作者感谢 António Cidadão、伊莎贝尔 Alcobia 和德里奥诺佩雷拉对手稿的批判性阅读, 对的视频叙事 Akkapeddi, 并体 Proa 从组织学服务 Histologia e Biologia 做Desenvolvimento, Faculdade de Medicina 里斯本, 里斯本 de 葡京, 为技术支持。我们特别感谢圣保罗 Caeiro 和雨果席尔瓦从 Unidade de audiovisuais (视听股), Faculdade de Medicina 德葡京, 里斯本 de 葡京, 他们对制作这部影片的杰出承诺。我们承认莱卡微系统的亲切提供了一个立体镜配备了视频体系和 Interaves-博彩农业 Pecuária, S. 为鹌鹑受精卵子贡献。这项工作得到葡京 Faculdade de Medicina、里斯本葡京 (FMUL) 的支持。

所有这些实验都遵循 Medicina Académico 中心的动物保育和道德准则。
1. 受精鹌鹑和鸡卵的孵化
<ol><li>孵化日本鹌鹑 (鹌鹑鹌鹑) 和鸡肉 (鸡鸡) 受精卵3和8天, 分别。<ol>
<li>放置鸡蛋与空气室 (鸡蛋钝端) 面对在一个湿润的孵化器在38摄氏度。</li>
		<li>用大约20只鹌鹑蛋和40只鸡蛋做这个实验。 注: 第一次建立此程序时, 这些数字应加倍。</li>
	</ol>
</li>
</ol>2. 在含有胸腺和甲状旁腺雏形的假定区域的鹌鹑胚胎区的分离
注: 使用水平层流罩和灭菌的仪器和材料, 在无菌条件下执行鸡蛋操作程序。
<ol><li>准备一个大的硼硅酸盐玻璃碗约3/4 填充冷磷酸盐缓冲盐水 (PBS) 解决方案。</li>
	<li>在3天的孵化后打开一个鹌鹑蛋, 通过敲壳和切割在其钝端与弯曲剪刀的另一侧的圆形开口。小心地取出外壳, 将胚胎转移到装有冷 PBS 的玻璃碗里。</li>
	<li>在薄钳的帮助下, 将鹌鹑 (q) 胚放在 E3 (qE3) (相当于鸡的21级的鹌鹑阶段)。用弯剪刀把卵黄膜包在蛋黄上。继续切割周围和外部的额外胚胎血管的周长。</li>
	<li>在薄钳的帮助下, 将胚胎移植到大约3/4 的小碗里, 用冷 PBS 填充。彻底地从剩下的蛋黄中冲洗胚胎。</li>
	<li>使用撇片将胚胎转移到一个100毫米玻璃培养皿与黑色基地 (见材料表) 包含10毫升的冷 PBS。</li>
	<li>把培养皿放在显微镜下。 注意: 从这一点向前, 执行显微手术程序在显微镜的渐进放大。作为照明光源, 建议使用显微镜或光纤中的 LED 灯, 考虑到有限的热负荷。</li>
	<li>用薄的昆虫别针把胚放在盘子的底部。放置四针形成一个正方形形状在额外胚胎区域。</li>
	<li>在薄钳的帮助下去除头区的额外胚胎膜, 并在那里放置第五针。 注: 如果胚胎正确定位, 那么耳囊, 心脏管, 1st, 2 和 3路咽弓 (PAs) 应该是可见的。</li>
	<li>用老爷车眼剪刀解剖感兴趣的胚胎区域,即3路和 4 咽弓区 (3/4 标准)。<ol>
<li>开始切割纵向和平行的胚胎轴, 之间的 somite/神经管区和 PAs。</li>
		<li>切除腹部定位心脏管。将剪刀保持在同一位置, 旋转培养皿, 根据切口的方向重新定位胚。</li>
		<li>切割之间的 2和 3路PAs 和低于4的 PA 。</li>
		<li>在薄钳的帮助下, 将剩余的膜从3/4 的标准中分离出来。</li>
	</ol>
</li>
	<li>吸入分离的组织 (3/4 标准), 并将其转移到一个玻璃盘3/4 填充冷 PBS 使用2毫升无菌塑料吸管。 注: 此后, 组织可在体外生长达48小时, 或立即嫁接到 E8 鸡胚的凸轮上。</li>
	<li>在体外试验制备过程中, 将含有隔离3/4 的玻璃器皿放在冰上。</li>
</ol>3.体外脊髓测定: 含有胸腺和甲状旁腺雏形的胚胎区域的培养
<ol><li>用 RPMI-1640 培养基制备培养基, 辅以10% 血清和1% 青霉素/链霉素。 注: 可溶性药理试剂可以添加到培养基中 (例如, LY-411.575 和 benzazepine 或环杷明和 vismodegib, 以抑制缺口和刺猬 (Hh) 信号通路, 分别。对于这种检测, 使用 50-200 nM 或5-15 µM 的 benzazepine, 以抑制缺口信号在3/4 的标准. 使用20µM 环杷明或10µM vismodegib, 以抑制 Hh 信号在3/4 面值5。</li>
	<li>在6井板上制备 3/4 pp外植体的试管培养。<ol>
<li>用5毫升培养基填充6井板中的一个井。在薄钳的帮助下, 在井下放置24毫米聚碳酸酯膜插入物 (见材料表)。</li>
		<li>在显微镜下, 将3/4 标准外植体从玻璃盘子转移到膜表面, 通过移植勺 (或刮刀) 和薄钳的帮助轻轻滑动。将外植体与腹侧向上和背侧与膜接触。每膜插入最多可添加七个外植体。继续执行步骤3.4。</li>
	</ol>
</li>
	<li>另外, 漂浮膜过滤器中的培养外植体。<ol>
<li>用5毫升培养基制备35毫米培养皿。借助薄钳, 浮膜过滤器 (见材料表), 保持干燥表面接触空气。</li>
		<li>在显微镜下, 通过移植勺 (或刮刀) 和薄钳的帮助, 将3/4 标准外植体从玻璃盘子转移到膜过滤器。将外植体与腹侧向上和背侧与膜接触。每个膜过滤器最多可添加8个外植体。</li>
	</ol>
</li>
	<li>小心地放置在步骤3.2 和3.3 中准备的外植体在37°c 与 5% CO2在一个湿润的孵化器。</li>
	<li>经过48小时的潜伏期, 从孵化器中取出6井板和35毫米培养皿。<ol>
<li>从6井板的膜插入物中收集培养的外植体。<ol>
<li>在室温下 (RT) 将 PBS 添加到膜插入。</li>
			<li>用2毫升不育的塑料吸管, 通过剧烈冲洗将外植体从膜中分离出来。</li>
			<li>在刮刀和薄钳的帮助下, 将培养的外植体转移到在 RT 中填充 PBS 的玻璃盘3/4。</li>
		</ol>
</li>
		<li>同样, 在35毫米培养皿中从浮膜过滤器中收集培养出来的外植体。<ol>
<li>将膜过滤器与薄钳转移到一个新的35毫米培养皿, 填充 PBS 在 RT。</li>
			<li>用2毫升不育的塑料吸管, 用有力的吹打从膜过滤器中分离出外植体。</li>
			<li>在薄钳的帮助下, 释放无外植体膜过滤器后, 确认没有外植体仍然附着在它。</li>
			<li>用刮刀和薄钳, 将外植体转移到在 RT 中填充 PBS 的玻璃盘中。</li>
		</ol>
</li>
	</ol>
</li>
	<li>用刮刀将培养的外植体转移到1毫升的试剂上, 用于 RNA 的分离和基因表达研究。 警告:接触此试剂 (见材料表) 可能是严重的健康危害。佩戴适当的防护眼镜、衣物和手套。按照操作说明, 阅读制造商提供的安全数据表。</li>
	<li>另外, 将培养的组织移植到 E8 鸡胚的凸轮上。执行步骤4。</li>
</ol>4. 凸轮的准备
<ol><li>从孵化箱中取出8天胚胎发育的鸡卵。 注: 在加湿的孵化器中, 用空气室在38摄氏度的温度下孵化出卵。</li>
	<li>用透明的塑料胶带盖住鸡蛋的钝端, 防止外壳的碎片落入空气室。轻敲外壳, 用弯剪刀在鸡蛋上切开圆形开口。空气室应该是可见的。</li>
	<li>慎用薄钳将空气室的白色膜取出。CAM 是可见的, 可用于异位组织移植。 注意: 在移植之前或之后, 不要使用 pbs 来水合凸轮, 因为 pbs 促进了外植体的滑动和错位。如果膜干燥, 丢弃鸡蛋。</li>
</ol>5. 将培养的外植体嫁接到凸轮上
<ol><li>在凸轮较小的血管中创建小的血管病变/伤口, microscalpel 在支架上。 注: 使用巴斯德吸管的尖端通过毛细现象在过量出血的情况下清除血液。</li>
	<li>使用刮刀和薄钳将培养的外植体转移到凸轮的受伤部位。</li>
	<li>切一张比外植体稍大一点的滤纸, 把它放在外植体的顶部。 注: 过滤纸在凸轮开发后, 有助于跟踪外植体位置。此外, 如果必要的话 (在步骤5.6 中描述), 它允许每天在蛋开发期间向外植体提供药物。</li>
	<li>用透明的塑料胶带封住鸡蛋窗, 用炭铅笔识别。 注: 塑料胶带可防止胚胎在潜伏期内脱水。</li>
	<li>在38摄氏度的湿润孵化箱中孵化10天的操作鸡蛋。执行步骤6。</li>
	<li>可选步骤: 在孵化期内每日用药管理。<ol>
<li>要访问过滤器, 请部分提起塑料胶带。添加100µL 的药物溶液, 下降, 在纸上。重新密封的窗口, 并把鸡蛋放回湿式孵化器在38摄氏度。 注: 对于这一检测, 剂量20µM 的环杷明将抑制 Hh 信号在蛋发展5。</li>
	</ol>
</li>
</ol>6.在蛋发育10天后 CAM 中的异位器官形成
<ol><li>经过10天的孵化, 从孵化箱取出鸡蛋, 并小心地取出塑料胶带。</li>
	<li>使用弯剪刀将凸轮导出的外植体与滤纸转移到一个小玻璃碗中, 用冷 PBS 填充大约3/4 块。</li>
	<li>在薄钳的帮助下, 将凸轮衍生的外植体转移到100毫米的培养皿中, 黑色底座含有10毫升的 PBS。用老爷车眼剪刀和薄钳轻轻地去除滤纸和多余的膜。</li>
	<li>将凸轮外植体转移到固定液 (PBS 中的3.7% 只粉煤灰) 上, 用撇嘴。弄死鸡胚, 而不将其从卵中取出, 通过在大剪刀的帮助下, 在胚胎的颈部区域进行精确的切割。</li>
	<li>用常规组织学和免疫组化法对凸轮衍生外植体石蜡切片的器官形成进行评价。</li>
</ol>

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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+of+the+third+pharyngeal+pouch+into+thymus/parathyroid+by+Six+and+Eya1.">Patterning of the third pharyngeal pouch into thymus/parathyroid by Six and Eya1.</a> Dev Biol. 293 (2), 499-512 (2006).</li><li>Uematsu, E., 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=Use+of+in+ovo.+chorioallantoic+membrane+engraftment+to+culture+testes+from+neonatal+mice.">Use of in ovo. chorioallantoic membrane engraftment to culture testes from neonatal mice.</a> Comp Med. 64 (4), 264-269 (2014).</li></ol>

作者没有什么可透露的。

这个方法的成功的一个关键方面是鸡和鹌鹑蛋的质量。考虑到长期的潜伏期, 特别是在蛋试验期间, 良好的鸡卵质量提高了生存率 (高达 90%) 的过程结束。要做到这一点, 测试鸡蛋从不同的供应商。长时间孵化 unmanipulated 卵 (最多16-17 天) 并检查其发育情况。要被认为是一个良好的质量批次, 超过80% 的胚胎应该呈现正常的发展。同样重要的是要确保每个孵化步骤提供可重现的同步发展阶段, 以?...

禽胚在精发育生物学研究中得到了广泛的应用。鸟类模型的主要优点包括打开卵子的可能性, 相对容易进入胚胎, 以及执行微操作系统的能力。一些例子包括经典的鹌鹑鸡嵌合体系统研究细胞命运1, 具体生长因子的应用到胚胎2, 以及异位细胞结构的增长在凸轮1,3, 4。
为了对器官形成的不同阶段有新的认识, 我们最近开发了一种将嫁接技术与胚胎组织体外操作相结合的方法5。这两个步骤的方法使对器官的早期和后期阶段的歧视和探索, 这往往是有限的, 因为高度动态和复杂的组织相互作用2。此外, 缺乏适当的组织特异标记经常限制使用基因改良动物模型6。这两步方法的新方法基本上克服了这种限制。
为了研究器官形成的早期阶段, 在第一步, 在 48 h 的体外脊髓系统中分离和培养出包括前瞻性器官雏形的鹌鹑胚胎区。在此期间, 特定信号通路的药理调控可以通过加入药物到培养基5,7来进行。此外, 培养组织可以收集在任何阶段的体外生长和探索基因表达 (使用方法, 如 qPCR, 微阵列等)。
第二步, 在胚胎日 (E) 8 (cE8) (汉堡和汉密尔顿 (HH)-阶段 33-35)8中, 将48个 h 培养组织移植到鸡 (c) 胚的凸轮上。该凸轮作为营养物质的血管供应商, 允许气体交换1,3,4到嫁接组织, 使其在蛋的发展更长一段时间。这一实验步骤特别适合研究器官的后期阶段, 因为完全形成的器官可以获得后10天的蛋发展5,9,10,11.形态学分析很容易通过传统的组织学来确认适当的器官形成和捐献者的细胞来源可以通过免疫组化使用特定物种的抗体 (即单克隆鹌鹑核周 (QCPN)) 鉴定。在凸轮潜伏期, 移植物也可以生长在药理剂的存在, 并收集在任何发展阶段, 以评估器官的进展。
这两步方法, 在这里深入描述, 已经在 Figueiredo等使用。5探讨禽甲状旁腺/胸腺共同普利摩顿的发展。因此, 在胸腺和甲状旁腺器官的形成过程中, 胚胎领土和发育阶段的固有特殊性将列于下文。
胸腺和甲状旁腺上皮, 虽然功能上不同, 从咽囊的内胚层 (PP)12。在禽类中, 这些器官的上皮来源于第三和第四 pp 内胚层 (3/4 pp)12, 而在哺乳动物中, 胸腺上皮来源于 3PP, 而甲状旁腺上皮则来源于老鼠和人类的3PP 和 3/4 PP,分别为13、14。
这些器官形成的最早阶段之一是在普通普利摩顿中出现离散胸腺和甲状旁腺领域。在鸡肉中, 这些领域可以通过原位杂交识别, 用特定的分子标记, 在 E4.515。随着发展的进行, 这些器官的雏形是个性化的, 并与咽分离, 而由神经嵴衍生细胞形成的薄间质胶囊 (在 E5;HH-阶段 27)。随后, 胸腺上皮由造血祖细胞 (E6.5) 所殖民;HH-阶段 30)12。
与经典鹌鹑鸡研究1,12, 两步方法是特别有用的研究造血/淋巴器官的形成, 即胸腺5。当鹌鹑外植体, 与器官雏形, 是嫁接在鸡胚前造血祖细胞殖民化, 一个嵌合胸腺形成与鸡血源祖细胞浸润鹌鹑胸腺上皮对应。因此, 该方法是探讨造血细胞在禽换血术/淋巴系统发展中的作用的有用工具。

<table><tbody><tr><td>Chicken fertilized eggs (&lt;em&gt;Gallus gallus&lt;/em&gt;)</td><td>Pintobar, Portugal</td><td></td><td>Poultry farm&amp;nbsp;</td></tr><tr><td>Quail fertilized eggs (&lt;em&gt;Coturnix coturnix&lt;/em&gt;)</td><td>Interaves, Portugal</td><td></td><td>Bird farm&amp;nbsp;</td></tr><tr><td>15 mL PP centrifuge tubes</td><td>Corning</td><td>430052</td><td></td></tr><tr><td>50 mL PP centrifuge tubes</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>60 x 20 mm pyrex dishes</td><td>Duran group</td><td>21 755 41</td><td></td></tr><tr><td>100 x 20 mm pyrex dishes</td><td>Duran group</td><td>21 755 48</td><td></td></tr><tr><td>Polycarbonate Membrane Insert&amp;nbsp;</td><td>Corning</td><td>3412</td><td>24 mm transwell with 0.4 mm Pore Polycarbonate Membrane Insert&amp;nbsp;</td></tr><tr><td>Membrane filter</td><td>Millipore</td><td>DTTP01300</td><td>0.6 mm Isopore membrane filter</td></tr><tr><td>6-well culture plates</td><td>Nunc, Thermo Fisher Scientific</td><td>140675</td><td></td></tr><tr><td>Petri dish, 35 x 10 mm</td><td>Sigma-Aldrich</td><td>P5112&amp;nbsp;</td><td></td></tr><tr><td>Pyrex bowls</td><td></td><td></td><td>from supermarket&amp;nbsp;</td></tr><tr><td>Transfer pipettes</td><td>Samco Scientific, Thermo Fisher Scientific</td><td>2041S</td><td>2 mL plastic pipet</td></tr><tr><td>Glass pasteur pipette</td><td>normax</td><td>5426015</td><td></td></tr><tr><td>Whatman&amp;nbsp;qualitative filter paper</td><td>Sigma-Aldrich</td><td>WHA1001090</td><td>Filter paper</td></tr><tr><td>Clear plastic tape</td><td></td><td></td><td>from supermarket&amp;nbsp;</td></tr><tr><td>Cytokeratin (pan; acidic and basic, type I and II cytokeratins), clone Lu-5</td><td>BMA Biomedicals</td><td>T-1302</td><td></td></tr><tr><td>Cyclopamine hydrate</td><td>Sigma-Aldrich</td><td>C4116</td><td>Pharmacological inhibitor of Hh signalling</td></tr><tr><td>Fetal Bovine Serum</td><td>Invitrogen, Thermo Fisher Scientific</td><td></td><td>Standart FBS</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Invitrogen, Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>GIBCO, Thermo Fisher Scientific</td><td>10010023</td><td></td></tr><tr><td>QCPN antibody</td><td>Developmental Studies Hybridoma Bank</td><td>QCPN</td><td></td></tr><tr><td>RPMI 1640 Medium, GlutaMAX Supplement&amp;nbsp;</td><td>GIBCO, Thermo Fisher Scientific</td><td>61870010</td><td></td></tr><tr><td>Bluesil RTV141A/B Silicone Elastomer 1.1Kg Kit</td><td>ELKEM/Silmid</td><td>RH141001KG</td><td>To prepare the back base for petri dish</td></tr><tr><td>Stemolecule LY411575</td><td>Stemgent</td><td>04-0054</td><td>Pharmacological inhibitor of Notch signalling</td></tr><tr><td>TRIzol Reagent</td><td>Invitrogen, Thermo Fisher Scientific</td><td>15596026</td><td>Reagent for total RNA isolation</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-30&amp;nbsp;</td><td>Thin forceps</td></tr><tr><td>Extra fine Bonn scissors, curved</td><td>Fine Science Tools</td><td>14085-08</td><td>Curved scissors</td></tr><tr><td>Insect pins&amp;nbsp;</td><td>Fine Science Tools</td><td>26001-30</td><td></td></tr><tr><td>Micro spatula&amp;nbsp;</td><td>Fine Science Tools</td><td>10087-12</td><td>Transplantation spoon</td></tr><tr><td>Minutien Pins</td><td>Fine Science Tools</td><td>26002-20</td><td>Microscalpel</td></tr><tr><td>Moria Nickel Plated Pin Holder</td><td>Fine Science Tools</td><td>26016-12</td><td>Holder</td></tr><tr><td>Moria Perforated Spoon</td><td>Fine Science Tools</td><td>10370-17</td><td>Skimmer</td></tr><tr><td>Wecker Eye Scissor</td><td>Fine Science Tools</td><td>15010-11</td><td></td></tr><tr><td>Camera</td><td>Leica Microsystems&amp;nbsp;</td><td>MC170 HD</td><td></td></tr><tr><td>Stereoscope</td><td>Leica Microsystems&amp;nbsp;</td><td>Leica M80</td><td></td></tr><tr><td>Microscope</td><td>Leica Microsystems&amp;nbsp;</td><td>DM2500</td><td></td></tr></tbody></table>

two-step approach to explore early- and late-stages of organ formation in the avian model: the thymus and parathyroid glands organogenesis paradigm

禽胚作为一种实验模型, 对发育生物学中的精发现具有极其重要的意义。在几种方法中, 鹌鹑鸡合体的形成和胚膜 (CAM) 用于维持异位组织发育的时间追溯到上个世纪。目前, 这些经典技术与最近的体外方法相结合, 为进一步探索器官形成提供了新的前景。
在这里, 我们描述了一个两个步骤的方法来研究早期和后期的器官。简言之, 含有该器官推定区域的胚胎区域与鹌鹑胚胎分离, 并在脊髓系统中体外生长 (最多48小时)。培养的组织随后被嫁接到鸡胚的凸轮上。经过10天的蛋发展, 完全形成的器官是从嫁接组织获得。这种方法还允许在整个体外和蛋发育步骤中, 通过药物的常规管理和组织遗传操作来调制信号通路。此外, 开发组织可以收集在任何时间窗口, 以分析其基因表达谱 (使用定量 PCR (qPCR), 微阵列等) 和形态学 (评估与常规组织学和免疫化学)。
所描述的实验程序可作为一种工具, 遵循鸟类胚胎外的器官形成, 从器官的早期阶段到完全形成和功能器官。

上述协议详细说明了一种方法, 它允许对器官的早期和后期阶段进行调查, 通常受复杂的细胞和分子相互作用的限制。
这种方法以前曾在 Figueiredo等地使用过。5解开缺口和 Hh 信号在禽甲状旁腺/胸腺共同普利摩顿发展中的作用。
在此,图 1</stro...

Watch this Scientific Journal Video about Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm at JoVE.co

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

鸟类模型中器官形成早期和晚期的两步探讨: 胸腺和甲状旁腺器官发生范式

本文通过结合新颖的体外和蛋实验程序, 为经典鹌鹑-鸡嵌合体系统的研究提供了一种最新的方法。

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...

two-step-approach-to-explore-early-late-stages-organ-formation-avian

Research

JoVE Journal

Developmental Biology

7.4K 次观看.  Universidade de Lisboa. 本文通过结合新颖的体外和蛋实验程序, 为经典鹌鹑-鸡嵌合体系统的研究提供了一种最新的方法。

视频: Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

3D器官联合培养模型支持胸腺髓质上皮细胞增殖，分化和混杂基因表达

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

科研

发育生物学

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

2 dGuo治疗胸腺器官培养的制备

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

生物学

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

脊髓培养法研究胚胎鸡组织的发展

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

胸腺标本分离和鸡刺激器官的形成

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

Creating Avian Forebrain Chimeras to Assess Facial Development

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the strengths of this model system is that the effect of, and interaction among, tissues can be directly assessed in chimeric embryos. We have previously shown that signals from the forebrain contribute to facial morphogenesis by regulating the shape of the expression domain of Sonic hedgehog (SHH) in the Frontonasal Ectodermal Zone (FEZ). In this article, the method of generating the forebrain chimeras and provide illustrations of the outcomes of these experiments is described.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of basal forebrain&#160;during craniofacial development.

创建鸟类前脑嵌合体以评估面部发育

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the ...

禽胚胎皮肤外植体培养方案的全面回顾

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

作者聚焦:深入了解当前的实验性禽皮外植体方法

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

使用禽皮肤外植体研究组织模式和器官发生

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

<meta charset="utf8"></head><body>使用 iPSC 来源的类器官重建胸腺微环境

Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the properties of its extracellular matrix (ECM). Thymic epithelial cells (TECs) are crucial in thymopoiesis, nurturing and selecting developing T cells by filtering self-reactive clones. TECs have been empirically demonstrated to be particularly sensitive to physical and chemical clues supplied by the ECM and classical monolayer cell culture leads to a quick loss of functionality until their death. Because of this delicate maintenance combined with relative rarity, and despite the high stakes in modeling thymus biology in vitro, models able to faithfully mimic the TEC niche at scale and over time are still lacking. Here, we describe the formation of a multicellular human thymic organoid model, in which the TEC compartment is derived from human induced pluripotent stem cells (iPSC) and reaggregated with primary early thymocyte progenitors in a three-dimensional (3D) fibrin-based hydrogel. This model answers current needs for a scalable culture system that reproduces the thymic microenvironment ex vivo and demonstrates functionality, i.e., the ability to produce T cells and to support thymus organoid growth over several weeks. Thus, we propose a practical in vitro model of thymus functionality through iPSC-derived organoids that would benefit research on TEC biology and T cell generation ex vivo.

<meta charset="utf8"></head><body>作者聚焦：利用人胸腺类器官推进胸腺上皮细胞和 T 细胞研究

Here, we describe a protocol for the formation of human iPSC-derived thymus organoids grown in 3D fibrin hydrogels aiming to support thymic epithelial cell (TEC) maturation and prolonged maintenance, as well as thymopoiesis in vitro.

在三维纤维蛋白水凝胶中形成人胸腺类器官

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the ...

鸟类模型中器官形成早期和晚期的两步探讨: 胸腺和甲状旁腺器官发生范式

摘要

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此视频中的章节

3D器官联合培养模型支持胸腺髓质上皮细胞增殖，分化和混杂基因表达

2 dGuo治疗胸腺器官培养的制备

脊髓培养法研究胚胎鸡组织的发展

胸腺标本分离和鸡刺激器官的形成

创建鸟类前脑嵌合体以评估面部发育

使用禽皮肤外植体研究组织模式和器官发生

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