これらの結果につながる研究がグラント契約の下で、欧州連合（EU）セブンス枠組み計画（FP7/2007-2013）から資金提供を受けたNフランス電力（EDF）からとL&#39;アジャン国立·デ·ラ·ルシェルシュから、323267を°ました - サンテ·エンバらサンテ - 苦労（ANR-SEST、Neurorad）。

1。動物の手順このプロトコルは、11月24日の欧州共同体理事会指令、1986（86/609/EEC）に準拠して設計されており、動物福祉（CETEA-CEA DSV IDF）で、当社の機関の委員会によって承認されている。 <ol><li>のEdU / BrdUおよびE14.5妊娠マウス（ 図2A）を照射して注射。 <ol><li> PBS中5ミリグラム/ mlで添加し、BrdUを1ミリグラム/ ATのEdUの溶液を調製する。妊娠中のマウスに25 G針を使用してのEdU溶液100μlを腹腔内注射（IP）を実行します。 1.5時間後、0グレイまたは2 Gyの（0.6線量/分）でマウス（全身照射）を照射する。直後に、妊娠中のマウスに25 G針を使用してのBrdU液（IP）200μLを注入。 </li></ol></li><li>胚の脳の冠状スライスの準備。 <ol><li> 1時間又は照射後4時間で、二酸化炭素によって妊娠マウスを安楽死させる。 </li><li>腹部CAVIを開くはさみを使用して3センチ出るTY。ホーンの両方四肢を区画し、子宮の残りの部分から2子宮角を分離する。 PBS、0.6％のグルコースを含むペトリ皿に子宮角を転送します。胚を単離するために鉗子で子宮角を開きます。鉗子を使用して、各胚の羊膜嚢を削除します。 </li><li>鉗子で胚の頭部を解剖し、4℃で4％パラホルムアルデヒド（PFA液、pH = 7.4）O / Nに浸漬することによってそれらを修正</li><li> 4℃で少なくとも一晩PBS中で胚の頭をすすぐヘッドは、数日間PBSに維持することができる。 </li><li> 表1に示すように真空浸透処理を用いてパラフィン中に埋め込 ​​むヘッド。ヘッド埋め込 ​​みはまた、エタノールおよびキシレン中浴の化学フード下で行われ、次いで、パラフィン浴65℃のオーブン中ですることができる。 </li><li>ミクロトームで5μmの厚さの冠状切片を準備します。 </li><li>ポリリジンでコーティングされた顕微鏡用スライド上にマウントします。&lt;/李&gt; <li>スライドを数ヶ月間、室温（RT）に維持することができる。 </li></ol></li></ol> 2。のEdU / BrdUの染色<ol><li>脱パラフィンおよび抗原マスク解除<ol><li> 5分間トルエン3浴にスライドを浸漬することによりパラフィン包埋した脳切片を脱パラフィン。 </li><li> 100％エタノール、95％エタノール、70％エタノール、5分間H 2 O中に次のようにエタノール濃度を低下させるの各溶液の2浴中で3分間再水和するスライドは、いくつかの時間脱イオン水に保つことができる。 </li><li>クエン酸溶液（10ミリモル、pH6）中で10分間スライスを沸騰し、次に5分間脱イオン水中でインキュベートする。 </li></ol></li><li> EdUの染色<ol><li>製造業者のプロトコルに従ってEdUの検出を行う。 10分間PBS中0.5％トリトンX-100で細胞を透過。 </li><li>脱イオン水に10倍の溶液を希釈することで1XのEDUバッファ添加物を準備します。このソリューションは、新たに作られ、同じDA上で使用する必要がありますY。 </li><li> EdUのアレクサフルーア488アジ化を含め、のEDU反応カクテルを準備します。それは、表2に示されている順序以下の成分を添加することが重要である、そうでなければ、反応が最適に進行しない。準備の15分以内のEDU反応カクテルを使用してください。 </li><li> PBSで2回洗った後、透過処理バッファー（ステップ2.1.1）を取り外します。 </li><li>のEDU反応カクテルの150μl加え、カバーグラスを入れて、室温で暗所で30分間インキュベートする。 </li><li>のEDU反応カクテルを削除した後、PBSで1回洗浄。 </li></ol></li><li> BrdU染色<ol><li> 7.5％ヤギ血清、PBS中の7.5％ウシ胎児血清を飽和バッファーを準備します。 、脳スライスに飽和バッファーの150μl加え、カバーグラスを入れて、非特異的抗体結合をブロックするために、室温で1時間インキュベートする。 </li><li>カバーガラスを取り外します。飽和バッファー中で1/300に希釈したマウス抗BrdU一次抗体150μl加え、/カバースリップを置くとOインキュベート4℃で、N </li><li>カバーガラスを外し、次にPBSで3回洗浄する。 </li><li>飽和バッファー中で1/400に希釈したヤギ抗マウスをAlexa594二次抗体150μl加え、カバーグラスを入れて、室温で1時間インキュベートする。 </li><li> PBSで一度洗った後、カバーガラスを取り外します。 </li><li>核染色：PBS中1μg/ mlの時に4 &#39;、6 - ジアミジノ-2 - フェニルインドール（DAPI）の150μLを加え、カバーグラスを入れて、室温で2分間インキュベートする。 </li><li>メディアをマウントスライドをマウントします。 </li></ol></li></ol> 3。分析<ol><li> 20Xの目的や共焦点顕微鏡を用いて蛍光顕微鏡下で脳切片を調べて、ファイルを分離として3チャンネル（488ナノメートル、594ナノメートルとUV）で画像を取得する。 </li><li> Photoshopソフトウェアで画像を積み重ねる。 </li><li>背内側脳壁の標準部門で脳切片を分析します。この部門では、その内側 - 横方向の寸法が100μmで、10μの18ビン（またはそれ以上）に分かれています先に説明したように31を画像に重畳グリッドを使用してその半径方向の寸法におけるmの高さ（ 図1）。 <ol><li>このような最初のビン、グリッドの位置を合わせ心室の境界にその長軸に平行で、心室表面である。その後、各ビンにラベルのEdU、BrdUおよび/または濃縮核（アポトーシス核に相当）の数。数の大部分が含まれているビン内、または核は2個のビンの中に等しい面積を占有している下のビン、中2のビンの境界に位置する核。 </li></ol></li><li>統計分析。 </li></ol>胚あたり少なくとも2皮質のスライスを分析します。別の仔から少なくとも3胚で実験を繰り返します。結果は、平均値の平均値±標準誤差（SEM）として与えられるべきである。統計分析は、双方向ANOVAとボンフェローニ多重比較ポストホック試験を用いてグラフパッドプリズムを用いて行われる。

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著者らは、利害関係のない。

のEdU 1.5時間の取り込みに基づいてここで説明する実験計画の前に照射が許可された直後に照射し、BrdUの取り込みNSPCsの後、SおよびG2 / Mチェックポイントを活性化することができるが、1 回目の時間の間に、G1 / Sチェックポイントではありませんデモ胎児のマウス脳における遺伝毒性ストレス。私たちは、私たちは、特にS期のエントリの割合を鑑賞することができ、EdUのはちょうど1?...

胚の脳の発達の際に、大脳皮質の投射ニューロンは、神経幹細胞および前駆細胞（NSPC）の様々なタイプで構成多列上皮はそのライン側脳室、脳室帯で生成されます。 NSPC、神経幹細胞として機能する放射状グリア細胞（RGC）の中で、interkinetic核移行（INM）を受ける：彼らは脳室帯（VZ）の基底限界で心室とS相の表面で有糸分裂を行う1、2,3。彼らはどちらか分けることができる対称的に2 RGCを発生させるか、非対称に1 RGCと1ニューロンまたは中間前駆細胞（IPC）4、5を生成する。 IPCは1、最後の対称分裂後、彼らは2未熟投射ニューロン6-8を生成する脳室下帯（SVZ）と呼ばれるその上に増殖層に移行します。 RGCに反して、IPCは（9に概説されている）INMを受けない。新しく生成されたニューロンMIGR皮質板（CP）10、8で最終的な宛先に到達するために中間ゾーン（IZ）を介してラジアル繊維に沿って放射状に食べました。すべてのこれらのイベントの完璧なタイミングが正しい皮質発達に必須である。例えば、神経前駆細胞集団の分化増殖のスイッチは、G1期の持続11,12によって制御される。 G1期の延長は、細胞の分化と相関するこうして。 このような（13に概説されている）、電離放射線、ひどく損なう脳の発達などの遺伝毒性ストレス、。私たちは、他はNSPCは、放射線誘導性アポトーシス14、15,16に非常に傾向があることが示されている。電離放射線は、増殖している細胞に最も深刻な被害であるDNA二本鎖切断を誘導する。サイクリング細胞におけるDNA損傷応答（DDR）の一つの必須成分は、G1 / SまたはG2 / M遷移における又はSの間の細胞周期チェックポイントの活性化である相（イントラSチェックポイント）17〜21。それらは、DNA損傷修復すぎたり損傷を受けた細胞の排除のための時間を提供するために、細胞周期の進行をブロックする。従って、細胞死、ならびに遅延、細胞周期の進行は、放射線曝露22-24電離に応答して、脳の発達を変更することができる。これは、照射したマウス胚性脳におけるNSPCにおける細胞周期チェックポイントの活性化を評価するための方法を開発することは興味深かった。 細胞周期の進行は、定期的に、チミジン類似体、5 - ブロモ-2&#39;-デオキシウリジン（BrdU）の取り込みを使用して追跡する。 DNAが複製されるときにBrdUは、細胞周期のS期の間に組み込まれる。のBrdUに対する抗体の使用は、その後のBrdUのパルスの間にS期にある細胞の検出を可能にする。 新規のチミジン類似体、5 - エチニル-2&#39;-デオキシウリジン（EdUの）は、蛍光アジドによって検出される。のEdUとBを検出するために様々な方法<html> RDUは、細胞周期の進行の研究に有用である両方のチミジン類似体の同時検出を可能にする、25に交差反応しない。通常の細胞は最初のEdUをパルスした後、両方の取り込み間の時間は、時間25、26のカップルを持続するのBrdUでパルスする。唯一のBrdUの取り込み27のメディアの結果と等モルまたはハーフモルBrdUによるEdUの25の同時添加しながら、EdUを排除してDNAへのBrdUの取り込みを優先におけるEdUの結果を含む培養培地中のBrdUの追加。これは、通常、前に第二の標識の添加培地から最初のラベルを削除するために必要とされる洗浄ステップを排除することによって、二重標識プロトコルを簡素化します。これはまた、細胞集団のS期の出入りの正確なタイミングを決定するために、洗浄工程が不可能なインビボ研究のために特に重要である。 t &quot;の&gt;最近、EdUのおよびBrdU 23、24,22を用いて、胚性マウス脳におけるデュアルパルス標識する方法は、 子宮内照射における後の細胞周期の進行およびNSPCのINMを分析するために開発された。また、それが実証されている間に、そのS期は、マウスの細胞をまずユークロマチン領域とし、ペリセントリックヘテロクロマチン28,29,30を複製します。興味深いことに、interphasic核内にクラスタ化された異なる染色体の挟動原体ヘテロクロマチンもchromocentersとして知られており、鮮やかな焦点とDAPI染色によって容易に検出可能なヘテロクロマチンフォーカスを形成する。そのため、ユークロマチンとchromocentersの差分のEdUおよびBrdU染色をより正確にNSPCのS期の進行​​を分析するために、私たちを助けた。 このメソッドは、このチェックポイントがゲノムの安定性にとって重要であると考えられるので、非常に驚くべきことであるNSCP 22〜24でのG1 / Sチェックポイントの見かけの欠如、のデモを許可された。いくつかのEXPのEdUおよびBrdUパルスのさまざまな組み合わせに基づいて、erimental設計は、腹側と背側終脳における細胞周期の進行を分析するために使用されてきた。ここでは、E14.5マウス胚の子宮内照射に続く最初の4時間以内でNSPCの急性DNA損傷の研究を可能にするプロトコルの例を与える。

<table border="0" cellpadding="0" cellspacing="0" width="727">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:228px;">EdU</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">A10044</td>
			<td style="width:163px;">1 mg/ml</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:228px;">BrdU</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">B5002</td>
			<td style="width:163px;">5 mg/ml</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:228px;">IBL 637</td>
			<td style="width:212px;">CIS BIO International</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:228px;">Tissu Tek VIP</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Microtome</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;">RM 2125 RT</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Triton X-100</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td align="right" style="width:125px;">93443</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:228px;">Click-iT EdU Alexa 488 imaging kit</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">C10083</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:228px;">Anti-BrdU</td>
			<td style="width:212px;">GE Healthcare</td>
			<td style="width:125px;">RPN202</td>
			<td style="width:163px;">1/300</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:228px;">Goat anti mouse-Alex594</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">A11001</td>
			<td style="width:163px;">1/400</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Fluoromount</td>
			<td style="width:212px;">SouthernBiotech</td>
			<td style="width:125px;">0100-01</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:228px;">Polysine slide</td>
			<td style="width:212px;">Thermo scientific</td>
			<td style="width:125px;">J2800AMNZ</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:228px;">Paraformaldehyde</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:125px;">P6148</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:228px;">PBS</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">20012-068</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:228px;">DAPI</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:125px;">D9542</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:228px;">Microscope BX51</td>
			<td style="width:212px;">Olympus</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:228px;">Confocal microscope SPE</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Prism software</td>
			<td style="width:212px;">Graphpad</td>
			<td style="width:125px;"></td>
			<td style="width:163px;">Version 5.0c</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:228px;">Photoshop software</td>
			<td style="width:212px;">Adobe</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
	</tbody>
</table>

assessing cell cycle progression of neural stem and progenitor cells in the mouse developing brain after genotoxic stress

大脳皮質の神経細胞は、胚の脳の側脳室の内側を覆う多列上皮を形成し、神経幹細胞および前駆細胞の様々なタイプ（NSPC）から脳の発達の間に生成される。このような電離放射線などの遺伝毒性ストレスは、NSPCの高感度に関係する脳の発達に大きく悪影響を及ぼす。関与する細胞および分子機構の解明は、損傷した組織における細胞周期を通してNSPCの進行を決定する正確な方法を必要とする細胞のこれらの特定の種類のDNA損傷応答の特性に依存する。ここで妊娠マウス胚性脳の冠状断面におけるこれら2チミジン類似体の更なる検出においてのEdUおよびBrdUの連続腹腔内注射に基づく方法が示されている。のEdUおよびBrdUの両方S期の間複製細胞のDNAに組み込まれ、2つの異なる技術（又はアジドによって検出されるそれらの同時検出を容易に特異的な抗体、それぞれ）。のEdUとBrdU染色は、その後、背側終脳の標準領域における心室余白からの距離の関数で各NSPC核について決定される。したがって、この二重標識技術は、DNA損傷に応答して細胞周期の停止をもたらす細胞周期チェックポイントを活性化したものから、細胞周期を通して進行する細胞を区別することができる。 実験の例はEdUのは直後に、照射およびBrdUの前に注入し、照射後4時間以内に行わ分析する、提示されている。このプロトコルは、それらが照射された時に細胞周期の位相の関数でNSPCの急性DNA損傷応答の正確な分析を提供する。この方法は、生体組織中の細胞周期の進行に対する特定の治療の影響を決定するために容易に転移他の多くのシステムにある。

図2に記載された実験では、EdUのちょうど照射後、照射前およびBrdU 1.5時間後に投与した。細胞の4つのタイプは、その後のEdUまたはBrdUの、両方かNoneの取り込みに応じて、1または4時間の照射後に調製皮 ​​質スライス識別された（ 図2Aおよび図2B）。重要なことに、EdUのも、BrdUの取り込みのいずれも、放射線誘導アポトーシス（データは示していない）...

Watch this Scientific Journal Video about Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress at JoVE.com

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress | Neuroscience | Video

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

妊娠マウスにおけるチミジンのEdUおよびBrdUの二類似体の投与は、胚性マウス脳の神経細胞および前駆細胞における細胞周期進行の分析を可能にする。この方法は、脳の発達の間に、電離放射線を含む遺伝毒性ストレスの効果を決定するために有用である。

神経幹細胞の細胞周期の進行を評価し、マウスでの前駆細胞は、遺伝毒性ストレス後の脳の開発

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

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15.2K ビュー.  CEA DSV iRCM SCSR. 妊娠マウスにおけるチミジンのEdUおよびBrdUの二類似体の投与は、胚性マウス脳の神経細胞および前駆細胞における細胞周期進行の分析を可能にする。この方法は、脳の発達の間に、電離放射線を含む遺伝毒性ストレスの効果を決定するために有用である。 

ビデオ: Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

開発ゼブラフィッシュ前脳におけるクローン関連の神経前駆細胞のタイムラプスライブイメージング

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

神経科学

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). ...

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 ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

発生中のマウス胚性皮質における有糸分裂のライブイメージング

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

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 ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

セル成体マウス脳室下帯から神経幹細胞および前駆細胞の選別とその細胞周期ダイナミクスのライブイメージング

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

脳オルガノイドを派生開発人間の脳の培養幹細胞を使用してジカ ウイルス感染症をモデル化

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

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.