作者承认该基金会Leducq（二尖瓣）和国民新电倒拉RECHERCHE（授予ANR Specistem）为这项研究提供资金。

1。准备动物的程序取得动物伦理委员会的批准，并按照工作的组织指导方针与病毒，HuESC和/或iPSC的（适用时），以及鼠标操作，获得小鼠胚胎，并进行小鼠手术。对于定时交配，插头的日子被认为是胚胎一天（五）0.5 / 0.5天后coitum。 <ol><li>显微注射针: 对于子宫外显微注射，用移液管拔出器/ beveller获得一个1或10微米的内部顶端直径和锋利和锥尖的形状，分别注入的DNA或细胞拉玻璃移液管（1.2 mm外径的硼硅酸盐毛细管）。对于超声介导注射，使用定制的无菌针头，其中有一个外/内直径为1.14 mm/0.53毫米（外径/内径），与50 /35μm的外径/内径一角。 </li><li>硅填充培养皿: 作为手册，准备硅。填一毫米直径的干净的玻璃60培养皿中〜1cm的弹性体层。避免产生气泡。 </li><li>工具: 保持在操作过程中之前和所有外科手术器械无菌的。 </li><li> DNA的制备: 新增3微克DNA和12微升的Opti-MEM中一管。加入3μL脂质体2000和12μL的Opti-MEM在另一试管中。孵育5分钟，在室温下，混合两种溶液。立即使用。 </li><li>细胞的制备: 制备10 6个细胞的悬浮液/ ml的DMEM（Dulbecco氏Eagle培养基，高葡萄糖，50％大鼠血清）。 50微升的最终细胞悬浮液是足够的8-10胚胎。 </li><li>染料的制备: 用绿色荧光CDCFDA-SE在25毫克/毫升DMSO中。准备20μL分装和储存在-20°C。在无菌生理盐水或PBS在使用前稀释1:100 / 1:200。 </li><li>病毒制剂: 使用商业第三代PGK-GFP表达慢病毒用〜10 9 -10 10 TRA滴度nsducing单位/ ml的DMEM。对于慢病毒的制备，见Tiscornia 等 14穿戴个人防护装备，安全地使用和处置的病毒。 </li></ol> E9.5和E10.5胚胎2。收集的体外注射<ol><li>安乐死怀孕小鼠在胚胎一天9.5或10.5。 </li><li>消毒鼠标的胸部和腹部，用70％的乙醇。 </li><li>使下腹正中切口，打开腹腔和检索子宫角在室温在PBS和两个PBS洗涤后，将其传送到一个​​硅胶填充的培养皿用M16媒介。 </li><li>针的子宫角与昆虫标签的硅氧烷层，使子宫血管的侧面是在底部。 </li><li>切细镊子子宫的表面上，并且蜕膜的盖在1毫米的表面之下。 </li><li>轻轻铺开蜕膜的墙壁检索胚胎卵黄囊。 </li><li>储存胚胎在37℃，C在M16培养基中的细胞注射前。对于注射，每个胚转移至聚硅氧烷盘充满M16培养基预先加热到37℃。 </li></ol> 3，DNA或细胞注射在立体显微镜下<ol><li>填补从背面玻璃吸管与Hamilton注射器和摇移液管，以在尖端除去气泡。 </li><li>将吸管上的显微注射持有人;设定的保压压力为50（DNA）或30（细胞）帕斯卡，而注射压力为150（DNA）到300（小区）帕斯卡。注射时间设定为0.2秒（DNA）或0.5秒（细胞）。这些设置可能会根据微量注射器和吸管的类型略有不同。 </li><li>通过卵黄囊引脚胚胎硅胶底脚不沾胚胎正确。关注心脏的区域，并打开囊略高于这一地区。 </li><li>添加围绕胚胎4个引脚，以防止细胞注射过程中的任何运动。 </li><li>使用显微操纵器（设置为手动），slowl以45°的心包以上的角度y的位置的移液管尖，只是将被注入（图1）心脏的区域的上方。 </li><li>使移液管进入感兴趣区域并触发注射。取出吸管尽快。确保心脏是正常的DNA /细胞注射后跳动。 </li></ol> 4，胚胎，离体心脏和外植体培养<ol><li>培养的胚胎E9.5在25％的M2培养基及75％大鼠血清，补充有青霉素/链霉素，预热，在37℃和氧化，用40％O 2。 </li><li>加入2ml培养基中25毫升玻璃管中，轻轻地添加胚胎和含氧化合物，用40％O 2的拧紧盖在管之前。孵育达36小时，在37℃，同时旋转或摇动（30转/分钟）。 </li><li>在期望的时间点（ 如 24小时），仔细解剖的心用细镊子，而不触及的心，首先去capitating胚胎;对心脏很容易消费使用远端流出道拉出。 </li><li>文化离体心脏再36-48小时的DMEM与基质胶涂层刀片在12孔板设置50％的FCS。见代尔和帕特森（2013年）对心脏文化15的改进协议。 </li><li>使该地区的利益的任何外植体。作为一个例子，解剖房室管（AVC）和它培养48小时胶原1型凝胶16。 </li></ol> 5，在心脏I N子宫内超声引导下注射<ol><li>超声波机及微量的设置: <ol><li>采用高分辨率超声系统具有40 MHz的换能器和显微注射设置在轨道上。 </li><li>在每次注射69 NL设定在微量的注射量。请参见制造商的注射手册，编程微量。 </li></ol></li><li>该核注射的制备njection设置和注射: <ol><li>放置一个玻璃微的微量。以确保有效注入，第一涂层与矿物油的吸移管的内侧，通过吸至最大容积。然后再弹出的油，留1-2毫米的油针内。 </li><li>吸出注射液，直到最大音量为止。要小心，不要触摸任何表面有针尖，因为这将钝尖，使注射变得更加困难。 </li><li>为了稳定注射过程中含有胚胎的子宫角，使用修改过的培养皿中，在中间的孔（直径2.5 cm），覆盖着薄薄的硅膜用小切口切中心，切口部位它上面放置。通过定位盘的边缘下3-4团块粘土稳定的鼠标处理表上的培养皿。 </li></ol></li><li>注射过程: <ol><li>刮胡子怀孕小鼠的腹部（在所需的萌芽阶段）之前anestheSIA去除毛发。治疗腹部用化学脱毛剂以除去残留的毛发和消毒用70％乙醇。 </li><li>通过一个面罩麻醉怀孕鼠标与氧/异氟醚。用3-5％的异氟醚在100％氧气初始麻醉，其次是1-1.5％，该过程的其余部分中。确保鼠标有足够的轻轻捏它的爪子和/或尾部麻醉。 </li><li>将鼠标处理表上的鼠标仰卧（设置为加热，在37℃）并覆盖眼睛用润滑剂以防止角膜脱水。 </li><li>应用电极凝胶在电极上并用胶带将爪子到电极来监测心脏速率，呼吸速率，和心电图检查。定位直肠温度计来监测体温。 </li><li>验证:第一，鼠标是通过怀孕和可视化超声计数的胚胎。应用超声凝胶上腹部和定位在膀胱以上的扫描头。为了保持一致性，首先形象化膀胱，和计数在左胚胎和右子宫角，从膀胱的位置开始。确保胚胎的心是正常跳动。 </li><li>如果鼠标是怀孕了，放置在培养皿未来的切口部位用粘土以上。按盘小幅上腹部，这样的菜是直接接触。 </li><li>取出盘子并再次消毒腹部。做一个1.5-2厘米腹正中切口，约0.75-1厘米以上的阴道，打开腹腔和腹膜。以免伤到内脏。 </li><li> Exteriorize左侧或右侧子宫角用钳子，轻轻走过的菜硅膜拉，并再次稳定在粘土的菜。防止大规模拉或操纵，因为这可能会引起子宫血管和女性和/或胚胎夭亡出血。 </li><li>再算上胚胎，以确保有足够的记录保存其中的胚胎注射。 </li><li>应用超声凝胶对Uterine牛角图像的心脏，防止脱水。 </li><li>图像的第一胚胎被注入。检查胚胎心脏的正常跳动，并保持胚胎的头脚和左右方向的记录。如有必要，请重新放置子宫角稍，以确保正确的方向。如果这被证明很难，就移动到下一个胚胎。 </li><li>用显微注射针头小心刺穿子宫，以45°角的胚胎上述定位针，（ 图3）略微外心包。心外膜细胞标记，图像的心脏在四腔心切和定位针，使其指向对房室沟（ 图3）。如果角度需要调整，拔出针头，并重新定位。请勿在子宫内针调整角度，因为这可能会破坏针。避免穿刺其它组织，如四肢或胎盘。 </li><li>移动针到心包壁，并用温和的，但迅速的运动刺穿它。在一般情况下，会有一些阻力，但移动针太快可能导致针尖刺穿心脏本身。小费应该结束了在房室沟的心包腔。在心包出血的情况下，血细胞将作为白色雪花状图案可见。如果是这样的情况下，停止注射胚胎和移动到另一个胚胎。 </li><li>注射注射液。最大限度地注入700 NL进入心包空间，以防止对心脏发育造成不利影响。监控适当注入心包轻微，颞肿胀。 </li><li>注射后，拔出注射针，并确认注射液仍然可以被喷射，以确保针没有被组织碎片堵塞。 </li><li>重新定位表处理图像并注入下一个胚胎。保持注射的胚胎的数量来最大限度3-4（在一个子宫角），以防止延长麻醉，以允许在相对的子宫角控制的胚胎，并确保该过程不干扰胚胎发育。 </li><li>注射胚胎的期望数目后，除去过量的超声凝胶，轻轻放置子宫角放回腹部在其适当的位置上。 </li><li>通过使用1层缝合腹膜和腹肌，和缝合线的第二层对皮肤闭合的母体腹部。 </li><li>注射用鼠标止痛药的首次剂量。使用一个注射0.05-0.1毫克/公斤丁丙诺啡15分钟结束麻醉和三个附加注射用12小时的时间间隔之前。 </li><li>停止麻醉和监测鼠标从过程充分恢复。房子的孕鼠在个别笼的后续所需的持续时间。 </li></ol></li></ol>

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作者什么都没有透露。

上述心内体外注射的协议是为了在中期阶段（E9.5-E11.5）的小鼠胚胎心肌保护作用至少48小时。这些注射的方法允许在空间上有针对性的注射DNA或细胞。在图1-3所示的几个实施例提供了概念证明划定离体和在所发生的受限制的心脏区域，例如心内膜或心外膜细胞的EMT的发育过程的生物体内的分子机制。最重要的是，这些协议提供了一个机会，以遵循注射的细?...

十年多前，人类胚胎干细胞（HuESCs）已被来自人类囊胚1。从那时起，这些细胞已成为一个重要的研究领域其中涉及在人类发育生物学问题未得到满足的对象。 HuESCs已进一步在再生医学提供了希望。近年来，人体诱导多能干细胞（iPS细胞）已经产生从患者特异性体细胞提供遗传病2的模型。许多体外协议对胚胎或诱导多能干细胞对各种细胞系，包括心脏谱系3的分化，已有报道。在分化的细胞通常是由RNA和蛋白质的表达，免疫染色和/或在体外功能性测试的表型分析。然而，多能干细胞衍生必须被放置在一个合适的胚胎的环境，以测试它们是否充分获得大公他们的胚胎对口升的命运，以及他们是否概括了真正的体内响应区域提示功能。而组织工程是有前途的，但还没有提供适当的体内所有已知的和未知的线索显影胚胎组织4,5。 在胚胎中，包括小鼠胚胎的染料或逆转录病毒载体的细胞标记，都带来了重要的信息，以细胞谱系的心脏发育6在胚胎起源。例如，染料注射到小鼠胚胎体外 ，接着在体外培养离体心脏的心包空间，用于标记心外膜细胞和它们的后代7。然而，染料和逆转录病毒细胞标记已大多应用于小鼠早期胚胎仍然不发达的器官，或鸡胚，这是更方便8。一个例外是大脑，这是比较容易瞄准在电子商务mbryos 9,10。这种方法还没有被应用到跳动的胚胎小鼠心脏。 为配合直接标记用染料或病毒，并进行血统跟踪更先进的阶段小鼠胚胎和成年小鼠的细胞标记方法已被结合利用Cre /液氧技术，转基因小鼠的分析。的酶Cre /液氧的方法11功能但是由于用来驱动重组酶表达的基因调控区域的时空特异性，与酶Cre /液氧重组12的效率一定的局限性。此外，这种方法并不能完全解决细胞迁移导向采集细胞命运的具体问题，因为它不仅可以激活用于驱动Cre重组酶的表达调控区域后标记一个前兆。它也不能适用于人类胚胎明显的道德问题。 由于这些限制，我们设计了一系列新的protocols到的目标区域的各种细胞标记剂，例如荧光染料，病毒，基因表达调节剂，如的shRNA和基于DNA的细胞标记的载体，或细胞在小鼠胚胎在E9.5和发展的后期阶段的注入心脏。 将DNA /细胞注射用立体显微镜和一个简单的微注射装置结合的体外胚胎培养到48小时，或离体心脏或胚胎外植体培养48-72小时。我们还报告在小鼠胚胎心脏在子宫内的超声介导的微注射协议。该技术允许监测胚胎13的发展，并允许长期随访injectates和/或标记的细胞。 我们发现，这些方法保存器官的功能，并提供了比在体外测试的干细胞潜能的比较有代表性的环境。它也提供了机会，跟随迁移的标记和/或注射的细胞来监测他们的命运。最终，这应该产生一个更好地了解区域组织图案和关键生物过程。

<table border="0" cellpadding="0" cellspacing="0" width="986">
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Setups / Hardware</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td style="width:411px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">40 MHz Transducer</td>
			<td style="width:175px;">VisualSonics</td>
			<td>MS550S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Microinjector</td>
			<td style="width:175px;">VisualSonics</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microinjector&#160;</td>
			<td style="width:175px;">Eppendorf</td>
			<td>5242</td>
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			<td height="21" style="height:21px;">Micromanipulator&#160;</td>
			<td style="width:175px;">Eppendorf</td>
			<td>5171</td>
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			<td height="21" style="height:21px;width:297px;">Nitrogen</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td>required to pressurize the injector</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Rail system</td>
			<td style="width:175px;">VisualSonics</td>
			<td style="width:104px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">rotator</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td>to rotate glass tube with embryos inside the incubator</td>
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			<td>5% CO2, 37 C</td>
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		<tr height="21">
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			<td style="width:104px;">Discovery. V8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Vevo 2100</td>
			<td style="width:175px;">VisualSonics</td>
			<td style="width:104px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:297px;">Microinjection</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">borosilicate capillary tubes</td>
			<td style="width:175px;">World Precision Instrument</td>
			<td style="width:104px;">KTW-120-6</td>
			<td>1.2 mm external diameter</td>
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		<tr height="21">
			<td height="21" style="height:21px;">pipette puller&#160;</td>
			<td style="width:175px;">Sutter</td>
			<td style="width:104px;">Model P87</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">microinjection needles</td>
			<td>Origio-Humagen&#160;</td>
			<td>C060609</td>
			<td>OD/ID 1.14mm/53mm, with 50/35 um OD/ID tip</td>
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			<td height="21" style="height:21px;">Hamilton syringes&#160;</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td></td>
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		<tr height="21">
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			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td>10 cm diameter</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Mineral oil&#160;</td>
			<td style="width:175px;">Sigma</td>
			<td>M8410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Silicon membrane</td>
			<td style="width:175px;">Visualsonics</td>
			<td style="width:104px;"></td>
			<td>4.3x4.3 cm</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Play-Doh</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Isoflurane</td>
			<td style="width:175px;">Vet One</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hair removal agent&#160;</td>
			<td style="width:175px;">Nair</td>
			<td style="width:104px;"></td>
			<td></td>
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		<tr height="24">
			<td height="24" style="height:24px;width:297px;">eye lubricant</td>
			<td style="width:175px;">Optixcare</td>
			<td>31779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Electrode gel (Signa)</td>
			<td style="width:175px;">Parker</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Suture</td>
			<td style="width:175px;">Sofsilk 5-0</td>
			<td style="width:104px;">S1173</td>
			<td></td>
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		<tr height="21">
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			<td style="width:175px;">Aquasonic</td>
			<td style="width:104px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;">Buprenex Buprenex (buprenorphine hydrochloride)</td>
			<td style="width:175px;">Reckitt Benckiser Pharmaceuticals Inc.</td>
			<td style="width:104px;">NDC 12496-0757-1</td>
			<td>0.05-0.1 mg/kg in saline</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone Elastomer</td>
			<td>Dow Corning</td>
			<td>Sylgard 184&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Glass petridishes</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:104px;"></td>
			<td>60mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">insect pins&#160;</td>
			<td>Fine Science Tools&#160;</td>
			<td>26002-20</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:297px;">Media and culture reagents</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optimem medium</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:104px;">51985026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M2 medium&#160;</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:104px;">M7167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Eagle Medium</td>
			<td style="width:175px;">Lonza</td>
			<td style="width:104px;">BE12-640F</td>
			<td>high glucose and 50% rat serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M16 medium&#160;</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:104px;">M7292</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">rat serum</td>
			<td style="width:175px;">Janvier</td>
			<td style="width:104px;">ODI 7158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pennicilin/streptomycin&#160;</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:104px;">15140-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">oxygen 40%</td>
			<td style="width:175px;">Air liquid</td>
			<td style="width:104px;"></td>
			<td>required to oxygenate the embryo culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">fetal calf serum</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:104px;">RVJ35882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">matrigel</td>
			<td style="width:175px;">BD</td>
			<td style="width:104px;">356230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">collagen type I</td>
			<td style="width:175px;">BD</td>
			<td style="width:104px;">354236</td>
			<td>to coat culture dishes for explant culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">culture dishes</td>
			<td style="width:175px;">Dutcher /Orange</td>
			<td style="width:104px;">131020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Injectates</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CDCFDA-SE&#160;</td>
			<td>Invitrogen/Molecular Probes&#160;</td>
			<td>C1165</td>
			<td>25mg/ml DMSO. Store at -20 C. Dilute 1:100-200 in saline before use.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PGK-GFP-expressing lentivirus&#160;</td>
			<td style="width:175px;"></td>
			<td style="width:104px;"></td>
			<td>~8E9 transducing units/ml DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lipofectamine 2000&#160;</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:104px;">11668019</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell labeling and injection in developing embryonic mouse hearts

测试或胚胎多能干细胞衍生体外协议的命运导致了有争议的结果并不一定反映其在体内的潜力。优选地，这些细胞应放置在一个适当的胚胎的环境，以获得它们的明确的表型。此外，细胞谱系与染料或逆转录病毒载体标记细胞后跟踪在小鼠的研究大多停留仅限于早期小鼠胚胎仍然具有发育不良的器官。为了克服这些局限性，我们设计的标准和超声介导微注射协议，在小鼠胚胎E9.5和发展的后期阶段注入不同药物在心脏的目标区域。胚胎植体或胚胎培养，然后向左或进一步发展在子宫内 。这些试剂包括荧光染料，病毒的shRNA，或干细胞来源的祖细胞。我们的方法允许保存的福器官同时监测迁移和标记和/或注射细胞命运nction。这些技术可以扩展到其他器官，将是非常有益的发展生物学重点解决生物学问题。

使用上述的注射协议，细胞可以被标记和/或注射到小鼠胚胎心脏。作为概念验证，几个实例示于其中的注射方案和体外 AVC的外植体，离体心脏，或全胚胎培养液合并（ 图1）。 图1显示了细胞注射前胚胎的制备。在E9.5胚胎从其蜕膜同时保持卵黄囊（ 图1A）的完整性除去。卵黄囊被打开略高于心脏（ 图1B）。移液器逼?...

Watch this Scientific Journal Video about Cell Labeling and Injection in Developing Embryonic Mouse Hearts at JoVE.com

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

我们描述了一系列的方法来注入染料，DNA载体，病毒和细胞，以监测来自于胚胎或多能干细胞内的小鼠胚胎在胚胎天（E）9.5和后期阶段内源性和移植细胞的两个细胞命运和表型的发展。

细胞标记和注射胚胎发展小鼠心脏

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

cell-labeling-and-injection-in-developing-embryonic-mouse-hearts

Research

JoVE Journal

Biology

14.0K 次观看.  Aix-Marseille University. 我们描述了一系列的方法来注入染料，DNA载体，病毒和细胞，以监测来自于胚胎或多能干细胞内的小鼠胚胎在胚胎天（E）9.5和后期阶段内源性和移植细胞的两个细胞命运和表型的发展。 

视频: Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

测量中使用FM标签的神经元的胞外分泌

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

科研

生物学

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

在人类胚胎干细胞体外培养的标签用于磁共振成像

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

在成年斑马鱼的眼窝射出

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

单从小鼠锥感光细胞吸录音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.
Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

分离的小鼠胚胎面外胚层和间充质

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...