这项工作是由纽约干细胞科学（NYSTEM C028107）格兰特GHT的支持。

1.温度，营养和光/暗周期调节动物<ol><li>来自世界各地的众多Nematostella实验室之一，还是一个非营利性的供应商获取Nematostella vectensis成人（ 表1） </li><li>在黑暗中在恒定温度（通常介于18和21℃）保持Nematostella，在"1/3×"人造海水（ASW）中的12份每千（ppt）的盐度。保持培养物在简单的钠钙玻璃培养皿，通常为250毫升或1.5升容量11。 注意:这些简单的培养条件通常是学习Nematostella实验室中使用，但文化的护理也可以自动的19。 </li><li>饲料Nematostella刚孵出的卤虫无节幼体2 - 4倍每星期。孵化丰年虫卵完全强度（36个百分点），或者1 / 3X ASW在30℃，在一个矩形浅玻璃盘20或任何一种规模小，商业或自制盐水虾孵化场的。如果一个孵化器不可用，虾孵化在室温下，但这样做的更慢。 注意:这通常需要完成超过24小时。 </li><li>每周更换海葵培养水至少一次。为了获得最佳成人健康，彻底清洁（不含肥皂）文化周累积粘液分泌物，这大衣碗，可以捕获吃剩的食物和垃圾，并套住动物碗一次。 </li></ol> 2.选择和营养学放松空调动物<ol><li>大致相同的长度的选择大小匹配息肉（3 - 5厘米，当自然松弛），并放置在从菌落中分离为截肢前三天碗。 注意:选择用于切割的动物的数量将通过实验正在进行当然，确定，但一般而言，我们推荐每个采样点的至少五只动物具有六个重复。从而，在典型的实验中的最低30的动物将被预选。一般情况下，这是明智的选择，因为相比那些不规则的（见下文）可以在以后影响得分截肢的最低数量（30）更多。 </li><li>从培养箱截肢之前去除所选动物的盘进入房间的灯的至少一小时。 注意:暴露于室内光线和振动的处理可能会导致动物收缩，所以他们需要适应或在实验台上孵化"轻松"。动物将变得难治触摸并曝光，并在该点可通过温和移液移动。 </li><li>可选:通过添加7.5％的MgCl 2（1/3×ASW）麻醉动物。轻轻地用标准的塑料5毫升吸管加入氯化镁溶液到碗里。 注意:虽然动物最终将成为习惯于光且物理操纵，它可能是有利的麻醉动物，以维持或"修理"吨他放松的状态后，他们已成为拉长16,21,22。 </li><li>使用宽孔（&gt; 0.5，厘米）的塑料吸管转移（在1/3×ASW）的五只动物从池中被截肢，到含有12个百分点ASW的直径100毫米的无菌玻璃切割盘的底部。放置培养皿上以10之间可变放大倍率立体显微镜的阶段 - 40X。 注意:如果动物没有被麻醉和轻松的切割，它们仍可能响应触摸和体视镜照明，因此可能需要几分钟的时间来重新变得轻松。 </li></ol> 3.截肢<ol><li>用无菌解剖刀，从每个息肉锯掉的反口physa，其目标是获得是只要大约是它的宽度和不含肠系膜physa的截面。 注:理想的切割位点就是反口的肠系膜终止。在切割面有来自肠系膜适当薄L的过渡对应于每个mesenterial插入（ 见图1，箭头）分级表。肠系膜缺席是至关重要的，因为它产生可便于&#39;堵&#39;的孔17,30粘液。 <ol><li>放置手术刀刀片在截肢的期望部位与动物接触。做到这一点无论求告无门（写意），或用5号钳（杜蒙风格或类似）轻轻地抓动物的身体。 </li><li>穿过组织通过利用手术刀的弯曲叶片横跨主体上的"摇摆"运动切割。 注:组织应断绝干净的手术刀震撼，解放physa从捐赠者的所需部分。然而，如果一个小块组织仍然连接在主体和physa，用手术刀切割。不要试图通过拉至所连接件分开，因为这可能会损坏physa。 </li></ol></li><li>从盘中取出每个截肢&#39;供体&#39;息肉 ，并返回到国家环保总局标有"汇集截肢者一碗率;迄今为止，碗，并返回到股市文化。 注意:离断息肉会愈合一天之内伤口反口，然后就可以正常喂食。他们将在两周在这一点上，如果需要，physa可以再次截肢内再生一个正常的期待physa。 </li><li>冲洗切除physa保留在12个百分点ASW切割盘，那么每个physa转移到单独的无菌孔中已经有10毫升中各12个百分点ASW的良好的多孔细胞培养板。 注:此示例使用一个六孔板，每孔持股10毫升海水和五个切除physa。一般来说海水中应涵盖physa足以避免暴露在空气中因处理和潜在蒸发的运动。该板或孔应具有一个盖子。 </li><li>重复步骤3.1 - 3.3，以收集至少5 physa在每口井保留每个实验治疗。 </li><li>孵育physa在将提供图片的温度下IDE再生的实验计划审问最好的速率。放置含physa成温度调节培养箱的板，在由再生的所希望的速率来确定一个固定的温度。 注:physa将重新生成缺少的组织，当温度15至27℃之间培养形成一个完整的息肉。再生的速率依赖于温度，除了前两个阶段。为达到第4阶段为所有的温度的平均天是第7天在切割之后，这也与在21℃再生重合。在27℃，第4阶段达到较早约3天，并在15℃，第4阶段是由大约3天在21℃（也参见参考文献10）延迟相比再生。 </li></ol> 4.评估与Nematostella再生分期系统再生（NRSS） <ol><li>采用可变庄重一个立体复式显微镜得分physa通货膨胀（10 - 80X）。比分新鲜切割Nematostella physa为0级，并继续得分在同一时间使用NRSS 10每一天后截肢（DPA）。 注意:对于关键的分期标准和详情，请参阅参考文献10。 <ol><li>如果一个新鲜切割physa显示为杯形肿块形似气球弛缓分数physa为0级（伤口），用一个开放的伤口部位可能是可见的。 注意:伤口边缘也可能会从一开始就粘在一起，但组织仍然会折叠和缺乏刚性。开放性伤口的边缘可以观察到发生径向收缩的伤口愈合。 </li><li>分数physa为第1阶段（伤口闭合），如果截肢伤口出现关闭。 注意:伤口位置将对应于未来的口服极。围绕未来口服磁极的外表面可开始显示对应于底层径向对称内胚mesenterial插入不同拱门。 </li><li>分数pH值YSA作为第2阶段（径向弓）如果口服极表面出现膨胀，露出排列在径向对称模式和沟分隔8上调拱门。在拱门的顶点观察小，半球形泡。他们将约高大宽，可能是瞬态的，一开始由单一的外胚层细胞层组成。 注:在某些情况下双层泡可能稳定下来。注:在这个或后期阶段粘膜层可能会出现封装physa（ 图2），在膜"鞘"。此封装材料应当被除去以促进得分。 </li><li>如果在至少一些径向弓的口腔端形成稳定的内胚层含有外胚层和组织层的触角芽分数physa为第3阶段（触须）。 注:触角更长比他们宽，是最低限度能动。该physa将显示上升，但通货膨胀变量使肠系膜雏形可能会变得可见IBLE从mesenterial插入延伸进入体腔（coelenteron）。 </li><li>得分physa如阶段4（线性肠系膜）如physa包含在体壁延伸进入coelenteron从插入，与口服-反口长度是比从那里出现连接两次测量它们的径向宽度更八个不同的，可见肠系膜在其反口端（enterostome）咽。 注:四个或更少的肠系膜有"褶"内部自由边。咽是可见的。超过八只触角是可见的，能动的，有时他们收缩到体内。 </li><li>得分physa如阶段5（主要百褶肠系膜）如physa有四个以上的肠系膜与打褶，并且打褶比在阶段4的动物用具有几乎"正常的"成人外观更充分和蜿蜒，但是没有可见的性腺细胞。 </li></ol></li></ol>

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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.

使用Nematostella的伤口愈合和再生的一个模型，变得越来越流行。因而，为了能够之前有效的细胞和分子分析，以可视特定协议的形态学模式是很重要的可以分配和比较。 Nematostella具有高度的再生"灵活性"，能够改革几乎在任何地点任何截肢缺少结构，在人生的浮浪幼虫后阶段。因此，各种调查人员检查从再生导致截肢或息肉的不同区域伤人，在不同的年龄和大小7-18。 <p cl...

再生在一个单一的水螅的观察是在生物学的出现为一门实验科学1,2开创性的事件。再生仍然非常广泛的吸引力生物学家的现象，都奠定人。对发育生物学家，医生，生物医学家和组织工程师了解并克服对人体再生的限制的潜力，使再生生物学超过内在有趣。 现在，利用新兴技术，如基因组测序和增益和功能的工具丢失，该领域有望梳理出的再生机制，并最终理解物种如何各种可以再生，有些则不能。在分子，细胞和形态响应的通用化程度有待阐明，但到目前为止，似乎动物中可再生基本的反应比本来意马更相似斯内德就在10年前3。 尤其是腔肠动物处于际形态多样性的广谱几乎所有的身体部位的再生简便。从孤淡水水螅，水螅与建造巨大的珊瑚礁，到复杂的殖民管水母，如葡萄牙文-O-战争的微小海洋息肉以来，再生往往是复制的模式，除了建立机制修复或改革免受伤害和掠夺造成损坏或丢失的身体部位。无论是刺胞动物门的物种多样使用类似或不同的机制再生是一个从根本上有趣的问题4-6。 我们和其他人已经开发了珊瑚虫，Nematostella vectensis作为再生7-17的典范。我们最近开发出一种分期系统描述从abora平分的形态均匀一块组织的整个身体的再生息肉10 l的结束。而当在任何级别等分Nematostella息肉可再生，我们选择在最形态简单地区，physa一个反口位置切割成人，部分是因为这是接近自然无性裂变18的正常平面，并且还因为它许可证观察和如何将整个体从最简单的形态部件重新组装分子分析。 所述Nematostella再生分期系统（NRSS）提供了一个相对简单的设置，可以被用于通过一个截去physa得分再生的任何方面的进展形态基准，正常培养条件下或在实验微扰的情况下，例如小分子治疗，基因操作或环境的改变。如预期的，在NRSS正在成为采用为在其上再生的细胞和分子事件可以被引用一个形态的支架10。 最后，我们的切割方法产生要比在最近的研究中使用的17针点刺更高的大洞几个数量级，但都是伤口愈合约有6小时。记录伤口闭合的视觉拦阻和不同的相应建议的实验方法来解释伤口的尺寸和花费，关闭时间的表观独立性。因此，反口截肢过程有更深入直观的了解，通过这种协议提供，将有助于进一步调查这种模式再生系统，拓宽使用Nematostella vectensis这种分期系统的应用。

<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

腔肠动物，特别水润 ，却显示出了再生受损的或损坏的结构，实际上这样的研究可以说是通过推出的托尼川伯利工作的现代生物调查超过250多年前，第一批动物。目前再生的研究已经看到了同时使用"经典"再生生物，如水螅 ，涡虫和有尾类的复苏，以及物种跨越后生动物的范围的扩大范围，从海绵通过哺乳动物。除了其内在的兴趣作为一种生物现象，了解再生多样的品种是如何工作的通知我们友情再生过程是否具有共同特点和/或种或上下文特定的细胞和分子机制。在明星海葵，Nematostella vectensis，是再生一个新兴的模式生物。如同水润 ，Nematostella是古门，刺胞动物门中的一员，但在T内他类珊瑚虫，一个妹妹分支到是更进化的基础水螅。在Nematostella再生因而方面将是有趣的比较和与九头蛇和其他腔肠动物的对比。在这篇文章中，我们提出要平分，观察和分类Nematostella成人，这就是所谓的physa的反口结束再生的方法。该physa自然发生裂变为无性繁殖的手段，无论是与自然裂变或physa手动触发截肢复杂形态的再生长和改造。在这里，我们编纂了Nematostella再生分期系统（在NRSS）这些简单的形态变化。我们用NRSS测试氯喹的影响，溶酶体功能阻断自噬的抑制剂。的结果表明，当自噬被抑制息肉的结构，特别是肠系膜，再生是不正常的。

在切断physa再生过程中的形态的事件的进程示于图1A中 ，其中包括在每个NRSS阶段physa的代表性的观点。典型physa切削部位被表示在成年（箭头）。 图1a中的照片显示，从新鲜切割physa通过完全形成息肉口头和身体结构的渐进式再生。 图1B，C示出内部隔垫，肠系膜的布置中，在阶段4和阶段5，分别。请注意，某些肠系膜阶段4将缺乏"打褶"?...

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.

从斯塔利特海葵的反口Physa完全诱导再生息肉<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 次观看.  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.

视频: 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.

视网膜色素上皮细胞的干细胞高效的推导

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 ...

科研

发育生物学

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.

的测定血细胞种群<em&gt;果蝇</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.

使用提顿体系的研究斑马鱼再生的分子机制

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.

完整的和更新的"轮虫混养法"饲养第一移送斑马鱼

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.

真涡虫的化学截肢和咽部再生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 ...

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.

Stentor再生的研究方法

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.

一种简便有效的成人皮肤组织中人角质形成细胞的分离方法

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.

从脐带血源性多能干细胞中提取三维皮肤有机细胞

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.

早期开发期间海葵基因分型

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.

小鼠肝外胆管有机体的产生

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

从斯塔利特海葵的反口Physa完全诱导再生息肉

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