このプロジェクトは、カナダの多発性硬化症協会からRKRWOへの助成金によって賄われていたカナダの多発性硬化症協会から学生の身分の受信者です。 SDRは、健康研究のカナダとカナダの研究所の多発性硬化症協会からポストドクトラルフェローシップの受賞者です。

<ol>
	<li>Avellana-Adalid, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+rat+oligodendrocyte+progenitors+into+proliferative+"oligospheres"+that+retain+differentiation+potential.">Expansion of rat oligodendrocyte progenitors into proliferative "oligospheres" that retain differentiation potential.</a> J Neurosci Res. 45, 558-570 (1996).</li><li>McCarthy, K. D., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+separate+astroglial+and+oligodendroglial+cell+cultures+from+rat+cerebral+tissue.">Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.</a> J Cell Biol. 85, 890-902 (1980).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+and+control+of+cell+survival+in+the+oligodendrocyte+lineage.">Cell death and control of cell survival in the oligodendrocyte lineage.</a> Cell. 70, 31-46 (1992).</li><li>Chan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGF+controls+axonal+receptivity+to+myelination+by+Schwann+cells+or+oligodendrocytes.">NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes.</a> Neuron. 43, 183-1891 (2004).</li><li>Camara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-mediated+axoglial+interactions+initiate+myelination+in+the+central+nervous+system.">Integrin-mediated axoglial interactions initiate myelination in the central nervous system.</a> J Cell Biol. 185, 699-712 (2009).</li><li>Ishibashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+promote+myelination+in+response+to+electrical+impulses.">Astrocytes promote myelination in response to electrical impulses.</a> Neuron. 49, 823-832 (2006).</li><li>Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nat Protoc. 2, 1044-1051 (2007).</li><li>Pedraza, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production,+characterization,+and+efficient+transfection+of+highly+pure+oligodendrocyte+precursor+cultures+from+mouse+embryonic+neural+progenitors.">Production, characterization, and efficient transfection of highly pure oligodendrocyte precursor cultures from mouse embryonic neural progenitors.</a> Glia. 56, 1339-1352 (2008).</li><li>Lewin, G. R., Ritter, A. M., Mendell, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+role+of+nerve+growth+factor+in+the+development+of+myelinated+nociceptors.">On the role of nerve growth factor in the development of myelinated nociceptors.</a> J Neurosci. 12, 1896-1905 (1992).</li><li>Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+vitro+studies+on+the+nerve+growth+factor+(NGF)+requirement+of+neurons.+II.+Sensory+neurons.">Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. II. Sensory neurons.</a> Dev Biol. 58, 106-113 (1977).</li></ol>

利害の衝突は宣言されません。

このレポートは、OL -濃縮培養物または共培養DRGN / OLの分化のためにマウスOPCを単離するための方法を説明します。単独で培養した場合、OPCはMBPに分化する+ VEの OLSを、膜状のシートミエリンのように生産。神経突起ベッドをDRGNに追加すると、OLSはMBPとDRGNの神経突起を包む+ VEの膜を。このモデルは、OL​​を介した軸索ensheathmentを支配する複雑な基盤の調査に利益をもたらします。 大きな値の間に、そのような文化の確立が技術的に困難です。特に、要求の厳しい側面が効率的な組織の消化/解離、バランスの培地のpHの維持、およびDRGNメディアの変更が含まれています。消化の長さは、組織の量を消化し、粉砕の量は組織の解離の効果と終了結果に影響されていることを考慮することが重要です。それは、経験豊かな研究者のために解離神経系の組織から、低細胞の収率を得ることが珍しくありません。さらに、マウスOPCは、特にアルカリ性の条件下で、培地のpHの変化に敏感になる傾向があります。 8.5％CO 2で培養の維持は、OPCはより基本的にやや酸性条件に耐えるように見えるので、これを防ぐことを目的としています。給餌DRGNsに関しては、メディアの変更は神経細胞を乾燥させるようにすることを迅速に実行する必要があります、しかし、発展途上神経突起のベッドを混乱させないよう穏やかにする必要があります。突然のメディアの変化は、基板からの神経突起ベッド、およびカバースリップから、その完全な解離の可能性が高い結果が外れる。 このモデルシステムの潜在的なメリットは大きく、その技術的な要求の性質を目立たなくします。このシステムの利点の一つは、胚組織を採取するために繁殖の女性を犠牲にする必要性を回避して、細胞培養の導出のための産後のマウスの使用です。もう一つの利点は、OPCの拡大のための増殖因子のための要件の欠如（GFS）です。混合グリア培養物は、アストロサイト由来の栄養因子の存在のためにおそらくOPCは、の伝播をサポートする環境を提供しています。このようなニューロスフェアを7,8を介して導出などの他の方法は、、そのような塩基性線維芽細胞増殖因子（bFGF）、上皮成長因子（EGF）とOPCの拡大のための血小板由来増殖因子（PDGF）などのGFSの分裂促進特性に依存しています。同様に、出生後（P5 - 10）DRGNsのためのマウスを使用すると、神経成長因子（NGF）、胚DRGNs 9、10のin vitroでの生存のために必要な神経栄養因子と培地を補充する要件を回避することができます。それはDRGNs 4で培養した場合にマイナスOLSのmyelinating能力に影響を与えるとして、NGFの使用を避けることは興味深い。大規模で使用すると、これらの試薬は高価になるとGF添加培地の使用を避けることにより、また、経済的利益を持っています。 おそらく、この培養モデルの最も重要な利点は、このようにトランスジェニックマウス系統の多種多様なOPCはとDRGNs両方を導出する機会を提供し、マウスのみの組織からの派生です。これはDRGNおよび/または髄鞘形成を支配するOPC固有のプロパティの両方の研究が可能になります。これは、軸索のOLを介した髄鞘形成を調節する受容体/リガンド相互作用を解明するために特に重要になります。すべてでは、こ​​の手法は、髄鞘形成の根底にある分子の手がかりを知るため、そのアプリケーションが原因で神経科学の研究に関しては大きな価値があります。

<html><table border="1"><tbody><tr><td> 製品名 </td><td> 会社 </td><td> 製品番号 </td></tr><tr><td>ダルベッコ改変イーグル培地（DMEM） </td><td>マルチセル</td><td> 319から005 - CL </td></tr><tr><td>ハンクス平衡塩類溶液（HBSS） </td><td>インビトロジェン</td><td> 14170-112 </td></tr><tr><td>最小必須培地（MEM） </td><td> GIBCO </td><td> 12360-038 </td></tr><tr><td>ウシ胎児血清（FBS） </td><td> GIBCO </td><td> 10091-148 </td></tr><tr><td>ペニシリン - ストレプトマイシン（ペン/連鎖球菌） </td><td> GIBCO </td><td> 15140-122 </td></tr><tr><td>グルタミン</td><td>インビトロジェン</td><td> 35050-061 </td></tr><tr><td>ポリ- L -リジン</td><td>シグマアルドリッチ</td><td> P2636 </td></tr><tr><td>ウシ血清アルブミン（BSA） </td><td>シグマアルドリッチ</td><td> A4503 </td></tr><tr><td>人間merosin精製タンパク質（LN2） </td><td>ミリポア</td><td> CC085 </td></tr><tr><td>組換えラット毛様体神経栄養因子（CNTF） </td><td>ぺプロ</td><td> 450から50 </td></tr><tr><td> L -チロキシン</td><td> Biochemika </td><td> 89430 </td></tr><tr><td>ホロトランスフェリン</td><td>シグマアルドリッチ</td><td> T0665 </td></tr><tr><td> B27サプリメント</td><td> GIBCO </td><td> 0080085 - SA </td></tr><tr><td>ウシインスリン</td><td>シグマアルドリッチ</td><td> I6634 </td></tr><tr><td> 3,3&#39;,5 -トリヨード- L -チロニン</td><td>シグマアルドリッチ</td><td> I6634 </td></tr><tr><td>プロゲステロン</td><td>シグマアルドリッチ</td><td> P8783 </td></tr><tr><td>プトレッシン</td><td>シグマアルドリッチ</td><td> P7505 </td></tr><tr><td>亜セレン酸ナトリウム</td><td>シグマアルドリッチ</td><td> S5261 </td></tr><tr><td> 5 - フルオロ-2&#39; - デオキシウリジン（FUDR） </td><td>シグマアルドリッチ</td><td> F0503 </td></tr><tr><td>パパインのソリューション</td><td>ワージントン</td><td> LS003126 </td></tr><tr><td> DNaseI </td><td>ロッシュ</td><td> 1010159001 </td></tr><tr><td> L -システイン</td><td>シグマアルドリッチ</td><td> C7352 </td></tr><tr><td> 24ウェル組織培養皿</td><td> Cellstar </td><td> 662から160 </td></tr><tr><td>ベントキャップ付T25 -組織培養フラスコ</td><td>コー​​ニング</td><td> 430639 </td></tr><tr><td> 10cmの組織培養皿</td><td>コー​​ニング</td><td> 430167 </td></tr><tr><td> 10cmペトリ皿</td><td>フィッシャーサイエンティフィック</td><td> 0875713 </td></tr><tr><td>コラゲナーゼ</td><td>ロッシュ</td><td> 103578 </td></tr><tr><td> CellTrics50μmのフィルター（オプション） </td><td> PARTEC </td><td> 04-004-2327 </td></tr><tr><td>ミエリン塩基性タンパク質（MBP）抗体</td><td> ABD Serotec </td><td> MCA409S </td></tr><tr><td> NG2抗体</td><td>ミリポア</td><td> AB5320 </td></tr><tr><td>ミエリン関連糖タンパク質（MAG）抗体</td><td>ミリポア</td><td> MAB1567 </td></tr><tr><td> 2&#39;、3&#39; -サイクリックヌクレオチド3&#39; -ホスホジエステラーゼ（CNP）抗体</td><td> Covance </td><td> SMI - 91R - 100 </td></tr><tr><td>アクチンパンAB - 5抗体</td><td>フィッツジェラルド</td><td> 10R - A106AX </td></tr><tr><td>ニューロフィラメント- 200（NF）抗体</td><td>シグマアルドリッチ</td><td> N4142 </td></tr><tr><td>チューブリンβ- 3チェーン（Tuj1）抗体</td><td>ミリポア</td><td> MAB5544 </td></tr><tr><td>のAlexa Fluor ® 488ヤギ抗ウサギIgG（H + L）二次抗体</td><td>インビトロジェン</td><td> A11008 </td></tr><tr><td>のAlexa Fluor ® 555ヤギ抗マウスIgG（H + L）二次抗体</td><td>インビトロジェン</td><td> A21422 </td></tr><tr><td>のAlexa Fluor ® 647ヤギ抗ラット抗体（IgG）（H + L）二次抗体</td><td>インビトロジェン</td><td> A21247 </td></tr><tr><td>ヤギ抗マウスIgG（H + L）- HRP標識二次抗体</td><td> BioRad社</td><td> 170-6516 </td></tr><tr><td>ヤギ抗ラットIgG（H + L）- HRP標識二次抗体</td><td>サンタクルスバイオテクノロジー</td><td> SC - 2065 </td></tr><tr><td> 4&#39;、6 - ジアミジノ-2 - フェニルインドール（DAPI） </td><td>シグマアルドリッチ</td><td> D9542 </td></tr></tbody></table>メディアのレシピ 100X OL -補足* <table border="1"><tbody><tr><td> 成分 </td><td> 追加する量 </td></tr><tr><td> DMEM </td><td> 100mLの</td></tr><tr><td> BSA </td><td> 1.02グラム</td></tr><tr><td>プロゲステロン</td><td> 0.6mgの</td></tr><tr><td>プトレッシン</td><td> 161 mgの</td></tr><tr><td>亜セレン酸ナトリウム</td><td> 0.05mgの</td></tr><tr><td> 3,3&#39;,5 -トリヨード- L -チロニン</td><td> 4mgを</td></tr></tbody></table> -80 * ° Cで保存250μL分注しT&quot;&gt; OLメディア <table border="1"><tbody><tr><td> 成分 </td><td> 追加する量 </td></tr><tr><td> DMEM </td><td> 23.75 mLの</td></tr><tr><td> 100X OL -サプリメント</td><td> 250μL </td></tr><tr><td>ウシインスリン（1 mg / mLの在庫から） </td><td> 125μL </td></tr><tr><td>グルタミン</td><td> 250μL </td></tr><tr><td>ホロトランスフェリン（33 mg / mlストックから） </td><td> 37.5μL </td></tr><tr><td> B27サプリメント</td><td> 500μL </td></tr><tr><td> FBS </td><td> 125μL </td></tr><tr><td> CNTF（50 ng /μLの在庫から） </td><td> 25μL </td></tr></tbody></table> 混合グリア培養培地（DMEMで構成される） <table border="1"><tbody><tr><td> 成分 </td><td> 最終濃度 </td></tr><tr><td> FBS </td><td> 10パーセント</td></tr><tr><td>ペン/咽頭（株式から0.33％） </td><td> 33ユニット/ mlペニシリン、33μg/ mlストレプトマイシン</td></tr><tr><td>グルタミン</td><td>一パーセント</td></tr></tbody></table> DRGN培地（DMEMで構成される） <table border="1"><tbody><tr><td> 成分 </td><td> 最終濃度 </td></tr><tr><td> FBS </td><td> 10パーセント</td></tr><tr><td>ペン/咽頭（株式から1％） </td><td> 100単位/ mlペニシリンおよび100μg/ mlストレプトマイシン</td></tr></tbody></table>消化溶液のレシピ： OPCパパイン溶液（MEMで構成される） <table border="1"><tbody><tr><td> 成分 </td><td> 最終濃度 </td></tr><tr><td>パパインのソリューション</td><td> 1.54 mg / mLの</td></tr><tr><td> L -システイン</td><td> 360μg/ mLの</td></tr><tr><td> DNaseI </td><td> 60μg/ mLの</td></tr></tbody></table> DRGパパイン溶液（HBSSで構成される） <table border="1"><tbody><tr><td> 成分 </td><td> 最終濃度 </td></tr><tr><td>パパイン</td><td> 1.54 mg / mLの</td></tr><tr><td> L -システイン</td><td> 360μg/ mLの</td></tr></tbody></table> DRGコラゲナーゼ溶液（HBSSで構成される） <table border="1"><tbody><tr><td> 成分 </td><td> 最終濃度 </td></tr><tr><td>コラゲナーゼ</td><td> 4 mg / mLの</td></tr></tbody></table>

derivation of enriched oligodendrocyte cultures and oligodendrocyte/neuron myelinating co-cultures from post-natal murine tissues

OL開発の根底にある分子機構を同定することはOL生物学の知識を深めるには重要であるだけでなく、このような多発性硬化症（MS）などの脱髄疾患の病因を理解するための意味を持っています。携帯電話の開発は、一般的に一次細胞培養モデルで研究されている。初代培養細胞は、 生体内に存在する余分な変数の自由、制御された環境を提供することにより、所定の細胞型の評価を容易にする。ラット由来OLの文化がOL生物学への洞察の膨大な量を提供しているが、マウスからOLの文化を確立することで、同様の努力が大きな障害に満たされている。文化マウス一次OLSする方法を開発することは可能なトランスジェニックマウス系統を利用するために不可欠です。 げっ歯類の組織からOPCを抽出するための複数のメソッドは、ニューロスフェアの導出、差動接着精製と1-3免疫精製に至るまで、記載されている。多くのメソッドが成功を提供していますが、ほとんどは大規模な文化の時間および/または高価な機器/試薬を必要とする。これを回避するために、当初はマッカーシー＆デVellis 2で説明した方法の適応とマウス組織からOPCを精製することが好ましい。このメソッドは、新生児げっ歯類皮質由来する混合グリア培養液から物理的に分離するOPCを伴います。結果は、OLに富んだ文化に分化することができる精製したOPCの人口です。このアプローチは、その比較的短い文化時間や成長因子またはimmunopanning抗体の不必要な要件のために魅力的です。 精製された文化の中でOL発展のメカニズムを探ることは有益ですが、それは、髄鞘形成を評価するための最も生理学的に関連する環境を提供していません。ニューロンとの共培養のOLSは、軸索のOLを介した髄鞘形成を制御する分子的基盤への洞察を貸すだろう。多くのOL /ニューロンの共培養の研究については、後根神経節ニューロンは（DRGNs）選択肢のニューロンのタイプであることが証明されている。彼らは、抽出、混入細胞の最小限の量、そして緻密な神経突起のベッドの形成の容易さのため、OLSとの共培養に最適です。ラット/マウスmyelinating xenoculturesを用いた研究が4-6を公開されているが、出生後のマウス組織から共培養をmyelinatingなど/ OL DRGNの導出のための方法が記載されていない。ここでは、効果的に予想される結果の例と共に、そのような文化を生産する方法に関する詳細な方法を提示する。これらのメソッドは、OL開発/ myelinating機能に関連する質問に対処するために有用であり、神経科学の分野で有用なツールです。

Watch this Scientific Journal Video about Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues at JoVE.com

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

この記事では、成熟オリゴデンドロサイト（OLS）を生成するために差別化初代培養におけるマウスのオリゴデンドロサイト前駆細胞体（OPC）の豊かな集団を、導出する方法を説明します。さらに、この報告書はマウス後根神経節ニューロン（DRGNs）の神経突起ベッドの上にマウスのOPCを播種によってマウスmyelinating共培養を生成する手法を説明します。

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-enriched-oligodendrocyte-cultures-oligodendrocyteneuron

Research

JoVE Journal

Neuroscience

36.0K ビュー.  Ottawa Hospital Research Institute. この記事では、成熟オリゴデンドロサイト（OLS）を生成するために差別化初代培養におけるマウスのオリゴデンドロサイト前駆細胞体（OPC）の豊かな集団を、導出する方法を説明します。さらに、この報告書はマウス後根神経節ニューロン（DRGNs）の神経突起ベッドの上にマウスのOPCを播種によってマウスmyelinating共培養を生成する手法を説明します。

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

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

高濃縮ニューロン培養の世代のための皮質組織ブロックの凍結保存

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

神経科学

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 ...

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 ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

からの同時プリとポストシナプスの電気生理学的記録<em&gt;アフリカツメガエル</em&gt;神経筋共培養

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

単セルの精度で神経共培養の調製

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

オリゴデンドロサイトダイナミクスと髄鞘形成を研究するための器官型スライス培養

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

マルチ電極アレイ上に成長させた器官脊髄共培養における機能的再生の調査

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

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 ...

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which macrophages are induced to produce conditioned medium (CM) that promotes neurite outgrowth. Adult dorsal root ganglion (DRG) neurons are acutely dissociated and plated. After the neurons are stably attached, peritoneal macrophages are co-cultured on a cell culture insert overlaid on the same well. Dibutyryl cyclic AMP (db-cAMP) is applied to the co-cultures for 24 h, after which the cell culture insert containing the macrophages is moved to another well to collect CM for 72 h. The CM from the co-cultures treated with db-cAMP, when applied to a separate adult DRG neuron culture, exhibits robust neurite outgrowth activity. The CM obtained from the db-cAMP-treated cultures consisting of single cell type alone, either DRG neuron or peritoneal macrophage, did not exhibit neurite outgrowth activity. This indicates that the interaction between neurons and macrophages is indispensable for the activation of macrophages secreting molecular factors with neurite outgrowth activity into CM. Thus, our co-culture paradigm will also be useful to study intercellular signaling in the neuron-macrophage interaction to stimulate the macrophages to be endowed with a pro-regenerative phenotype.

The current protocol presents experimental procedures to stimulate cultured macrophages to be endowed with capacity to release molecular factors that promote neurite outgrowth. Treatment of cAMP to the neuron-macrophage co-cultures induces the macrophages to produce conditioned medium that possesses strong neurite outgrowth activity.

アクティブに大食細胞の神経突起伸展作用と分子因子を分泌する神経細胞・ マクロファージ共培養

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

準備や髄脊髄小脳スライス培養の免疫染色

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...