笔者想承认多能干细胞基金和共焦成像的核心在辛辛那提儿童医院。这项工作是由R21AI119236（HJ），R21AI101375（HJ），NIH / NCATS 8UL1TR000077-04（HJ），U19 AI070412（HJ）和2U19AI70235（GKKH）的支持。

以下协议遵循机构的人力研究伦理委员会的指导方针。 1.采样鼻粘膜注:从科目谁是免费的呼吸道病毒感染的迹象索取样品。 <ol><li>参加者访问前准备一个15毫升的锥形新鲜，加2ml BEGM（支气管上皮细胞生长培养基）加20微升无菌青​​霉素/链球菌/两性霉素B（P / S / F）（0.01％）。 </li><li>打开细胞刷只是把样品面前，一定要保持无菌刷，不要触摸它的任何表面除了鼻腔表面。 </li><li>请参加者坐不​​住了朝天花板倾斜的头。有较小的或更多的孩子躁动坐在自己的手和/或躺着，以免乱打刷掉。 </li><li>在那里的通道变窄（看起来像一个小的黑洞）鼻背部瞄准刷。滑动刷下来，因为刷从nostri删除扭动手腕湖</li><li>将刷子在圆锥形和BEGM淹没。切掉多余的刷子和管更换帽。不要旋涡振荡。 注意:如果参与者愿意，获得相同的方式，但在第二个鼻孔的第二刷。获得两个鼻孔的刷涂将导致较高的可能性，即采样将是成功的。采样同一鼻孔可能导致出血和不太可能提高样品。 </li><li>样本保持接近37℃，尽量使用温水的烧杯运输过程中。 </li></ol> 2.细胞计数和细胞离心涂片<ol><li>轻轻搅动刷在BEGM和取10微升样品用于使用血细胞计数器的细胞计数。添加活/死染色的10微升，数只活在显微镜下的细胞。 </li><li>取50微升样品稀释至200μl的用1×PBS。添加到附着到玻璃载玻片和旋转以300rpm持续2分钟离心涂片漏斗。让干滑动O / N和染色的幻灯片跟随着G制造商的说明。晾干O / N，最好是在黑暗中。 </li><li>染色幻灯片与喝骂3染色<ol><li>转移每个解决方案（HEMA固定液，浅蓝色;喝骂3解决方法，粉红色;而喝骂3溶液II，深蓝色）到染色皿;包括保持在不使用时。幻灯片插入幻灯片染色船</li><li>浸幻灯片连续固定液30秒，然后让多余的流失。浸在溶液中连续幻灯片我30秒，然后让多余的流失。浸在不断解决方案II幻灯片30秒，然后让多余的排出</li><li>用去离子水冲洗，直到水是清水。晾干O / N，优选在黑暗中</li></ol></li><li>看在显微镜下和计数细胞幻灯片，确定上皮细胞的百分比。应在90％以上，如果采样是好的。 </li></ol> 3.种子细胞注:在适当的和认证的组织培养进行所有的细胞培养过程罩使用无菌技术。 <ol><li>与牛真皮层胶原蛋白（BDC）涂层板，型1.将0.5ml 3毫克/毫升BDC的49.5毫升的1X PBS稀释。制备更少，如果将用于只有几个平板。涂层，T25培养瓶中用2ml的BDC和在无菌罩õ孵育/ N或在37℃2小时，如果必要的。 </li><li>孵育后，吸出任何残留的液体。暴露烧瓶于UV光在无菌罩30分钟。瓶储存在4°长达一个月C，但种子细胞之前给他们带来RT或37℃。 </li><li>保持画笔和细胞BEGM在37℃，直到准备播种。轻轻地内圆锥沙沙刷，但不要旋涡。 <ol><li>板7×10 5个细胞在T25烧瓶中。如果有较少的，使用较小的容器中，一个6孔板的诸如一个阱。这个大小板需要大约3×10 5个细胞。 注意:如果不存在这许多单元，这是不可取的继续。 </li></ol></li><li>在无菌罩，带刷出管和GE的ntly涂层瓶表面上的旋转刷，注意不要划伤涂层。在适当的生物危害容器丢弃画笔。 </li><li>缓慢吸取在BEGM中剩余的细胞从圆锥形的并进入T25培养瓶中。观察显微镜下的细胞。一些漂浮细胞的将观察到，并有可能是从鼻子，这是在此阶段（ 图3A）上可接受的一些碎片。 </li></ol> 4.细胞培养<ol><li>种子细胞后，让他们满足于48小时不中断（ 图3）。 48小时后，慢慢地和轻轻加入2毫升温BEGM和20μl无菌青霉素/链霉素/两性霉素B（P / S / F）。 注意:不要吸原来的2毫升的媒体。 </li><li>上播种后第 4天，小心吸所有液体，并用4毫升新鲜替换，温热BEGM加1×青霉素/链霉素（两性霉素B不再是必要的）。更换介质每两天和评估附着细胞换货，增长和融合。当细胞达到约80％汇合时，通常在2-3周，它们已准备好进行传代。 </li><li>一个T25培养瓶中（约2.5×10 6个细胞时汇合）可以传代成三个T25烧瓶或一种T75烧瓶中。第一通道之后，细胞应该是健壮和健康，达到汇合约每三天。 </li></ol> 5.传代细胞<ol><li>轻轻吸从板的介质。每添加T25瓶1毫升0.05％胰蛋白酶溶液在孵化器板和地点约4分钟，或直至细胞分离板。检查细胞每2分钟支队的水平，不要离开胰蛋白酶的时间超过了10分钟。 </li><li>加入5 ml的胰蛋白酶中和溶液（TNS），或含有血清的培养基中以中和酶。在15ml的锥形管中取出所有从烧瓶并置于液体和细胞。 </li><li>离心机在200 xg离心（加速度0减速0）5分钟以获得细胞沉淀。吸superna坦和悬浮细胞在BEGM + P / S为新烧瓶所需的体积（4毫升T25培养瓶中，将10毫升的T75烧瓶） </li><li>用活/死染色进行细胞计数上述（2.1）中所述。添加BEGM和细胞的适量涂布烧瓶，并返回到培养箱中。维持如上详细或在每毫升0.5-2×10 6个细胞的浓度冷冻介质（70％BEGM，20％FBS和10％DMSO）中冷冻保存。 </li></ol> 6.重新编程为iPS细胞注:生成iPS细胞前，请确保遵守有关产生和使用人iPS细胞的所有机构的规定。无菌技术用于IPSC的培养物尤其重要，因为培养基不经常含有抗生素。至关重要的是，NEC的出现健康是有力增生成功重新编程。 <ol><li>板每一个6孔组织培养板的孔5×10 5 NEC的。 24小时后，加入聚凝胺到BEGM通过增加表达的Oct4，Sox2的，Klf4的，c-Myc的媒体的慢病毒颗粒8微克/毫升和转导NEC的终浓度。旨在实现感染（MOI）的多重〜5 27。 <ol><li>要确定MOI，准备集中慢病毒股票的连续稀释，并转导HT1080细胞与这些稀释。首先明确地检测在HT1080细胞dTomato表达后，通过计分dTomato阳性细胞/在每个稀释的细胞小团块的数量计算的"转​​导单位/ ml"的数目。 注意:通常情况下，将有一个太高的稀释液（一切dTomato正）和过低（没有或只有几个阳性细胞）。 </li><li>从其中的细胞分离阳性细胞/小团块被识别的稀释液进行打分。通过校正转导单位/ ml的与适当的稀释因子的数量确定的效价。 MOI然后数的比率的转导的细胞对病毒粒子27的数量。 注:每个dTomato阳性细胞/小丛假定从一个单一的病毒颗粒出现。 </li></ol></li><li>约3小时转导后，取出媒体和新鲜BEGM取代。丢弃使用制度上批准的程序包含慢病毒介质。返回转NEC的向培养箱3天。 </li><li>在第3天，用新鲜的BEGM替换介质，然后孵育另外3天。吸用过的培养基，洗涤细胞用1×PBS和加入1ml 0.05％胰蛋白酶溶液到孔中。用胰蛋白酶传代细胞如上面写的（第5）。小心吸上清，悬浮细胞在4毫升BEGM + P / S。细胞可以传代涂有BDC 6孔板的一个新井，或者加入MEF文化，如果准备（见下一步）。 </li><li>准备MEF涂层板。第4天（1毫升/孔）加0.1％的明胶溶液至2口井6孔的（执行+/- Thiazovivin（SPT）见步骤6.6）。上的下一个当天，解冻灭活的MEF 28的小瓶中。 </li><li> MEF中放置到10ml MEF培养基（DMEM + 1×NEAA + 10％DFCS）的。旋，在200×g离心5分钟以沉淀。悬浮在MEF媒体。来自6孔板吸明胶和每明胶包被6孔板的孔加入1.87×10 5的MEF。孵育O / N，37°C，或最少24小时。 </li><li>传输2毫升BEGM含转导的细胞每孔成2口井以前包被的6孔板孵育O / N。在第二天，每含有4纳克/毫升碱性成纤维细胞生长因子的标准的hESC媒体的井2.5毫升替换BEGM。日采食细胞与新鲜胚胎干细胞媒体+/- SPT 10天 注:可以添加小分子（SB431542，PD0325901和Thiazovivin（SPT）的鸡尾酒）的鸡尾酒，加强对长者邻舍23的效率和重编程的动力学。 </li><li>十天后，继续每天喂用胚胎干细胞媒体没有SPT。日采食文化，直到人类胚胎干细胞样菌落出现（ 图5A，P0菌落）。确定菌落他们的高核窝藏公认的iPS细胞:质比和核仁明显。 </li><li>与人类胚胎干细胞样形态手动切除和转移的菌落用于培养和扩展如在无饲养系统分开线分开的孔中。切除菌落应迅速适应这些条件。 <ol><li>电镀切除菌落后这些离散推定iPSC系现在被认为是通道1（P1）。饲料培养每天mTeSR1。通道和扩大使用标准程序iPSC系。这可能需要数天至一周为P1殖民地到达在他们应该传代的尺寸。 </li></ol></li></ol> 7.保持文化iPS细胞<ol><li>饲料和日常评估文化。手动消费领域用无菌玻璃吸管或微量移液管尖端刮表现出明显的差异化。每天采摘后更换介质。 </li><li>传代细胞approxima后tely每四天:轻轻抽吸媒体，添加每一个6孔板（半馈送体积）以及1毫升温分散酶（1毫克/毫升），然后在37℃下约4分钟。殖民地的边缘将开始解除，它看起来像菌落周围白色轮廓。轻轻吸了分散酶和温暖DMEM / F12洗3次。 </li><li>加2ml mTeSR1并使用细胞升降机从板抬起菌落。使用5毫升血清吸管或微量P1000轻轻磨碎的细胞，直到菌落分解成更小的簇。 <ol><li>避免产生非常小的簇或单个细胞，存活和随后的增殖将是次优的。当重新电镀菌落，以1:4至1:6拆分将保持最佳菌落密度。轻轻地从板取出殖民地和媒体，并添加新的，基质胶包被的孔。 </li></ol></li></ol>

The authors have no conflicts of interest to disclose.

鼻粘膜上皮细胞（长者邻舍中心）是研究气道疾病可访问的平台，NEC-iPS细胞提供了一条康庄大道，探讨疾病的发展，治疗及治疗1,31,32。长者邻舍中心可以轻松无压力或潜在有害程序6,23获得。根据我们的经验，鼻粘膜如在本协议中所描述的采样似乎是更少的压力比采血儿童感知。因此，这种方法可以是在儿科患者人群特别有用的和相关性研究的气道疾病。在NEC的的另一个优点是，一旦他们在培养细胞，上皮细胞被选择用于与媒体和培养条件而成为均匀。这比使用一个复杂的组织如血细胞有利的，并且可以更容易地评价细胞起源中所得用转录和后生方法iPSC系的影响。 什么时候采样鼻粘膜，重要的是鼻子的正确的区域进行采样，使得大多上皮细胞被检索，用显微镜（ 图2）根据观看细胞离心涂片所证明。正确的采样将产生≥90％的上皮细胞。关键是要获得来自健康患者的样品，并确保样品，以便最大限度地获得并接种细胞数目适当地收集。在协议（第1.5节）中描述的扭转运动确保了刷接触到尽可能多的鼻表面尽可能的接触。它是一种自然反射把一个人的头，当外来物体正朝着自己的脸前进。因此，在一个子转动他或她的头或拉远的情况下，至关重要的是，采样快速，准确地发生。使用这些技术会增加协议的每一个其他步骤的效率，并确定成功重新编程的可能性。询问孩子躺下，甚至把他们的手在他们之下，或有父母或兄弟姐妹握住他们的手是鼓励他们静静地躺着，并帮助确保平稳，无痛的样本收集的好方法。 最近，一些其他的抽样技术已被评估为的舒适性，便于和样品的结果的基础上，细胞含量和组成，以及RNA和DNA产生33。的组审查取样用细胞学刷与聚酯拭子，并且它们的采样都下鼻甲以及通过剧烈涡旋围绕鼻孔刷或拭子的前鼻孔。用刷子采样鼻甲被发现是略微不舒服，虽然它产生最佳的RNA产量和组成最纤毛上皮细胞相比，聚酯拭子。因此，在这个协议提出的方法被验证为采样鼻上皮细胞，以此来研究呼吸气道的最可靠的方法上皮。 一旦获得样品，它必须被保持在37℃。其中一个最有效的方法就是温暖的水的烧杯中，以大于37°C和雀巢入安全运输发泡胶容器中，同时准备迎接参与者。在采集样本之前，加冷水，使温度升至约37℃，并与媒体添加锥形管中。当电刷被加入到锥形管，立即返回管至水浴，并保持不动，直至它可以返回到实验室，在该点处管应放在一个调节37℃水浴中直至细胞接种到培养瓶中。在所有点，无菌技术应用于避免将外来代理到培养。 当细胞放入培养液看到变异样本当中，有相当数量。这种变化似乎从两个供体来源，制止以及样品质量本身。以增加样品的质量的一种方法是，以获得从每个鼻孔一个电刷，或每名参与者的两个电刷。播种新鲜样品后，并随着细胞传代，将细胞的条件应定期监测，以确保它们是适合进行再编程。细胞，让您的最佳人选是采取文化和快速增殖的人。目前还不知道为什么某些供体细胞比那些来自其他小区的更健壮的，但它可能是由于各种因素，如年龄，鼻子的大小和供体的健康在采样的时间。当样品的产量非常少的细胞，或仅少数细胞附着一次镀，样品是不可能生长至汇合。如果一个挣扎的文化确实达到汇合，细胞往往是软弱和传代过程中许多细胞死亡。这种类型的样品是用于重新编程一个非常差的候选，因为它是必不可少的，这些细胞是迅速，鲁棒增殖，并且它们在尽可能早的通道号转导。这是不可取的转染正逐渐生长的细胞，这是否是由于供体变异，取样不佳，或传代培养太薄。如果重新编程试图用一个贫穷的细胞培养，这是不可能继续取得成果。 制备细胞转导是最困难的准备的协议的一部分。基于所述细胞的生长，将2×10 5至5×10 5细胞被建议用于转导，这可能依赖于特定的患者样品。因此，它可能需要镀有不同的细胞数，以获得理想数目的几个井。 这个协议概括不仅便于采样的，但也相对容易，并建立原代细胞培养的时间表，和那些原代细胞培养至iPSC系更可持续的，多功能的研究机会的后续编程。整个从过程诊所在文化建设离散iPS细胞大约需要3个月，缩短重新编程时间的新方法正在开发6,34,35。此字段快速发展找到更有效，更彻底的方式来原代细胞转化为iPS细胞，并随后iPS细胞分化为特定的细胞和组织类型的兴趣。但是，对于组织中气道疾病的重要分化的协议，如肺上皮细胞，仍在摸索出36-38。 从NEC的产生iPSCs的具有细胞简单和相对无痛隔离的优点。此外，可能的是NEC-iPSCs的提供超过从可被利用其它类型的组织衍生的iPSC优点。在随后的iPSC分化的iPSC表观遗传学的影响尚不完全清楚，但有证据表明，小鼠和人类iPS细胞怀有自己的体细胞的转录和表观遗传记忆起源和这个选择性有利于它们分化 ​​成与供体细胞类型，同时限制其他命运5,26相关的谱系。由于起源组织的重要性，而这种影响的iPSC的功能特性，特别是相对于组织特异性表观遗传标记39-45的保持如何，iPSC系需要，以便更清楚地研究，以更好地理解起源组织的影响。先前已表明，NEC衍生的iPSC怀有指示其来源23的组织的后生存储器的保留的基因组甲基化的签名。保持在NEC-iPS细胞如后生存储器可以是多能干细胞到气道细胞分化有益的，因为以前的研究已经表明，血细胞来源的iPSC呈现原点的血细胞的后生存储器更容易被分化成血细胞比成纤维细胞或平滑肌的iPSCs的缺乏布卢ð细胞特异性后生存储器5,26。鉴于源自iPSC的呼吸道细胞模型肺部疾病的巨大潜力，将研究是否NEC的代表最佳细胞类型iPS细胞分化成气管细胞/组织是重要的。此外，此稳定地保持存储器可代表供体细胞的疾病状态的染色体位置，以重新编程抗性和候选人。如协议转换的iPSC到近端和远端肺细胞类型的开发36-38，这些NEC-iPS细胞可具有在建模气管疾病，如哮喘，慢性阻塞性肺病，囊性纤维化，和许多其他与在研究对肺发育环境影响的优点和动态平衡。

从人体样本（iPS细胞）诱导多能干细胞是干细胞研究的一个快速发展的技术。他们提供少得多的伦理和道德的缺点1,2胚胎干细胞（胚胎干细胞）研究的替代品。尽管它们不是表观遗传学上相同的人类胚胎干细胞3-5，人​​iPS细胞提供建模发育和疾病的表型的独特方式，并且可以从相关的疾病状态5-8的组织中获得。正在不断探索产生人iPS细胞的新方法，以确定最佳的细胞类型，开始时，作为一种方法来制备适用于移植的GMP质量iPSCs的，并且也增加了重编程过程6,9-11的及时性和效率。 呼吸道上皮细胞在过敏性炎症12的发展是至关重要的，而上皮的过敏反应和哮喘气道重塑的主要驱动力通过与与免疫相互作用e和间质细胞。气道上皮起着起源和肺疾病如哮喘的持久性的重要作用。然而，较低的气道上皮细胞是难以得到在临床环境，特别是从健康对照患者和小孩。从几个研究的数据支持的前提下，从鼻腔粘膜上皮细胞可用于下呼吸道上皮细胞13-20的有效和实际的代理，学习到的空气污染物和过敏原的反应时尤其如此。鼻粘膜由90％以上的纤毛呼吸道上皮细胞和取样这些鼻上皮细胞（NEC的）可以在儿童年仅四岁或五个可以容易地进行的，因为它比其他细胞/组织取样技术微创和是与不良事件，如感染20-23的最小的风险相关联。它提供了一个快速，简单的方法来品尝健康和患病的孩子不长，不必要的，往往是痛苦的支气管镜procedu使得需要镇静资源。先前的研究已经发现，与哮喘的严重程度的疾病亚型可以在两个鼻粘膜以及哮喘的儿童采取支气管细胞样品区分开，并且两个组织类型之间的基因表达在非无所不在基因22约90％的相似24。为iPS细胞的来源，NEC的报价比其他经常使用的细胞类型的优势。成纤维细胞通常用于的iPSC产生，但尽管这些细胞可以很容易地从皮肤活检培养，这个过程通常需要局部麻醉，切开和缝合线，并与感染的一些风险。因此，获得来自患者的知情同意对于这种类型的活检可能是困难的25。一个替代的成纤维细胞是外周血单核细胞（PBMC）。然而，可能难以从小儿患者获得足够的血液的iPSC产生。此外，还有下游AP的局限性褶皱成纤维细胞和血细胞来源的iPS细胞，特别是它们的分化能力，某些细胞类型5,26。因此，考虑到相对可及性和副作用跟随他们收集低风险，NEC的代表来自儿童人群的产生的iPSC一个理想的细胞来源。 iPS细胞已经收到了很多关注最近在研究人类的发展，产生新的疾病模型的平台，并作为细胞的个性化​​疗法的潜在来源。可实现这一技术的全部潜力之前，重编程过程的分子基础需要被阐明，但现在这个协议和内概述的程序将阐明集中在呼吸道暴露的调查研究，以及提供一种用于平台研究涉及的iPSCs个性化医疗的效果。 几个实验室的协同工作导致了generatio不仅取样鼻粘膜，而且还培养NEC的，和重编程这些细胞的iPSC 23成功技术的n个。本文提供了最佳的取样，培养和重新编程条件的协议的轮廓。

<table border="0" cellpadding="0" cellspacing="0" width="655">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">15mL conical</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:108px;">14-959-49D</td>
			<td style="width:139px;">Protocol Step 1.1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">BEGM</td>
			<td>Lonza</td>
			<td>CC-3170</td>
			<td>Protocol Step 1.1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Penn/Strep/Fungicide</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td>Protocol Step 1.1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Penn/Strep</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Protocol Step 4.a.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">cytosoft cytology brush</td>
			<td>Fisher Scientific</td>
			<td>22-263-357</td>
			<td>Protocol Step 1.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">trypan blue</td>
			<td>Fisher Scientific</td>
			<td>MT-25-900-CI</td>
			<td>Protocol Step 2.1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>02-671-54</td>
			<td>Protocol Step 2.1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">PBS</td>
			<td>Fisher Scientific</td>
			<td>BP2438-4</td>
			<td>Protocol Step 2.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Cytology Funnel Clips</td>
			<td>Fisher Scientific</td>
			<td>10-357</td>
			<td>Protocol Step 2.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">cytospin funnel</td>
			<td>Fisher Scientific</td>
			<td>23-640-320</td>
			<td>Protocol Step 2.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Cytospin 4</td>
			<td>Fisher Scientific</td>
			<td>A78300003</td>
			<td>Protocol Step 2.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">blank slide</td>
			<td>Fisher Scientific</td>
			<td>S95933</td>
			<td>Protocol Step 2.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">hema 3 stain kit</td>
			<td>Fisher Scientific</td>
			<td>22-122-911</td>
			<td>Protocol Step 2.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Bovine Dermal Colagen, type 1</td>
			<td>Life Technologies</td>
			<td>A1064401</td>
			<td>Protocol Step 3.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">T25 flask</td>
			<td>Fisher Scientific</td>
			<td>08-772-45</td>
			<td>Protocol Step 3.3.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Trypsin</td>
			<td>Lonza</td>
			<td>CC-5012</td>
			<td>Protocol Step 5.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Trypsin Neutralizing Solution</td>
			<td>Lonza</td>
			<td>CC-5002</td>
			<td>Protocol Step 5.2.</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:207px;">Fetal Bovine Serum (FBS), heat sterilized at 65&#176;C for 30min</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td>Protocol Step 5.5.</td>
		</tr>
		<tr height="82">
			<td height="82" style="height:82px;width:207px;">Dimethyl sulfoxide Hybri-Max&#8482;, sterile-filtered, BioReagent, suitable for hybridoma, &#8805;99.7% -&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td>Protocol Step 5.5.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">polycistonic lentivirus*</td>
			<td>e.g. Millipore</td>
			<td>SCR511</td>
			<td>Protocol Step 6.4.&#160; 
			A commercial source of reprogramming vector is listed. We routinely use the 4-in-1 plasmid reported by Voelkel et al (PMID: 20385817) to generate VSV-G-pseudotyped polycistronic reprogramming lentivirus in-house. This plasmid can be obtained by contacting&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">polybrene</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-134220</td>
			<td>Protocol Step 6.4.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Irradiated CF1 MEFs</td>
			<td>GlobalStem&#160;</td>
			<td>GSC-6301G</td>
			<td>Protocol Step 6.4.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">hESC media</td>
			<td>See recipe included in protocol</td>
			<td></td>
			<td>Protocol Step 6.11.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">SB431542</td>
			<td>Stemgent</td>
			<td>04-0010</td>
			<td>Protocol Step 6.11.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">PD0325901</td>
			<td>Stemgent</td>
			<td>04-0006</td>
			<td>Protocol Step 6.11.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Thiazovivin&#160;</td>
			<td>Stemgent</td>
			<td>04-0017</td>
			<td>Protocol Step 6.11.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">hESC-qualified Matrigel</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>Protocol Step 6.13.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Corning plate, 6 well</td>
			<td>Fisher Scientific</td>
			<td>08-772-1B</td>
			<td>Protocol Step 6.13.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">mTeSR1</td>
			<td>StemCell</td>
			<td>5850</td>
			<td>Protocol Step 6.13.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">250mL disposable filter flask (0.22&#181;m)</td>
			<td>Fisher</td>
			<td>SCGP-U02-RE&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">dispase</td>
			<td>StemCell</td>
			<td>7923</td>
			<td>Protocol Step 7.3.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11320-033</td>
			<td>Protocol Step 7.3.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">cell lifter</td>
			<td>Fisher Scientific</td>
			<td>08-100-240</td>
			<td>Protocol Step 7.4.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hESC Media**</td>
			<td></td>
			<td></td>
			<td>Protocol Step 6.11. 
			components should be mixed and then filter sterilized. Media can be kept at 4&#176;C for up to two weeks. When warming media, do not leave at 37&#176;C longer than 15 min</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:207px;">DMEM-F12 50/50 media</td>
			<td>Invitrogen</td>
			<td>&#160;11330-032</td>
			<td>Final Concentration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">KO replacement serum (KO-SR)</td>
			<td>Invitrogen</td>
			<td>10828-028</td>
			<td align="right">0.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">200mM L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030-081</td>
			<td>1mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">55mM &#223;-mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985-023</td>
			<td>0.1mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">100x non-essential amino acids</td>
			<td>Invitrogen</td>
			<td>11140-050</td>
			<td>1x</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">2ug/mL Basic-Fibroblast Growth Factor (b-FGF)</td>
			<td>Invitrogen</td>
			<td>13256-029</td>
			<td>4ng/mL</td>
		</tr>
	</tbody>
</table>

cultivate primary nasal epithelial cells from children and reprogram into induced pluripotent stem cells

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

鼻子的初始部分，称为鼻前庭，是由软骨29包围的区域。刷需要顺利晃过此区域鼻子，超越鼻瓣（口internum，或"黑洞"，在鼻孔的背面看到的），并且是从下鼻甲（ 图1）获得的样品。鼻甲的骨性结构会增加鼻部29的表面积，使他们采样的理想地点。该地区还通过血管粘膜组织，它非常类似于下呼吸道的衬里，也活跃在与环境暴露的免疫反应内衬。 采样正确的组织保证被重新编程的细胞是上皮细胞。这可以通过在初始细胞计数和细胞离心涂片进行评价，而且还通过在培养观察细胞。该细胞离心涂片，一旦离心下来，干燥并染色，示出了样品的组成。大多数样品包括上皮细胞，这是柱状纤毛的，具有一个圆形核。与赫马3染色试剂盒，它们染色浅蓝色与较暗的紫色核（ 图2），以及常时从它们被看作成群一起鼻子直接取出。虽然鼻腔细胞样本最初的细胞类型的混合，以及碎片从鼻子，用在文化和媒体与时间的变化，上皮细胞占据主导地位（ 图3A，B）。当上皮细胞生长至汇合，它们的形状像一个新鲜裂解卵，其中所述核是大，并且在中心和细胞质是一种"伸出"和拉伸的作为细胞准备分裂（ 图3B）。随着细胞共同成长，他们开始承担更多的足球形状，并紧贴在一起的菜（ 图3C）。 一旦细胞被转导，它们将表示在细胞核中的tdTomato荧光标记物。 图4示出了细胞一旦被转导，在亮视野，荧光，并与两个图像合并。不是每一个细胞表达的标记物，因此不是每个细胞转导，但每个孔应具有约90％的转导效率，而这将增加的可能性，一些细胞将在MEF中进行镀覆后实现多能性。 一旦共培养用的MEF和作为细胞开始形成菌落，它们将逐渐停止表达tdTomato。然而，这不是集落形成的最佳指示。集落形成的最佳适应症是视觉确认。人类胚胎干细胞克隆，这是"黄金标准"多能性的，大多是圆形，是由许多小而圆的细胞的LARGË核和高细胞核浆比例。每个小区出现主要由细胞核和其它细胞器不清晰可见。继IPSC的殖民地收获，和文化标准无饲养条件下，新成立的iPSC菌落看起来很像他们的人类胚胎干细胞的同行，并且单个细胞几乎相同的人类胚胎干细胞的表型， 如图5B可见。 在实验中，从鼻上皮细胞（NEC-iPS细胞）中产生利用的iPSC之前，建议进行评估的新线路的质量。这通常涉及核型，多潜能标志物的表达，和iPS细胞的分化成各3胚层（​​内胚层，中胚层和外胚层）的能力的评估。分化能力可以以多种方式进行测试，但目前，最严格的检测是畸胎瘤测定2,23,30。更全面的测定，如转录<html>与表观基因组分析是确定是否有任何染色体异常被重新编程23介绍有用。 <img alt="图1" src="/files/ftp_upload/53814/53814fig1.jpg" /> 图1.下鼻甲，采样运动和抽样对象，下鼻甲，用黑色箭头指示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53814/53814fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53814/53814fig2.jpg" /> 图2.细胞离心涂片。大多数样品包括上皮细胞，这是长柱状，在一端纤毛和在另一圆核。当从鼻孔直接采样他们往往成群在一起。箭头指示单个上皮细胞。"https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53814/53814fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53814/53814fig3.jpg" /> 图3.原发性鼻上皮细胞中培养。（A）中漂浮细胞和碎片样品被收集和镀之后。 （B）细胞在一天后传代。细胞形态是一贯的，文化主要包括鼻粘膜上皮细胞，放大10倍，和（C）放大5倍的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53814/53814fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53814/53814fig4.jpg" /> 图4.转邻舍中心。细胞<html>被转导扛起了载体和表现出红色荧光标记tdTomato。因为他们对多能状态进步此标记应该消失。 （A）转染细胞的明视场图像。 （B）转导的细胞的荧光图像。 （C）覆盖亮场和荧光图像（板A和B）的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53814/53814fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53814/53814fig5.jpg" /> 图5的iPSC干细胞样集落。（A）生长在MEF中，菌落多呈圆形的边缘，并显示几乎没有区别。 （ 二 ）个别细胞显示典型的人类胚胎干细胞样形态，小圆细胞细胞核大。5large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells at JoVE.com

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

从培养儿童原发性鼻腔上皮细胞和重新编程为诱导多能干细胞

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

cultivate-primary-nasal-epithelial-cells-from-children-reprogram-into

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

11.1K 次观看.  Cincinnati Children's Hospital. This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

视频: Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

科研

发育生物学

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

从小鼠羊水细胞多能干细胞使用转座子系统的生产

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

在人多能的体外干细胞向滋养层细胞

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

分化软骨细胞与外周血源性人诱导多能干细胞

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

高效可扩展的人多潜能干细胞临床相容角膜缘上皮干细胞的定向分化

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...