脳腫瘍の治療のためのプレストンA.ウェルズジュニアセンター、この作品は、脳腫瘍研究のためのフロリダセンターからの補助金によって支えられている。

1。プライマリGBMのティッシュのコレクション<ol><li>神経膠芽腫mutliforme（GBM）の腫瘍はがんと診断され手術を受けた患者から取得されます。 </li><li>手配は脳神経外科チームと行う必要があります。神経外科医は、神経幹細胞（NSC）10〜15％の抗生物質（Peniciline / Streptomycine）を添加した基礎培地を含む適切なサイズのチューブに切除されたGBMの腫瘍を配置します。冷たい培地は実験室で手術室に提供する必要があります。また、冷HEPES緩衝最小必須培地（HEM）またはリン酸塩は、抗生物質の高濃度で生理食塩水（PBS）もこの目的のために使用できるバッファ。 </li><li>切除した腫瘍組織は、氷上でラボに配信され、ボンネットの下に配置されます。 </li></ol> 2。シングルセル懸濁液中に原発腫瘍の解離<ol><li>オリジナルメディア/ PBSの過剰は、ファルコンチューブから削除され、サンプルは5〜10メートルに2〜3回洗浄し、PBS / NSC基礎培地のlは、血液や破片を除去する。 PBS /培地を除去されており、GBM腫瘍の組織をシャーレに入れられます。 </li><li>組織を小片に切断し、トリプシン処理プロセスのための表面積を大きくする小さな部分に第10号手術用メスの刃で刻まれている。ミンチは、腫瘍の大きさに応じて1〜3分かかる場合があります。 </li><li>みじん切り組織は、予め温めておいた％0.05 37℃の水浴で10〜15分間トリプシン- EDTAの3〜5ミリリットルでトリプシン処理しています。電子ピペットは、15ミリリットルファルコンチューブに小さな腫瘍の部分とトリプシンを転送するために使用されます。 </li><li>大豆トリプシン阻害剤の等量は、潜伏期間の後に酵素のトリプシンを加えて反応を停止されている。 </li><li>トリプシンの不活性化は、上下に数回の中断をピペッティングすることにより確保されている。その後、懸濁液は、5分間約800 rpm（110グラム）で遠心分離することによってダウンしてペレット化です。 </li><li>上清を捨て、組織片を無菌NSC BA 1mlの中に再懸濁されているサル媒体。塊は優しく滑らかな乳白色の単細胞懸濁液が得られるまで（3-7回）上下にピペッティングして解離する。ピペッティングステップ数は、直接ミンチ組織中の粒子のサイズによって異なります。それが細胞死と球形成の減少につながる可能性があるので長いと積極的な機械的解離は避けてください。 </li><li>非解離片や破片を除去するために、基礎培地の10〜15 mlのチューブに添加し、細胞懸濁液を50ミリリットルチューブに40ミクロ​​ンのセルストレーナーで濾過する。 </li><li>フィルタ処理された懸濁液を5分間約800 rpm（110グラム）で遠心分離する。上清はその後破棄されます。 </li><li> Pellettedその後、細胞を細胞計数のための完全なNSCの培地の1〜2ミリリットル中に再懸濁されている。 </li></ol> 3。セル数とめっき<ol><li>細胞懸濁液の10μLを1ミリリットルエッペンドルフチュー​​ブ0.04％トリパンブルーの90μLに追加されます。 注：他の適当なセルdilutionsを使用することもできます。 </li><li>サスペンションを混在させる上下にピペッティングし。細胞の10μL/トリパンブルー液は細胞密度をカウントするために、血球計数器に転送されます。 </li><li>細胞は0.2％のヘパリンの20ng/ml EGF、10ng/ml bFGFおよび1μl/mlを添加した完全なNSCの培地（9:1の比率でNSC基礎培地とNSC増殖サプリメントの混合物）（2μg/ml）でメッキされています適切な組織培養容器。培地の5、20及び40mlをそれぞれ、T25、T80およびT175フラスコに使用されます。抗生物質は、汚染の可能性を減らすために1:100の濃度で培地に添加してもよい。 </li><li>フラスコを37℃、5％CO 2インキュベーターセットに置かれます。 </li></ol> 4。継代とGBM派生球の拡大： <ol><li>ニューロスフェアは、直径150から200ミクロンの平均サイズに達したときに、文化はサブカルチャーの準備ができています。各フラスコの内容が適切なサイズ滅菌tissで削除して配置されますUEは文化チューブ、そして室温で5分間、800回転（110 g）で遠心分離。 </li><li>上清を除去し、ペレットを％0.05トリプシン- EDTAを1mlに再懸濁されている。 </li><li>最適なトリプシン処理を達成するために、細胞懸濁液を37℃で2〜3分間水浴中でインキュベートされる。トリプシン活性を停止するには大豆トリプシン阻害剤の等量の細胞懸濁液に添加し、細胞懸濁液を静かに上下にピペッティングしています。 </li><li>細胞懸濁液を5分間800回転（110グラム）で遠心分離する。その後、上清を除去し、細胞はNSCの培地1mlに再懸濁する。 </li><li>前述のように細胞数が実行されます。 </li><li>細胞は、増殖因子や、前の部分で説明した適切なサイズの組織培養容器内のメッキを補充した完全なNSCの培地で5 × 10 4細胞/ mlの濃度で播種されています。 </li><li>多湿で37℃でインキュベートしたときの二次ニューロスフェアは7〜10日に形成されています5％CO 2と化されたインキュベーター。 </li></ol> 5。代表的な結果： GBMの腫瘍組織から採取した単一の細胞を播種した後、腫瘍幹様細胞は増殖と（ビデオと図1を参照）3-4日で数個の細胞から成る細胞の小さなクラスターを生成する。これらのクラスターが成長するにつれ、彼らは150から200ミクロンの形式（ビデオおよび図2を参照）の平均粒径が7〜8日でように、より球形、適切な位相明るい球を獲得する。より高い倍率で、健康な球は通常、その周囲にmicrospikesを示しています。培養開始後1-2日以内に、クラスタと同じような大きな球を持つことは、文化の一気飲みで非解離塊の存在によるもので、真球と誤解されるべきではない。原発腫瘍の球培養での残骸の量は、組織の最初のソースで、それは周囲の脳組織を含むかどうかによって異なります。適切な組織の調製技術培地の十分な量の酵素的および機械的解離、およびサンプルのその後のろ過を含むことは文化の小さい破片になる可能性があります。 <img src="/files/ftp_upload/3633/3633fig1.jpg" alt="図1" /> 図1 4日メッキ後のプライマリGBMの球の文化。腫瘍幹様細胞は増殖と3-4日で細胞の小さなクラスターを生成する。オリジナルの倍率、20倍。 <img src="/files/ftp_upload/3633/3633fig2.jpg" alt="図2" /> 図2。8日間めっき後のパッセージone GBM球の文化を。オリジナルの倍率、20倍。

<ol>
	<li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Siebzehnrubl, F. A., Vedam-Mai, V., Azari, H., Reynolds, B. A., Deleyrolle, L. P. <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+Characterization+of+Adult+Neural+Stem+Cells.">Isolation and Characterization of Adult Neural Stem Cells.</a> Methods Mol Biol. 750, 61-77 (2011).</li><li>Azari, H., Rahman, M., Sharififar, S., Reynolds, 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=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J. Vis. Exp. (45), e2393-e2393 (2010).</li><li>Azari, H., Sharififar, S., Rahman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J. Vis. Exp. (47), e2457-e2457 (2011).</li><li>Eramo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+expansion+of+the+tumorigenic+lung+cancer+stem+cell+population.">Identification and expansion of the tumorigenic lung cancer stem cell population.</a> Cell Death Differ. 15, 504-514 (2008).</li><li>Patrawala, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+purified+CD44++prostate+cancer+cells+from+xenograft+human+tumors+are+enriched+in+tumorigenic+and+metastatic+progenitor+cells.">Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells.</a> Oncogene. 25, 1696-1708 (2006).</li><li>Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+resistance+of+tumorigenic+breast+cancer+cells+to+chemotherapy.">Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy.</a> J Natl. Cancer. Inst. 100, 672-679 (2008).</li><li>Galli, R. <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+characterization+of+tumorigenic,+stem-like+neural+precursors+from+human+glioblastoma.">Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.</a> Cancer Res. 64, 7011-7021 (2004).</li><li>Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+label-retaining+tumour-initiating+cells+in+human+glioblastoma.">Evidence for label-retaining tumour-initiating cells in human glioblastoma.</a> Brain. 134, 1331-1343 (2011).</li></ol>

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神経幹細胞および正常成人と胎児の脳1、2、3、4から前駆細胞を単離するために、幹は、同じようにこのような肺5、前立腺6、乳房7、脳8、9のような癌組織由来の細胞ニューロスフェアのアッセイはまた、腫瘍されている頻繁に選択の方法として使用。 ex vivoでの多能、注入時に新しい腫瘍を作成する機能、および自己再生、このシンプルで再現性のアッセイを使用して、一つは体性幹細胞と同様の特性を示す切除した腫瘍組織からの細胞の無限数を生成することができます。これらの細胞は、細胞間相互作用、および分化、遊走、浸潤および細胞死を含む基本的な癌の細胞生物学を研究するために使用することができる。さらに、孤立した腫瘍幹様細胞はどのように腫瘍の形態、進行、および再発とも勉強し、最終的に治療するために洞察力を提供できる基盤となる派生する細胞メカニズムを解明する貴重なツールを提供オプション。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> タイプ </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> NeuroCult NSC基礎培地（ヒト） </td><td>中</td><td>セル技術を幹</td><td> 05750 </td><td></td></tr><tr><td> NeuroCult NSC増殖サプリメント（人間） </td><td>培地サプリメント</td><td>セル技術を幹</td><td> 05753 </td><td></td></tr><tr><td> ％0.05トリプシン- EDTA </td><td>試薬</td><td> GIBCO </td><td> 25300-062 </td><td></td></tr><tr><td> * MEM </td><td>試薬</td><td> GIBCO </td><td> 41500-018 </td><td> HEMのコンポーネント</td></tr><tr><td> * HEPES </td><td>試薬</td><td>シグマ</td><td> H4034 </td><td> HEMのコンポーネント</td></tr><tr><td> *蒸留水</td><td>試薬</td><td&gt;ギブコ</td><td> 15230-147 </td><td></td></tr><tr><td> ** DNase Iを</td><td>試薬</td><td>ロッシュ</td><td> 104159 </td><td></td></tr><tr><td> **大豆トリプシンインヒビター</td><td>試薬</td><td>シグマ</td><td> T6522 </td><td></td></tr><tr><td>ペン/連鎖球菌</td><td>試薬</td><td> GIBCO </td><td> 15140-122 </td><td></td></tr><tr><td> 10番メスの刃</td><td>手術道具</td><td> BD </td><td> 371610 </td><td></td></tr><tr><td>ペトリ皿</td><td>文化ウェア</td><td> BDファルコン</td><td> 353003 </td><td></td></tr><tr><td>小鉗子</td><td>手術器具</td><td>ファイン科学ツール</td><td> 11050〜10 </td><td></td></tr><tr><td>セルストレーナー</td><td>ふるい</td><td> BDファルコン</td><td> 352340 </td><td></td></tr><tr><td> T25フラスコ</td><td>文化ウェア</td><td> NalgeニュースキンNC国際</td><td> 136196 </td><td></td></tr><tr><td> T80フラスコ</td><td>文化ウェア</td><td> Nalge Nunc社国際</td><td> 178905 </td><td></td></tr><tr><td> 15 mlチューブ</td><td>文化ウェア</td><td> BDファルコン</td><td> 352096 </td><td></td></tr><tr><td> 50mlチューブ</td><td>文化ウェア</td><td> BDファルコン</td><td> 352070 </td><td></td></tr><tr><td> EGF </td><td>成長因子</td><td> R＆D </td><td> 2028 - EG </td><td></td></tr><tr><td> B - FGF </td><td>成長因子</td><td> R＆D </td><td> 3139 - FB </td><td></td></tr><tr><td>ヘパリン</td><td>成長因子</td><td>シグマ</td><td> H4784 </td><td> PBSで再構成</td></tr></tbody></table> * HEMを行うには、1M HEPESのMEM and160mlの1x10Lパケットを混在させると蒸留水を用いて8.75 Lにボリュームをもたらす。 7.4に最終的なPHを設定するD 4℃で保管してください **トリプシンインヒビター溶液を調製するには、まずDNase I溶液（100mgのDNaseがHEMの100mlに溶解）の10mlを確認してから、DNase溶液にトリプシンインヒビターの0.14 gを追加し、最終的にHEMを使用して1リットルにボリュームを構成する。 -20℃の冷凍庫で最終製品のアリコートをしてください。

isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay

幹様細胞は、乳がん、肺がん、大腸、前立腺、脳などの腫瘍に分離されている。すべてのこれらの腫瘍の重要な問題は、特に神経膠芽腫のmutliforme（GBM）で、腫瘍形成、進行、および再発における役割を調べるために腫瘍開始細胞集団を同定し、分離することです。細胞集団を開始する腫瘍を理解することはこれらの腫瘍に対する効果的な治療法を見つけるための手がかりを提供します。そのシンプルさと再現性に起因するロスフェアアッセイ（NSA）は、この腫瘍細胞の多くの単離および増殖のための選択の方法として使用されています。このプロトコルは、外科的に切除したヒトGBMの腫瘍組織に幹細胞様細胞を分離して展開するニューロスフェア培養法を示しています。手順は、初期の化学的消化と腫瘍組織の機械的解離、およびその後NSAの文化の中で、その結果、単一の細胞懸濁液をめっきなどがあります。 7〜10日後に、150から2の一次ニューロスフェア直径00μmのが観察され、さらに継代と拡大のための準備が整いましたことができます。

Watch this Scientific Journal Video about Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay at JoVE.com

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

このビデオプロトコルは、ニューロスフェアアッセイ培養法を用いて外科的に切除したヒト神経膠芽腫mutliforme（GBM）の腫瘍組織からの細胞のような分離と茎の拡大を示しています。

ニューロスフェアのアッセイを用いたヒト多形性膠芽腫の腫瘍細胞の単離と拡大

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

isolation-expansion-human-glioblastoma-multiforme-tumor-cells-using

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JoVE Journal

Neuroscience

29.7K ビュー.  University of Florida. このビデオプロトコルは、ニューロスフェアアッセイ培養法を用いて外科的に切除したヒト神経膠芽腫mutliforme（GBM）の腫瘍組織からの細胞のような分離と茎の拡大を示しています。

ビデオ: Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

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

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

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

ニューロスフェアのアッセイを使用して成体マウス神経幹細胞の単離と拡大

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

神経科学

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

ニューロスフェアのアッセイを使用してマウス胎児神経幹細胞培養の確立

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

人間の茸状の味覚乳頭細胞の単離および培養

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

誘導された神経細胞への成人の脳の周皮細胞由来細胞の系統の再プログラミング

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

最適化されたNを使用して急性脳スライスの準備-メチル-D-glucamine 保護回復法

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

F9 RARE-lacZをセルベースレポーターアッセイを使用したE8.5胚および神経球培養における相対的なレチノイン酸レベルの定量的測定

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

マイクロインジェクションを用いたヒトヘラ細胞におけるスプリセオソームsnRNA局在化の解析

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

出生後(P1-3)マウス神経起源ニッチからの神経球の分離と拡大

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

ヒト人工多能性幹細胞由来脳微小血管内皮細胞の誘導、拡大、凍結保存および特性評価

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

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

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

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

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

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