这项工作是从Overstreet基金会的资金支持。

1。需要细胞电镀之前准备的项目: <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> NeuroCult NCFC无血清培养基的无细胞因子等分解冻。 </li><li>在37℃水浴介质预热。 </li><li>表皮生长因子（EGF），碱性成纤维细胞生长因子（b - FGF的）在浓度为10微克/毫升和0.2％肝素联合解决方案，提前准备。 </li><li>根据实验的大小，需要几个35mm组织培养的菜肴板的细胞和一个150至200cm的塑料培养皿中，还需要举行重复的35mm菜和第三水35毫米菜。 </li></ol> 2。细胞制备: 根据你的实验，细胞可从成人或胚胎的来源（主要分离组织或游离神经球）的准备。剖析从成人/胚胎小鼠中枢神经系统（CNS）组织或游离于前1,2，然后描述成人/胚胎源性神经球: <ol><li>的是通过一个40微米大小的网状过滤器通过的单细胞悬液，以消除非游离团块。 </li><li>台盼蓝90μl，进行细胞计数细胞悬液10μL 混合注 :也可用于其他合适的细胞稀释。 </li><li>如果使用的是初级胚胎或成人的神经细胞，稀释的细胞悬液浓度6.5 × 10 5细胞/毫升在完成国科会培养基。如果使用的是从成人或胚胎神经细胞产生的游离神经球细胞，稀释细胞悬液浓度2.2 × 10 5细胞/毫升在完成国科会培养基。 </li></ol> 3。半固态的N - CFCA中等电镀细胞: <ol><li>混合，以适当的音量以下几部分组成，上复制的数量而定。在这里，我们混合了两所需要的金额复制或重复的菜肴。复制其他号码，请参阅表1。 <ul><li> 1.7毫升NeuroCult NCFC无血清培养基，无细胞因子</li><li> 330μLNeuroCult NSC增殖辅料</li><li> 6.6μL表皮生长因子（10μg/mL） </li><li> 32μL青霉素/链霉素。 </li><li> 3.3μLB - FGF（10μg/mL）时才需要培养成人神经细胞产生的细胞。 </li><li> 3.3μL肝素（0.2％）时才需要培养成人神经细胞产生的细胞 。 </li><li> 25μL细胞悬液（2.2 × 10 5细胞/ ml的分离神经球或6.5 × 10 5细胞/毫升原代细胞从组织分离的中枢神经系统细胞）注:镀金的最后一个单元格的数字应约2500每35毫米培养皿中的细胞衍生的神经干细胞和7500细胞的原代细胞每35 mm培养皿。在某些情况下，最终细胞电镀密度要通过执行一个单元滴定实验调整。对于统计分析，经过21天的文化检测菌落总数应在范围内至少有50 - 150菌落。 </li></ul></li><li>轻轻地，然后是混合细胞的培养基中含有1.3毫升冷胶原溶液转移到细胞悬液，轻轻吹打混合，以避免任何气泡。 </li><li>的混合溶液配发到每个35mm的培养皿（〜1.5毫升/皿）的中心和菜都轻轻放倒，使用打圈，让分布在菜的表面均匀的混合物。 。 </li><li>重复35毫米培养皿中放入一个100毫米的Petri板。新的35毫米培养皿的盖子被删除，开放的菜也是相同的100毫米的培养皿中。无菌水添加到这个开放的35毫米盘保持在孵化期间的最佳湿度。广场生物测定板（245毫米），使用时复制35毫米的菜已经建立起来。同样，包括2个或3开放35 mm培养皿无菌水。 </li><li>板块转移一个孵化器设定在37℃，5％CO2和95％的湿度。通过增加温度和凝胶形成的胶原蛋白凝结会发生在大约一个小时。的文化不应该在此期间受到干扰。 </li><li>培养的细胞培养21天（群体规模的差异，在21天以后可以清楚地分辨）。 </li></ol> 4。准备增资的中型和喂养文化: 孵育时间（21天）的延长期的N - CFCA文化，文化应投喂与适当的完整NeuroCult 增资中期准备新鲜如下: <ul><li> NeuroCult NSC基础培养基的4.5毫升混合0.5米NeuroCult NSC增殖辅料，然后生长因子的L是: </li><li> 250μL10μg/mL表皮生长因子（给予终浓度为0.5μg/ mL的EGF） </li><li> 125μLB - FGF10μg/mL（给予终浓度为0.25μg/mLB - FGF） 时才需要培养成人神经细胞产生的细胞。 </li><li> 125μL0.2％肝素， 只有培养成人神经细胞产生的细胞。 </li><li> 60μL适当完成NeuroCult补货中（胚胎或成年细胞）添加到每个NCFC含量菜每7天一次，在整个NCFC含量文化潜伏期（21天）的中心。 </li></ul> 5。得分的N - CFC含量派生殖民地和真正的神经干细胞和神经祖细胞的频率计算: <ul><li>每个35 mm培养皿放置在一个2.0毫米× 2.0毫米的网格大小的网格得分菜，两个菜，然后放在显微镜舞台上。 </li><li>使用低功率（2.5倍 - 5倍）物镜，每道菜扫描，并根据它们的大小的殖民地都取得了。 </li><li>殖民地分为两大类: <ol><li>直径小于2毫米</li><li>直径≥2毫米</li></ol></li></ul> 6。代表性的成果: 由于在检测神经球，在镀上的N - CFC检测的细胞开始增殖，使细胞内的小菌落电镀后3 - 7天（图1）。在两个星期的时间，可以区分不同大小的菌落。虽然大多数的殖民地往往在14天后停止增长，一些殖民地继续扩大规模。第21天，菌落可分为四类:1）直径小于0.5毫米，2）0.5 - 1毫米直径，3）1 - 2毫米直径4）直径≥2毫米。实际上，小于2毫米直径的殖民地被称为祖派生（图2）和殖民地≥直径2mm被称为国科会派生（图3）。殖民地≥2毫米每镀细胞总数的直径，代表长期的自我更新和多潜能的能力，实际真正的神经干细胞的频率。据估计总的神经球，在一个特定的细胞群形成后7-8天在一个平行的国家安全局实验的频率类似的总菌落形成后21天在一个N - CFCA实验相同的细胞群的频率。的N - CFCA然而，提供了一个更宽松的条件，使每个单元可以充分显示其增殖潜力。 <table border="1"><tbody><tr><td> 组件 </td><td> 2复制 </td><td> 重复3次 </td><td> 4个重复 </td></tr><tr><td> NeuroCult NCFC无细胞因子的无血清培养基</td><td> 1700 </td><td> 2550 </td><td> 3400 </td></tr><tr><td> NeuroCult国科会扩散辅料</td><td> 330 </td><td> 495 </td><td> 660 </td></tr><tr><td> EGF（10微克/毫升） </td><td> 6.6 </td><td> 9.9 </td><td> 13.2 </td></tr><tr><td>碱性成纤维细胞生长因子（10微克/毫升）， 仅适用于成年细胞 </td><td> 3.3 </td><td> 4.95 </td><td> 6.6 </td></tr><tr><td>肝素溶液（0.2％）， 仅适用于成年细胞 </td><td> 3.3 </td><td> 4.95 </td><td> 6.6 </td></tr><tr><td>青霉素/链霉素（1:100） </td><td> 32 </td><td> 48 </td><td> 64 </td></tr><tr><td>细胞在: 2.2 × 10 5培养细胞/ mL或 6.5 × 10 5的主要细胞/ ml </td><td> 25 </td><td> 37.5 </td><td> 50 </td></tr><tr><td>胶原溶液</td><td> 1300 </td><td> 1950年</td><td> 2600 </td></tr></tbody></table> 表1。完整的N - CFC检测文化的组成部分。 <img src="/files/ftp_upload/2639/2639fig1.jpg" alt="图1" /> 图1 CFCA的N -通道之一，E14小鼠的NSCs文化的代表殖民地后7天电镀。殖民地可能会显示不同的形态和大小。原始的放大倍率; 4X <img src="/files/ftp_upload/2639/2639fig2.jpg" alt="图2" /> 图2不同大小的N - CFCA通过文化之一，E14电镀后21天的小鼠的NSCs的代表祖派生殖民地。如图所示，殖民地有不同的形态，但都少2毫米大小。原始的放大倍率; 4X <img src="/files/ftp_upload/2639/2639fig3.jpg" alt="图3" /> 图3。代表真正的干细胞衍生的N - CFCA通过电镀后21天之一，E14小鼠的NSCs的文化殖民地。干细胞衍生的殖民地，可能会显示不同的形态（见视频），但所有≥2毫米直径。原始的放大倍率; 4X

<ol>
	<li>Azari, H., Rahaman, M., Sharififar, S., Reynolds, B. A. <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>Azari, H., Sharififar, S., Rahaman, M., Ansari, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J Vis Exp. , (2011).</li><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>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. , (2008).</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>Louis, S. A., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosphere+and+Neural+Colony-Forming+Cell+Assays.">Neurosphere and Neural Colony-Forming Cell Assays.</a> Protocols for Neural Cell Culture. 10, 1-28 (2010).</li></ol>

作者之一，沙龙A.路易斯，是隶属于与干细胞技术公司，在这项研究中所使用的一些试剂生产商。

虽然神经干检测3,1,2是最常用的方法，隔离和扩大像成人和胚胎的中枢神经系统组织的各种来源的神经干细胞，它不能准确地测量在混居的神经前体细胞的NSC频率（茎祖细胞），因为没有神经球的数量和真正的干细胞 4之间的一对一关系。为了解决这个限制，原国家安全局已被改编，让神经干细胞和祖细胞成长为三个星期5-7充分发挥其扩散能力。与液体NSA的不同的是，在N - CFCA殖民地实际上是克隆而得，半固态的胶原基质防止单细胞镀和菌落聚集的迁移。对于这个实验的结果一致，我们建议: <ol><li>确保一个单细胞悬液，在原来的细胞悬液。穿过40微米大小的网状过滤器，你的单细胞悬液，以消除非游离团块。 </li><li>保持胶原蛋白溶液在4 ° C冰箱冰或。胶原蛋白是最后一个项目被添加到混合细胞悬液，因为它通过增加温度凝结。 </li><li>不要忘了喂文化每周。添加补充培养基中含有生长因子（S）每7天一次，将使细胞继续增殖扩展文化时期。 </li><li>最后的细胞电镀密度进行了优化，从小学或培养的神经细胞衍生的细胞，使经过21天的文化在检测菌落总数的NCFC内的范围内至少有50 - 200菌落。此范围内允许直径样品中罕见的NSC派生殖民地&gt;2毫米检测，同时提供足够数量的统计分析的殖民地。根据起始细胞源，显着减少（&lt;50）和更高的数字（250）菌落有时获得。将导致菌落&gt;2毫米太少菌落直径低于检测水平，而太多的殖民地，会造成人满为患anddepletion生长介质中产生的组件在一个不准确的菌落计数。因此细胞电镀密度将需要作相应的调整（即增加或减少总数的细胞镀） </li></ol>

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NeuroCult NSC Basal Medium</td>
<td>Medium</td>
<td>STEMCELL</td>
<td>05700</td>
<td>&nbsp;</td>
<tr>
<td>NeuroCult NSC Proliferation Supplements</td>
<td>Medium supplement</td>
<td>STEMCELL</td>
<td>05701</td>
<td>&nbsp;</td>
<tr>
<td>NeuroCult NCFC medium</td>
<td>Medium</td>
<td>STEMCELL</td>
<td>05720</td>
<td>&nbsp;</td>
<tr>
<td>0.05% 	trypsin-EDTA</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25300-062</td>
<td>&nbsp;</td>
<tr>
<td>Soybean trypsin inhibitor </td>
<td>Reagent</td>
<td>Sigma</td>
<td>T6522</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer </td>
<td>Sieve</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>Pen/Strep </td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>T25 flask</td>
<td>Culture ware</td>
<td>Nalge Nunc international</td>
<td>136196</td>
<td>&nbsp;</td>
<tr>
<td>T80 flask</td>
<td>Culture ware</td>
<td>Nalge Nunc international</td>
<td>178905</td>
<td>&nbsp;</td>
<tr>
<td>Collagen</td>
<td>Reagent </td>
<td>STEMCELL</td>
<td>04902</td>
<td>&nbsp;</td>
<tr>
<td>EGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>2028-EG</td>
<td>&nbsp;</td>
<tr>
<td>b-FGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>3139-FB</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>Growth factor</td>
<td>Sigma</td>
<td>H4784</td>
<td>Reconstituted in PBS</td>
</tr>
<tr>
<td>15 ml tubes</td>
<td>Culture ware</td>
<td>BD Falcon</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>50 ml tubes</td>
<td>Culture ware</td>
<td>BD Falcon</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>35 mm culture dishes</td>
<td>Culture ware</td>
<td>STEMCELL</td>
<td>27100</td>
<td>&nbsp;</td>
<tr>
<td>Gridded scoring dishes</td>
<td>Culture ware</td>
<td>STEMCELL</td>
<td>27500</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

neural-colony forming cell assay: an assay to discriminate bona fide neural stem cells from neural progenitor cells

神经球吸附试验（NSA）是最常用的方法，隔离开来，扩大，并计算出神经干细胞（NS​​Cs）的频率之一。此外，本无血清培养系统也被扩大干细胞，并确定它们的频率从各种肿瘤和正常组织。最近，它已被证明，神经球的形成和神经干细胞之间不存在一个以一对一的关系。这表明，目前适用的国家安全局，高估了一个混合的人口从胚胎和成年哺乳动物大脑的神经前体细胞神经干细胞的频率。实际上该视频演示了一种新的胶原蛋白为基础的半固体含量，神经细胞集落形成细胞分析（N CFCA），它具有歧视基于其长期的增殖潜能的祖细胞的干的能力，从而提供了一种方法枚举国科会的频率。在N - CFCA殖民地≥2毫米，直径是来自细胞，满足了NSC的所有功能标准，而殖民地&lt;2毫米祖细胞衍生。可用于从不同的来源，包括小学和培养成人或胚胎小鼠中枢神经系统的细胞准备细胞的N - CFCA程序。在这里，我们使用准备通过从胚胎14天小鼠大脑产生执行N - CFCA一个神经球的细胞。培养增殖培养基补充了三个星期，每7天允许镀细胞表现出充分的增殖潜能，然后神经祖和真正的神经干细胞的频率计数的菌落&lt;2毫米数分别计算和那些在最初镀细胞数量≥2毫米。

Watch this Scientific Journal Video about Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells at JoVE.com

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

这个视频协议演示了如何真正的神经干细胞在一个混合使用的神经细胞集落形成细胞分析的神经前体细胞人口歧视和枚举。

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). ...

neural-colony-forming-cell-assay-an-assay-to-discriminate-bona-fide

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JoVE Journal

Neuroscience

22.0K 次观看.  University of Florida. 这个视频协议演示了如何真正的神经干细胞在一个混合使用的神经细胞集落形成细胞分析的神经前体细胞人口歧视和枚举。

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

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 ...

科研

神经科学

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

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 span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

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 ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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 ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

人类神经的聚集体cGMP的适用展开法干细胞和祖细胞来源从多能干细胞或胎儿脑组织

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

从人多能神经嵴前体细胞的无饲养推导干细胞

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

神经干细胞和祖细胞来自成年小鼠脑室下区细胞分选和实时成像的细胞周期动力学

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

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. ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.