Name: generation of a human ipsc-based blood-brain barrier chip
Uploaded: 2020-03-02T14:30:00
Description: Watch this Scientific Journal Video about Generation of a Human iPSC-Based Blood-Brain Barrier Chip at JoVE.com

我们要感谢索沙娜·斯文德森博士的批判性编辑。这项工作得到以色列科学基金会第1621/18年赠款、科学和技术部（MOST）、以色列3-15647、加州再生医学研究所IDDISC1-08800、谢尔曼家庭基金会、NIH-NINDS赠款1UG3NS105703和ALS协会赠款18-SI-389的支持。AH由瓦伦堡基金会资助（赠款编号2015.0178）。

1. 生成 iPSC 衍生神经祖细胞 （iNPC）
<ol><li>生产EZ球从iPSC殖民地如下所述，并如先前出版的20，21，22。<ol>
<li>培养 iPSC 菌落在 mTESR1 或其他商业介质中，在基底膜基质涂层 6 孔板 （0.5 mg/板） 上汇合（参见材料表）。</li>
		<li>取出 iPSC 介质，用 2 mL 的 EZ 球介质 [ESM] 替换;DMEM：F12 7：3 补充100纳克/mL基本成纤维细胞?...

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NVU 中的器官片上技术和 iPSC 衍生细胞的结合为人类 BBB 的精确建模带来了希望。在这里，我们提供了一个详细的协议，用于简单和稳健地应用最近发布的基于iPSC的BBB芯片16。种子设定范式的概述和时间如图3所示。要获得和维护适合 BBB 建模的屏障函数，生成均质 iBMEC 单层并保持其完整性至关重要。迈向功能单层的第一步包括非极性PDMS表面的化学激活，允?...

BBB是一种高度选择性的屏障，将中枢神经系统与循环血液分离。它保护关键的大脑功能免受潜在的破坏性物质，因子和异种生物，同时也允许营养物质和其他代谢物的流入，以保持大脑平衡1。BBB 是一种多细胞 NVU，其中圆细胞、星形细胞端脚和神经元过程直接接触脑微血管内皮细胞 （BMECs）。这些交互允许 BMEC 形成由紧密和附着点2、3支持的专用屏障属性。这种屏障的形成限制了分子的准细胞通道，但它含有偏振的传输器，以主动将分子输送到CNS或回到血液1。由于这些独特的屏障特性，BBB是生物药物输送到大脑的主要障碍，据估计，不到5%的FDA批准的小分子可以达到CNS4。
动物模型已广泛用于研究BBB渗透和BBB开发中涉及的分子机制5。虽然动物模型忠实地表现了复杂的多细胞在体内环境，BBB运输者的表情和活性的差异，以及不同物种的基质特异性往往排除了动物数据准确地推断给人类6。因此，基于人的模型对于研究人类BBB和用于开发针对CNS的药物至关重要。随着生物、人类专用药物在制药开发领域的日益占据主导地位，这种需求变得更加明显。累积的证据表明，受损的BBB与一些严重的中枢神经系统疾病有关，包括脑肿瘤和神经系统疾病7，8，9。忠实地反映这些疾病的人类模型有可能1）确定可能针对药物开发的新途径，2）预测CNS渗透，从而减少临床前研究的时间和资源，并可能降低临床试验的失败率。
体外模型已被广泛应用，以研究BMEC与NVU其他细胞之间的相互作用，并为潜在的BBB渗透药物10进行筛选。为了重建人类BBB的关键方面，体外模型必须显示生理相关特性（即，低副细胞渗透性和生理相关的跨端面电阻[TEER]穿过内皮单层）。此外，体外系统的分子轮廓必须包括代表性功能传输系统的表达。通常，体外模型由内皮细胞组成，这些细胞在半渗透膜上与其他NVU细胞的组合共同培养，以增强BBB特性11。这种方法允许对阻隔功能和分子渗透性进行简单和相对快速的评估。这种基于细胞的BBB模型可以建立与动物或人类细胞源，包括从手术切除或永生BMEC线分离的细胞。
最近，将人类多能细胞分化为BMEC的协议被引入，作为体外人类BBB模型12、13的诱人来源。诱导多能干细胞（iPSC）衍生的BMECs（iBMECs）具有高度的可扩展性，表现出人类BBB的关键形态和功能特征，并携带患者的遗传学。在文化中，iBMECs 形成一个单层，表示紧密的交汇点标记，并在体内显示类似紧密的交汇点复合物。这些细胞也表达BBB标记，包括BBB葡萄糖转运器，葡萄糖转运器1（GLUT1）。重要的是，与人类BMECs的其他替代细胞源不同，iBMECs获取的屏障特性与在体内14测量的值一样高，沿巴索边轴极化，并表达功能外泄泵。此外，使用不同科目的 iPSC 1） 欢迎有机会以个性化医学方式测试 BBB 的各个方面，2） 为生成 NVU 的其他细胞类型提供了灵活的来源。从异源细胞源生成这些细胞以产生个性化的BBB芯片也有助于了解药物反应的个体间差异，这是导致抗药性或对临床研究中观察到的治疗反应受损的主要原因。
在盘式或半透射式跨井插入物中，将 iBMEC 用作单层，是 BBB 建模的有力方法。这些系统往往坚固、可重复且经济高效。此外，TEER 和渗透性等功能分析执行起来相对简单。然而，二维（2D）系统无法概括体内组织的3D特性，它们缺乏循环血液和血细胞提供的生理剪切应激力量。这限制了这些模型中血管内皮开发和维护内在BBB特性和功能的能力。
由活细胞排列的微工程系统已经实现，以模型各种器官功能的概念称为器官在芯片上。通过重建体内类似多细胞的架构、组织-组织界面、物理化学微环境和血管灌注，这些微工程平台产生无法与传统的 2D 文化系统。它们还支持高分辨率、实时成像和分析与体内组织和器官环境中的活细胞相似的生化、遗传和代谢特征。然而，芯片上器官的一个特别挑战是，这些微工程芯片的设计、制造和应用需要专业工程专业知识，而这些专业知识在生物学学术实验室中通常缺乏。
我们最近将iPSC和器官芯片技术相结合，生成了15、16的个性化BBB芯片型号。为了克服上述技术挑战，商用Chip-S1与培养模块一起使用，该仪器旨在以简单可靠的方式自动维护芯片（Emulate Inc.）。BBB芯片再现了神经细胞和内皮细胞之间的相互作用，并实现了生理相关的TEER值，该值由定制的器官芯片与集成的金电极17测量。此外，BBB芯片显示低副细胞渗透性，响应器官级别的炎症提示，表示活性排泄泵，并表现出可溶性生物标志物和生物药物的预测传输。值得注意的是，由几个人产生的BBB芯片捕获了健康个体与神经系统疾病患者之间的预期功能差异15。
下面详述的协议描述了一种可靠、高效和可重复的方法，用于在动态流条件下生成基于 iPSC 的 BBB 芯片。提供有关可以直接在 BBB 芯片或从取样流出物中执行的测定和端点分析类型的指导。因此，该协议演示了可用于评估人类相关模型中的生物和功能特性和响应的技术范围。
此处简要介绍了基于 iPSC 的 BBB 芯片。人类iPSC最初在组织培养瓶中作为神经祖体的自由浮动聚集体（称为EZ球体）进行分化和繁殖。芯片-S116，18，19的顶部通道被种子与分离的EZ球，形成芯片的"大脑侧"，因为细胞分化在7天的神经祖细胞（iNPCs），iAstrocyt和iNeurons的混合培养。人类 iPSC 在组织培养板中也区分为 iBMEC。芯片的底部通道与iBMECs一起播种，形成"血侧"，形成内皮管（图1）。多孔细胞外基质 （ECM） 涂层膜可分离顶部和底部通道 1），允许通道和 2 之间形成细胞与细胞之间的相互作用，允许用户使用传统的光学显微镜在任一通道中运行渗透性测定和图像单元。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase</td>
			<td>EMD Millipore</td>
			<td>SCR005</td>
			<td>Dissociation solution</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bfgf</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Chip-S1</td>
			<td>Emulate Inc</td>
			<td>Chip-S1</td>
			<td>Organ-Chip</td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma</td>
			<td>C5533</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran-FITC</td>
			<td>Sigma</td>
			<td>46944</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM: F12</td>
			<td>Thermo Fisher Scientific</td>
			<td>31330038</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Emulate Reagent 1 (ER-1)</td>
			<td>Emulate Inc</td>
			<td>ER-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Emulate Reagent 2 (ER-2)</td>
			<td>Emulate Inc</td>
			<td>ER-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>Glial Fibrillary Acidic Protein (GFAP)</td>
			<td>Dako</td>
			<td>Z0334</td>
			<td></td>
		</tr>
		<tr>
			<td>GLUT-1</td>
			<td>Invitrogen</td>
			<td>MA5-11315</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Life Technologies</td>
			<td>35050038</td>
			<td>Glutamine supplement</td>
		</tr>
		<tr>
			<td>hBDNF</td>
			<td>Peprotech</td>
			<td>450-02</td>
			<td></td>
		</tr>
		<tr>
			<td>KOSR</td>
			<td>Thermo Fisher Scientific</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>StemCell Technologies, Inc.</td>
			<td>85851</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Biological industries</td>
			<td>01-340-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Nestin</td>
			<td>Millipore</td>
			<td>MAB353</td>
			<td></td>
		</tr>
		<tr>
			<td>NutriStem</td>
			<td>Biological industries</td>
			<td>05-100-1A</td>
			<td>Alternate media</td>
		</tr>
		<tr>
			<td>PECAM-1</td>
			<td>Thermo Fisher Scientific</td>
			<td>10333</td>
			<td></td>
		</tr>
		<tr>
			<td>Platelet-poor plasma-derived bovine serum (PPP)</td>
			<td>Biomedical Technologies</td>
			<td>J64483AB</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid (RA)</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>S100&#946;</td>
			<td>Abcam</td>
			<td>ab6602</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip-GP Sterile Centrifuge Tube Top Filter Unit</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>ZO-1 Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>33-9100</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;III-tubulin (Tuj1&#945;)</td>
			<td>Sigma</td>
			<td>T8660</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>31350010</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of a human ipsc-based blood-brain barrier chip

血脑屏障 （BBB） 由神经血管单元 （NUS） 形成，这些单元保护中枢神经系统 （CNS） 免受血液中发现的一系列可能破坏大脑功能的因素的影响。因此，BBB是向国家癌症系统提供治疗药物的主要障碍。积累的证据表明，BBB在神经系统疾病的发病和进展中起着关键作用。因此，非常需要BBB模型，该模型可以预测中枢神经系统靶向药物的渗透，并阐明BBB在健康和疾病中的作用。
我们最近将芯片上的器官和诱导多能干细胞（iPSC）技术相结合，以产生完全个性化对人类的BBB芯片。该新平台显示细胞、分子和生理特性，适用于预测药物和分子在人体BBB中的传输。此外，我们使用患者特有的BBB芯片，生成了神经系统疾病模型，并展示了个性化预测医学应用的潜力。这里提供了一个详细的协议，演示如何生成iPSC衍生的BBB芯片，从iPSC衍生的脑微血管内皮细胞（iBMECs）的分化开始，并产生包含神经祖子的混合神经培养，分化神经元和星形细胞。还描述了将细胞播种到器官芯片和在受控层流下培养BBB芯片的过程。最后，对BBB芯片分析进行了详细说明，包括用于评估药物和分子渗透性的准细胞渗透性测定，以及用于测定芯片内细胞类型成分的免疫细胞化学方法。

图6A，B，C表示一个BBB芯片，在"大脑侧"顶部通道上播种EZ球体，在"血侧"底部通道上使用iBMEC。iBMEC 先播种，并可在一夜之间附加，之后 EZ 球体被播种。然后，在静态条件下培养芯片，每天更换介质7天。然后，在 RT 下使用 4% PFA 固定 BBB 芯片 10 分钟，并用 DPBS 清洗 3 倍。免疫细胞化学在BBB芯片上进行，使用1）内丁作为神经祖细胞的标记，2）S100+或GFAP作为星形细?...

Watch this Scientific Journal Video about Generation of a Human iPSC-Based Blood-Brain Barrier Chip at JoVE.com

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

血脑屏障（BBB）是一种多细胞神经血管单元，严密调节大脑平衡。通过结合人类 iPSC 和芯片上器官技术，我们生成了个性化的 BBB 芯片，适用于疾病建模和 CNS 药物渗透性预测。介绍了BBB芯片的生成和运行的详细方案。

生成基于人类 iPSC 的血脑屏障芯片

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

该协议将展示如何分化诱导多能干细胞在片上器官上播种，以产生一个完全人性化的、个性化的微流体血脑屏障，可用于预测中枢神经系统药物的渗透性，并研究神经系统疾病。使用市售的片上器官，我们将演示任何面向生物的实验室如何使用这项技术来生成个性化的微流体血脑芯片屏障。累积的证据表明，BBB通过产生源自遗传神经系统疾病个体的 iPSC 的个性化 BBB 芯片，在神经系统疾病中发挥作用。研究BBB在健康和疾病中的作用是可能的。这种方法对制药公司也很有用，它可以使用这个人类BBB平台来筛选候选神经药物进入人脑的可接受性。在片上应用器官技术通常需要专门的工程技能。使用市售平台可在任何面向生物的实验室中应用此技术。首先，将装有准备好的薯条的盘子带到生物安全柜。使用 P200 移液器，通过将 200 微升神经分化介质加入入口并从出口中拉出液体，轻轻清洗两个通道。避免与端口接触，小心地从芯片表面吸入多余的介质液滴。轻轻搅拌细胞悬浮液，以确保细胞均匀悬浮液。要将 iPSC 衍生的神经祖细胞播种到顶部通道中生成大脑侧，请添加一个 P200 尖端，其中包含 30 到 100 微升细胞悬浮液，浓度为每毫升 10 倍至 6 个细胞到顶部通道入口，然后轻轻地从移液器中释放尖端。取一个空的P200移液器，压压柱塞，插入顶部通道出口，并小心地将单细胞悬浮液拉过尖端。将芯片转移到显微镜上，检查顶部通道内细胞的种子密度和均匀分布。在确认细胞密度为20%覆盖后，立即将芯片在37摄氏度的培养箱中放置两小时。之后，通过向入口中加入新的神经分化介质，然后通过移液器从出口中拉出液体，洗去不连接的细胞。将细胞在静态条件下保持37摄氏度，每日更换神经分化介质。在 iNPC 连接后或在播种后的第二天，将含有已准备的芯片的盘子带到生物安全柜。用 200 微升内皮细胞介质轻轻清洗底部通道。避免与端口接触，小心地从芯片表面吸入多余的内皮细胞介质液滴，确保将介质留在两个通道中。轻轻搅拌细胞悬浮液，以确保细胞均匀悬浮液。使用P200移液器，在浓度为14至20倍的10倍至每毫升6个细胞时，绘制30至100微升的 iPSC 衍生脑微血管内皮细胞悬浮液，然后将尖端放入底部通道入口。实现功能屏障特性的最关键步骤是脑微血管内皮细胞的播种。以适当的密度播种细胞，以避免气泡形成，以验证器官芯片内的均匀分布至关重要。用空尖端压压 P200 移液器上的柱塞，插入底部通道出口，然后缓慢释放移液器柱塞，小心地将单单元悬架拉过底部通道。从芯片表面吸出多余的电池悬浮液。避免与进气口和出口端口直接接触，以确保没有从通道中吸气。将芯片转移到显微镜上以检查播种密度。底部通道充满了单元格，两者之间没有可观察到的间隙。确认正确的细胞密度后，在剩余的芯片中播种细胞。将电池连接到位于底部通道顶部的多孔膜上，反转每个芯片，让它们放在芯片底座中。将一个含有无菌 DPBS 的小储液罐放在 150 毫米的培养皿内，为细胞提供湿度。在37摄氏度下孵育芯片约3小时，或直到底部通道中的细胞附着。一旦 iPSC 衍生的脑微血管内皮细胞连接，将芯片翻转回直立位置，使细胞附着到底部通道的底部。iPSC衍生脑微血管内皮细胞播种48小时后，在37摄氏度的水浴中加热一小时，平衡内皮细胞介质的温度。然后，在真空驱动的过滤系统下通过孵化将介质除气15分钟。接下来，用70%乙醇对便携式模块包装和托盘的外部进行消毒，擦拭，然后转移到生物安全柜。打开包装盒，将模块放入托盘中，将模块与储液罐朝向托盘背面的方向。向每个进气储层提供三毫升预平衡的暖介质。将内皮细胞培养介质添加到底部通道的入口储液罐中，将神经分化介质添加到顶部通道的顶部通道进储液罐中。然后，移液器300微升的预平衡，温暖的介质到每个出口储液罐，直接在每个出口端口。在每个托盘上放置多达六个便携式模块。将托盘带到培养箱中，然后完全滑入培养模块，托盘手柄朝外。在区域性模块上选择并运行主要周期。关闭孵化器门，让培养模块为便携式模块准备约一分钟。当状态栏读取就绪时，启动周期完成。从培养模块中取出托盘，并将其带到生物安全柜。检查生物安全柜中每个便携式模块的底面，以验证便携式模块是否已成功起底。查找所有四个端口是否存在小液滴。如果任何便携式模块未显示液滴，请在这些模块上重新运行主要循环。如果任何介质滴到托盘上（更频繁地出现在出口端口上），请用 70% 乙醇清洁托盘。接下来，用温暖、平衡的细胞特异性培养基轻轻清洗每个芯片的两个通道，以去除通道中任何可能的气泡，并在每个入口和出口端口的顶部放置小介质滴。将带托架的芯片插入便携式模块，并在每个托盘上放置最多 6 个。将纸盒插入培养模块。在培养模块上编程适当的器官芯片培养条件，如流速和拉伸。一旦调节周期完成，大约需要两个小时，编程条件就开始。之后，培养模块在预设的器官芯片培养条件下开始流动。基于 iPSC 的血脑屏障芯片上免疫细胞化学在播种后七天显示 EZ 球体分化成顶部大脑侧通道的混合神经细胞群，包括 S100 β 阳性星细胞、内丁阳性神经祖细胞以及 GFAP 阳性星细胞和 β III 图布蛋白阳性神经元。IPSC衍生的脑微血管内皮细胞在底部血侧通道中播种，表达胶质-1和PECAM-1。对血脑屏障芯片渗透性的评价表明，与仅用 iPSC 衍生脑微血管内皮细胞和 EZ 球体播种的器官芯片相比，仅用 iPSC 衍生脑微血管内皮细胞播种的器官芯片具有紧密的屏障。单独用EZ球体播种的器官芯片没有显示任何屏障特性。从通道的初始表面处理到每天的介质切换，避免通道中形成气泡。在整个协议中，需要通过通道插入表面活化试剂、细胞外基质或含细胞介质，因此仔细验证是否未发现气泡非常重要。可进行渗透性测定，以评估候选药物的人体大脑渗透性。这种方法可用于测试潜在的治疗方法。基于 iPSC 的 BBB 芯片的应用允许研究 BBB 在各种神经系统疾病中的作用。将来，这种平台可能有助于预测性、个性化的医学应用。

generation-of-a-human-ipsc-based-blood-brain-barrier-chip

Research

JoVE Journal

Developmental Biology

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

通过成熟的脂肪细胞在天花板文化去分化代人脂肪干细胞

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

科研

发育生物学

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 ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

界定和由人多能干细胞的肝细胞样细胞的可扩展代

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

源自iPSC的人脑组织体的一代新型早期神经发育障碍

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

成人皮肤角质形成细胞的生成与培养

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

人肠道 Organoids 上皮屏障通透性的实时测量

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

果蝇黑色素血脑屏障完整性分析

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

多细胞人类原发性子宫内膜器官的生成

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...