Name: lineage-reprogramming of pericyte-derived cells of the adult human brain into induced neurons
Uploaded: 2014-05-12T21:45:00
Description: Watch this Scientific Journal Video about Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons at JoVE.com

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?...

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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;

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			<td style="width:80px;">Sigma-Aldrich</td>
			<td style="width:80px;">A9418</td>
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</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

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

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。

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

Research

JoVE Journal

Neuroscience

Studying the Integration of Adult-born Neurons

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

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 ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

中脑多巴胺神经元的数目在成年小鼠环境的调变

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

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

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

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

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

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

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

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

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 ...