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/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>

<ol>
	<li>Keller, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+embryonic+stem+cells.">In vitro differentiation of embryonic stem cells.</a> Current Opinion in Cell Biology. 7, 862-869 (1995).</li><li>Kaufman, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+colony-forming+cells+derived+from+human+embryonic+stem+cells.">Hematopoietic colony-forming cells derived from human embryonic stem cells.</a> Proceedings of the National Academy of Sciences. 98, 10716-10721 (2001).</li><li>Kaufman, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+clinical+therapies+using+hematopoietic+cells+derived+from+human+pluripotent+stem+cells.">Toward clinical therapies using hematopoietic cells derived from human pluripotent stem cells.</a> Blood. 114, 3513-3523 (2009).</li><li>Ng, E. S., Davis, R., Stanley, E. G., Elefanty, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+describing+the+use+of+a+recombinant+protein-based,+animal+product-free+medium+(APEL)+for+human+embryonic+stem+cell+differentiation+as+spin+embryoid+bodies.">A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies.</a> Nature Protocols. 3, 768-776 (2008).</li><li>Ng, E. S., Davis, R. P., Azzola, L., Stanley, E. G., Elefanty, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forced+aggregation+of+defined+numbers+of+human+embryonic+stem+cells+into+embryoid+bodies+fosters+robust,+reproducible+hematopoietic+differentiation.">Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation.</a> Blood. 106, 1601-1603 (2005).</li><li>Vodyanik, M. A., Bork, J. A., Thomson, J. A., Slukvin, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+CD34++cells:+efficient+production+in+the+coculture+with+OP9+stromal+cells+and+analysis+of+lymphohematopoietic+potential.">Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential.</a> Blood. 105, 617-626 (2005).</li><li>Woll, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cells+differentiate+into+a+homogeneous+population+of+natural+killer+cells+with+potent+in+vivo+antitumor+activity.">Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity.</a> Blood. 113, 6094-6101 (2009).</li><li>Woll, P. S., Martin, C. H., Miller, J. S., Kaufman, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+NK+cells+acquire+functional+receptors+and+cytolytic+activity.">Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity.</a> Journal of Immunology. 175, 5095-5103 (2005).</li><li>Ni, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Pluripotent+Stem+Cells+Produce+Natural+Killer+Cells+That+Mediate+Anti-HIV-1+Activity+by+Utilizing+Diverse+Cellular+Mechanisms.">Human Pluripotent Stem Cells Produce Natural Killer Cells That Mediate Anti-HIV-1 Activity by Utilizing Diverse Cellular Mechanisms.</a> Journal of Virology. 85, 43-50 (2011).</li><li>Ng, E. S., Davis, R. P., Hatzistavrou, T., Stanley, E. G., Elefanty, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+embryonic+stem+cells+as+spin+embryoid+bodies+and+a+description+of+the+hematopoietic+blast+colony+forming+assay.">Directed differentiation of human embryonic stem cells as spin embryoid bodies and a description of the hematopoietic blast colony forming assay.</a> Current Protocols in Stem Cell Biology. Chapter 1, Unit 1D.3 (2008).</li><li>Denman, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-Bound+IL-21+Promotes+Sustained+Ex+Vivo+Proliferation+of+Human+Natural+Killer+Cells.">Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells.</a> PLoS ONE. 7, e30264 (2012).</li><li>Somanchi, S. S., Senyukov, V. V., Denman, C. J., Lee, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion,+Purification,+and+Functional+Assessment+of+Human+Peripheral+Blood+NK+Cells.">Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells.</a> J. Vis. Exp. (48), e2540 (2011).</li><li>Tian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+imaging+demonstrates+that+transplanted+human+embryonic+stem+cell-derived+CD34++cells+preferentially+develop+into+endothelial+cells.">Bioluminescent imaging demonstrates that transplanted human embryonic stem cell-derived CD34+ cells preferentially develop into endothelial cells.</a> Stem Cells. 27, 2675-2685 (2009).</li><li>Wilber, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+stable+transgene+expression+in+human+embryonic+stem+cells+using+transposon-mediated+gene+transfer.">Efficient and stable transgene expression in human embryonic stem cells using transposon-mediated gene transfer.</a> Stem Cells. 25, 2919-2927 (2007).</li><li>Shcherbo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+fluorescent+proteins.">Near-infrared fluorescent proteins.</a> Nature Methods. 7, 827-829 (2010).</li><li>Nguyen, V. T., Morange, M., Bensaude, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Firefly+luciferase+luminescence+assays+using+scintillation+counters+for+quantitation+in+transfected+mammalian+cells.">Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells.</a> Analytical Biochemistry. 171, 404-408 (1988).</li><li>Sadikot, R. T., Blackwell, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+Imaging.">Bioluminescence Imaging.</a> Proceedings of the American Thoracic Society. 2, 537-540 (2005).</li><li>Santos, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+in+vivo+imaging+of+T+cells+using+a+membrane-bound+Gaussia+princeps+luciferase.">Sensitive in vivo imaging of T cells using a membrane-bound Gaussia princeps luciferase.</a> Nature Medicine. 15, 338-344 (2009).</li><li>Chudakov, D. M., Matz, M. V., Lukyanov, S., Lukyanov, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Proteins+and+Their+Applications+in+Imaging+Living+Cells+and+Tissues.">Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues.</a> Physiological Reviews. 90, 1103-1163 (2010).</li><li>Knorr, D. A., Bock, A., Brentjens, R. J., Kaufman, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+Human+Embryonic+Stem+Cell-Derived+Lymphocytes+to+Study+In+Vivo+Trafficking+and+Immunotherapy.">Engineered Human Embryonic Stem Cell-Derived Lymphocytes to Study In Vivo Trafficking and Immunotherapy.</a> Stem Cells Dev. 28, 28 (2013).</li><li>Germain, R. N., Robey, E. A., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Decade+of+Imaging+Cellular+Motility+and+Interaction+Dynamics+in+the+Immune+System.">A Decade of Imaging Cellular Motility and Interaction Dynamics in the Immune System.</a> Science. 336, 1676-1681 (2012).</li></ol>

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...

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 border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>BPEL media for Spin EB generation</td>
		</tr>
		<tr>
			<td>Iscove's Modified Dulbecco's Medium (IMDM)</td>
			<td>Fisher Scientific</td>
			<td>SH3022801</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>F-12 Nutrient Mixture w/ Glutamax I</td>
			<td>Invitrogen</td>
			<td>31765035</td>
			<td>&nbsp;</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>&nbsp;</td>
		</tr>
		<tr>
			<td>Linoleic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>L1012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Linolenic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>L2376</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Synthechol 500X solution</td>
			<td>Sigma-Aldrich</td>
			<td>S5442</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>a-monothioglycerol (a-MTG)</td>
			<td>Sigma-Aldrich</td>
			<td>S5442</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Protein-free hybridoma mix II</td>
			<td>Invitrogen</td>
			<td>12040077</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ascorbic Acid 2-phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glutamax I</td>
			<td>Invitrogen</td>
			<td>35050061</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Insulin, Transferrin, Selenium 100X solution (ITS)</td>
			<td>Invitrogen</td>
			<td>41400-045</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen-Strep</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>NK Differentiation Media </td>
		</tr>
		<tr>
			<td>Dubecco's Modified Eagle Medium (DMEM)</td>
			<td>Invitrogen</td>
			<td>11965-118</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>F-12 Media</td>
			<td>Invitrogen</td>
			<td>CX30315</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15% Human AB serum</td>
			<td>Valley Biomed</td>
			<td>HP1022HI</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5 ng/ml Sodium Selenite</td>
			<td>Sigma-Aldrich</td>
			<td>S5261</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 uM ethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>E9508</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20 ng/ml ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>25 uM 2-mercaptoethanol (BME)</td>
			<td>Gibco</td>
			<td>21985</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2 mM L-glutamine</td>
			<td>Gibco</td>
			<td>CX30310</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1% Pen-Strep</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Cytokines</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>(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;D Systems</td>
			<td>314-BP</td>
			<td>20 ng/ml in BPEL media</td>
		</tr>
		<tr>
			<td>rhVEGF</td>
			<td>R&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 105 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>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>In vivo Imaging</td>
		</tr>
		<tr>
			<td>D-Luciferin Sodium Salt</td>
			<td>Gold BioTechnology</td>
			<td>Lucna-500</td>
			<td>&nbsp;</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>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>IVIS Spectrum Imaging System</td>
			<td>Caliper Life Sciences</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Cells</td>
		</tr>
		<tr>
			<td>Membrane bound IL-21 expressing artificial antigen presenting cells</td>
			<td>MD Anderson, Houston, TX</td>
			<td>&nbsp;</td>
			<td>contact: Dean A. Lee. <a href="http://www.jove.com/video/2540/expansion-purification-functional-assessment-human-peripheral-blood" target="_blank">http://www.jove.com/video/2540/expansion-purification-functional-assessment-human-peripheral-blood</a></td>
		</tr>
		<tr>
			<td>Firefly luciferase expressing hESCs</td>
			<td>University of Minnesota, Minneapolis, MN</td>
			<td>&nbsp;</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' end of a GFP:zeocin fusion construct.</td>
		</tr>
		<tr>
			<td>TurboFP650 expressing K562 cells</td>
			<td>University of Minnesota, Minneapolis, MN</td>
			<td>&nbsp;</td>
			<td>contact: Dan S. Kaufman. Description under plasmid comments section</td>
		</tr>
	</tbody>
</table>
Materials 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...

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

Research

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

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

科研

生物工程

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

订货单细胞和单胚胎的三维约束:高内涵筛选新设备

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

在化学定义条件下，成熟肾细胞与人类诱导多能干细胞的引导分化

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

维持静止人类造血干细胞的文化方法

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

体外 人体牙浆干细胞向胰腺血统的诱导

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

开发，扩展，

摘要

探索更多视频

此视频中的章节

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

订货单细胞和单胚胎的三维约束:高内涵筛选新设备

利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成

在化学定义条件下，成熟肾细胞与人类诱导多能干细胞的引导分化

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

维持静止人类造血干细胞的文化方法

体外人体牙浆干细胞向胰腺血统的诱导

开发，扩展，

摘要

探索更多视频

此视频中的章节

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

订货单细胞和单胚胎的三维约束:高内涵筛选新设备

利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成

在化学定义条件下，成熟肾细胞与人类诱导多能干细胞的引导分化

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

维持静止人类造血干细胞的文化方法

体外 人体牙浆干细胞向胰腺血统的诱导

体外人体牙浆干细胞向胰腺血统的诱导