L.M.E. 得到了 DFG 研究培训计划的支持，该计划GRK2157题为"用于研究人类病原体微生物感染的 3D 组织模型"，授予 A. S-U.B.J.K. 得到了亚历山大·冯·洪堡基金会的博士后奖学金的支持。此外，我们感谢 Lena Wolter 在培养物中生成 iPSC-BEC 方面的技术援助。

注:所有培养基/试剂制备、干细胞维护和分化步骤均改编自 Stebbins 等人 22。
1.制备iPSC培养和BEC分化所需的材料。
<ol><li>用于 IMR90-4 iPSC 培养的组织培养 （TC） 塑料基质包被<ol><li>将基底膜基质凝胶（例如基质胶）等分成2.5mg等分试样，并储存在-20°C。 注意:在冰上处理基质凝胶和等分试样时，请快速工作，因为它在4°C以上形成凝胶，一旦凝固就不能分装。</li>
		<li>对于涂覆 TC 塑料，在 50 mL 锥形管中快速将一等分的基质凝胶加入 30 mL Dulbecco 改良 Eagle 培养基 （DMEM）/F12 培养基中。将 1 mL 培养基加入冷冻基质凝胶上，上下移液直至解冻，然后立即将剩余培养基转移到 50 mL 锥形管中。</li>
		<li>在 6 孔板的每孔中使用 1 mL 的基质凝胶包被溶液，每个 T75 培养瓶使用 12 mL。 注意:基质凝胶涂层的 TC 塑料可在使用前两周制备。然而，避免基质凝胶溶液变干至关重要，这可能需要偶尔在孔顶部添加更多的 DMEM/F12。</li>
	</ol></li>
	<li>在生物安全柜的无菌环境中，将 50 mL 50x 补充剂加入 450 mL 干细胞维持基础培养基中，制备干细胞维持培养基。 注:其他干细胞维持培养基（mTeSR 和 E8）已用于其他研究 13,14,15,22,23,24,25。 </li>
	<li>要制备 500 mL 未调节培养基 （UM），将 392.5 mL DMEM/F12 与 100 mL 敲除血清替代物 （KOSR）、5 mL 非必需氨基酸、终浓度为 1 mM 的 L-谷氨酰胺和 3.5 μL β-巯基乙醇混合。过滤灭菌并在4°C下储存长达2周。</li>
	<li>要制备 200 mL 内皮细胞 （EC） 培养基加视黄酸 （RA） 加 bFGF，请混合 198 mL 人内皮血清游离培养基 （hESFM）、2 mL 过滤灭菌的贫血小板衍生血清 （PDS） 和 20 ng/mL bFGF。过滤灭菌并在4°C下储存长达2周。 在添加到细胞之前，向 EC 培养基中加入 10 μM RA。 注意:由于PDS已停产，因此可能受到限制，因此该协议已使用B27代替PDS 15,23,26成功进行。</li>
	<li>要制备不含 RA 或 bFGF 的 200 mL EC 培养基，请合并 198 mL hESFM 和 2 mL 过滤灭菌的 PDS 并过滤灭菌。在4°C下储存长达4周。</li>
</ol>2. 维持IMR90-4细胞培养
注:这里我们以IMR90-4细胞系为例，但其他诱导多能干细胞系如CC3、CD10、CD12、DF19-9-11T、83iCTR、00iCTR和CS03iCTRn2已成功用于分化为BECs13,14,15,16,17,23,27,28。
<ol><li>在37°C的5%CO2 中培养iPSC，通常以不同的密度在6孔板上维持，每孔2mL干细胞维持培养基。</li>
	<li>为了维持 iPSC 培养物，选择一个不汇合且菌落之间有开放空间的通道。<ol><li>吸出培养基，加入1mL非酶促细胞解离试剂，并在37°C下孵育7分钟。在孵育过程中，用每孔 2 mL 新鲜干细胞维持培养基替换新的 6 孔板上的基质凝胶溶液。</li>
		<li>小心地吸出非酶促细胞解离试剂，注意不要吸出仍附着在平板上的细胞。</li>
		<li>加入 6 mL 干细胞维持培养基，冲洗孔底几次，直至所有细胞完全分离。然后，接种具有不同密度的新 6 孔板，通常为 1:6 或 1:12 以进行正常维护。 注意:使用上述比例，细胞大约每周分裂两次。</li>
		<li>将板移至培养箱中，通过来回和从左到右摇动板，在交替摇动之间暂停直到培养基沉淀，将接种的细胞均匀地分布在孔中。</li>
	</ol></li>
</ol>3. 脑内皮细胞与人iPSC的分化
<ol><li>用于分化的 iPSC 铺板（分化方案的第 -3 天 ）<ol><li>每个IMR90-4维持培养孔用于分化，吸出培养基，每孔加入1mL酶促细胞解离试剂，并在37°C下孵育7分钟。</li>
		<li>通过将 1 mL 解离的细胞悬液转移到 15 mL 锥形管中，每 1 mL 细胞至少含有 2 mL 新鲜干细胞维持培养基，使酶促细胞解离试剂失活。将细胞悬液以1,500× g 旋转5分钟。</li>
		<li>将细胞沉淀重悬于每孔使用的 IMR90-4 细胞的 1 mL 干细胞维持培养基中，并使用血细胞计数器对细胞进行计数。 注意:在计数时，用 0.4% 台盼蓝以 1:1 稀释以区分活细胞和死细胞可能会有所帮助。根据 iPSC 的密度，6 孔板的一个孔通常产生 1-10 个6 个细胞。</li>
		<li>为了在 T75 烧瓶中分化，将 7.5 x 105 个细胞加入 12 mL 干细胞维持培养基和 ROCK 抑制剂（Y27632 二盐酸盐）中，终浓度为 10 μM。 从 T75 烧瓶中吸出基质凝胶包被溶液并将细胞悬浮液转移到烧瓶中。通过来回和从左到右摇动烧瓶（参见步骤2.2.4）均匀分布细胞，并在37°C和5%CO2下孵育。 注意:在此步骤中添加ROCK抑制剂以增强解离的单个干细胞的存活率至关重要25,29。细胞应均匀分布在烧瓶中，并且由于 ROCK 抑制剂处理而表现出扩散的间充质样形态的单体中22。</li>
	</ol></li>
	<li>在第 -2 天 和第 -1 天，将培养基更换为新鲜的干细胞维持培养基;每 T75 12 mL。</li>
	<li>在第 0 天，通过将培养基更改为 UM 开始分化;每 T75 12 mL。</li>
	<li>每天更改 UM（第 1 天 - 第 5 天）。 注意:细胞通常在 UM 中 2 至 3 天后达到汇合，可以用肉眼或通过倒置明场显微镜观察。随着分化的进展，巢蛋白+"神经束"变得可见，中间有PECAM-1+细胞，如前所述13,14。</li>
	<li>通过切换到含有 20 ng/mL bFGF 和 10 μM 视黄酸 （RA）（第 6 天）的 EC 培养基并孵育两天，选择性地扩增内皮细胞群。含 bFGF 的 EC 培养基可在两周前制备。RA在使用当天从冷冻原液中添加（例如，每1mL EC + bFGF加入1μL 10 mM RA原液）。 注意:在第 6 天和第 8 天无需补充 RA 也可以实现成功分化。然而，省略 RA 将产生 TEER降低 12、13、14 的 BEC。</li>
	<li>用胶原 IV 和纤连蛋白（第 7 天）包被细胞培养板和膜插入物（例如 Transwell），用于纯化 BEC 和后续实验。<ol><li>对于膜插入物的包被，将 4 份 IV 型胶原蛋白（1 mg/mL 溶于 0.5 mg/mL 乙酸中）、1 份纤连蛋白 （1 mg/mL） 和 5 份无菌组织级水混合。ECM 溶液可以按 1:5 的比例稀释用于包被细胞培养板（即 4 份 IV 份胶原蛋白、1 份纤连蛋白、45 份水）。与包衣溶液在37°C孵育过夜。 注:在纯化步骤的同一天，在传代培养之前，也可以在传代培养前包被板和膜插入物至少4小时。</li>
	</ol></li>
	<li>通过在胶原 IV 和纤连蛋白包被的平板或膜插入物上传代培养分化的细胞来纯化 BEC（第 8 天）。<ol><li>吸出 EC 培养基并加入酶促细胞解离试剂（每 T75 12 mL）。在37°C孵育，直到90%的细胞从烧瓶中分离出来。 注意:细胞解离可能需要长达1小时。</li>
		<li>在孵育期间，从先前制备的板/插入物中取出胶原蛋白IV /纤连蛋白涂层溶液，并在无菌罩中干燥。插入物干燥大约需要 20 分钟。</li>
		<li>细胞分离后，使用 10 mL 移液管将它们从烧瓶中洗掉。上下移液器以实现单细胞悬浮液。 注:单细胞悬浮液对于可靠的细胞计数和实现固体单层非常重要。</li>
		<li>在 50 mL 锥形管中用至少等体积的新鲜 hESFM 稀释，并使用血细胞计数器计数细胞。</li>
		<li>将细胞以1,500×g沉淀10分钟。</li>
		<li>将细胞重悬于适当体积的新鲜制备的 EC + bFGF + RA 中，以达到 2 x 106 个细胞/mL 的悬浮液，用于接种在膜插入物上。在 12 孔插入物顶部加入 500 μL（1 x 106 个细胞），在底部加入 1.5 mL EC + bFGF + RA 培养基。对于接种在 24 和 48 孔板上，以 1:2 稀释细胞悬液，每孔分别加入 500 μL（5 x 10 5 个细胞）和 250 μL（2.5 x 105 个细胞）。将细胞均匀分布在孔/插入物上（参见步骤2.2.4），并在37°C下在5%CO2下孵育。</li>
	</ol></li>
	<li>将平板/转孔上的培养基更换为 不含 bFGF 或 RA 的 EC（第 9 天）。</li>
	<li>进行感染实验、TEER 测量和免疫荧光染色，如以下各节所述（第 10 天）。 注:成功分化和纯化的 BEC 通常在第 10 天达到峰值 TEER，并表达脑内皮细胞的特征标志物，例如 PECAM-1 （CD31） 和 VE-cadherin、葡萄糖转运蛋白 GLUT-1、外排转运蛋白（如 p-糖蛋白）和紧密连接成分 ZO-1、Occludin 和 Claudin-5 13,14,16,17,19,22 .有关分化过程中细胞类型、形态和细胞类型特异性标志物表达的更多详细信息和图像，请参阅 Lippmann 等人、Stebbins 等人13、14、15、16、17、19、22。IMR90-4细胞在BEC分化过程不同阶段的代表性图像可以在补充图1中找到。</li>
</ol>4. 跨内皮电阻 （TEER） 作为屏障密封性的量度
注:通常在分化第 9 天和第 10 天在膜插入物上读取 TEER，以确认成功产生形成屏障的 iPSC-BEC（图 1A）。
<ol><li>将上皮伏欧表 （EVOM） 置于生物安全罩的无菌环境中，并将电极连接到 EVOM。</li>
	<li>将电极浸入 70% EtOH 中至少 5 分钟，然后使其完全干燥，从而对电极进行消毒。 注意:如果需要，可以在 70% EtOH 中孵育更长时间或使用 5% 次氯酸盐溶液对电极进行去污。</li>
	<li>从培养箱中取出膜插入物上的iPSC-BEC并测量TEER。 注意: 从培养箱中取出后快速读取 TEER 很重要，因为温度变化可能会影响 TEER 测量。</li>
	<li>通过将电极浸入介质中来读取 TEER，以便将较短的电极放置在插入物的顶部，而较长的电极伸入插入插入物周围的介质中。 注意: 确保"筷子"尖端的电极完全被液体覆盖。如果需要，在再次放下板进行测量之前，倾斜孔以达到此目的。</li>
</ol>5. 免疫荧光 （IF） 染色以验证 BEC 表型
注:为了验证完全分化和纯化细胞的质量，如前所述，在分化过程的第10天对iPSC-BEC单层的脑内皮细胞特征标志物进行染色（图1B - G）13,14,15,16,17,19,22。 
<ol><li>吸出培养基并用PBS洗涤1x。</li>
	<li>在室温（RT）下用冰冷的甲醇固定细胞15分钟。</li>
	<li>用磷酸盐缓冲盐水（PBS）洗涤3次，并在室温下用10%胎牛血清（FBS）在PBS中封闭1小时。</li>
	<li>吸出并加入在封闭液中稀释的一抗。在4°C孵育过夜。 注:有关抗体的来源和稀释度的信息，请参阅 材料表 和Stebbins等人22。</li>
	<li>用 PBS 洗涤 3 次，然后加入在封闭液中稀释的二抗。在室温下孵育1小时。从现在开始保护样品免受光线照射。</li>
	<li>用 PBS 洗涤 2 次。然后，在PBS中以1:5,000稀释度加入DAPI，并在室温下染色15分钟。</li>
	<li>洗涤 1 次，保持 PBS 开启状态，并使用荧光显微镜拍摄图像。</li>
</ol>6. 细菌的制备和iPSC-BECs的感染
<ol><li>在感染实验的前一天（分化的第9天 ），开始从冷冻原液中培养细菌过夜。对于Nm感染，用5%绵羊血（血琼脂）将细菌条纹到哥伦比亚琼脂上。将细菌培养物在37°C和5%CO2 下孵育过夜。</li>
	<li>第二天（第 10 天），通过每 10 mL 补充 50 μL 2 M MgCl 2、50 μL2 M NaHCO3 和 100 μL 凯洛氏补充剂来制备新鲜的 PPM+。使用无菌棉签将 10 mL PPM + 培养基接种在 50 mL 锥形管中，其中含有来自过夜培养板的 Nm。在37°C下以200rpm振荡孵育1.5小时（即，直到细菌处于对数生长阶段）。</li>
	<li>在细菌孵育期间，通过用每孔/膜插入物顶部用 400 μL 新鲜 EC 培养基（不含 bFGF 或 RA）替换旧培养基，在 24 孔板或膜插入物上制备用于感染的 iPSC-BEC。</li>
	<li>将细菌培养物以4000× g 离心10分钟并吸出培养基。将细菌沉淀重悬于 250 μL PBS 中。</li>
	<li>在 4 mL 新鲜 PBS 中，使用一部分细菌细胞悬液并调节至 OD600 为 0.4（约 1 x 108 CFU/mL）。</li>
	<li>然后，根据所需的感染多重性（MOI）稀释细胞培养基（EC培养基）中的细菌。 注:例如，对于 MOI 为 10，在 24 孔板或膜插入物中感染 iPSC-BEC 时，分别稀释 1:10 或 1:5（24 孔板中每单层 1 x 105 个细胞）。正如其他手稿中所描述的那样，添加人血清不包括在感染的 Nm 的制备中，因为它被观察到对 TEER 测量的 iPSC-BEC 屏障表型有有害影响（数据未显示）30,31。然而，无论是否使用人血清，Nm 和 iPSC-BEC 的相互作用都不会受到影响（数据未显示）。</li>
	<li>用 100 μL 制备的细菌悬浮液感染每个孔中的 iPSC-BEC，并孵育所需的感染时间。 注:当感染 Nm 时，iPSC-BEC 中许多促炎细胞因子和趋化因子的表达升高，最突出的是感染 8 小时后，如 Martins-Gomes 等人先前描述的那样（图 2）19。</li>
</ol>7. 通过定量PCR激活先天免疫
注:使用优选的RNA分离、cDNA合成和qPCR方案，收集样品并对选定的细胞因子进行qPCR。
<ol><li>7.1. 在分化方案的第 10 天 感染后，使用市售 RNA 分离试剂盒中的试剂从 BEC 样品中收集 RNA（参见 材料表）。 注意: 避免核酸酶污染 样品 在清洁和消毒的环境中小心工作。<ol><li>制备裂解缓冲液，并向每个孔/单层 iPSC-BEC 中加入 350 μL。</li>
		<li>通过上下移液多次（例如，20x）并将悬浮液转移到无菌微量离心管中来收集样品。 注:样品可以储存在-80°C，直到准备进行RNA分离。</li>
		<li>按照 RNA 分离试剂盒提供的方案从培养的细胞和组织中纯化 RNA。</li>
		<li>在无核酸酶的水中洗脱后，将RNA样品保持在冰上，以尽量减少任何潜在的RNase活性。</li>
		<li>在分光光度计（例如，Nanodrop）上估计RNA浓度。</li>
	</ol></li>
	<li>使用cDNA合成试剂盒从收集的RNA生成cDNA文库（参见 材料表）。<ol><li>按照cDNA合成试剂盒的方案所述，在规定的总反应体积中建立由cDNA合成预混液、至少200 ng（理想情况下为500 ng）样品RNA和无核酸酶水组成的反应。</li>
		<li>在标准热循环仪上，运行适用于所用cDNA合成试剂盒试剂的程序。例如:25°C下2分钟，55°C下10分钟，95°C下1分钟。</li>
		<li>合成后，在无核酸酶的水中将cDNA稀释至1:10，并将样品移至4°C进行短期或-20°C进行长期储存。 注:如果RNA浓度低（例如，低于50ng），则可能需要较低的稀释度。稀释的cDNA可以在-20°C下储存长达一年。</li>
	</ol></li>
	<li>使用精心设计和验证的引物，对靶向先天免疫应答基因（如细胞因子和趋化因子）转录本的 cDNA 样品进行 qPCR。 注:由于引物设计对qPCR结果的质量非常重要，因此应进行稀释系列测试引物效率，并应在DNA凝胶上确认不存在多种产物。<ol><li>每 25 μL 反应，使用 0.5 μL 正向和 0.5 μL 反向引物（10 mM 无核酸酶水中）、1 μL cDNA、10.5 μL 无核酸酶 H2O 和 12.5 μL qPCR 预混液。</li>
		<li>使用以下循环仪方案在qPCR机器上进行反应:（a）4°C2分钟;（b） 95°C下15分钟;（c） 95 °C下15 s;（d） 60°C下1分钟;循环 （c） 和 （d） 45 次;（e） 可选熔体曲线:30-99 °C，增量为 1 °C;25°C5分钟。</li>
		<li>对于数据分析，使用 ΔΔCT 计算将细胞因子和趋化因子表达水平与参考管家基因（如 18S 或 GAPDH）进行比较。</li>
	</ol></li>
</ol>

<ol>
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作者没有什么可透露的。

BECs和BBB的建模遇到了挑战，因为在体外，原发性和永生化的人BEC往往缺乏强大的屏障表型。人类干细胞技术的出现允许产生 iPSC 衍生的 BEC 样细胞，这些细胞保留了预期的标志性 BBB 表型，例如内皮标志物、紧密连接表达、屏障特性、对其他 CNS 细胞类型的反应和功能性外排转运蛋白 12、13、14、15、22、24 </su...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61400">Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells Larvae</a>. The Authors section was updated from:
Leo M. Endres1 
Alexandra Schubert-Unkmeir1 
Brandon J. Kim1,2 
1Institute for Hygiene and Microbiology, University of W&#252;rzburg 
2Department of Biological Sciences, University of Alabama
to:
Leo M. Endres1 
Sarah F. Hathcock2 
Alexandra Schubert-Unkmeir1 
Brandon J. Kim1,2 
1Institute for Hygiene and Microbiology, University of W&#252;rzburg 
2Department of Biological Sciences, University of Alabama

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61400">Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells Larvae</a>. The Authors section was updated.

Erratum: Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

血脑屏障 （BBB） 和脑膜血脑脊液屏障 （mBCSFB） 是将循环与中枢神经系统 （CNS） 分开的极其紧密的细胞屏障，主要由高度特化的脑内皮细胞 （BEC） 组成1,2。BECs通过调节大脑内外的营养物质和废物来维持适当的大脑稳态，同时排除许多毒素、药物和病原体1,2。当血源性细菌能够与BECs相互作用并穿透BECs形成的屏障并引起炎症时，就会发生细菌性脑膜炎。脑膜炎奈瑟菌（Nm，脑膜炎球菌）是一种革兰氏阴性细菌，定植于10-40%健康个体的鼻咽部，但在某些情况下可引起严重的全身性疾病3。在受影响的个体中，Nm 可以进入血流，在那里它可以引起暴发性紫癜，并穿透 BEC 进入中枢神经系统，从而引起脑膜炎3。Nm 是全球细菌性脑膜炎的主要原因，尽管接种了疫苗，但仍是脑膜炎的主要原因 4.现代医疗干预，如抗生素治疗，使这些疾病得以生存，但那些患有脑膜炎的人往往会留下永久性的神经损伤5,6。
先前的研究已经确定了有助于 Nm-BEC 相互作用的细菌因子和宿主信号转导 7,8,9,10,11。已鉴定的粘附蛋白和侵入蛋白，如混浊蛋白Opc和IV型菌毛，以及受体如CD147，已经在体外对各种BEC模型进行了研究，但这些模型缺乏许多定义的BBB特性7,9,11,12。对 Nm-BEC 相互作用的完全理解仍然难以捉摸，部分原因是无法利用体内模型、不完全的疫苗接种保护以及体外缺乏强大的人类 BEC 模型。
由于 BEC 的独特特性，在体外模拟 hBEC 一直具有挑战性。与外周内皮细胞相比，BEC 具有许多增强其屏障特性的表型，例如由于复杂的紧密连接而导致的高跨内皮电阻 （TEER）12。一旦从大脑微环境中移除，BECs就会迅速失去其屏障特性，从而限制了仅形成弱屏障的原代或永生化体外模型的有用性12,13。Nm 感染的人类特异性、缺乏稳健的体内模型以及体外模拟人类 BEC 的挑战相结合，需要更好的模型来理解 Nm 和 BEC 之间复杂的宿主-病原体相互作用。最近，使用模型人诱导多能干细胞 （iPSC） 技术，BEC 样细胞来源于 iPSC，可在体内更好地模拟 BEC 12、13、14、15。iPSC-BECs是人类来源的，易于扩展，并且与原代或永生化对应物相比具有预期的BEC表型12,13,14,15。此外，我们和其他人已经证明 iPSC-BEC 可用于模拟中枢神经系统的各种疾病，例如宿主-病原体相互作用、亨廷顿舞蹈症和导致 Allan-Hurndon-Dudley 综合征的 MCT8 缺乏症 16,17,18,19,20,21.在这里，我们展示了如何从可再生的 iPSC 来源获得 iPSC-BEC，以及用 Nm 感染 iPSC-BEC 导致先天免疫反应的激活。我们认为，该模型可用于研究宿主-病原体相互作用，这在其他体外模型中无法概括，并且在检查与人类特定病原体（如Nm）的相互作用时特别有用。

<table border="0" cellpadding="0" cellspacing="0" style="width:1301px;" width="1300">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Accutase (1x)</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:161px;">A6964</td>
			<td style="width:465px;">Enzymatic cell dissociation reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Acetic acid</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:161px;">A6283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">All-trans retinoic acid (RA)</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:161px;">R2625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Anti-CD31 (PECAM-1)</td>
			<td style="width:216px;">Thermo Scientific (Labvision)</td>
			<td style="width:161px;">RB-10333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Anti-Claudin-5</td>
			<td style="width:216px;">Invitrogen</td>
			<td style="width:161px;">4C3C2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Anti-Glut-1</td>
			<td style="width:216px;">Thermo Scientific (Labvision)</td>
			<td style="width:161px;">SPM498 (MA5-11315)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Anti-Occludin</td>
			<td style="width:216px;">Invitrogen</td>
			<td style="width:161px;">33-1500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Anti-VE-cadherin</td>
			<td style="width:216px;">Santa Cruz</td>
			<td style="width:161px;">sc-52751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Anti-ZO-1</td>
			<td style="width:216px;">Invitrogen</td>
			<td style="width:161px;">33-9100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Proteose Peptone</td>
			<td style="width:216px;">BD</td>
			<td style="width:161px;">211684</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">b-Mercaptoethanol</td>
			<td style="width:216px;">Merck (Sigma-Aldrich)</td>
			<td style="width:161px;">805740</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Cell culture plates and flasks</td>
			<td style="width:216px;">Sarstedt</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Centrifuge (Heraeus Megafuge 1.0R)</td>
			<td style="width:216px;">Thermo Scientific</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Class II biosafety cabinet</td>
			<td style="width:216px;">Nuaire</td>
			<td style="width:161px;">NU-437-400E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">CO2 Incubator (DHD Autoflow CO2 Air-Jacketed Incubator)</td>
			<td style="width:216px;">Nuaire</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Collagen IV</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:161px;">C5533</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Columbia ager + 5 % sheep blood</td>
			<td style="width:216px;">Biomerieux</td>
			<td style="width:161px;">43049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Costar Transwell polyester filters (12- or 24-well)</td>
			<td style="width:216px;">Corning</td>
			<td style="width:161px;">3460, 3470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">D(+)-Glucose</td>
			<td style="width:216px;">Merck (Sigma-Aldrich)</td>
			<td style="width:161px;">G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">DAPI</td>
			<td style="width:216px;">Invitrogen</td>
			<td style="width:161px;">D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">DMEM/F12</td>
			<td style="width:216px;">Gibco</td>
			<td style="width:161px;">31330-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">DMSO</td>
			<td style="width:216px;">ROTH</td>
			<td style="width:161px;">A994.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td style="width:216px;">Gibco</td>
			<td style="width:161px;">21600-069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Epithelial Volt-Ohm Meter (Millicell ERS-2) with STX electrode</td>
			<td style="width:216px;">Merck (Millipore)</td>
			<td style="width:161px;">MERS00002</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:459px;">Fe(NO3)3</td>
			<td style="width:216px;">ROTH</td>
			<td style="width:161px;">5632.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Fibronectin</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:161px;">F1141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Fluoresence microscope (Eclipse Ti)</td>
			<td style="width:216px;">Nikon</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Hemacytometer (Neubauer)</td>
			<td style="width:216px;">A. Hartenstein</td>
			<td style="width:161px;">ZK06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Human basic fibroblast growth factor (bFGF)</td>
			<td style="width:216px;">PeproTech</td>
			<td style="width:161px;">100-18B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Human Endothelial Serum Free Medium (hESFM)</td>
			<td style="width:216px;">Gibco</td>
			<td style="width:161px;">11111-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Inverted microscope (Wilovert)</td>
			<td style="width:216px;">Hund (Will Wetzlar)</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">iPS(IMR90)-4 cells</td>
			<td style="width:216px;">WiCell</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:459px;">Kellogg&#39;s supplement</td>
			<td style="width:216px;"></td>
			<td style="width:161px;"></td>
			<td>To prepare 110 ml of Kellogg&#39;s supplement, prepare 100 ml of 4 g/ml glucose, 0.1 g/ml glutamine, and 0.2 mg/ml thiamine pyrophosphate and 10 ml of 5 mg/ml Fe(NO3)3 and combine the solutions. Filter sterilize and store aliquoted at -20 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Knockout serum replacement (KOSR)</td>
			<td style="width:216px;">Gibco</td>
			<td style="width:161px;">10828-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">L-glutamine (GlutaMAX)</td>
			<td style="width:216px;">Invitrogen</td>
			<td style="width:161px;">35050-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LunaScript RT SuperMix Kit</td>
			<td style="width:216px;">NEB</td>
			<td style="width:161px;">E3010L</td>
			<td>cDNA synthesis kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Matrigel Matrix</td>
			<td style="width:216px;">Corning</td>
			<td style="width:161px;">354230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Methanol</td>
			<td style="width:216px;">ROTH</td>
			<td style="width:161px;">4627.5</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgCl2</td>
			<td style="width:216px;">ROTH</td>
			<td style="width:161px;">KK36.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Micropipettes (Research Plus)</td>
			<td style="width:216px;">Eppendorf</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3</td>
			<td style="width:216px;">ROTH</td>
			<td style="width:161px;">6329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Nonessential amino acids (NEAA)</td>
			<td style="width:216px;">Gibco</td>
			<td style="width:161px;">11140-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NucleoSpin RNA isolation kit</td>
			<td>Machery-Nagel</td>
			<td style="width:161px;">740955</td>
			<td>RNA isolation kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Pipette boy (Accu-Jet Pro)</td>
			<td style="width:216px;">Brand</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Platelet poor plasma-derived serum, bovine (PDS)</td>
			<td style="width:216px;">Fisher</td>
			<td style="width:161px;">50-443-029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PowerUp SYBR Green Master Mix</td>
			<td>Applied Biosystems</td>
			<td style="width:161px;">A25742</td>
			<td>qPCR master mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">qPCR film (MicroAmp Optical Adhesive Film)</td>
			<td style="width:216px;">Applied Biosystems</td>
			<td style="width:161px;">4211971</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">qPCR plates (MicroAmp Fast 96-well)</td>
			<td style="width:216px;">Applied Biosystems</td>
			<td style="width:161px;">4346907</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">ROCK inhibitor, Y27632 dihydrochloride</td>
			<td style="width:216px;">Tocris</td>
			<td style="width:161px;">1254</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">RT-PCR thermo cycler (StepOnePlus)</td>
			<td style="width:216px;">Applied Biosystems</td>
			<td style="width:161px;">4376600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Serological pipettes</td>
			<td style="width:216px;">Sarstedt</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">StemFlex basal medium + 50x StemFlex supplement</td>
			<td style="width:216px;">Gibco</td>
			<td>A3349401</td>
			<td>Stem-cell maintenance medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Swinging Bucket Rotor (Heraeus #2704)</td>
			<td style="width:216px;">Thermo Scientific</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Thiamine pyrophosphate</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:161px;">C8754-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:216px;">Gibco</td>
			<td style="width:161px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Versene</td>
			<td style="width:216px;">Gibco</td>
			<td style="width:161px;">15040-033</td>
			<td>Non-enzymatic cell dissociation reagent (EDTA)</td>
		</tr>
	</tbody>
</table>

neisseria meningitidis infection of induced pluripotent stem-cell derived brain endothelial cells

脑膜炎球菌性脑膜炎是一种危及生命的感染，当脑膜炎 奈瑟菌（脑膜炎球菌 ，Nm）可以通过穿透高度特化的脑内皮细胞 （BEC） 进入中枢神经系统 （CNS） 时发生。由于 Nm 是一种人类特异性病原体，由于缺乏强大的 体内 模型系统，因此对 Nm 和 BEC 之间的宿主-病原体相互作用的研究具有挑战性，并需要一种模拟天然 BEC 的基于人类的模型。与外周内皮细胞相比，BEC 具有更紧密的屏障特性，其特征是复杂的紧密连接和升高的跨内皮电阻 （TEER）。然而，许多 体外 模型，如原发性BEC和永生化BECs，在从天然神经微环境中移除后，要么缺乏或迅速失去其屏障特性。人类干细胞技术的最新进展已经开发出从诱导多能干细胞 （iPSC） 中提取类脑内皮细胞的方法，与其他体 外 人类模型相比，该方法具有更好的表型 BEC。使用 iPSC 衍生的 BEC （iPSC-BEC） 对 Nm-BEC 相互作用进行建模具有使用具有 BEC 屏障特性的人类细胞的好处，可用于检查屏障破坏、先天免疫激活和细菌相互作用。在这里，我们演示了除了细菌制备、感染和样本采集分析外，还如何从 iPSC 中衍生 iPSC-BEC。

这里描述的方案改编自Stebbins等人，并强调了将iPSC分化为具有BBB特性的脑样内皮细胞的过程，以及如何利用该模型进行使用具有Nm19,22的iPSC-BEC的感染研究。当分化得当时，iPSC-BEC表现出通过TEER测量的紧密屏障特性，通常大于2000 Ω·cm 2，并表达内皮标志物，如VE-钙粘蛋白和CD31（PECAM）（图1A\u2012C）。此外，它们...

Watch this Scientific Journal Video about Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells at JoVE.com

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

这里描述的方案强调了分化诱导多能干细胞衍生的脑样内皮细胞、用于感染的 脑膜炎奈瑟菌 的制备以及用于其他分子分析的样本收集的主要步骤。

脑膜炎奈瑟菌 诱导多能干细胞来源的脑内皮细胞的感染

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...

neisseria-meningitidis-infection-induced-pluripotent-stem-cell

Research

JoVE Journal

Immunology and Infection

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

2.3K 次观看.  University of Würzburg. 这里描述的方案强调了分化诱导多能干细胞衍生的脑样内皮细胞、用于感染的 脑膜炎奈瑟菌 的制备以及用于其他分子分析的样本收集的主要步骤。

视频: Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

胞内相互作用的可视化和量化脑膜炎奈瑟菌和人类α-辅肌动蛋白共聚焦成像

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

科研

免疫与感染

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

选择恶性疟原虫</emCytoadhesion>寄生虫对人脑血管内皮细胞

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

临床应用<em&gt;睡美人</em和人工抗原呈递细胞基因修饰T细胞从外设和脐带血中

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

类器官的模型传染病:人类的文化与小鼠胃组织体与幽门螺杆菌的显微注射

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

利用人类诱导的多能干细胞衍生的肠道有机体研究和改性上皮细胞对沙门氏菌和其他病原体的保护

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

人类干细胞衍生的伊拉尔肠细胞单层中M细胞样细胞的诱导分化

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8+ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

干细胞衍生病毒Ag特异性T淋巴细胞抑制小鼠HBV复制

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

恒流中原发性人类内皮细胞的肺炎球菌感染

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

脑膜炎奈瑟菌 诱导多能干细胞来源的脑内皮细胞的感染

脑膜炎奈瑟菌诱导多能干细胞来源的脑内皮细胞的感染