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，...

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><tbody><tr><td>15 ml conical</td><td>Fisher Scientific</td><td>14-959-49D</td><td>Protocol Step 1.1.</td></tr><tr><td>BEGM</td><td>Lonza</td><td>CC-3170</td><td>Protocol Step 1.1.</td></tr><tr><td>Penn/Strep/Fungicide</td><td>Life Technologies</td><td>15240-062</td><td>Protocol Step 1.1.</td></tr><tr><td>Penn/Strep</td><td>Life Technologies</td><td>15140-122</td><td>Protocol Step 4.a.</td></tr><tr><td>cytosoft cytology brush</td><td>Fisher Scientific</td><td>22-263-357</td><td>Protocol Step 1.2.</td></tr><tr><td>trypan blue</td><td>Fisher Scientific</td><td>MT-25-900-CI</td><td>Protocol Step 2.1.</td></tr><tr><td>hemacytometer</td><td>Fisher Scientific</td><td>02-671-54</td><td>Protocol Step 2.1.</td></tr><tr><td>PBS</td><td>Fisher Scientific</td><td>BP2438-4</td><td>Protocol Step 2.2.</td></tr><tr><td>Cytology Funnel Clips</td><td>Fisher Scientific</td><td>10-357</td><td>Protocol Step 2.2.</td></tr><tr><td>cytospin funnel</td><td>Fisher Scientific</td><td>23-640-320</td><td>Protocol Step 2.2.</td></tr><tr><td>Cytospin 4</td><td>Fisher Scientific</td><td>A78300003</td><td>Protocol Step 2.2.</td></tr><tr><td>blank slide</td><td>Fisher Scientific</td><td>S95933</td><td>Protocol Step 2.2.</td></tr><tr><td>hema 3 stain kit</td><td>Fisher Scientific</td><td>22-122-911</td><td>Protocol Step 2.2.</td></tr><tr><td>Bovine Dermal Colagen, type 1</td><td>Life Technologies</td><td>A1064401</td><td>Protocol Step 3.2.</td></tr><tr><td>T25 flask</td><td>Fisher Scientific</td><td>08-772-45</td><td>Protocol Step 3.3.</td></tr><tr><td>Trypsin</td><td>Lonza</td><td>CC-5012</td><td>Protocol Step 5.2.</td></tr><tr><td>Trypsin Neutralizing Solution</td><td>Lonza</td><td>CC-5002</td><td>Protocol Step 5.2.</td></tr><tr><td>Fetal Bovine Serum (FBS), heat sterilized at 65 &amp;deg;C for 30 min</td><td>Sigma-Aldrich</td><td>F2442</td><td>Protocol Step 5.5.</td></tr><tr><td>Dimethyl sulfoxide Hybri-Max&amp;trade;, sterile-filtered, BioReagent, suitable for hybridoma, &amp;ge;99.7%</td><td>Sigma-Aldrich</td><td>D2650</td><td>Protocol Step 5.5.</td></tr><tr><td>polycistonic lentivirus*</td><td>&lt;em&gt;e.g.&lt;/em&gt; Millipore</td><td>SCR511</td><td>Protocol Step 6.4.&amp;nbsp;&lt;br/&gt; 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&amp;nbsp;</td></tr><tr><td>polybrene</td><td>Santa Cruz Biotechnology</td><td>sc-134220</td><td>Protocol Step 6.4.</td></tr><tr><td>Irradiated CF1 MEFs</td><td>GlobalStem&amp;nbsp;</td><td>GSC-6301G</td><td>Protocol Step 6.4.</td></tr><tr><td>hESC media</td><td>See recipe included in protocol</td><td></td><td>Protocol Step 6.11.</td></tr><tr><td>SB431542</td><td>Stemgent</td><td>04-0010</td><td>Protocol Step 6.11.</td></tr><tr><td>PD0325901</td><td>Stemgent</td><td>04-0006</td><td>Protocol Step 6.11.</td></tr><tr><td>Thiazovivin&amp;nbsp;</td><td>Stemgent</td><td>04-0017</td><td>Protocol Step 6.11.</td></tr><tr><td>hESC-qualified Matrigel</td><td>BD Biosciences</td><td>354277</td><td>Protocol Step 6.13.</td></tr><tr><td>Corning plate, 6 well</td><td>Fisher Scientific</td><td>08-772-1B</td><td>Protocol Step 6.13.</td></tr><tr><td>mTeSR1</td><td>StemCell</td><td>5850</td><td>Protocol Step 6.13.</td></tr><tr><td>250 ml disposable filter flask (0.22 &amp;micro;m)</td><td>Fisher</td><td>SCGP-U02-RE&amp;nbsp;</td><td></td></tr><tr><td>dispase</td><td>StemCell</td><td>7923</td><td>Protocol Step 7.3.</td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>11320-033</td><td>Protocol Step 7.3.</td></tr><tr><td>cell lifter</td><td>Fisher Scientific</td><td>08-100-240</td><td>Protocol Step 7.4.</td></tr><tr><td>hESC Media**</td><td></td><td></td><td>Protocol Step 6.11.&lt;br/&gt; components should be mixed and then filter sterilized. Media can be kept at 4 &amp;deg;C for up to two weeks. When warming media, do not leave at 37 &amp;deg;C longer than 15 min</td></tr><tr><td>DMEM-F12 50/50 media</td><td>Invitrogen</td><td>&amp;nbsp;11330-032</td><td>Final Concentration</td></tr><tr><td>KO replacement serum (KO-SR)</td><td>Invitrogen</td><td>10828-028</td><td>0.2</td></tr><tr><td>200 mM L-glutamine</td><td>Invitrogen</td><td>25030-081</td><td>1 mM</td></tr><tr><td>55 mM &amp;szlig;-mercaptoethanol</td><td>Invitrogen</td><td>21985-023</td><td>0.1 mM</td></tr><tr><td>100x non-essential amino acids</td><td>Invitrogen</td><td>11140-050</td><td>1x</td></tr><tr><td>2 &amp;micro;g/ml Basic-Fibroblast Growth Factor (b-FGF)</td><td>Invitrogen</td><td>13256-029</td><td>4 ng/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...

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

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

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

使用非整合附加型质粒从冷冻血沉棕黄层诱导多能干细胞的产生

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

科研

发育生物学

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

Expansion of human pediatric esophageal epithelial cells utilizing conditional reprogramming provides investigators with a patient-specific population of cells that can be utilized for engineering esophageal constructs for autologous implantation to treat defects or injury and serve as a reservoir for therapeutic screening assays.

小儿食管上皮细胞重编程的条件在组织工程和疾病使用调查

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

无异条件下多原代羊水和膜细胞重新编程

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

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 ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

神经科学

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research.
Induced pluripotent stem cells (iPSCs) are readily generated by "reprogramming" somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells.
Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-&#223; and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects.
Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.

Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures.

从人多能干细胞中产生气道上皮细胞气液界面培养物

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

生物学

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 ...

生物工程

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...