イメージングおよび硬化環境代謝疾患、ヘルムホルツ協会のイニシアチブとネットワーク基金を通じ、エルスKroener-フレゼニウス財団からの助成金、およびドイツ学術振興（からの助成金 - この作品は、ヘルムホルツアライアンスICEMEDによって（部分的に）資金を供給したSFB 655：組織への細胞）。

仕事はHelsinskiの宣言とARVO動物ステートメントに準拠しています。ドレスデン大学の動物施設の指示に従って、動物は組織収集のために使用された、関連するすべてのメソッドに従った。動物は処理され、収容された実験動物の使用とケアのためのドイツ連邦ガイドラインに従って、および研究はLandesdirektionドレスデンで承認されたされた。適切な安楽死の方法論について、あなたの機関の獣医ポリシー（動物実験委員会または他のボード）にご相談ください。 特定の株式や試薬の使用濃度は、このプロトコルに付随する試薬、表に記載されています。 1。培養皿の準備<ol><li>一晩細胞培養インキュベーター中で75μg/ mlのポリ-L-オルニチン（6ウェルプレートのつまり 2ミリリットル/ウェル）の十分なボリュームのあるコートディッシュ2日前までにスケジュールされた解剖日まで。 </li><li&gt;吸引し、ポリ-L-オルニチン、次の日および5分間のPBS 3mlで各ウェル2Xを洗う。 </li><li>最後の洗浄を削除し、プレートにフィブロネクチン（希釈1:250、PBS中の4μg/ mlの）を追加し、一晩培養器に料理を置く。 </li><li>解剖の日にフィブロネクチンを吸引し、皿をPBSで2回洗浄します。まだ解離した組織は、培養のための準備ができるまで、インキュベーターに洗って、最終的なPBSを含むプレートを返します。その時に、前に組織をメッキに、PBSを除去します。 </li><li>ストア·プレートは、最大3週間、インキュベーターをポリ-L-ornithineinで被覆。フィブロネクチンでコーティングしたプレートは1週間まで保存することができる。緊急の場合は、フィブロネクチンコーティングは、わずか2時間で行うことができる。フィブロネクチンは、変性を受けやすい。ソリューションを撹拌や食器を乾燥させてください。 </li></ol> 2。解剖/メッキ<ol><li>このプロトコルは、3成体ラット（生後3〜6ヶ月）の使用に適用されます。 </li><li>子どもラットを安楽死させる前に、L氷上で脳切片のブロック。 </li><li>氷上で追加されたbFGFを持つN2培地の5ミリリットルが含まれている15ミリリットルファルコンチューブを配置します。 </li><li>あなたの機関の獣医ポリシーに従ってラットを安楽死させる。 </li><li>頭の正中切開下にすることで頭蓋骨から脳を取り出します。慎重に抽出されるべき脳を可能に骨を離れて剥離する鉗子を使用してください。 </li><li>氷冷PBSを含む10 10cmペトリ皿に脳を置きます。これは残留髪と血液を除去します。 （ 図1A） </li><li>チルド切片ブロックに脳を置きます。 （ 図1B） </li><li> 図1Cに写真のようにブロックに1新しいクリーンかみそりの刃を挿入します。 </li><li> 図1（d）に示すように、第1、第2のクリーンかみそりの刃3ミリメートルの尾側を挿入します。 </li><li>彼らと関心のある部分を取って、ブロックからブレードを慎重に持ち上げます。 </li><li>秒フロートPBSに浸漬することにより、ブレードオフTION。 （ 図1E） </li><li>関心領域から収穫組織は＃5ピンセットを使用して（この例では、前交連、（前方）ACA、 図1Fに使用される）およびbFGFを含む1ミリリットルN2培地を含む15ミリリットルコニカルチューブに集める。 </li><li>無菌条件下で、プロトコルの残りを継続する、層流細胞培養フードにチューブを移動させる。 </li><li>機械的にP1000ピペッターに装着1ミリリットルピペットチップを用いて組織を解離する。ピペットは、上下に数回（約20倍、毎秒1分注サイクルの割合で）円錐管の底部にピペットチップの開口部を押しながら（1G 1-3図 ）。溶液が均一になったときにチュレーションを停止します。 </li><li>ソリューションは、任意の大きな非解離組織が底に沈殿することができるように2分間静置する。 （ 図1G 4） </li><li> homogenを集めるeous上清とN2培地の8.5ミリリットルを加えた20 ng / mlのbFGFを、500 ng / mLのDll4の、500 ng / mLのAng2は、200 nMJAK阻害剤を含むコニカルチューブに入れます。 </li><li> /ウェルの希釈した細胞溶液3mlを用いて、6ウェルプレートの3ウェルにそれらをプレート。 </li><li> 37°C、5％CO 2、5％O 2の加湿細胞インキュベーター中にプレートを置き。 </li></ol> 3。毎日のケア<ol><li>めっき後24時間後に阻害剤のbFGF、Ang2は、Dll4の、およびJAKを含むN2培地で培養皿上でメディアを交換してください。 </li><li> bFGFは、Dll4の、Ang2は、次の日に、JAK阻害剤のボーラスを追加します。 </li><li>メディアの変化と、10〜14日の計因子添加のこの交互のプロセスを継続する。 </li></ol> 4。分化誘導<ol><li>培地を含む阻害剤のbFGF、Dll4の、Ang2は、およびJAKを撤回し、任意の追加要素が含まれていないN2培地に交換してください。 </li><li>隔日の媒体を変更します。 </li><li> 10日後に細胞を固定し、免疫細胞化学を行う。 </li></ol> 5。免疫細胞化学的検出<ol><li>培地を吸引し、20分間、4％パラホルムアルデヒド2mlの細胞培養プレートを固定する。 </li><li> 5分ごとに、3mlのPBSで洗浄2X。 </li><li> PBSを吸引除去し、次に20分間、5％正常ロバ血清（NDS）および0.1％トリトンX-100を含有するPBS 2 mlを添加することにより細胞を透過し、遮断する。 </li><li> 5％、NDSを含むPBS中の一次抗体ミックスを調製する。のTuJ1（クラスIIIβ-チューブリン）、GFAP、およびCNPアーゼ抗体は分化マーカーの三重染色のために一緒に使用することができ、一方、抗ネスチン抗体は、SCのを識別するために使用することができる。 </li><li>ブロッキング溶液を吸引し、各ウェルに一次抗体混合物の1.5ミリリットルを追加。 90分間室温でインキュベートする。 </li><li>一次抗体を除去し、3mlのPBSで2回洗浄し、5分ごとに5％のNDSを含むPBS 3mlで1X。 </li><li>た準備5％NDSを含有するPBSで二次抗体を電子。 </li><li>最終洗浄を吸引し、各ウェルに二次抗体溶液1.5mlを加える。室温で40分間、暗所でインキュベートする。 </li><li>二次抗体溶液を除去し、新たに調製されたDAPI染色（PBS中500 ng / mL）を2 mlを加える。 3分間インキュベートする。 </li><li> DAPI溶液を吸引し、5分間ずつ3mlのPBSで3回洗浄します。細胞は、今や蛍光顕微鏡で可視化することができる。 </li></ol>

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我々は、開示することは何もありません。

成功した分離、拡大、及び成体脳由来の神経幹細胞の分化にとって重要なステップの多くは、標準的な組織培養技術に共通に共有される。心に留めて目的は、成人の脳から単離されたNSCは、できるだけそれらのインビボ特性（ 図2J）の多くを維持するべきであるということです。多くの場合、必要​​な注意と適切な速度のバランスが打たれる必要がある。頭蓋骨から脳?...

神経生物学のセントラルドグマは、前世紀以上ラモン·Y.カハールによって行われた脳の細胞構築の基本的な観測が定めた、神経新生はCNS 1で見つかったニューラルネットワークの複雑さを考えると、思春期の後にそうだったと判示した。 1960年代のアルトマンの作品、およびそれ以降のカプランは、3 H-チミジンは、実際にはニューロンは、成体脳の異なる領域で生成されていたことを示している成熟した神経細胞で見つけることができることを実証しているにもかかわらず、定説は2、3を保持し続けた。証拠は小鳥の脳4に存在するニューロンの数の季節変化を記述するノッテボームの研究をマウントし続けた。これはグールドらはラットの連合学習課題の性能だけでなく、Kornackとラキッチdemonstraの観察と海馬が増大するニューロンの生成に作品を発表1999年までではなかったティンは剛性が低く、より多くのプラスチック、脳の概念は、5認識された大人のマカク、6で神経新生を続けた。 これらの新たに生成された神経細胞のための細胞源の探索はニッチ7と呼ばれる脳の領域に存在する幹細胞（SCS）の離散的な人口の発見につながる。脳室下帯と海馬のサブ粒状ゾーンは二つの主要な神経性の地域8,9であると考えられている。これらの場所から単離された細胞は、古典的な胚または胎児に由来するSCの特徴的な、自己再生および分化能を表示します。神経幹細胞（NSC）の場合、それらは、ニューロン、アストロサイト、およびオリゴデンドロサイトに分化させることができる。さらに、これらのSCは、例えば、中間径フィラメントタンパク質ネスチン10胎児NSCマーカーについて陽性に染色。 SCSはこれらのTに限定されていない可能性があり、より最近の研究のハイライトエリア、WOとは、実際にはしっかりと血管系11に関連する細胞の大部分は静止人口として脳全体にローカライズされています。 のSCが傷害に応答して動員される観察は、神経変性疾患および脳卒中12,13からの回復を助けるために回生目的のために、これらの細胞を利用することができるという可能性を示唆している。これは、間葉系幹細胞（MSC）は、骨芽細胞、軟骨細胞、および脂肪細胞14になる可能性がある血管周囲細胞として見出される結合組織の治癒において果たす役割とは違っていません。しかし、NSCは、ルーチン吸引および密度勾配遠心法によって骨髄からのMSCと同様にして採取することができないし、続いて自己細胞に基づく治療に利用する。結果として、そのようなEMBに由来する胎児ニューロンのNSC又は前駆体の使用などの細胞の他の源ryonic幹細胞が広範囲に成功15の様々な程度で、動物の病気や怪我モデルで検討されている。体細胞の供給源を利用して人工多能性幹細胞技術は、限られた在庫および胚細胞および胎児組織16の使用に関する倫理的な問題を克服する、広範囲の用途のために治療的に有用な細胞ベースの治療を製造するための別の潜在的な手段を提供する。しかし、これらの知見の臨床的変換は、SC-ベースの治療アプローチ17,18と同様に、規制当局の認可を曲がりくねった経路で種々の神経学的状態を治療する闘争に示されているように、困難な作業であることが証明されている。別のアプローチとして、特定の薬理学的治療の導入は、NSC数を調節し、パーキンソン病および脳卒中19のモデルの回復を促進することができる。戦略は、効果的にこれらの細胞を操作する方法を理解することがありますどのようなアクセス可能なin vitroの系を必要とします。 NSCの培養物はまた、ニューロスフェアとしても知られている凝集体の培養物として、または単層8,20のいずれかとして行うことができる。両方の技術は、広く、規定された培養条件の確立を可能にする、多能性前駆体の膨張を提供するマイトジェン源として上皮成長因子（EGF）または塩基性線維芽細胞成長因子（bFGFの） すなわち使用が使用されてきた。ニューロスフェア培養物が単離された細胞型のクローン増殖能力を研究するためのより適しているかもしれないが、システム21は、膨張時に細胞の混合集団を産生することが示されている。また、ニューロスフェアの密閉構造は、細胞の薬理学的操作が非現実的になり、これらの要因が持つ影響力の解釈は、ニューロスフェア自体の中で確立された微小環境に混乱する可能性があります。単層培養物は、他の一方で、あることができるSCの増殖および分化を調節し、特にこの細胞集団を標的とする新規化合物を発見する機会を開くシグナル伝達機構を探索するための強力なツールを提供する、小分子ライブラリーのハイスループットスクリーニングに用いられる。 その結果、再現可能脳内の関心の異なる領域からの成体NSCの培養物を生成する能力は、中枢神経系（CNS）の発達の研究からの新規な再生医療アプローチを探索するに至るまで、研究用途の広いスペクトルにおいて使用することができる。ここで紹介するプロトコルは、成人の齧歯類脳から単離されたCNS SCの分化能力を分析し、評価する方法を示しています。

<table border="0" cellpadding="0" cellspacing="0" width="596">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">6-well tissue culture dishes</td>
			<td style="width:95px;">BD Biosciences</td>
			<td style="width:119px;">353934</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="190" rowspan="1" style="height:190px;width:216px;">Poly-L-ornithine</td>
			<td rowspan="1" style="width:95px;">Sigma-Aldrich</td>
			<td rowspan="1" style="width:119px;">P-3655</td>
			<td style="width:167px;">
			5 mg/ml stock solution prepared in double distilled water (Stable for several months at -20 &#176;C).
			Working concentration 0.5 mg/ml in water (Stable for 1 month at 4 &#176;C).
			</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fibronectin</td>
			<td style="width:95px;">R&#38;D Systems</td>
			<td style="width:119px;">1030-Fn</td>
			<td style="width:167px;">Do not agitate stock solution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Filtration Apparatus</td>
			<td style="width:95px;">Corning Life Sciences</td>
			<td style="width:119px;">430769</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">DMEM/F-12</td>
			<td style="width:95px;">Mediatech</td>
			<td style="width:119px;">10-090-CV</td>
			<td style="width:167px;">See note below for complete N2 media preparation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Apo-transferrin</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">T-2036</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Insulin</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">I-0516</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:216px;">Putrescine</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">P-5780</td>
			<td style="width:167px;">1 M stock solution in ddH2O (Stable at 20 &#176;C for 6 months)</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:216px;">Sodium Selenite</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">S-5261</td>
			<td style="width:167px;">500 &#181;M stock solution in ddH2O (Stable at -20 &#176;C for 6 months)</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Progesterone</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">P-8783</td>
			<td style="width:167px;">100 &#181;M stock solution in ethanol (Stable at -20&#176;C for 6 months)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:95px;">Invitrogen</td>
			<td style="width:119px;">15140-122</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:95px;">Mediatech</td>
			<td style="width:119px;">21-040-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Basic Fibroblast Growth Factor (bFGF)</td>
			<td style="width:95px;">R&#38;D Systems</td>
			<td style="width:119px;">233-FB</td>
			<td style="width:167px;">Working concentration 20 ng/ml&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Delta4 (Dll4)</td>
			<td style="width:95px;">R&#38;D Systems</td>
			<td style="width:119px;">1389-D4</td>
			<td style="width:167px;">Working concentration&#160; 500 ng/ml</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Angiopoetin 2 (Ang2)</td>
			<td style="width:95px;">R&#38;D Systems</td>
			<td style="width:119px;">623-AN</td>
			<td style="width:167px;">Working concentration&#160; 500 ng/ml</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">JAK Inhibitor</td>
			<td style="width:95px;">Calbiochem</td>
			<td style="width:119px;">420099</td>
			<td style="width:167px;">Working concentration 200 nM&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">A-2058</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">15- and 50-ml Conical tubes</td>
			<td style="width:95px;">Corning Life Sciences</td>
			<td style="width:119px;">430053, 430829</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="243">
			<td colspan="4" height="243" style="height:243px;width:596px;">Other necessary items include: General dissection instruments, including razor blade and forceps, Adult rat (3-6 months old; Sprague-Dawley or Long Evans), CO2 intoxication chamber, Laminar flow hood for cell culture and incubator Incubator (humidified, 37 &#176;C, 5% CO2, 5% O2). Note: For complete N2 media preparation, to one bottle of DMEM/F-12 (500 ml) add 0.05 g of apotransferin, 0.0125 g of insulin (freshly predissolved in 1 ml of 10 mM NaOH), 50 &#956;l of putrescine, 30 &#956;l of sodium selenite, 100 &#956;l of progesterone stocks, and 5 ml of penicillin/streptomycin solution.&#160; Adjust pH to 7.2, if needed. Filter-sterilize and store at 4 &#176;C for up to 3 weeks and protect from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td>[header]</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td>Immunofluorescence Reagents Table</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Paraformaldehyde</td>
			<td style="width:95px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15719</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Normal Donkey Serum (NDS)</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">D-9663</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Triton X-100</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">T-8787</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">4,6-Diamidino-2-phenylindole (DAPI)</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:119px;">D-8417</td>
			<td style="width:167px;">5 mg/ml stock solution in methanol</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;"></td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Primary Antibody Table</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">Nestin</td>
			<td rowspan="1" style="width:95px;">Chemicon</td>
			<td rowspan="1" style="width:119px;">MAB353</td>
			<td style="width:167px;">
			Dilution Factor: 1:400
			Species: Mouse IgG1
			</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">Hes3</td>
			<td rowspan="1" style="width:95px;">Santa Cruz</td>
			<td rowspan="1" style="width:119px;">sc-25393</td>
			<td style="width:167px;">
			Dilution Factor: 1:100
			Species: Rabbit IgG
			</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">Sox2</td>
			<td rowspan="1" style="width:95px;">R&#38;D Systems</td>
			<td rowspan="1" style="width:119px;">MAB2018&#160;</td>
			<td style="width:167px;">
			Dilution Factor: 1:100
			Species: Mouse IgG2a
			</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">CNPase</td>
			<td rowspan="1" style="width:95px;">Chemicon</td>
			<td rowspan="1" style="width:119px;">MAB326</td>
			<td style="width:167px;">
			Dilution Factor: 1:200
			Species: Mouse IgG1
			</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">&#946;-tubulin III (TUJ1)</td>
			<td rowspan="1" style="width:95px;">R&#38;D Systems</td>
			<td rowspan="1" style="width:119px;">MAB1195</td>
			<td style="width:167px;">
			Dilution Factor: 1:500
			Species: Mouse IgG2a
			</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="1" style="height:43px;width:216px;">Glial Fibrillary Acidic Protein (GFAP)</td>
			<td rowspan="1" style="width:95px;">Dako North America</td>
			<td rowspan="1" style="width:119px;">Z0334</td>
			<td style="width:167px;">
			Dilution Factor 1:500
			Species: Rabbit
			</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;"></td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Secondary Antibody Table</td>
		</tr>
		<tr height="21">
			<td height="64" rowspan="1" style="height:64px;width:216px;">Alexa&#160;&#160; 568</td>
			<td rowspan="1" style="width:95px;">Invitrogen</td>
			<td rowspan="1" style="width:119px;">A-21124</td>
			<td style="width:167px;">
			Dilution Factor: 1:200
			Species: Goat anti Mouse IgG1
			</td>
		</tr>
		<tr height="21">
			<td height="64" rowspan="1" style="height:64px;width:216px;">Alexa 488</td>
			<td rowspan="1" style="width:95px;">Invitrogen</td>
			<td rowspan="1" style="width:119px;">A-21131</td>
			<td style="width:167px;">
			Dilution Factor: 1:200
			Species: Goat anti Mouse IgG2a
			</td>
		</tr>
		<tr height="21">
			<td height="64" rowspan="1" style="height:64px;width:216px;">Cy5</td>
			<td rowspan="1" style="width:95px;">Jackson ImmunoResearch</td>
			<td rowspan="1" style="width:119px;">59883</td>
			<td style="width:167px;">
			Dilution Factor: 1:200
			Species: Goat anti Rabbit
			</td>
		</tr>
	</tbody>
</table>

growing neural stem cells from conventional and nonconventional regions of the adult rodent brain

最近の研究は、中枢神経系（CNS）腫瘍形成は、再生および幹細胞の集団（SCの）成体の脳内に存在することを含むことを示している。しかし、これらの正常静止細胞は、神経回路網の適切な機能を確保するだけでなく、神経変性過程の傷害や緩和からの回復におけるそれらの役割に採用するメカニズムはほとんど理解されています。これらの細胞は、血管系および免疫系の両方から調節シグナルを含む維持環境を提供する「ニッチ​​」と呼ばれる地域に存在します。未知の要因を除外する規定された培養条件の下での分離、メンテナンス、およびCNS SCの分化は、従って、それらのin vivoでの行動への洞察を提供し、薬理学的または遺伝的手段による治療にそれらにアクセスできるようになります。ここでは、成人の脳の異なった領域から中枢神経系のSCの文化を生成する方法に関する詳細な情報を提供し、そのDを評価するために近づいたニューロン、アストロサイト、およびin vitroでオリゴデンドロサイトへのifferentiationポテンシャル。この技術は、個々のSCおよびその子孫を研究するために可視化することができる単層培養として均一な細胞集団が得られる。さらに、損傷した成体の神経系において回生応答を予測するために以前に使用されて、異なる動物モデル系および臨床試料の両端に印加することができる。

神経幹細胞を採取するための関心領域を識別することは重要な第一歩であり、それは細胞のコンフルエントなプレートを取得するのにかかる時間の量を定義する。例えば、SVZは、古典的な神経原性領域であり、従って、神経幹細胞のより高い相対的割合を有している。しかしながら、ここに記載した技術は、多くの場合、成人期にロバスト神経原性ポテンシャルを有すると認識されていない?...

Watch this Scientific Journal Video about Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain at JoVE.com

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

成人脳から採取した神経幹細胞は神経系の発達の基礎研究から再生医療の潜在的な臨床応用を探索範囲の用途に利用が増加している。これは実験的な成果を鳴らす重要なこれらの細胞を成長させるために使用される分離と培養条件に厳密な制御を行う。

大人の齧歯類の脳の従来の従来にない地域から神経幹細胞を増殖させる

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

growing-neural-stem-cells-from-conventional-nonconventional-regions

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JoVE Journal

Neuroscience

12.1K ビュー.  University of Dresden. 成人脳から採取した神経幹細胞は神経系の発達の基礎研究から再生医療の潜在的な臨床応用を探索範囲の用途に利用が増加している。これは実験的な成果を鳴らす重要なこれらの細胞を成長させるために使用される分離と培養条件に厳密な制御を行う。 

ビデオ: Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

成人海馬ホッキョクジリス神経幹細胞の増殖と分化

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

神経科学

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

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

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

ニューロスフェアのアッセイを使用して成体マウス神経幹細胞の単離と拡大

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

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 ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

マウスを一回、二つの文化：個々のマウスの二つの神経原性のゾーンから成体神経幹細胞の単離および培養

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

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. ...

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

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

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

マウス梁下ゾーンから成体神経幹細胞の単離および培養

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

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

さまざまな地域と発展途上と大人の脳のラミナを明らかにするシナプス亜鉛化学の使用

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

下流解析のための凍結げっ歯類の脳領域の収集

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

ラットおよびマウス脳への神経幹細胞の動脈内デリバリー:脳虚血への応用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...