Differentiation Of Neurospheres Research Articles

Generation and Differentiation of Primary Neurospheres from Zebrafish Neural Stem Cells

Generation and Differentiation of Primary Neurospheres from Zebrafish Neural Stem Cells

Generation and Differentiation of Primary Neurospheres from Zebrafish Neural Stem Cells

Generation and Differentiation of Primary Neurospheres from Zebrafish Neural Stem Cells

SFlt-1 impairs neurite growth and neuronal differentiation in SH-SY5Y cells and human neurons.

Pre-eclampsia (PE) is a hypertensive disorder of pregnancy which is associated with increased risk of neurodevelopmental disorders in exposed offspring. The pathophysiological mechanisms mediating this relationship are currently unknown, and one potential candidate is the anti-angiogenic factor soluble Fms-like tyrosine kinase 1 (sFlt-1), which is highly elevated in PE. While sFlt-1 can impair angiogenesis via inhibition of VEGFA signalling, it is unclear whether it can directly affect neuronal development independently of its effects on the vasculature. To test this hypothesis, the current study differentiated the human neural progenitor cell (NPC) line ReNcell® VM into a mixed culture of mature neurons and glia, and exposed them to sFlt-1 during development. Outcomes measured were neurite growth, cytotoxicity, mRNA expression of nestin, MBP, GFAP, and βIII-tubulin, and neurosphere differentiation. sFlt-1 induced a significant reduction in neurite growth and this effect was timing- and dose-dependent up to 100 ng/ml, with no effect on cytotoxicity. sFlt-1 (100 ng/ml) also reduced βIII-tubulin mRNA and neuronal differentiation of neurospheres. Undifferentiated NPCs and mature neurons/glia expressed VEGFA and VEGFR-2, required for endogenous autocrine and paracrine VEGFA signalling, while sFlt-1 treatment prevented the neurogenic effects of exogenous VEGFA. Overall, these data provide the first experimental evidence for a direct effect of sFlt-1 on neurite growth and neuronal differentiation in human neurons through inhibition of VEGFA signalling, clarifying our understanding of the potential role of sFlt-1 as a mechanism by which PE can affect neuronal development.

Methamphetamine exposure drives cell cycle exit and aberrant differentiation in rat hippocampal-derived neurospheres.

Introduction: Methamphetamine (METH) abuse by pregnant drug addicts causes toxic effects on fetal neurodevelopment; however, the mechanism underlying such effect of METH is poorly understood. Methods: In the present study, we applied three-dimensional (3D) neurospheres derived from the embryonic rat hippocampal tissue to investigate the effect of METH on neurodevelopment. Through the combination of whole genome transcriptional analyses, the involved cell signalings were identified and investigated. Results: We found that METH treatment for 24h significantly and concentration-dependently reduced the size of neurospheres. Analyses of genome-wide transcriptomic profiles found that those down-regulated differentially expressed genes (DEGs) upon METH exposure were remarkably enriched in the cell cycle progression. By measuring the cell cycle and the expression of cell cycle-related checkpoint proteins, we found that METH exposure significantly elevated the percentage of G0/G1 phase and decreased the levels of the proteins involved in the G1/S transition, indicating G0/G1 cell cycle arrest. Furthermore, during the early neurodevelopment stage of neurospheres, METH caused aberrant cell differentiation both in the neurons and astrocytes, and attenuated migration ability of neurospheres accompanied by increased oxidative stress and apoptosis. Conclusion: Our findings reveal that METH induces an aberrant cell cycle arrest and neuronal differentiation, impairing the coordination of migration and differentiation of neurospheres.

Characterization of Bovine Bone Marrow Derived Mesenchymal Stem Cells and Immunostaining of Differentiated Neurospheres

Stem cell differentiation has been a key element for the therapeutical applications for various cases. The novel anti-inflammatory and angiogenic findings and potential applications for both autologous and allogenic studies have emphasized about the usage of mesenchymal stem cells in the clinical trials. The neurosphere differentiation studies and their role for peripheral nerve injuries have gained momentum. Following study emphasized on the isolation of stem cells from bovine bone marrow, their characterization by PCR and their differentiation into mesodermal lineages and into neurospheres under specific induction media. The generated neurospheres were durther investigated for their potential expressions of neuroprogenitor markers Sox2, Nestin and neurofilament protein β-III Tubulin by immunofluorescence staining. The results of following study highlight the use of bone marrow derived mesenchymal stem cells for translational research and regenerative therapy.

Chikungunya virus infection induces ultrastructural changes and impaired neuronal differentiation of human neurospheres.

Chikungunya virus (CHIKV) is an arthropod-borne virus recently associated with large outbreaks in many parts of the world. Infection is typically manifested as a febrile and self-limited illness, characterized by joint pain and myalgia, albeit severe neurological manifestations are also reported. Although CHIKV is not recognized as a truly neurotropic virus, neurons, astrocytes, and oligodendrocytes are susceptible to infection in vitro. Here we employed a model of 3D cell culture to obtain neurospheres from ATRA/BNDF differentiated human neuroblastoma cells. We demonstrate that CHIKV is able to establish a productive infection, resulting in ultrastructural changes in cell morphology and impaired neuronal differentiation. Ultrastructural analysis of neurospheres infected with CHIKV during neuronal differentiation revealed diminished neuron dendrite formation, accumulation of viral particles associated with the plasma membrane, numerous cell vacuoles, and swollen mitochondria. Apoptotic cells were significantly increased at 72 h post-infection. Compared to Zika virus, a well-characterized neurotropic arbovirus, CHIKV infection resulted in a more discrete, albeit detectable upregulation of IL-6 levels. Finally, we found that CHIKV infection resulted in an altered profile expression, mainly downregulation, of a group of transcription factors named Hox genes. Altogether our findings highlight important features of CHIKV in the CNS, as well as the feasibility of neurospheres as robust experimental models that can support further studies for novel pharmacological interventions.

The Mechanical Properties of Neurospheres

Stem cell‐derived 3D tissues such as spheroids are excellent models for investigating mechanisms of tissue formation and responses to physiological and mechanical cues. Neuronal spheroids, also known as neurospheres, have attracted particular interest. A lot is now known about the differentiation and maturation of neurospheres, as well as their responses to biochemical cues. However, understanding about their mechanical properties pales in comparison, which is all the more galling in light of newfound insights about how mechanical stimuli trigger the onset of neurodegenerative conditions. Herein, formative steps are taken to fill this knowledge gap. Neurospheres are generated from murine neural stem cells and are subjected to compressive forces. It is observed that neurospheres exhibit stress relaxation under static compression and viscoelastic behavior at low strains. The suitability of the Tatara model for characterizing the mechanical properties of neurospheres is also evaluated. The study is the first study of its kind to investigate the mechanical properties of in vitro 3D tissues. Moreover, the methodologies developed in the study can also be used to improve the quality and safety of cell and tissue biomanufacturing processes.

Quantitative profiling of axonal guidance proteins during the differentiation of human neurospheres

Axon guidance is required for the establishment of brain circuits. Whether much of the molecular basis of axon guidance is known from animal models, the molecular machinery coordinating axon growth and pathfinding in humans remains to be elucidated. The use of induced pluripotent stem cells (iPSC) from human donors has revolutionized in vitro studies of the human brain. iPSC can be differentiated into neuronal stem cells which can be used to generate neural tissue-like cultures, known as neurospheres, that reproduce, in many aspects, the cell types and molecules present in the brain. Here, we analyzed quantitative changes in the proteome of neurospheres during differentiation. Relative quantification was performed at early time points during differentiation using iTRAQ-based labeling and LC-MS/MS analysis. We identified 6438 proteins, from which 433 were downregulated and 479 were upregulated during differentiation. We show that human neurospheres have a molecular profile that correlates to the fetal brain. During differentiation, upregulated pathways are related to neuronal development and differentiation, cell adhesion, and axonal guidance whereas cell proliferation pathways were downregulated. We developed a functional assay to check for neurite outgrowth in neurospheres and confirmed that neurite outgrowth potential is increased after 10 days of differentiation and is enhanced by increasing cyclic AMP levels. The proteins identified here represent a resource to monitor neurosphere differentiation and coupled to the neurite outgrowth assay can be used to functionally explore neurological disorders using human neurospheres as a model.

CXCR4 antagonism sensitizes cancer cells to novel indole-based MDM2/4 inhibitors in glioblastoma multiforme

Glioblastoma Multiforme (GBM) is a highly invasive primary brain tumour characterized by chemo- and radio-resistance and poor overall survival. GBM can present an aberrant functionality of p53, caused by the overexpression of the murine double minute 2 protein (MDM2) and its analogue MDM4, which may influence the response to conventional therapies. Moreover, tumour resistance/invasiveness has been recently attributed to an overexpression of the chemokine receptor CXCR4, identified as a pivotal mediator of glioma neovascularization. Notably, CXCR4 and MDM2-4 cooperate in promoting tumour invasion and progression. Although CXCR4 actively promotes MDM2 activation leading to p53 inactivation, MDM2-4 knockdown induces the downregulation of CXCR4 gene transcription.Our study aimed to assess if the CXCR4 signal blockade could enhance glioma cells’ sensitivity to the inhibition of the p53-MDMs axis. Rationally designed inhibitors of MDM2/4 were combined with the CXCR4 antagonist, AMD3100, in human GBM cells and GBM stem-like cells (neurospheres), which are crucial for tumour recurrence and chemotherapy resistance. The dual MDM2/4 inhibitor RS3594 and the CXCR4 antagonist AMD3100 reduced GBM cell invasiveness and migration in single-agent treatment and mainly in combination. AMD3100 sensitized GBM cells to the antiproliferative activity of RS3594. It is noteworthy that these two compounds present synergic effects on cancer stem components: RS3594 inhibited the growth and formation of neurospheres, AMD3100 induced differentiation of neurospheres while enhancing RS3594 effectiveness preventing their proliferation/clonogenicity. These results confirm that blocking CXCR4/MDM2/4 represents a valuable strategy to reduce GBM proliferation and invasiveness, acting on the stem cell component too.

Enriched Environment Minimizes Anxiety/Depressive-Like Behavior in Rats Exposed to Immobilization Stress and Augments Hippocampal Neurogenesis (In Vitro).

Chronic exposure to stress disturbs the homeostasis of the brain, thus, deleteriously affecting the neurological circuits. In literature, there are investigations about the stress-related alterations in behavioral response and adult neurogenesis; however, an effective combating strategy to evade stress is still at stake. Hence, the present study is designed to investigate the effect of an enriched environment in alleviating the anxiety/depressive-like behavioral response and enhancing the adult neurogenesis in the hippocampal region of rats exposed to chronic immobilization stress. The rats were exposed to chronic immobilization stress (IS) for 4 h/day followed by the enriched environment (EE) for 2 h/day for 28days, and finally, the hippocampal region was dissected out after the behavioral analyses. IS group showed increased behavioral despair to tail suspension test, decrement in the activity for light/dark box test, and less grooming activity towards splash test. In contrast, IS + EE rats exhibited a decrease in the activity of tail suspension test and an increase in the behavioral response to light/dark box test and splash test. Thein vitro assessment of primary cultures of neurospheres from the IS group resulted in decreased levels of proliferation in the cell number and metabolic activity of both MTT assay and lactate levels. IS + EE group revealed an increase in the growth curve of neurospheres and higher metabolic activities of MTT and lactate. The IS cultures had reduced neurite length, while the neurite outgrowths were increased in IS + EE group. The IS group showed significant reduction in the protein and mRNA levels of nestin, GFAP, CD11b, MOG, and synaptophysin, whereas the IS + EE cultures exhibited significant increase in the levels of these stem cell markers. Our data highlight the positive impact of EE against stress-related behavioral changes in rats exposed to chronic immobilization stress perhaps by interfering with the differentiation of neurospheres and neurogenesis.

Effects of primary microglia and astrocytes on neural stem cells in in vitro and in vivo models of ischemic stroke.

Transplantation of neural stem cells (NSCs) can protect neurons in animal stroke models; however, their low rates of survival and neuronal differentiation limit their clinical application. Glial niches, an important location of neural stem cells, regulate survival, proliferation and differentiation of neural stem cells. However, the effects of activated glial cells on neural stem cells remain unclear. In the present study, we explored the effects of activated astrocytes and microglia on neural stem cells in vitro stroke models. We also investigated the effects of combined transplantation of neural stem cells and glial cells after stroke in rats. In a Transwell co-culture system, primary cultured astrocytes, microglia or mixed glial cells were exposed to glutamate or H2O2 and then seeded in the upper inserts, while primary neural stem cells were seeded in the lower uncoated wells and cultured for 7 days. Our results showed that microglia were conducive to neurosphere formation and had no effects on apoptosis within neurospheres, while astrocytes and mixed glial cells were conducive to neurosphere differentiation and reduced apoptosis within neurospheres, regardless of their pretreatment. In contrast, microglia and astrocytes induced neuronal differentiation of neural stem cells in differentiation medium, regardless of their pretreatment, with an exception of astrocytes pretreated with H2O2. Rat models of ischemic stroke were established by occlusion of the middle cerebral artery. Three days later, 5 × 105 neural stem cells with microglia or astrocytes were injected into the right lateral ventricle. Neural stem cell/astrocyte-treated rats displayed better improvement of neurological deficits than neural stem cell only-treated rats at 4 days after cell transplantation. Moreover, neural stem cell/microglia-, and neural stem cell/astrocyte-treated rats showed a significant decrease in ischemic volume compared with neural stem cell-treated rats. These findings indicate that microglia and astrocytes exert different effects on neural stem cells, and that co-transplantation of neural stem cells and astrocytes is more conducive to the recovery of neurological impairment in rats with ischemic stroke. The study was approved by the Animal Ethics Committee of Tongji University School of Medicine, China (approval No. 2010-TJAA08220401) in 2010.

Maternal vitamin A deficiency impairs cholinergic and nitrergic neurons, leading to gastrointestinal dysfunction in rat offspring via RARβ

AimsMany gastrointestinal (GI) disorders are developmental in origin and are caused by abnormal enteric nervous system (ENS) formation. Maternal vitamin A deficiency (VAD) during pregnancy affects multiple central nervous system developmental processes during embryogenesis and fetal life. Here, we evaluated whether maternal diet-induced VAD during pregnancy alone can cause changes in the ENS that lead to GI dysfunction in rat offspring. Main methodsRats were selected to construct animal models of normal VA, VA deficiency and VA supplementation. The fecal water content, total gastrointestinal transmission time and colonic motility were measured to evaluate gastrointestinal function of eight-week-old offspring rats. The expression levels of RARβ, SOX10, cholinergic (ChAT) and nitrergic (nNOS) enteric neurons in colon tissues were detected through western blot and immunofluorescence. Primary enteric neurospheres were treated with retinoic acid (RA), infection with Ad-RARβ and siRARβ adenovirus, respectively. Key findingsOur data revealed marked reductions in the mean densities of cholinergic and nitrergic enteric neurons in the colon and GI dysfunction evidenced by mild intestinal flatulence, increased fecal water content, prolonged total GI transit time and reduced colon motility in adult offspring of the VAD group. Interestingly, maternal VA supplementation (VAS) during pregnancy rescued these changes. In addition, in vitro experiments demonstrated that exposure to appropriate doses of RA promoted enteric neurosphere differentiation into cholinergic and nitrergic neurons, possibly by upregulating RARβ expression, leading to enhanced SOX10 expression. SignificanceMaternal VAD during pregnancy is an environmental risk factor for GI dysfunction in rat offspring.

Photobiomodulation-Induced Differentiation of Immortalized Adipose Stem Cells to Neuronal Cells.

Transdermal differentiation of human adipose stem cells (ASCs) to other cell types is still a challenge in regenerative medicine. Studies using primary ASCs are also limited as they may undergo replicative senescence during repeated passages in vitro. However, ASCs immortalized (iASCs) with human telomerase enzyme expressing plasmid exhibits a uniform population suitable for differentiation in vitro. A right combination of biological and physical stimuli may induce transdermal differentiation of iASCs into neurons in vitro. iASCs were differentiated to free-floating neural stem cell aggregates (neurospheres) using a combination of growth inducers. Cells in these spheres were induced to differentiate into neurons using low-intensity lasers by a process called photobiomodulation (PBM). Laser at the near infrared (NIR) wavelength 825 nm and fluences 5, 10, and 15 J/cm2 was capable of increasing the differentiation of neurospheres to neurons. Precisely, there was a statistically significant increase in the early neuronal marker at 5 J/cm2 and a much appreciable increase at 15 J/cm2 in correlation with the biphasic dose response of PBM. However, these differentiated cells failed to express late neuronal markers in vitro. Comparison of these differentiating iASCs with the primary ASCs revealed a sharp distinction between the metabolic processes of the primary ASCs, neurospheres, and newly differentiated neurons. We found that PBM increased the yield of neurons and effected stem cell differentiation through modulation of cellular metabolism and redox status. Our study also identifies that iASCs are an excellent model for analysis of stem cell biology and for performing transdermal differentiation. This study demonstrates that a combination of biological and physical inducers can advance the differentiation of adipose stem cells to neurons. We were able to establish the optimal energy for the neuronal differentiation of iASCs in vitro. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

NeuroCore formation during differentiation of neurospheres of mouse embryonic neural stem cells.

Neural stem cells (NSCs) in the embryonic neocortex have the potential to generate a well-organized laminar architecture of the cerebral cortex through precise regulation of the proliferation, differentiation, and migration of neural cells. NSCs can be isolated in vitro and expanded as cell clusters, called neurospheres, which are primarily related to the proliferation ability of NSCs. Conversely, the tissue-organizing properties of NSCs via regulated differentiation and migration of the cells are not well understood. In this study, we established a three-dimensional (3D) differentiation model of neurospheres, which produce unique neuronal clusters, termed NeuroCore (NC). NC formation was initiated by the aggregation of young neurons. Upon maturation of the neurons and the establishment of radial glia-like structures, the initial organization of the NCs transformed into a glomeruli-like arrangement of cortical neurons. These neurons expressed multiple markers of upper and deep cortical neurons. Taken together, we propose that NSCs in vitro maintain some aspects of their original in vivo tissue-organizing properties, providing an alternative opportunity to explore the fundamental components of brain histogenesis in vitro.

ShRNA-based POLD2 expression knockdown sensitizes glioblastoma to DNA-Damaging therapeutics.

Glioblastoma (GBM) has limited therapeutic options. DNA repair mechanisms contribute GBM cells to escape therapies and re-establish tumor growth. Multiple studies have shown that POLD2 plays a critical role in DNA replication, DNA repair and genomic stability. We demonstrate for the first time that POLD2 is highly expressed in human glioma specimens and that expression correlates with poor patient survival. siRNA or shRNA POLD2 inhibited GBM cell proliferation, cell cycle progression, invasiveness, sensitized GBM cells to chemo/radiation-induced cell death and reversed the cytoprotective effects of EGFR signaling. Conversely, forced POLD2 expression was found to induce GBM cell proliferation, colony formation, invasiveness and chemo/radiation resistance. POLD2 expression associated with stem-like cell subsets (CD133+ and SSEA-1+ cells) and positively correlated with Sox2 expression in clinical specimens. Its expression was induced by Sox2 and inhibited by the forced differentiation of GBM neurospheres. shRNA-POLD2 modestly inhibited GBM neurosphere-derived orthotopic xenografts growth, when combined with radiation, dramatically inhibited xenograft growth in a cooperative fashion. These novel findings identify POLD2 as a new potential therapeutic target for enhancing GBM response to current standard of care therapeutics.

P.638 Evaluation of disease control and QoL in patients with major depressive disorder with or without generalized anxiety disorder in Greece

Glioblastoma (GBM) has limited therapeutic options. DNA repair mechanisms contribute GBM cells to escape therapies and re-establish tumor growth. Multiple studies have shown that POLD2 plays a critical role in DNA replication, DNA repair and genomic stability. We demonstrate for the first time that POLD2 is highly expressed in human glioma specimens and that expression correlates with poor patient survival. siRNA or shRNA POLD2 inhibited GBM cell proliferation, cell cycle progression, invasiveness, sensitized GBM cells to chemo/radiation-induced cell death and reversed the cytoprotective effects of EGFR signaling. Conversely, forced POLD2 expression was found to induce GBM cell proliferation, colony formation, invasiveness and chemo/radiation resistance. POLD2 expression associated with stem-like cell subsets (CD133+ and SSEA-1+ cells) and positively correlated with Sox2 expression in clinical specimens. Its expression was induced by Sox2 and inhibited by the forced differentiation of GBM neurospheres. shRNA-POLD2 modestly inhibited GBM neurosphere-derived orthotopic xenografts growth, when combined with radiation, dramatically inhibited xenograft growth in a cooperative fashion. These novel findings identify POLD2 as a new potential therapeutic target for enhancing GBM response to current standard of care therapeutics.

ER stress and UPR activation in glioblastoma: identification of a noncanonical PERK mechanism regulating GBM stem cells through SOX2 modulation

Patients with aggressive brain tumors, named glioblastoma multiforme (GBM), have a poor prognoses. Here we explored if the ER stress/unfolded protein response (UPR) is involved in the pathophysiology of GBM and may provide novel therapeutic targets. Immunohistochemical analyses of a tissue microarray containing primary GBM specimens showed strong variability in expression of the UPR markers GRP78/BiP, XBP1, and ATF4. Interestingly, high ATF4 expression was associated with poor overall survival suggesting involvement of PERK signaling in GBM progression. In vitro experiments using patient-derived neurospheres, enriched for GBM stem cells (GSCs), showed high sensitivity for the ER stressor thapsigargin (Tg) mainly via PERK signaling. In contrast, neurospheres-derived differentiated GBM cells were less sensitive likely due to lower UPR activity as indicated by comparative transcriptional profiling. Tg and Tunicamycin strongly reduced neurosphere forming ability of GSCs that was linked with potent PERK-dependent downregulation of SOX2 protein. Interestingly, SOX2 downregulation occurred directly via PERK, not requiring downstream activation of the PERK-UPR pathway. Moreover, PERK inactivation resulted in aberrant serum-induced differentiation of GBM neurospheres accompanied by persistent SOX2 expression, delayed upregulation of GFAP and reduced cell adherence. In conclusion, we provide evidence that PERK signaling contributes to the prognoses of primary GBM patients and identified PERK as a novel regulator of SOX2 expression and GSC differentiation. The role of PERK appeared to be pleiotropic involving UPR-dependent, as well as novel identified noncanonical mechanisms regulating SOX2. ER stress and PERK modulation appear to provide promising therapeutic targets for therapy in GBM.

Trypanosoma evansi impacts on embryonic neural progenitor cell functions

Trypanosoma evansi appears to have a significant tropism for brain tissue in its chronic and acute phases. The most common symptoms of this brain infection are motor incoordination, meningoencephalitis, demyelination, and anemia. There have only been few studies of the effects of T. evansi infection on neuronal differentiation and brain plasticity. Here, we investigated the impact of the congenital T. evansi infection on brain development in mice. We collected telencephalon-derived neural progenitor cells (NPCs) from T. evansi uninfected and infected mice, and cultivated them into neurospheres. We found that T. evansi significantly decreased the number of cells during development of neurospheres. Analysis of neurosphere differentiation revealed that T. evansi infection significantly increased neural migration. We also observed that T. evansi promoted expression of glial fibrillary acidic protein (GFAP) in infected cells. These data suggest that congenital T. evansi infection may affect embryonic brain development.

The systemic exercise-released chemokine lymphotactin/XCL1 modulates in vitro adult hippocampal precursor cell proliferation and neuronal differentiation

Physical exercise has well-established anti-inflammatory effects, with neuro-immunological crosstalk being proposed as a mechanism underlying the beneficial effects of exercise on brain health. Here, we used physical exercise, a strong positive modulator of adult hippocampal neurogenesis, as a model to identify immune molecules that are secreted into the blood stream, which could potentially mediate this process. Proteomic profiling of mouse plasma showed that levels of the chemokine lymphotactin (XCL1) were elevated after four days of running. We found that XCL1 treatment of primary cells isolated from both the dentate gyrus and the subventricular zone of the adult mice led to an increase in the number of neurospheres and neuronal differentiation in neurospheres derived from the dentate gyrus. In contrast, primary dentate gyrus cells isolated from XCL1 knockout mice formed fewer neurospheres and exhibited a reduced neuronal differentiation potential. XCL1 supplementation in a dentate gyrus-derived neural precursor cell line promoted neuronal differentiation and resulted in lower cell motility and a reduced number of cells in the S phase of the cell cycle. This work suggests an additional function of the chemokine XCL1 in the brain and underpins the complexity of neuro-immune interactions that contribute to the regulation of adult hippocampal neurogenesis.

NG2 and GFAP co-expression after differentiation in cells transfected with mutant GFAP and in undifferentiated glioma cells

IntroductionAlexander disease is a rare disorder caused by mutations in the gene coding for glial fibrillary acidic protein (GFAP). In a previous study, differentiation of neurospheres transfected with these mutations resulted in a cell type that expresses both GFAP and NG2. ObjectiveTo determine the effect of molecular marker mutations in comparison to undifferentiated glioma cells simultaneously expressing GFAP and NG2. MethodsWe used samples of human glioblastoma (GBM) and rat neurospheres transfected with GFAP mutations to analyse GFAP and NG2 expression after differentiation. We also performed an immunocytochemical analysis of neuronal differentiation for both cell types and detection of GFAP, NG2, vimentin, Olig2, and caspase-3 at 3 and 7 days from differentiation. ResultsBoth the cells transfected with GFAP mutations and GBM cells showed increased NG2 and GFAP expression. However, expression of caspase-3-positive cells was found to be considerably higher in transfected cells than in GBM cells. ConclusionsOur results suggest that GFAP expression is not the only factor associated with cell death in Alexander disease. Caspase-3 expression and the potential role of NG2 in increasing resistance to apoptosis in cells co-expressing GFAP and NG2 should be considered in the search for new therapeutic strategies for the disease.