Name: derivation of adult human fibroblasts and their direct conversion into expandable neural progenitor cells
Uploaded: 2015-07-29T13:00:00.000+00:00
Duration: 13 min 58 s
Description: Watch this Scientific Journal Video about Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells at JoVE.com

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><tbody><tr><td>Astrocyte media</td><td>ScienCell</td><td>1801</td><td></td></tr><tr><td>B27</td><td>LifeTechnologies</td><td>17504-044</td><td></td></tr><tr><td>BDNF</td><td>Peprotech</td><td>450-02</td><td></td></tr><tr><td>Biopsy Punch</td><td>pfm medical</td><td>48301</td><td></td></tr><tr><td>cAMP</td><td>Sigma</td><td>A6885</td><td></td></tr><tr><td>CHIR99021</td><td>Axon medchem</td><td>1386</td><td></td></tr><tr><td>Collagenase Type2</td><td>Worthington Biochemical</td><td>LS004177</td><td></td></tr><tr><td>Dispase</td><td>PAN&amp;nbsp;</td><td>P10-032100</td><td></td></tr><tr><td>DMEM</td><td>LifeTechnologies</td><td>41966-029</td><td></td></tr><tr><td>DMEM F12</td><td>LifeTechnologies</td><td>11320-033</td><td></td></tr><tr><td>DMSO</td><td>Roth</td><td>4720</td><td></td></tr><tr><td>DPBS</td><td>LifeTechnologies</td><td>14190-094</td><td></td></tr><tr><td>EGF</td><td>Life Technologies</td><td>PHG0313</td><td></td></tr><tr><td>FCS</td><td>Biochrom AG</td><td>50115</td><td></td></tr><tr><td>GDNF</td><td>Peprotech</td><td>450-10</td><td></td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G1397</td><td></td></tr><tr><td>GFAP-antibody</td><td>Dako</td><td>Z0334</td><td></td></tr><tr><td>GlutaMAX</td><td>LifeTechnologies</td><td>35050-038</td><td></td></tr><tr><td>hLIF</td><td>Peprotech</td><td>300-05</td><td></td></tr><tr><td>Insulin</td><td>Seralab</td><td>GEM-700-112-P</td><td></td></tr><tr><td>Ki67-antibody</td><td>NeoMarkers</td><td>RM-9106-S</td><td></td></tr><tr><td>L-Glutamine</td><td>LifeTechnologies</td><td>25030-024</td><td></td></tr><tr><td>Laminin</td><td>Sigma</td><td>L2020</td><td></td></tr><tr><td>N2</td><td>LifeTechnologies</td><td>17502-048</td><td></td></tr><tr><td>Nestin-antibody</td><td>R&amp;amp;D Systems</td><td>MAB1259</td><td></td></tr><tr><td>Neurobasal</td><td>LifeTechnologies</td><td>21103-049</td><td></td></tr><tr><td>Non-essential amino acids</td><td>LifeTechnologies</td><td>11140-050</td><td></td></tr><tr><td>NSC freezing media</td><td>Sigma</td><td>C6295</td><td></td></tr><tr><td>Oct4-antibody</td><td>Santa Cruz</td><td>sc9081</td><td></td></tr><tr><td>Pax6-antibody</td><td>Covance</td><td>PRB-278P</td><td></td></tr><tr><td>SB431542</td><td>Invivogen</td><td>inh-sb43</td><td></td></tr><tr><td>Sendai virus: CytoTune Sendai Reprogramming Kit</td><td>&amp;nbsp;LifeTechnologies</td><td>A1378001</td><td></td></tr><tr><td>Sodiumpyruvate</td><td>Life Technologies</td><td>11360-039</td><td></td></tr><tr><td>Sox1-antibody</td><td>R&amp;amp;D Systems</td><td>AF3369</td><td></td></tr><tr><td>Sox2-antibody</td><td>R&amp;amp;D Systems</td><td>MAB2018</td><td></td></tr><tr><td>T3</td><td>Santa Cruz</td><td>sc-205725</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Life Technologies</td><td>15400-054</td><td></td></tr><tr><td>TUJ1-antibody</td><td>Covance</td><td>MMS-435P-250</td><td></td></tr><tr><td>Vitamin C</td><td>Sigma</td><td>A4544</td><td></td></tr><tr><td>Y27632</td><td>Calbiochem</td><td>CAS 146986-50-7</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>LifeTechnologies</td><td>21985023</td><td></td></tr></tbody></table>

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

15.4K 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

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

科研

发育生物学

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

神经科学

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 ...

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 ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

生物工程

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

生物学

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

使用从纤维细胞直接转换为神经元祖先细胞和分化为星形细胞的神经系统疾病体外建模

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

Isolating Induced Neural Progenitor Cell Colonies Derived From Adult Human Fibroblasts

Source: Meyer, S. et al., <a href="https://app.jove.com/v/52831">Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells.</a>&#160;J. Vis. Exp. (2015).
This video demonstrates a technique for isolating induced neural progenitor cells (iNPCs) derived from adult human fibroblasts. It outlines the steps involved in isolating the iNPC colonies and culturing them in laminin-coated multi-well plate wells in the presence of suitable culture media that facilitate the continued proliferation and expansion of the iNPC population.

分离源自成人成纤维细胞的诱导神经祖细胞集落

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of Putative Induced ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Differentiating Induced Neural Progenitor Cells into Astrocytes

Source: Meyer, S., et al., Edenhofer, F. <a href="https://app.jove.com/v/52831">Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells.</a> J. Vis. Exp. (2015)
This video demonstrates the process for differentiating induced neural progenitor cells (iNPCs) into neurons and astrocytes using specialized media.

将诱导的神经祖细胞分化为星形胶质细胞

1. Differentiation of iNPCs

	Differentiation towards neuronal lineage&#8203;
	
		Culture cells on laminin-coated plates as described above. Change media ...