Name: laser capture microdissection of neurons from differentiated human neuroprogenitor cells in culture
Uploaded: 2013-09-16T14:30:00
Description: Watch this Scientific Journal Video about Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture at JoVE.com

这项工作是由退伍军人管理局优异审查补助金（NEUD-004-07F）（以SP）的支持。

1。扩展人类的NPC（ 图1） <ol><li>重振胎儿脑（龙沙，Walkersville，MD，USA），并将它们悬浮培养中的T-75瓶中含有增殖补充，表皮生长因子（10毫微克/毫升）的神经球（ 图1A）的Neurobasal培养基人类的NPC的冷冻股票和FGF（10纳克/毫升）。 </li><li>后在培养3天后，将神经球转移到15毫升管中，离心分离机，在500 rpm离心5分钟。 </li><li>弃去上清液留下〜100μl的培养基上的细胞沉淀?...

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	<li>Doetsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+identity+of+neural+stem+cells.">The glial identity of neural stem cells.</a> Nat Neurosci. 6, 1127-1134 (2003).</li><li>Galli, R., Gritti, A., Bonfanti, L., Vescovi, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells:+an+overview.">Neural stem cells: an overview.</a> Circ. Res. 92, 598-608 (2003).</li><li>Kim, S. U., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+cell+therapy+in+neurological+diseases:+a+review.">Stem cell-based cell therapy in neurological diseases: a review.</a> J. Neurosci. Res. 87, 2183-2200 (2009).</li><li>Breier, J. 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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection:+molecular+analysis+of+tissue.">Laser capture microdissection: molecular analysis of tissue.</a> Science. 278, 1481-1483 (1997).</li><li>Vincent, V. A., DeVoss, J. J., Ryan, H. S., Murphy, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+neuronal+gene+expression+with+laser+capture+microdissection.">Analysis of neuronal gene expression with laser capture microdissection.</a> J. Neurosci. Res. 69, 578-586 (2002).</li><li>Robles, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+gene+expression+profiling+in+spatial+discrimination+learning.">Hippocampal gene expression profiling in spatial discrimination learning.</a> Neurobiol. Learn Mem. 80, 80-95 (2003).</li><li>Su, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+ethanol+versus+formalin+fixation+on+preservation+of+histology+and+RNA+in+laser+capture+microdissected+brain+tissues.">Comparison of ethanol versus formalin fixation on preservation of histology and RNA in laser capture microdissected brain tissues.</a> Brain Pathol. 14, 175-182 (2004).</li><li>Shimamura, M., Garcia, J. M., Prough, D. S., Hellmich, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+and+analysis+of+amplified+antisense+RNA+from+distinct+cell+populations+of+the+young+and+aged+rat+brain:+effect+of+traumatic+brain+injury+on+hippocampal+gene+expression.">Laser capture microdissection and analysis of amplified antisense RNA from distinct cell populations of the young and aged rat brain: effect of traumatic brain injury on hippocampal gene expression.</a> Brain Res. Mol. Brain Res. 122, 47-61 (2004).</li><li>Pugazhenthi, S., Wang, M., Pham, S., Sze, C. I., Eckman, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downregulation+of+CREB+expression+in+Alzheimer's+brain+and+in+Abeta-treated+rat+hippocampal+neurons.">Downregulation of CREB expression in Alzheimer's brain and in Abeta-treated rat hippocampal neurons.</a> Mol. Neurodegener. 6, 60 (2011).</li><li>Dworkin, S., Mantamadiotis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+CREB+signalling+in+neurogenesis.">Targeting CREB signalling in neurogenesis.</a> Expert Opin. Ther. Targets. 14, 869-879 (2010).</li><li>Trujillo, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+perspectives+of+neural+stem+cell+differentiation:+from+neurotransmitters+to+therapeutics.">Novel perspectives of neural stem cell differentiation: from neurotransmitters to therapeutics.</a> Cytometry A. 75, 38-53 (2009).</li><li>Pugazhenthi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Varicella-zoster+virus+infection+of+differentiated+human+neural+stem+cells.">Varicella-zoster virus infection of differentiated human neural stem cells.</a> J. Virol. 85, 6678-6686 (2011).</li><li>Kamme, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+microarray+analysis+in+hippocampus+CA1:+demonstration+and+validation+of+cellular+heterogeneity.">Single-cell microarray analysis in hippocampus CA1: demonstration and validation of cellular heterogeneity.</a> J. Neurosci. 23, 3607-3615 (2003).</li></ol>

我们在本研究中通过自我更新的人neuroprogenitor细胞分化神经元丰富的细胞培养模型和方法，通过激光捕获显微切割分离神经元的纯人口。我们已经使用了NGF，BDNF，DBC和视黄酸对的NPC的神经元分化的组合。 DBC是用来激活CREB，增强神经发生12的转录因子。维甲酸诱导细胞周期退出和减少胶质人口13。在这项研究中所描述的方法采用修改过我们之前的报告14。我们早先用捣碎的?...

寿命长的神经发生是已知发生在侧脑室的脑室下区和在成年哺乳动物脑1的齿状回的颗粒下层。 Neuroprogenitor细胞（的NPC）来自这些地区的起源是多能细胞，它可以分化成神经元，星形胶质细胞和少突胶质细胞2。的NPC所产生的，因为其潜在的患者的各种神经变性疾病，包括帕金森氏病，肌萎缩性侧索硬化，中风和阿尔茨海默氏病（AD）的3到被移植的兴趣。与研究的NPC一般都集中在这个移植的角度，但NPC来源的神经元的细胞培养模型，以确定神经退行性病变的机制的潜力还没有得到充分发挥。以前的研究一般用有丝分裂后的神经元从啮齿动物脑组织中需要被分离为每个实验中分离，因为它们是不自我更新。虽然人类成神经细胞瘤细胞系包括SH-SY5Y和SK-N-MC细胞可以扩展，它们不具有原代神经元的特征。人类的NPC，在另一方面，提供了两者的优点，因为它们可以被扩展为多通道，并且可以分化产生的细胞群体与原代神经元4,5的特性。在目前的研究中，我们描述了一种新的分化方案，从人胎脑分离市售的NPC获得一致的神经元的富人口。因为这些文化都包含一个小的百分比胶质细胞，我们需要更多的方法来分离神经元的分子鉴定纯人口。激光捕获显微切割（LCM）是一种新颖的技术，其中来自组织切片的细胞同源性的群体可以被选择性地捕获用于基因表达分析6。大脑是一个异类组织包括神经元，神经胶质细胞和其他细胞TYPES。 LCM被用于确定神经元特异性基因表达分析7-10。之前我们已经进行海马神经元的LCM从公元（Tg2576转）鼠脑切片，专门用于显示在海马神经元11表达降低的环磷酸腺苷反应元件结合蛋白（CREB）和BDNF。在本研究中，我们描述了人类的NPC的扩大，神经元分化，免疫荧光染色神经元标志物及LCM神经元对PEN膜玻片培养的分离程序。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Neuroprogenitor cells (NPCs) </td>
			<td>Lonza</td>
			<td>PT-2599</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A human basal medium</td>
			<td>Stem cell technology</td>
			<td>5750</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A Proliferation supplement</td>
			<td>Stem cell technology</td>
			<td>5753</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A Differentiation supplement</td>
			<td>Stem cell technology</td>
			<td>5754</td>
			<td></td>
		 </tr>
		 <tr>
			<td>B-27 Supplement (50x) </td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Human epidermal growth factor</td>
			<td>Stem cell technology</td>
			<td>2633</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fibroblast growth factor </td>
			<td>Sigma</td>
			<td>F0291</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Brain Derived Neurotrophic Factor </td>
			<td>Cell signaling </td>
			<td>3897S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Nerve Growth Factor </td>
			<td>Invitrogen</td>
			<td>13257-019</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dibutyryl cyclic AMP</td>
			<td>Sigma</td>
			<td>D-0627</td>
			<td></td>
		 </tr>
		 <tr>
			<td>poly-L-lysine </td>
			<td>Sigma</td>
			<td>P-5899</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mouse laminin </td>
			<td>Sigma</td>
			<td>L-2020</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R-2625</td>
			<td></td>
		 </tr>
		 <tr>
			<td>STAT3 antibody</td>
			<td>Cell signaling</td>
			<td>9132</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GFAP antibody</td>
			<td>Cell signaling</td>
			<td>3670</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GAP43 antibody</td>
			<td>Transduction laboratories</td>
			<td>612262</td>
			<td></td>
		 </tr>
		 <tr>
			<td>BDNF antibody</td>
			<td>Millipore</td>
			<td>AB1534SP</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Acetyl cholinesterase antibody </td>
			<td>Santz cruz</td>
			<td>Sc-11409</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Synaptophysin antibody</td>
			<td>Abcam</td>
			<td>ab18008-50</td>
			<td></td>
		 </tr>
		 <tr>
			<td>NeuN antibody</td>
			<td>Chemicon</td>
			<td>MAB377</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Synapsin antibody</td>
			<td>Novus</td>
			<td>NB300-104</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti mouse FITC</td>
			<td>Jackson Immuno </td>
			<td>115-095-146</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td>Research Laboratories</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti Rabbit Cy3</td>
			<td>Jackson Immuno </td>
			<td>711-165-152</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td>Research Laboratories</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A1653</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Triton-X 100</td>
			<td>Acros</td>
			<td>21568-2500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Paraformaldehye </td>
			<td>Fisher</td>
			<td>4042</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Coverslip (Big circle cover slip)</td>
			<td>Fisherbrand</td>
			<td>12-545-102</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mounting medium (Prolong Gold)</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pen membrane</td>
			<td>Applied biosystems</td>
			<td>LCM0521</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Histogene LCM frozen section staining kit</td>
			<td>Applied biosystems</td>
			<td>KIT0401</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RiboAmp RNA Amplification kit</td>
			<td>Applied biosystems</td>
			<td>KIT0201</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Picopure RNA Isolation kit</td>
			<td>Applied biosystems</td>
			<td>KIT0202</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CapsureMacro LCM caps</td>
			<td>Applied biosystems</td>
			<td>LCM0211</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

laser capture microdissection of neurons from differentiated human neuroprogenitor cells in culture

Neuroprogenitor细胞（的NPC）从人胎脑分离出的表皮生长因子（EGF）和成纤维细胞生长因子（FGF），以提供细胞的大量供应的存在下增殖的条件下展开。的NPC分化中的神经生长因子（NGF）的新组合的存在下，脑源性神经营养因子（BDNF），丁酰腺苷（DBC）与上餐盘涂有聚-L-赖氨酸和小鼠层粘连蛋白 - 视黄酸，以获得神经元丰富的文化。的NPC也有区别在缺乏神经营养因子，DBC和视黄酸和睫状神经营养因子（CNTF），得到星形胶质细胞培养丰富的存在。差异化的NPC的特点是免疫荧光染色的神经元标志物，包括的NeuN，突触素，乙酰胆碱酯酶，突触素及GAP43面板。胶质纤维酸性蛋白（GFAP）和STAT3，星形胶质细胞标志物，在分化的NPC的10-15％进行检测。为了便于细胞类型特异性分子鉴定，激光捕获显微切割进行分离神经元的聚乙烯酯（PEN）膜的载玻片培养。在这项研究中所描述的方法提供了有价值的工具，以促进我们的神经变性的分子机制的理解。

扩展的NPC（ 图1） 当神经球被分解为单细胞悬浮液通过研磨，枪头需要触摸在管的底部，以便有当将悬浮液用移液管上下一些阻力。中研磨的次数将个体，需要通过反复试验通过在显微镜下检查所得到的细胞悬液决定之间变化。关键是要提供细胞悬液足够的密度，因为扩散会以更快的速度，当的NPC都在密切接触。如果的NPC不生长，细胞密度应增加减少瓶中的数量。?...

Watch this Scientific Journal Video about Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture at JoVE.com

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

人类neuroprogenitor细胞（的NPC）增殖的条件下扩大。的NPC分化成神经营养因子中的组合的存在下神经元的富培养物。用免疫组化法检测神经元标志物。为了分离神经元的纯人口的NPC分化的PEN膜幻灯片并进行激光捕获显微切割。

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The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

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

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

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