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1。基本的には、解剖に進む前に設定します。 <ol><li>完全なNSC媒体の適切な量はそれぞれ、9:1の比率でNeuroCult NSC基礎培地とNeuroCult NSC増殖サプリメントを混合して調製される。 </li><li>培地は37℃の水浴で暖めています。 </li><li>抗生物質の高濃度（10％）とコールドHEPES緩衝最小必須培地（HEM）は、解剖や洗浄を目的として作成されています。また、抗生物質の補充とNSC基礎培地はまた、この目的のために使用されることがあります。 </li><li>抗生物質を含む冷HEMの25〜30 mLを胚の収集のための滅菌50mLのチューブに分注しています。 </li><li>いくつかの10cmのプラスチック製のペトリ皿を解剖中の胚や脳を保持するため、また、解剖組織を保持するために必要とされる。 </li><li>胚（大はさみ、小さな尖ったハサミ、大型ピンセット、小さな湾曲鉗子）または胚の脳の解剖のための（小さな鉗子、湾曲した微細な鉗子、45 °傾斜細かい鉗子、そして小さなはさみ）を削除するために必要な手術器具は、使用して滅菌されています250ガラス玉滅菌器° Cまたは他の利用可能なオートクレーブ方法。 </li><li>解剖顕微鏡は、70％アルコールで拭き取ると層​​流またはPC2のフード内に設定されています。 </li></ol> 2。 E14マウス脳およびマイクロダイセクションを収穫。 <ol><li>妊娠中のマウスを交配時間は一日に自分の機関の承認された動物のプロトコルに従って、妊娠第 14 回麻酔です。 </li><li>頸椎脱臼は、動物が苦痛を被ることはないか確認されます。 </li><li>麻酔マウスは、吸収性ティッシュペーパーで、その後ろにレイアウトされ、そして腹部はエリアを殺菌するために70％エタノールでリンスする。 </li><li>腹部の皮膚は、大規模な鉗子を用いて把握しているし、皮膚および基礎となる筋膜は、腹腔内および子宮角を公開する大型ハサミで切断されています。 </li><li>子宮は小さな鉗子とはさみで除去し、50 mlコニカルチューブを含有する冷HEMをに転送されます。 </li><li>子宮組織は、フードに転送され、血液や髪の毛のような可能性汚染物質を除去するために、新鮮な無菌冷たいHEMの十分な量で一度か二度洗浄する。 </li><li>子宮組織は、その後コールドHEMを含む10センチメートルペトリ皿に移しています。 </li><li>子宮角を小さく湾曲鉗子や鋏を使用して開かれ、胚は冷たいHEMを含む新しい10cmの皿に転送されます。 </li><li>胚の頭部は、頚髄レベルで分離し、冷HEMを含む別のペトリ皿に移しています。 </li><li>ペトリ皿は、頭蓋骨から脳を削除するには解剖顕微鏡下で転送されます。 </li><li>ヘッドは、ヘッドの背側が上向きに直面しているように尾側から細かい曲がったピンセットで保持されます。マイクロはさみを使用して、最初の水平カットは目の上に作られ、次に頭の後ろに向かって額から正中線に続いた。皮膚や頭蓋骨を介してカットすると、基礎となる脳を損傷しないようにしてください。 </li><li>脳は、湾曲鉗子を使用して後方にモーションで切断面の端を押して、頭蓋骨から削除されます。すべて脳が頭蓋骨から削除されるまでこの手順が続行されます。 </li><li>脳は、背側が上向きに直面してmicroscissorsカットが神経節隆起を公開するために、嗅球から半球の後ろに伸びる各半球の皮質を介して実行されるを使用しているように湾曲した鉗子を使用して着実に開催されます。 </li><li>片手で湾曲鉗子やその他の45 °傾斜鉗子を用いて、皮質のカットフラップが広がっていると神経節隆起が出解剖されています。 </li><li>解剖組織を無菌10センチメートルペトリ皿に配置され、すべて脳がマイクロ解剖されるまで、この手順が繰り返される。 </li><li>組織片は、1 mLのピペットチップでNSCの培地1mLを用いて採取し、15 mLの遠心管に移しています。 </li><li>組織はその後、徹底的に、かつ慎重にチューブの底にピペットチップを押すと上下サスペンションをピペッティングにより解離している。この方法では、凝集塊は、単一の細胞に分割されています。ピペッティングは乳白色のような懸濁液が得られるように3-4回行われます。その後、懸濁液を非解離塊が沈殿するように1〜2分間静置しています。 </li><li>ほぼ全体の細胞懸濁液は、他のチューブに転送され、その後NSC媒体の別の1 mLを説明されているように、残りの塊と単一細胞に解離するために追加されます。 </li><li>チューブの含有量は、700rpm（110グラム）で、室温で5分のためにプールし、遠心分離されています。 </li><li>上清は、実際のペレットにダウンオフ真空引きされ、細胞は完全なNSCの培地1mLに再懸濁する。 </li><li>培地を穏やかに上下にピペッティングしています均質な単一の細胞懸濁液を持っている。 </li><li>細胞懸濁液の10μLを、細胞数を数えるために、トリパンブルーの90μLと混合される。 </li><li>最後に、細胞は、20ng / mLの上皮成長因子（EGF）を添加した完全なNSCの培地で2 × 10 5細胞/ mLの密度でプレーティングされています。 5 mLの培地は、T175フラスコ用T75および40mLのためのT25、20 mLのために使用されます。 </li><li> 37℃加湿インキュベーター中、5％のCO 2でインキュベートしたときにニューロスフェアは、5〜7日間に形成されています。 6-7日に、球は150から200ミクロンの間で測定する必要がありますし、サブカルチャー（通路）への準備が整います。 </li></ol> 3。継代と胚NSCの拡張： <ol><li>ニューロスフェアは、サブカルチャー（直径150から200ミクロン）のための準備ができたら、一時停止球と培地は、フラスコから除去し、適切なサイズの滅菌組織培養チューブに入れ、そしてで5分間、700rpmで（110 g）で遠心分離され室温。 </li><li>上清を捨て、球は％0.05トリプシン- EDTAを1 mLに再懸濁する。 </li><li>細胞懸濁液を2〜3分間37℃の水浴中でインキュベートされている場合、大豆トリプシンインヒビターの等量のトリプシン活性を停止するために使用されます。 </li><li>細胞懸濁液を穏やかにトリプシンが完全に不活化されていることを保証するために上下にピペッティングしています。 </li><li>細胞懸濁液を5分間700rpmで（110グラム）で遠心分離する。その後、上清を除去し、細胞は完全なNSCの培地1mLに再懸濁する。 </li><li>細胞懸濁液の10μLを、細胞数を数えるために、トリパンブルーの90μLと混合される。 </li><li>細胞は、適切なサイズの組織培養フラスコで20ng / mLのEGFを補充した完全なNSCの培地で5 × 10 4細胞/ mLの濃度で播種されています。 5 mLの培地は、T175フラスコ用T75および40mLのためのT25、20 mLのために使用されます。 </li><li> 37℃加湿インキュベーター中、5％のCO 2でインキュベートしたときの二次ニューロスフェアは、5〜7日間に形成されています。 </li></ol> 4。代表的な結果： 一次胚NSCの文化では、細胞の大部分は肥大になり、メッキ時に組織培養皿に付着する。細胞の大多数はどちらか死ぬかを区別しますが、2〜3日後、増殖細胞は、基板（図1）から切り離されて、細胞の小さなクラスターを作る。文化の最初の48時間の大球状凝集体の形成は、主球と誤解されるべきではない。凝集体形成は、主に破片や文化における非解離組織の塊の量に依存します。真のニューロスフェアは、位相明るく、サイズが大きくなる（図2）として、より球形になる。小さなmicrospikesは生存と健康な球体の外面（図3）に表示されます。 5〜7日後、球は丸いですが圧縮してはいけません、そして、直径150〜200μmを測定する必要があります。ニューロスフェアは、（文化9〜10日後）が大きすぎるを成長させるために許可されている場合、それらは、凝集体を形成するか（ビデオを参照）球の中心にいるため細胞死の色で暗くなることがあります。大規模なニューロスフェアは、最終的に（基板に装着し、周辺に向かって移行する） その場で差別化を開始することがあります。また、大規模なニューロスフェアを解離することは困難であり、それらをサブカルチャー。 <img src="/files/ftp_upload/2457/2457fig1.jpg" alt="図1" /> 図1。プライマリE14 NSCの文化は、めっき後の3日間。矢印は、NSCの増殖クラスタを示しています。オリジナルの倍率、20倍<img src="/files/ftp_upload/2457/2457fig2.jpg" alt="図2" /> 図2。プライマリE14 NSCの文化めっき後の7日間。オリジナルの倍率、10倍<img src="/files/ftp_upload/2457/2457fig3.jpg" alt="図3" /> 図3。めっき後のパッセージone E14ニューロスフェアを5日間を。球の周囲にマイクロスパイクを（矢印）に注意してください。オリジナルの倍率、20倍

<ol>
	<li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Morshead, C. M. <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+in+the+adult+mammalian+forebrain:+a+relatively+quiescent+subpopulation+of+subependymal+cells.">Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.</a> Neuron. 13, 1071-1082 (1994).</li><li>Craig, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+growth+factor+expansion+of+endogenous+subependymal+neural+precursor+cell+populations+in+the+adult+mouse+brain.">In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain.</a> J Neurosci. 16, 2649-2658 (1996).</li><li>Golmohammadi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+the+frequency+and+distribution+of+stem+and+progenitor+cells+in+the+adult+mouse+brain.">Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain.</a> Stem Cells. 26, 979-987 (2008).</li><li>Azari, H., Rahman, M., Sharififar, S., BA, R. e. y. n. o. l. d. s. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J Vis Exp. , (2010).</li><li>Reynolds, B. A., Rietze, R. 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+and+neurospheres--re-evaluating+the+relationship.">Neural stem cells and neurospheres--re-evaluating the relationship.</a> Nat Methods. 2, 333-336 (2005).</li><li>Louis, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+neural+stem+and+progenitor+cells+in+the+neural+colony-forming+cell+assay.">Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay.</a> Stem Cells. 26, 988-996 (2008).</li></ol>

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ニューロスフェアのアッセイは、そのシンプルさと再現性の神経幹細胞1-5の分離と拡大のための選択の方法です。このアッセイは、in vitroおよびin vivo試験の両方に使用できる未分化のCNS ​​前駆細胞、の大規模な世代のための貴重なツールです。それは、ニューロスフェアは、善意の神経幹細胞とより限定された前駆細胞の両方から生成することができることが強調さ?...

* HEMを行うには、1M HEPESのMEM and160mlの110Lパケットを混在させると蒸留水を使用して、8.75 Lのボリュームをもたらす。 7.4に最終pHを設定し、それは4℃で保存する

establishing embryonic mouse neural stem cell culture using the neurosphere assay

哺乳類では、幹細胞は、動物の寿命の間にさまざまな組織や器官の細胞の起源と更新を維持するために未分化細胞のソースとして機能します。彼らは潜在的に老化の過程で、または負傷し、疾病の過程で失われた細胞を置き換えることができます。神経幹細胞（NSC）の存在が最初の小説、無血清培養系を用いて成体哺乳類の中枢神経系（CNS）、ニューロスフェアアッセイ（NSA）にレイノルズとWeiss（1992）によって記述されていた。このアッセイを使用して、それは胚の中枢神経系のさまざまな地域からのNSCを分離し、拡大することも可能です。 in vitroでの拡張のNSC でこれらは多能性であり、中枢神経系の三大細胞型を生じさせることができる。 NSAが実行するのが比較的簡単に思えますが、信頼性と一貫性のある結果を達成するために必要とされるここで示す手順に注意が必要です。このビデオでは、実質的にNSAは、胚性14日目マウスの脳組織からのNSCを生成し、拡大を示し、1つが再現可能なニューロスフェア培養を達成できるように技術的な詳細を提供します。手順は、単一の細胞懸濁液を得るために神経節隆起、NSCの培地で収穫した組織の解離を収穫するために、脳の顕微解剖をE14マウス胚を収穫し、最終的にNSAの培養中の細胞をプレーティングが含まれます。文化の中で5〜7日後、得られた一次ニューロスフェアは、さらに将来の実験のためのNSCの数を拡大して継代されています。

Watch this Scientific Journal Video about Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay at JoVE.com

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

このビデオプロトコルは、胚14日目マウスの脳の神経節隆起から神経幹細胞の単離と拡大のためのニューロスアッセイのアプリケーションを示しています。

ニューロスフェアのアッセイを使用してマウス胎児神経幹細胞培養の確立

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

establishing-embryonic-mouse-neural-stem-cell-culture-using

Research

JoVE Journal

Neuroscience

24.6K ビュー.  Shiraz University of Medical Sciences, Shiraz, Iran. このビデオプロトコルは、胚14日目マウスの脳の神経節隆起から神経幹細胞の単離と拡大のためのニューロスアッセイのアプリケーションを示しています。

ビデオ: Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

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

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

ニューロスフェアのアッセイを使用して成体マウス神経幹細胞の単離と拡大

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

3D神経幹細胞培養におけるローカライズたんぱく質：ハイブリッド可視化手法

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

神経コロニー形成細胞アッセイ：神経前駆細胞から善意の神経幹細胞を区別するアッセイ

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

神経向性マウス肝炎ウイルスに感染したマウスの脊髄にマウス神経幹細胞の外科的移植

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

神経芽細胞アッセイ：フローサイトメトリーを用いて神経幹細胞の子孫を差別から神経前駆細胞の生成と濃縮のためのアッセイ

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

胎児マウス神経管から神経堤細胞の単離および培養

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

クローンのアッセイを用いて神経幹細胞の列挙

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

マウス梁下ゾーンから成体神経幹細胞の単離および培養

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Methods Article

ニューロスフェアのアッセイを使用してマウス胎児神経幹細胞培養の確立