恶性胶质瘤中干细胞分离与细胞周期检测

目的从多形性胶质母细胞瘤组织标本中分离、培养、鉴定胶质瘤干细胞,并检测其所处的细胞周期。方法采用逐步递减培养基中血清含量的方式从多形性胶质母细胞瘤组织标本中获得悬浮生长的肿瘤球,应用免疫荧光染色检测胶质瘤干细胞及其分化细胞表面标志物的表达,免疫组化检测裸鼠颅内移植瘤表型。免疫磁珠分选多形性胶质母细胞瘤组织标本中的CD133阳性细胞后立即检测细胞周期。结果在多形性胶质母细胞瘤组织标本中成功分离出胶质瘤干细胞,在无血清培养液中呈悬浮生长,具有很强的自我更新与繁殖能力,免疫荧光染色显示该细胞表达CD133和巢蛋白,诱导分化后可分化成为神经元、星形胶质细胞与少突胶质细胞,裸鼠颅内移植后可重现亲本肿瘤表型。位于其中的胶质瘤干细胞大多处于G0～G1期。结论多形性胶质母细胞瘤组织中存在胶质瘤干细胞,相对处于静止状态。     

The presence of stem cell marker‐expressing cells is not prognostically significant in glioblastomas

Glioblastoma is one of the most frequent primary brain tumors and is characterized by aggressive clinical behavior and biologic heterogeneity. To evaluate the prognostic implication of cancer stem cell markers in glioblastoma, the expression of these markers was investigated in a large series of glioblastoma patients in relation to the survival rate. This series includes 88 cases of glioblastoma that were diagnosed at the Chonnam University Hwasun Hospital from 2004 to 2009. The expression of newly established stem cell markers (nestin, CD133 and CD15) was detected using immunohistochemical analysis. The presence of immunopositive tumor cells was evaluated and interpreted in comparison with the patients' survival data. The expression of nestin was high in 60 cases (68.2%). CD133 and CD15 were positive in 52 cases (59.1%) and 40 cases (45.5%), respectively. No statistically significant difference in patient survival according to stem cell marker expression was observed (P > 0.05). However, gross total resection or combined radiation therapy and chemotherapy significantly prolonged survival (P = 0.04 and P = 0.04). Cox's proportional hazards model showed that the gross total resection and combined radiation therapy and chemotherapy were independent prognostic factors. Although the correlation of stem cell marker expression with clinical outcome in glioma is of considerable interest, the data do not support their prognostic value in glioblastoma. Identification of the key cells in the glioblastoma population in the context of clinical outcomes will provide insight into the mechanism of brain tumorigenesis and will be of paramount importance in determining therapeutically appropriate targets.     

Lentivirus介导RNAi抑制人胶质瘤干细胞STAT3基因生物学效应观察

目的了解慢病毒载体对胶质瘤干细胞的亲嗜性及对靶基因表达的干扰效率，初步探索干扰STAT3基因对胶质瘤干细胞增殖的影响。方法构建STAT3基因shRNA的慢病毒表达载体，经293T细胞包装后，获得可表达STAT3基因shRNA的慢病毒颗粒；活性载体病毒感染人原代胶质瘤干细胞后流式细胞分析细胞感染效率；实时定量多聚酶链反应（PCR）和Westernblot检测细胞STAT3基因mRNA和蛋白表达及活化水平；增殖分析试剂盒测定细胞生长曲线，流式细胞分析细胞周期分布。结果在病毒感染比率值20：1时慢病毒载体对人原代胶质瘤干细胞的感染效率为98．6％；细胞感染可表达STAT3基因shRNA的载体慢病毒后，STAT3基因mRNA显著下降，抑制率为84．3％，STAT3蛋白表达下降81．5％，活化的pSTAT3下降97．9％；胶质瘤干细胞STAT3表达、活化受抑后细胞生长显著变慢，G1期细胞比例显著增高。结论慢病毒载体对人原代胶质瘤干细胞有着很高的感染效率，介导的RNAi可显著抑制靶基因的表达与活化，是对人胶质瘤干细胞基因功能研究的理想工具。人胶质瘤干细胞STAT3基因受抑后细胞生长显著变慢，出现G1期阻滞。     

脂肪源性γ-氨基丁酸能神经元移植治疗帕金森病大鼠的疗效观察

目的 研究脂肪来源的间充质干细胞诱导为γ-氨基丁酸(GABA)能神经元的方法 ,探讨GABA能神经元移植治疗帕金森病模型大鼠的疗效. 方法 取大鼠脂肪组织.利用本单位自行配制的神经干细胞培养基诱导分化为神经干细胞,利用GABA能神经元定向诱导培养基对神经干细胞进行二次定向诱导,并对其进行特异性鉴定.将诱导成功的神经干细胞、GABA能神经元分别移植入帕金森病大鼠模型的丘脑底核,在移植后2周、4周、8周观察大鼠行为学变化情况.结果 体外扩增的脂肪间充质干细胞经过神经干细胞培养基培养后,细胞定向诱导并表达巢蛋白、神经元烯醇化酶(NSE)等神经干细胞标志.经GABA能神经元定向分化培养基二次诱导后,免疫荧光鉴定细胞GAD65阳性.立体定向移植细胞4周后,神经干细胞组与GABA能神经元组的大鼠行为学均得到改善,且GABA能神经元组的疗效更加显著. 结论 脂防来源的间充质干细胞可诱导分化为GABA能神经元,将其移植人大鼠的丘脑底核可以明显改善帕金森病大鼠的运动功能.     

不同代数的成人骨髓间充质干细胞体外转化为神经细胞的实验研究

目的　探讨不同代数的成人骨髓间充质干细胞 ( h MSCs)体外向神经元细胞转化的效率 ,为骨髓间充质干细胞应用于临床提供可靠的实验数据。方法　采用 β-巯基乙醇做为诱导剂 ,选用第 2、4、6、8代 h MSCs在体外诱导 6 h后 ,用细胞化学及免疫组织化学检测神经元细胞、星形胶质细胞标记蛋白的表达。结果　第 2、4、6代h MSCs诱导后胞浆中均可见深蓝色的颗粒状尼氏体 ,第 8代 h MSCs诱导 6 h后胞浆中未见明显的深蓝色尼氏体结构。不同代数的 h MSCs经诱导 6 h后均表达 NSE、NF- M,不表达 GFAP;第 2、4、6代的阳性率无显著性差异 ( P>0 .0 5 ) ,第 8代与第 2、4、6代的阳性率有显著性差异 ( P<0 .0 5 )。结论　 β-巯基乙醇在体外可定向诱导 h MSCs转化为神经元细胞 ,第 2、4、6代的阳性率明显高于第 8代。     

Type 1 collagen as a potential niche component for CD133‐positive glioblastoma cells

Cancer stem cells are thought to be closely related to tumor progression and recurrence, making them attractive therapeutic targets. Stem cells of various tissues exist within niches maintaining their stemness. Glioblastoma stem cells (GSCs) are located at tumor capillaries and the perivascular niche, which are considered to have an important role in maintaining GSCs. There were some extracellular matrices (ECM) on the perivascular connective tissue, including type 1 collagen. We here evaluated whether type 1 collagen has a potential niche for GSCs. Imunohistochemical staining of type 1 collagen and CD133, one of the GSCs markers, on glioblastoma (GBM) tissues showed CD133‐positive cells were located in immediate proximity to type 1 collagen around tumor vessels. We cultured human GBM cell lines, U87MG and GBM cells obtained from fresh surgical tissues, T472 and T555, with serum‐containing medium (SCM) or serum‐free medium with some growth factors (SFM) and in non‐coated (Non‐coat) or type 1 collagen‐coated plates (Col). The RNA expression levels of CD133 and Nestin as stem cell markers in each condition were examined. The Col condition not only with SFM but SCM made GBM cells more enhanced in RNA expression of CD133, compared to Non‐coat/SCM. Semi‐quantitative measurement of CD133‐positive cells by immunocytochemistry showed a statistically significant increase of CD133‐positive cells in Col/SFM. In addition, T472 cell line cultured in the Col/SFM had capabilities of sphere formation and tumorigenesis. Type 1 collagen was found in the perivascular area and showed a possibility to maintain GSCs. These findings suggest that type 1 collagen could be one important niche component for CD133‐positive GSCs and maintain GSCs in adherent culture.     

胶质母细胞瘤肿瘤干细胞的分离培养与生物学特性研究

目的从人脑胶质母细胞瘤中分离培养脑肿瘤干细胞（brain tumorstem cells,BTSC）,并研究其生物学特性。方法利用无血清培养基和悬浮培养方法,从6例人胶质母细胞瘤标本中分离培养BTSC,并通过单克隆形成实验观察脑肿瘤干细胞球（brain tumor sphere,BTS）的形成过程。将BTSC接种于含血清培养基,观察其在体外的分化特点。将BTSC子代分化细胞更换培养条件,观察其在无血清培养基中的逆向分化现象。应用细胞免疫荧光染色对BTSC及其分化细胞进行鉴定。结果在人胶质母细胞瘤中成功分离出BTSC,其在无血清培养基中呈悬浮球状生长,具有很强的自我更新和增殖能力;免疫荧光显示其表达CD133。BTSC在含血清培养基中发生贴壁分化。分化后的子代细胞可表达CD133、神经元特异性烯醇化酶（NSE）和胶质纤维酸性蛋白（GFAP）。BTSC子代分化细胞在无血清条件下培养,能够再次增殖形成BTS,呈逆向分化现象。结论人胶质母细胞瘤中存在BTSC,其具有自我更新、无限增殖、多向分化以及逆向分化等生物学特性。     

Membranous expression of glucose transporter-1 protein (GLUT-1) in embryonal neoplasms of the central nervous system

The human erythrocyte GLUT-1 is a transmembrane protein which facilitates transport of glucose in the cell in an energy-independent fashion. Neuroectodermal stem cells show strong membrane immunoreactivitry with this marker at early developmental stages in rodents. Membranous expression by undifferentiated neuroectodermal cells gradually decreases while GLUT-1 becomes confined to the endothelial cells, when these acquire blood-brain barrier function. We thus sought to determine whether GLUT-1 expression was limited to embryonal neoplasms of the central nervous system (CNS) which are presumably derived from developmentally arrested neuroectodermal stem cells. Archival material of 40 primary CNS neoplasms were examined for immunoreactivity with anti-GLUT-1. This included both non-embryonal neoplasms (18 astrocytic tumours, one ependymoma and three oligodendroglioma) and embryonal neoplasms (12 cerebellar medulloblastomas, four supratentorial PNETs and two atypical teratoid/rhabdoid tumours (AT/RhT)). In addition, cell lines and nude mice xenografts derived from both undifferentiated and differentiated tumours were assessed for GLUT-1 immunoreactivity by both immunohistochemistry and Western blotting. All embryonal tumours, MBs and PNET xenografts consistently showed GLUT-1 membrane staining. Non-embryonal neoplasms were negative except for vascular staining. Membrane protein fraction of embryonal tumours cell lines immunoreacted by immunoblot with GLUT-1, whereas the glioblastoma cell line was negative. Expression of GLUT-1 supports the stem cell nature of the cells of origin of MBs, supratentorial PNET and AT/RhTs. As a result, GLUT-1 is a useful marker to define the embryonal nature of CNS neoplasms.     

胶质瘤干细胞抗凋亡和多重耐药基因的表达

目的 从人脑胶质瘤组织中分离、培养胶质瘤干细胞,并探讨其生物学特性及其抗凋亡和多重耐药基因的表达差异.方法 人脑胶质瘤组织经过原代细胞培养后,用无血清培养方法获得胶质瘤干细胞球,用10%的胎牛血清培养诱导分化,分化前后分别做nestin、tubulin-β、GFAP免疫细胞化学荧光染色,并观察胶质瘤干细胞形态学改变.同时,应用实时荧光定量PCR技术检测livin,livinot,livinβ,survivin,MRPI和MRP3 mRNA的表达.结果 有2例分离培养出干细胞球体,这些球体具有典型的干细胞特性,nestin染色为阳性;在无血清培养基中呈悬浮球样生长,能够自我更新和增殖,在有血清培养基中能够分化,tubulin-β、GFAP染色为阳性.胶质瘤干细胞球livin、livinα、livinβ、survivin和MRP-1 mRNA表达量比较胶质瘤组织表达量都有不同程度的升高,而MRP-3mRNA表达量降低.结论 胶质瘤干细胞抗凋亡和MRP-1基因表达量较胶质瘤表达量有不同程度的升高,提示胶质瘤干细胞比较其同源的胶质瘤细胞具有更强的耐药性,这可能是肿瘤耐药的机制.     

Response of human glioblastoma cells to recombinant interleukin-2

We investigated the ability of glioma cells to respond to T cell-derived lymphokines. The growth of astrocytoma and mixed glioblastoma cell lines, as assessed by DNA synthesis, was inhibited in the presence of supernatants derived from mitogen-stimulated human T cells, an HTLV-II-transformed human T cell line, Mo, and human interleukin-2 (IL-2). The mixed glioblastoma cell line, 138-MG-C, was subjected to limiting dilution analysis, and two cell lines (5D7, 5C5) were derived which were homogeneous with respect to staining for galactocerebroside (GalC) (100%). These two GalC+ glioblastoma cell lines proliferated in the presence of high concentrations of recombinant human interleukin-2 (RIL-2). Additionally, these cell lines bear receptors for the IL-2 molecule as determined by immunofluorescent staining with various anti-IL-2 receptor antibodies.     

The origins of glioma: E Pluribus Unum?

Malignant glioma is among of the most devastating, and least curable, types of cancer. Since the re-emergence of the cancer stem cell hypothesis, much progress has been made towards elucidating the cellular origin of these tumors. The hypothesis that tumors are hierarchically organized, with a cancer stem cell at the top that shares defining features with somatic stem cells and provides therapeutic refractoriness properties, has put adult stem cells into the limelight as prime suspect for malignant glioma. Much confusion still exists, though, as to the particular cell type and processes that lead to oncogenic transformation. In this review, we will discuss recent developments and novel hypotheses regarding the origin of malignant gliomas, especially glioblastoma. In particular, we argue that glioblastoma is the result of different pathways originating in multiple sources that all ultimately converge in the same disease. Further attention is devoted to potential scenarios leading to transformation of different stem/progenitor cell types of the brain, and the probability and relevance of these scenarios for malignant tumorigenesis.     

Apoptosis in glioma-bearing rats after neural stem cell transplantation

Abnormal activation of the Ras/Raf/Mek/Erk signaling cascade plays an important role in glioma. Inhibition of this aberrant activity could effectively hinder glioma cell proliferation and promote cell apoptosis. To investigate the mechanism of gJioblastoma treatment by neural stem ceiJ trans- plantation with respect to the Ras/Raf/Mek/Erk pathway, C6 glioma cells were prepared in sus- pension and then infused into the rat brain to establish a glioblastoma model. Neural stem cells isolated from fetal rats were then injected into the brain of this glioblastoma model. Results showed that Raf-1, Erk and Bcl-2 protein expression significantly increased, while Caspase-3 protein expression decreased. After transplantation of neural stem cells, Raf-1, Erk and Bcl-2 protein expression significantly decreased, while Caspase-3 protein expression significantly in-creased. Our findings indicate that transplantation of neural stem cells may promote apoptosis of glioma cells by inhibiting Ras/Raf/Mek/Erk signaling, and thus may represent a novel treatment approach for glioblastoma.     

PiB对胶质瘤细胞增殖与侵袭能力的影响

目的探讨PIN1抑制剂Pi B对U87胶质瘤细胞系增殖、迁移和侵袭能力的影响。方法人胶质瘤细胞系U87常规培养,取对数生长期细胞进行试验,以1.0μmol/L Pi B处理U87细胞48 h为实验组,未经任何处理的U87细胞作为对照组。RT-PCR和Western blotting检测PIN1基因的m RNA和蛋白表达。免疫荧光检测阳性细胞数。Transwell实验检测细胞迁移和侵袭能力的改变。MTT比色法检测Pi B对细胞增殖的抑制作用。结果与对照组相比,实验组细胞PIN1基因的m RNA和蛋白表达水平明显下调,PIN1阳性细胞数明显减少,细胞抑制率明显,且与药物剂量和作用时间成相关性,细胞迁移和侵袭能力下降,差异有统计学意义（P〈0.05）。结论 Pi B可靶向抑制PIN1基因的m RNA和蛋白水平表达,进而抑制胶质瘤细胞增殖、迁移和侵袭能力。     

人胎儿肝干细胞在聚酸酐-氨基葡聚糖三维载体内的生长活性◆

目的：观察聚酸酐-氨基葡聚糖三维载体材料对胎儿肝干细胞黏附及增殖的影响。 方法：采用改进的两步胶原酶灌注消化法加percoll液不连续密度梯度离心的方法,分离胎儿肝干细胞。选取胎儿肝干细胞的第3代细胞,种植于聚酸酐-氨基葡聚糖三维载体材料上。倒置显微镜下观察细胞的黏附和生长状况;计算细胞贴壁率、细胞增殖活力、计数细胞数。取细胞-载体进行组织学切片,苏木精-伊红染色光镜下观察细胞在载体中生长情况。在细胞培养第7天进行免疫荧光化学染色和流式细胞仪检测标志物表达。 结果：聚酸酐-氨基葡聚糖三维载体能促进肝干细胞在材料内黏附并保持其在机体内的形态。载体材料内的肝干细胞功能活跃,在材料表面和三维空间内部培养的肝干细胞均能持续增殖。经过连续10 d共同培养,聚酸酐-氨基葡聚糖三维载体对干细胞无毒性,人胎儿肝干细胞可以很好的贴附于聚酸酐-氨基葡聚糖三维载体支架上,细胞增殖活力良好,标志物持续表达,培养7 d得到的细胞数量增多19.7%。 结论：聚酸酐-氨基葡聚糖三维载体能促进肝干细胞的增殖,有可能作为肝干细胞的载体应用于肝脏组织工程。 关键词：聚酸酐;氨基葡聚糖;胎儿肝干细胞;人工肝;组织工程 doi:10.3969/j.issn.1673-8225.2009.47.022     

60^Coγ射线对多形性胶质母细胞瘤中的肿瘤干细胞的影响

目的研究不同生存条件下多形性胶质母细胞瘤中肿瘤干细胞对60^Coγ射线的敏感性。方法60^Coγ射线分别照射多形性胶质母细胞瘤中的肿瘤干细胞及从多形性胶质母细胞瘤中分离、培养的肿瘤干细胞后,流式细胞仪检测细胞周期的变化。采用TUNEL、Annexin-FITC等方法检测其凋亡及观察照射后上述肿瘤干细胞裸鼠颅内移植的致瘤性的变化。结果单独体外存活的脑肿瘤干细胞处于活跃的细胞周期中,对射线敏感,经60^Coγ射线照射后凋亡细胞明显增多,裸鼠颅内移植后未引起肿瘤。在多形性胶质母细胞瘤中存活的肿瘤干细胞处于G0-G1期,对射线抗拒,裸鼠颅内移植后继续分化成为多形性胶质母细胞瘤。结论多形性胶质母细胞瘤经过放射治疗后原位复发可能是由于其中的脑肿瘤干细胞对射线抗拒而导致。     

人胶质瘤干细胞内在自我更新能力

目的 了解胶质瘤干细胞内在的自我更新和增殖能力。方法 观察原代胶质瘤细胞在单纯改良Eagle/F12培养液（DMEM/F12）中胶质瘤干细胞球的形成,并检测其CD133、胶质纤维酸性蛋白（GFAP）、微管相关蛋白（MAP2）、髓磷脂碱性蛋白（MBP）的表达。通过二代球体形成、细胞增殖测定、分化实验分析其自我更新、增殖、多能分化能力。通过裸鼠移植瘤实验观察所分离细胞球细胞与原代培养胶质瘤细胞成瘤能力的差异。结果 在单纯DMEM/F12培养液中形成的胶质瘤细胞球细胞表达神经干细胞标记CD133,不表达分化标志GFAP、MAP2,少数细胞表达MBP。分离出的胶质瘤细胞球细胞可在单纯DMEM/F12培养基中增殖,并能形成CD133阳性的二代细胞球,可分化为GFAP、MAP2、MBP阳性表达的肿瘤细胞。裸鼠成瘤实验显示其成瘤能力显著高于原代胶质瘤细胞。结论 胶质瘤干细胞能在无血清、无外源性细胞因子培养基中形成肿瘤干细胞球,胶质瘤干细胞的自我更新和增殖不依赖于外源性生长因子,它可能拥有自我更新的自身活化机制。     

人脂肪源干细胞分离培养及向成骨细胞的诱导分化

背景：脂肪源干细胞可分泌众多的免疫调节因子,不引起T细胞的细胞毒作用,并可通过调整T淋巴细胞的种类和数量。 目的：探讨人脂肪源干细胞在体外分离培养扩增的方法及向成骨细胞诱导分化的能力。 方法：以0.1%的Ⅰ型胶原酶通过组织消化的方法分离人脂肪组织中的干细胞,体外扩增培养至第2代后检测其表面抗原的表达,并在成骨诱导液中促进其向成骨细胞的分化,通过碱性磷酸酶染色、茜素红染色及对碱性磷酸酶的RT-PCR检测来明确其分化能力。 结果与结论：体外分离培养的脂肪源干细胞生长稳定,扩增速度快。流式细胞仪检测结果显示其高表达干细胞相关抗原。向成骨细胞诱导后经免疫组化染色可见矿化结节形成,RT-PCR检测发现碱性磷酸酶表达阳性。提示脂肪源干细胞在体外分离培养方法简单,扩增速度快,并具有定向分化的能力,是可靠的组织修复和细胞治疗的种子细胞来源。     

A protocol for the differentiation of human embryonic stem cells into dopaminergic neurons using only chemically defined human additives: Studies in vitro and in vivo

Our ability to use human embryonic stem (hES) cells in cell replacement therapy for Parkinson's disease depends on the discovery of ways to simply and reliably differentiate a dopaminergic (DA) phenotype in these cells. Although several protocols exist for the differentiation of DA traits in hES, they involve the prolonged use of complex media with undefined components, cell conditioned media and/or co-culture with various cells, usually of animal origin. In this study, several well-characterized (H9, BG01) and several new uncharacterized (HUES7, HUES8) hES cell lines were studied for their capacity to differentiate into DA neurons in culture using a novel rapid protocol which uses only chemically-defined human-derived media additives and substrata. Within 3 weeks, cells from all 4 cell lines progressed from the undifferentiated state to beta-tubulin III positive cells expressing DA markers in vitro. Moreover, transplantation of these cells into the striata of 6-hydroxydopamine-treated rats at the neuronal progenitor stage resulted in the appearance of differentiated DA traits in vivo 2-3 weeks later.     

Functional Differences of miR-125b on the Invasion of Primary Glioblastoma CD133-Negative Cells and CD133-Positive Cells

MicroRNAs (miRNAs) are small noncoding RNAs whose function as modulators of gene expression is crucial for the proper control of cell development, differentiation, and homeostasis. The total number and composition of miRNAs expressed per cell at different stages of development varies widely, and the same miRNA may function differently at different stages of development. In this prospective study, we evaluated the function of miR-125b at different developmental stages of glioblastoma cells, such as primary glioblastoma cells and the corresponding stem cells. CD133 is an important surface marker in glioblastoma stem cells. We found that the upregulation of miR-125b had no effects on the invasion of primary glioblastoma CD133-negative cells but that it could inhibit the invasion of corresponding CD133-positive cells; however, the downregulation of miR-125b also had no effects on the invasion of primary glioblastoma CD133-negative cells but promoted the invasion of CD133-positive cells. Further research into the underlying mechanism demonstrated that the effects of miR-125b on the invasion of glioblastoma CD133-positive cells were associated with the alteration of the expression of MMPs (MMP-2 and MMP-9) and corresponding inhibitors (RECK and TIMP3). Our results demonstrate that miR-125b expression plays an essential role in the invasion of glioblastoma CD133-positive cells but not CD133-negative cells. Therefore, miR-125b may represent a novel target for therapy targeting the invasion of glioblastoma stem cells in the future.     

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.