我们愿干细胞和维尔茨堡大学的再生医学集团有益的建议和Martina Gebhardt的以及海克Arthen的所有成员感谢优秀的技术支持。这项工作是由德意志研究联合会DFG（ED79 / 1-2），教育的德国与研究部BMBF（01 GN 0813），巴伐利亚研究网络诱导多能干细胞“forIPS”和“基金会西比勒Assmus资金支持“ 图1是使用施维雅医疗艺术，从www.servier.com生产。

在这项研究中所用的人成纤维细胞是从皮肤穿刺活检获得由维尔茨堡，德国的大学伦理委员会得到知情同意和伦理过关后（道德报告编号：日期为2011年6月10日一十一分之九十六）。 1.冲床活检<ol><li>消毒患者的皮肤。麻醉用0.5至1ml盐酸甲哌卡因皮肤皮内其中一部分活检将取自（优先不太阳光曝晒区），并用无菌3毫米活检打孔准备皮肤活检，活检冲洗用DPBS + 1微升/1毫升庆大霉素和删除脂肪。活检冲洗两次DPBS +庆大霉素，吸DPBS完全和覆盖活检分散酶II（2.4 U / ml）和孵育16 - 18小时在4℃。 </li><li>吸分散酶完全，用DPBS冲洗两次，去除表皮用镊子。用DPBS洗涤两次活检，添加2毫升胶原酶类型2（500 U / ml的CDU），转印在15ml管中，加入Collagena本身最多到5ml总体积。孵育45分钟，在37℃，5％的CO 2。 </li><li>离心5分钟180 XG事后弃上清。重悬沉淀在10ml的DMEM-FCS-庆大霉素培养基（10％FCS，1微升/毫升庆大霉素）和离心再进行5分钟，在180×g下。弃上清，重悬沉淀在1.5ml DMEM-FCS-庆大霉素媒体。转移到T25粘附组织培养瓶和孵育在37℃，5％的CO 2。 </li><li>更换介质每3天。 </li><li>后约2周，当细胞周围的皮肤部位，使用胰蛋白酶/ EDTA（0.05％）分裂细胞汇合：洗涤细胞一次用DPBS，孵育5分钟，在37℃，5％CO 2的加入2ml胰蛋白酶/ EDTA的后（0.05％），加入2ml的DMEM-FCS-庆大霉素介质，转移至15ml管中并离心，在180×g离心5分钟;丢弃DMEM-FCS-庆大霉素媒体适量上清，重新悬浮颗粒。 </li><li>通过改变培养基每3 R进一步扩大细胞d日和分裂时细胞汇合如上所述;此外庆大霉素可以通道2之后被停止。 </li><li>采用细胞直接转化实验之前，请务必通过标准检测，以排除支原体污染。 </li><li>当细胞已被扩大，您可以直接开始转换或用10％DMSO和90％FCS冷冻细胞下来。细胞应贮存在-80℃下进行2天，然后转移至液氮长期贮存。 </li><li>为了制备转化，用DPBS，吸DPBS洗涤一次细胞，并加入2.5 ml胰蛋白酶/ EDTA（0.05％）。保持5分钟，在37℃，5％的CO 2。轻轻拍打瓶分离细胞，悬浮于2.5毫升的成纤维细胞培养基（DMEM含L-谷氨酸+ 10％FCS + 1％NEAA + 1％丙酮酸钠），并转移到15ml试管。离心在180×g离心5分钟，吸出上清液。悬浮细胞在1ml成纤维细胞培养基和吸管上下以产生单细胞悬浮液。&lt;/ LI&gt; <li>算使用福克斯-Rosenthal-或纽鲍尔细胞计数室和板每一个24孔板的孔3×10 4个细胞的细胞数。孵育O / N在37℃，5％的CO 2。 </li></ol> 2.感染人成纤维细胞与仙台病毒和转分化注：为了确保安全，处理病毒以下步骤必须在生物安全柜和一个层流罩，并用适当的个人安全设备，特别是外科口罩，防止粘膜曝光进行。 <ol><li>开关单元至100μl成纤维细胞介质（步骤1.9）。解冻Oct4-，Klf4-，Sox2-的等分试样和c-myc-仙台病毒和悬浮每种病毒在1毫升的成纤维细胞的介质。添加每种病毒以3至细胞的MOI（病毒滴度从很多变化到很多，但是每种病毒的一个等份一般可以用于10孔24孔板的感染），轻轻混匀。孵育O / N在37℃，5％CO 2 </sUB&gt;。 </li><li> 24小时后吸出培养基并加入500μlneuroinduction介质（1：1的DMEM / F-12：的Neurobasal，1×N 2，1×27，1％含有GlutaMAX，10纳克/毫升hLIF，3μMCHIR99021，2微米SB431542）的和文化在39℃，5％CO 2，从现在开始。隔日更换介质。 </li><li>在第6天感染后制备层粘连蛋白包被的6孔板：稀释的层粘连蛋白至1微克/毫升在DPBS，加入1毫升溶液到6孔板，并保持在4℃24小时。 </li><li>在第7天感染后拆分使用DPBS / EDTA将细胞：吸媒体和用DPBS洗涤一次细胞，然后加入500μl的DPBS / EDTA和孵育10分钟，在37℃，5％的CO 2。 </li><li>加入500μl的DMEM / F12的和从盘在15ml管中，离心除去悬浮液在180×g离心5分钟。 </li><li>吸出上清液和悬浮颗粒在1.5 neuroinduction媒体，板上层粘连蛋白涂层6孔板中并添加ROCK抑制剂Y27632的最终conce为10μM至细胞，培养在39℃ntration，5％的CO 2。 </li><li>隔日更换介质。 </li><li>从感染培养后第14天将细胞在37℃，5％的CO 2。神经上皮感染菌落用相差显微镜后，成为各地17天明显。它们应该足够大约20天感染后要被拾取。 </li></ol> 3.隔离推定的INPC殖民地<ol><li>一天采摘前，涂布一层48孔板与层粘连蛋白：稀释的层粘连蛋白至1微克/毫升在DPBS，加入300微升溶液到板，并保持在4℃24小时。一天后吸层粘连蛋白从板，并添加200微升neuroinduction介质的孔中。 </li><li>每孔洗涤6孔板的6孔板要被拾取一次用DPBS含有菌落并加入2ml的DPBS。挑菌落机械：围绕刮用细针摆脱周围细胞的克隆;设置2001; 50微升升移液器，并挑选使用相应的枪头殖民地。 </li><li>传送每一个菌落成层粘连蛋白包被的48孔板的一个孔并通过移液器上下吹吸10次产生单细胞悬浮液机械。添加ROCK抑制剂Y27632在10μM至细胞的终浓度。 </li><li>生长在48孔板的细胞中，在37℃，5％CO 2下2天。每隔一天更换培养基，直到细胞达到80 - 90％汇合（约3 - 4天）。 </li></ol> 4.扩展和冷冻保存iNPCs的<ol><li>传代和扩展iNPCs的<ol><li>吸出培养基并用DPBS洗涤细胞。吸出DPBS并添加200微升DPBS / EDTA孵育10分钟，在37℃，5％的CO 2，添加200μl的DMEM / F12的和从板在15毫升管除去悬浮液中，离心，在180×g离心5分钟。 </li><li>吸出上清液和悬浮颗粒在0.5 neuroi毫升nduction媒体（步骤2.2），盘上的层粘连蛋白包被的12孔板，并添加ROCK抑制剂Y27632在10μM的终浓度向细胞，培养在37℃，5％的CO 2。经过细胞达到80 - 90％汇合，他们既可以进一步扩大或冻结列如下。 </li></ol></li><li>冻结iNPCs的<ol><li>吸出培养基并用DPBS洗涤细胞。吸出的DPBS和添加300μLDPBS / EDTA孵育10分钟，在37℃，5％的CO 2中，添加300μl的DMEM / F12的和从板在15毫升管除去悬浮液中，离心，在180×g离心5分钟。 </li><li>吸出上清液和悬浮颗粒在0.5 NSC商用冷冻培养基中。转移悬浮液到冷冻小瓶中并置于细胞冷冻集装箱。储存在-80℃下进行2天，然后转移到液氮长期贮存。 </li></ol></li></ol> 5.分化iNPCs的<ol><li>对神经元分化人的血统<ol><li>关于层粘连蛋白包被的平板培养细胞如上所述。改变媒体的神经差异媒体（DMEM / F-12，1个N2，1X B27，300毫微克/毫升的阵营中，200μM的维生素C，10纳克/毫升BDNF，10纳克/毫升GDNF）当细胞约70％汇合。三个星期更改媒体隔日1次。分化过程中，不要分裂的细胞。 </li><li>三周以后细胞应该表达的神经元标记TUJ1。为了获得更多的成熟神经元和神经元亚型继续分化长达3个月。 </li></ol></li><li>对胶质细胞谱系分化<ol><li>改变媒体胶质感应介质（1：1的DMEM / F-12：的Neurobasal，1×N 2，1×B27，10μMβ巯基乙醇，100μM的非必需氨基酸，1.5mM的L-谷氨酰胺，5微克/毫升胰岛素， 40毫微克/毫升T3），包括20毫微克/毫升表皮生长因子诱导分化成神经胶质前体细胞。删除媒体的一半，并用新媒体取代隔日约2周。在此期间，细胞应被分割1：3在10μMROCK抑制剂Y27632存在当达到100％汇合。 </li><li> 2周后分化细胞进一步为星形胶质细胞：当细胞几乎汇合改变媒体的商业可用的星形胶质细胞的媒体，改变媒体每隔一天。后约7天细胞开始表达的星形胶质细胞标记物（ 例如，GFAP）。在10μMROCK抑制剂Y27632存在2比例：如果细胞获得的分化方案在100％汇合，将它们分割在一个1。 </li></ol></li></ol>

The authors declare that they have no competing financial interests.

这里，我们显示了隔离和直接转换人成纤维细胞的成膨胀转基因的无神经祖细胞和它们的分化后代作为细胞替代疗法或药物筛选分析应用的推定依据。直接谱系体细胞转化成的NPC已经由谱系特异性转录的强制表达因子26,33,34来实现的。然而，在许多情况下，胎儿成纤维细胞用于转实验33,34。用于生物医学应用中，这些细胞的用法是不适宜的，因为这单元源排除了临床使用自体细胞?...

在2006年山和同事可以显示首次体细胞的重编程的可能性成多能状态1。此去分化是由四个转录表达在小鼠成纤维细胞获得因子的Oct4，Sox2的，Klf4和c-Myc的。所产生的所谓的诱导多能干细胞（iPS细胞）显示功能等同于胚胎干细胞（ESCs），并因此可以分化成成年生物体的所有细胞类型。一年后重新编程为iPS细胞也可以实现对人成纤维细胞2。在动物模型中的实验表明，源自iPSC的细胞中通常可以用于细胞替代疗法，例如 ，在帕金森氏病（PD）3-5。然而，与使用iPS细胞相关的一些限制表示路障，为全面实现其治疗的潜力。首先，细胞重编程为多能状态和随后的质量控制通常是一个耗时和低效的过程，得到了广泛的，因而昂贵的细胞培养过程。第二，iPSCs的需要被重新分化成感兴趣的生物医学应用之前所需的细胞类型和残留多能干细胞在分化的人口的概率藏着显著瘤潜力，从而显示细胞移植6后的高风险。第三，重编程过程通常是由通过lenti-或逆转录病毒感染诱导的重编程因子来实现的。这些病毒进入宿主基因组的整合可能导致插入突变和/或所述转基因7,8-不受控制活化。非整合系统已被开发以提供重编程因子的靶细胞，其最小化插入诱变和转基因激活的风险。这些转基因无方法的实例是细胞使用非整合的重新编程腺或仙台病毒9,10，基于DNA的载体11或不含DNA的方法的应用，如合成的mRNA的重组蛋白13,14的12或转导的转染。另一种很有前途的方法对转基因自由的iPSC的推导是利用loxP的修饰慢病毒重新编程结构和随后删除使用的Cre-loxP的DNA重组系统15,16转基因的。 一个更简单的方法来生成神经细胞的细胞替代疗法代表直接转换成纤维细胞进入有丝分裂后神经元17-20。 Vierbuchen 等人报告说，转录的表达因子ASCL1，Brn2和Myt1l结果在20％的神经元的产生来自鼠成纤维细胞17。在2011年它被示出，即在相同的三个转录因子与NEUROD1的过表达的组合使人成纤维细胞成神经的转罗恩的19。人类诱导的神经元也可以通过ASCL1和Ngn2下双SMAD-和GSK3β-抑制过度20产生。值得注意的是，直接转换成纤维细胞的成神经细胞的产生非增殖，有丝分裂后的细胞群，不允许进一步扩张和生物库。 最近，直接转换成纤维细胞的成增殖神经干/祖细胞群报道21-26。为了清楚起见，所有这些类型的细胞将被命名为这份报告中诱导神经祖细胞（iNPCs）。 Han 等过度Brn4，Sox2的，c-Myc的和Klf4来生成iNPCs。像他们无论从小学组织或多能干细胞源性神经干细胞，这些同行都是iNPCs和三电位可以分化为神经元，星形胶质细胞和少突胶质细胞21。我们的团队报道涉及SOX2，Klf4的过表达略有不同的转换协议，和c-Myc诱导的Oct4表达为仅5天。通过这种方法，我们可以生成表现出完全沉默的重编程因子的小鼠22胚胎和成年成纤维细胞增殖稳定iNPCs。在对比的iPSC，iNPCs不移植27后表现出致瘤的潜力。我们使用在脱髓鞘的动物模型，髓鞘缺陷大鼠成功转化的细胞，这表明iNPCs在临床上是有用的22。在此之前，筹备可能只能从多能干细胞或原代神经组织28-32生成的。 iNPCs是稳定地膨胀的室，可以冷冻保存，并且能够分化成神经元，星形胶质细胞和少突胶质细胞。很大的努力已取得能适应从小鼠的直接转换协议到人类细胞23,26,33,34。在2012年它被出版，在成纤维细胞的单因子Sox2的过度表达足以产生小鼠和人iNPCs <s达&gt; 33。作者报告生成胎儿包皮成纤维细胞的人类iNPCs的特点和他们通过对染色和Sox2的巢。然而，用于重编程靶细胞表示，将不会在临床实践中可用的并有转化细胞中进行的动物模型中没有功能表征通过移植一个非常特定的细胞类型。更近的出版物Sox2的，c-Myc的，要么Brn2或Brn4 34的过量表达描述了从人胎儿成纤维细胞的神经元限制性祖细胞的产生。所产生的细胞系表现出的自我更新能力并且可以分化成不同类型的终端的神经元。然而，胎儿成纤维细胞的使用是不利的，因为这些细胞是异质的原点和一个不能排除残余例如，神经嵴干细胞的制剂的存在。在2014年，朱等人报道了直接成人和新的转换产后的成纤维细胞进入三电位神经祖细胞通过过表达Sox2的连同的Oct4或Oct4的单独和除了小分子的细胞培养介质。值得注意的是，根据他们的研究，Sox2的独显不足以诱导直接转化26。最近，Lu 等人报告说，在山中的过表达因子Oct4-，Sox2-，Klf4-，c-Myc的仙台病毒24小时的病毒和随后的失活温度导致膨胀三电位神经的产生增加前体细胞23。总之，虽然公布了人类细胞的转化的协议迄今具有在至少一个所述山因素的一个或多个，通常是一个及时限制的方式的共同过表达，有需要用来驱动直接最小分子因素没有明确指示转换成iNPCs。 Oct4的的及时过度限制或者通过遗传手段，转染的mRNA合成，或cELL-透膜蛋白与SOX2，KLF-4，和c-Myc的组成型表达没有造成稳定的人力INPC行呢。因此，仙台病毒的应用以过表达所有山因素和及时由病毒23的热灭活限制其活动一起优化神经介质诱导条件22,31表示的优选策略迄今。 一些研究表明神经干细胞或在不同的动物疾病模型的区别同行的细胞功能。从人多能干细胞的神经祖细胞已移植在神经炎性病症多发性硬化35,36的小鼠模型。人类胚胎干细胞衍生的神经祖细胞中的EAE的适用性（实验性自身免疫性脑脊髓炎）-mice最早于2008年35所示。多能神经前体细胞注射到小鼠脑的脑室和移植结果编在EAE的临床症状的减少。 Kim 等生成由hESCs少突胶质细胞前体和脑室内移植那些成EAE-小鼠。虽然移植了10多天没有生存，小鼠表现出显著改善神经功能和白质减少36促炎免疫细胞的。干细胞疗法也已应用于临床前靶向帕金森氏病的作为的NPC可以被成功地用于在相应的动物模型。对于这一点，祖细胞无论是来源于胎儿脑组织37从多能干细胞38-40或间质干细胞41被用于鉴别。在2012年，我们表明，小鼠iNPCs能够产生蛋白脂质蛋白，主要的髓鞘蛋白成分，移植后成髓鞘缺陷（MD）大鼠22的大脑。通过验证-的原则，实验治疗applicabiliNPCs的性被首次证明和另一项研究32很快证实。然而，治疗用途的人类iNPCs的全部潜力还有待探索。 这里，我们显示了从通过皮肤活检的成年患者的人原代细胞（ⅰ）推导一个坚固的一体化方法，（ⅱ）直接转换人成纤维细胞的成神经祖状态和iNPCs的（ⅲ）的能力也可以分化成神经元和神经胶质细胞系。这个协议的使用，将有助于加快新一代自体的人类细胞用于治疗应用。

<table border="0" cellpadding="0" cellspacing="0">
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			<td>Astrocyte media</td>
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			<td>17504-044</td>
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			<td>BDNF</td>
			<td>Peprotech</td>
			<td>450-02</td>
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			<td>Biopsy Punch</td>
			<td>pfm medical</td>
			<td align="right">48301</td>
			<td></td>
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			<td>cAMP</td>
			<td>Sigma</td>
			<td>A6885</td>
			<td></td>
		</tr>
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derivation of adult human fibroblasts and their direct conversion into expandable neural progenitor cells

代从成人皮肤成纤维细胞诱导多能干细胞（iPS细胞）和随后的分化成体细胞为规避组织相容性障碍的自体移植的推导引人入胜的前景。然而，通过多能状态和随后的全面分化成所需的谱系进展仍然是一个障碍为IPSC的技术，因为相关的肿瘤潜力和基因组不稳定性的临床翻译。近来，我们和其他人发现，体细胞不仅可以通过使用确定的因子，从而通过多能状态绕过级数变换为iPS细胞，但也为不同类型的多能成体干细胞。特别是，直接由人类成纤维细胞进入诱导神经祖细胞（iNPCs）的转化预示着一个新的自体细胞来源的各种应用，如细胞替代，疾病建模的可能性和药物筛选。这里，我们描述成人原发性成纤维细胞通过皮肤活检及其有效的直接转换的隔离成iNPCs通过的Oct4，Sox2的，Klf4的及时限制性表达，以及c-Myc的。 17天后诱导SOX2阳性神经上皮菌落出现，INPC线可以有效地单克隆分离和扩增成立。感染和补充的白血病抑制因子的病毒多样性的在诱导期精确调整代表实现高达0.2％的转换效率的关键因素。迄今为止，针对具体患者INPC线可以扩大超过12的通道和均匀地显示神经干/祖细胞的形态学和分子的特性，如Nestin和Sox2的表达。该INPC线可以分化成神经元和星形胶质细胞作为判断染色针对T​​UJ1和GFAP分别。总之，我们提出一个强大的协议推导和可怕人成纤维细胞的克拉转换成可以提供用于生物医学应用的蜂窝源诸如自体神经细胞替代和疾病建模稳定地膨胀神经祖细胞。

这里，我们提出一个综合过程，允许一代从人成纤维细胞已经由从皮肤钻取活组织检查内小于8周（ 图1）中获得诱导的神经祖细胞（iNPCs）的描述。病人specifc iNPCs可以进一步分化为神经元和神经胶质细胞系和海港的巨大潜力细胞替代治疗和疾病建模。 与Oct4-，Klf4-，Sox2-和c-myc-仙台的成纤维细胞感染病毒的neuroinductive介质条件下22,31导致成纤维细胞和殖民地...

Watch this Scientific Journal Video about Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells at JoVE.com

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

诱导多能干细胞的产生提供了用于自体移植的推导引人入胜的前景。然而，通过多能状态和艰苦的再分化仍然进展阻碍临床翻译。在这里，我们描述了成年人成纤维细胞的衍生和其直接转化为诱导神经祖细胞和随后的分化成神经谱系。

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

derivation-adult-human-fibroblasts-their-direct-conversion-into

Research

JoVE Journal

Neuroscience

15.3K 次观看.  University of Würzburg. 诱导多能干细胞的产生提供了用于自体移植的推导引人入胜的前景。然而，通过多能状态和艰苦的再分化仍然进展阻碍临床翻译。在这里，我们描述了成年人成纤维细胞的衍生和其直接转化为诱导神经祖细胞和随后的分化成神经谱系。 

视频: Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

科研

神经科学

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). ...

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 ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

谱系重编程的成人大脑的周细胞源性细胞诱导成神经细胞

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

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. ...

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.

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Interneuron 前体 Homochronic 移植早期小鼠脑的研究

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

通过Sialoglycan的代谢标记识别主要神经干细胞和祖细胞的细胞表面标记

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

利用 PiggyBac 载体将人诱导的多能干细胞 (Ipsc) 转化为功能性脊柱和颅内运动神经元

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...