我们非常感谢马格达莱纳格茨博士对本协议的发展过程中她的投入。我们感谢马吕斯Wernig博士（斯坦福大学）慷慨地为我们提供了SOX2编码序列。我们也非常感谢亚历山大Lepier博士病毒的生产。这项工作是由德意志研究联合会（BE 4182/2-2）和BMBF（01GN1009A）至BB，和科学的巴伐利亚州部，研究和艺术，以MK和BBCS收到资金来自两个民族SYSTHER-的资金支持INREMOS虚拟学院（德国和斯洛文尼亚联邦部委教育及研究）及东风集团（SFB 824）。

1，分离和成人的大脑细胞培养涉及人体组织的实验应按照有关人类使用材料的研究目的，所有相关政府及机构规定进行。本协议是按照批准慕尼黑大学的医学院的伦理委员会，并从所有患者的书面知情同意开发。 使用的两性从颞叶性癫痫或其他深层次的非创伤性的，非恶性病变痛苦的患者标本已经建立的人成体大脑皮质制备培养物的该协议。从手术室内由专门的接入信道的脑损伤得到，因此，组织被认为是健康的。这些病人的年龄范围为19-70岁。 <ol><li>加入热灭活的胎牛血清的制备生长培养基（FCS），以高糖DMEM含有GlutaMAX，得到20％FCS的终浓度。加入5 ml青霉素/链霉素，以总500毫升生长培养基。执行此并要求无菌培养条件下，在适当的层流罩所有后续步骤。 </li><li>保持从手术室在Hanks&#39;用的CaCl 2和MgCl 2（HBSS）介质包括HEPES（10mM终浓度），于冰上，直到处理平衡盐溶液中得到的成年人脑活检。开始尽快处理。 </li><li>要开始解离成单细胞，转移到组织为65mm培养皿和剁碎成小块，通过使用两个无菌一次性使用的手术刀。对于酶消化用3-6毫升的TrypLE在15毫升锥形管和水浴孵育15-30分钟，在37°C。 </li><li>加入1倍体积预热的生长培养基中，以促进分解，并轻轻磨碎的溶液含有组织块向上和向下通过首先使用5毫升的一次性吸管，然后用玻璃巴氏吸管直至细胞悬浮液的均匀化。通常情况下，一些残留的组织片，大多是由白质，将保持在悬浮液中。 </li><li>降速在157×g离心5分钟，沉淀重悬在培养基中适当的量（每未涂覆的T75培养瓶10ml）中。使用一个T75培养瓶中为5-10毫米直径大小的一个切片检查，并从这种情况下的较大的标本推断。 </li><li>将细胞注入37℃培养箱中，5％的CO 2和5％O 2。 </li><li>更换一半培养基每3-4天直到细胞达到汇合。这通常需要长达两周（被称为通道0 [P0]）。 </li></ol> 2， 在体外扩增成人人脑细胞及表征<ol><li> 在体外扩增 : <ol><li>吸出培养基，洗涤用磷酸盐缓冲盐水（PBS），保持在室温吨emperature，加入3ml的TrypLE之前。 </li><li>在37℃下进行5〜10分钟，直至大多数细胞分离。轻轻拍打瓶，方便细胞的吊装;以下加入3倍体积的生长培养基，转移细胞到15毫升锥形管中并离心以157×g离心5分钟，重悬细胞在生长培养基中的适当的量。 </li><li>通道中的细胞以1:3为最佳产量成新的T75细胞培养瓶中的比率。 注:本报告并不降低重编程效率的任何迹象传这些细胞，直到小五。而在P0文化中包含了相当部分内皮细胞（作为评估CD34的表达），在随后的传代其数量可忽略不计。 </li></ol></li><li>成年人脑培养物的表征: <ol><li>免疫组化: <ol><li>对于细胞，种子〜60,000个细胞的表征到聚-D-赖氨酸包被的玻璃盖玻片于500μl新鲜的生长mediu米在24孔组织培养板中，孵育在37℃，5％CO 2和5％O 2。 </li><li>对培养的人脑细胞的免疫细胞化学分析取出介质，并用1ml的PBS洗涤3次。 </li><li>通过加入500μl的4％多聚甲醛的PBS中15分钟，在室温下固定细胞。 </li><li>传输玻璃盖玻片到适当的加湿室染色并用含有80微升PBS中的3％牛血清白蛋白（BSA）和0.5％的Triton X-100的1小时在室温下阻断。 </li><li>添加稀释在同一溶液中的一抗（稀释因子见表特定的试剂和设备），并孵育过夜，在室温下4℃或1小时。 注意:细胞在此阶段培养中的相当，但不同的部分是免疫血小板衍生生长因子受体β（PDGFRβ），分化146（CD146），NG2硫酸软骨素健体的集群聚糖，和平滑肌肌动蛋白（SMA），微血管相关的周细胞特征，即标记。只有少数（&lt;1％）表达星形胶质细胞标志物胶质纤维酸性蛋白（GFAP）和S100β。此外，在这一阶段的文化应该是缺乏表达神经元标记βIII-微管蛋白的任何细胞。使用多个主要抗体组合不同的标记物的联合标识。可以通过反转录和定量实时聚合酶链反应（在本协议未描述）获得的神经胶质，神经元，内皮细胞和周细胞标记物的表达的进一步确认。 </li><li>孵育后与初级抗体洗三次，用1ml的PBS和相应的二抗（在适当的稀释液）加入到染色溶液孵育1小时，在室温下在黑暗中。用PBS洗涤细胞3倍。如果需要的话，前安装包括染色用DAPI 5分钟，在室温温度。 </li><li>使用抗衰落的安装中，使用萤光显微镜或共聚焦显微镜分析盖玻片。 </li></ol></li><li>流式细胞仪: <ol><li>对于使用流式细胞仪检测表面标志物表达分析，细胞生长至汇合在无涂层的T25或T75细胞培养瓶中。 </li><li>使用的TrypLE如在由PBS +0.5％BSA的染色液和之前描述的重悬细胞分离。每染色反应，重悬在100μl的染色溶液100,000-200,000细胞。准备使用（CD146-FITC，CD140b-PE，CD34-APC，CD13-FITC，各自同型对照抗体）每单抗体染色反应。 </li><li>添加荧光标记的抗体对每个染色溶液（稀释度见表特定的试剂和设备）直接进入细胞悬液孵育在4℃下在黑暗中30分钟。 </li><li>用500μlPBS洗细胞三次，自旋向下的各洗涤步骤在157×g离心5分钟。 </li><li>通过细胞过滤器（70微米）至FACS管重悬细胞沉淀在0.5-1毫升的染色溶液和转移的细胞。从暴露于光保护样品。 </li><li>涡样品放置在流式细胞仪的仪器前。 </li><li>分析活细胞和排除碎片和细胞聚集，门对FSC-A和SSC-A。细胞小芯片是专门由FSC-A和FSC-W。要确定正确的门控条件设置与门同型匹配的抗体对照结合到各自的荧光的细胞表面标志物。然后确定抗体结合的细胞的比例。 </li></ol></li></ol></li></ol> 3，用于富集通过荧光激活细胞周细胞衍生的细胞分选<ol><li>通过使用70微米的喷嘴流式细胞仪分选纯化的周细胞的细胞群（悬浮于生长培养基）之前转导。使用CD34-APC与C组合D140b-PE和CD146-FITC选择的周细胞。排序为CD34阴性，CD140b-和CD146阳性细胞。 </li><li>收集的分选的细胞在生长培养基中和板到聚-D-赖氨酸包被的玻璃盖玻片在24孔组织培养板中，孵育在37℃，5％CO 2和5％O 2。 </li><li>分选后的48小时用新鲜的生长培养基更换培养基，并在协议4所述的方法进行转导。 </li></ol> 4，逆转录病毒周细胞衍生的细胞转导和重编程为诱导神经元注:请参阅处理逆转录病毒颗粒时，当地的生物安全准则。在德国，使用这里所使用的逆转录病毒需要生物安全等级2。对于生产逆转录病毒颗粒是指协议6。对用于重编程的逆转录病毒构建体，参考的Karow 等人（2012）。逆转录病毒是假型的VSV-G（水泡性口炎病毒糖蛋白）5。对于生产请参阅GAVRILESCU 等[6]。 <ol><li> 24小时前，逆转录病毒转导的种子〜60,000个细胞到聚-D-赖氨酸包被的玻璃盖玻片于500μl新鲜的生长培养基中的24孔组织培养板中，孵育在37℃，5％CO 2和5％O 2的。 </li><li>第二天，用500μl新鲜预热的生长培养基更换生长培养基，并加入1微升含有逆转录病毒颗粒（pCAG-IRES-DsRed的控制，特别是在建立协议在实验室中），pCAG-ASCL1各自的浓缩上清液的-IRES-DsRed的，pCAG-SOX2-IRES-GFP。 </li><li>一天后，除去包括从细胞中的病毒颗粒的介质，并加入1 ml新鲜，预热的B27-分化培养基中，49的10ml DMEM高糖含有GlutaMAX和1ml B27无血清的补充构成。在5保持培养中的细胞在37℃下％CO 2和5％O 2为4-8周不改变介质的重复介质的改变成为有害的，因为INS成熟。 </li></ol>诱导神经元5。通过免疫细胞化学鉴定<ol><li>以确定转导细胞的细胞同一性，特别是展示了一个获得的神经元表型，进行免疫细胞化学的抗细胞抗原如BIII-微管蛋白，微管相关蛋白和神经元核抗原（抗体和它们各自的稀释液，见表特定试剂和设备;遵循相同的程序，在2.2.1所示）。 </li><li>用于改善PdiNs，从E14小鼠大脑皮质衍生的神经元共培养这些细胞的成熟。为此，解剖大脑皮质，用火抛光的玻璃巴斯德移液管机械分离组织。添加10,000-50,000小鼠神经元周细胞源性细胞2-3周的培养下转导与SOX2和ASCL1编码重troviruses并继续培养。转导的细胞可从小鼠的神经元来区分他们的记者（GFP和DsRed）的表达，无论是现场或落射荧光免疫组化之后的GFP和DsRed。 </li><li>为了评估突触的形成到PdiNs通过小鼠神经元，对水疱性神经递质受体，如囊泡膜谷氨酸转运体1（VGLUT1）进行免疫。 </li><li>探测转导的细胞其电气特性（ 即产生动作电位的能力），并通过膜片钳电生理建立功能性突触。有关步骤的详情，请参阅海因里希等人 （2011）。 </li></ol>

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作者什么都没有透露。

本协议描述了在体外扩增和富集周细胞来源的细胞从以下的成年人的大脑皮层分离和随后转化成INS由神经源性转录的逆转录病毒介导的表达Sox2的因素和ASCL1。这种协议提供了实验体外系统研究谱系转换的脑居民细胞向神经元可能还有神经胶质细胞，用共同的目标，最终把这种直接转换方法为成人大脑的体内环境。 技术考虑在我们的研究?...

相对于体细胞成诱导多能干细胞（iPS细胞），这反过来又被赋予了过多的分化潜能的重新编程，直接重编程的目的是为一个特定的信元类型到另一个的直的转换。相对于它们在疾病建模和潜在的基于细胞的疗法的上下文中的应用程序，无论是重新编程的方法具有特殊的优点和缺点。重编程为iPS细胞（i）项规定的细胞几乎无限的来源; （二）允许基因工程; （三）赋予了几乎无限的分化潜能。然而，iPS细胞的主要缺点涉及在未分化的细胞（畸胎瘤形成体内 ）的致瘤性和需要进行离体培养和随后的移植的风险，如果这些细胞将被用于基于细胞的疗法。相反，直接谱系重编程是由所需的细胞的产量较低限制这直接关联与起始种群的靶细胞的数量，但具有该谱系重编程的细胞出现移植1,2时表现出无致瘤性风险的优点;此外，直接重新编程，甚至可以在原位实现， 即在其中将需要这些细胞，从而避免移植的必要性的器官。 考虑到这一点，我们的实验室一直奉行谱系重编程脑固有细胞的可能性为插件作为对神经退行性疾病的细胞疗法的一种新方法。这可能会潜在地视为谱系重编程细胞靶点脑固有细胞包括不同类型胶质细胞的（星形胶质细胞，NG2细胞和少突胶质细胞），小胶质细胞和微血管相关的细胞（内皮细胞和周细胞）。我们已经广泛地研究了大脑皮质的星形胶质细胞的体外重编程的潜力TEX出生后早期小鼠3-5。在寻找类似的合适的细胞来源，可直接谱系重编程在成人的大脑，我们遇到了一个细胞群，可以成功地重编程为INS和周的展览标志。这里，我们描述了如何从成人脑活检收获这些细胞中，以扩大和体外富集这些细胞，并最终成功地重新编程这些体外扩增的细胞的主要部分（在25-30％的范围内）到一个协议INS。重新编程可通过同时逆转录病毒介导的共表达两种转录因子，SOX2和ASCL1来实现。这些PdiNs被发现收购重复性动作电位射击的能力，并作为突触目标，其他神经元表明其整合到神经网络的能力。我们的协议提供了一个简单程序的隔离和传承转换成人的大脑周细胞成文件。 </P&gt;

<table border="0" cellpadding="0" cellspacing="0" width="320">
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			<td style="width:80px;">use 1:400</td>
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			<td style="width:80px;">12-4301-81</td>
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			<td style="width:80px;">17-4301-81</td>
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			<td height="64" style="height:64px;width:80px;">Penicillin-Streptomycin</td>
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			<td height="106" style="height:106px;width:80px;">HBSS (Hanks Balanced Salt Solution)</td>
			<td style="width:80px;">Invitrogen</td>
			<td style="width:80px;">24020-091</td>
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			<td height="198" style="height:198px;width:80px;">Dulbecco&#39;s PBS without CaCl2 and MgCl2 (D-PBS, 1&#215;; Invitrogen, cat. no. 14190)</td>
			<td style="width:80px;">Invitrogen</td>
			<td style="width:80px;">14190</td>
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			<td height="67" style="height:67px;width:80px;">75 cm2 culture flasks</td>
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		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:80px;">Disposable pipettes 10 ml</td>
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			<td align="center" style="width:80px;">#########</td>
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		<tr height="85">
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			<td height="64" style="height:64px;width:80px;">Surgical disposable scalpels&#160;</td>
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			<td style="width:80px;">Olympus</td>
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			<td height="64" style="height:64px;width:80px;">Confocal microscope LSM710</td>
			<td style="width:80px;">Zeiss</td>
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		</tr>
	</tbody>
</table>

lineage-reprogramming of pericyte-derived cells of the adult human brain into induced neurons

直接谱系重编程非神经元细胞分化为神经元的诱导（INS）可以提供深入了解相关的神经发生的分子机制，使在体外模型或修复患病的大脑新策略。识别脑居民非神经元细胞类型适合于直接转换成文件可能允许开展这样的做法在原地，IE中的受损脑组织。在这里，我们描述了在确定从成人的大脑是满足这一前提源性干细胞的尝试开发的协议。这个协议包括:人体细胞（1）由从成人的脑活检得到的大脑皮质培养; （2） 体外扩增 （大约需要2-4周），并培养免疫细胞化学表征和流式细胞仪; （3）通过荧光激活细胞分选（FACS），使用抗PDGF受体-β和抗CD146抗体的富集;（4）逆转录病毒介导转导的神经源性转录因子SOX2和ASCL1; （5），最后将得到的周细胞源性诱导的神经元（PdiNs）通过免疫细胞化学的特性（14天至8周下述的逆转录病毒转导）。在这个阶段，INS可以通过膜片钳记录被探测其电气性能。该协议提供了一个高度可重复的程序，对大脑居民周细胞的体外血统转化为功能性人类INS。

这一协议后，成功地从人类成人大脑皮层的标本建立文化的第一个成果包括在文化的细胞组成的鉴定。免疫细胞化学细胞类型特异性的蛋白质揭示了相当程度不同的患者（ 图1A）的样本得出不同文化之间的异质性。作为定量用流式细胞仪总是有细胞表达PDGFRβ（平均〜75％，从30到最多99％）的主要组分;周细胞如CD146，NG2和CD13的其它标记物一般以较低的范围（ 图1B）。同样S...

Watch this Scientific Journal Video about Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons at JoVE.com

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

针对脑固有细胞直接谱系重编程提供了脑修复的新观点。在这里，我们描述了如何准备丰富的文化从成人大脑皮质脑居民周细胞，并通过转录逆转录病毒介导因子的表达Sox2的和ASCL1将这些转换引起的神经元的协议。

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 ...

lineage-reprogramming-pericyte-derived-cells-adult-human-brain-into

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JoVE Journal

Neuroscience

12.2K 次观看.  Ludwig Maximilians University Munich. 针对脑固有细胞直接谱系重编程提供了脑修复的新观点。在这里，我们描述了如何准备丰富的文化从成人大脑皮质脑居民周细胞，并通过转录逆转录病毒介导因子的表达Sox2的和ASCL1将这些转换引起的神经元的协议。 

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

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

成人出生的神经元的整合研究

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

科研

神经科学

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

从人类多能干细胞的规范在前脑谷氨酸能神经元

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

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

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

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 ...

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 ...

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 ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

来自成人大脑的短期自由浮动切片培养

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

人类干细胞衍生内皮细胞和GABAergic神经元的迁移、化学吸引和共培养测定

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

从成人人类脑组织获取人类微胶质

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

人类诱导多能干细胞脑微血管内皮细胞的衍生、扩张、冷冻保存和特征化

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

从多能干细胞生成人类神经元和少突胶质细胞，用于模拟神经元-少突胶质细胞相互作用

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...