笔者想感谢梅林达·Hexum为我们的实验室内旋EB协议开始。我们想感谢实验室的其他成员，包括劳拉E. Bendzick，迈克尔Lepley，和他们这项工作的技术援助的振亚镍。作者还要感谢布拉德·泰勒在他的专家技术咨询卡尺生命科学。

1。适应TrypLE自旋EB培养的人类胚胎干细胞或iPS细胞<ol><li> TrypLE的初始分解效果最好，如果胶原酶传代培养的ES / IPS殖民地都比较小。开始ES / IPS的人群应该是细胞通过不超过4-5天前。 TrypLE通道前4-5天开始，通过ES细胞的密度，使细胞〜70％汇合，在4天的时间。使用常规ES媒体文化的TrypLE传代的胚胎干细胞。在这里，我们使用人类胚胎干细胞的稳定结构（ 材料表 ）荧光素酶报告修改。该协议还适用于iPS细胞，利用未修改的iPS细胞，造血祖细胞和NK细胞的发育比较。 </li><li> TrypLE通道开始前四天，通过ES / iPS细胞胶原酶，后正常通行协议。我们通常通过TrypLE在第一通道，1:1。事先展开ES / iPS细胞。 </li><l&gt;前一天在0.9-1×10 5细胞/孔6孔板起始的TrypLE通道，板块MEF中。难以适应细胞系或为密度较小文化）中的第一通道与TrypLE通过1:1（或2:1。因此，准备1的MEF板每ES / IPS板你打算投入TrypLE文化的。 </li><li>仔细挑选有区别的殖民地（那些缺乏一个外接边框或成纤维细胞/上皮特点），然后再进行分离TrypLE。分化的细胞可以接管的文化比较容易TrypLE通道，所以重要的是要确保你的人口开始很干净。 </li><li>吸井加1.0毫升预暖的的TrypLE选择每以及媒体。 </li><li> TrypLE 5分钟，在37℃培养箱孵育ES / iPS细胞。 5分钟后，轻轻移液管关闭的井的底部的ES细胞。 </li><li> TrypLE细胞悬液井收集并转移至锥形管。移液器上下GEntly 2-3次，以鼓励掰开最大的团块。至少有1等量ES媒体和1等量DPBS稀释TrypLE。 TrypLE培养基骤冷，所以重要的是从板中去除细胞后，与媒体和DPBS稀释TrypLE。我们使用DPBS洗涤+媒体保存ES媒体。 </li><li>降速细胞在1500转5分钟，在冷冻离心机（8°C）。 </li><li>吸出尽可能多的上清液未能去除TrypLE。 </li><li>重悬细胞在4毫升ES媒体加4毫升DPBS和重复自旋摆脱TrypLE残留。 </li><li>吸上清，重悬在适当的音量ES媒体电镀。 </li><li>板ES细胞1:1（或2:1，如果需要的话）到MEF中低密度ES媒体。 </li><li> 24小时后，喂新鲜的ES细胞媒体。将最有可能是许多单细胞不重视。每天饲料ES细胞与ES媒体。 </li><li>通过TrypLE到新鲜的MEF（0.9-1×10 <suP&gt; 5个细胞/孔），每3-4天（ 例如周二和周五），使用上述相同的基本过程。通常情况下，你不应该挑细胞TrypLE通过。 </li><li>对于第几个通道（通道5-10），细胞将需要通过在1:1。这使得细胞扩张困难。根据我们的经验，电镀ES细胞的效率急剧下降通道4-5左右，然后回来一对夫妇的通道内。通道5-7后，你可能会开始通过细胞1:2，最终1:3。除了通道20，您可能需要开始传递细胞在1:5或1:6。确保板高密度第5-10通道的ES细胞。一旦你得到一个点的细胞板有效地下降，以至于他们可以在两天内达到〜70％汇合通过后，您可以尝试在1:2开始传递，并最终应该能够通过1:3或1:4 iPSCs的，尤其是，如果没有足够密集的传代才完全一dapted，往往分化。 </li></ol> 2。设置旋转EB文化，准备联合解决方案<ol><li> 10％BSA溶液的IMDM:挂起4克BSA在35毫升的IMDM（50毫升锥形）。允许溶液在室温下放置1-2小时，以使BSA，充分溶解。调整总体积为40毫升的IMDM，得到最终浓度为10％。市售的牛血清白蛋白可以是ES细胞的细胞毒性。脱离子的解决方案，可以减少潜在的细胞毒性。去离子，加入BSA溶液40毫升〜1.3 g树脂珠，摇匀混合。 </li><li>地点BSA溶液（用树脂珠）在4℃，直到珠改变颜色从蓝绿色到黄色（，表明交换容量已经用完）。 </li><li>摇的解决方案，每次20-30分钟，以方便交流</li><li>每个交换都可能需要长达2小时。交换完成后，离心溶液以1,000 rpm持续2分钟沉淀在管的底部有孔玻璃珠。 </li><li>点子到一个新的50毫升锥形ETTE公司BSA溶液，留下被丢弃的有孔玻璃珠。 </li><li>重复步骤2.2-2.5至少有两个额外的时间总共有3个或更多的交流。在过去的交换，有孔玻璃珠的能力，可能无法完全用尽即使经过两个小时的培养的有孔玻璃珠。 </li><li>无菌过滤BSA溶液和删除任何剩余珠。 </li><li> BSA溶液应在4℃下保持稳定至少2个月。 </li><li> 5％的PVA溶液:量取100毫升Millipore公司H 2 O划分为125毫升玻璃瓶。 </li><li>添加5克PVA水。重要的是要加入的PVA的PVA水，使得到充分的"湿"。如果加水，PVA，这将需要很长一段时间的PVA进入溶液。 </li><li> PVA将立即解散。名次的PVA /水溶液中，在4℃下过夜。 </li><li>第二天早晨，地点PVA /水溶液在37°C水浴4-8小时。 ，PVA应该大多在年底解决方案孵化。 </li><li>广场瓶早在4°C为24-48小时，或直至完全溶解。解决方案可能会略有粘性。 </li><li> PVA的解决方案应该是稳定在4℃至少6周。 </li><li> 亚油酸和亚麻酸（10,000×解决方案）:200防乙醇稀释至1毫克/毫升。添加10毫升纯油10毫升乙醇。分装和储存在-20°C </li><li> α-MTG原液稀释13微升α-MTG在1毫升IMDM。 </li><li> 抗坏血酸2 -磷酸溶液（100X）:5毫克/毫升的溶液。 250毫克抗坏血酸-2 -磷酸在50ml无菌的，组织培养级H 2 O的易溶。 </li></ol> 3。大会的BPEL媒体（〜200毫升总体积） <ol><li>的PVA-脂质混合物（此步骤可防止形成在介质中，浏览滤出不溶性液滴的PVA）。 </li><li> IMDM加入20毫升和20毫升的F-12营养混合物50毫升锥形管。 </li><li>加入10毫升5％的PVA库存，0.4毫升，20微升亚麻酸synthechol，，和20μl亚油酸在200毫升BPEL媒体。摇管很好地混合。抛开，而其他媒体组件组装。 </li><li>将所有剩余的媒体顶部，250毫升Stericup过滤单元组件。添加所需的IMDM中的平衡和F-12体积（66毫升），牛血清白蛋白，MTG，100X，GLUTAMAX I，青/链霉素，无蛋白的杂交瘤的第二混合物，和抗坏血酸。加入的PVA脂质混合物Stericup从步骤1的顶部。过滤介质。介质应该是稳定在4℃至少2周。使用旧介质洗涤步骤。 </li></ol> 4。设置我旋转到舞台的TrypLE的传代的ES细胞胚使用TrypLE已经适应了通过MEF中密度较低（即ES / iPS细胞已传TrypLE至少5-7倍）的ES / iPS细胞。如果细胞可以通过在〜1:3，并成为70-80％汇合在3-4天细胞应该是很好用。 <ol><li>前两天自旋EB分化（-2天）:TrypLE适应ES / iPS细胞传递到新鲜的MEF中的密度，使他们能够70-80％汇合分化设置的日子。我们通常通过前48小时旋转EB设置。 </li><li> 日分拆EB设置阶段朕的分化（第0天）:要准备分拆EB电镀，移液管加入150μl无菌水到36外每个96 -孔板的孔中，以尽量减少蒸发。只使用圆底，低附着96孔板。 </li><li>吸ES / iPS细胞的培养基，并添加1.0毫升预暖的的TrypLE选择到每口井。 </li><li>将板在培养箱（37℃）5分钟。轻轻吸取细胞的板块。 </li><li>收集分离的细胞在锥形管，吸管向上和向下掰开团块。 1等体积的BPEL媒体和至少为1的等体积的DPBS稀释TrypLE。 </li><li>取出上清，悬浮细胞在5毫升BPEL媒体加5毫升的DPBS。重复旋转。 </li><li>取出上清，重悬细胞在5-10毫升BPEL媒体，取决于预期的细胞数量。通过细胞通过70微米的过滤器（BD隼＃352350）到一个新的50毫升锥形，以去除团块（这可能会干扰EB形成）。 </li><li>过滤细胞计数。分装到50毫升锥形和自旋细胞用于电镀。电镀:3000 ES细胞/孔，60孔/板/板= 1.8×10 5 ES细胞。 </li><li>离心后，将需要被重新悬浮细胞以3×10 4个细胞/ ml（体积100微升/孔，3000 ES细胞/孔）。 </li><li>准备一个体积的BPEL媒体+重悬细胞（这将是6毫升/板）的足够的细胞因子。为第一阶段的分化，我们使用SCF（40毫微克/毫升），BMP4（20毫微克/毫升），血管内皮生长因子（20毫微克/毫升）的造血祖细胞的推导。 </li><li>板100微升细胞/孔（= 3000个细胞/孔）到各内部部件60的孔中prepar编的96孔板。这是最简单的使用多通道移液器和无菌槽时。可以肯定，混合细胞通过移液敷上低谷和工作细胞很快，所以没有机会解决。 </li><li>为4分钟〜1,500 rpm下，8℃下冷冻离心机旋转的96孔板</li><li>小心转移板从离心机到37℃培养箱。避免扰乱板尽可能地。查看板，以确保细胞在孔的底部形成均一的粒子。 </li><li>第二阶段我旋转直到胚胚不应被送入或以其他方式扰乱通过3-4天的分化已经形成了更耐用的外层。如果EBS超过10-12天的阶段，我会留下，我们有时喂EBS 50微升新鲜BPEL媒体与细胞因子，每孔（从每口井，先拿出50微升的老媒体）。在24小时内，细胞开始形成的3-D EB结构，由3-5天应该有一个明显的外层，和启动德韦扑击囊性结构。 </li></ol> 5。游离阶段我EBS流式细胞仪分析收集around18-36旋EB流式细胞仪分析。如果分析前6天的细胞，更的井需要获得足够的细胞数量。 <ol><li>准备必要的体积的胰蛋白酶用2％鸡血清。放置在37°C水浴，直到使用。 </li><li>自旋的EBS和媒体从96孔板转移至15 ml锥形管。 </li><li>允许EBS解决锥形管的底部。用5毫升吸管，小心吸取上清液，并转移到一个单独的管（你可以所有的上清液进入一个15毫升或50毫升的锥形管）。由于一些造血细胞已经被释放，从自旋EB 9和11天之间，含有这些细胞的上清液分离，防止过量的胰蛋白酶消化。 </li><li>重悬EBS中的胰蛋白酶结合+ 2％鸡血清3-4毫升，每次15毫升锥形管。将EBS与胰蛋白酶在37°C年末3-7分钟rbath。轻轻摇晃或涡每隔1-2分钟。在较早的时间点（8天前），EBS将掰开更容易，可以采取少3分钟。在稍后的时间点（8天）可兑换债券可能需要更长的时间才能分解。我们通常不会让他们胰蛋白酶消化超过5分钟，在37°C。请注意，这些时间是仅供参考。对于任何特定的细胞株或EB分化，可能需要更多或更少的时间来分解胚。密切监察分解。重要的是不要离开细胞中的胰蛋白酶结合太久。这是更好地停止胰蛋白酶分解，只有几个小团块时仍然可见，而不是试图消除所有团块。 </li><li>淬火胰蛋白酶与至少1等量的FBS含媒体。降速细胞（1500转，5分钟，8°C）。 </li><li>重悬细胞在适当体积的介质中进行计数（通常为3-5毫升）。 </li><li>通过100微米的细胞过滤筛选细胞。取出等分细胞​​计数。继续与细胞到标准实验室FACS协议。 </li></ol> 6。 HPSC源性祖细胞的NK细胞的发展，扩展和表征由于自旋胚体含有高比例的造血祖细胞，它们可以被直接转移到NK细胞培养的第11天。自旋胚体可能被转移到24孔板，带或不带供料器。我们已经表明在每个条件下NK细胞的表型或功能之间没有差异。因此，我们一般会转移到无馈线文化NK细胞的发展有一个完全定义的系统。如果使用符合最理想的造血分化的人类胚胎干细胞/ iPS细胞饲养层的使用推荐7,8。 <ol><li>使用多道移液器设置为自旋胚体，转让6到24孔板的各孔中加入100μl。 </li><li>随着馈线转移自旋EBS含有100,000照射（3000 CGY）EL08-1D2（小鼠embryoni的24孔板丙肝细胞株）细胞每口井。无馈线传输EBS直接涂层，24孔板。 </li><li>中加入400μl的NK细胞分化的培养基中的细胞因子（IL-3，IL-15，IL-7，SCF，Flt3L，所有自Peprotech公司），使各孔中的总体积为1毫升。 </li><li>将细胞与NK细胞的分化培养基含有所有细胞因子在6.3除IL-3步在孵化器和饲料每5-7天。 </li><li>在第28天以下转移到NK细胞分化培养（ 图2），NK细胞表达的成熟表型。 </li><li>要测试NK细胞的细胞毒作用，4小时铬释放法，可以进行8。 </li><li>如果需要大量的细胞在体内研究，人工抗原提呈细胞（AAPCS）共培养可以产生日志或更大的扩展。如需详细的协议，访问<a href="https://www-jove-com.remotexs.ntu.edu.sg/video/2540/expansion-purification-functional-assessment-human-peripheral-blood" target="_blank">http://www.jove</a>.com/video/2540/expansion-purification-functional-assessment-human-peripheral-blood 11,12。 </li></ol> 7监视来自人类胚胎干细胞的NK细胞和肿瘤细胞的免疫缺陷小鼠体内荧光和生物发光成像我们使用非肥胖糖尿病/重症联合免疫缺陷与γ-链淘汰赛（NOD / SCID /γC - / - ）小鼠在所有我们的实验中，6〜8周龄。我们使用双重成像模型，同时跟踪的肿瘤进展（记者turboFP650）和胚胎干细胞的NK细胞的动力学（涨落记者表示在父ES细胞） 在体内 。这种成像机制是广泛适用于抗肿瘤模型以外的其他系统。 IVIS频谱（卡尺生命科学）是同时体内荧光和生物发光成像的最佳选择。 <ol><li>肿瘤细胞注射前24小时，照射小鼠（225-250剂量）。 </li><li>重悬所需数量的肿瘤细胞（1×10 6个K562细胞示出）在200μl的Iscove改良的Dulbecco培养基（IMDM）补充有20％FBS的。可以改变肿瘤细胞的剂量，这取决于所使用的机型。 TurboFP650也可以表示在非肿瘤类型。这是最好的定位到一个单一的解剖位置与细胞成像。 </li><li>注射肿瘤细胞皮下注射到小鼠使用27或28号针头的上左胸廓，使嫁接4天。根据鼠标的皮肤细胞应形成气泡。剃光小鼠可以在该部分的身体更好地可视化注射。它还暴露出的区域更好地在体内荧光成像。使用这种模式，因为胸部周围的老鼠被注射腹腔注射NK细胞是最好的可视化，而鼠标是仰卧。的替代方法，包括皮下投的背面或侧面，压力较小的动物，应予以考虑。 </li><li>重悬所需的人类胚胎干细胞派生粘弹性阻尼器的NK细胞（10×10 6个NK细胞示出）中加入300μl的Iscove改良的Dulbecco培养基（IMDM）补充有20％FBS的无抗生素。 </li><li>每只小鼠腹腔（IP）使用的是27或28号针头，注射胚胎干细胞来源的NK细胞。对于所有的实验中，包括没有肿瘤细胞或NK细胞输注小鼠作为阴性对照。 </li><li>为了维持体内的 NK细胞群，给小鼠250微升腹腔注射IL-2（1×10 4 U /鼠标）和IL-15（10毫微克/鼠标）在无菌DPBS随后的第7天，每天由IL- 2只，每2至3天，直到牺牲的时候。 </li><li>监控植入人类胚胎干细胞衍生的NK细胞通过生物发光成像。注入每只小鼠的IP与120μl的D-荧光素（150毫克/千克）。图像小鼠D-荧光素注射后10分钟。 </li><li>麻醉小鼠，使用允许在0.8升/分钟的异氟醚的框中，然后进入一个腔室。确保到室也有氧气流量。 </li><li>将麻醉IVIS频谱和安全小鼠的小鼠在他们的后面使用磁带或魔术贴，并允许在0.5升/分钟，以保持麻醉异氟烷进入盒。 </li><li>设置IVIS机校正成像平台（D为4-5只，C为3个或更少的小鼠），设置分级介质，F /停止1和1分钟获取图像。 </li><li>要执行为turboFP650 +细胞的荧光成像，使用成像向导设置测量探头的利益，以及作为背景信号发射扫描。从探查列表中选择输入EM / EX，并输入正确的激发和发射。对于turboFP650，使用605激发和660-720排放来衡量肿瘤信号和570激发640-720排放的测量背景信号。使用自动曝光设置，并选择所需的成像平台。整个成像序列通常会采取收购的3-5分钟。 </li><li>允许老鼠完全从麻醉中恢复运输前从成像系统。分析图像中，使用锂咏图像软件包4.2版（卡尺生命科学）。 </li><li>量化使用设置总光通量（光子/秒）或平均四射（光子/秒/厘米2 / SR）的所有图像，并设置相同的比例（ 图3）的感兴趣区域（ROI）的发光信号。在这里，有代表性的图像被用来证明每只小鼠的两个细胞群（ 图3）。从我们的实验室的其他研究已经证明使用这种技术20共存。共定位的时间将需要进行优化的基础上所用的实验模型。 </li><li>为了量化荧光信号从发射扫描序列使用"光谱分离"功能的生活图像。选择包括在分析中的图像绘制掩模鼠标包含感兴趣的信号的区域，在这种情况下，上部的胸廓。选择组织autofluorescense的和未知的探头纯一不杂。如果小鼠食物中含有紫花苜蓿，粮食信号也可以为unmixed利益和组织的自体荧光探针。 </li><li>如果所产生的图像不显示组织的自体荧光的均匀分布（包括信号位于的区域）的限制可以被设置，以获得一致的分布和未混合的组件的更好的分离。这是典型的，往往需要对探针不是在软件中，当特定的信号是低，或接近背景信号。设置一个高通滤波器，根据"更新"选项卡，在640 nm处，其对应于发射曲线的下端turboFP650。也可以设置一个低通滤波器约束，但是这是不必要的，此处所示的成像分析。 </li><li>量化荧光信号未混合图像的感兴趣区域（ROI）的辐射效率（光子/秒/厘米2 / SR）/μW）的所有图像，并设置相同的比例（ 图3）。 </li><li>所需的时间点，老鼠可以牺牲和分析ENgraftment人类胚胎干细胞衍生的细胞通过流式细胞仪。腹腔注射NK细胞，我们通常分析外周血（面静脉出血），脾和腹膜。腹腔冲洗液可进行，只是腹膜暴露和内侧切口，其宽度足以使的玻璃巴斯德移液管，以访问腹膜腔。使用无菌PBS腹腔确保彻底混合的解决方案，通过旋转鼠标，执行多次洗涤。收集足够的液体，直到你有一个可见的颗粒离心后（通常为5-10毫升洗）。 </li></ol>

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这项工作是由NIH / NHLBI R01-HL77923（DSK）和NIH MSTP授予T32 GM008244（DAK），干细胞生物学培训格兰特T32（DAK）（T32HD060536），本科生研究机会计划（UROP）格兰特，美国明尼苏达大学的支持（AMB），明尼苏达大学癌症中心和威廉·L和布兰奇·休斯基金会白血病研究基金。

人类胚胎干细胞是一个理想的平台，研究不同的细胞类型，并保持卓越的潜力为临床翻译。我们使用一个定义，自旋EB的方法，对造血祖细胞分化的人类胚胎干细胞/ iPS细胞。的自旋EB的方法已经取得了一致的推导造血祖细胞和NK细胞的分化，变化仍然存在跨细胞系的分化效率，并可能需要进行修改，以生成其他的造血细胞谱系。虽然比较结果可以得到从其他EB或基质培养方法的推导，这个系统是无血清和代表更明确的方法与其他方法相比，造血祖细胞。我们也有标准化的有效方法得出类似的无饲养层的方式，克服一个主要关注临床翻译这些造血祖细胞的NK细胞。它还提供了一个定义的系统，NK细胞研究发展的t 在体外 。广泛的临床前， 在体内的造血细胞，如NK细胞的特性，也需要专门定义这些实验得到的细胞类型和它们的临床试验的实施。 在成像和显微技术的进展已使长期和非侵入体内监测细胞表达生物发光和荧光记者15-17,19,21。最值得注意的是使用的萤火虫荧光素酶构建体，它已被用于可视化不同的细胞类型，因为它的初始特性。 在活体成像，这是一个理想的报告，但被限制为只监视一个细胞种群。一个双报告系统的最大优点是能够按照两种不同的细胞群之间的相互作用。然而，很少有记者已经开发了既易于在体内的图像，并有足够的，可量化的信号，背景。 TurboFP650，远红荧光蛋白在我们的双重成像计划，转移，这给不同背景的自体荧光信号。然而，利用光谱分离方法，在生活图像软件（卡尺生命科学）是必要的，以最大限度地在我们的系统中的信号背景比。在生活图像软件有许多功能能够帮助荧光蛋白成像的内在挑战。尽管如此，信号渗透率仍然是一个挑战。小细胞群体或分散的细胞成像成像系统需要进一步优化，将肿瘤细胞的情况下，如果被注入的IP。但是，荧光成像的一致性和易用性使得它的使用使用双重成像模型，一个理想的平台。将动物在麻醉状态下为更短的时间内，就没有必要提供一第二基板，与大多数双荧光素酶系统。 这种方法推导，扩展和双重成像在体内提供了一个一致的方法生产人类胚胎干细胞衍生的iPSC衍生的NK细胞和他们的评价，这是必要的，以改善当前NK细胞过继疗法。的的双重荧光和生物发光成像模型是广泛适用于长期研究的两种不同的细胞群以外的抗肿瘤模型，我们已经表明。

是未分化的人类胚胎干细胞（hESCs）和诱导多能干细胞（iPS细胞），多能干细胞能无限的自我更新和多谱系分化。人胚胎干细胞已被成功地分化为成熟和功能的子集的每一个胚层，包括细胞的造血系统1-3。自然杀伤（NK）细胞是先天免疫系统的淋巴细胞，可以是来自于人类胚胎干细胞形成胚状体（胚体）4,5或共培养基质细胞系1,2,6-8。 NK细胞具有抗病毒和抗肿瘤能力，有可能可以有效对抗广泛的恶性肿瘤，因为它们不执行需要事先抗原刺激的效应子功能。因此，NK细胞的人类胚胎干细胞衍生的是一个有吸引力的免疫细胞来源。此外，推导出人类胚胎干细胞的NK细胞基因提供了一个适合的系统来研究正常发展&lt;EM&gt;的体外。 因为它们提供了基因听话的系统，人类胚胎干细胞来源的NK细胞可以通过实验修饰以表达荧光和生物发光记者提供一个最佳的模型来研究在体外和体内的 NK细胞的效应子功能。人类胚胎干细胞衍生的NK细胞具有活性，对一系列目标，包括艾滋病毒，白血病（K562）和其他类型的癌症7,8。然而，能够有效地获得足够的NK细胞能够治疗患者仍然是一个重要的障碍的临床翻译是，在较小的程度，广泛的临床前体内研究的NK细胞的发育和抗癌功能的限制。在这里，我们使用一个自旋EB的方法来获得4,5,10从人类胚胎干细胞的造血祖细胞。之后11天的胚体的自旋转移到NK细胞培养28天，带或不带供料器。 NK细胞培养基中，4周后，NK细胞被转修饰以表达膜结合的白细胞介素21（IL-21），作为人工抗原呈递细胞（AAPCS）与K562细胞共培养sferred。适应扩张外周血NK细胞，使用这些人工装甲运兵车11,12的协议，我们能够扩大NK细胞日志，同时保留一个成熟的表型和细胞毒性的能力。 这个过程的发展和扩张提供了足够的人类胚胎干细胞衍生的NK细胞在体内表征广泛。对于体内研究，我们能够以非侵入性监测长期植入，注入萤火虫荧光素酶的动力学表达（涨落+），人类胚胎干细胞来源的NK细胞，利用生物发光成像。此外，我们都能够按照使用双，生物发光或荧光成像计划的与肿瘤细胞的NK细胞间的相互作用。我们先前的研究组利用生物发光成像的抗肿瘤模型遵循肿瘤进展和CLEarance涨落+ K562细胞体内 7。现在，我们的人类胚胎干细胞表达萤火虫荧光素酶13,14工程，我们可以遵循的分布和贩运NK细胞对K562肿瘤细胞表达最近特征的荧光蛋白，turboFP650 15。我们选择了这个双报告系统，以同时跟随两个淋巴细胞的增殖（ 图1）的。大多数双成像机型已经双荧光素酶系统，但这些系统可以在技术上具有挑战性，由于腔肠素的交付要求，表达最海肾和Gaussia的荧光素酶记者16-18所需的基板。荧光报告在体外的许多细胞系和构造允许简单的监控，但由于组织和皮毛的自体荧光和许多共同的发射光谱之间的重叠部分在体内成像了有限的成功mmonly使用荧光记者，包括红色荧光蛋白，绿色荧光蛋白，和TdTomato 15,19。这种担忧鼓励发展的远红荧光蛋白，它可以更好的组织渗透率和较高的特定信号比背景15,19。 TurboFP650，在本系统中所示的荧光蛋白，远红移，克服了许多与成像的荧光蛋白在活的动物所涉及的问题。 这种发展和扩大是来自于人类胚胎干细胞的NK细胞的方法，使我们能够进一步表征人类胚胎干细胞来源的NK细胞， 在体外和体内 ，这是必要的，以便更好地了解NK细胞功能和临床上重要的，以改善电流NK细胞过继疗法。它也适合于iPS细胞衍生的NK细胞的推导和扩展。双荧光和生物发光成像方案广泛适用于系统以外的抗肿瘤模型中，我们已经在这里显示。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Materials&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Media&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;BPEL media for Spin EB generation&lt;/strong&gt;</td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>Fisher Scientific</td><td>SH3022801</td><td></td></tr><tr><td>F-12 Nutrient Mixture w/ Glutamax I</td><td>Invitrogen</td><td>31765035</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A3311</td><td>Commercially available BSA can be cytotoxic to ES cells. Deionizing the solution can reduce the potential for cytotoxicity.</td></tr><tr><td>Poly(vinyl alcohol)</td><td>Sigma-Aldrich</td><td>P8136</td><td></td></tr><tr><td>Linoleic Acid</td><td>Sigma-Aldrich</td><td>L1012</td><td></td></tr><tr><td>Linolenic Acid</td><td>Sigma-Aldrich</td><td>L2376</td><td></td></tr><tr><td>Synthechol 500X solution</td><td>Sigma-Aldrich</td><td>S5442</td><td></td></tr><tr><td>a-monothioglycerol (a-MTG)</td><td>Sigma-Aldrich</td><td>S5442</td><td></td></tr><tr><td>Protein-free hybridoma mix II</td><td>Invitrogen</td><td>12040077</td><td></td></tr><tr><td>Ascorbic Acid 2-phosphate</td><td>Sigma-Aldrich</td><td>A8960</td><td></td></tr><tr><td>Glutamax I</td><td>Invitrogen</td><td>35050061</td><td></td></tr><tr><td>Insulin, Transferrin, Selenium 100X solution (ITS)</td><td>Invitrogen</td><td>41400-045</td><td></td></tr><tr><td>Pen-Strep</td><td>Invitrogen</td><td>15140122</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;NK Differentiation Media &lt;/strong&gt;</td></tr><tr><td>Dubecco&#039;s Modified Eagle Medium (DMEM)</td><td>Invitrogen</td><td>11965-118</td><td></td></tr><tr><td>F-12 Media</td><td>Invitrogen</td><td>CX30315</td><td></td></tr><tr><td>15% Human AB serum</td><td>Valley Biomed</td><td>HP1022HI</td><td></td></tr><tr><td>5 ng/ml Sodium Selenite</td><td>Sigma-Aldrich</td><td>S5261</td><td></td></tr><tr><td>50 uM ethanolamine</td><td>Sigma-Aldrich</td><td>E9508</td><td></td></tr><tr><td>20 ng/ml ascorbic acid</td><td>Sigma-Aldrich</td><td>A8960</td><td></td></tr><tr><td>25 uM 2-mercapt&amp;#8211;thanol (BME)</td><td>Gibco</td><td>21985</td><td></td></tr><tr><td>2 mM L-glutamine</td><td>Gibco</td><td>CX30310</td><td></td></tr><tr><td>1% Pen-Strep</td><td>Invitrogen</td><td>15140122</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Cytokines&lt;/strong&gt;</td></tr><tr><td colspan="4">(all cytokines used fresh from frozen aliquots)</td></tr><tr><td>SCF</td><td>PeproTech, Inc.</td><td>300-07</td><td>40 ng/ml in BPEL media; 20 ng/ml in NK medium). As noted by the Elefanty protocol, and as we discovered with a bad lot of BMP4, there can be lot differences with cytokines in regards to hematopoietic differentiation.Especially if buying cytokines in bulk, obtain a sample of the lot to test prior to purchase. Compare head-to-head with old lot.</td></tr><tr><td>rhBMP-4</td><td>R &amp;amp; D Systems</td><td>314-BP</td><td>20 ng/ml in BPEL media</td></tr><tr><td>rhVEGF</td><td>R&amp;amp;D Systems</td><td>293-VE</td><td>20 ng/ml in BPEL media</td></tr><tr><td>IL-2</td><td>PeproTech, Inc.</td><td>200-02</td><td>1 x 10&lt;sup&gt;5&lt;/sup&gt; U/ml given to mice after NK cell injection; 50 U/ml used in NK cell expansion protocol</td></tr><tr><td>IL-15</td><td>PeproTech, Inc.</td><td>200-15</td><td>10 ng/ml given to mice after NK cell injection (first 7 days only)</td></tr><tr><td>IL-3</td><td>PeproTech, Inc.</td><td>200-03</td><td>5 ng/ml in NK media</td></tr><tr><td>IL-7</td><td>PeproTech, Inc.</td><td>200-07</td><td>20 ng/ml in NK media</td></tr><tr><td>Flt-3-Ligand</td><td>PeproTech, Inc.</td><td>300-19</td><td>10 ng/ml in NK media</td></tr><tr><td colspan="4">&lt;strong&gt;&lt;em&gt;In vivo&lt;/em&gt; Imaging&lt;/strong&gt;</td></tr><tr><td>D-Luciferin Sodium Salt</td><td>Gold BioTechnology</td><td>Lucna-500</td><td></td></tr><tr><td>TurboFP650 plasmid</td><td>Evrogen, Moscow, Russia</td><td>FP731</td><td>Subcloned into a Sleeping Beauty transposon based plasmid driven by the mCAGs promoter. Cells were then sorted on their expression of turboFP650 by FACS. Cells and plasmids can be obtained from our lab.</td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>IVIS Spectrum Imaging System</td><td>Caliper Life Sciences</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Cells&lt;/strong&gt;</td></tr><tr><td>Membrane bound IL-21 expressing artificial antigen presenting cells</td><td><a href="https://www-jove-com.remotexs.ntu.edu.sg/video/2540/expansion-purification-functional-assessment-human-peripheral-blood" target="_blank"><img src="/img/new_window.png" /> MD Anderson, Houston, TX</a></td><td></td><td>contact: Dean A. Lee. &lt;a href=&quot;https://www-jove-com.remotexs.ntu.edu.sg/video/2540/expansion-purification-functional-assessment-human-peripheral-blood&quot; target=&quot;_blank&quot;&gt;https://www-jove-com.remotexs.ntu.edu.sg/video/2540/expansion-purification-functional-assessment-human-peripheral-blood&lt;/a&gt;</td></tr><tr><td>Firefly luciferase expressing hESCs</td><td>University of Minnesota, Minneapolis, MN</td><td></td><td>contact: Dan S. Kaufman . H9 cells modified with a Sleeping Beauty transposon based method (references 13 and 14). Expression of firefly luciferase is driven by the mCAGGS promoter. Following the firefly luciferase gene is an IRES element at the 5&#039; end of a GFP:zeocin fusion construct.</td></tr><tr><td>TurboFP650 expressing K562 cells</td><td>University of Minnesota, Minneapolis, MN</td><td></td><td>contact: Dan S. Kaufman. Description under plasmid comments section</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;Materials Table.&lt;/strong&gt;&lt;/p&gt;</td></tr></tbody></table>

development, expansion, and in vivo monitoring of human nk cells from human embryonic stem cells (hescs) and induced pluripotent stem cells (ipscs)

我们提出一个方法推导自然杀伤（NK）细胞未分化的人类胚胎干细胞和iPS细胞使用馈线的方法。此方法产生高水平的NK细胞培养4周后，可以进行进一步的人工抗原呈递细胞的2 - 日志扩展。在本系统中开发的人类胚胎干细胞和iPS细胞来源的NK细胞有成熟的表型和功能。基因修改的NK细胞的大量生产适用于两个基本机理以及抗肿瘤的研究。萤火虫荧光素酶在人类胚胎干细胞衍生的NK细胞可表达一种非侵入性的方法，遵循NK细胞植入，分布和功能。我们还描述了一种双重成像计划，允许独立的监控两种不同的细胞群，以更清楚地描述他们在体内的相互作用。推导，膨胀， 在体内成像和双此方法提供了可靠的制造NK细胞的方法，其EVALU这是必要的，以改善当前NK细胞过继疗法通报BULLETIN。

造血祖细胞的产生使用自旋EB方法允许最佳的NK细胞从人胚胎干细胞和iPS细胞的发展。这表现在图2中，11天自旋胚体含有高百分比的祖细胞表达CD34，CD45，CD43，和CD31。高水平的CD34和CD45 NK条件允许直接传输，无需进行排序或支持基质细胞。如果有不理想的自旋EB分化，它是建议基质细胞，例如EL08-1D2中使用的二次NK条件。自旋胚体培养4周后产生大量的NK细胞（约1-2×10 6细胞的24孔板每孔）。文化也可以在较早的时间点看表型NK细胞发育的逐步推进，在这个系统中7。 NK细胞维持，然后可以随后被改装成父ES /的iPS线，因此对报告基因的表达<em&gt;在体内。在这里，我们已经使用了IVIS频谱NK细胞和肿瘤细胞中的相同的鼠标（ 图3）说明了同时成像。使用组织的自体荧光光谱分离功能的生活图像软件，可以减去以优化信号从荧光标记的细胞。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50337/50337fig1highres.jpg" src="/files/ftp_upload/50337/50337fig1.jpg" /> 图1。双的成像计划和发展的时间表。一）示意图体内模型。小鼠腹腔注射荧光素，并按照正确的孵化时间，萤火虫荧光素酶+细胞生物发光信号成像。继收购生物发光图像，荧光信号从TurboFP650 +细胞可以成像，B）时间的开发和测试的人类胚胎干细胞DERIV编NK细胞。 <img alt="图2" fo:content-width="5.5in" fo:src="/files/ftp_upload/50337/50337fig2highres.jpg" src="/files/ftp_upload/50337/50337fig2.jpg" /> 图2。自旋EBS来自人类胚胎干细胞和iPS细胞和NK细胞的表型。 A）以下11天在旋EB文化，细胞可以通过流式细胞仪分析。 11天的时间点给出了高比例的CD34，CD43，CD45，和CD31的表达细胞最佳的NK细胞的分化。 乙）可以被检测到NK细胞，NK细胞培养4周后，用流式细胞仪检测。细胞内的淋巴细胞门（FSC / SSC曲线）分析CD56的表达。 CD56 +细胞可以进一步分析的标记，如CD117，CD94，NKp46，或其他的共同表达。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50337/50337fig2large.jpg" target="_blank">点击这里查看大图</a> 。 图3。荧光和生物发光成像在体内使用IVIS频谱。 在体内监测是此协议的主要目的。因此，老鼠没有遵循肿瘤间隙。我们先前的报告（使用200,000 K562细胞）表明，肿瘤的间隙发生在三个星期7）生物发光成像的波动+人类胚胎干细胞来源的NK细胞NK细胞注射后第0天，第7和14的顶行显示。荧光成像TurboFP650 + K562细胞的NK细胞注射后的第0天，第7和14示出在底行。获取荧光图像，紧随生物发光图像采集。B）序列，显示组织的自体荧光和荧光信号turboFP650 +细胞，产生的生活图像软件使用光谱分离功能。鼠标在每个图像最左边是一个非喷射控制，只显示背景信号。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50337/50337fig3large.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs at JoVE.com

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

本协议描述的发展，扩张和来自人类胚胎干细胞和iPS细胞的NK细胞在体内成像。

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

development-expansion-vivo-monitoring-human-nk-cells-from-human

Nanyang Technological University

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JoVE Journal

Bioengineering

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

16.7K 次观看.  University of Minnesota, Minneapolis. 本协议描述的发展，扩张和来自人类胚胎干细胞和iPS细胞的NK细胞在体内成像。

视频: Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

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

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells 

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

免疫与感染

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

多能高效分化干细胞NKX6-1<sup&gt; +</sup&gt;胰腺祖

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, particularly CAR-NK cell, is one of the most promising 'off-the-shelf' development without severe life-threatening toxicity. However, the bottleneck for developing a successful CAR-NK therapy is achieving sufficient numbers of non-exhaustive, long-lived, 'off-the-shelf' CAR-NK cells from a third party. Here, we developed a new CAR-NK expansion method using an Epstein-Barr virus- (EBV) transformed B cell line expressing a genetically modified membrane form of interleukin-21 (IL-21). In this protocol, step-by-step procedures are provided to expand NK and CAR-NK cells from cord blood and peripheral blood, as well as solid organ tissues. This work will significantly enhance the clinical development of CAR-NK immunotherapy.

Natural Killer NK and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells.

使用膜结合IL-21修饰B细胞系的自然杀伤（NK）和CAR-NK细胞扩增方法

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, ...

癌症研究

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Generation of Natural Killer Cells from Human Expanded Potential Stem Cells

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular products for immunotherapy. Described here is a two-phase protocol that uses a serum-free commercial medium and a cocktail of cytokines (interleukin [IL]-3, IL-7, IL-15, stem cell factor [SCF], and FMS-like tyrosine kinase 3 ligand [Ftl3L]) to differentiate human expanded potential stem cells (hEPSCs) into cells that possess NK cell properties in vitro with both 3-dimensional (3D) and 2-dimensional (2D) culture technology. Following this protocol, CD3&#8722;CD56+ or CD45+CD56+ NK cells are consistently generated. When cocultured with tumor targets for 3 h, the differentiated products display mild cytotoxicity as compared to an IL-2-independent permanent cell line, NK92mi cells. The protocol preserves the complexity of the differentiation microenvironment by the generation of 3D structures, thus facilitating the study of the spatial relationships between immune cells and their niches. Meanwhile, the 2D culture system enables the routine phenotypical validation of cell differentiation without harming the delicate differentiation niche.

The present protocol shows how to differentiate CD3&#8722;/CD45+CD56+ cells with mild cytotoxicity from human expanded potential stem cells (hEPSCs) under both 3D and 2D culture conditions. This allows for routine phenotypical validation without the destruction of the complex microenvironment.

从人类扩增的潜在干细胞中产生自然杀伤细胞

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

生物学

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

另类文化的人多能干细胞的生产，维护和遗传分析

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of human induced pluripotent stem cell (iPSC)-derived chimeric antigen receptor (CAR) expressing NK cells has emerged as a promising avenue for "off the shelf" cancer immunotherapy. Here, we utilized an NK cell-optimized CAR construct that includes the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3&#950; signaling domain, which has been demonstrated to stimulate robust antigen-specific NK cell-mediated antitumor activity. The use of iPSCs for CAR NK cell generation offers several advantages, including homogenous CAR expression, scalability, reproducibility, and the potential for clinical application. This detailed step-by-step protocol from cell engineering to differentiation enables the generation of NK cell-optimized iPSC-derived CAR-expressing NK cells, providing a standardized and targeted cancer immunotherapy with improved antitumor activity and highlighting their potential as a promising treatment option for various malignancies.

Here, we present a method to differentiate and expand human iPSC-derived chimeric antigen receptor (CAR)-expressing natural killer cells with improved killing against various malignancies. This protocol demonstrates the differentiation and expansion of natural killer (NK) optimized iPSC-derived CAR-NK cells and the measurement of antitumor activity against various tumor cell lines.

人诱导多能干细胞衍生的嵌合抗原受体表达自然杀伤细胞的研究进展

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of ...

开发，扩展，

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