この研究は、ヴァンダービルト - イングラムがんセンターサポート助成金（P30 CA068485）、小児脳腫瘍財団と米国立衛生研究所（NS042205）からの補助金によって支えられている。

1。担癌小脳の微小解剖、腫瘍組織及びめっきの解離<ol><li>腫瘍組織の検索<ol><li>病気のマウスのベアリングの髄芽腫は、しばしばruntedされ、水頭症と後部麻痺と覆さときに姿勢を取り戻すために、障害を含む典型的な神経学的症状は、表示されます。腫瘍組織を取得するには、二酸化炭素の吸入によりマウスを安楽死させる。頸椎脱臼、後頭骨に圧力を発生し、腫瘍組織の整合性が損なわれる可能性が手順を実行しないことが重要です。 </li><li>断頭は、頭蓋骨の良い可視化のために髪と筋肉組織を可能な限り除去し、はさみを使って死の直後に実行されます。 95％エタノールを浸したキムワイプで頭蓋骨の表面を清掃してください。 </li><li>担癌小脳を含む脳全体が露出している時点で、頭蓋骨の正中線に沿って開口部をカットし、細かいピンセットを使って頭蓋骨の組織を除去するために微細なハサミを使用してください。 </li><li>健康な成人の小脳は、明確に定義された半球と虫部を表示しながら、担癌マウスの小脳では、多​​くの場合、滑らかな表面と顕著な血管を持つアモルファス拡大です。無菌技術を用いて、氷冷PBSでピンセットと場所を使用して小脳の腫瘍を取り出すのMg2せずに+とCa 2 +。 </li></ol> 注：すべての楽器が使用前に95％エタノールで滅菌されています。 </li><li>腫瘍組織の解離<ol><li> PBSから腫瘍組織の約4倍の体積が50％のAccutase（PBSで希釈）に腫瘍組織を転送、37℃でインキュベーションに続いて、室温で3分間細かいハサミで組織をミンチ℃で4分間、そのあと組織は、さらに3分間1 mLのPipetmanで反復的なペッティングを受ける。このメソッドは、単一細胞と小さな細胞の集合体の混合物を生成する必要があります。 </li><li> PBSとペレットに1000gで5分間遠心し、細胞と細胞懸濁液3倍に希釈する。ゼラチン60 mmのPrimariaの組織培養皿に新鮮な神経幹細胞培養培地とプレートで細胞ペレットを再懸濁します。我々は、第1のめっきで強化された添付ファイルのPrimaria皿を使用して、その後の継代は、通常の組織培養皿上に播種されることがあります。 </li></ol></li></ol>注：少なくとも30分間、0.1％ゼラチンでコーティング培養皿。グルタミン、ペン球菌、N2、B27、ヒトEGF（25 ng / ml）および塩基性FGF（25ng / mL）でNeurobasal媒質で構成された新鮮な神経幹細胞培養の培地を準備します。 2。連続継代による濃縮、メンテナンスおよび髄芽腫の腫瘍細胞の増殖我々は通常、初期メッキの1週間（図1）の後に多くのコロニーを得る。これらのコロニーは解離し、腫瘍細胞集団の濃縮のための新たな糊皿の上に色を保ち続けることができます。新しい培地への最初の変更は、その後、単に機械的に細胞懸濁液（不要Accutase）を得た4分間反復ペッティングに続いてコロニーを分離するために、1 mLのPipetmanを使用してください。大きな細胞塊が解離されていることを確認するために顕微鏡下で確認してください。その後、さらに培養するための新たな糊化ディッシュに細胞懸濁液を分注し、追加の通路のための同じ手順を通過。アタッチされていない腫瘍細胞、血液細胞や他の細胞型は、接続されている腫瘍細胞が急速に拡大して残して、再めっきシリアルによって除去されています。文化の最初のシードに続く二日目の培地、及びその後一日おきに変更します。 3。免疫蛍光染色と培養細胞の検討 1。ガラス製カバースリップ上で腫瘍細胞を成長させる 1日目、6ウェルプレートの各々の場所のガラスカバースリップ上で、その後37℃で30分間、0.1％ゼラチンを加える℃にmL当たりの種子は約2 - 4X 10 5腫瘍細胞。細胞は一晩固定してくださいと神経幹細胞培地は3日目に変更。染色は4日目に行われます。 2免疫蛍光染色氷冷したPBSで2回培養液および洗浄細胞を取り除く。 15分間たての4％PFAで細胞を固定してください。簡潔にPBSで細胞を2回洗浄し、5分間、0.3％トリトン（PBS中）でpermeablize。簡単に言うと40分間10％ヤギ血清を含むPBSとブロックで細胞を2回洗浄する。 90分間一次抗体で細胞をインキュベートし、各5分間、PBSで細胞を3回洗浄する。 PBS、5分ごとで3回洗浄し、60分間二次抗体で細胞をインキュベートします。水性マウンティング培地5mLを持つスライドにカバースリップをマウントし、共焦点顕微鏡を行います。 4。腫瘍細胞の分化 nを削除するeural幹細胞の培地と簡単に二回PBSで細胞を洗浄する。 1日目の分化培地を追加し、4日目に培地を変更し、7日目に免疫蛍光染色により分化のレベルを決定します。分化培地は、DMEM、ペン球菌および10％ウシ胎児血清で構成されています。 5。代表的な結果形態の結果 Accutaseの治療と穏やかな反復ペッティングした後、腫瘍組織は、小さな細胞凝集と単一細胞に主に解離してください。多くのかなりのコロニーは、図1で示すように、初期のめっき後の5日には早くも観察することができます。高度に増殖性の腫瘍細胞はしばしば高い核/細胞質比の二極です。これらの細胞は通常、ゼラチン表面に付着した小さな細胞の集合体から放射状に見られている。小さいと丸い血液細胞は、通常、その後のメディアの変化とシリアルめっきによって削除されます。いくつかの継代後、1つは他の細胞タイプ（図2）で均一に分布し、増殖Ki67 +腫瘍細胞と少し汚れを観察することができます。 神経幹細胞マーカー、クローン解析し、複数の系統への分化の発現増殖細胞は、腫瘍幹細胞の解離小脳腫瘍の組織のショー特性に由来するかどうかを判断するために、我々は、幹細胞マーカーの発現、クローン解析と分化能によりさらに特性評価を実施。我々は、これらの孤立した腫瘍細胞が高度にSox2、及びネスチン（図3）などの神経幹細胞マーカーを発現することを発見。我々はSmoM2 - YFP腫瘍から組織を取得したとして、すべての腫瘍細胞はYFP。一次繊毛は、ソニックヘッジホッグ経路に依存する髄芽腫12の開発のために重要であることを報告する最近の研究と一致し、SmoM2 - YFPの発現は明らかにSox2のの繊毛+腫瘍細胞（図3）に局在していた。培養液のmL当たり300個の細胞のクローン密度で播種したとき、我々は単一の腫瘍細胞からかなりのコロニー（図4）にクローン増殖を観察した。さらに、これらの細胞はプロ分化の条件（図5）で培養したとき、様々な神経細胞およびグリア細胞型に分化することができた。 <img src="/files/ftp_upload/2086/2086fig1.jpg" alt="図1" /> 図1。解離髄芽腫の組織の第1のめっき後の高度の増殖性コロニーの形態 。解離腫瘍組織の初期播種後5日以内にコロニーの形態一般的に、かなり大きな、そして代表的な1つは、明視野でここに示されています。高密度コアのセルの集合体からバイポーラ、細長い細胞が放射状に。徐々にシリアルめっきによって枯渇している小さな、丸い赤の血液細胞は、ある腫瘍細胞に添付。 <img src="/files/ftp_upload/2086/2086fig2.jpg" alt="図2" /> 図2。 3継代を超えて確立された髄芽腫細胞の形態 。この明視野像には、いくつかの継代後に確立された髄芽腫細胞の典型的な外観を示しています。細胞は主に高い核/細胞質比の二極のまま。我々は強いKi67の発現によって示されるようにサイクリングしているそのほとんどが腫瘍細胞のアセチル化チューブリンの染色を示す。 <img src="/files/ftp_upload/2086/2086fig3.jpg" alt="図3" /> 図3。確立された髄芽腫細胞は神経幹細胞マーカーを発現する 。 SmoM2 - YFPは、構成的に活性なSmoと、それ故にShhの経路活性を発現する細胞をマーク。確立された髄芽腫細胞株のすべてのセルは、YFPを発現しており、それらのほとんどがそのようなネスチンとSox2などの様々な神経幹細胞マーカー、高レベルの同時発現。興味深いことに、我々は共焦点顕微鏡の間に最小限のレーザパワーとYFPのシグナルの検出を実行したときに、我々はSox2の+細胞の繊毛の集中YFPのシグナルを検出しました。この観察は、ソニックヘッジホッグ経路に依存する髄芽腫の開発に繊毛の重要な役割を説明する最近の報告書12と整合的である。 <img src="/files/ftp_upload/2086/2086fig4.jpg" alt="図4" /> 図4。確立された腫瘍細胞がクローン性増殖を受ける 。解離した後、単一の細胞懸濁液に、腫瘍細胞は、培養液のmL当たり300個の細胞のクローン密度で24ウェルプレート上に播いた。各ウェルでは、クローン拡大コロニーを観察した。明視野画像のこのシリーズは、文化の10日間で典型的な変化を示しています。 <img src="/files/ftp_upload/2086/2086fig5.jpg" alt="図5" /> 図5。確立された髄芽腫細胞の分化の解析 。腫瘍細胞は、EGF / bFGFがDMEM/10％FBS、YFP +腫瘍細胞は有意にその形態を変え、様々な細胞型、株式会社に分化する無血清培地を含むからスイッチングされたときTuj1瀘定+またはNeuN +の神経細胞、GFAP +アストログリア細胞またはCNPase +オリゴデンドロサイト。

<ol>
	<li>Rossi, A., Caracciolo, V., Russo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma:+From+Molecular+Pathology+to+Therapy.">Medulloblastoma: From Molecular Pathology to Therapy.</a> Clin Cancer Res. 14 (4), 971-971 (2008).</li><li>Wechsler-Reya, R. J., Scott, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Neuronal+Precursor+Proliferation+in+the+Cerebellum+by+Sonic+Hedgehog.">Control of Neuronal Precursor Proliferation in the Cerebellum by Sonic Hedgehog.</a> P. Neuron. 22 (1), 103-103 (1999).</li><li>Dahmane, N., Ruiz, A., Altaba, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+regulates+the+growth+and+patterning+of+the+cerebellum.">Sonic hedgehog regulates the growth and patterning of the cerebellum.</a> Development. 126 (14), 3089-3089 (1999).</li><li>Wallace, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purkinje-cell-derived+Sonic+hedgehog+regulates+granule+neuron+precursor+cell+proliferation+in+the+developing+mouse+cerebellum.">Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum.</a> Curr Biol. 9 (8), 445-445 (1999).</li><li>Fan, X., Eberhart, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medullablastoma+Stem+Cells.">Medullablastoma Stem Cells.</a> J Clin Oncol. 26 (17), 2821-2821 (2008).</li><li>Yoon, J. W., Gilbertson, R., Iannaccone, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+Role+for+Hedgehog+Pathway+Activation+in+Desmoplastic+Medulloblastoma+by+Identifying+GLI1+Target+Genes.+International+journal+of+cancer.">Defining a Role for Hedgehog Pathway Activation in Desmoplastic Medulloblastoma by Identifying GLI1 Target Genes. International journal of cancer.</a> 124 (1), 109-109 (2009).</li><li>O'Dorisio, M. S., Khanna, G., Bushnell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+anatomic+and+molecularly+targeted+imaging+in+the+diagnosis+and+surveillance+of+embryonal+tumors+of+the+nervous+and+endocrine+systems+in+children.+Review.">Combining anatomic and molecularly targeted imaging in the diagnosis and surveillance of embryonal tumors of the nervous and endocrine systems in children. Review.</a> Cancer metastasis reviews. 27 (4), 665-665 (2008).</li><li>Corcoran, R. B., Bachar Raveh, T., Barakat, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+Growth+Factor+2+Is+Required+for+Progression+to+Advanced+Medulloblastoma+in+patched1+Heterozygous+Mice.">Insulin-like Growth Factor 2 Is Required for Progression to Advanced Medulloblastoma in patched1 Heterozygous Mice.</a> Cancer research. 68 (21), 8788-8788 (2008).</li><li>Gilbertson, R. J., Ellison, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Origins+of+Medulloblastoma+Subtypes.">The Origins of Medulloblastoma Subtypes.</a> Annual review of pathology. 3, 341-341 (2008).</li><li>Ward, R. J., Lee, L., Graham, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+CD15++Cancer+Stem+Cells+in+Patched-1+Deficient+Mouse+Medulloblastoma.">Multipotent CD15+ Cancer Stem Cells in Patched-1 Deficient Mouse Medulloblastoma.</a> Cancer research. 69 (11), 4682-4682 (2009).</li><li>Mao, J., Ligon, K. L., Rakhlin, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Somatic+Mouse+Model+to+Survey+Tumorigenic+Potential+Applied+to+the+Hedgehog+Pathway.">A Novel Somatic Mouse Model to Survey Tumorigenic Potential Applied to the Hedgehog Pathway.</a> Cancer research. 66, 10171-10171 (2006).</li><li>Han, Y. G., Kim, H. J., Dlugosz, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+and+opposing+roles+of+primary+cilia+in+medulloblastoma+development.">Dual and opposing roles of primary cilia in medulloblastoma development.</a> Nature medicine. 15 (9), 1062-1062 (2009).</li></ol>

利害の衝突は宣言されません。

我々は、構成的に活性なヘッジホッグシグナル伝達と変異マウスから取得した原発腫瘍組織からの髄芽腫の幹細胞を、分離豊かにし、維持するための効率的な方法を説明します。私たちは正常に健康な腫瘍の幹細胞株を確立する上でひとつの重要なステップは、原発腫瘍の組織の解離の際に使用Accutase治療法であることがわかった。我々の経験では、ときに小脳からの最初の解離腫瘍組織、37...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Neurobasal medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103049</td>
<td>&nbsp;</td>
<tr>
<td>hEGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0311</td>
<td>25ng/ml</td>
</tr>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0023</td>
<td>25ng/ml</td>
</tr>
<tr>
<td>N2</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17502048</td>
<td>1X</td>
</tr>
<tr>
<td>B27(-RA)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12587010</td>
<td>1X</td>
</tr>
<tr>
<td>Accutase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A1110501</td>
<td>50%</td>
</tr>
<tr>
<td>Gelatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1393</td>
<td> 0.1%</td>
</tr>
<tr>
<td>Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030081</td>
<td>2mM</td>
</tr>
<tr>
<td>Primaria dishes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>087724C</td>
<td>&nbsp;</td>
<tr>
<td>Sox2 antibody</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MAB4343</td>
<td>Mouse, 1:1000</td>
</tr>
<tr>
<td>Nestin antibody</td>
<td>&nbsp;</td>
<td>DSHB</td>
<td>rat401</td>
<td>Mouse, 1:200</td>
</tr>
<tr>
<td>YFP antibody</td>
<td>&nbsp;</td>
<td>Molecular Probes</td>
<td>A11122</td>
<td>Rabbit, 1:2000</td>
</tr>
<tr>
<td>GFAP antibody</td>
<td>&nbsp;</td>
<td>Neuromics</td>
<td>CH22102</td>
<td>Chicken, 1:1000</td>
</tr>
<tr>
<td>Tuj1 antibody</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T5076</td>
<td>Mouse, 1:2000</td>
</tr>
<tr>
<td>NeuN antibody</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MAB377</td>
<td>Mouse, 1:2000</td>
</tr>
<tr>
<td>CNPase antibody</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MAB326</td>
<td>Mouse, 1:1000</td>
</tr>		</tbody>
		</table>
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isolation, enrichment, and maintenance of medulloblastoma stem cells

脳腫瘍は、腫瘍形成の根本原因である幹細胞の小集団を持つことが示唆されている。ニューロスフェアのアッセイは、一般的に、正常および腫瘍組織由来のものを含む神経幹細胞の性質を研究するために採用されています。しかし、分化と細胞死のかなりの量は、培地への球の集合内のすべてのセルのアクセシビリティなど次善の状態に可能性が高いため、培養ニューロスフェアでは一般的です。 髄芽腫、最も一般的な小児中枢神経系の腫瘍は、その急速な進行や全体の脳脊髄陰気な臨床転帰との軸に沿って広がる傾向によって特徴づけられる。髄芽腫は、それぞれ小脳の神経上皮腫瘍、20％を占めると小児期の頭蓋内および後頭蓋窩腫瘍の40％、1です。それは今もソニックヘッジホッグシグナル伝達が小脳発達2月4日中に小脳顆粒神経前駆細胞（CGNPs）の増殖を刺激することが確立されている。マウスモデルを用いて多くの研究では、Shhの経路が構成的に活性化されている、髄芽腫5月9日とソニックヘッジホッグシグナル伝達をリンクされています。 最近の報告では、Patched1 のLacZ / +マウス由来の髄芽腫細胞のサブセットは、腫瘍の10を開始し、伝播することが可能ながん幹細胞が、あることが示されている。ここでは、平滑化の変異（11、ここに至ってはSmoM2と呼ばれる）のために構成的に活性化さShhの経路で、Shhのために重要なGPCRの髄芽腫のいくつかのモデルマウス由来の腫瘍幹細胞を、分離豊かにし、維持するために効率的な方法を記述する経路の活性化。すべての孤立した髄芽腫の組織では、我々は数々の高度に増殖したコロニーを確立することができた。確実にネスチンとSox2などのいくつかの神経幹細胞マーカーを発現するこれらの細胞は、連続継代を経て（20以上）とクローン原性であったこともできます。これらの培養腫瘍幹細胞は、幹細胞の増殖に有利な条件下で培養すると細胞質比が高いの核としばしばbipoar比較的小規模でしたが、彼らは劇的に、その形態を変化させた複数の細胞プロセスを拡張し、平らにし、セルへの切り替え時に細胞周期から撤退培地は10％ウシ胎児血清を補充した。さらに重要なことは、これらの腫瘍幹細胞は、このように、そのマルチ効力を強調、Tuj1 +またはNeuN +の神経細胞、GFAP +アストロサイトとCNPase +オリゴデンドロサイトに分化した。さらに、これらの細胞は、同所宿主マウスに移植すると、二次髄芽腫を伝播することができた。

Watch this Scientific Journal Video about Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells at JoVE.com

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

このプロトコルは、分離、濃縮、および異所性ソニックヘッジホッグ経路の活性をもつ変異マウス由来の髄芽腫の腫瘍幹細胞の維持を説明します。

髄芽腫の幹細胞の単離、濃縮、およびメンテナンス

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been ...

isolation-enrichment-and-maintenance-of-medulloblastoma-stem-cells

Research

JoVE Journal

Neuroscience

16.2K ビュー.  Vanderbilt University. このプロトコルは、分離、濃縮、および異所性ソニックヘッジホッグ経路の活性をもつ変異マウス由来の髄芽腫の腫瘍幹細胞の維持を説明します。

ビデオ: Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron precursors (CGNPs) as the cell-of-origin for medulloblastoma1-4. However, the diverse clinical presentations of medulloblastoma subtypes in human patients (nodular, desmoplastic, classical and large cell/anaplastic), and the fact that medulloblastoma is found in a subset of human patients with no ectopic expression of CGNP marker5, suggest that the cellular and molecular origins of medulloblastoma are more complex and far from being completely deciphered. Therefore, it is essential to determine whether there is an alternative medulloblastoma tumor cell-of-origin based on which cell-type specific therapeutic modality can be developed. To this end, intracranial orthotopic allografting of genetically marked tumor cell types followed by subsequent analyses of secondary tumor development in recipients will allow determination of the cellular origin of tumor-initiating cells. Here we describe the experimental protocol for intracranial orthotopic allografting of medulloblastoma cells derived from primary tumor tissue, and this procedure can also be used for transplanting cells from established cell lines.

This protocol describes the isolation and dissociation of mouse medulloblastoma tissue, and subsequent allografting of the tumor cells into immunocompromised recipient mice in order to initiate secondary medulloblastoma.

免疫不全マウスにおける髄芽腫細胞の頭蓋内同所Allografting

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron ...

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 Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

人間の毛包から神経堤幹細胞の単離および培養

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

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 ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

胚性齧歯類の脳から単離、プライマリ脳ドーパミンニューロンの文化と長期メンテナンス

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

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 ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

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

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

マウス萌芽期の神経幹細胞の分離と培養

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

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.

成虫ゼブラフィッシュにおける鈍力損傷の影響を研究するスケーラブルモデル

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

成人ゼブラフィッシュを用いた学習・記憶研究における認知評価のための連想学習ツールとしてのシャトルボックスアッセイ

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...