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1。細胞のプレーティングに進む前に準備が必要な項目： <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>サイトカインなしNeuroCult NCFC無血清培地のアリコートを解凍しています。 </li><li>培地は37℃の水浴で暖めています。 </li><li> 0.2％の10μg/ mLのとヘパリンの濃度で上皮成長因子（EGF）、基本的な線維芽細胞成長因子（β- FGF）のストック溶液は、先に用意されています。 </li><li>実験のサイズに応じて、いくつかの35ミリメートルの組織培養皿、プレート、細胞一150〜200センチメートルプラスチックシャーレにも重複して35ミリメートル皿を保持するために必要とされており、水のためのサード35mmディッシュに必要とされる。 </li></ol> 2。細胞の準備： あなたの実験に応じて、細胞は、成体または胎児ソース（主に解離組織または解離ニューロスフェア）から調製することができる。大人/マウス胎児中枢神経系（CNS）から組織を細かく分析またはその後1,2の前に説明したとのように成人/胚由来のニューロスフェアを解離する。 <ol><li>単一の細胞懸濁液は、非解離塊を除去するために、40μmのサイズのメッシュフィルターを通過させる。 </li><li>細胞懸濁液の10μlをは、細胞数を数えるために、トリパンブルーの90μlと混合し、 注 ：他の適切な細胞の希釈液を使用することもできます。 </li><li>一次胚や成体由来の神経細胞を用いた場合、完全なNSCの培地で6.5 × 10 5細胞/ mLの濃度に細胞懸濁液を希釈する。成人または胎児の神経細胞に由来する解離ニューロスフェアから細胞を使用する場合は、完全なNSCの培地で2.2 × 10 5細胞/ mLの濃度に細胞懸濁液を希釈する。 </li></ol> 3。半固体のN -シミュレーションプロジェクトの培地で細胞をメッキ： <ol><li>次のコンポーネントの適切​​な量は、複製の数に応じて、順番に混合される。ここでは二つの複製や皿を複製するために必要な量を混ぜる。複製の追加の番号については、 表1を参照してください。 <ul><li>サイトカインなしNeuroCult NCFC無血清培地1.7 mLを</li><li> NeuroCult NSC増殖サプリメント330μL </li><li> EGFの6.6μL（10μg/mL） </li><li>ペニシリン/ストレプトマイシンの32μL </li><li> B - FGF（10μg/mL）3.3μLの培養細胞は成体神経細胞由来の場合にのみ必要です。 </li><li>ヘパリン（0.2％）3.3μLの培養細胞は成体神経細胞由来の場合にのみ必要です。 </li><li>細胞懸濁液の25μlの（解離ニューロスフェアまたは解離CNS組織から6.5 × 10 5細胞/ mLの初代細胞から2.2 × 10 5細胞/ mLの細胞の） 注 ：播種、最終的な細胞数が35 mmの培養皿当たり約2500細胞でなければならないニューロスフェア由来細胞および初代培養細胞を35 mmの培養皿当たり7500細胞のための。いくつかのケースでは、最終的な細胞の播種密​​度は細胞の滴定実験を行うことにより調整する必要があります。 150コロニー - 統計分析のために、文化の21日後に検出されたコロニーの総数は少なくとも50の範囲内でなければなりません。 </li></ul></li><li>細胞を含む培地を穏やかに混合され、コールドコラーゲン溶液の後、1.3mlの細胞懸濁液に移して、任意の気泡を導入しないように穏やかなピペッティングによりよく混合する。 </li><li>この混合溶液を各35ミリメートルの培養皿（〜1.5 mLの/皿）の中心に分注していると料理を穏やかに混合物は、皿の表面に均等に分散させるために円形の動きを使ってチップを渡したされています。 。 </li><li>重複した35mmの培養皿を100mmペトリ皿に入れられます。新しい35mm皿のふたが除去され、オープン皿も同じ100ミリメートルペトリ皿に配置されます。滅菌水は、潜伏期間中に最適な湿度を維持するために、このオープン35mm皿に追加されます。正方形のバイオアッセイプレート（245 mm）をより多くの複製35mmディッシュが設定されている場合に使用されています。再び、滅菌水を含む2つまたは3つのオープン35mmディッシュが含まれています。 </li><li>プレートを37℃インキュベーターにセットして転送される° C、5％CO2、湿度95％。温度の上昇やゲル形成によるコラーゲンは固まるには約1時間以内に発生します。培養物は、この時間の間乱されるべきではない。 </li><li>培養細胞は21日（コロニーの大きさの違いが明確に21日後に区別することができる）インキュベートする。 </li></ol> 4。補充培地を準備し、文化を食べさせる。 N -シミュレーションプロジェクトの文化が長期間（21日間）インキュベートされているように、培養物を、適切な完全なNeuroCultを供給する必要がある 補充培地は、次のように新しく調製： <ul><li> NeuroCult NSC基礎培地4.5 mLを0.5メートルと混合され、NeuroCult NSC増殖サプリメントとその後の成長因子のLが追加されます。 </li><li> 10μg/mLEGFの250μL（0.5μg/ mLのEGFの最終濃度を与えるために） </li><li> 10μg/mLB - FGF（0.25μg/mLB - FGFの最終濃度を与えるために）、125μLの培養細胞は成体神経細胞由来の場合にのみ必要です 。 </li><li> 0.2％ヘパリンの125μLは、 培養細胞は成体神経細胞由来の場合にのみ必要です 。 </li><li>適切な完全なNeuroCult補充培地（胚、大人の細胞のための）60μLを全体NCFCアッセイ培養インキュベーション（21日）中に7日ごとに一回、各NCFCアッセイ皿の中央に追加されます。 </li></ul> 5。 N - CFCアッセイ由来のコロニーをスコアリングし、 善意のNSCと神経前駆細胞の頻度を計算する： <ul><li>各35mmの培養皿は、2.0ミリメートル× 2.0 mmのグリッドサイズとグリッド得点皿上に配置され、その後、両方の皿は、顕微鏡ステージ上に配置されています。 </li><li>低消費電力を（2.5倍 - 5X）を使用して、対物レンズ、各皿がスキャンされ、コロニーは、そのサイズに基づいて採点されます。 </li><li>コロニーは2つの主要カテゴリに分類されます。 <ol><li>直径2mm未満</li><li> ≥直径2mm </li></ol></li></ul> 6。代表的な結果： めっき後の7日間（図1） - ニューロスアッセイと同様に、N - CFCアッセイで播種細胞が増殖し、3内の細胞の小さなコロニーを作り始める。 2週間後に、異なるサイズのコロニーを区別することができます。コロニーの大半は14日後に成長を停止する傾向がある一方で、いくつかのコロニーのサイズが増加し続ける。 1mmの直径、3）1 - - 2 mmの直径と4）≥2ミリメートル、直径0.5mm未満の直径、2）0.5）：1日21で、コロニーが4つのカテゴリのいずれかに分類することができます。実質的に、2mmの直径よりも小さいコロニーが派生前駆と呼ばれます（図2）とNSCの派生として、直径コロニー≥2ミリメートルは（図3）と呼ばれます。メッキの総細胞あたりの直径はコロニー≥2mmの数は、長期の自己再生およびマルチ潜在的な能力を持つ実際の善意の神経幹細胞の頻度を表します。並列NSAの実験7-8日後に特定の細胞集団の頻度を構成する総ニューロスフェアは、N -シミュレーションプロジェクトの実験で21日後に同じ細胞集団の頻度を構成する総コロニーに類似すると推定されている。 N -シミュレーションプロジェクトでは、しかし、各セルは、その完全な増殖能を示すことができるように、より寛容な条件を提供します。 <table border="1"><tbody><tr><td> コンポーネント </td><td> 2は、複製 </td><td> 図3は、複製 </td><td> 4複製 </td></tr><tr><td> NeuroCult NCFCサイトカインのない無血清培地</td><td> 1700 </td><td> 2550 </td><td> 3400 </td></tr><tr><td> NeuroCult NSC増殖サプリメント</td><td> 330 </td><td> 495 </td><td> 660 </td></tr><tr><td> EGF（10μg/ mL）を</td><td> 6.6 </td><td> 9.9 </td><td> 13.2 </td></tr><tr><td> 唯一の大人の細胞のためのbFGF（10μg/ mL）を、 </td><td> 3.3 </td><td> 4.95 </td><td> 6.6 </td></tr><tr><td> 唯一の大人の細胞のためのヘパリン溶液（0.2％）、 </td><td> 3.3 </td><td> 4.95 </td><td> 6.6 </td></tr><tr><td>ペニシリン/ストレプトマイシン（1:100） </td><td> 32 </td><td> 48 </td><td> 64 </td></tr><tr><td>で細胞： 2.2 × 10 5の培養細胞/ mLまたは 6.5 × 10 5一次細胞/ mLの</td><td> 25 </td><td> 37.5 </td><td> 50 </td></tr><tr><td>コラーゲン溶液</td><td> 1300 </td><td> 1950 </td><td> 2600 </td></tr></tbody></table> 表1。完全なN - CFCアッセイの文化のコンポーネント。 <img src="/files/ftp_upload/2639/2639fig1.jpg" alt="図1" /> 図1の通路のN -シミュレーションプロジェクトの文化の代表コロニーone E14マウスのNSCの7日間めっき後。コロニーは、さまざまな形態とサイズが表示される場合があります。オリジナルの倍率; 4倍<img src="/files/ftp_upload/2639/2639fig2.jpg" alt="図2" /> 図2の通路のN -シミュレーションプロジェクトの文化の異なるサイズ21日メッキ相次いE14マウスのNSCの代表前駆細胞由来のコロニー。図に示すように、コロニーは異なる形態を持っていますが、すべてのサイズは2未満mmである。オリジナルの倍率; 4倍<img src="/files/ftp_upload/2639/2639fig3.jpg" alt="図3" /> 通過のN -シミュレーションプロジェクトの文化one E14マウスのNSCの21日めっき後にコロニー由来の図3。代表善意の幹細胞。幹細胞由来のコロニーは、（参照は異なる形態が表示されることがあります。ビデオ）が、すべては、直径≥2ミリメートルです。オリジナルの倍率; 4倍

<ol>
	<li>Azari, H., Rahaman, M., Sharififar, S., Reynolds, B. A. <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>Azari, H., Sharififar, S., Rahaman, M., Ansari, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J Vis Exp. , (2011).</li><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>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. , (2008).</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>Louis, S. A., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosphere+and+Neural+Colony-Forming+Cell+Assays.">Neurosphere and Neural Colony-Forming Cell Assays.</a> Protocols for Neural Cell Culture. 10, 1-28 (2010).</li></ol>

著者の一人、シャロンA.ルイは、StemCellテクノロジーズ株式会社、本研究で使用されるいくつかの試薬の生産者と提携しています。

ニューロスフェアの検定3,1,2は、大人と胚中枢神経系の組織のようなさまざまなソースからの神経幹細胞を分離し、拡大する最も一般的な方法ですが、それは正確に神経前駆細胞（幹細胞の混合集団でNSCの周波数を測定することができないとニューロスフェアの数と善意の幹細胞の数は4との間の一対一の対応関係がないとして前駆細胞）。この制限に対処するため、元NSAは、神経幹細胞および前駆細胞5-7三週間、完全な増殖能に成長させるように適応されています。半固体コラーゲンマトリックスは、コロニーのメッキと凝集単一細胞の遊走を妨げるとして液体NSAとは異なり、N -シミュレーションプロジェクトではコロニーは実際にクローン的、派生しています。このアッセイで一貫性のある結果を得るためにお勧め： <ol><li>オリジナルの細胞懸濁液に単一の細胞懸濁液を確認してください。非解離塊を除去するために、40μmのサイズのメッシュフィルターを通過して、単一の細胞懸濁液を渡します。 </li><li>氷の上または+4℃の冷蔵庫内のコラーゲン溶液を保管してください。コラーゲンは、温度を上昇させることにより固まるように細胞懸濁液の混合物に追加される最後の項目です。 </li><li>毎週の文化を供給することを忘れないでください。 7日ごとに一度成長因子（s）を含む補充培地を追加すると、細胞が拡張された培養期間にわたって増殖させ続けることが可能となります。 </li><li>最終的な細胞播種密度は、プライマリまたは培養神経細胞由来の細胞用に最適化されているように文化の21日後に検出されたコロニーの総数NCFC -少なくとも50の範囲内にある - 200個のコロニー。統計分析のためのコロニーの十分な数字を提供しながら、この範囲は、試料の直径は珍しいNSC由来のコロニー&gt; 2mmの検出を可能にする。開始セルのソースに応じて、有意に少なかった（&lt;50）とコロニーの高い数字が（&gt; 250）時に得られる。あまりにも多くのコロニーは、不正確なコロニー計数の結果、増殖培地の成分の過密anddepletionをもたらす間、少なすぎるのコロニーは、直径が検出レベル以下であることで植民地&gt; 2ミリメートルになります。したがって、細胞播種密度は（すなわち、細胞メッキの合計数を増加または減少）に応じて調整する必要があります。 </li></ol>

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NeuroCult NSC Basal Medium</td>
<td>Medium</td>
<td>STEMCELL</td>
<td>05700</td>
<td>&nbsp;</td>
<tr>
<td>NeuroCult NSC Proliferation Supplements</td>
<td>Medium supplement</td>
<td>STEMCELL</td>
<td>05701</td>
<td>&nbsp;</td>
<tr>
<td>NeuroCult NCFC medium</td>
<td>Medium</td>
<td>STEMCELL</td>
<td>05720</td>
<td>&nbsp;</td>
<tr>
<td>0.05% 	trypsin-EDTA</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25300-062</td>
<td>&nbsp;</td>
<tr>
<td>Soybean trypsin inhibitor </td>
<td>Reagent</td>
<td>Sigma</td>
<td>T6522</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer </td>
<td>Sieve</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>Pen/Strep </td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>T25 flask</td>
<td>Culture ware</td>
<td>Nalge Nunc international</td>
<td>136196</td>
<td>&nbsp;</td>
<tr>
<td>T80 flask</td>
<td>Culture ware</td>
<td>Nalge Nunc international</td>
<td>178905</td>
<td>&nbsp;</td>
<tr>
<td>Collagen</td>
<td>Reagent </td>
<td>STEMCELL</td>
<td>04902</td>
<td>&nbsp;</td>
<tr>
<td>EGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>2028-EG</td>
<td>&nbsp;</td>
<tr>
<td>b-FGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>3139-FB</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>Growth factor</td>
<td>Sigma</td>
<td>H4784</td>
<td>Reconstituted in PBS</td>
</tr>
<tr>
<td>15 ml tubes</td>
<td>Culture ware</td>
<td>BD Falcon</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>50 ml tubes</td>
<td>Culture ware</td>
<td>BD Falcon</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>35 mm culture dishes</td>
<td>Culture ware</td>
<td>STEMCELL</td>
<td>27100</td>
<td>&nbsp;</td>
<tr>
<td>Gridded scoring dishes</td>
<td>Culture ware</td>
<td>STEMCELL</td>
<td>27500</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

neural-colony forming cell assay: an assay to discriminate bona fide neural stem cells from neural progenitor cells

ニューロスアッセイ（NSA）は、分離拡大し、また、神経幹細胞（NSC）の周波数を計算するために最も頻繁に使用される方法の1つです。また、この無血清培養システムは、幹細胞を拡大し、腫瘍と正常組織のさまざまなから自分の周波数を決定するために採用されている。それは一対一の関係はニューロスフェアの形成とNSCの間に存在していないことが最近示されている。これはNSAとして現在適用されているが、胚と成体哺乳類の脳の両方から分離された神経前駆細胞の混合集団でNSCの頻度を過大評価することを示唆している。このビデオでは、実質的に半固形アッセイ、彼らの長期的な増殖能に基づいて、前駆細胞から幹細胞を識別する能力を持つニューラルコロニー形成細胞アッセイ（N -シミュレーションプロジェクト）を、考慮した、全く新しいコラーゲンを示し、そのための方法を提供するNSCの周波数を列挙する。コロニーが&lt;2ミリメートルの前駆細胞から派生している間、N -シミュレーションプロジェクトでは、直径のコロニー≥2 mmを、NSCのすべての機能の基準を満たす細胞から派生しています。 N -シミュレーションプロジェクトの手順は、プライマリおよび培養成体または胎児マウスの中枢神経系の細胞を含むさまざまなソースから調製した細胞に使用することができます。ここでは、通路からN -シミュレーションプロジェクトを実行するために胚性14日目マウスの脳から生成されたニューロスフェアを準備セルを使用してください。培養は、播種した細胞がその完全な増殖能を示すことができるように、増殖培地で3週間、7日ごとに補充されていますし、神経前駆細胞と善意の神経幹細胞の頻度は、&lt;2ミリメートルであるコロニーの数をカウントすることで、それぞれ計算されます。と≥2ミリメートルが最初に播種した細胞の数を参照しているもの。

Watch this Scientific Journal Video about Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells at JoVE.com

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

このビデオプロトコルは、神経コロニー形成細胞アッセイを用いて神経前駆細胞の混合集団で善意の神経幹細胞を区別して列挙する方法を示しています。

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). ...

neural-colony-forming-cell-assay-an-assay-to-discriminate-bona-fide

Research

JoVE Journal

Neuroscience

22.0K ビュー.  University of Florida. このビデオプロトコルは、神経コロニー形成細胞アッセイを用いて神経前駆細胞の混合集団で善意の神経幹細胞を区別して列挙する方法を示しています。

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

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 ...

神経科学

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

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 span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

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 ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

無傷の感覚器官の前駆細胞の生細胞イメージングショウジョウバエ蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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 ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

ヒト多能性幹細胞からの神経堤前駆細胞のフィーダーフリー導出

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

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. ...

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.

強膜網膜移植ひと網膜色素上皮細胞のラットの目の洗練されたプロトコルの開発

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

マウス萌芽期の神経幹細胞の分離と培養

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

シアログリカンの代謝標識による原発神経幹および前駆細胞の細胞表面マーカーの同定

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Methods Article

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