Development of this protocol was funded by private donations directed to the Parkinson's Institute Brain Donation Program.

脳の寄付登録が寄付する意思の登録者によってドキュメントが含まれています。法律で認められているように、組織の検索のために剖検許可は近親者により提供されます。収集した剖検標本を用いた調査研究は、医療保険の携行性と責任に関する法律（HIPAA）規制の遵守を確保するために、治験審査委員会（IRB）によって検討されています。 注：軟膜試料は脳解剖中脳切開または神経病理学者によって収集され、25〜30 mLの髄膜増殖培地を含有する50mLのコニカルチューブに格納されています。試料は、試料調製まで4℃で保存します。細胞の生存率は、時間死後と減少する処理が可能な限り早く行われるべきです。 1.セットアップ軟膜解剖を開始する前に注：1.1ステップ - 1.3は、生物学的安全キャビネットの内部で実行されるべきです。 <ol><li>ダルベッコ改変イーグル培地、ウシ胎仔血清を100mLの375ミリリットルを組み合わせることにより、髄膜増殖培地を準備し、5 mLの10 mMの非必須アミノ酸、5 mLの200 mMのL-アラニル-L-グルタミン、5mlの100mMのピルビン酸ナトリウム、 5mLの万U / mLのペニシリン/ストレプトマイシンおよび5 mLの250 / mlのアムホテリシンBを500mLのフィルターユニットです。フィルタと混ぜます。 4週間までのメディアを使用してください。 </li><li>解剖を開始する前に、バイオセーフティキャビネット内のすべての材料を配置する：4倍ペトリ皿、2×6ウェルプレート、2倍メス、15-20滅菌15 mmのカバースリップ、ピペットを吸引するリン酸緩衝生理食塩水（PBS）、血清学的ピペット、。 </li><li> 6ウェルプレートの各ウェルに滅菌オートクレーブ0.1％ゼラチン溶液1mLを加えます。 30〜60分間脇にプレートを設定します。 2×6ウェルプレートを準備します。 </li></ol>軟膜サンプルの調製注：2.1ステップ - 2.3は、生物学的安全キャビネットの内部で実行されるべきです。 <ol><li>にPBSの約10 mLを加え滅菌10 cmの組織培養皿と皿に髄膜サンプルを配置します。鉗子を使用して、静かに血液を除去するためにPBSで軟膜を洗います。 </li><li>新鮮な10mLのPBSを含む新しい10-cmの組織培養皿に髄膜を移し、洗浄を続けます。できるだけ多くの血液を除去するために、必要に応じて繰り返します。 </li><li> 2メスを使用して、X 6センチメートル約6センチメートルに大部分を軟膜の組織を切断します。 </li><li>培養皿と水平層流フード内で解剖顕微鏡への転送の上に置いて蓋。 </li></ol>軟膜組織の3解剖注：3.1手順 - 3.3解剖顕微鏡を使用して、水平層流フード内で実行されます。 <ol><li>少なくとも20-25 3ミリメートル×3ミリメートルの作品を作成するのに十分な大き軟膜ピース（ステップ2.3）上の領域から血管を削除します。 1メスでの場所で軟膜を持ち、他に、穏やかな浅い掻きカ月を使用ションは、血管を分離します。 </li><li>準備された領域から組織の小さな正方形をカット。最初の大きい作品を作り、その後、小さなセクションにそれらをスライスして、段階的プロセスでカットします。 </li><li>揺動運動と組織を切断する代わりに、組織と別のメスを保持するために1メスを使用してください。組織の一貫性のために、互いに対してメスをスライドすることは困難です。これは離れて組織を引き裂くとギザギザのエッジになります。 </li><li>約20〜25 3ミリメートル×3ミリメートル片が存在するまで、半分に作品を切断続けます。 </li></ol>組織培養プレート上に解剖髄膜個の4譲渡注：ステップ4は、生物学的安全キャビネットの内部で行われるべきです。 <ol><li> （ステップ1.3から）6ウェルプレートから吸引除去ゼラチン。各ウェルに、髄膜増殖培地の1 mLを加え。滅菌ピンセットを用いて、各ウェルに3-4生検片を配置します。 </li></ol> Mの上カバースリップの5配置eninges小品注：5.1手順 - 5.2解剖顕微鏡を使用して、水平層流フード内で実行されます。 <ol><li>ウェル当たり1-2滅菌15-mmの円形カバースリップで作品をカバーしています。 </li><li>片がプレートの底に触れていることを確認するために鉗子でしっかりと押し下げます。これは、井戸の底に組織片の付着を可能にします。 </li><li> 37℃、5％CO 2インキュベーターに6ウェルプレートに置き。 </li></ol> 6.細胞培養のメンテナンス注：6.1ステップ - 6.3は、生物学的安全キャビネットの内部で実行されるべきです。カバースリップは、髄膜片を移動させ、取り除くべきではないよう注意して培養皿を扱います。 <ol><li>文化の中で2日後、各ウェルに新鮮な髄膜増殖培地の追加の1 mLを加え。 </li><li> 7日目に、慎重にメディアを吸引し、各ウェルに新鮮な培地の2 mLを加え。 </li><li> 9日後、増殖培地毎に変化し続けます文化まで、他の日は、コンフルエントになります。 </li></ol> 7.継代注：すべてのステップは、生物学的安全キャビネットの内部で実行されるべきです。髄膜線維芽細胞は、髄膜組織片の外に移行し、より大きな培養皿に髄膜線維芽細胞を、培養容器の縁に向かって成長拡大を開始した後。 <ol><li>継代を開始する前に、コート15-30分間1％ゼラチンで培養皿。 </li><li> 6ウェル皿から培地を吸引し、2mLのPBSで細胞を洗浄。 </li><li>各ウェルにトリプシン/ EDTAを1 mLを加え、5〜10分間37℃でインキュベートします。カバースリップまたはこのステップの髄膜組織を除去する必要はありません。 </li><li>細胞は丸みを帯びていると、デタッチ培地2mLのを追加して起動すると。 </li><li>ピペット細胞最大とまでは完全にすべての塊を破壊し、より大きな培養皿に細胞溶液を移します。 </li><li>三6ウェル皿から細胞を結合するために4：1〜3：1の比率を使用し1 T75培養皿に。 </li><li>髄膜線維芽後細胞を、細胞をトリプシン処理、T75培養フラスコ中でコンフルエントに、バイアル当たり1×10 6細胞/ mlで凍結保存培地中で凍結保存します。コンフルエント髄膜線維芽細胞の一つのT75培養フラスコは、約5〜7万個の細胞が含まれています。 </li></ol>免疫染色により髄膜線維芽細胞の8キャラクタリゼーション注：手順を開始する前に、4％パラホルムアルデヒド溶液を調製し、PBS中の5％正常ヤギ血清（ブロッキング緩衝液）、0.3％トリトンX-100 PBS（透過化緩衝液）、一次および二次抗体溶液中で、およびを1μg/ mLでのヘキスト33342は、10mg / mLのストックからPBSで希釈しました。 <ol><li> 0.1％ゼラチンでコーティングした8ウェルチャンバースライドに15,000〜30,000髄膜線維芽細胞をシード。 </li><li>細胞が付着している場合、24時間後、室温で10分間、ウェルあたり100μLの4％パラホルムアルデヒドで細胞を固定します。 1×PBSでウェルを3回洗浄。 </li><li>室温で5分間、0.3％トリトンX-100150μlで細胞を透過性。 1×PBSでウェルを3回洗浄。 </li><li>室温で1時間ブロッキング溶液200μL（PBS + 5％正常ヤギ血清）を加えます。 </li><li>ブロッキング溶液を吸引除去し、ブロッキング溶液中に希釈し、所望の一次抗体の150μLを追加します。二重染色のために、次のように氷上で別々の遠心分離管中の一次抗体の作業希釈液を準備します。 <ol><li>抗フィブロネクチン+ 1の300倍希釈液：ブロッキング溶液中の抗ネスチンの200倍希釈を準備します。抗SERPINH1 + 1の250倍希釈液：ブロッキング溶液中の抗TUJ1 1,000希釈1を準備します。抗SERPINH1 + 1の250倍希釈液：ブロッキング溶液中の抗SOX2 100希釈の1を準備します</li></ol></li><li> 4℃で一晩インキュベートします。 </li><li> 1×PBSでウェルを3回洗浄。 </li><li>抗ウサギ（緑ヤギ二次抗体を蛍光標識の400希釈; EX /エム2：作業1を準備します</sアップ&gt; 519分の495 nm）をヤギ抗マウス（赤; EX /エム2 617分の590 nm）をブロッキング溶液中で、各ウェルに100μLを追加します。室温、暗所で1時間インキュベートします。その後、1×PBSで一回、ウェルを洗浄します。 </li><li>各ウェルへの解決策を、3分間、暗所でインキュベートし; 100を1μg/ mLのヘキスト33342のμL（例/エム2 461分の358 nmの青に）を追加します。ウェルの最終1×PBS洗浄に残して、1×PBSでウェルを3回洗浄し、アルミ箔でスライドをカバーしています。 </li><li>蛍光顕微鏡下で細胞を分析します。 </li></ol>

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The authors have nothing to disclose.

このプロトコルは、脳の寄付金と合わせて採取したヒト死後の軟膜から髄膜線維芽細胞培養を導出するためのシンプルかつ堅牢なプロトコルを記述します。死後の人間の材料から細胞培養物を導出するプロトコルの非常にいくつかの説明があります。二つの研究は、皮膚由来線維芽細胞培養物7を説明する 8、9、</...

硬膜、くも膜と軟膜：髄膜は脳を保護する3膜で構成されています。より最近には、髄膜も脳の発達と脳のホメオスタシス1で重要な役割を果たすことが認識されています。髄膜は、間葉系および神経堤由来細胞から誘導され、興味深いことに、髄膜内に存在する前駆細胞は、in vitroおよびin vivo 2、3、4 における移植後の神経細胞を生じさせることができることが示されています。それらはドーパミン作動性ニューロン5に、胚性幹細胞の分化のための間質細胞由来誘導活性を有するような髄膜培養物はまた、成功裏に、フィーダー層として使用されてきました。また、軟膜は直接虚血状態6の下で、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する能力を持っています。 このプロトコルでは、人間の死後髄膜サンプルをまとめて、くも膜と軟膜から収集された軟膜と呼ばれ、研究目的のために人間の脳の寄付金の一部として調達されています。脳の解剖死の24時間以内に行われ、このプロトコルでここに示されているように軟膜サンプルは、次の6~8時間以内にさらなる処理のために冷増殖培地中に置かれます。 このプロトコルは、患者の一次軟膜細胞培養の開発のための解剖と人間の髄膜試料の調製を記載します。組織は、約3ミリメートル×3ミリメートルの正方形の25-30片に切断されています。三枚は各6ウェルに配置されたゼラチンでコーティングされたウェルと丸いガラスカバースリップでダウンして開催されました。髄膜切開は、約25-35分かかります。この文化の主な課題は、脳の調達、輸送、および解剖などの無菌性は、一般的に無菌条件下で行われていないです。 Therefore、ペニシリン、ストレプトマイシン、およびアムホテリシンBのカクテルで培地を補足し、別々に培養組織片にマルチウェルディッシュを使用することが重要です。 髄膜線維芽細胞の増殖は、最初の週以内に通常発生します。細胞がコンフルエントになるまで培地を2〜3日ごとに変化させ、細胞を酵素的に継代します。髄膜線維芽細胞は、凍結保存培地mLの/バイアルあたり100万個の細胞で凍結保存します。このプロトコルでは、20から30000000髄膜線維芽細胞は、凍結保存のため6-8週間で導出することができます。これらの髄膜線維芽細胞の下流のアプリケーションが、疾患の研究、直接神経分化または病気のメカニズムの理解のため、および薬物開発のための軟膜から誘導された多能性幹細胞の誘導のための一次培養物です。

<table border="0" cellpadding="0" cellspacing="0" width="1256">
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		<tr height="22">
			<td height="22" style="height:22px;width:459px;">Corning Petri dishes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:127px;">351029</td>
			<td style="width:485px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc 6-well plate</td>
			<td>Fisher Scientific</td>
			<td>14-832-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-mm cover slips</td>
			<td>Fisher Scientific</td>
			<td colspan="2">12-545-83 15CIR-1D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpels, sterile blade, No. 15</td>
			<td>Miltex</td>
			<td>4-415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved precision tip forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serological pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteur pipettes</td>
			<td>Fisher Scientific</td>
			<td>22-230490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin</td>
			<td>Sigma</td>
			<td>G1890-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Solution</td>
			<td>Fisher Scientific</td>
			<td>SH30264.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning 500 mL filter unit</td>
			<td>Fisher Scientific</td>
			<td>430770</td>
			<td>Combine media components and filter.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Nunc Cell Culture Treated Flasks with Filter Caps, T175 cm2&#160;</td>
			<td>Thermo Scientific</td>
			<td>178883</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Growth Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Hyclone DMEM</td>
			<td>Fisher Scientific</td>
			<td>SH30081.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone FBS</td>
			<td>Fisher Scientific</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution (100x)</td>
			<td>Thermo Fisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX Supplement (100x)</td>
			<td>Thermo Fisher</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate (100 mM)</td>
			<td>Thermo Fisher</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphotericin B (Yellow Solution/250 &#181;g/mL)</td>
			<td>Fisher Scientific</td>
			<td>BP264520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bambanker Freeze 120 mL</td>
			<td>Fisher Scientific</td>
			<td>NC9582225</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Fibronectin Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">8 well chamber slides&#160;</td>
			<td>Fisher Scientific</td>
			<td>1256518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20% paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100&#160;</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% Glycerol&#160;</td>
			<td>BioRad</td>
			<td>9455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% normal goat serum&#160;</td>
			<td>Fisher Scientific</td>
			<td>101098-382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Fibronectin antibody [F1]</td>
			<td>Abcam</td>
			<td>ab32419</td>
			<td>1:300 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SERPINH1</td>
			<td>Sigma</td>
			<td>S5950-200ul</td>
			<td>1:250 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SOX2</td>
			<td>Millipore</td>
			<td>MAB4343</td>
			<td>1:100 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Nestin</td>
			<td>Millipore</td>
			<td>MAB5326</td>
			<td>1:200 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TUJ1</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td>1:1000 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 anti-rabbit</td>
			<td>Thermo Fisher</td>
			<td>A11029</td>
			<td>1:400 dilution in blocking solution; (green channel; Ex/Em2 495/519 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 555 anti-mouse</td>
			<td>Thermo Fisher</td>
			<td>A21424</td>
			<td>1:400 dilution in blocking solution; (red channel; Ex/Em2 590/617 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342 stain</td>
			<td>Thermo Fisher</td>
			<td>H3570</td>
			<td>dilute to a final concentration of 1.0 &#956;g/ml; (blue channel; Ex/Em2 358/461 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Suppliers are suggestions, similar products from alternative vendors can be used as well.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

derivation of leptomeninges explant cultures from postmortem human brain donors

大きな進歩は、パーキンソン病の臨床的特徴付けてなされたものであっても、いくつかの研究は、パーキンソン病の診断は病理学的に、臨床的に診断されたパーキンソン病の25％までで確認されていないことを報告しています。したがって、特発性パーキンソン病と臨床的に診断された患者から採取した組織は、誤診率の高さを持つことができます。したがって、前臨床モデルとして、パーキンソン病を研究するために、このような組織からのin vitro試験で無駄になることができます。 パーキンソン病の確認神経病理学的診断と死後の人間の軟膜を収集し、黒質線条体細胞損失およびレビー小体と呼ばれる細胞内タンパク質封入体によって特徴づけられることで、1は、臨床的に観察されたパーキンソニズムは、他の基礎疾患プロセス（ 例えば、腫瘍、動脈硬化症）によって引き起こされていないことを確信することができます。 このプロトコルPRESEN髄膜線維芽細胞培養の導出のための死後の人間の軟膜の解剖と準備TS。この手順は、堅牢であり、高い成功率を有します。脳の調達は、一般的に無菌条件下で行われていないとして、文化の課題は、無菌性です。従って、ペニシリン、ストレプトマイシン、およびアンホテリシンBのカクテルで培地を補充することが重要ですパーキンソン病の剖検で確認例から髄膜線維芽細胞の導出は、パーキンソン病のin vitroモデル化のための基盤を提供します。髄膜線維芽細胞は3-9日、試料調製後に表示され、約20〜30万個の細胞を6〜8週間で凍結保存することができます。髄膜線維芽細胞培養物は、均質であり、細胞は、フィブロネクチン、髄膜を識別するために一般的に使用されるマーカーを発現します。

軟膜の処理プロトコルが成功したときに、これは脳のための死後変化の長さに依存することができますが、髄膜線維芽細胞の増殖は最初、3〜9日切開後に観察されます。 図1は、4つの異なるドナーの髄膜線維芽細胞培養物を示しています。 図1Aは、4日10時間20分の死後変化と88歳のドナーからの処理後の組織の周りにガラスカバー?...

Watch this Scientific Journal Video about Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors at JoVE.com

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

人間の死後脳から軟膜外植片培養プロトコルは6-8週間以内にフィブロネクチン陽性髄膜線維芽細胞を導出し、約20から30000000細胞を凍結保存するための技術的に堅牢かつ簡単な方法です。

死後ヒト脳ドナーから軟膜植片培養物の導出

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

derivation-leptomeninges-explant-cultures-from-postmortem-human-brain

Research

JoVE Journal

Neuroscience

8.2K ビュー.  Parkinson’s Institute and Clinical Center. 人間の死後脳から軟膜外植片培養プロトコルは6-8週間以内にフィブロネクチン陽性髄膜線維芽細胞を導出し、約20から30000000細胞を凍結保存するための技術的に堅牢かつ簡単な方法です。 

ビデオ: Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

げっ歯類からの神経細胞とヒト以外の霊長類の脳スライスのDiOLISTIC標識

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

神経科学

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

器官培養における神経雪崩のマルチ電極アレイの録音

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト神経前駆細胞と神経細胞の効率的な導出

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

出生後のマウス組織から培養オリゴデンドロサイトと共培養Myelinatingニューロン/オリゴデンドロサイトエンリッチドの導出

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

E13マウスからグリア制限前駆体の導出

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

からの神経外植文化<em&gt;アフリカツメガエル</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

ヒト胎児脳から由来オリゴデンドロサイト前駆細胞培養システム

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

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 ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

ヒト皮質下の高分解能構造磁気共鳴イメージング<I&gt;インビボ</I&gt;と死後

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

新鮮な人間の脳から脳の毛細血管の分離

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

死後ヒト脳ドナーから軟膜植片培養物の導出

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