我々は、画像処理と解析アドバイスデニスFlipoに感謝し、デビッド·W·シルバーサイズが実験室でGata4p [5キロバイト]-GFPマウスラインが生成されました。ピロンの実験室での研究はCIHR、NSERC、FRQSとFRQNTによって資金を供給される。

倫理声明マウスを含む実験は医学研究に使用される動物の世話や操作のための動物実験ガイドラインのカナダの評議会後に実施された。動物の操作を含むプロトコルはモントリオールのケベック大学の施設内倫理委員会によって承認された（化委員会Institutionnelデ·保護DES Animaux、参照番号0512-R3-650から0513）。 1。コラーゲンゲルの調製組織培養フードの下で、無菌方式で動作します。 <ol><li>標準的な抗生物質を含む完全な5X DMEM（イーグル改変必須培地）を準備します。のDMEM粉末3.37グラムと水20ml中のNaHCO 3の0.925グラムを溶解する。 0.22μmのフィルターを通すことにより滅菌する。無菌100Xペニシリン/ストレプトマイシン、および滅菌熱不活性化ウシ胎児血清25ミリリットルの2.5ミリリットルを追加します。 4℃で保存する</li><li>氷の上に、800μLをミックス完全5XのDMEM600μlのと1N NaOHを17μlのコラーゲンI溶液（0.02 N酢酸、濾過滅菌中3.77 mg / ml）を。 3ミリリットルの最終容量に滅菌水で希釈します。ミックス内の関連する成長因子が含まれる。私たちは通常、腸NCCの移行を刺激するために10 ngの/ mlでGDNFを使用しています。 </li><li> 24ウェルプレート内の単一行の各ウェルに沈着約480μL。泡を避けてください。残りの行は、細胞遊走に対する他の成長因子の効果を試験するために使用することができる。 </li><li>コラーゲンは、37℃で滅菌インキュベーター中で少なくとも1時間重合しましょ</li></ol> 2。動物16の解剖<ol><li>交配を設定し、翌朝膣栓を確認してください。デイE0.5が膣栓が検出されたその日の正午であること、女性を隔離し、E12.5まで12日待ちます。 </li><li>イソフルランで妊娠マウスを麻酔して、CO 2吸入によって安楽死させる。 </li><li> 70％エタノールでマウスをスプレーします。腹部Sを持ち上げる親族や解剖ハサミで腹腔を開きます。 </li><li>氷冷PBS（リン酸緩衝生理食塩水）を充填したガラスペトリ皿に子宮を除去する。各胚の移植部位を分離するために、個々のdeciduum腫脹との間で横方向に子宮をカット。 </li><li>氷冷PBSで満たされた別のガラスシャーレに別々に移植部位に取り組んでいます。解剖顕微鏡下で、子宮の筋肉層を除去するために細かい鉗子を使用しています。 </li><li>胚を明らかにするために、内臓卵黄嚢と羊膜を開きます。彼らが開発し、腸と密接に絡み合っているように、胎盤/内臓卵黄嚢に胚を結ぶ血管を切断する際には注意してください。 </li><li>首に胚の頭を切断。 </li><li>ただ濃い赤色の肝臓の上に、胚の腹腔内に、閉じた鉗子を挿入し、腹腔内の横方向の開口部を作るために（圧力をかけて停止する）自体の鉗子を開いてみましょう。に向けて開かれた鉗子を引き下げ完全に腹部を開くには、胚の後端。 </li><li>肝臓の背後にある結合組織をつかみ、（コロンは肛門に添付されている）、腸を壊さないように注意しながら、腹部から内臓を引き出します。 </li><li>胚の他の部分から勇気を解放するためにコロンをカット。カットは結腸に沿った任意の位置で行うことができる。後でテール部分を予約。 </li><li>盲腸から結合組織、腸の後、残りの部分を引き出す。そうしながら、腸を巻かないように注意してください。 </li><li>いくつかが存在する場合、肝臓と胃（小腸の吻側端にある）だけでなく、中腎と生殖隆起を切り取る。 </li><li>小腸を分離します。繰り返しますが、腸をニップしないように注意してください。今後は、腸組織のrosrrocaudal向きは頭側の端に存在鋭い曲率を使用して追跡することができます。実施する前に、できるだけ短い時間、室温でPBSで小腸を残す寝具（ステップ3.3を参照）。 </li><li>最終的に、正確に胚発生の段階を決定するために、各胚のための尾体節の数を記録します。 </li></ol> 3。胚性腸の切片<ol><li>胚の解剖を続行する前に100mlのPBS（リン酸緩衝生理食塩水）中に1.5グラムアガロースを融解し、50℃で維持</li><li>埋め込 ​​み型内に注ぐ溶融アガロース（ 例えば、aは約1/4の管壁を切除するために縦に切断された2mlのマイクロ遠心チューブを閉じた状態）。アガロースは、約42〜45℃に冷ましてみましょう、それは触るとわずかに暖かいはずです。 </li><li>ただ、アガロースが固化する前に、胚性腸に埋め込む（これは鉗子の先端を用いて評価することができ、36〜38℃付近が発生します）。折り畳んだ吻側年末までにピンセットで腸を持ち、金型の長さに沿ったアガロースを通​​して非常にゆっくりと引き出します。これはストレートアガロースセットしながら、腸を維持するのに役立ちます。 Tをリリースとすぐに移動しているレジストをし始めると問題。腸の吻側 - 尾側の向きを追跡する。 </li><li>アガロースが完全に設定されていることを確認するために、冷蔵庫、2〜3分で金型を置く。 </li><li> （開かれたエッペンドルフチュー​​ブの外にスライドさせて）金型からアガロースを取り出します。刃で、腸に垂直カットを作り、両端に余分なアガロースを取り出します。 </li><li> /アガロース振動刃ミクロトームの金属ステージを下にブロックし、腸の吻側端を接着します。腸/アガロースブ​​ロックの側面に余分なアガロースをトリミング。 </li><li>振動ミクロトーム室に金属ステージをマウントします。必要に応じて腸をできるだけ垂直になるように（したがって、ブレードに垂直）ステージの角度を調整する。氷冷PBSで標本をカバーしています。バッファ面の下に数ミリまでミクロトームの刃を持参してください。 </li><li>その電子を確保し、尾最も小腸の200ミクロンビブラトーム横カットを作るACHアガローススライスは完全な腸セクションが含まれています。 </li></ol> 4。腸管外植片の文化<ol><li>静かに、各ウェルの中央に向かって1スライスを置く、ピンセットでコラーゲンゲルに新たに切断した腸/アガローススライス、フラットを堆積させる。 </li><li>外植片からNCCの移動を可能にするために、湿った5％CO 2雰囲気中、37℃で3日間インキュベートする。 </li><li>ピンセットで非常に優しくコラーゲンゲルから腸/アガローススライスを取る。以下のゲルを損傷しないように注意してください。 </li><li>細胞移動パターンが妨げられないことを確実にするために、その後のインキュベーションおよび洗浄ステップの間に直接コラーゲンゲルに触れないようにしてください。室温でよく、1時間あたりの（PBS中）を4％PFA（パラホルムアルデヒド）500μlで固定してください。 </li><li>ウェルあたり500μlのDAPI（4 &#39;、6 - ジアミジノ-2 - フェニルインドール）溶液（PBS中の5μg/ ml）を用いて固定液を交換し、室温で10分間インキュベートする。 </li><li>各ウェル3Xを洗う5分間のPBS500μlで。 </li><li>各ウェル内のコラーゲンゲル内に埋め込まれた蛍光細胞（GFPおよびDAPIチャネル）写真。 </li></ol> 5。画像解析私たちは、移植片培養した後に生成された画像を処理し、定量化するためにImageJの17の大規模な使用をした。 <ol><li>蛍光細胞写真と同じ倍率でマイクロスライドを画像化することから始めます。 （ストレートライン·ツールを使用して）ピクセル単位で1ミクロンの長さを測定します。 </li><li> （/設定スケールを分析、ピクセル/ミクロンの入力数）スケールを設定します。 </li><li>各GFP蛍光写真は、8ビットグレースケール（Image/Type/8-bit）に画像形式を変更します。 </li><li>信号（画像/調整/明るさ/コントラスト）の強さを調整します。 </li><li>必要に応じて（;ローリングボールの半径を調整プロセス/背景を引く）、バックグラウンドノイズを差し引く。 </li><li>細胞および細胞塊を（画像/ /しきい値を調整）、ハイライト表示し、しきい値を設定するN塊（プロセス/バイナリ/流域）を分割するために流域を適用します。 </li><li>関心領域（ROI）を指定し、細胞数の統計情報を生成する粒子を分析（粒子を分析/分析、設定されたサイズの最小値は、残りのピクセルを除外する）。 </li><li>フェレ径（/セット測定の分析）を含むように、測定オプションを設定します。 </li><li>グループのROIと、フェレ径を決定するために、全体として細胞の広がり（ANALYZE /ツール/ ROIマネージャー/詳細/ OR、そしてROIマネージャー/追加し、ROIマネージャー/測定）の指標を測定します。 </li></ol>

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著者らは、開示することは何もありません。

我々のex vivoでの外植片培養技術を正確GDNFの存在下で腸溶性NCCの移行の可能性を定量化するために使用することができる方法を示す。このような正確な定量が大幅に以前に11,12,15を説明するように、厚さ200μmのビブラトーム腸のセクションを使用しての代わりに、おおよその大きさの大部分によって容易になる。確かに、これは、私たちは再現性の高い環境でのセルの合理的な?...

神経堤細胞（NCC）は、胚発生の間に多くのデリバティブを形成している脊椎動物に特有の過渡的な細胞型である。この細胞集団は、非神経外胚葉1に隣接して、神経板の境界で生じる。神経胚形成の間に、神経板の曲げ成形神経管の背側縁に沿って、NCCが配置されます。 NCCは、その後分離し、離れて神経管からの移行、上皮間葉転移を受ける。 NCCは、彼らが全体の腸管神経系（ENS）、腸の壁に埋め込まれた神経の神経節の相互接続されたネットワークを形成し、消化管を含む様々な胚の構造を、植民地化。最近2,3日のように、多くの遺伝子がこの複雑な構造の開発に携わってきました。 ENSのほとんどは（ つまり、将来の後脳/脊髄の境界付近）迷走神経管からのNCC発信の小さなプールに由来している4。これらの神経前駆細胞は、マウスでは胎生（E）9.0を中心に前腸に到達してから、全体胚性腸にコロニーを形成するのに約e15.0までの消化管間葉内で尾側に移行します。結腸の神経前駆細胞の小サブセットはまた、最大盲腸4と反対方向に後方腸に侵入仙骨NCCによって提供される。迷走神経と仙骨の両方NCCは、ENSの完全な形成を確実にするために、複数の移行、増殖、生存および分化を促進する手がかりが必要になります。 GDNF（グリア細胞由来神経栄養因子）、エンドセリン-3、ニューロトロフィン3、BMPは（骨形成タンパク質）、ネトリン：この点では、動物モデル - - 特に遺伝子改変マウスは、いくつかの不可欠な細胞外リガンドの同定に尽力されてきただけでなく、ソニックとインディアンヘッジホッグ（ShhおよびIhhは）5月10日 。これらのうち、（トランスフェクション中に再配列）チロシンキナーゼ膜貫通受容体を介したシグナル伝達RET GDNFは番目として認識される胚性腸へと内魅力とNCCの指導のための電子の最も重要な経路。 GDNFは消化管間葉から分泌され、RET 11,12を表現腸NCCに直接化学誘引である時空間的に制御rosrrocaudal勾配を形成している。 他の機能の中でも、ENSは、腸壁内の平滑筋との相互作用を介して、消化管内での移動を規制する。ヒルシュスプルング病における腸結果の末端領域の神経の神経節が存在しない場合：影響を受けたセグメントの強壮収縮が詰まり、消化された材料の上流に蓄積し、腸や腹部の大規模な膨満につながる。ヒルシュスプルング病は、およそ1 5000での出生数を発生します。腸溶NCCの吻側 - 尾側移行パターンは、ヒルシュスプルング病の病因に主要な寄与因子であると考えられている。コロン、NCCの移行のソースから最も遠いとBの最後の部分植民地化されるowelは、ENS形成における欠陥の影響を最も受けている。腸溶NCC遊走におけるその重要な役割に応じて、GDNF / RETシグナル伝達の破壊は、ヒルシュスプルング病13の主既知の遺伝的原因である。 [5キロバイト]-GFP 14 Gata4p命名- -回遊NCCは、緑色蛍光タンパク質（GFP）で標識された良好なNCCとENSの発達を研究するために、我々は、トランスジェニックマウス系統を生成した。我々は次に、現在では、GDNFのような種々の成長因子の存在下で腸溶性NCC移行電位の正確な定量化を可能にする他のグループ11,12,15によって発表された研究から適合ex vivoでの細胞遊走アッセイを、完成。

<table border="1">
	<tbody>
		<tr>
			<td>DMEM powder</td>
			<td>Wisent</td>
			<td>219-010-XK</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Steriflip vacuum filtration system (0.22 micron)</td>
			<td>EMD Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin solution, 100x</td>
			<td>Wisent</td>
			<td>450-201-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Wisent</td>
			<td>095-150</td>
			<td>High quality grade</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>BD biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Bioshop</td>
			<td>SHY700</td>
			<td>Diluted from 10 N stock then sterile-filtered</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Cedarlane</td>
			<td>CLCYT305</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Plate</td>
			<td>BD biosciences</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>Fisher Scientific</td>
			<td>089515</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dish</td>
			<td>VWR</td>
			<td>89000-306</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>Cell culture grade</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica</td>
			<td>M125</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioshop</td>
			<td>AGA001</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Feather</td>
			<td>21</td>
			<td></td>
		</tr>
		<tr>
			<td>All Purpose Instant Krazy Glue Pen</td>
			<td>Krazy Glue</td>
			<td>KG824</td>
			<td></td>
		</tr>
		<tr>
			<td>HM 650V Vibrating-Blade Microtome</td>
			<td>Thermo Scientific</td>
			<td>920110</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative cell migration assay for murine enteric neural progenitors

神経堤細胞（NCC）は、背側神経管に由来し、開発脊椎動物の胚全体で広範囲に移動し、一過性および多能細胞集団である。末梢グリアおよびニューロンを提供することに加えて、NCCはメラノサイトだけでなく、頭蓋顔面骨格の大部分を生成します。 NCC遊走および分化は、神経管に沿った軸方向の原点と地域の異なる細胞外の合図への曝露との組み合わせによって制御される。細胞外のリガンドのような貢献は、腸神経系（ENS）、ローカルに（特に）を制御する神経核の複雑な相互接続されたネットワーク腸の筋肉の動きや腸の運動を形成する際に特に顕著である。尾ファッションに吻側に - - 将来の腸の全長にENSのほとんどが植民地化するために長い旅に着手するNCCの小さな初期プールに由来している。知られているいくつかのシグナル伝達経路のうち、腸NCCのコロニー形成に影響を与え、GDNF / RETシグナル伝達は、最も重要なものとして認識されている。確かに、消化管間葉によるRETリガンドGDNFの時空間的に制御された分泌は、胚性腸へと内誘引およびRET発現腸NCCの指導のために主に責任があります。ここでは、GDNFを含む様々な成長因子の存在下で腸溶性NCC移行電位の正確な定量化を可能にする蛍光標識されたNCCを有するトランスジェニックマウス系統を利用して、ex vivoでの細胞遊走アッセイを記載している。

以下の結果は、ここで記載された技術（ 図1）で得られるものの代表である。増殖因子の使用（ すなわち、GDNF）は、腸の外植片からコラーゲンゲル（ 図2）にGFPを発現する腸溶性NCCの遊走を刺激する。一部の細胞は、成長因子の非存在下で外植片から出てくるが、これらは主にGFP-標識されず、パッシブエントリーを表す。この組織はまだ頻繁に蛍光細胞によ?...

Watch this Scientific Journal Video about A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors at JoVE.com

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

我々は、様々な成長因子の存在下での腸溶神経堤細胞の遊走電位の正確な定量を可能にex vivoで細胞遊走アッセイを提示する。

マウス腸の神経前駆細胞の定量細胞遊走アッセイ

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

a-quantitative-cell-migration-assay-for-murine-enteric-neural

Research

JoVE Journal

Neuroscience

9.2K ビュー.  UQAM. 我々は、様々な成長因子の存在下での腸溶神経堤細胞の遊走電位の正確な定量を可能にex vivoで細胞遊走アッセイを提示する。 

ビデオ: A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

慢性二光子用薄型頭蓋骨のウィンドウテクニック in vivoで</em神経炎症のモデルのマウスミクログリアの>イメージング

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

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 ...

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). ...

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

修正Zigmondチャンバーアッセイを使用してトランク神経冠細胞移動の解析

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

ホールマウントマッスル分析による再現性のあるマウスの坐骨神経クラッシュと再生のその後の評価

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

クロス種の移植による神経堤の移行および分化の解析

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

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 ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

外部から印加された直流電界における神経前駆細胞の移行動態の解析のための走電性アッセイ

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

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.