神经球的异质性、发生方式及神经球方法的适用性

神经球是神经干细胞在体外扩增培养中的一般表现形式.早先认为神经球是神经干细胞的单克隆细胞团,并在这一假设基础上形成了目前广为应用的神经球方法.但最近神经球被证实并非神经干细胞的单克隆群体,在神经球内部和神经球之间存在着普遍的异质性;神经球的发生除了细胞增殖外,还包括细胞重团聚,神经球融合等方式;在神经球的形成过程中,亦有诸多因子参与影响.这些研究结果说明目前神经球方法的精确性需要重新定义,神经球方法的适用性还需要进一步探讨.     

人胎儿脊髓神经干细胞的分离培养

本文旨在探讨是否能够从低温保存的流产儿分离培养出脊髓神经干细胞。将14周流产儿在4℃下保存,2、6和12h后取脊髓,将颈段、胸段、腰骶段分别进行无血清培养,并用胎牛血清诱导分化。用克隆培养的方法验证培养细胞的干细胞特性;用免疫荧光细胞化学的方法检测神经干细胞标志nestin及干细胞诱导分化后神经元标志MAP2、星形胶质细胞标志GFAP、胆碱能标志ChAT,并比较不同时间点以及不同部位分离的神经T细胞的差异。在各个时间点,从颈段、胸段、腰骶段脊髓均分离培养出具有连续增殖能力的神经球,其中腰骶段分离出的神经球数量最多,12h组各段分离出的神经球较2、6h组显著减少。各段培养中的神经球均为nestin阳性,诱导分化后均能够产生GFAP阳性星形胶质细胞、MAP2阳性神经元以及ChAT阳性胆碱能神经元。各段培养中的神经干细胞的克隆形成能力相似。以上结果表明,从低温保存的人胎儿能够分离培养出脊髓神经干细胞,这为基础研究以及未来治疗应用提供了新的细胞来源。     

神经干细胞移植在神经退行性疾病中的研究进展

神经退行性疾病是一类可导致感觉丧失、运动功能丧失和记忆衰竭等症状的难治性疾病,传统治疗方法虽能延缓疾病进展,但局限性明显。而神经干细胞移植作为一种潜在的新型治疗方式能够有效促进神经细胞的功能恢复及组织再生,在神经退行性疾病的治疗应用方面前景广阔。因此,本文通过对神经干细胞的现有来源及其在神经退行性疾病治疗中的研究进展进行综述,以期为神经干细胞移植在神经退行性疾病治疗中的应用提供新的思路。     

体外神经干细胞克隆球的超微结构-透射电镜观察

为观察培养的神经干细胞克隆球内部的超微结构特征,采用无血清培养技术,在体外进行小鼠纹状体神经干细胞克隆球的培养传代,经过免疫细胞化学鉴定后,对单一的神经干细胞克隆球进行固定,常规透射电镜观察。结果表明,神经干细胞可以在bFGF等生长因子存在的情况下,在无血清培养液内增殖生成悬浮状态的神经干细胞克隆球,这种克隆可被诱导生成神经细胞和神经胶质细胞,电镜下,神经干细胞克隆球内部细胞相互间可形成特化的膜性结构,细胞内可有小泡出现,部分细胞有凋亡的形态。     

重组腺相关病毒转染神经干细胞球的实验研究

目的:探讨重组腺相关病毒2型(rAAV2)对神经干细胞球的转染能力.方法:①将FITC标记的rAAV2(FITC-rAAV2)分成两组,A组直接与神经干细胞球混合,B组与肝素混匀后再与神经干细胞球混合,孵育30 min后在荧光显微镜下观察;②含有GFP报告基因的rAAV2(rAAV2-GFP)与神经干细胞球孵育30 min后,分成两组:A组继续在培养箱内培养,B组分散成单细胞后移植到大鼠脑内,一个月后分别在荧光显微镜下观察神经干细胞球和大鼠脑组织切片中报告基因的表达情况;③将含有低氧启动子(低氧应答元件,HRE)、VEGF和GFP的rAAV2(rAAV2-HRE-VEGF-GFP)转染神经干细胞球后分为两组:A组在低氧条件下培养,B组在常规条件下培养,72 h后观察报告基因的表达情况.结果:①FITC-rAAV2转染神经干细胞球的结果:A组有明亮的绿色荧光,B组基本无绿色荧光;②rAAV2-GFP转染神经干细胞球后一个月,A、B两组均可以看到绿色荧光;③rAAV2-HRE-VEGF-GFP转染神经干细胞球后72 h,A组可见绿色荧光,B组无绿色荧光.结论:rAAV2可以与神经干细胞球特异性结合,rAAV2携带的外源基因在体内和体外均可以有效表达,rAAV2携带外源基因的表达可以人为调控.     

大鼠胚胎神经干细胞单克隆化及单层化培养和鉴定

采用原代培养SD胎鼠神经干细胞,在形成神经球之后,传代至0.1%明胶包被的培养皿,显微镜下挑取一个神经球贴壁后的细胞团,吹打后贴壁培养．同样方法挑细胞团并传代培养5～6次,得到纯化的由一个神经干细胞扩增的克隆,对得到的神经干细胞进行鉴定以及分化能力的评估,证明得到的细胞就是神经干细胞．结果表明,成功分离了SD胎鼠的神经干细胞,进行单克隆化单层培养,神经干细胞和分化后的细胞标志基因都可以检测到．上述工作为疾病模型大鼠治疗及相关基础研究提供细胞来源及形态标准．     

神经干细胞克隆球中干细胞的比例变化

为了定量研究神经干细胞体外产生的克隆结构“neurospheres”中干细胞的比例变化,利用无血清培养、细胞克隆培养技术及免疫细胞化学染色方法,观察不同代数神经干细胞克隆球中nestin阳性细胞的比例。发现随着传代次数增加,克隆球中nestin阳性细胞的比例也在显著减少(P<0.001)。提示在体外培养体系中,形成的克隆球具有异质性,并且在不同代数间神经干细胞的比例也显著不同。     

神经干细胞研究进展

神经干细胞(neural stem cells, NSCs)是中枢神经系统中保持分裂和分化潜能的细胞,对它的研究和应用已成为近年来脑科学研究的一个重要领域.神经干细胞体外培养技术的建立提供了对其进行研究的有力手段.目前的研究主要集中于神经干细胞在脑中的起源、分布及在中枢神经系统疾病治疗中的应用等方面.     

钙离子与缺氧性神经干细胞凋亡的相关性研究

目的检测钙离子浓度在缺氧性神经干细胞凋亡过程中的变化,以探讨缺氧性神经干细胞凋亡的发生机制。方法从大鼠胚胎神经管获取神经干细胞,经无血清悬浮培养技术获得神经球。对所获神经球行干细胞克隆试验、Br-dU掺入标记试验、nestin、NSE和GFAP免疫荧光染色,以确认神经干细胞的生物学特性。三气培养箱予以缺氧干预,按缺氧程度分为5%O2组、10%O2组和正常对照组(20%O2),每个实验组又依缺氧干预时间的不同,分为24h、48h、72h、96h、120h5个亚组。用激光扫描共聚焦显微镜和Fluo-3荧光探针标记技术检测神经干细胞内钙离子浓度;采用An-nexinⅤ-FITC/PI检测细胞凋亡率。结果5%O2120h组和10%O2120h组中神经干细胞凋亡率显著高于正常对照组和其他缺氧干预组,并且伴随有胞内钙超载。结论细胞内钙超载可能是缺氧性神经干细胞凋亡机制中的一个重要环节。     

不同浓度的添加剂 N2和 B27对神经干细胞增殖的影响

目的：通过比较添加不同浓度的无血清培养添加剂N2、B27对神经干细胞增殖的影响,探讨一种最优的体外培养大鼠胚胎神经干细胞的方法。方法：分离培养胚胎13．5dSD大鼠的神经干细胞,采用CCK-8细胞计数、BrdU掺入、测量神经球直径的方法,比较不同浓度N2、B27对神经干细胞增殖的影响。结果：N2与B27合用对神经干细胞增殖和成球的作用最为明显,单独添加2％B27的作用优于1％B27和N2组。结论：1％N2和1％B27同时添加更有利于神经干细胞的增殖和自我更新,此方法为神经干细胞的应用提供了实验依据。     

人胚胎神经干细胞的基础及应用研究进展

神经干细胞是中枢神经系统中具有增殖、自我更新能力以及多种分化潜能的细胞,对它的研究已经成为神经生物学、发育生物学以及脑科学研究的一个热点。随着神经干细胞（特别是胚胎神经干细胞）的分离、培养成功,神经干细胞移植已被尝试用于神经系统损伤等疾病的治疗。但是,关于胚胎神经干细胞的研究尚处于初级阶段,特别是人胚胎神经干细胞的研究、报道还比较少。本文对国内、外近几年来关于人胚胎神经干细胞的基础及应用研究进展作了综述。     

Strengths and limitations of the neurosphere culture system

After the initial reports of free-floating cultures of neural stem cells termed neurospheres (1,2), a wide array of studies using this promising culture system emerged. In theory, this was a nearperfect system for large-scale production of neural cells for use in cell replacement therapies and to assay for and characterize neural stem cells. More than a decade later, after rigorous scrutiny and ample experimental testing of the neurosphere culture system, it has become apparent that the culture system suffers from several disadvantages, and its usefulness is limited for several applications. Nevertheless, the bulk of high-quality research produced over the last decade has also shown that under the right circumstances and for the appropriate purposes, neurospheres hold up to their initial promise. This article discusses the pros and cons of the neurosphere culture system regarding its three major applications: as an assay for neural stem cells, as a model system for neurogenesis and neural development, and for expansion of neural stem cells for transplantation purposes.     

Biology and clinical application of neural stem cells

Neural stem cells, which exist in various regions of the CNS throughout the mammalian lifespan, can be expanded and induced to differentiate into neurons and glia in vitro and in vivo. Because of these characteristics, there has been increasing interest in the identification and characterization of neural stem cells and neural progenitor cells both for basic developmental biology studies and for therapeutic applications to the damaged brain. Transplantation of neural stem cells or their derivatives into a host brain and the proliferation and differentiation of endogenous stem cells by pharmacological manipulations are potential treatments for many neurodegenerative diseases and brain injuries, such as Parkinson's disease, brain ischemia and spinal cord injury. Continued progress in neural stem cell research is providing a new future for brain repair.     

Neural stem cells could serve as a therapeutic material for age-related neurodegenerative diseases

Progressively loss of neural and glial cells is the key event that leads to nervous system dysfunctions and diseases. Several neurodegenerative diseases, for instance Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, are associated to aging and suggested to be a consequence of deficiency of neural stem cell pool in the affected brain regions. Endogenous neural stem cells exist throughout life and are found inspecific niches of human brain. These neural stem cells are responsible for the regeneration of new neurons to restore, in the normal circumstance, the functions of the brain. Endogenous neural stem cells can be isolated, propagated, and, notably, differentiated to most cell types of the brain. On the other hand, other types of stem cells, such as mesenchymal stem cells, embryonic stem cells, and induced pluripotent stem cells can also serve as a source for neural stem cell production, that hold a great promise for regeneration of the brain. The replacement of neural stem cells, either endogenous or stem cell-derived neural stem cells, into impaired brain is highly expected as a possible therapeutic mean for neurodegenerative diseases. In this review, clinical features and current routinely treatments of agerelated neurodegenerative diseases are documented. Noteworthy, we presented the promising evidence of neural stem cells and their derivatives in curing such diseases, together with the remaining challenges to achieve the best outcome for patients.     

Gene delivery to adult neural stem cells

Neural stem cells may present an ideal route for gene therapy as well as offer new possibilities for the replacement of neurons lost to injury or disease. However, it has proved difficult to express ectopic genes in stem cells. We report methods to introduce genes into adult neural stem cells using viral and nonviral vectors in vitro and in vivo. Adenoviral and VSV-G-pseudotyped retroviral vectors are more efficient than plasmid transfection or VSV-G lentiviral transduction in vitro. We further show that adult neural stem cells can be directed to a neuronal fate by ectopic expression of neurogenin 2 in vitro. Plasmids can be delivered in vivo when complexed with linear polyethyleneimine, and gene expression can be targeted specifically to neural stem or progenitor cells by the use of specific promoters. These techniques may be utilized both to study the function of various genes in the differentiation of neural stem cells to specific cell fates and, ultimately, for gene therapy or to generate specific differentiated progeny for cell transplantation.     

Isolation and characterization of neural stem cells from human fetal striatum

This paper described that neural stem cells (hsNSCs) were isolated and expanded rapidly from human fetal striatum in adherent culture. The population was serum- and growth factor-dependent and expressed neural stem cell markers. They were capable of multi-differentiation into neurons, astrocytes, and oligodendrocytes. When plated in the dopaminergic neuron inducing medium, human striatum neural stem cells could differentiate into tyrosine hydroxylase positive neurons. hsNSCs were morphologically homogeneous and possessed high proliferation ability. The population doubled every 44.28h and until now it has divided for more than 82 generations in vitro. Normal human diploid karyotype was unchanged throughout the in vitro culture period. Together, this study has exploited a method for continuous and rapid expansion of human neural stem cells as pure population, which maintained the capacity to generate almost fifty percent neurons. The availability of such cells may hold great interest for basic and applied neuroscience.     

Regenerative therapy for neuronal diseases with transplantation of somatic stem cells

Pluripotent stem cells, which are capable of differentiating in various species of cells, are hoped to be donor cells in transplantation in regenerative medicine. Embryonic stem (ES) cells and induced pluripotent stem cells have the potential to differentiate in approximately all species of cells. However, the proliferating ability of these cells is high and the cancer formation ability is also recognized. In addition, ethical problems exist in using ES cells. Somatic stem cells with the ability to differentiate in various species of cells have been used as donor cells for neuronal diseases, such as amyotrophic lateral sclerosis, spinal cord injury, Alzheimer disease, cerebral infarction and congenital neuronal diseases. Human mesenchymal stem cells derived from bone marrow, adipose tissue, dermal tissue, umbilical cord blood and placenta are usually used for intractable neuronal diseases as somatic stem cells, while neural progenitor/stem cells and retinal progenitor/stem cells are used for a few congenital neuronal diseases and retinal degenerative disease, respectively. However, non-treated somatic stem cells seldom differentiate to neural cells in recipient neural tissue. Therefore, the contribution to neuronal regeneration using non-treated somatic stem cells has been poor and various differential trials, such as the addition of neurotrophic factors, gene transfer, peptide transfer for neuronal differentiation of somatic stem cells, have been performed. Here, the recent progress of regenerative therapies using various somatic stem cells is described.     

猪神经干细胞分离培养研究进展

神经干细胞用于神经学临床修复和基础理论研究的前提是首先完成神经干细胞的体外分离、培养、纯化并大量扩增。鼠、人、猪中都已成功分离出神经干细胞并已尝试用于动物神经系统损伤等疾病的治疗,尽管在鼠和人上的研究很多,相对于鼠神经干细胞在神经学临床应用上的局限和人神经干细胞在材料来源上的不便,猪作为神经干细胞临床应用和基础研究的模式动物有很大的潜力。但关于猪神经干细胞体外分离培养的研究非常少,本文对这方面的研究进展做一综述。     

Neural stem cells—trends and advances

For many years, accepted dogma held that brain is a static organ with no possibility of regeneration of cells in injured or diseased human brain. However, recent preclinical reports have shown regenerative potential of neural stem cells using various injury models. This has resulted in renewed hope for those suffering from spinal cord injury and neural damage. As the potential of stem cell therapy gained impact, these claims, in particular, led to widespread enthusiasm that acute and chronic injury of the nervous system would soon be a problem of the past. The devastation caused by injury or diseases of the brain and spinal cord led to wide premature acceptance that “neural stem cells (NSCs)” derived from embryonic, fetal or adult sources would soon be effective in reversing neural and spinal trauma. However, neural therapy with stem cells has not been realized to its fullest extent. Although, discrete population of regenerative stem cells seems to be present in specific areas of human brain, the function of these cells is unclear. However, similar cells in animals seem to play important role in postnatal growth as well as recovery of neural tissue from injury, anoxia, or disease. J. Cell. Biochem. 114: 764–772, 2013. © 2012 Wiley Periodicals, Inc.