We would like to acknowledge Connecticut Children's Medical Center Strategic Research Funding for supporting this work.

后从儿科患者的家长/监护人，并根据机构审查委员会（IRB＃13-094）获得知情同意，获得食管活检。 1.消毒仪器和明胶溶液<ol><li>高压灭菌镊子，刀片和前处理组织，以防止污染剪刀。 </li><li>使200毫升的0.1％明胶溶液，结合的蒸馏水200毫升含有0.2 1g明胶。高压灭菌并冷却后才能使用。 </li></ol> 2.镀层组织培养板<ol><li>前细胞分离约2小时，添加足够的0.1％明胶覆盖组织培养皿（100毫米），并在细胞培养培养箱中于37℃孵育。 注意:这些板也可以由提前时间和在使用前保持在培养箱中。 </li></ol> 3.使酶液解离<ol><li>大约前细胞分离30分钟，掂量出基于每毫克浓度的单位上的小瓶的平衡分散酶的10个单位。放置在一个15毫升锥形管的粉末与10的Dulbecco改良的Eagle培养基（DMEM）中的溶液，并发生在37℃水浴中。 </li></ol> 4.进行细胞培养基<ol><li>让50毫升的条件重新编程培养基中，将以下成分混合入无菌锥形管:35毫升F12的（咸的培养基），11.5毫升的DMEM，0.5毫升L-谷氨酰胺，0.5毫升100×青霉素/链霉素，2.5毫升的胎牛血清（FBS），人胰岛素的250微克，500纳克人表皮生长因子（EGF）的。 </li><li>让500毫升3T3培养基中，混合50毫升胎牛血清，5毫升100×青霉素/链霉素和5mL L-谷氨酰胺用500mL的DMEM中。 </li><li>让500毫升角质细胞无血清培养基的，解冻EGF和牛垂体提取物（BPE）补充剂（提供媒体）。补品加入到一瓶500毫升基础培养基中，加入5毫升100X青霉素/链霉素</li></ol> 5.培养及辐照3T3细胞作为馈线源<ol><li>制备3T3介质，包括DMEM，10％FBS，1×青霉素/链霉素和2mM L-谷氨酰胺。 </li><li> 文化3T3细胞在组织处理的T-75瓶患者样本到来前至少3天。 <ol><li>之前，为了患者样品的到来，trypsinize使用5的每烧瓶0.25％胰蛋白酶-EDTA溶液与3T3细胞孵育在37℃下5分钟，或直至大多数细胞是浮动</li></ol></li><li>添加5毫升3T3介质，使反应停止。吸出细胞悬浮液，并转移到15毫升锥形管中。旋在300×g下5分钟。 </li><li>吸出介质并悬浮细胞在台盼蓝的细胞计数一个限定的体积。结合细胞的10微升台盼蓝的10微升排除死细胞。放入10μL对细胞计数血球这一解决方案。 注:对于这个原山口，辐照3T3细胞接种密度为每厘米2，这等于每100mm板600,000个单元格10,900细胞。 </li><li>分装必要到板每名患者样品和重悬100mm的板在25mL 3T3介质的在50mL锥形管中，用3000拉德照射细胞的数目。 </li><li>照射之后，旋下来的细胞在300 XG在每100毫米的钢板5毫升5分钟，悬浮在有条件的重编程网上平台。 </li><li>抛开管，直到患者细胞镀在协议发生后（见步骤7.6）。 </li></ol> 6.获取，保存和运输小儿食管活检<ol><li>添加5毫升100微克/毫升primocin到15毫升锥形管中完整角质细胞无血清培养基中，并使其在冰上的医疗设施。 注意:介质的这些管子可被储存在4℃下长达2个月。 </li><li> 在TI获得约8食管活检我内窥镜单独的和，如下所示: <ol><li>放置在含有完整角化细胞的无血清培养基用100微克/毫升primocin和地点在湿冰上的15毫升锥形6活检。 </li><li>放置一个活检5毫升湿冰上福尔马林和地点。 </li><li>放置一个活检与最佳切削温度复合cryomold小活检。 <ol><li>这个模具立即放到置于液氮的异戊烷的烧杯中。一旦冷冻，将cryomold一个标本袋，并存储在一个聚苯乙烯泡沫塑料容器干冰。 </li></ol></li></ol></li><li>将含密封的袋子里，然后到含有湿冰包装箱在中等或福尔马林活检管。封并用生物危害标签标记框，并将其运送至实验室。 注:包含干冰的容器和冷冻活检也密封并用生物危害标签标记。 </li></ol> 7.处理病人组织对下游文化与分析<ol><li>一旦在实验室后，在干冰上转移冰冻活检，-80℃冷冻切片下游或RNA提取。放置在福尔马林样品。储存在4℃冰箱，并允许以固定过夜。 </li><li>旋转剩余活检下来300 XG，并吸出网上平台。加入10 mL分散酶溶液的锥形管，密封件，并允许它在37℃的水浴中孵育15分钟。 </li><li>孵育后，旋下活检以300×g的。吸中期和活检悬浮在5毫升0.05％胰蛋白酶EDTA的。 </li><li>转移5毫升含活检到100毫米的无菌板胰蛋白酶-EDTA，并剁碎使用2-灭菌刀片尽可能细的活检。 </li><li>传输含有剁碎活检胰蛋白酶-EDTA的5毫升到一个新的15毫升锥形。用另外5毫升0.05％胰蛋白酶-EDTA的冲洗所述板两次，并加入到锥形管。孵育管在37℃水浴中10分钟。 </li><li>继温育后，旋活检向下在300 XG（1.4 RPM）5分钟，并吸出培养基。加入5毫升的条件重新编程介质到锥形管以及包含经照射的饲养细胞的条件重新编程介质的5毫升（见步骤5.7）。 </li><li> ROCK抑制剂（1微升/毫升）添加到10毫升细胞悬浮液和板从组织培养皿抽吸明胶后。在37℃。 </li></ol> 8.培养和扩大细胞<ol><li>后约5天内培养的，吸含有切碎活检片起始培养基，用5ml无菌PBS冲洗培养皿2-3倍。新介质添加到板，包括在10μMROCK抑制剂。 </li><li>当达到70％汇合，展开的细胞。加入5毫升0.05％胰蛋白酶-EDTA的到板，并在37℃培养箱中孵育不超过2.5分钟，除去第Ë饲养细胞。以下该温育后，吸出胰蛋白酶-EDTA，并用5毫升的PBS冲洗板。 </li><li>吸出PBS中并加入5毫升0.25％胰蛋白酶-EDTA的到板并孵育5-10分钟，或直至所述细胞是从板松动。介质等量添加到板以停止反应，所有的流体转移到50毫升锥形管中并离心，在300×g离心5分钟。 </li><li>悬浮细胞在限定的条件重新编程介质的体积并用台盼蓝10μL的混合悬浮液的10微升。指望血球测定总细胞数。 </li><li>扩大小区100万人食管癌细胞添加至150mm的板沿与120万在15毫升的总体积照射3T3细胞。在10μM添加新鲜ROCK抑制剂和细胞悬浮液添加至明胶涂层150毫米菜。 </li></ol> 9.冷冻和保存人类食管上皮细胞<ol><li>为了使冷冻剂，加10％DMSO有条件重新编程网上平台。胰蛋白酶及数量后，分装细胞冻结到一个新的50毫升锥形管中并离心，在300×g离心5分钟。 </li><li>吸出培养基，并添加在每离心管2毫升体积冷冻介质。一旦进入细胞悬浮液是均匀的，分装10 6个细胞在预标记cyrovials。发生在在-80℃的冷冻容器之前移动所述小瓶长期液氮储存至少24小时。 </li></ol>

<ol>
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The authors declare they have no competing financial interests.

为了从患者的活检隔离和扩大食管上皮细胞的最重要的步骤是:1）充分游离活检组织以最小的细胞死亡; 2）确保ROCK抑制剂加入到在每次更换培养基的细胞培养基; 3）不要使用更多的饲养层细胞比建议; 4）保持干净无菌文化; 5）传代细胞之前，为了达到汇合。 由于为条件重新编程得到的活检样品中的患者相关的差异，这是合理的预期生长动力学，表型和扩展到多个通道的能力的差异。通常情况下，4食管活检从每个患者为细胞分离得到的。一旦分离，我们净赚在第0天约300,000-600,000细胞这平均由第3代扩展到超过10万个细胞。形成应该类似于上皮细胞系中描述的鹅卵石形态菌落形态&lt;SUP类="外部参照"&gt; 25（ 图2）。培养中的细胞的样品应通过定量RT-PCR进行分析，流式细胞术和免疫荧光对上皮标记，以确保扩大种群确实上皮在原点（ 图3）。应在细胞中不表达上皮标记（E-钙粘蛋白，EpCAM的，TJP1），该细胞系应终止并丢弃。据预计，大于90％的存在的细胞是EpCAM的正面的，如果它们在原点上皮。为了如果文化正在改变的表型或失去上皮表达，免疫荧光评估或流式细胞仪应该用来评估上皮标记（E-cadherin的），祖表达式（P63），以及上皮细胞紧密连接蛋白（TJP1）。随着细胞传代，预期的表型不应该改变，增殖不应降低，并且这些细胞应显示上皮表达以及祖表达（ 图4 </strong&gt;）。此外，即解离并接种在条件重新编程培养正常食管组织似乎具有大约36-40小时（ 图5）的群体倍增时间。被发现不加倍为5天以上的细胞很可能是衰老和应该被丢弃。 它已经描述了从患者分离与存在炎性过程，如EOE的细胞，具有具有降低的增殖速率的上皮细胞，（ 图3例26和28日）和细胞凋亡26的一个高速率降低E-钙粘蛋白的表达。因此，它是不是不合理的难以培养细胞使用此方法严重患者。这是可能的细胞将只承受几段甚至只是初始通路。这应该仍然提供调查者有比原先孤立的更大数目的细胞。然而，这个数字可能并不足够所有建议的研究。因为这些细胞被用来最终研究食管疾病或构建体食道置换，要记住这些细胞仅用于粘膜再生负责是重要的。其他类型的细胞如成纤维细胞，神经细胞，平滑肌细胞和血管也仅有食管疾病的研究和用于组织工程应用的重要。 该技术提供了用于研究疾病的发病机制，以及作为用于食管组织工程应用的患者特异性的细胞的电位储层的宝贵工具患者特异性的细胞来源。播种生物材料植入手术使用的电池必须是自由的基因异常，病毒序列，而不是形成畸胎瘤。利用干细胞（多能或多能）的产生以下分化过多不需要的细胞类型，其中一些可形成畸胎瘤，如果他们不经受differentiation。此外，这种细胞来源的表型是一致的，并且不需要纯化或除去不需要的细胞的。提供了理想的细胞来源将是在正被工程化在相同解剖位置中发现的细胞。在这种方法中，这些食管上皮细胞被用来重新填充食管支架的粘膜。所需的食道再生额外的细胞类型;然而，该技术的焦点是确保粘膜再接种，而不是不完整，因为这可能导致泄漏，狭窄和穿孔。 除了利用这些细胞工程的食道新的手术方案，这些细胞也可用于研究食管疾病进程以及筛选新的治疗方法来治疗这些条件。嗜酸性粒细胞性食管炎（EOE）被描述为食道的变态反应性疾病，具有确切机制和发病机理尚未充分描述。目前治疗章iments包括消除饮食，口服激素和多种内镜。不存在检测，以评估什么触发为每一个病人，这导致医生使用一种"猜测和检查"的做法，是漫长的，往往令人沮丧的病人的炎症过程。因为患者特异性细胞扩增的该方法不利用慢病毒转导或遗传修饰，以在细胞从重编程过程的基因型永久性变化的可能性是最小的。这意味着细胞理论上保持从体内环境不变。测试患者细胞在体外的能力，以确定所述引发剂将是新颖的和可能最终导致的新的诊断或治疗剂对这些儿童患者发展。

食管癌组织工程和嗜酸性粒细胞性食管炎（EOE）一直是研究在许多实验室在过去十年中的焦点。先天性缺陷，如食管闭锁，被认为在约1 4,000活产，这导致在食道导致无力的发育不完全吃1。 EOE的发病率和患病率一直在自从疾病实体于1993年鉴定EOE的发病率从0.7变化到每人10年/ 10万和患病率0.2范围到43/10万2的崛起。一种新的有吸引力的手术方法来治疗长差距食管闭锁在于生成的组织构建了利用患者自身的细胞植入。这些细胞在用合成脚手架结合将产生自体构造，不需要免疫抑制。一些团体已经开始调查美国食管组织工程3以及使用本地食管上皮细胞的干细胞样细胞电子重新填充粘膜4 - 7。疾病存在于儿童患者食道往往难以诊断或不干预研究。此外，利用动物模型或体外永生化细胞系模型的儿科疾病，如EOE不包括确切的发病机制或患者的具体差异8。因此，为了研究患者的疾病过程在体外的能力，以确定特定疾病触发抗原，评估潜在的机制和调查药物治疗将是新颖的和提供可在患者的治疗辅助信息临床医生。 已经有已被建议用于Tissu酒店使用许多自体或患者特定的细胞类型机电工程和研究人类疾病的发病机制。然而，这些类型的细胞在它们的能力是有限的，以产生一个特定的表型的足够的细胞接种大型支架或体外研究进行高吞吐量。使用多潜能或多能的干细胞已经大量的研究讨论的话题，但是，局限性和缺点对于使用这些细胞已被很好地描述9。利用人类胚胎干细胞的高度辩论，提出了许多伦理问题。最重要的是，这些细胞形成畸胎瘤，其类似于一个肿瘤，如果他们不从它们的多能性状态提供它们到生物主机10之前区分。此外，使用胚胎干细胞的不会是患者特异性的，并可能引起同种异体反应和需要免疫抑制10。诱导多能干细胞（iPS细胞）是多能干细胞，它可以从患者自身的细胞。体细胞，如皮肤细胞，可以使用各种一体化和非整合的技术来诱导多能状态。这些细胞然后作为用于组织工程或疾病调查患者特异性细胞来源。不需要的遗传物质整合到这些细胞是很多人所描述即使序列完全去除iPS细胞似乎节省了后生"记忆"对从它们所衍生11细胞类型值得关注。这些细胞还将形成畸胎瘤体内如果不是移植前11区分。许多分化的协议已经研究专注于上皮谱系12，13，14，但是，要注意的是在分化结束时所产生的细胞类型不均匀和O这是非常重要的唯一一句具有感兴趣的细胞类型的一小部分。这导致低收率和需要纯化所需的细胞类型。虽然iPS细胞是一个潜在的患者特定的细胞来源，该方法以获得对任何组织工程或疾病调查感兴趣的细胞类型是非常低效的。 肺15，乳房16，小肠17，结肠18，膀胱19和食道20:人上皮细胞已经从各种人体包括患病和非患病组织的被成功分离。要注意的是人的原代细胞具有其中表型被保持21,22通道的一个有限数量是很重要的。不幸的是，这意味着需要的疾病调查或播种的工程支架的细胞数用于植入可能无法达到。因此，需要新的技术来扩大患者的细胞，同时仍保持上皮表型。利用饲养细胞和ROCK抑制剂正常和癌性上皮细胞的有条件的重编程被刘等人在2012年描述。 2 3。这个技术被用于扩展从利用照射饲养细胞，ROCK抑制剂和有条件的重新编程介质前列腺癌和乳腺癌的活检获得的癌细胞的上皮细胞。我们的目标是产生足够的细胞用于在体外测定，如药物筛选。该技术是能够通过"重新编程"这些细胞干细胞或祖状态，这是高度增殖性无限期膨胀的上皮细胞的。已经证实，这些细胞是非致瘤性和不具有形成畸胎瘤23,24的能力。此外，没有染色体异常或遗传操作是使用这种技术23,24在培养传代这些细胞后存在。最重要的是，这些细胞仅能够分化成感兴趣的天然细胞类型。因此，这种技术提供了一个大的贮存器的患者特异性上皮细胞疾病调查或组织工程无需永生化。 从一个特定的器官，以研究疾病过程获得上皮组织往往有限，并不总是可能的，因为病人的风险。对于那些从食管疾病或缺陷的患者，内镜活检检索是用于获得可以解离，并有条件地重新编程，以提供特定于该病人的食管粘膜无限期细胞源的上皮组织微创方法。然后，这允许在体外研究上皮细胞，以评估疾病过程和屏幕为潜在治疗剂。一种疾病过程，可以极大地从该方法中受益是嗜酸性食管炎，已被描述为食道8的过敏性疾病。过敏试验以及治疗方法可以在体外使用患者自身的上皮细胞进行评估，然后这个数据可以被传递到治疗医师来开发个性化的治疗方案。条件重新编程的与来自儿科患者获得内窥镜活检一起使用时的技术提供了从任何患者无限膨胀正常食管上皮细胞的能力。此单元源因此可以用天然或合成的脚手架一起合作，以提供缺陷，疾病或创伤患者专用手术选择。有一个不确定的细胞数量将有助于工程师是拥有一个完全重新接种食道的构造为了与食管上皮细胞流明帮助促进剩余的细胞类型的再生。

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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.

在分离来自患者活检食管上皮细胞中的关键步骤的简要总结于图1。上皮细胞的集落将形成在约4-5天，将由成纤维细胞饲养细胞（ 图2A）所包围。作为这些菌落扩大他们将与其他的菌落合并，以形成较大的集落（ 图2B）。一旦培养物都成为70％汇合时，它们需要被膨胀（ 图2C）。以确保成纤维细胞从板中除去上皮细胞之前除去，0.05％胰蛋白酶-EDTA用于高达2.5分钟以去除馈线。缺乏馈线应类似于图 2D。粘附上皮细胞，然后用0.25％胰蛋白酶-EDTA胰蛋白酶消化并重新接种在新的辐照饲养细胞（ 图2E）。 <p class="jove_content" fo:keep-together.wi薄页="1"&gt;通过定量RT-PCR检测这些细胞的表型特征表明P63，祖细胞在流路1的端部的标记的上调，相比于初始活检样本，同时仍保持E-钙粘蛋白和TJP1表达。这表明，细胞已被"重新编程"到相对于非重编程的细胞是更增殖更祖/干细胞样的状态。当这些细胞被从条件重新编程条件而采取并接种在正常组织培养板上，P63降低，TJP1和E-钙粘蛋白的（CDH1）的表达增加表明从祖细胞中的表型的转变，以一个更加分化的上皮细胞（ 图3A ）。这些细胞也分析在流路1用于通过流式细胞仪上皮标记和大于90％存在从三个患者细胞呈阳性的EpCAM（ 图3B）。细胞也通过免疫荧光在通道1和3分析<html>以确保表型对这些3代未发生变化。染色表明上皮标志物E-cadherin的持久性（ 图4A-D），祖标记P63（ 图4E-H），增殖标志物KI-67（ 图4I-L）和上皮细胞紧密连接蛋白TJP1（ 图4M-P ）。 最后，生长曲线，使用的细胞系从非患病患者，以评估这些细胞在条件重新编程培养的倍增时间作图。细胞在第0天100,000个细胞铺板并被发现是稍稍超过500,000个细胞在第4天（ 图5）。倍增时间经计算为约36-40小时。预计患病细胞将具有较慢的倍增时间，但是，每一个病人，应分别评估以考虑个体差异。 1"&gt; <img alt="图1" src="/files/ftp_upload/55243/55243fig1.jpg" /> 图1:小儿活检分离培养人食管上皮细胞的概述。活检从知情同意后儿科患者获得，在角质形成细胞无血清培养基中到实验室输送。这些活检然后用1U / mL的分散酶，随后用无菌刀片切碎组织并最后用0.05％胰蛋白酶-EDTA处理处理。然后将细胞悬浮液加入到在条件重新编程介质照射饲养细胞。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55243/55243fig1large.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/55243/55243fig2.jpg" /> 图2:培养和拓展人食管上皮细胞。之后在条件重新编程5天明介质，食管上皮细胞开始形成小鹅卵石菌落（A）中，最终收敛于形成较大的菌落（B）。一旦组织培养皿达到70％汇合（C）中 ，将板处理一个非常短的时间用0.05％胰蛋白酶-EDTA除去饲养细胞，同时仍维持上皮细胞（D）的粘合性。上皮细胞，然后用0.25％胰蛋白酶-EDTA除去，并与新的饲养细胞再接种。分裂，这些细胞就会形成新的殖民地，并继续增殖（E）后仅24小时。放大倍数为100倍。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55243/55243fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55243/55243fig3.jpg" /> 图3:流式细胞仪和定量RT-P在扩大培养细胞CR分析。 （A）在条件重编程的通道1在培养细胞的代表性基因表达证明了P63表达一个6倍的增长，祖细胞的标志物，以及在上皮标记CDH1和TJP1相比活检基因表达为下降。这表明细胞群是在一个更祖状态，因此是更高度增生。基因表达后24小时重新编程（除去饲养细胞和ROCK抑制剂后）显示提高的CDH1和TJP1表达，但在P63表达的减少。 （B）流式细胞术从通道1获得的多个病人的细胞分析表明大于90％的表达EpCAM的细胞。病人27表示非EOE患者细胞，同时患者26和28是EOE患者的细胞。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55243/55243fig3large.jpg" target="_blank">请点击此处Ť</a>Ø查看此图的放大版本。 <img alt="图4" src="/files/ftp_upload/55243/55243fig4.jpg" /> 图4:1传代在条件重编程培养基中生长与照射的饲养层细胞的细胞和细胞3.免疫荧光染色表达E-钙粘蛋白（A - D）以及祖标记P63（E - H）。这些细胞仍处于第3代（I - L）的增殖。最后，这些细胞表达紧密连接蛋白1（TJP1），这也与上皮表型（M - P）一致。细胞核复染用DAPI（蓝色）和使用的Alexa Fluor 546抗体（橙色）检测抗体。 第二抗体（抗体）控制表示非特异性的二级抗体结合于组织（Q，R）。比例尺= 50微米。放大倍数为200倍。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55243/55243fig4large.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/55243/55243fig5.jpg" /> 图5:在有条件的重编程细胞的生长曲线。在从非患病患者的条件重新编程介质细胞在限定的密度接种并复制三个计数，每天4天。使用这些细胞计数和倍增时间的基础上，在4日和0天，这些细胞的倍增时间所获得的数字，计算近似36-40 h的产生的生长曲线。误差棒表示标准误差。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55243/55243fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation at JoVE.com

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

利用条件重新编程人类小儿食管上皮细胞的扩张提供了可用于工程食管构造为自体移植来治疗缺损或损伤，并作为治疗性筛选测定的储存器单元的特定病人群调查。

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 ...

conditional-reprogramming-pediatric-human-esophageal-epithelial-cells

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.9K 次观看.  UConn Health. 利用条件重新编程人类小儿食管上皮细胞的扩张提供了可用于工程食管构造为自体移植来治疗缺损或损伤，并作为治疗性筛选测定的储存器单元的特定病人群调查。 

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

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

BMPR-IB +快速分离脂肪来源的使用基质细胞在颅骨缺损愈合模型

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

科研

发育生物学

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

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 ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

人肠道 Organoids 上皮屏障通透性的实时测量

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

一种简便有效的成人皮肤组织中人角质形成细胞的分离方法

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is to efficiently revert lineage-primed, conventional human pluripotent stem cells (hPSC) maintained on either feeder-free or feeder-dependent conditions to a na&#239;ve-like pluripotency with improved functionality. This chemical na&#239;ve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3&#946;, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only a tankyrase inhibitor XAV939 (LIF-3i). LIF-3i reverts conventional hPSC to a stable pluripotent state adopting biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. This LIF-3i method requires minimal cell culture manipulation and is highly reproducible in a broad repertoire of human embryonic stem cell (hESC) and transgene-free human induced pluripotent stem cell (hiPSC) lines. The LIF-3i method does not require a re-priming step prior to the differentiation; N-hPSC can be differentiated directly with extremely high efficiencies and maintain karyotypic and epigenomic stabilities (including at imprinted loci). To increase the universality of the method, conventional hPSC are first cultured in the LIF-3i cocktail supplemented with two additional small molecules that potentiate protein kinase A (forskolin) and sonic hedgehog (sHH) (purmorphamine) signaling (LIF-5i). This brief LIF-5i adaptation step significantly enhances the initial clonal expansion of conventional hPSC and permits them to be subsequently na&#239;ve-reverted with LIF-3i alone in bulk quantities, thus obviating the need for picking/subcloning rare N-hPSC colonies later. LIF-5i-stabilized hPSCs are subsequently maintained in LIF-3i alone without the need of anti-apoptotic molecules. Most importantly, LIF-3i reversion markedly improves the functional pluripotency of a broad repertoire of conventional hPSC by decreasing their lineage-primed gene expression and erasing the interline variability of directed differentiation commonly observed amongst independent hPSC lines. Representative characterizations of LIF-3i-reverted N-hPSC are provided, and experimental strategies for functional comparisons of isogenic hPSC in lineage-primed vs. na&#239;ve-like states are outlined.

We present a protocol for efficient, bulk, and rapid chemical reversion of conventional lineage-primed human pluripotent stem cells (hPSC) into an epigenomically-stable na&#239;ve preimplantation epiblast-like pluripotent state. This method results in decreased lineage-primed gene expression and marked improvement in directed multilineage differentiation across a broad repertoire of conventional hPSC lines.

改良多向分化效力的常规人多能干细胞的化学逆转与幼稚样态

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch&#39;s membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models.

从人胚胎干细胞中提取合适的视网膜色素上皮组织的工程移植

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

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 ...