O(2) treatment significantly reduces Atm(+/+) neurosphere formation and proliferation. In Atm(-/-) neurosphere cells, the Akt and Erk1/2 pathways are disrupted, together with enhanced activity of the p38 mitogen-activated protein kinase (MAPK). Treatment of these cells with the antioxidant N-acetyl-L-cysteine (NAC) or with a p38 MAPK inhibitor restores normal proliferation and reduced expression of p21(cip1) and p27(kip1) in the Atm(-/-) NSCs. These observations indicate that ATM plays a crucial role in NSC proliferation, by activating Akt and Erk1/2 pathways and by suppressing ROS-p38 MAPK signaling. Together, our results suggest that p38 MAPK signaling acts as a negative regulator of NSC proliferation in response to oxidative stress. These findings suggest a potential mechanism for neuronal cell loss as a result of oxidative stress in NSCs in progressive neurodegenerative diseases such as A-T.