HD054347とVLSへNS063997 - 01：この作品は、国立衛生研究所によって部分的にサポートされていました。この作品はまた、保健契約＃C024324 VLSへのニューヨーク国務省を通じて、エンパイアステート幹細胞の基金によって部分的にサポートされていました。ご意見は、ここに表明し、執筆者の個人的な見解であり、必ずしもエンパイアステート幹細胞委員会、ニューヨーク州保健局、または、ニューヨーク州の見解を反映するものではありません。 VLSはドリスデューク臨床科学発達受賞者です。我々はまた、抗O1、抗O4抗体の彼の贈り物のために教授ティモシーVartanianに感謝。

1。解剖および神経幹細胞培養の維持のためのソリューションと材料の準備<ol><li>事前に100mlの解剖媒体（KNOCKOUT DMEM/F12、Invitogen）を準備して冷蔵庫に。 </li><li> Prepare100 mlの培地（ステムプロNSC SFM、Invitrogen社）と37℃保管℃の水浴でC。 </li><li>細胞の長期凍結保存のための細胞凍結培地を（KNOCKOUT DMEM/F12 +10％FBS + 5％DMSO）の準備。 </li><li>必要に応じて、組織固定のために4％パラホルムアルデヒド（PFA）を準備する。 </li><li>ハンドル付き滅菌、高圧蒸気滅菌ピンセットとメスのブレードは、解剖のために使用されます。 </li><li>ピペットの銃を脇に置いて、10 mlの転送ピペット、および解離は40μMのセルストレーナー（BDファルコン352340）。 </li><li>組織のストレージおよび凍結細胞のための凍結バイアル（BD）のために解剖のためのいくつかの10cmの培養皿（BD）、解離の50 ml遠心チューブ（BD）、1.5 mlの遠心チューブを脇に置きます。 </li></ol> 2。人間の胎児の脳からヒト神経幹細胞を単離する<ol><li>すぐに胎児の終了後に実行可能な組織の収穫は、手続きの成功に不可欠です。選択科目の手順については、サンプルを取得するタイミングは、胎児の死亡の後の時間を最小化するように事前に配置することができます。受胎産物は、2時間後にプロシージャ内で収穫されますが、理想的には数分以内に選択科目の手続きに達成することができます。胎児組織は、しばしば断片化されています。しかし、一般的には脳のかなりの部分は、視覚的に識別するためそのまま残ります。在胎週数（GA）の制限事項が制定法によって決定されますが、18〜22週GAの間にこのプロトコルを使用して行われている。 </li><li>胎児の脳を氷冷KNOCKOUT DMEM/F12ソリューションを含む10mlのペトリ皿に配置されます。解剖学的ランドマークが皮質の異なる部分を識別します。前頭葉と頭頂後頭野は中心溝とシルビウス裂の外挿した交点が配向している。ための境界心室はそのままと破損していないように注意しながら、中心溝の前部前頭葉皮質からと外科ブレードでシルビウス裂の境界に沿って組織を細かく分析。 </li><li>前頭皮質の区切られたブロックからの残留血液および髄膜を取り外します。サンプルの品質が十分であれば、それは様々な目的のためにいくつかの小さなサンプルにブロックを分析するのに最適です。セクション（PFA、4％パラホルムアルデヒドで固定）とタンパク質/ mRNAのアッセイ（高速-80℃で凍結）。 </li><li> 50 ml遠心チューブに、選択された脳のブロックを転送し、組織の体積の約3倍で、氷冷KNOCKOUT DMEM/F12ソリューションを追加します。すべての組織が（一般的に20〜30倍）断片化し、単一または近くに、単一のセルを取得するために40μmのセルストレーナー（BDファルコン352340）を介して細胞をフィルタリングするようになるまでゆっくりと10 mlの転送ピペットで機械的なピペッティングにより組織を解離するサスペンション。 </li><li> 5分間2000rpmで細胞懸濁液と室温を遠心、10 mlの新鮮な暖かい培地（ステムプロNSC SFM）に細胞ペレットを再懸濁し、そして血球計算盤で細胞数を数える。 </li><li>それぞれ25cm 2の培養フラスコに5ミリリットル暖かい培地を追加し、各フラスコに2X10 6細胞を移す。培養物は、分析前に1週間、37℃/ 5％CO 2インキュベーター中で維持されます。さらに文化や実験のために週に一度half培養液を交換する。 </li></ol> 3。さらなる特徴またはexperimentationsのための神経幹細胞のManipulaton <ol><li>ニューロスフェアは、通常200〜400μmの間の範囲のNSCの直径推奨培養条件下で1〜2週間で形成する。この段階でニューロスフェアは単一細胞を得るために15分間37℃で、カルシウムとマグネシウムを含まないハンクス培地（ハンクス）に0.2 g / LのEDTA ° Cで解離させることができる。細胞懸濁液を新鮮なハンクスにすすぎ、2000回転、で紡糸、とサブカルチャーのための暖かい培地中で白い色を保ちされています。 </li><li>分化を開始するには、解離細胞はカバースリップ当たり1X10 5細胞（24mmX24mm）の密度でpoly-D-lysine/laminin 1コーティングカバーガラス上に播種されています。オリゴデンドロサイト分化がためにKNOCKOUT DMEM/F12（Invitrogen社、メイン、MD）2パーセントB27（50X、Invitrogen社、メイン、MD）+10 ng / mlのbFGFを100 ng / mlのSHH + 10ng/ml PDGF - AAで細胞を維持することによって達成され2日は、その後、別の5日間増殖因子なしで同じ培地に切り替え。神経分化は7日のノックアウトでDMEM/F12 2パーセントB27を（50X）細胞を維持することによって達成される。アストロサイト分化が週にKNOCKOUT DMEM/F12 1パーセントFBSで細胞を培養することによって行われます。 </li><li>遺伝子による神経幹細胞のトランスフェクションは、予め形成されたニューロスフェアから解離細胞で行うことができます。ここで、我々は、電気穿孔法によりEGFP - C1をトランスフェクトした神経幹細胞を示した。 EGFP - C1構造のエレクトロポレーションは、マウス神経幹細胞（VPG -用AMAXA Nucleofectorキットを使用して行われました会社の命令に続くAMAXA Nucleofectorデバイス（ロンザAAD - 1001）と1004）、。一言で言えば、1 X 10 6細胞を5μgのDNAを100μlのトランスフェクション培地と混合し、パルスのエレクトロポレーション後、細胞はさらに培養用培地を維持する神経幹細胞に再懸濁した。トランスフェクションされた細胞の分化は、ステップ3.2で説明した同じ手順に従って、エレクトロポレーション後、ニューロスフェア3-4日の解離で処理した。 </li></ol> 4。凍結神経幹細胞とサブカルチャー<ol><li>解離細胞懸濁液は1X10 7細胞/バイアル/ mlの濃度で遠心分離し、凍結培地に再懸濁する。徐々に-20℃、-80で細胞を凍結° C、長い時間の貯蔵のために液体窒素に移す。 </li><li>細胞は、高速37℃の水浴で解凍し、中に再懸濁した凍結培地を除去するため遠心分離洗浄のための血清DMEM/F12 +10％を、加温し、温めておいた培地に再懸濁する。 </li></ol> 5。代表的な結果： 18週で正常胎児から神経幹細胞は在胎週数が記載される方法およびニューロスフェアが丸い、滑らかな境界線とはかなり均一なサイズ（Fig.2A）と一週間後に見られる以下の培養した。これらのニューロスフェアは、EGFP - C1または他の構築物でトランスフェクトし、蛍光顕微鏡（図2b、）で追跡することができる。確立されたニューロスフェアは、EDTAとの解離とコーティングされたカバースリップ上に分散した細胞として播種した。それぞれのプロトコルの下で分化した細胞を4％parafamaldehydeで固定し、異なる細胞タイプ特異的なマーカーで染色した。多能は、ニューロン（Fig2C、D、ローダミン）アストロサイトを示すマーカーの発現（Fig2E、F、ローダミン）、オリゴデンドロサイト（Fig2G、H、ローダミン）で観察される。 EGFPのエレクトロポレーションを受けていない細胞はまた、異なる細胞型に分化して異なる細胞特異的なマーカーで染色した。多能は（Fig3C、ローダミン及びFig3D、フルオレセイン）アストロサイト（Fig3A、B、フルオレセイン）ニューロンを示すマーカーとオリゴデンドロサイト（Fig3E、F、ローダミン）の発現が観察される。 <img src="/files/ftp_upload/2681/2681fig1.jpg" alt="図1"/> 図1。破棄されたヒト胎児脳から神経幹細胞を分離する実験手順の概略図<img src="/files/ftp_upload/2681/2681fig2.jpg" alt="図2"/> 図2。 試験管内で propogated未分化および分化したヒト神経細胞。 （A）ニューロスフェアは、滑らかな、円形の境界線を示し、1週間以上の培養後の急速な成長を位相差顕微鏡の下に表示されます。 （B）、様々なプラスミド及び構造の導入はトランスフェクションによって達成することができます。 EGFP - C1トランスフェクション後3日には、複数のセルはフルオレセインの免疫染色と蛍光顕微鏡下で見られるように緑色蛍光タンパク質の発現を示す。 EGFP - C1トランスフェクションしたニューロスフェアは、ニューロン（C、D）、アストロサイト（E、F）、オリゴデンドロサイト（G、H）に異なる条件下で解離し、分化しており、ローダミンの蛍光下で見る。同時に、トランスフェクトされたEGFP陽性細胞はフルオレセイン蛍光の下に表示されます。トランスフェクトした細胞（白矢印の頭）はトランスフェクトされていない細胞（白矢印）と区別がつかない。細胞核をHoechst33342で染色されています。スケールバーは用は200μm、BとCHの場合は25ミクロンでは100μmです。 <img src="/files/ftp_upload/2681/2681fig3.jpg" alt="図3"/> 図3。トランスフェクションなしのニューロスフェアは神経細胞（A、B、フルオレセイン）、アストロサイト（C、ローダミン、D、フルオレセイン）およびオリゴデンドロサイト（E、F、ローダミン）への異なる条件下で解離と区別されています。細胞核をHoechst33342で染色されています。スケールバーは、AFは25μmです。

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利害の衝突は宣言されません。

文化新鮮な組織と生ヒト細胞株に向けたさまざまなアプローチがある。歴史的に、新鮮な組織を採取され、中枢神経系における種々の細胞型を生成するために直ちに培養。このアプローチは、しかしはっきりと得られたヒトサンプルの場合には、通常非常に小さいことができるサンプルの数によって制限されます。操作の最小限の度合いを考えると、新鮮な培養神経細胞では、拡張培養から?...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td> KNOCKOUT DMEM/F12 </td><td>インビトロジェン</td><td> 12660-012 </td><td>解離培地</td></tr><tr><td>幹プロNSC SFM </td><td>インビトロジェン</td><td> A10509 - 01 </td><td>培地</td></tr><tr><td>ウシ胎仔血清</td><td>インビトロジェン</td><td> 10091-148 </td><td>凍結媒体</td></tr><tr><td>ハンクス溶液（-のCa 2 +、 -のMg 2 +） </td><td>インビトロジェン</td><td> 14175-095 </td><td>解離培地</td></tr><tr><td> DMSO </td><td>シグマアルドリッチ</td><td> D2650 </td><td>凍結媒体</td></tr><tr><td> EDTA </td><td>シグマアルドリッチ</td><td> 431788 </td><td>解離培地</td></tr><tr><td>パラホルムアルデヒド</td><td>シグマアルドリッチ</td><td> 158127 </td><td>固定液</td></tr><tr><td> bFGFの</td><td> R＆D </td><td> 234 - FSE </td><td>分化培地</td></tr><tr><td> SHH </td><td> R＆D </td><td> 1845 - SH </td><td>分化培地</td></tr><tr><td> PDGF - AA </td><td> R＆D </td><td> 221 - AA </td><td>分化培地</td></tr><tr><td> B27 </td><td>インビトロジェン</td><td> 17504-044 </td><td>分化培地</td></tr><tr><td>マウス抗MAP2 </td><td>シグマアルドリッチ</td><td> M2320 </td><td> 1:200 </td></tr><tr><td>ウサギ抗DCX </td><td>細胞シグナリング</td><td> 4604s </td><td> 1:200 </td></tr><tr><td>ウサギ抗GFAP </td><td> DAKO </td><td> Z0334 </td><td> 1:200 </td></tr><tr><td>ウサギ抗- S100B </td><td> DAKO </td><td> Z0311 </td><td> 1:200 </td></tr><tr><td>ウサギ抗- O1 </td><td>教授ティモシーVartanianの贈り物* </td><td></td><td>午前1時50分</td></tr><tr><td>ウサギ抗O4 </td><td>教授ティモシーVartanianのギフト* </td><td></td><td>午前1時50分</td></tr><tr><td> 40μmのセルストレーナー</td><td> BDファルコン</td><td> 352340 </td><td></td></tr></tbody></table> *ティモシーVartanian、MD、PhDは、神経と神経科、ワイルコーネル医科大学、ニューヨーク、アメリカ合衆国

generation of neural stem cells from discarded human fetal cortical tissue

神経幹細胞（NSC）は、皮質板の開発中に脳室帯の神経上皮に沿って存在する。これらの初期の前駆細胞は、最終的に、中間前駆細胞を生じさせると後で、大脳皮質を形成する様々な神経細胞およびグリア細胞のサブタイプ。廃棄された正常な胎児組織からヒトNSCを（そのニューロスフェアと呼ばれる）を生成し、拡大する能力は、直接ヒトの正常なNSC開発1-5の機能的側面 ​​を研究するために使用する手段を提供します。このアプローチはまた、それによって前駆細胞増殖、遊走および分化6-9を変更する病気のプロセスを識別する機会を得た、既知の神経疾患からのNSCの世代に向けて送ることができます。我々は、高速化、アルツハイマー病の表現型10,11に貢献するかもしれない人間のダウン症候群のNSCでの病理学的メカニズムを識別するに焦点を当てている。 生体内でも、in vitroでのマウスモデルのどちらのヒト21番染色体上に位置する遺伝子の同一のレパートリーを複製することができます。 ここでは、中止された人間の胎児皮質から症候群のNSCをダウン分離し、文化の中で、それらを成長させる、シンプルで信頼性の高いメソッドを使用します。方法論は、ヒトNSCのめっき及び継代、限られた解剖学的ランドマーク、細胞ソーティングと解剖、組織の収穫の特定の側面を提供します。また、より選択的な細胞のサブタイプにヒトNSCの分化を誘導するためのいくつかの基本的なプロトコルを提供します。

Watch this Scientific Journal Video about Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue at JoVE.com

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

捨てられた人間の胎児皮質組織から神経幹細胞の単離および培養上シンプルで信頼性の高い方法が記載されている。既知のヒト神経疾患から派生した培養物は、病理学、細胞及び分子過程の特性だけでなく、薬理学的有効性を評価するためのプラットフォームを提供するために使用することができます。

廃棄されたヒト胎児脳組織から神経幹細胞の生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

generation-neural-stem-cells-from-discarded-human-fetal-cortical

Research

JoVE Journal

Neuroscience

15.4K ビュー.  Beth Israel Deaconess Medical Center. 捨てられた人間の胎児皮質組織から神経幹細胞の単離および培養上シンプルで信頼性の高い方法が記載されている。既知のヒト神経疾患から派生した培養物は、病理学、細胞及び分子過程の特性だけでなく、薬理学的有効性を評価するためのプラットフォームを提供するために使用することができます。

ビデオ: Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

神経コロニー形成細胞アッセイ：神経前駆細胞から善意の神経幹細胞を区別するアッセイ

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

神経科学

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 ...

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 ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

ヒト神経の集合体のためのcGMP適用可能な展開法は、幹細胞および前駆細胞は、多能性幹細胞や胎児の脳組織由来

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

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 ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

ヒト多能性幹細胞からドーパミン作動性ニューロンの分化を監督

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

モデル星細胞腫発症機序<em&gt;体外</em&gt;と<em&gt;インビボ</em&gt;皮質アストロサイトまたは条件付き、遺伝子改変マウスから神経幹細胞を使用した

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

クローンのアッセイを用いて神経幹細胞の列挙

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...