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1。基本设置，然后再进行剥离: <ol><li>完成国科会培养基的准备适当的音量NeuroCult NSC基础培养基和NeuroCult NSC增殖辅料，分别在9:1的比例混合。 </li><li>在37℃水浴介质预热。 </li><li>冷战的HEPES缓冲的最低限度的基本介质与高浓度抗生素（10％）（HEM）准备剥离和清洗的目的。另外，国科会与抗生素补充培养基，也可能被用于此目的。 </li><li> 25-30毫升含抗生素的冷HEM分装到无菌的胚胎收集50 mL管。 </li><li>几个10厘米塑料培养皿需要清扫期间举行的胚胎和大脑，还举行解剖组织。 </li><li>消毒用的手术工具，需要删除的胚胎（大剪刀，小尖剪刀，大钳，小弯钳）或胚胎脑夹层（小镊子，弯细钳，45 °角的精细镊子，小剪刀）玻璃珠的灭菌器在250 ° C或其他可用高压灭菌方法。 </li><li>解剖镜下用70％酒精擦拭和层流或PC2罩内设立。 </li></ol> 2。收获E14，小鼠大脑和显微解剖: <ol><li>交配孕鼠一时间天麻醉14 次妊娠的机构批准的动物协议。 </li><li>颈椎脱位执行，以确保动物不遭受的痛苦和悲伤。 </li><li>麻醉鼠标是奠定其吸水纸巾的背面，腹部是用70％乙醇冲洗，消毒面积。 </li><li>在腹部的皮肤是掌握使用大钳，大剪刀切割，然后在皮肤底层筋膜暴露腹腔和子宫角。 </li><li>用小镊子和剪刀的子宫被删除，转移到50 ml锥形管中冷下摆。 </li><li>子宫组织转移到引擎盖和足够量的新鲜无菌冷下摆一次或两次，然后冲洗，以消除可能的污染物，如血液和头发。 </li><li>子宫组织，然后转移到一个培养皿中含有冷HEM10厘米。 </li><li>子宫角用小弯钳和剪刀打开，胚胎被转移到一个新10厘米的菜，其中包含冷HEM。 </li><li>胚胎的头是分开的脊髓型颈椎病水平，并转移到另一个培养皿中含有冷HEM。 </li><li>在解剖显微镜下去除颅骨脑转移培养皿。 </li><li>从尾侧用细弯钳，使首脑背侧朝上元首举行。采用微型剪刀，是第一个横向切上面的眼睛，然后继续在中线从额头对的后脑勺。确保穿过皮肤和颅骨，不要损害底层的大脑。 </li><li>大脑是从头骨，推动在一个落后的议案，用弯钳切条的边缘。此过程继续，直到所有的大脑是从头骨中删除。 </li><li>大脑是稳定的使用，使背侧朝上，然后使用microscissors降息是通过每个从嗅球半球半球揭露节后金榜背面的皮质执行的弯钳举行。 </li><li>一方面使用弯钳和在其他的45 °直角钳，皮质切断皮瓣的传播和解剖，神经节金榜。 </li><li>解剖组织是放置在一个无菌10厘米的培养皿中，此过程重复进行，直到所有的大脑已经微解剖。 </li><li>收集组织块用1毫升的NSC培养基在1毫升枪头，转移到15 mL离心管中。 </li><li>组织彻底分离，但轻轻按下枪头，试管底部，吹打暂停向上和向下。这种方式的团块被分解成单个细胞。移液是使银河系一样悬挂，是取得了3-4倍。然后，暂停允许定居1-2分钟，使沉淀的非游离团块。 </li><li>几乎整个细胞悬液转移到其他管，然后再1毫升的NSC培养基添加到剩余的团块和单细胞分离所述。 </li><li>池，离心管的内容为5分钟，在室温下，在700rpm（110克）。 </li><li>上清液真空断下降到实际的颗粒，并在1毫升完成国科会培养基悬浮细胞。 </li><li>介质轻轻吸管和向下有一个均匀的单细胞悬液。 </li><li> 10μL的细胞悬液混合90μL台盼蓝进行细胞计数。 </li><li>最后，这些细胞是镀在国科会与20ng / mL表皮生长因子（EGF）在完全培养基2 × 10 5细胞/ mL密度。用于T25，T75和T175烧瓶40毫升20毫升5毫升培养基。 </li><li>在5-7天，在37 ° C的5％，在培养箱CO 2培养时形成神经球。在6-7天，球应措施150-200微米之间，将准备的亚文化（通过）。 </li></ol> 3。胚胎神经干细胞传代和扩展: <ol><li>当神经球准备的亚文化（150-200微米，直径），悬浮领域的介质是从烧瓶中删除，放置在一个适当大小的无菌组织培养试管，离心5分钟700转（110克）室温。 </li><li>上清被丢弃和重悬于1毫升的0.05％胰蛋白酶EDTA的领域。 </li><li>细胞悬液，然后在37℃水浴2-3分钟中孵育，然后同等体积的大豆胰蛋白酶抑制剂是用来阻止胰蛋白酶的活性。 </li><li>细胞悬液轻轻吸管，以确保已完全灭活，胰蛋白酶。 </li><li>细胞悬液，离心5分钟700转（110克）。然后，取上清液被删除，在1毫升完成国科会培养基悬浮细胞。 </li><li> 10μL的细胞悬液混合90μL台盼蓝进行细胞计数。 </li><li>在完成国科会与20ng / mL的EGF在一个适当大小的组织培养瓶中补充的中等浓度的5X10 4细胞/ mL接种于细胞。用于T25，T75和T175烧瓶40毫升20毫升5毫升培养基。 </li><li>中学神经球形成5-7天，在37 ° C的5％，在培养箱CO 2培养时。 </li></ol> 4。代表性的成果: 初级胚胎NSC文化，将成为大多数细胞肥厚和附加到组织培养皿后，电镀。虽然多数细胞死亡后2-3天，或分化，增殖细胞，使细胞脱离基板（图1）的小集群。文化的第一个48小时的大型球状聚合形成的，不应该被误认为是首要领域。总结形成主要取决于在文化的碎片和非游离组织团块金额。真正的神经球是相明亮，变得更加大小的增加（图2）球形。小microspikes上可行的和健康的领域（图3）的外表面出现。经过5-7天，球必须是圆的，但不板结;和测量直径150至200微米。如果神经球是允许的增长过大（后9-10天在文化），他们可能会形成聚集体或颜色较暗，在球体的中心，因为细胞死亡（见视频）。大神经球，最终可能开始分化原位 （附加基板和向外围迁移）。这也是难以解离大神经球传代培养他们。 <img src="/files/ftp_upload/2457/2457fig1.jpg" alt="图1" /> 图1。小学E14，NSC电镀后3天的文化。箭头显示神经干细胞的增殖集群。原放大; 20X <img src="/files/ftp_upload/2457/2457fig2.jpg" alt="图2" /> 图2。小学E14，NSC电镀后第7天的文化。原始的放大倍率; 10倍<img src="/files/ftp_upload/2457/2457fig3.jpg" alt="图3" /> 图3。电镀后的通道之一，E14神经球5天。注意在外围的领域微尖峰（箭头）。原放大; 20X

<ol>
	<li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Morshead, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+in+the+adult+mammalian+forebrain:+a+relatively+quiescent+subpopulation+of+subependymal+cells.">Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.</a> Neuron. 13, 1071-1082 (1994).</li><li>Craig, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+growth+factor+expansion+of+endogenous+subependymal+neural+precursor+cell+populations+in+the+adult+mouse+brain.">In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain.</a> J Neurosci. 16, 2649-2658 (1996).</li><li>Golmohammadi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+the+frequency+and+distribution+of+stem+and+progenitor+cells+in+the+adult+mouse+brain.">Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain.</a> Stem Cells. 26, 979-987 (2008).</li><li>Azari, H., Rahman, M., Sharififar, S., BA, R. e. y. n. o. l. d. s. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J Vis Exp. , (2010).</li><li>Reynolds, B. A., Rietze, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+and+neurospheres--re-evaluating+the+relationship.">Neural stem cells and neurospheres--re-evaluating the relationship.</a> Nat Methods. 2, 333-336 (2005).</li><li>Louis, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+neural+stem+and+progenitor+cells+in+the+neural+colony-forming+cell+assay.">Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay.</a> Stem Cells. 26, 988-996 (2008).</li></ol>

神经球检测神经干细胞，因为它的简单性和可重复性1-5隔离和扩张的首选方法。这个实验是一种未分化的中枢神经系统的前体细胞，这可能是在体外和体内研究的大型代的宝贵工具。应该强调的，可以从无论是真正的神经干细胞和更受限制的祖细胞生成神经球。因此，计算神经球的形成频率，简单地高估了真正的神经干细胞在任何特定的神经细胞人口6。要估计真正的神经干细胞的频率，这是强烈建议使用的神经细胞集落形成细胞分析（N CFCA），并已为此 7开发。 为了有一个一致的高品质的E14，神经干细胞的神经干文化，我们建议: <ol><li>前的准备开始收获的胚胎需要的物品。 </li><li>为了保持在寒冷的HEM或整个解剖基础NSC培养基中的胚胎。 </li><li>为了尽可能快地执行清扫（1 h内），作为组织变得柔软，粘随着时间的推移，可能难以剖析。 </li><li>不要让太多的领域成长。通常情况下，他们应子培养5-7天，每一个球体的大小而定。 </li><li>要trypsinize在2-3分钟150-200微米大小的球。留下超过3分钟的胰蛋白酶，导致细胞受损和球体形成效率会降低，细胞往往会附加培养皿和分化。 </li></ol>

*为了使下摆，混合1M HEPES的MEM and160ml 110L包和带卷到8.75 l使用蒸馏水。设置最终pH至7.4，并存储在4 ° C

establishing embryonic mouse neural stem cell culture using the neurosphere assay

在哺乳类动物中，干细胞作为一种未分化的细胞来源，动物的寿命期间保持在不同组织和器官的细胞起源和重建。他们有可能取代细胞在衰老过程中或在损伤和疾病的的过程中丢失。神经干细胞（NS​​Cs）的存在，最初是由雷诺和Weiss（1992），在成年哺乳动物的中枢神经系统使用一种新型的无血清培养系统，神经球检测（NSA）（中枢神经系统）。使用这个实验中，它也是可行的隔离和扩大来自胚胎中枢神经系统的不同地区的神经干细胞。这些在体外培养扩增神经干细胞是多能的，可以引起中枢神经系统的三个主要类型的细胞。虽然NSA似乎相对简单的执行，注意这里展示的程序是必需的，以实现可靠的和一致的结果。实际上该视频演示NSA的产生和扩大神经干细胞从胚胎一天14鼠脑组织和提供技术细节，因此可以实现重复性的神经球文化。过程包括收获，E14小鼠胚胎脑显微切割收获神经节金榜，在国科会中期收获的组织分解，从而获得单细胞悬液，并终于在NSA的文化电镀细胞。经过5-7天的文化，由此产生的初级神经球传代，以进一步扩大，为今后的实验中的神经干细胞的数量。

Watch this Scientific Journal Video about Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay at JoVE.com

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

这个视频协议演示应用程序的神经球实验胚胎每天14鼠脑的神经节金榜，神经干细胞的分离和扩张。

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

establishing-embryonic-mouse-neural-stem-cell-culture-using

Nanyang Technological University

Research

JoVE Journal

Neuroscience

24.6K 次观看.  Shiraz University of Medical Sciences, Shiraz, Iran. 这个视频协议演示应用程序的神经球实验胚胎每天14鼠脑的神经节金榜，神经干细胞的分离和扩张。

视频: Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

本地化的蛋白质三维神经干细胞培养:一个混合的可视化方法

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

小鼠神经干细胞移植手术与器官的诱向性鼠肝炎病毒感染小鼠的脊髓

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

人类胶质母细胞瘤肿瘤细胞的分离和扩大使用的神经球含量

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

神经母细胞含量:从区分神经干细胞后代使用流式细胞仪，神经祖细胞的生成和富集含量

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

使用F9稀土LACZ基于Cell的报告基因检测在E8.5胚胎和神经球文化相对维甲酸水平的定量测量

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

使用克隆测定神经干细胞的计数。

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...