この作品は、（SPに）退役軍人からメリットレビュー助成金（NEUD-004-07F）によってサポートされていました。

1。人間のNPCの拡大（ 図1） <ol><li> 、増殖サプリメントを含有する神経基礎培地でニューロスフェア（ 図1A）のように、T-75フラスコ中の懸濁液中の胎児の脳（ロンザ、ウォーカー、MD、USA）と文化からそれらを人間のNPCの凍結ストックを復活させるEGF（10 ng / mL）をし、 FGF（10 ng / mL）を。 </li><li>培養3日後、5分間、500rpmで15mlチューブと遠心分離機に神経球を移す。 </li><li>細胞ペレットの上に〜100μlの培地を残して上清を捨て、エッペンドルフチュー​​ブに移す。 100μlの細胞ペレットを2 T-75フラスコに分割するのに十分である。神経前駆細胞が薄い分割した場合に増殖することができないように少なく、フラスコの数を減らした場合。 </li><li>チューブの底部に200μlの先端を保持しながら200μlの先端50Xでペレットを粉砕する。二つの部分に均等に細胞懸濁液を分割し、2のT-75フラスコ（1-2スプリット）、継続のための1に追加します差別化のための拡張および他の。 </li><li>ニューロスフェアは、300〜500ミクロンの元のサイズになるまで4〜5日毎に培地を変更することで、拡張のための神経前駆細胞の培養（第一フラスコ）を継続。 （500 rpmで、5分間）遠心分離により培地を変更し、古い培地を廃棄し、新しいメディアを追加します。 </li></ol> 2。ニューロンに富んだ文化に人間のNPCの分化（ 図2） <ol><li>神経豊かな文化への神経前駆細胞の分化のために、各ウェルに500μLを添加することにより、ポリ-L-リジン100μgの/ mlの24ウェルプレートとコートの各ウェルに、単一のカバーガラスを配置します。室温で30分間料理をインキュベートする。 </li><li>ポリ-L-リジンソリューションを吸引し、滅菌水でプレートをすすぐ。 </li><li>次に、5μg/ mlのマウスラミニン500μlでコートプレートのウェルを、30分間インキュベートする。インキュベーション後、PBSでウェルを洗浄する。 </li><li> 4日後にsplitti細胞（ステップ1.4）にNGの24ウェルプレートのウェルあたり〜500ニューロスフェアの密度でコーティングされた皿に「差別化のため」とラベル付けされたフラスコの小さなニューロスフェアを転送します。 </li><li> 6時間後、ニューロスフェアは、皿に付着した場合、増殖培地を除去し、神経基礎培地からなる分化培地を追加し、B27サプリメント、NGF（20 ng / mL）を、BDNF（10 ng / mL）を、DBC（100mm）に、レチノイン酸（2μM）。 </li></ol> 3。差別化されたNPCの免疫蛍光染色（ 図3） <ol><li> 2週間の神経分化培地中で神経前駆細胞の培養に続いて、神経細胞に富む培養物が得られる。 PBSで1回ニューロンを洗い流してから30分間、4％パラホルムアルデヒドで固定します。固定したら、PBSで細胞を3回すすいでください。 </li><li> 60分間室温で透過化緩衝液（5％BSAおよびPBS中の0.2％トリトンX-100）で細胞をインキュベートする。 </li><li> 4℃のIでの料理のO / Nインキュベート;アセチルコリンエステラーゼ（1:500）とシナプトフィジン（1:250）; BDNF（1のNeuN（1:250）およびシナプシン（1:250）：PBS中の3％BSA中のポリクローナルおよびモノクローナル抗体の組み合せでシェーカーNA ：500）およびGAP43（1:250）; STAT3（1:500）およびGFAP（1:1,000）。 </li><li>カバーガラスをPBSで3回洗浄し、次いで、90分間、暗所でRTにて抗ウサギCy3および抗マウスFITC二次抗体でそれらをインキュベートする。インキュベーション後、PBSでカバーグラスを3回洗浄する。 </li><li>ピンセットで培養皿からカバーグラスを取り出し、封入剤に逆さに置き、スライドガラス上に封入剤を10μlを置きます。やさしく、余分な封入剤を乾いた布で拭き取ってくださいとマニキュアでエッジを封印。 </li><li>蛍光顕微鏡での免疫染色されたニューロンを調査します。 </li></ol> 4。ニューロンのレーザーキャプチャーマイクロダイセクション（ 図4） <ol><li> PEN膜スライド上に神経細胞に富んだ文化にNPCのを差別図2に記載の手順に従って、ポリ-L-リジンおよびマウスラミニンで被覆sの。 </li><li> RNA分解酵素を含まない条件下で、以降のすべてのステップを実行します。 </li><li>細胞を可視化するためにHistoGeneステイン（アルクトゥルス）を使用して、文化を染色。 </li><li>ベリタスLCMシステムとスライド全体のロードマップイメージを使用して、アストロサイトを欠い純粋なニューロン集団の領域を見つける。描画ツールを使用して神経細胞をマーク。 </li><li> 2段階のプロセスでコンピュータ制御の精度と自動化を使用して、これらの領域のレーザーキャプチャーを実行します。まず、IR（赤外線）は、膜がキャップに付着し得るために70 Mwおよび2500秒のパルスのレーザーパワー設定で複数のスポットで焼成する。次に、マークされた領域は10 mVでの低レベル設定でUV切削工具を用いて切除される。 </li><li>これらのプロセスの組み合わせを選択的CapSure LCMマクロのキャップの上にペン膜からマークされた領域をキャプチャします。キャップが持ち上げられると、Nとメンブレンeuronsがキャップに付着している。 </li><li>のPicoPure RNA単離キット（アルクトゥルス）を使用してLCMサンプルから全RNAを分離し、DNA分解酵素で処理する。 </li><li>キットの指示に従ってRiboAMP RNA増幅キットを使用して単離されたRNAを増幅する。 </li><li>人間の神経フィラメント重鎖（hNFHc）を検出するためのTaqManプローブを用いたリアルタイムRT-PCR解析を行う。 </li></ol>略語： AD、アルツハイマー病、BDNF、脳由来神経栄養因子、CREB、サイクリックAMP応答エレメント結合タンパク質; DBC、ジブチリル環状AMP、EGF、上皮成長因子、FGF、線維芽細胞増殖因子、GFAP、グリア線維性酸性タンパク質、LCM、レーザマイクロダイセクションをキャプチャ、NGF、神経成長因子、NPC、神経前駆細胞、ペン、ポリエチレンナフタレート。

<ol>
	<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. 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=Neural+progenitor+cells+as+models+for+high-throughput+screens+of+developmental+neurotoxicity:+state+of+the+science.">Neural progenitor cells as models for high-throughput screens of developmental neurotoxicity: state of the science.</a> Neurotoxicol. Teratol. 32, 4-15 (2010).</li><li>Christie, V. B., 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=Retinoid+supplementation+of+differentiating+human+neural+progenitors+and+embryonic+stem+cells+leads+to+enhanced+neurogenesis+in+vitro.">Retinoid supplementation of differentiating human neural progenitors and embryonic stem cells leads to enhanced neurogenesis in vitro.</a> J Neurosci Methods. 193, 239-245 (2010).</li><li>Bonner, R. 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>

宣言するためには、特別の利害関係。

我々は、この研究において自己複製ヒト神経前駆細胞の分化による神経細胞に富む細胞培養モデルおよびレーザーキャプチャーマイクロダイセクションによってニューロンの純粋な集団を単離する方法を記載している。我々は、NGF、BDNF、DBCとのNPCの神経分化のためのレチノイン酸の組み合わせを使用している。 DBCはCREB、神経発生12を強化する転写因子を活性化するために使用される...

生涯神経新生は、側脳室の脳室下帯にと大人の哺乳類の脳1の歯状回の顆粒下の層に発生することが知られている。神経前駆細胞（NPCの）は、これらの地域から発信は、ニューロン、アストロサイトおよびオリゴデンドロサイト2に分化することができる多能性細胞である。 NPCがあるため、パーキンソン病、筋萎縮性側索硬化症、脳卒中およびアルツハイマー病（AD）3を含む種々の神経変性障害を有する患者に移植されるべき潜在能力の関心を生成した。 NPCを用いた研究は、一般的に、この移植角度に焦点を当てているが、神経変性の機構を決定するための細胞培養モデルとしてNPC由来のニューロンの電位が十分に利用されていない。以前の研究は、一般的にそうでないように、各実験のために単離する必要が齧歯類の脳組織から単離した有糸分裂後ニューロンを使用している自己再生。 SH-SY5Y、およびSK-N-MC細胞を含むヒト神経芽腫細胞株を拡大することができるが、それらは一次ニューロンの特性を持っていない。彼らは複数の継代のために拡張することができ、一次ニューロン4,5の特性を有する細胞集団を生成するために分化させることができるので、人間のNPCは、一方、両方の利点を提供する。現在の研究では、ヒト胎児脳から単離されたNPCから商業的に入手可能な一貫性のあるニューロンの豊富な集団を得るための新たな分化プロトコールを記載する。これらの培養は、グリア細胞の小さな割合を含んでいないため、我々は、分子特性のために神経細胞の純粋な集団を単離するための追加のメソッドを必要としています。レーザーキャプチャーマイクロダイセクション（LCM）は、組織切片からの細胞の均質な集団を選択的遺伝子発現解析のために6捕捉することのできる新規な技術である。脳は、ニューロン、グリアおよび他の細胞TYからなる異質な組織であるPES。 LCMは、ニューロン特異的遺伝子発現を決定するために使用されている7-10を解析する。我々は以前に具体的に海馬ニューロン11において減少サイクリックAMP応答エレメント結合タンパク質の発現（CREB）およびBDNFを示すためにAD（のTg2576）マウス脳切片から海馬ニューロンのLCMを行った。本研究では、PEN膜スライド上で培養した神経細胞を単離するための、人間のNPCの拡大、神経分化、神経細胞のマーカーの免疫蛍光染色およびLCMのための手順について説明します。

<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

ヒト胎児脳から単離された神経前駆細胞（NPCの）は、上皮成長因子（EGF）、細胞の豊富な供給を提供するために、線維芽細胞増殖因子（FGF）の存在下で増殖条件下で増殖させた。のNPCは、ニューロンを得るために、ポリ-L-リジンおよびマウスラミニンでコーティングしたディッシュ上で、神経成長因子（NGF）、脳由来神経栄養因子（BDNF）の新しい組み合わせの存在下で分化ジブチリルcAMPの（DBC）およびレチノイン酸た豊富な文化。 NPCはまた、ニューロトロフィンの非存在下、DBC及びレチノイン酸およびアストロサイトに富む培養物を得た毛様体神経栄養因子（CNTF）の存在下で分化させた。差別化されたNPCはNeuNを、シナプシン、アセチルコリンエステラーゼ、シナプトフィジンとGAP43を含む神経細胞のマーカーのパネルのための免疫蛍光染色によって特徴付けられた。グリア線維性酸性タンパク質（GFAP）及びSTAT3、星状細胞マーカーは、分化したNPCの10〜15％で検出された。促進するために、細胞型特異的分子特性は、レーザーキャプチャーマイクロダイセクションは、ポリエチレンナフタレート（PEN）膜スライド上で培養したニューロンを単離するために実施した。この研究に記載された方法は、神経変性の分子機構の理解を進めるための貴重なツールを提供しています。

NPCは拡大します（ 図1） ニューロスフェアは、粉砕により単一細胞懸濁液に分解されるときに、ピペットチップは、懸濁液を上下にピペットである程度の抵抗があるように、チューブの底に触れる必要がある。トリチュレーションの回数は、個人や顕微鏡下で得られた細胞懸濁液を検査することによって、試行錯誤によって決定される必要がある間で変化する...

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

人間の神経前駆細胞（NPCの）が増殖している条件の下で拡大した。のNPCは、ニューロトロフィンの組み合わせの存在下での神経細胞が豊富な培養物に分化させた。ニューロンのマーカーは、免疫蛍光染色により検出した。ニューロンの純粋な集団を単離するために、PENのNPCは、メンブレンスライドに分化し、レーザーキャプチャーマイクロダイセクションを行った。

文化における分化したヒト神経前駆細胞からの神経細胞のレーザーキャプチャーマイクロダイセクション

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

laser-capture-microdissection-neurons-from-differentiated-human

Research

JoVE Journal

Neuroscience

12.1K ビュー.  Denver VA Medical Center. 人間の神経前駆細胞（NPCの）が増殖している条件の下で拡大した。のNPCは、ニューロトロフィンの組み合わせの存在下での神経細胞が豊富な培養物に分化させた。ニューロンのマーカーは、免疫蛍光染色により検出した。ニューロンの純粋な集団を単離するために、PENのNPCは、メンブレンスライドに分化し、レーザーキャプチャーマイクロダイセクショ?...

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

胚の脊髄から交連ニューロンの解剖と文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

神経科学

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

のレーザーキャプチャーマイクロダイセクションショウジョウバエ末梢ニューロン

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

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.

小分子の誘導と多能性ヒト胚性幹細胞からヒト神経前駆細胞と神経細胞の効率的な導出

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

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

外傷性脳損傷後の神経細胞や遺伝子発現解析のための単一ニューロンの濃縮された集団のレーザーキャプチャーマイクロダイセクション

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

レーザー捕捉マイクロ解離によるマウス脊髄運動ニューロン細胞体からの転写解析のためのRNAの生成

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

レーザーキャプチャーマイクロダイセクションで鼻生検併用して得られた嗅覚ニューロン：精神疾患における治療応答を研究するために潜在的なアプローチ

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

レーザーキャプチャーマイクロダイセクション - 個々のドーパミンニューロンの単離と全体の腹側被蓋領域の実証

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

根神経節ニューロンと差別化された脂肪由来幹細胞を背：<em&gt;インビトロ</em末梢神経再生を研究するために&gt;共培養モデル

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

外傷によって影響を受ける脳のレーザーキャプチャーマイクロダイ セクション定位アトラス誘導レーザー

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

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.

ひと成人皮膚線維芽細胞から誘導される神経細胞の高収率文化の生成の単純です

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 ...