マルクスPajtlerとロバートボーラーのサポートやアドバイスは、このプロトコルの可視化のための非常に貴重でした。

動物実験は動物実験で定められている実験的な手続きと規制のための動物の管理と使用に関するNI​​Hのガイドラインに従って、欧州共同体理事会指令（86/609/EEC）に準拠して行われたという著者らの状態ケアとデュースブルク - エッセン大学（ドイツ）での委員会（IACUC）を使用してください。 パート1：（ビデオで示されていない）の設定 （インキュベータから卵を取る前に）材料と溶液の調製<ul><li>卵は37℃で11日間の加湿インキュベーター中でインキュベートする必要があります° C（100 ° F）。 </li><li> 70％エタノールのスプレー、クリーンプレート、正確な清潔なピンセットを用い、4℃（40 ° F）冷滅菌氷上でDMEM、60 mLの滅菌シリンジ</li><li> 50 mLの滅菌コーニングチューブには、° C、コールドDMEMは1:1の比率（：DMEM浸軟質量）で浸軟した胚を混在させる4を半分埋める必要があります。早く卵がニワトリ胚のインキュベータの解剖から取り出されているとして起動する必要があります。 </li><li>オートクレーブや解剖の日に解剖ツールを殺菌、20分間70％エタノールでツールを浸す。 </li></ul>第2部：ニワトリ胚の解剖（ビデオで実証） <ol><li>少なくとも30秒間、70％エタノールを噴霧インキュベート卵を湿らせ、過度に卵を振っていないがきれいで柔らかなペーパータオルで慎重にそれらを乾燥させます。 注：私たちは、同時に60以上のニワトリ胚を解剖することはお勧めしません。 </li><li>鋭いエッジに対して破線で慎重に卵を開いて、卵黄の袋またはニワトリ胚自体を破壊しない、注意して胚を取り出す。 </li><li>すぐに臍帯の開始のための検索と卵黄の袋または少し船を破壊することなく明確なラッピングを開きます。その後、正確な清潔なピンセットを用い、へその緒を解剖。 </li><li>卵黄の袋から胚を取り出す。 注：胚が（ビデオに示されていない）すぐに断頭してください。 </li><li> 4で最小必須培地で胚を収集℃に</li></ol>パート3：ニワトリ胚のマセラシオン<ol><li> 60 mLの滅菌注射器の外プランジャーを取る。 </li><li> one 60mLのシリンジに約10胚を転送します。 </li><li>浸軟物質を失うリスクにしないために慎重に戻ってシリンジにプランジャーを置く。 </li><li>プランジ​​ャーを押し下げることによって痩せ衰えさせる。 </li><li>準備コーニングチューブ1:1（：DMEM浸軟質量）の比でDMEMで半分満たさで浸軟した質量を収集する。約25 mLの量が生産される10の胚を使用する。 </li><li> 4℃45分℃でシェーカーで混合物を置かない</li></ol>第4部：ニワトリ胚の遠心分離 - DMEMサスペンション<ol><li>ニワトリ胚を移す - DMEM懸濁液を遠心管に。 </li><li>胚の25 mg当たり1ミリグラムの濃度で滅菌ヒアルロニダーゼを追加。 </li><li>非常に正確に風袋遠心分離チューブ。 注：遠心操作は非常に高いg力および材料または不均衡なチューブを使用する場合は、遠心機が発生する可能性の損傷時に実行されるため、この手順は慎重に行われるべき。すべてのチューブの小数点以下をDMEMで不足額を調整することで受信する必要があります後に3つのポジションについて、マシンに基づいて秤量精度の高い。 </li><li> 4に遠心分離機の温度を下げる℃に 注：また、遠心分離ローターは、一晩冷却室または冷蔵庫に保管してください。 </li><li>少なくとも180.000 xgx時間にわたって6時間を遠心注：単相の分離に関する優れた結果を得るためには266.000 xgx hを使用する可能であれば、バキューム調整を使用してください。 </li></ol>第5部：ニワトリ胚抽出物のろ過遠心分離後、3つのフェーズは気づいたことができる：脂肪のフラグメントを持つ上層薄汚い液相、中央にある透明な液体の相と遠心チューブの下部にあるペレットを注：遠心混合物の振動や移動活発になる必 ​​要がありますこれは明確な中心的な液相​​を失うことによって遠心分離のステップの結果を破壊するように厳密に、避けて。 注：すべての次の手順では、層流のベンチを使用して無菌条件下で実行する必要があります。 <ol><li> 0.45μmの細孔を有するフィルターシステムに明確な液相をピペット。その後、接続された真空ポンプのスイッチをオンにします。 注：下部にあるペレットに触れないでください。これは、抽出液とフィルターシステムの閉塞の汚染につながる。 </li><li> 0.22μmのと接続された真空ポンプのスイッチを再度の細孔を持つフィルターシステムに事前に抽出して転送する。 </li><li>注し達成ニワトリ胚エキス滅菌15mlのコーニングチューブインチ</li><li>迅速° C（-62 ° F）-80分注して保存する。注：-80で保存した場合最長2年間の最後のCEE℃に</li></ol>

<ol>
	<li>Bronner-Fraser, M., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+analysis+reveals+multipotency+of+some+avian+neural+crest+cells.">Cell lineage analysis reveals multipotency of some avian neural crest cells.</a> Nature. 335, 161-164 (1988).</li><li>Stemple, D. L., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+stem+cell+for+neurons+and+glia+from+the+mammalian+neural+crest.">Isolation of a stem cell for neurons and glia from the mammalian neural crest.</a> Cell. 71, 973-985 (1992).</li><li>Rao, M. S., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+and+controlled+in+vitro+differentiation+of+murine+multipotent+neural+crest+stem+cells.">Immortalization and controlled in vitro differentiation of murine multipotent neural crest stem cells.</a> J Neurobiol. 32, 722-746 (1997).</li><li>Paratore, C., Goerich, D. E., Suter, U., Wegner, M., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+glial+fate+acquisition+of+neural+crest+cells+are+regulated+by+an+interplay+between+the+transcription+factor+Sox10+and+extrinsic+combinatorial+signaling.">Survival and glial fate acquisition of neural crest cells are regulated by an interplay between the transcription factor Sox10 and extrinsic combinatorial signaling.</a> Cell. 128, 3949-3961 (2001).</li><li>Kim, J., Lo, L., Dormand, E., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX10+maintains+multipotency+and+inhibits+neuronal+differentiation+of+neural+crest+stem+cells.">SOX10 maintains multipotency and inhibits neuronal differentiation of neural crest stem cells.</a> Neuron. 38, 17-31 (2003).</li><li>Anderson, D. J., Groves, A., Lo, L., Ma, Q., Rao, M., Shah, N. M., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+determination+and+the+control+of+neuronal+identity+in+the+neural+crest.">Cell lineage determination and the control of neuronal identity in the neural crest.</a> Review Cold Spring Harb Symp Quant Biol. 62, 493-504 (1997).</li><li>Dorsky, R. I., Moon, R. T., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+signals+and+cell+fate+specification+in+premigratory+neural+crest.">Environmental signals and cell fate specification in premigratory neural crest.</a> Review Bioessays. 22, 708-716 (2000).</li><li>Sieber-Blum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+controlling+lineage+specification+in+the+neural+crest.">Factors controlling lineage specification in the neural crest.</a> Int Rev Cytol. 197, 1-33 (2000).</li><li>Dupin, E., Real, C., Ledouarin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+crest+stem+cells:+control+of+neural+crest+cell+fate+and+plasticity+by+endothelin-3.">The neural crest stem cells: control of neural crest cell fate and plasticity by endothelin-3.</a> Int Rev Cytol. 73, 533-545 (2001).</li><li>Maurer, J., Fuchs, S., J&#228;ger, R., Kurz, B., Sommer, L., Schorle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+controlled+differentiation+of+neural+crest+stem+cell+lines+using+conditional+transgenesis.">Establishment and controlled differentiation of neural crest stem cell lines using conditional transgenesis.</a> Differentiation. 75, 890-891 (2007).</li><li>Wu, Y. Y., Mujtaba, T., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+stem+and+precursor+cells+from+fetal+tissue.">Isolation of stem and precursor cells from fetal tissue.</a> Methods Mol Biol. 198, 29-40 (2002).</li></ol>

利害の衝突は宣言されません。

このプロトコルは過去10,11に記載されている手順の変更に基づいています。前者の出版物に加えて、我々はここで、プロセスの個々の異なるステップより多くの拡張を示します。これが正しい手続きで信頼性の高い成果を保証する重要な詳細と実験者をサポートしています。さらに、解剖プロセスとして我々は細部に卵黄の袋から胚を取り出すの手順を記述するCEEの抽出の重要な部分です。以下に述べるように注意することは、いくつかの重要な点があります。 インキュベーションが終了した後のニワトリ胚の解剖はできるだけ早く開始する必要があります。前の解剖に死んでいる胚に悪影響結果に影響を与える可能性がある酵素活性とタンパク質の分解など、このプロトコルのいずれかのステップには使用しないでください。 いくつか手の訓練を始める前にすることは解剖のプロセスのすべてのステップとして実行する必要があります迅速かつ正確に実施する必要があります。それは否定的に干渉するタンパク質の顕著な量を伴っていた卵黄と汚染を避けるために、手順全体の成功にとって重要です。胚は卵黄の袋から取り出した後、彼らは（ビデオには示されていない）すぐに断頭してください。 温度だけでなく、ローターのセレリティは、あまりにも、非常に重要です。酵素活性化とNCSCMに必要な要因の質と量の減少を避けるために遠心分離し、ローターの温度を4に減らす必要が℃に遠心分離工程の後に透明な中央の液相はNCSCMに不可欠な成長因子を含む重要なプリエキスです。異なる画分と要因の濃縮のかなりの分離を達成するために我々は180.000 xgx hよりではない低いセレリティを推薦が必要透明な液相と遠心チューブの上部にある薄汚い液相間の密度の違いとして、任意の揺れを回避する必要がある二つの部分のリミックスを妨げるものではない。それの脂肪の部分が非常に早くフィルターシステムの目詰まりにつながるとして、薄汚い液相の成分は、0.45μmのフィルターシステムを介して濾過することができます。それの部分はフィルタシステムの目詰まりにつながるとしても、遠心分離機の下部にあるペレットは、全く触れてはいけません。 最後のステップでNCSCsの栽培のためのCEEの可能性が検証される必要があります。幹細胞のこの多能一時的な人口は、十分な生産CEEを追加せずにメディアに急速にその実行可能性を失います。したがって、導入遺伝子活性依存的に早期のNCSCのマーカーを発現し、NCSC生物学を研究するために使用されるトランスジェニックマウス系統（JoMa1）は、CEE 10のすべての新たに得られた電荷で栽培されている。 NCSCsの成功した栽培は、CEEの適切な検証を表します。

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>11 days incubated chicken eggs</td>
<td>&nbsp;</td>
<td>S&ouml;rries-Trockels Vermehrungszucht GmbH &amp; Co.KG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Alcoholic disinfection spray</td>
<td>&nbsp;</td>
<td>Sch&uuml;lke &amp; Mayr GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DMEMGlutamax</td>
<td>&nbsp;</td>
<td>GIBCO Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Syringe sterile (60 ml)</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tubes sterile (50; 15 ml)</td>
<td>&nbsp;</td>
<td>Greiner Bio-One GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipet tips sterile</td>
<td>&nbsp;</td>
<td>Starlab GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Hyaluronidase typeIV-S</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Milipore stericups sterile (0.45; 0.22 &mu;l)</td>
<td>&nbsp;</td>
<td>Fisher Scientific AG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipettes sterile (25; 10; 5 ml)</td>
<td>&nbsp;</td>
<td>Greiner Bio-One GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge L5-50</td>
<td>&nbsp;</td>
<td>Rotor type Ti-45</td>
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production of chick embryo extract for the cultivation of murine neural crest stem cells

神経堤は、胚発生中に神経外胚葉から発生し、一時的にしか保持されます。初期の実験は、すでに神経堤1の重要部分であると多能性前駆細胞をへのドア。表現型、神経堤幹細胞（NCSC）を同時に神経堤2,3,4,5から、その移行中にP75（低アフィン神経成長因子受容体、LNGFR）とSOX10の発現によって定義されます。これらの前駆細胞は平滑筋細胞、クロム親和性細胞、神経細胞とグリア細胞だけでなく、メラノサイト、軟骨や骨6,7,8,9に分化することができる。 in vitroで NCSCを育成するために、特別な神経堤幹細胞の培地（NCSCM）が10必要です。 NCSCMの最も複雑な部分は、NCSCのためだけでなく、神経外植片の他の種​​類の成長因子の本質的なソースを表すニワトリ胚抽出物（CEE）の準備です。 CEEの横にある他のNCSCM成分は市販されている。それは分離、浸軟、遠心分離、およびろ過プロセスなどの挑戦的な詳細を知るために非常に重要であるCCE使用して実験室の標準機器を生産。このプロトコルでは、純粋かつ高品質CEEの最大化の量を生産するために、正確なテクニックを説明します。

Watch this Scientific Journal Video about Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells at JoVE.com

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

in vitroで神経堤幹細胞（NCSC）を育成するために、特殊な培地（NCSCM）が必要です。 NCSCMの本質的な部分は、ニワトリ胚抽出物（CEE）です。ここでは、分離、浸軟、遠心分離、およびろ過プロセスなどの詳細を含む、純粋で高品質CEEの最大化量を、生成する正確なテクニックを説明します。

マウス神経堤幹細胞の培養のためのニワトリ胚抽出物の製造

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

production-chick-embryo-extract-for-cultivation-murine-neural-crest

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.7K ビュー.  University Children's Hospital Essen. in vitroで神経堤幹細胞（NCSC）を育成するために、特殊な培地（NCSCM）が必要です。 NCSCMの本質的な部分は、ニワトリ胚抽出物（CEE）です。ここでは、分離、浸軟、遠心分離、およびろ過プロセスなどの詳細を含む、純粋で高品質CEEの最大化量を、生成する正確なテクニックを説明します。

ビデオ: Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

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 ...

神経科学

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 ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

ニワトリ胚における軸索軌道とシナプスターゲットを追跡する後脳のエレクトロ

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

大人の齧歯類の脳の従来の従来にない地域から神経幹細胞を増殖させる

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within 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 ...

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. ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

強膜網膜移植ひと網膜色素上皮細胞のラットの目の洗練されたプロトコルの開発

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

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

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

マウス萌芽期の神経幹細胞の分離と培養

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

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

シアログリカンの代謝標識による原発神経幹および前駆細胞の細胞表面マーカーの同定

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...