Empowering sonic hedgehog to rescue brain cells after ischemic stroke

Ischemic stroke occurs when blood supply to the brain is interrupted. This can cause irreversible injury to the cen- tral nervous system （CNS） tissue. Each year in the United States almost 800,000 people experience a new or recur- rent stroke. 15% of stroke patients die shortly after insult and only 10% recover completely, leaving the majority of surviving stroke patients with disabilities. Tissue-type plasminogen activator （tPA） is the only available therapy for stroke but its clinical use is limited because of associ- ated danger of intracranial hemorrhage. Therefore, there is an emergent need for stroke therapeutics that are safe and effective when administered at a later time point after insult.

Effects of Chuanxiongqin hydrochloride on increasing the fluidity of brain cell membrane and scavenging free radicals in model rats with ischemia/reperfusion injury

BACKGROUND: The fluidity of cell membrane can be affected by various factors. Many experiments have confirmed that the ischemia/reperfusion of organic tissue can increase the contents of free radicals, which lead to high rigidity and low fluidity of cell membrane, and the conditions can be changed by Chuanxiongqin. OBJECTIVE: To observe the effect and mechanism of Chuanxiongqin hydrochloride on the fluidity of brain cell membrane in rat models of ischemia/reperfusion. DESIGN: A completely randomized controlled animal trial. SETTINGS: Institute of Brain Sciences; Department of Physiology, Medical College, Datong University. MATERIALS: Twenty male grade Ⅰ Wistar rats of 170-220 g were randomly divided into model group (n =10) and control group (n =10). Chuanxiongqin hydrochloride (molecular mass was 172.2) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (batch number: 0817-9803); Spin labelers: 5-doxyl-stearlic acid methylester (5DS), 16-doxyl-stearlic acid methylester (16DS), xanthine, xanthine oxidase (XOD) and 5,5-dimeth-1-pyrroline- N-oxide (DMPO) from Sigma Company; Bruker ESP 300 electron paramagnetic resonance (EPR) spectrometer by Bruker Company (Germany). METHODS: The experiments were carried out in the State Key Laboratory of Natural and Biomimetic Drugs, Peking University from June 2001 to July 2002. In the model group, rats were made into models of cerebral ischemia by 30-minute ligation and 2-hour reperfusion of common carotid arteries; The rats in the control group were not made into models. The order parameter (S) and rotational correlation time (τc) were detected with the ESR spectrometer by means of spin labeling. The greater the S and τc, the smaller the fluidity. Meanwhile, the clearance rate of free radicals was detected with ESR spin trapping. The measurement data were compared using the t test. MAIN OUTCOME MEASURES: The S, τc and clearance rates of O2 · and OH· free radicals were compared between the model group and control group. RESULTS: The S and τc in the model group [0.738 4±0.003 5; (8.472±0.027)×10-10 s/circle] were obviously different from those in the control group [0.683 9±0.008 3; (7.945±0.082)×10-10 s/circle, t =5.731, 5.918, P < 0.05], which suggested that ischemia/reperfusion injury decreased the fluidity of brain cell membrane. After adding Chuanxiongqin hydrochloride, there were no obvious differences between the model group [0.688 5±0.030 5; (7.886±0.341)×10-10 s/circle] and control group (P > 0.05), indicating that Chuanxiongqin hydrochloride could recover the fluidity of brain cell membrane after ischemia/reperfusion injury close to the level in the normal control group. Chuanxiongqin hydrochloride could directly scavenge the O2 · and OH· free radicals, and the maximal clearance rates were 83.92% and 44.99% respectively. CONCLUSION: Chuanxiongqin hydrochloride increases the fluidity of membrane of ischemia-injured brain cell by scavenging both O2 ·and OH· free radicals.

Neuroanatomy and morphological diversity of brain cells from adult crayfish Cherax quadricarinatus

As in vertebrates, brains play key roles in rhythmic regulation, neuronal maintenance, diff erentiation and function, and control of the release of hormones in arthropods. But the structure and functional domains of the brain are still not very clear in crustaceans. In the present study, we reveal the structural details of the brain in the redclaw crayfish using hematoxylin-eosin staining and microscopic examination, firstly. The brain of crayfish is consist of three main parts, namely, protocerebrum, deutocerebrum, and tritocerebrum, including some tracts and commissures, briefly. Secondly, at least 9 kinds of brain cells were identified on the basis of topology and cell shapes, as well as antibody labeling. We also provide morphological details of most cell types, which were previously un-described. In general, four types of glia and three types of neurosecretory cells were described except cluster 9/11 and cluster 10 cells. Glia were categorized into another three main kinds:(1) surface glia;(2) cortex glia; and(3) neuropile glia in addition to astrocytes identified by GFAP labelling. And neurosecretory cells were categorized into I, Ⅱ and III types based on morphological observation. Finally, cluster 9/11 and 10 cells derived from the brain of crayfish, could be used for primary culture about 7–9 d under the optimized conditions. There results provide a resource for improving the knowledge of the still incompletely defined neuroendocrinology of this species. Using the crayfish as an animal model, we are easy to carry out further research in manipulating their endocrine system, exploring cellular and synaptic mechanisms so much as larval production on a small scale, such as in a cell or tissue.

Brain cell apoptosis and enhancement of nervous excitability in pregnant rats with high plasma levels of homocysteine

Hyperhomocysteinemia is an important risk factor for preeclampsia-eclampsia. This study established a pregnant rat model of hyperhomocysteinemia, in which blood plasma homocysteine concentrations were twice or three times greater than that of normal pregnant rats. TUNEL revealed an increase in the number of apoptotic cells in the frontal cortex of pregnant rats with hyperhomocysteinemia. In addition, immunohistochemical staining detected activated nuclear factor-KB-positve cells in the frontal cortex. Reverse transcription-PCR detected that mRNA expression of the anti-apoptotic gene bcl-2 diminished in the frontal cortex. In situ hybridization and western blotting revealed that N-methyi-D- aspartate receptor 1 mRNA and protein expression was upregulated in the frontal cortex and hippocampus. These results indicate that hyperhomocysteinemia can induce brain cell apoptosis, increase nerve excitability, and promote the occurrence of preeclampsia in pregnant rats.

Neural stem cell-derived exosomes regulate cell proliferation,migration,and cell death of brain microvascular endothelial cells via the miR-9/Hes1 axis under hypoxia

Background:Our previous study found that mouse embryonic neural stem cell(NSC)-derived exosomes(EXOs)regulated NSC differentiation via the miR-9/Hes1 axis.However,the effects of EXOs on brain microvascular endothelial cell(BMEC)dysfunction via the miR-9/Hes1 axis remain unknown.Therefore,the current study aimed to determine the effects of EXOs on BMEC proliferation,migration,and death via the miR-9/Hes1 axis.Methods:Immunofluorescence,quantitative real-time polymerase chain reaction,cell counting kit-8 assay,wound healing assay,calcein-acetoxymethyl/propidium iodide staining,and hematoxylin and eosin staining were used to determine the role and mechanism of EXOs on BMECs.Results:EXOs promoted BMEC proliferation and migration and reduced cell death under hypoxic conditions.The overexpression of miR-9 promoted BMEC prolifera-tion and migration and reduced cell death under hypoxic conditions.Moreover,miR-9 downregulation inhibited BMEC proliferation and migration and also promoted cell death.Hes1 silencing ameliorated the effect of amtagomiR-9 on BMEC proliferation and migration and cell death.Hyperemic structures were observed in the regions of the hippocampus and cortex in hypoxia-induced mice.Meanwhile,EXO treatment improved cerebrovascular alterations.Conclusion:NSC-derived EXOs can promote BMEC proliferation and migra-tion and reduce cell death via the miR-9/Hes1 axis under hypoxic conditions.Therefore,EXO therapeutic strategies could be considered for hypoxia-induced vascular injury.

Crosstalk among Oxidative Stress,Autophagy,and Apoptosis in the Protective Effects of Ginsenoside Rb1 on Brain Microvascular Endothelial Cells:A Mixed Computational and Experimental Study

Objective Brain microvascular endothelial cells (BMECs) were found to shift from their usually inactive state to an active state in ischemic stroke (IS) and cause neuronal damage. Ginsenoside Rb1 (GRb1),a component derived from medicinal plants,is known for its pharmacological benefits in IS,but its protective effects on BMECs have yet to be explored. This study aimed to investigate the potential protective effects of GRb1 on BMECs. Methods An in vitro oxygen-glucose deprivation/reperfusion (OGD/R) model was established to mimic ischemia-reperfusion (I/R) injury. Bulk RNA-sequencing data were analyzed by using the Human Autophagy Database and various bioinformatic tools,including gene set enrichment analysis (GSEA),Gene Ontology (GO) classification and enrichment analysis,Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis,protein-protein interaction network analysis,and molecular docking. Experimental validation was also performed to ensure the reliability of our findings. Results Rb1 had a protective effect on BMECs subjected to OGD/R injury. Specifically,GRb1 was found to modulate the interplay between oxidative stress,apoptosis,and autophagy in BMECs. Key targets such as sequestosome 1 (SQSTM1/p62),autophagy related 5 (ATG5),and hypoxia-inducible factor 1-alpha (HIF-1α) were identified,highlighting their potential roles in mediating the protective effects of GRb1 against IS-induced damage. Conclusion GRbl protects BMECs against OGD/R injury by influencing oxidative stress,apoptosis,and autophagy. The identification of SQSTM1/p62,ATG5,and HIF-1α as promising targets further supports the potential of GRb1 as a therapeutic agent for IS,providing a foundation for future research into its mechanisms and applications in IS treatment.

β-Estradiol 17-acetate enhances the in vitro vitality of endothelial cells isolated from the brain of patients subjected to neurosurgery

In the current landscape of endothelial cell isolation for building in vitro models of the blood-brain barrier,our work moves towards reproducing the features of the neurovascular unit to achieve glial compliance through an innovative biomimetic coating technology for brain chronic implants.We hypothesized that the autologous origin of human brain mic rovascular endothelial cells(hBMECs)is the first requirement for the suitable coating to prevent the glial inflammato ry response trigge red by foreign neuroprosthetics.Therefo re,this study established a new procedure to preserve the in vitro viability of hBMECs isolated from gray and white matter specimens taken from neurosurge ry patients.Culturing adult hBMECs is generally considered a challenging task due to the difficult survival ex vivo and progressive reduction in proliferation of these cells.The addition of 10 nMβ-estradiol 17-acetate to the hBMEC culture medium was found to be an essential and discriminating factor promoting adhesion and proliferation both after isolation and thawing,suppo rting the well-known protective role played by estrogens on microvessels.In particular,β-estradiol 17-acetate was critical for both freshly isolated and thawed female-derived hBMECs,while it was not necessary for freshly isolated male-derived hBMECs;however,it did countera ct the decay in the viability of the latter after thawing.The tumo r-free hBMECs were thus cultured for up to 2 months and their growth efficiency was assessed befo re and after two periods of cryopreservation.Des pite the thermal stress,the hBMECs remained viable and suitable for re-freezing and storage for several months.This approach increasing in vitro viability of hBMECs opens new perspectives for the use of cryopreserved autologous hBMECs as biomimetic therapeutic tools,offering the potential to avoid additional surgical sampling for each patient.

An Eperimental Study on the Transplantation of Human Fetal Brain Cells into Monkey Models of Parkinson's Disease.

Unilateral administration of methly-phenyl-tetrahydropyridine(MPTP) into the common carotid artery

New Brain Cells Love to Learn

俗话说,脑子越用越灵。最近,加拿大多伦多的研究者对老鼠进行实验,发现成年鼠在学习一项新任务时,它们新生成的脑细胞会比原有的细胞活跃3倍之多。研究同时还显示,与其他时段相比,细胞形成后6个星期时细胞活动最为活跃。

Targeting brain microvascular endothelial cells: a therapeutic approach to neuroprotection against stroke

Brain microvascular endothelial cells form the interface between nervous tissue and circulating blood, and regulate central nervous system homeostasis. Brain microvascular endothelial cells differ from peripheral endothelial cells with regards expression of specific ion transporters and receptors, and contain fewer fenestrations and pinocytotic vesicles. Brain microvascular endothelial cells also synthesize several factors that influence blood vessel function. This review describes the morphological characteristics and functions of brain microvascular endothelial cells, and summarizes current knowledge regarding changes in brain microvascular endothelial cells during stroke progression and therapies. Future studies should focus on identifying mechanisms underlying such changes and developing possible neuroprotective therapeutic interventions.

In vitro model of the blood-brain barrier established by co-culture of primary cerebral microvascular endothelial and astrocyte cells

Drugs for the treatment and prevention of nervous system diseases must permeate the bloodbrain barrier to take effect.In vitro models of the blood-brain barrier are therefore important in the investigation of drug permeation mechanisms.However,to date,no unified method has been described for establishing a blood-brain barrier model.Here,we modified an in vitro model of the blood-brain barrier by seeding brain microvascular endothelial cells and astrocytes from newborn rats on a polyester Transwell cell culture membrane with 0.4-μm pores,and conducted transepithelial electrical resistance measurements,leakage tests and assays for specific bloodbrain barrier enzymes.We show that the permeability of our model is as low as that of the bloodbrain barrier in vivo.Our model will be a valuable tool in the study of the mechanisms of action of neuroprotective drugs.

LncRNA SNHG12 ameliorates brain microvascular endothelial cell injury by targeting miR-199a

Long non-coding RNAs regulate brain microvascular endothelial cell death, the inflammatory response and angiogenesis during and after ischemia/reperfusion and oxygen-glucose deprivation/reoxygenation（OGD/R） insults. The long non-coding RNA, SNHG12, is upregulated after ischemia/reperfusion and OGD/R in microvascular endothelial cells of the mouse brain. However, its role in ischemic stroke has not been studied. We hypothesized that SNHG12 positively regulates ischemic stroke, and therefore we investigated its mechanism of action. We established an OGD/R mouse cell model to mimic ischemic stroke by exposing brain microvascular endothelial cells to OGD for 0, 2, 4, 8, 16 or 24 hours and reoxygenation for 4 hours. Quantitative real-time polymerase chain reaction showed that SNHG12 levels in brain microvascular endothelial cells increased with respect to OGD exposure time. Brain microvascular endothelial cells were transfected with pc DNA-control, pc DNA-SNHG12, si-control, or si-SNHG12. After exposure to OGD for 16 hours, these cells were then analyzed by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide, trypan blue exclusion, western blot, and capillary-like tube formation assays. Overexpression of SNHG12 inhibited brain microvascular endothelial cell death and the inflammatory response but promoted angiogenesis after OGD/R, while SNHG12 knockdown had the opposite effects. miR-199a was identified as a target of SNHG12, and SNHG12 overexpression reversed the effect of miR-199a on brain microvascular endothelial cell death, the inflammatory response, and angiogenesis. These findings suggest that SNHG12 suppresses endothelial cell injury induced by OGD/R by targeting miR-199a.

Hypoxia inducible factor-1alpha mediates protection of DL-3-n-butylphthalide in brain microvascular endothelial cells against oxygen glucose deprivation-induced injury

Studies have demonstrated that DL-3-n-butylphthalide can significantly alleviate oxygen glucose deprivation-induced injury of human umbilical vein endothelial cells at least partly associated with its enhancement on oxygen glucose deprivation-induced hypoxia inducible factor-1α expression.In this study,we hypothesized that DL-3-n-butylphthalide can protect against oxygen glucose deprivation-induced injury of newborn rat brain microvascular endothelial cells by means of upregulating hypoxia inducible factor-1α expression.MTT assay and Hoechst staining results showed that DL-3-n-butylphthalide protected brain microvascular endothelial cells against oxygen glucose deprivation-induced injury in a dose-dependent manner.Western blot and immunofluorescent staining results further confirmed that the protective effect was related to upregulation of hypoxia inducible factor-1α.Real-time RT-PCR reaction results showed that DL-3-n-butylphthalide reduced apoptosis by inhibiting downregulation of pro-apoptotic gene caspase-3 mRNA expression and upregulation of apoptosis-executive protease bcl-2 mRNA expression;however,DL-3-n-butylphthalide had no protective effects on brain microvascular endothelial cells after knockdown of hypoxia inducible factor-1α by small interfering RNA.These findings suggest that DL-3-n-butylphthalide can protect brain microvascular endothelial cells against oxygen glucose deprivation-induced injury by upregulating bcl-2 expression and downregulating caspase-3 expression though hypoxia inducible factor-1α pathway.

Establishment and characterization of an astroglial cell line derived from the brain of half-smooth tongue sole(Cynoglossus semilaevis)

An astroglial cell line was established from the brain of half smooth tongue sole （Cynoglossus semilaevis） and was designated as CSAC. CSAC shows the morphological homogeneity of epithelial cells. The cell identity was tested by the presence of glial fibrillary acidic protein （GFAP）, which was revealed by RT-PCR and immunofluorescence. The cell line was optimally maintained at 24 ℃ in minimum essential medium supplemented with HEPES, antibiotics, 20% fetal bovine serum, 2- Mercaptoethanol （2-Me） and basic fibroblast growth factor. Chromosome analysis revealed that the CSAC cells maintained a normal diploid chromosome number （2n=42）. The fluorescent signals were observed in CSAC after the cells were transfected with green fluorescent protein （GFP） reporter plasmids. The CSAC cell line may serve as a valuable tool for studies on the potential functions of fish astroglial cells.

Differentiation profile of brain tumor stem cells:a comparative study with neural stem cells

大脑肿瘤干细胞(BTSC ) 的区别侧面的理解，在肿瘤房间人口之中的关键的，将通过有神经干细胞(NSC ) 的比较借卓见进 glioma 的起源并且最终产出新途径与这难处理的疾病作斗争。这里，我们从外科的 glioma 标本的有教养、净化的 BTSC 和 NSCsfrom 人的胎儿的大脑织物，并且进一步分析了他们的细胞的生物行为，他们的特别区别性质。是期望，区分进成熟神经显型的 NSC。然而， BTSC 展出了的 Inthe 一样的区别状况区分了差别。词法上，房间变得变平并且属于第一个星期，但是逐渐地此后聚集了并且改过漂浮的肿瘤范围。在相应时期期间，无差别的房间标记 CD133 和巢在的表示率在 BTSC 不停地减少，但是 1 个星期以后，他们重获上升趋势。有趣地，区分的房间标记 GFAP 和 beta-tubulinIIIshowed 表示变化逆到无差别的房间标记的。总起来说， BTSCswere 揭示了拥有一个能力抵抗区别，它实际上代表 glioma 的恶意的行为。

WJSC 6^(th) Anniversary Special Issues(2):Mesenchymal stem cells Brain mesenchymal stem cells:The other stem cells of the brain?

Multipotent mesenchymal stromal cells(MSC),have the potential to differentiate into cells of the mesenchymal lineage and have non-progenitor functions including immunomodulation.The demonstration that MSCs are perivascular cells found in almost all adult tissues raises fascinating perspectives on their role in tissue maintenance and repair.However,some controversies about the physiological role of the perivascular MSCs residing outside the bone marrow and on their therapeutic potential in regenerative medicine exist.In brain,perivascular MSCs like pericytes and adventitial cells,could constitute another stem cell population distinct to the neural stem cell pool.The demonstration of the neuronal potential of MSCs requires stringent criteria including morphological changes,the demonstration of neural biomarkers expression,electrophysiological recordings,and the absence of cell fusion.The recent finding that brain cancer stem cells can transdifferentiate into pericytes is another facet of the plasticity of these cells.It suggests that the perversion of the stem cell potential of pericytes might play an even unsuspected role in cancer formation and tumor progression.

Similarity on neural stem cells and brain tumor stem cells in transgenic brain tumor mouse models

Although it is believed that glioma is derived from brain tumor stem cells, the source and molecular signal pathways of these cells are still unclear. In this study, we used stable doxycycline-inducible transgenic mouse brain tumor models （c-myc/SV40Tag＋/Tet-on＋） to explore the malignant trans- formation potential of neural stem cells by observing the differences of neural stem cells and brain tumor stem cells in the tumor models. Results showed that chromosome instability occurred in brain tumor stem cells. The numbers of cytolysosomes and autophagosomes in brain tumor stem cells and induced neural stem cells were lower and the proliferative activity was obviously stronger than that in normal neural stem cells. Normal neural stem cells could differentiate into glial fibrillary acidic protein-positive and microtubule associated protein-2-positive cells, which were also negative for nestin. However, glial fibrillary acidic protein/nestin, microtubule associated protein-2/nestin, and glial fibrillary acidic protein/microtubule associated protein-2 double-positive cells were found in induced neural stem cells and brain tumor stem cells. Results indicate that induced neural stem cells are similar to brain tumor stem cells, and are possibly the source of brain tumor stem cells.

Collagen-chitosan scaffold impregnated with bone marrow mesenchymal stem cells for treatment of traumatic brain injury

Combinations of biomaterials and cells can effectively target delivery of cells or other therapeutic factors to the brain to rebuild damaged nerve pathways after brain injury.Porous collagen-chitosan scaffolds were prepared by a freeze-drying method based on brain tissue engineering.The scaffolds were impregnated with rat bone marrow mesenchymal stem cells.A traumatic brain injury rat model was established using the 300 g weight free fall impact method.Bone marrow mesenchymal stem cells/collagen-chitosan scaffolds were implanted into the injured brain.Modified neurological severity scores were used to assess the recovery of neurological function.The Morris water maze was employed to determine spatial learning and memory abilities.Hematoxylin-eosin staining was performed to measure pathological changes in brain tissue.Immunohistochemistry was performed for vascular endothelial growth factor and for 5-bromo-2-deoxyuridine(BrdU)/neuron specific enolase and BrdU/glial fibrillary acidic protein.Our results demonstrated that the transplantation of bone marrow mesenchymal stem cells and collagen-chitosan scaffolds to traumatic brain injury rats remarkably reduced modified neurological severity scores,shortened the average latency of the Morris water maze,increased the number of platform crossings,diminished the degeneration of damaged brain tissue,and increased the positive reaction of vascular endothelial growth factor in the transplantation and surrounding areas.At 14 days after transplantation,increased BrdU/glial fibrillary acidic protein expression and decreased BrdU/neuron specific enolase expression were observed in bone marrow mesenchymal stem cells in the injured area.The therapeutic effect of bone marrow mesenchymal stem cells and collagen-chitosan scaffolds was superior to stereotactic injection of bone marrow mesenchymal stem cells alone.To test the biocompatibility and immunogenicity of bone marrow mesenchymal stem cells and collagen-chitosan scaffolds,immunosuppressive cyclosporine was intravenously injected 12 hours before transplantation and 1-5 days after transplantation.The above indicators were similar to those of rats treated with bone marrow mesenchymal stem cells and collagen-chitosan scaffolds only.These findings indicate that transplantation of bone marrow mesenchymal stem cells in a collagen-chitosan scaffold can promote the recovery of neuropathological injury in rats with traumatic brain injury.This approach has the potential to be developed as a treatment for traumatic brain injury in humans.All experimental procedures were approved by the Institutional Animal Investigation Committee of Capital Medical University,China(approval No.AEEI-2015-035)in December 2015.

Polylactic Acid Nanoparticles Targeted to Brain Microvascular Endothelial Cells

In this work, blank polylactic acid （PLA） nanoparticles with unstained surface were prepared by the nano-deposition method. On the basis of the preparation, the effect of surface modification on brain microvascular endothelial cells （BMECs） targeting was examined by in vivo experiments and fluorescence microscopy. The results showed that PLA nanoparticles are less toxic than PACA nanoparticles but their BMECs targeting is similar to PACA nanoparticles. The experiments suggest that drugs can he loaded onto the particles and become more stable through adsorption on the surface of PLA nanoparticles with high surface activity. The surface of PLA nanoparticles was obviously modified and the hydrophilicity was increased as well in the presence of non-ionic surfactants on PLA nanoparticles. As a targeting moiety, polysobate 80 （T-80） can facilitate BMECs targeting of PLA nanoparticles.

Isolation,cultivation and identification of brain glioma stem cells by magnetic bead sorting

This study describes a detailed process for obtaining brain glioma stem cells from freshly dissected human brain glioma samples using an immunomagnetic bead technique combined with serum-free media pressure screening. Furthermore, the proliferation, differentiation and self-renewal biological features of brain glioma stem cells were identified. Results showed that a small number of CD133 positive tumor cells isolated from brain glioma samples survived as a cell suspension in serum-free media and proliferated. Subcultured CD133 positive cells maintained a potent self-renewal and proliferative ability, and expressed the stem cell-specific markers CD133 and nestin. After incubation with fetal bovine serum, the number of glial fibrillary acidic protein and microtubule associated protein 2 positive cells increased significantly, indicating that the cultured brain glioma stem cells can differentiate into astrocytes and neurons. Western blot analysis showed that tumor suppressor phosphatase and tensin homolog was highly expressed in tumor spheres compared with the differentiated tumor cells. These experimental findings indicate that the immunomagnetic beads technique is a useful method to obtain brain glioma stem cells from human brain tumors.