1.建议开始之前<ol><li>不要使用被污染的产品分成合同与支原体，因为他们不会分化成正常窦房结细胞。启动协议之前，测试支原体污染。用PCR试剂盒进行快速，灵敏地检测支原体做到这一点，并按照manufacturer`s协议。 </li><li>对于每个培养皿（步骤2.3.4），涂层1 10cm 2的细胞培养皿用无菌7毫升0.1％明胶从冷水鱼皮1小时，在37℃。除去明胶，并让在无菌条件下将培养皿干燥在无菌台上。 </li><li>在你开始分化协议，你需要一个双稳定转染的小鼠胚胎干细胞系克隆包含以下特点:一）构TBX3使用哺乳动物表达载体过表达。 ⅱ）MYH6启动子5的控制下的G418抗性基因。 </li><li>未分化细胞的物业服务公司应共同培育ð与标准条件如所述1下照射饲养细胞。 </li></ol> 2.分化过程<ol><li>两天之前分化 - 从饲养层细胞中取出产品分成合同。 <ol><li>吸出介质，用10毫升的磷酸盐缓冲盐水PBS中洗涤细胞，添加7毫升胶原酶IV溶液，并在37℃下孵育细胞10分钟。放置在50ml管中的无菌40μm的过滤器。 </li><li>仔细冲洗PSC殖民地（避免去除饲养层细胞）吹打胶原酶溶液向上和向下的5倍。 </li><li>细胞悬液转移到一个40微米的过滤器，冲洗过滤器三次用8ml PBS中。转身过滤器，并将其放置在培养皿倒挂。吹打10毫升PBS中至过滤器的底部取出的PSC细胞集落。 </li><li>细胞悬液转移到15毫升管离心5分钟，在300×g下。 </li><li>取出PBS，悬浮细胞1毫升的Accutase和我ncubate在37℃下5分钟。 </li><li>加入10毫升的PBS，吹打它向上和向下的5倍，离心5分钟，在300×g下混合的细胞溶液。 </li><li>除去PBS，悬浮细胞在10ml培养基中并测定细胞数。 </li><li>种子15000细胞/ cm 2上的75cm 2的过滤器烧瓶中，并加以培养2天，在37℃，5％CO 2。 2天后将烧瓶应50-70％汇合。 </li></ol></li><li> 0天 - 开始分化<ol><li>除去培养基并用10ml的PBS洗涤细胞。 </li><li>吸出PBS，加2ml的Accutase孵育将细胞在37℃下5分钟。放置在50ml管中的无菌40μm的过滤器。 </li><li>加入10毫升的PBS，将细胞悬浮液转移到40微米的过滤器，通过加入10ml PBS中，离心冲洗过滤器流过5分钟，在300×g下。 </li><li>悬浮细胞在10ml的分化培养基，并确定细胞数。 </li><li>淡化吨他的细胞悬液与分化培养基中至20000个细胞/ ml的终浓度。 </li><li>吸管20ml水和5ml挂在一个二次培养皿，以避免干燥出加氢脱硫的压降（HD）的溶液中。 注:对于每个24以及含板iSABs（见2.8.6.4/2.8.7）启动16个培养皿。 </li><li>吸取最多至50ml细胞悬浮液中的托盘。 </li><li>转身培养皿的盖子。将180日立数据系统每片含20微升（400个细胞/ HD）细胞悬液到使用12道移液器的盖子。 </li><li>小心地将周围的盖子，将它放到培养皿中。 注:扭转盖子的速度是很重要的。如果盖子被打开围绕太慢或太快，将细胞悬浮液的表面张力不够高，保留悬滴在盖。在试图产生日立数据系统首次练习扭转盖子用中的20微升滴。 </li><li>在3培养细胞2天7°C，5％CO 2，让他们形成EB。 注:将一个培养皿盛满水的一叠5培养皿的顶部。否则，HD在上培养皿就会枯竭。 </li></ol></li><li>第2天<ol><li>小心地将周围的二次培养皿的盖子，并从2培养皿（360 EBS）衍生的胚转移至50ml管。 </li><li>等待10分钟，让胚定居在试管底部（不要离心EBS）。 </li><li>吸出尽可能多的介质成为可能，暂停在10ml分化培养基中的胚，悬浮液转移到10cm的培养皿中。 </li></ol></li><li>一天2-6悬浮培养<ol><li>培养在悬浮液4天的EB中，在37℃，5％CO 2，2天后改变介质。 注意:即使在陪替氏培养皿不涂（而不是一个细胞培养皿）的胚往往附加到表面。控制培养皿每天附着胚，并在必要时分离的胚从表面通过温和移液。可选:在悬浮培养期间不断摇晃培养皿。小心振动筛的速度。 EBS的既不应积累在中间，也没有浮到的培养皿边界。 </li></ol></li><li> 6天<ol><li>从一个培养皿转移EBS的一个明胶涂10厘米2细胞培养皿。 </li></ol></li><li> 7-12日<ol><li>细胞应该开始后8-12天打。检查每天在显微镜下跳动的细胞灶。细胞开始形成的层。 </li><li>改变媒体每天。 注意:在改变培养基后，将细胞需要3-4小时，以恢复和重新开始跳动。 </li></ol></li><li> 12-15日选择窦淋巴结细胞。 <ol><li>所述细胞已经开始跳动（约12-15天）后三天，吸新鲜分化培养基含有400微克/毫升G418的细胞的培养基和吸管10毫升</li></ol></li><lI&gt;的选择的开始后的第4天解离的细胞层。 注意:这是可能的细胞层已经从表面脱离。 <ol><li>小心取出介质不吸细胞层。 </li><li>加入10毫升的PBS，并小心地取出PBS不吸细胞层。 </li><li>加入10毫升胶原酶IV溶液，将细胞转移到50毫升管中并孵育所述细胞在37℃下5分钟。 </li><li>大力通过移液器上下吹吸10次混合的悬浮液，并培育的细胞在37℃下5分钟。应该发生的小簇暂停。如果仍有离开层的庞大零件，然后重复步骤2.8.4两次以上。 </li><li>加入20毫升的PBS和离心机在300×g下10分钟。 </li><li>小心吸出PBS。 注:如果iSABs要生成继续步骤2.8.7。如果ISAB衍生窦结单细胞是要产生继续步骤2.9。 </li><li>悬浮细胞于12ml分化培养基中含有400微克/毫升G418和种子它们上6个孔的明胶包被的24孔（2毫升/孔）。 </li></ol></li><li>代单结电池。 <ol><li>暂停从步骤2.8.6在5毫升的Accutase细胞孵育在37℃下5分钟。 </li><li>大力通过移液器上下吹吸10次混合的悬浮液，并培育的细胞在37℃下5分钟。 </li><li>加入20毫升的PBS，离心5分钟，在300×g下。 </li><li>暂停含有400微克/毫升G418的细胞于12ml分化培养基和种子它们上6个孔一个明胶包被的24孔（2毫升/孔）。 </li></ol></li><li>第2天解离后，吸出培养基，用PBS洗涤细胞三次。加入1毫升的分化培养基/孔。 </li><li> 4-8天解离后，改中，每2 次一天。上解离使用iSABs或ISAB衍生窦后第10天的节点的单细胞可用于进一步的分析。 </li></ol>

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的能力产生干细胞来源的心脏起搏细胞可允许适当心律重建中的"生物起搏器"的感觉。同样，在体外药物测试将受益于他们的可用性。的PSCs可以引起哺乳动物体包括与起搏器电池特性8,9,10,11,12,13心肌的任何细胞类型。然而，通常为"胚体"内的细胞群体是高度异质的，这不可避免地导致了可靠的选择和分离的策略的要求 - 这尤其适用于心脏结细胞的非常罕见的细胞类型。基于外源性和内源性表面多种方法标志物14，在小分子15,16,17药理管理和直接重新编程的策略18,19已经接踵而至。此外，非干细胞为基础的方法，旨在模拟生物起搏器通过操作终端效应分子功能齐全的节点细胞20,21底层肌膜电，而不是从头生成。 然而，这些都不想出了所需的高自发性收缩频率和细胞纯度。与此相反，我们的协议导致细胞不仅表现出的电振荡，也微妙电生理和钙信号特性以及内源性起搏细胞7的鲜明的形态特征。这种技术有可能成为替代电子起搏设备的重要前提。 虽然该协议的发展过程中进行了优化，但仍有一些步骤，可能会导致问题:起点为ISAB的选择是至关重要的协议的。细胞的跳动为αMHC-表达在分化细胞的指标。 MYH6-表达伴随着前PRESSION的G418抗性基因。太早开始选择将熄灭很多这可能会分化为心肌细胞，因此将减少窦结细胞的总产率的细胞。另一个关键步骤是细胞层（步骤2.8）的离解。这样做的原因是，这些细胞形成一个强有力的细胞外基质和强大的力是必要的，以解离的细胞层。如果需要的话，步骤2.8.4应重复直到小簇细胞已经从细胞层的发展。避免细胞层由如胰蛋白酶直接解离，因为它被证明是非常低效率的。 对于未来的临床应用中，以下的障碍仍需要解决:所述的方法来产生人类iSABs的可能成为朝向未来细胞治疗和体外药物检验的一个关键步骤的转移。此外，该技术仍是基于稳定的遗传modifi的PSCs和因此的阳离子将需要被修改，才可以被应用到病人。

术语"病态窦房结综合征"总结了多种疾病导致的心脏起搏器系统的恶化。它包括病理，对症窦性心动过缓，窦房传导阻滞，窦性停搏以及心动过速，心动过缓综合征。由此，一个"病态窦房结综合征"常伴一般心脏疾病如缺血性心脏疾病，心肌病或心肌炎。目前，治疗方法是基于电起搏器的植入。然而，这伴随着一个数字，如感染和电池故障的风险。总体而言，并发症的发生率还是非常高的患者已经植入人工心脏起搏器。此外，相对于内源性心脏起搏器，这些设备不响应​​激素刺激。 未来的另一种可能依赖于哪些产品分成合同可能成为的"生物起搏器"的可用性作为合适的细胞来源，这也将是用于体外药物测试非常有价值的。然而，一个主要的问题出在胚体（EBS）中非常罕见的外观窦结细胞-这通常不超过约0.5％1。 此前，它表明"向前编程"朝特定心肌亚型是通过不同的早期心血管转录因子如中胚层特异性-后1（MesP1）和NK2转录因子相关的，轨迹5（的Nkx2.5）2的过表达是可行的， 3。对于正常尺寸和窦房结（SAN）的功能，在T-box转录因子TBX3是至关重要的，这已被证明以引发起搏器基因程序，并控制在SAN 4的分化。而这种增强的功能性起搏细胞的外观，内容依然没有整个cardiomyocytic细胞群中超过〜40％。 因此，一个额外的MYH6，促进基于抗生素选择步骤5中我们进行了介绍。这最终导致尚未未观察到心肌细胞聚集体（"诱导窦房机构;"iSABs"），其在体外表现出高度增加的跳动的频率（&gt; 400 BPM），首次近似那些鼠心脏的和可比较的体外培养窦结细胞从鼠心脏6隔离。在异丙肾上腺素管理甚至殴打的550 BPM频率来实现。值得注意的是，iSABs包括超过80％的功能节点细胞从粗放生理得到印证分析7。最近，一些方法来使用直接重新编程19产生窦淋巴结细胞，表面标记14或药物治疗与小分子16,17进行了描述。然而，这些方法导致起搏细胞的这种高纯度和跳动频率接近鼠他艺术在iSABs观察。 此外，在已失去了他们的自发活动的跳动种植成年小鼠脑室片的体外模型中，iSABs能够集成到切片组织，因此剩余的自发主动和有力起搏心脏切片收缩7。这些iSABs的生成一个详细的协议，本文介绍。

<table border="0" cellpadding="0" cellspacing="0" width="597">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Name of the Material/Equipment</td>
			<td style="width:132px;">Company</td>
			<td style="width:131px;">Catalog Number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iscove&#39;s liquid medium with stable glutamine</td>
			<td>Biochrom AG</td>
			<td>FG 0465</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM liquid medium without Na-pyruvate, with stable glutamine</td>
			<td>Biochrom AG</td>
			<td>FG 0435</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CLS type IV, CLS IV</td>
			<td>Biochrom AG</td>
			<td>C4-22</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Non-essential amino acids</td>
			<td>Biochrom AG</td>
			<td>K 0293</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS Superior</td>
			<td>Biochrom AG</td>
			<td>S 0615</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium pyruvate (100 mM)</td>
			<td>Biochrom AG</td>
			<td>L 0473</td>
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		<tr height="20">
			<td height="20" style="height:20px;">G 418-BC liquid (ready-to-use solution), sterile</td>
			<td>Biochrom AG</td>
			<td>A 2912</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Reagent reservoir PP f.multichannel pip. 60ml,sterile</td>
			<td>Brand</td>
			<td>703409</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Accutase</td>
			<td>eBioscience,Inc.</td>
			<td>00-4555-56</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Eppendorf Xplorer/Xplorer plus, electronic pipette</td>
			<td>Eppendorf</td>
			<td>4861000155</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Falcon Cell Strainer 40&#181;m</td>
			<td>Falcon</td>
			<td>352340</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin&#160;</td>
			<td>GE Healthcare</td>
			<td>P11-010</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri Dishes</td>
			<td>Greiner BioOne</td>
			<td>663102</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS without Ca and Mg</td>
			<td>PAN-Biotech</td>
			<td>P04-36500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>PAN-Biotech</td>
			<td>P30-3302</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leukemia inhibitory factor</td>
			<td>Phoenix Europe GmbH</td>
			<td>LIF-250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Quadratic petri dishes</td>
			<td>Roth</td>
			<td>PX67.1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin from cold water fish skin</td>
			<td>SigmaAldrich</td>
			<td>G7765</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-Mercaptoethanol</td>
			<td>SigmaAldrich</td>
			<td>M3148&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Thioglycerol</td>
			<td>SigmaAldrich</td>
			<td>M6145</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue culture dishes</td>
			<td>TPP</td>
			<td>93100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue culture flask</td>
			<td>TPP</td>
			<td>90076</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue culture test plates (24 well)</td>
			<td>TPP</td>
			<td>92424</td>
		</tr>
	</tbody>
</table>

generation of murine cardiac pacemaker cell aggregates based on es-cell-programming in combination with myh6-promoter-selection

"病窦综合征"的治疗是根据人工心脏起搏器。这些熊的危害，如电池故障和感染。此外，它们缺乏激素响应性和总体过程是成本密集型的​​。从生成的PSCs"生物起搏器"有可能成为一种替代方法，还起搏细胞的胚状体的典型含量（EBS）是非常低的。所描述的协议将通过窦房结诱导TBX3与MYH6，促进基于抗生素选择"前进编程"鼠产品分成合同。这就产生心肌聚集一致&gt; 80％生理功能起搏细胞。这些"诱导窦房结-体"（"iSABs"）被自发收缩在对应于节点的细胞从小鼠心脏中分离但未得频率（400-500 BPM），并能踱步鼠心肌体外 。使用所描述的协议的高纯度窦可以生成节点单个细胞，其例如可以用于在体外药物测试。此外，根据该协议所产生的iSABs可能成为朝向心脏组织工程的关键步骤。

所描述的协议允许代iSABs与大约450 BPM的产品分成合同（在电影中所示），一个跳动频率附近是对小鼠心脏跳动频率。 iSABs的解离（步骤2.8.8）后所观察到的单个细胞显示窦房结（主轴和蜘蛛细胞）的细胞的典型形状如图1，这些细胞是已知的高表达的蛋白质是该功能所必需像超极化激活环核苷酸门控阳离子通道4（HCN4），连接蛋白（Cx45的），Connexin30.2（Cx30.2）和MYH6（ - C图2A）窦房结的。治疗ISAB来源的细胞与药物后，将细胞显示预期的行为:像在窦房结，滑稽通道阻滞剂ZD7288（ 图3A）以及毒蕈碱受体激动剂卡巴胆碱（ 图3B），都可以导致一个显著降低打浆ISAB DERIV频率编细胞。将β肾上腺素受体 ​​激动剂异丙肾上腺素导致升高的跳动频率（ 图3C）。 <img alt="图1" src="/files/ftp_upload/52465/52465fig1highres.jpg" /> 图1:主轴和蜘蛛细胞 ISAB衍生主轴（A）和蜘蛛（B）的结电池的典型的蜂窝形状的蜂窝形状 。比例尺为20μm。 <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/52465/52465fig2highres.jpg" width="600" /> 图2:窦房结标志物的表达。 （A）HCN4（黄色）和Cx45的（洋红色），（B）在窦房结细胞Cx45的（品红色）和Cx30.2（黄色）的表达和（C）MYH6（品红色）的表达。中核（turquois）复染。比例尺为20μm。 图3:iSABs代表的药理治疗前（对照）跳动iSABs的频率和ZD7288（A），卡巴胆碱（B）和异丙肾上腺素（C）的处理之后。数据代表八个独立iSABs并表示为平均值±标准差。 *** P &lt;0.001 <img alt="图4" src="/files/ftp_upload/52465/52465fig4highres.jpg" /> 图4:示意图分化协议 。简化卡通反射实验流程图为ISAB生成。 电影:解离后殴打的典型致窦房体（ISAB）的iSABs殴打<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52465/Rimmbach et al movie.wmv" target="_blank">抗辩</a>SE点击此处观看该视频。

Watch this Scientific Journal Video about Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection at JoVE.com

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

本协议描述了如何制作从小鼠多能干细胞（PSC）功能性鼻窦淋巴组织。 T-BOX3（TBX3）表达以及心肌肌球蛋白重链（MYH6）启动抗生素的选择导致了高纯度的起搏细胞聚集。这些"诱导窦房结-机构"（"iSABs"）含有80％以上的起搏细胞，显示高度增加跳动的速度，并能步伐心肌体外 。

基于ES-Cell的编程组合MYH6启动子，选择小鼠心脏起搏器细胞聚集一代

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

generation-murine-cardiac-pacemaker-cell-aggregates-based-on-es-cell

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.6K 次观看.  University of Rostock. 本协议描述了如何制作从小鼠多能干细胞（PSC）功能性鼻窦淋巴组织。 T-BOX3（TBX3）表达以及心肌肌球蛋白重链（MYH6）启动抗生素的选择导致了高纯度的起搏细胞聚集。这些"诱导窦房结-机构"（"iSABs"）含有80％以上的起搏细胞，显示高度增加跳动的速度，并能步伐心肌体外 。 

视频: Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

基于发育毒性测定化学品安全筛选和系统生物学数据生成人类多能干细胞

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

科研

发育生物学

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/&#946;-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol to generate aggregates of mouse embryonic stem cells that display self-organization, symmetry breaking and elongation paralleling axial development. This technique allows the study of axial developmental processes and the generation of cell types that are otherwise difficult to perform in monolayer culture.

小鼠胚胎干细胞的聚集体的产生，显示对称破缺，极化和突发集体行为<em&gt;体外</em

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

转基因器官皮肤培养使用灭活人真皮代

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

在微孔聚集尺寸优化人类多能干细胞的基于悬浮心脏分化

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

小鼠心脏场特定心脏祖细胞的体外生成

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

通过微分试穿素生成马赛克乳腺有机体

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...