我々は、ビデオ援助をマークウォズニアックを承認したいと思います。また、培養ラットNC細胞の元のプロトコルのためのUTの南西で、ショーン·モリソンを承認したいと思います。この作品は、ヴァンダービルト大学医療センターアカデミックプログラムのサポートを支持し、NIH（HD36720とHD036720-11S109）とPALにAHA 11GRNT7690040、NAMにAHA（0615209B）とNIH（NS065604）から博士号を取得する前の奨学金と、ERPからの補助金によってであったNIH訓練助成金T32HD007502によってサポートされています。

1。プレートの準備<ol><li>すべての回で無菌テクニックを使用しています。 </li><li>ダルベッコPBS（DPBS）3.3 mLの最終容量にヒト血漿FNの株式の100μLを希釈することによってフィブロネクチン（FN）を準備します。最終濃度は30μg/ mLであり、これは1週間に4℃で保存することができます。 </li><li> FN溶液を滅菌組織培養4ウェルプレートの各ウェルの底をカバーし、15分間放置します。表面全体が覆われていることを確認してください。この時間（手順2および3）の間にメディアを作成します。 </li><li>ソリューションを、FN、プレートを乾燥することができます削除します。静かに500μLDMEM、削除DMEMでウェルを洗浄し、自己再生（SR）培地（下記参照）の500μLを追加します。 CO 2 5％を含む加湿インキュベーター中で37℃でインキュベートします。プレートは使用前に乾燥されるように解剖する前に、この約1時間を完了します。 </li></ol> 2。 SRメディアの準備<ol><li>迷走神経とトランクNCの培養10胚（約1リットル、マウスラインの遺伝的背景に依存します）から、SR培地の25mLを準備します。 12.5 mLの低グルコースDMEM、7.5 mLのNeurobasal中、25μLレチノイン酸（117μM最終濃度）、25μLの2 - メルカプトエタノール（50mMの最終濃度）を兼ね備えています。よく混ぜる。 </li><li> 3.75 mLの鶏胚エキス、250μLN 2塩サプリメント、500μLB27サプリメント、250μLペニシリン-ストレプトマイシン（1％最終濃度）を追加します。 0.22μmのフィルターを介してメディアをフィルタリングします。 </li><li> 10μLの滅菌IGF1（20μg/ mLの最終濃度）及び20μlの滅菌のbFGF（20μg/ mLの最終濃度）を追加します。転倒混和する。 4℃で保管してください</li></ol> 3。洗浄媒体の準備<ol><li>約10胚は洗浄培地の50mLを準備します。 35 mLの低グルコースDMEM、15 mLのNeurobasal Medium、および500μLペニシリン - ストレプトマイシン（1％最終濃度）で50mgのBSAを兼ね備えています。 0.22の滅菌フィルターμmのフィルター。 </li></ol> 4。コラゲナーゼ/ディスパーゼを準備する<ol><li> 5 mLのDPBS〜100 mg / mLのコラゲナーゼ/ディスパーゼの50μLを追加します。よく混ぜる。 </li><li>シリンジは、0.2μmのフィルターとフィルターと12ウェルプレートの3つのウェルの各々に1.5 mLを加える。残りのウェルにピペットで約1 mLの洗浄媒体。解剖組織を消化する準備が整うまで、氷上にプレート全体を格納します。 </li><li>滅菌したカミソリの刃とP20とP1000ピペットフィルターチップの先端をカットします。ちょうど先端の斜めのエッジの下にカットします。 P20は、孤立した組織片を転送するために使用されている間P1000のカットオフは、全胚を転送するために使用されます。 </li></ol> 5。 9.5 DPCの胚から単離する迷走神経およびトランクの神経管<ol><li>タイミング妊娠のために、膣栓とダムは、プラグが観察された朝昼で0.5 DPCと見なされます。 9.5 DPCで子宮を犠牲にして削除します。 </li><li>子宮から脱落膜を除去し、やさしくR脱落膜から胚を残したまま削除する。一度、このプロトコルで経験、滅菌DPBSの時間で3〜4 9.5 DPCの胚を分離します。無菌操作と滅菌解剖器具を使用しています。インスツルメンツは、オートクレーブ、加熱殺菌するか、またはエタノール中でのインキュベーションによって滅菌することができる。 </li><li>前迷走神経のNCの場合：インスリン針を使用して、半ばに耳プラコードで神経管を切った。 4 番目の体節の後縁で再びカットします。咽頭弓と心臓を削除するには、神経管の腹側組織をトリミングします。 </li><li>トランクNCの場合：インスリン針を使用して、体節16から22（または胚以前の22体節期より発達であれば最後の体節）間の神経管の一部を削除します。 </li><li>卵黄嚢とジェノタイピングのために残りの胚組織を保持します。 </li></ol> 6。非神経外胚葉と中胚葉の除去<ol><li> 10分間室温でコラゲナーゼ/ディスパーゼにセグメントを含む神経管を配置だ。直ちに洗浄培地で洗ってください。 </li><li>滅菌DPBSに組織を返します。無菌のインスリン針を使用して、静かに組織からの非神経外胚葉を除去し、離れて神経管から体節を分離します。体節中胚葉組織からの最後の残りの部分はカットダウンP20チップを用いて穏やかに摩砕することによって削除できます。神経管を損傷しないように注意してください。粉砕中に密接にこれを守ってください。 </li><li>洗浄媒体の2番目と3番目の30秒の洗浄によって分離された神経管を配置します。 </li><li> SR培地で一度洗浄し、（ステップ1.3）以前に調製したFN-コートしたウェルの中央に孤立した神経管を配置します。滅菌水の入ったお皿で低酸素室を加湿。低酸素室に皿を置き、3％混合ガスとO 2をチャンバーをフラッシュ（1％O 2、6％CO 2、93％N 2の混合物を含有するタンクを使用します）。 </li><li>常に股関節を確保するために非常に慎重にチャンバーを扱うtの外植片を邪魔されずにおり、井戸の中心に残っています。 </li><li> 37℃で手順5-6の概要を図1に示されています。 </li></ol> 7。神経管の除去<ol><li> 24時間のインキュベーションの後、穏やかに滅菌インスリン注射針を使用して移行細胞から神経管の端をからかうことで神経管を削除します。削除し、ステップ6.2のように無菌P20のカットを使用して媒体から神経管を破棄し、新鮮なSR培地（ 図2）で培地を交換してください。これは最も簡単に倒立顕微鏡を使用して行われます。 </li></ol> 8。代表的な結果低酸素状態で°C、NC細胞はほぼ純粋な集団（ 図3a）は神経管から移行した37℃で24時間のインキュベーションに続いて。時には、理想的な文化未満は、堅牢なoutgrowthsが得られません。たとえば、その24時間後にトンが可能です彼神経管は、それ自体に丸くなっているだろうとNCは神経管（ 図3b）から移行されません。時折神経管は、フィブロネクチンでコートしたプレートに取り付けられません。 我々の経験では、サブ最適なNCの移行や神経管の添付ファイル付きの問題が悪影響をそれぞれ、正常酸素条件またはフィブロネクチンの濃度によって影響を受けることができます。コラゲナーゼ/ディスパーゼの酵素活性を適切に調整する必要がありますバッチと消化時間によって若干異なりますが、しかし、15分以上の組織を消化していません。コラゲナーゼ/ディスパーゼの組織を含む神経管のOverdigestionも欠損outgrowthsになります。体節組織は簡単にコラゲナーゼ/ディスパーゼでインキュベートした後、神経管から削除されていない場合は、神経管は10分以上インキュベートすることができます。時には神経管は、基板に取り付けられません。このような場合は、fibronecをダブルチェックスズ濃度と低酸素条件。 正常酸素条件が文化野生型NCに使用できますが、低酸素状態は、より密接に生体内環境29,30で模倣する。我々の経験では、低酸素条件が培養するとき変異NC危険な状態になりました。たとえば、Foxd3変異NCは正常酸素条件下で培養した場合、トランクNCは、コントロールに比べて大幅に減少し、細胞伸長しました。植が低酸素状態（ 図4）で培養したときに突起の大きさのこの格差は削除されました。さらに、野生型の神経管の外植片は、正常酸素状態で培養した場合、カスパーゼ陽性細胞の数（データは示されていない）低酸素状態で培養された類似した外植片のことをして大きかった。低酸素状態にあるすべてのNCの文化を維持することによって、比較がより容易に制御および変異体の文化のダイナミクスとの間で行うことができます。 <img src="/files/ftp_upload/4134/4134fig1.jpg" alt="図1" /> <strong&gt;図1。NC分離の全体的な概略図。 A）胚から関心領域を解剖。 B）（15分を超えない）10分間コラゲナーゼ/ディスパーゼで神経管のダイジェスト。 C）洗浄培地で洗浄します。 D）非神経外胚葉と中胚葉の距離を解剖。 EF）は洗浄培地で2回洗浄する。自己再生培地中のG）プレート。 3パーセントO 2低酸素条件で37℃でインキュベートします。 <img src="/files/ftp_upload/4134/4134fig2.jpg" alt="図2" /> 図2片からの神経管の段階的除去。神経管は、非NCの細胞の混入を防ぐために、24〜48時間後に削除する必要があります。 A）神経管とNCの伸長（実線）との境界に注目してください。 B）インスリン注射針で神経管の端に沿って切り取ります。 C）神経管を破棄し、新鮮な自己再生培地で培地を交換してください。破線は、伸長の度合いを示します。略称：NT、神経管。 図3代表的な結果の例。低酸素条件下で自己再生培地中で24時間インキュベートした後A）典型的な片の伸長は、（破線）は、伸長の度合いを示します。 B）培養48時間後にNC伸長のビューを拡大。低伸長の収率でC）少ない理想的な文化。とCは同じ条件下で培養した。画像は文化の堅牢性で自然な範囲を示しています。これは、神経管の分離、FNの濃度、低酸素状態、およびコラゲナーゼ/ディスパーゼ消化の時間効率によって影響を受けることができます。 <img src="/files/ftp_upload/4134/4134fig4.jpg" alt="図4" /> 図4：正常酸素に対する低酸素症におけるNC植培養のin vitroでの解析。コントロール（野生型）NC細胞は、正常酸素培養条件下で48時間後に神経管の外植片から移行した。対照的に、Foxd3変異体NCが大幅に正常酸素の細胞outgrowthsを（NCのoutgrowthsの赤いアウトラインマークエッジ）減少した。同等の外植片は、低酸素条件下で栽培した場合は、Foxd3変異NC植は、その後の分析を可能にする、コントロールと同等になりました。 （注）、この動作は、in vivoでの変異体Foxd3 NCの動作とよく相関した。

<ol>
	<li>Le Douarin, N., Kalcheim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The neural crest. , (1999).</li><li>Kulesa, P. M., Gammill, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+migration:+patterns,+phases+and+signals.">Neural crest migration: patterns, phases and signals.</a> Developmental biology. 344, 566-568 (2010).</li><li>Saint-Jeannet, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neural crest induction and differentiation. , (2006).</li><li>Plank, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+and+timing+of+arrival+of+murine+neural+crest+on+pancreatic+beta+cell+development+and+maturation.">Influence and timing of arrival of murine neural crest on pancreatic beta cell development and maturation.</a> Developmental biology. 349, 321-330 (2011).</li><li>Nekrep, N., Wang, J., Miyatsuka, T., German, 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=Signals+from+the+neural+crest+regulate+beta-cell+mass+in+the+pancreas.">Signals from the neural crest regulate beta-cell mass in the pancreas.</a> Development. 135, 2151-2160 (2008).</li><li>Freem, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intrinsic+innervation+of+the+lung+is+derived+from+neural+crest+cells+as+shown+by+optical+projection+tomography+in+Wnt1-Cre;YFP+reporter+mice.">The intrinsic innervation of the lung is derived from neural crest cells as shown by optical projection tomography in Wnt1-Cre;YFP reporter mice.</a> J. Anat. 217, 651-664 (2010).</li><li>Cohen, A. M., Konigsberg, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clonal+approach+to+the+problem+of+neural+crest+determination.">A clonal approach to the problem of neural crest determination.</a> Developmental biology. 46, 262-280 (1975).</li><li>Sieber-Blum, M., Cohen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+of+quail+neural+crest+cells:+they+are+pluripotent+and+differentiate+in+vitro+in+the+absence+of+noncrest+cells.">Clonal analysis of quail neural crest cells: they are pluripotent and differentiate in vitro in the absence of noncrest cells.</a> Developmental biology. 80, 96-106 (1980).</li><li>Baroffio, A., Dupin, E., Douarin, N. M. L. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+precursors+for+neural+and+mesectodermal+derivatives+in+the+cephalic+neural+crest.">Common precursors for neural and mesectodermal derivatives in the cephalic neural crest.</a> Development. 112, 301-305 (1991).</li><li>White, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+stem+cells+undergo+cell-intrinsic+developmental+changes+in+sensitivity+to+instructive+differentiation+signals.">Neural crest stem cells undergo cell-intrinsic developmental changes in sensitivity to instructive differentiation signals.</a> Neuron. 29, 57-71 (2001).</li><li>Morrison, S. J., White, P. M., Zock, C., 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=Prospective+identification,+isolation+by+flow+cytometry,+and+in+vivo+self-renewal+of+multipotent+mammalian+neural+crest+stem+cells.">Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells.</a> Cell. 96, 737-749 (1999).</li><li>Bronner-Fraser, M., Sieber-Blum, M., Cohen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+of+the+avian+neural+crest:+migration+and+maturation+of+mixed+neural+crest+clones+injected+into+host+chicken+embryos.">Clonal analysis of the avian neural crest: migration and maturation of mixed neural crest clones injected into host chicken embryos.</a> J. Comp. Neurol. 193, 423-434 (1980).</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>Corpening, J. C. <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+live+imaging+of+enteric+progenitors+based+on+Sox10-Histone2BVenus+transgene+expression.">Isolation and live imaging of enteric progenitors based on Sox10-Histone2BVenus transgene expression.</a> Genesis. 49, 599-618 (2011).</li><li>Biernaskie, J. A., McKenzie, I. A., Toma, J. G., Miller, F. D. <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+skin-derived+precursors+(SKPs)+and+differentiation+and+enrichment+of+their+Schwann+cell+progeny.">Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny.</a> Nat. Protoc. 1, 2803-2812 (2006).</li><li>Chung, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+property+of+postmigratory+cranial+neural+crest+cells+and+their+utility+in+alveolar+bone+regeneration+and+tooth+development.">Stem cell property of postmigratory cranial neural crest cells and their utility in alveolar bone regeneration and tooth development.</a> Stem Cells. 27, 866-877 (2009).</li><li>Biernaskie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKPs+derive+from+hair+follicle+precursors+and+exhibit+properties+of+adult+dermal+stem+cells.">SKPs derive from hair follicle precursors and exhibit properties of adult dermal stem cells.</a> Cell Stem Cell. 5, 610-623 (2009).</li><li>Hagedorn, L., Suter, U., 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=P0+and+PMP22+mark+a+multipotent+neural+crest-derived+cell+type+that+displays+community+effects+in+response+to+TGF-beta+family+factors.">P0 and PMP22 mark a multipotent neural crest-derived cell type that displays community effects in response to TGF-beta family factors.</a> Development. , 126-3781 (1999).</li><li>Heanue, T. A., Pachnis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+and+isolation+of+enteric+nervous+system+progenitors+using+Sox2.">Prospective identification and isolation of enteric nervous system progenitors using Sox2.</a> Stem Cells. 29, 128-140 (2011).</li><li>Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+in+reduced+levels+of+oxygen+promotes+clonogenic+sympathoadrenal+differentiation+by+isolated+neural+crest+stem+cells.">Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells.</a> J. Neurosci. 20, 7370-7376 (2000).</li><li>Ito, K., Morita, T., 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=In+vitro+clonal+analysis+of+mouse+neural+crest+development.">In vitro clonal analysis of mouse neural crest development.</a> Developmental biology. 157, 517-525 (1993).</li><li>Etchevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+chick,+mouse+or+human+neural+crest+cells.">Primary culture of chick, mouse or human neural crest cells.</a> Nat. Protoc. 6, 1568-1577 (2011).</li><li>Mundell, N. A., Labosky, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+stem+cell+multipotency+requires+Foxd3+to+maintain+neural+potential+and+repress+mesenchymal+fates.">Neural crest stem cell multipotency requires Foxd3 to maintain neural potential and repress mesenchymal fates.</a> Development. 138, 641-652 (2011).</li><li>Gammill, L. S., Gonzalez, C., Gu, C., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+of+trunk+neural+crest+migration+requires+neuropilin+2/semaphorin+3F+signaling.">Guidance of trunk neural crest migration requires neuropilin 2/semaphorin 3F signaling.</a> Development. 133, 99-106 (2006).</li><li>Gammill, L. S., Gonzalez, C., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropilin+2/semaphorin+3F+signaling+is+essential+for+cranial+neural+crest+migration+and+trigeminal+ganglion+condensation.">Neuropilin 2/semaphorin 3F signaling is essential for cranial neural crest migration and trigeminal ganglion condensation.</a> Dev. Neurobiol. 67, 47-56 (2007).</li><li>Kasemeier-Kulesa, J. C., Bradley, R., Pasquale, E. B., Lefcort, F., Kulesa, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eph/ephrins+and+N-cadherin+coordinate+to+control+the+pattern+of+sympathetic+ganglia.">Eph/ephrins and N-cadherin coordinate to control the pattern of sympathetic ganglia.</a> Development. 133, 4839-4847 (2006).</li><li>Osborne, N. J., Begbie, J., Chilton, J. K., Schmidt, H., Eickholt, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semaphorin/neuropilin+signaling+influences+the+positioning+of+migratory+neural+crest+cells+within+the+hindbrain+region+of+the+the+chick.">Semaphorin/neuropilin signaling influences the positioning of migratory neural crest cells within the hindbrain region of the the chick.</a> Dev. Dyn. 232, 939-949 (2005).</li><li>Schwarz, Q., Maden, C. H., Vieira, J. M., Ruhrberg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropilin+1+signaling+guides+neural+crest+cells+to+coordinate+pathway+choice+with+cell+specification.">Neuropilin 1 signaling guides neural crest cells to coordinate pathway choice with cell specification.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 6164-6169 (2009).</li><li>Simon, M. C., Keith, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+oxygen+availability+in+embryonic+development+and+stem+cell+function.">The role of oxygen availability in embryonic development and stem cell function.</a> Nat. Rev. Mol. Cell Biol. 9, 285-296 (2008).</li><li>Ivanovic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+or+in+situ+normoxia:+The+stem+cell+paradigm.">Hypoxia or in situ normoxia: The stem cell paradigm.</a> J. Cell Physiol. 219, 271-275 (2009).</li></ol>

我々は、開示することは何もありません。

細心の注意は、このアプローチの成功を確実にするために胚の発達段階に支払わなければなりません。マウス初期胚の体節を数えると、ごみの中に胚を照合し、絶縁のための神経管の適切な領域を決定する段階の両方が重要です。胚の間に1つまたは2つの体節のバリエーションが行った実験の解像度に応じて、発生のタイミングの合理的な範囲内である。 9と9.5の間に胚は、DPCは17〜25体節が?...

<table border="1"><tbody><tr><td> 試薬 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> DMEM（低グルコース） </td><td> GIBCO / Invitrogen社</td><td> 11885 </td><td></td></tr><tr><td> Neurobasal中</td><td>ギブコ</td><td> 21103 </td><td></td></tr><tr><td> BSA </td><td>シグマ</td><td> A3912-10G </td><td></td></tr><tr><td> DPBS </td><td>ギブコ</td><td> 14190-144 </td><td></td></tr><tr><td> IGF1 </td><td> BDバイオサイエンス</td><td> 354037 </td><td> -20℃で50μg/ mLのアリコートでストア</td></tr><tr><td> bFGFの</td><td> BDバイオサイエンス</td><td> 354060 </td><td> -20℃で25μg/ mLのアリコートでストア</td></tr><tr><td>フィブロネクチン</td><td>ギブコ</td><td> 33016-015 </td><td> 1mg/mLらに格納されている-20℃でiquots </td></tr><tr><td>レチノイン酸</td><td>シグマ</td><td> R2625 </td><td> -20℃でエタノールに再溶解後35μg/ mlのアリコートでストア</td></tr><tr><td> 2 - メルカプトエタノール</td><td>シグマ</td><td> D-5637 </td><td></td></tr><tr><td> N 2の補足</td><td>ギブコ</td><td> 17502-048 </td><td></td></tr><tr><td> B27のサプリメント</td><td>ギブコ</td><td> 17504-044 </td><td></td></tr><tr><td> Steriflip0.22μmのフィルター</td><td>ミリポア</td><td> SCGP00525 </td><td></td></tr><tr><td>ペニシリン - ストレプトマイシン</td><td>インビトロジェン</td><td> 15140122 </td><td></td></tr><tr><td> 0.20μmのフィルター</td><td>コー​​ニング</td><td> 431219 </td><td></td></tr><tr><td>シリンジ（ろ過用） </td><td> BDバイオサイエンス</td><td> 301604 </td><td></td></TR&gt; <tr><td>四ウェルプレート</td><td>サーモフィッシャーサイエンティフィック</td><td> 176740 </td><td></td></tr><tr><td>コラゲナーゼ/ディスパーゼ</td><td>ロシュ社</td><td> 269​​ 638 </td><td>活動は、バッチによって異なります。 -20℃で100 mg / mLのアリコートでストア</td></tr><tr><td>インスリンの針 （29½ゲージ） </td><td>ベクトン·ディクソン</td><td> 309306 </td><td></td></tr><tr><td>低酸素チャンバー</td><td>ビラップス - ローゼンバーグ</td><td></td><td></td></tr><tr><td>酸素分析装置</td><td>ビラップス - ローゼンバーグ</td><td></td><td></td></tr><tr><td>鉗子＃5 </td><td>ファイン科学ツール</td><td></td><td>子宮脱落膜とを除去するために。 </td></tr><tr><td>トリプシン-EDTA（0.25％） </td><td>ギブコ</td><td> 25200 </td><td></td></tr></tbody></table>

isolation and culture of neural crest cells from embryonic murine neural tube

胚の神経堤（NC）は、神経管の背側面に由来する多能性前駆細胞集団である間葉移行（EMT）に上皮を受け、多様な細胞の種類1-3に上昇を与えて、胚全体に移行します。 NCはまた、標的器官4-6の分化と成熟に影響を与えるユニークな能力を持っています。 in vitroで外植する場合は、NC前駆細胞が自己再生を受けて、移行し、神経細胞、グリア細胞、平滑筋細胞、軟骨および骨を含む組織、さまざまな種類の細胞に分化する。 NCの多能性は、最初の鳥の神経管7-9の外植片から記述されていた。NC細胞の in vitro での分離では増殖、移行、および多能含むNCダイナミクスの研究を容易にします。胚10に戻って移植時に植NC細胞は、そのNCの可能性を保持することを明らかに鳥とネズミシステムの更なる作業-13。これらの固有の細胞の特性が植NC前駆細胞に保持されているため、神経管植アッセイは、in vitroでの NCを研究するための魅力的なオプションを提供しています。 哺乳類のNCのより良い理解を達成するために、多くのメソッドは、NC集団を分離するために採用されています。 NC-由来の前駆細胞がポスト渡り鳥NC前駆11,14-20のダイナミクスを研究するための胚と成人の両方で、ポスト渡り鳥の場所から培養することができる、しかし、彼らは神経管からの移住としてNC前駆細胞の分離は、最適な保全を提供していますNCの潜在的な細胞や渡り鳥プロパティ13,21,22。いくつかのプロトコルは、特定の前駆細胞11,13,14,17を濃縮NC人口を分離するために蛍光活性化細胞選別（FACS）を採用しています。しかし、初期段階の胚から始まるときは、分析のための十分な細胞数は初期のNC populatioの分離を複雑にし、FACSを得ることが困難である個々の胚からNS。ここでは、Wnt1-Creリコンビナーゼ活性化系レポーター23に基づいて約96％純粋なNC集団のFACS、その結果に依存しないアプローチを説明します。 ここで紹介する方法は、ラットNC 11,13の培養に最適化されたプロトコルから構成されている。このプロトコルは、従来の方法に比べての利点は、1）細胞はFACS））は、比較的純粋なNCの人口、3を取得する必要はありませんpremigratory NC細胞が分離されている、4）フィーダー層2上に成長されていない結果が簡単にあることです。定量化した。さらに、このプロトコルは、異なる遺伝的操作とNC特性の研究を促進し、任意の変異マウスモデルからNCの分離に使用することができます。このアプローチの制限は、NCはNC 2,24-28の生存、遊走、分化に影響を及ぼすことが知られている胚のコンテキストから削除されていることです。

Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube at JoVE.com

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

神経管から胚の神経堤の分離の使用を容易に<em&gt; in vitroで</em&gt;移行、自己再生し、神経堤の多能性を研究するための方法。

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 ...

isolation-culture-neural-crest-cells-from-embryonic-murine-neural

Research

JoVE Journal

Neuroscience

17.1K ビュー.  Vanderbilt University Medical Center. 神経管から胚の神経堤の分離の使用を容易に in vitroで移行、自己再生し、神経堤の多能性を研究するための方法。

ビデオ: Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

胚の脊髄から交連ニューロンの解剖と文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

神経科学

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

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

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 ...

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

マウス全胚培養における神経管閉鎖

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

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 ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

ラット胚神経細胞の分離と文化：クイックプロトコル

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

胚前脳からの内皮細胞の単離および培養

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

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 ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

マウス梁下ゾーンから成体神経幹細胞の単離および培養

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

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 ...

<meta charset="utf8"></head><body>マウス脳室内領域から単離されたNSCの効率的なヌクレオフェクション

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>著者スポットライト:マウス脳室下ゾーン由来神経幹細胞培養における高効率遺伝子導入のためのヌクレオフェクションの改善

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

成体マウス脳室下領域からの神経幹細胞の単離、増殖、およびヌクレオフェクション

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...