この作品は、GHTにニューヨーク幹細胞科学（NYSTEM C028107）グラントによってサポートされていました。

温度、栄養および明/暗サイクルのための動物の1コンディショニング<ol><li>世界中の数多くのNematostellaラボの1、または非営利業者からNematostella vectensis成人を得る（ 表1） </li><li>千あたり12部（PPT）の塩分で「1/3× &quot;人工海水（ASW）で、暗闇の中で（典型的には18と21°C）一定温度でNematostellaを維持します 。一般的に250 mlまたは1.5 L容量11、シンプルなソーダライムガラス培養皿で培養を維持します。 注：これらの単純な培養条件は、一般的にNematostellaを研究ラボの中で使用されているが、文化のケアも19を自動化することができます。 </li><li>週4倍- 2ノープリウスNematostella新しく孵化したアルテミアを養います。フル強度（36 PPT）または1/3倍の浅い長方形のガラス皿20内またはで30℃、でASWでハッチアルテミアシスト小規模、商業や自家製ブラインシュリンプ孵化場の数のいずれか。インキュベーターが使用できない場合は、エビはRTで孵化が、よりゆっくりとそうなります。 注：これは、多くの場合、完了のために24時間以上を必要とします。 </li><li>少なくとも週に一度アネモネ培養水を交換してください。最高の大人の健康のために、徹底的にクリーン（石鹸なし）文化が一度コートボウルと缶トラップ食べ残しや廃棄物の蓄積粘液分泌、の週をボウル、動物を絡ませます。 </li></ol>栄養的に馴化動物の2.選択とリラクゼーション<ol><li>ほぼ同じ長さの大きさがマッチしたポリープを選択（3〜5センチメートルを、ときに自然にリラックスした）前切断に3日間コロニーから分離ボウルに配置します。 注：切断のために選択された動物の数は、実験によって決定されますもちろん、行われているが、一般的に、我々は6反復でサンプル点当たり少なくとも5匹の動物をお勧めします。このように、典型的な実験では30匹の動物の最小値は、事前に選択されたことになります。 （下記参照）不規則な切断術の後スコアに影響を与えることができるので、一般には、より多くの最小数（30）より選択することが賢明です。 </li><li>切断する前に少なくとも1時間室内光にインキュベーターから選択した動物の食器を削除します。 注：室内光とハンドリングの振動への曝露は、おそらく動物が収縮する原因となりますので、実験台上でのインキュベーションによって適合または「リラックス」する必要があります。動物は触れるために、耐火及び露光となり、その時点での穏やかなピペッティングして移動することができます。 </li><li>オプション：（1/3×ASWに）7.5％のMgCl 2を追加することにより、動物を麻酔。静かに標準的なプラスチック5 mLのピペットでボウルへのMgCl 2溶液を追加します。 注：動物は、最終的に光へと物理的な操作に慣れになるだろうが、維持またはトンを「修正」するために動物を麻酔することが有利な場合があります彼らは細長い16,21,22となった後、彼は状態を緩和しました。 </li><li> 12 PPTのASWを含む直径100mmの滅菌ガラス切断皿の底に、切断されるように、プールから5匹の動物（1/3×ASWに）転送するために広い口径（&gt; 0.5センチメートル）プラスチックピペットを使用してください。 40X - 10の間に可変倍率の実体顕微鏡のステージに上の皿を置きます。 注：動物は、切断のために麻酔をかけ、リラックスされていない場合、彼らはまだ触れないように対応し、ステレオスコープの照明ので、再びリラックスになるために数分を必要とする場合があります。 </li></ol> 3.切断術<ol><li>滅菌メスを使用して、約限り、それは広いと全く腸間膜を含まないあるようですphysaのセクションを得ることを目標に、各ポリープから尾方physaを切断します。 注：理想的な切断部位は、腸間膜の終了にちょうど尾方です。切断面に薄いリットルに対して適切腸間膜からの移行がありますINESは、各腸間膜挿入（ 図1参照、矢印）に対応します。それは穴17,30 &#39;を差し込む」容易にすることができる粘液を生成するので、腸間膜の不在は重大です。 <ol><li>切断の所望の部位での動物と接触したメスの刃を配置します。 （フリーハンド）補助なし、または静かに＃5鉗子（デュモンスタイルまたは類似）で動物の体を把握することでこのいずれかの操作を行います。 </li><li>体全体の「ロッキング」運動にメスの湾曲した刃を活用することで、組織を通ってカット。 注：組織は、メスが揺動されるようにきれいに切断し、ドナーからphysaの所望の部分を解放する必要があります。組織の小片がまだ体とphysaを接続する場合は、メスでそれをカット。これはphysaに損傷を与える可能性として、引っ張ることにより、接続された作品を分離しようとしないでください。 </li></ol></li><li>皿から各切断「ドナー」ポリープを削除し、SEPAに戻します「プールされた切断者」の標識率ボウル。日付ボウルとストック培養物に戻します。 注：切断ポリープ日以内に尾方の傷を癒してくれるし、その後正常に供給することができます。彼らは、必要に応じてphysaが再び切断することができ、その時点で2週間以内に通常見physaを再生成します。 </li><li> 12 PPT ASWで切断皿に残って切除したphysaをすすぎ、その後、すでに各ウェルに12 PPT ASWの10ミリリットルを持つマルチウェル細胞培養プレートに別々の無菌ウェルに各physaを転送します。 注：この例では、各ウェル海水の10 mL及び5切除したphysaを保持して、6ウェルプレートを使用しています。一般海水でハンドリングし、潜在的な蒸発に起因する動きに空気への暴露を避けるために十分physaをカバーする必要があります。プレートまたはウェルは、蓋を持っている必要があります。 </li><li>各実験治療のために、各ウェルの予約で少なくとも5 physaを収集するために、3.3 - 繰り返して、3.1を繰り返します。 </li><li>箴ます温度でphysaをインキュベート計画された実験的な取調べのための再生の最高の速度をIDE。再生の所望の速度によって決定される一定の温度で、温度調節インキュベーターにphysaを含むプレートを置きます。 注：physaが欠落した組織を再生し、15と27℃の間の温度でインキュベートした場合、完全なポリープを形成することになります。再生の速度は、最初の2つのステージを除いて、温度依存性です。全ての温度のための段階4に到達するための平均日は、切断後7日であり、これはまた、21℃における再生と一致します。 27℃では、ステージ4は、約3日早く到達し、15℃で、ステージ4は、21°C（参照文献10）で再生と比較して約3 dだけ遅延されます。 </li></ol> 4. Nematostella再生ステージングシステム（NRSS）で再生を評価します<ol><li>変数壮麗なステレオ-化合物顕微鏡を用いてphysaスコアエーション（10 - 80X）。ステージ0のように新鮮にカットNematostellaの physaスコアと同時に、NRSS 10を使用して、毎日ポスト切断術（DPA）をスコアリングを続けます。 注：キーのステージング基準や詳細についてはリファレンス10を参照してください。 <ol><li>オープン創傷部位で、弛緩性バルーンに似ているcup-形の質量はそう見えるようたてカットphysaが表示された場合、ステージ0（開いた傷口）としてphysaスコア。 注：傷のエッジがまた最初から一緒に固執する可能性がありますが、組織はまだ崩壊され、剛性が不足しています。開放創のエッジは、創傷治癒のような半径方向の収縮を受けて観察することができます。 </li><li>切断術創が閉じて表示された場合（閉鎖した創傷）第1段階としてphysaスコア。 注：創傷場所は、将来の経口極に対応させていただきます。将来の経口ポールの周りの外表面は、基礎となる放射対称内胚葉腸間膜の挿入に対応する明確なアーチを表示し始める可能性があります。 </li><li>スコアのpHYSAステージ2（ラジアルアーチ）などの経口極の表面が放射状に対称なパターンに配置された8つの隆起したアーチを明らかにし、膨張した溝によって分離された表示されます。アーチの頂点に小さな半球状のブレブを守ってください。彼らは広い、おそらく一過性のように背の高い、と最初は単一の外胚葉細胞層によって構成についてになります。 注：いくつかのケースでは二重層ブレブを安定化させることができます。注：この以降の段階で粘液層がphysa膜状の「鞘」で（ 図2）をカプセル化するために表示されることがあります。このカプセル化材料は、スコアリングを容易にするために削除する必要があります。 </li><li>内胚葉と外胚葉組織層を含む触手の芽を安定的に、少なくともいくつかの半径方向のアーチの経口端部に形成されている場合には、ステージ3（触手）としてphysaスコア。 注：触手は、彼らが広く、最小限の運動性よりも長くなっています。腸間膜の基礎が可視になるようにphysaは増加したが、変数インフレ率が表示されます体腔（腔腸）に腸間膜の挿入から延びるible。 </li><li> physaは、彼らが接続されているように見えたところから測定その2倍の半径方向の幅よりもある口腔尾方の長さで、体壁に挿入から腔腸中に延びる8明確な、目に見える腸間膜が含まれている場合は、ステージ4（リニア腸間膜）としてphysaスコアその尾方端（enterostome）で咽頭へ。 注：4つ以下の腸間膜には、「プリーツ」内部の自由縁を持っています。咽頭が表示されます。以上8触手は、運動性表示され、時には彼らは体内に収縮します。 </li><li> physaはプリーツとつ以上の腸間膜を有しており、プリーツは、動物はほとんど &quot;通常の&quot;大人の外観を持っていますが、目に見えるはありませんステージ4でより完全かつしなやかである場合、ステージ5（主にプリーツ腸間膜）としてphysaスコア生殖細胞。 </li></ol></li></ol>

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M., Walzl, M., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+epithelial+organisation+of+muscle+cells+in+the+sea+anemone+Nematostella+vectensis.">Development and epithelial organisation of muscle cells in the sea anemone Nematostella vectensis.</a> Front Zool. 11, 44 (2014).</li><li>Kelava, I., Rentzsch, F., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+eumetazoan+nervous+systems:+insights+from+cnidarians.">Evolution of eumetazoan nervous systems: insights from cnidarians.</a> Philos Trans R Soc Lond B Biol Sci. 370 (1684), (2015).</li><li>Nakanishi, N., Renfer, E., Technau, U., Rentzsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nervous+systems+of+the+sea+anemone+Nematostella+vectensis+are+generated+by+ectoderm+and+endoderm+and+shaped+by+distinct+mechanisms.">Nervous systems of the sea anemone Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct mechanisms.</a> Development. 139 (2), 347-357 (2012).</li><li>Richards, G. S., Rentzsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+analysis+of+a+SoxB+gene+reveals+neural+progenitor+cells+in+the+cnidarian+Nematostella+vectensis.">Transgenic analysis of a SoxB gene reveals neural progenitor cells in the cnidarian Nematostella vectensis.</a> Development. 141 (24), 4681-4689 (2014).</li><li>DuBuc, T. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+Nematostella+vectensis+embryogenesis+and+late+development+using+fluorescent+probes.">In vivo imaging of Nematostella vectensis embryogenesis and late development using fluorescent probes.</a> BMC Cell Biol. 15, (2014).</li><li>Kaur, J., Debnath, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+at+the+crossroads+of+catabolism+and+anabolism.">Autophagy at the crossroads of catabolism and anabolism.</a> Nat Rev Mol Cell Biol. 16 (8), 461-472 (2015).</li><li>Carroll, B., Korolchuk, V. I., Sarkar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acids+and+autophagy:+cross-talk+and+co-operation+to+control+cellular+homeostasis.">Amino acids and autophagy: cross-talk and co-operation to control cellular homeostasis.</a> Amino Acids. 47 (10), 2065-2088 (2015).</li><li>Glick, D., Barth, S., Macleod, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+cellular+and+molecular+mechanisms.">Autophagy: cellular and molecular mechanisms.</a> J Pathol. 221 (1), 3-12 (2010).</li><li>Rodolfo, C., Di Bartolomeo, S., Cecconi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+stem+and+progenitor+cells.">Autophagy in stem and progenitor cells.</a> Cell Mol Life Sci. 73 (3), 475-496 (2016).</li><li>Guan, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+stem+cells.">Autophagy in stem cells.</a> Autophagy. 9 (6), 830-849 (2013).</li><li>Phadwal, K., Watson, A. S., Simon, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tightrope+act:+autophagy+in+stem+cell+renewal,+differentiation,+proliferation,+and+aging.">Tightrope act: autophagy in stem cell renewal, differentiation, proliferation, and aging.</a> Cell Mol Life Sci. 70 (1), 89-103 (2013).</li><li>Varga, M., Fodor, E., Vellai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+zebrafish.">Autophagy in zebrafish.</a> Methods. 75, 172-180 (2015).</li><li>Varga, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+is+required+for+zebrafish+caudal+fin+regeneration.">Autophagy is required for zebrafish caudal fin regeneration.</a> Cell Death Differ. 21 (4), 547-556 (2014).</li><li>Gonzalez-Estevez, C., Salo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+apoptosis+in+planarians.">Autophagy and apoptosis in planarians.</a> Apoptosis. 15 (3), 279-292 (2010).</li><li>Buzgariu, W., Chera, S., Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+investigate+autophagy+during+starvation+and+regeneration+in+hydra.">Methods to investigate autophagy during starvation and regeneration in hydra.</a> Methods Enzymol. 451, 409-437 (2008).</li><li>Tettamanti, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+invertebrates:+insights+into+development,+regeneration+and+body+remodeling.">Autophagy in invertebrates: insights into development, regeneration and body remodeling.</a> Curr Pharm Des. 14 (2), 116-125 (2008).</li></ol>

The authors have nothing to disclose.

創傷治癒および再生のモデルとしてNematostellaの使用は、ますます人気が高まっています。したがって、効果的な細胞および分子分析は、割り当てと比較することができる前に、特定のプロトコルの形態学的パターンを可視化できることが重要です。生活の後プラヌラの段階で、ほぼすべての場所で切断し、不足している構造を改革することができるという、回生「柔軟性」の高い学位?...

単一ヒドラにおける再生の観察は、実験科学の1,2のような生物学の出現で精液のイベントでした。再生は、生物学者に非常に広くアピールの現象のままと同様に人を置きます。人間の再生の限界を理解し、克服する発生生物学者、臨床医、生物医学科学者や組織のエンジニアの可能性はより本質的に興味深いより再生生物学になります 。 さて、このようなゲノム配列決定およびゲインと機能ツールの損失などの新たな技術を使用して、フィールドが回生機構を離れていじめるために、最終的に他の人がすることはできませんが再生成することができますどのように様々な種を理解する態勢を整えています。 、分子細胞性および形態学的応答における共通の度合いはまだ解明されていないが、今のところ再生することができる動物の間の基本的な応答がimagiであったであろうよりも類似していると思われますNEDのみ一昔前3。 特に、刺胞動物は、形態的多様性の広いスペクトルの中で、ほぼすべての身体の部分のを再生するに容易です。孤独な淡水ポリープから、ハイドラは、ポルトガル人-O-戦争のような複雑な植民地siphonophoresに、広大なサンゴ礁を構築小さな海洋ポリープと共に、再生はのための機構に加えて、多くの場合、再生モードであり、怪我や捕食に起因する破損や紛失の体の部分を修理または改。刺胞動物門の多様な種が再生のために類似または異なるメカニズムを使用するかどうかを根本的に興味深い質問4-6です。 私たち、そして他の人が再生7-17のモデルとして花虫類、Nematostella vectensisを開発してきました。我々は最近、aboraから二分組織の形態学的に均一な片から体全体の再生を記述するためのステージングシステムを開発しましたポリープ10のリットルの終わり。任意のレベルで二分したときNematostellaポリープを再生成することができますが、我々はこれが自然な無性分裂18の通常の平面に近いため一部では、ほとんどの形態学的に単純な地域、physaで尾方の位置で大人をカットすることを選んだ、ともあるため体全体が最も単純な形態学的構成要素から組み立て直されているかを観察し、分子解析を可能にします。 Nematostella再生ステージングシステム（NRSS）は、通常の培養条件下で、切断physaによって再生のいずれかの態様の進捗状況を採点するために使用することができる形態学的ベンチマークの比較的単純なセットを提供したり、実験的にそのような小分子治療、遺伝子操作などの状況を摂動しました、または環境の変化。予想通り、NRSSは再生の細胞および分子事象を参照することが可能な形態学的足場として採用されつつあります10。 最後に、切断の我々の方法は、最近の研究17で使用されるピンポイント穿刺よりも数桁大きいぽっかりと穴を生成し、まだ両方の傷は6時間の周りで癒します。創傷閉鎖の視覚的に逮捕した別個のフェーズを文書化することは傷の大きさと、それを閉じるのにかかる時間の見かけ上の独立性を説明するために実験的アプローチを示唆している必要があります。したがって、このプロトコルによって提供される尾方切断プロセスのより深い視覚的理解は、このモデル再生システムにさらなる研究を支援し、Nematostella vectensisのを使用して、この病期分類システムの適用を拡大します。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Nematostella vectensis, adults</td>
			<td>Marine Biological Lab (MBL)</td>
			<td></td>
			<td>non-profit supplier</td>
		</tr>
		<tr>
			<td>Glass Culture Dish, 250 ml</td>
			<td>Carolina Biological Supply</td>
			<td>741004</td>
			<td>250 ml</td>
		</tr>
		<tr>
			<td>Glass Culture Dish, 1,500 ml</td>
			<td>Carolina Biological Supply</td>
			<td>741006</td>
			<td>1,500 ml</td>
		</tr>
		<tr>
			<td>Polyethylene transfer pipette, 5ml&#160;</td>
			<td>USA Scientific&#160;</td>
			<td>1022-2500</td>
			<td>narrow bore, graduated</td>
		</tr>
		<tr>
			<td>Polyethylene transfer pipet, tapered</td>
			<td>Samco</td>
			<td>202-205</td>
			<td>cut off 1 inch of tip to make wide bore</td>
		</tr>
		<tr>
			<td>Disposable Scalpel</td>
			<td>Feather Safety Razor Co. Ltd</td>
			<td>no. 10</td>
			<td>blade should be curved</td>
		</tr>
		<tr>
			<td>#5 Dumont Fine point tweezers</td>
			<td>Roboz</td>
			<td>RS5045</td>
			<td>alternative suppliers available</td>
		</tr>
		<tr>
			<td>Pyrex petri dish, 100 mm diameter</td>
			<td>Corning&#160;</td>
			<td>3160</td>
			<td>can substitute other glass petri plates</td>
		</tr>
		<tr>
			<td>Sterile 6 well plate</td>
			<td>Corning Falcon&#160;</td>
			<td>353046</td>
			<td>or similar from other manufacturer</td>
		</tr>
		<tr>
			<td>Sterile 12 well plate</td>
			<td>Nunc&#160;</td>
			<td>150628</td>
			<td>or similar from other manufacturer</td>
		</tr>
		<tr>
			<td>Sterile 24 well plate</td>
			<td>Cellstar, Greiner bio-one</td>
			<td>662-160</td>
			<td>or similar from other manufacturer</td>
		</tr>
		<tr>
			<td>Brine shrimp hathery kit</td>
			<td>San Francisco Bay; drsfostersmith.com</td>
			<td>CD-154005</td>
			<td>option for growing brine shrimp</td>
		</tr>
		<tr>
			<td>pyrex baking dish</td>
			<td>common in grocery stores</td>
			<td></td>
			<td>option for growing brine shrimp</td>
		</tr>
		<tr>
			<td>artificial seawater mix 50 gal or more&#160;</td>
			<td>Instant Ocean; drsfoster-smith.com</td>
			<td>CD-116528</td>
			<td>others brands may suffice</td>
		</tr>
		<tr>
			<td>Plastic tub for stock ASW preparation</td>
			<td>various</td>
			<td></td>
			<td>common 25 gallon plastic trash can OK</td>
		</tr>
		<tr>
			<td>Polypropylene Carboy</td>
			<td>Carolina Biological Supply</td>
			<td>716391</td>
			<td>For working stock of ASW @ 12 ppt</td>
		</tr>
		<tr>
			<td>Beaker, Graduated, 4,000ml</td>
			<td>PhytoTechnology Laboratories</td>
			<td>B199</td>
			<td>For dilution of 36 ppt ASW to 12 ppt</td>
		</tr>
		<tr>
			<td>Stereomicroscope and light source</td>
			<td>various&#160;</td>
			<td></td>
			<td>with continuous 1 - 40x magnification&#160;</td>
		</tr>
	</tbody>
</table>

inducing complete polyp regeneration from the aboral physa of the starlet sea anemone nematostella vectensis

刺胞動物、具体的にHydraは 、損傷を受けたり、切断構造を再生するために示した第一の動物であった、そして実際にこのような研究は間違いなく250年以上前にトレンブレーの仕事を通じて、現代生物学的調査を開始しました。現在再生の研究では、両方の「クラシック」の再生などヒドラ 、プラナリアやUrodelesのような生物、ならびに海綿から哺乳動物を介して、後生動物の範囲を、スパニング種の拡大スペクトルを用いて復活を見ています。生物学的現象としての本質的な関心に加えて、再生は様々な種でどのように機能するかを理解することは、再生プロセスは、共通の特徴および/または種またはコンテキスト固有の細胞および分子メカニズムを共有しているかどうかをお知らせします。女優のイソギンチャク、Nematostella vectensisは 、再生のための新たなモデル生物です。 ヒドラのように、Nematostellaは古代の門、刺胞動物のメンバーですが、トン内彼のクラス花虫綱、進化的により基礎であるヒドロ虫の姉妹クレード。したがってNematostellaでの再生の側面は、比較し、 ヒドラ及びその他の刺胞動物のものと対比するために興味深いものになります。この記事では、我々は、二等分観察し、physaと呼ばれるNematostella大人の尾方端の再生を分類する方法を提示します。 physaは自然に無性生殖の手段として、核分裂を起こし、自然核分裂またはphysaの手動切断のいずれかは、複雑な形態の再成長と改革をトリガします。ここでは、Nematostella再生ステージングシステム（NRSS）におけるこれらの単純な形態学的変化を成文化しています。私たちは、クロロキンの効果、リソソーム機能ブロックのオートファジーの阻害剤をテストするためにNRSSを使用しています。結果は、自食作用が阻害されたときにポリープ構造、特に腸間膜の再生が異常であることを示しています。

切断されたphysaで再生中の形態学的イベントの進行は、各NRSS段階でphysaの代表的な景色を備えて、図1Aに示されています。典型的なphysa切断部位は、成人（矢じり）に示されています。 図1Aの写真は完全に形成されたポリープを介して新鮮にカットphysaから口頭と身体構造のプログレッシブ再生を示しています。 図1B、Cにはそれぞ?...

Watch this Scientific Journal Video about Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis at JoVE.com

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

スターレットイソギンチャクの尾方Physaから完全ポリープ再生を誘導<em&gt; Nematostella vectensis</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

inducing-complete-polyp-regeneration-from-aboral-physa-starlet-sea

Research

JoVE Journal

Developmental Biology

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

8.4K ビュー.  Stony Brook University. Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

ビデオ: Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

幹細胞から網膜色素上皮細胞の効率的な導出

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

発生生物学

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

の血液細胞集団のアッセイ<em&gt;キイロショウジョウバエ</em&gt;幼虫

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

ゼブラフィッシュ再生の分子機構を研究するためにテトンシステムの利用

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science. One of the most demanding technical aspects of culture of this species in the laboratory is rearing first-feeding larvae to the juvenile stage with high rates of growth and survival. The central management challenge of this developmental period revolves around delivering highly nutritious feed items to the fish on a nearly continuous basis without compromising water quality. Because larval zebrafish are well-adapted to feed on small zooplankton in the water column, live prey items such as brachionid rotifers, Artemia, and Paramecium are widely recognized as the feeds of choice, at least until the fish reach the juvenile stage and are able to efficiently feed on processed diets. This protocol describes a method whereby newly hatched zebrafish larvae are cultured together with live saltwater rotifers (Brachionus plicatilis) in the same system. This polyculture approach provides fish with an "on-demand", nutrient-rich live food source without producing chemical waste at levels that would otherwise limit performance. Importantly, because the system harnesses both the natural high productivity of the rotifers and the behavioral preferences of the fish, the labor involved with maintenance is low. The following protocol details an updated, step-by-step procedure that incorporates rotifer production (scalable to any desired level) for use in a polyculture of zebrafish larvae and rotifers that promotes maximal performance during the first 5 days of exogenous feeding.

Larval zebrafish are adapted to feed on zooplankton. It is possible to capitalize on this natural feature in the laboratory by growing first feeding fish together in the same system with live saltwater rotifers. This "polyculture" strategy promotes high growth and survival with minimal labor and disturbance to the larvae.

飼育第1の給電ゼブラフィッシュの完全および更新された「ワムシPolyculture方法」

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science.  One of the most demanding technical aspects of ...

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to any type of injury. Common injury models in these animals remove large amounts of tissue, which damages multiple organs. To overcome this broad tissue damage, we describe here a method to selectively remove a single organ, the pharynx, in the planarian Schmidtea mediterranea. We achieve this by soaking animals in a solution containing the cytochrome oxidase inhibitor sodium azide. Brief exposure to sodium azide causes extrusion of the pharynx from the animal, which we call &#34;chemical amputation.&#34;&#160;Chemical amputation removes the entire pharynx, and generates a small wound where the pharynx attaches to the intestine. After extensive rinsing, all amputated animals regenerate a fully functional pharynx in approximately one week. Stem cells in the rest of the body drive regeneration of the new pharynx. Here, we provide a detailed protocol for chemical amputation, and describe both histological and behavioral methods to assess successful amputation and regeneration.

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

The planarian Schmidtea mediterranea is an excellent model for studying stem cells and tissue regeneration. This publication describes a method to selectively remove one organ, the pharynx, by exposing animals to the chemical sodium azide. This protocol also outlines methods for monitoring pharynx regeneration.

化学の切断とSchmidtea メディテラネアプラナリアの咽頭再生

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

ラッパムシの再生の研究のための方法

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

大人の皮膚組織から人間ケラチノ サイト主を特定する簡易で効率的な方法

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

臍帯血由来から 3 D 肌 Organoid の生成誘導多能性幹細胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Genotyping of Sea Anemone during Early Development

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the life of the animal. Following in vitro fertilization and de-jellying, zygotes are allowed to develop for 24 h at room temperature to reach the early- to mid-gastrula stage. The gastrula embryos are then placed on an agarose gel bed in a Petri dish containing seawater. Under the dissecting microscope, a tungsten needle is used to surgically separate an aboral tissue fragment from each embryo. Post-surgery embryos are then allowed to heal and continue development. Genomic DNA is extracted from the isolated tissue fragment and used as a template for locus-specific PCR. The genotype can be determined based on the size of PCR products or presence/absence of allele-specific PCR products. Post-surgery embryos are then sorted according to the genotype. The duration of the entire genotyping process depends on the number of embryos to be screened, but it minimally requires 4&#8211;5 h. This method can be used to identify knockout mutants from a genetically heterogeneous population of embryos and enables analyses of phenotypes during development.

The goal of this protocol is to genotype the sea anemone Nematostella vectensis during gastrulation without sacrificing the embryo.

初期開発におけるアネモネのジェノタイピング

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

マウス肝外胆管からオルガノイドの生成

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...