Name: conditional reprogramming of pediatric human esophageal epithelial cells for use in tissue engineering and disease investigation
Uploaded: 2017-03-22T13:00:00
Description: 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

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?...

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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...

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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			<td height="21" style="height:21px;width:385px;">ROCK Inhibitor (Y-27632)</td>
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			<td height="21" style="height:21px;width:385px;">F-12 Medium&#160;</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">11765054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Fetal Bovine Serum</td>
			<td style="width:172px;">Denville Scientific</td>
			<td style="width:119px;">FB5001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Dispase</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">17105041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">0.05% Trypsin-EDTA</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">25300062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">0.25% Trypsin-EDTA</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">25200072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">100mm Dishes</td>
			<td style="width:172px;">Denville Scientific</td>
			<td style="width:119px;">T1110-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">150mm Dishes</td>
			<td style="width:172px;">Denville Scientific</td>
			<td>T1115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">50ml Conicals</td>
			<td style="width:172px;">Denville Scientific</td>
			<td style="width:119px;">&#160; C1062-9&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Phosphate Buffered Saline Tablets</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td style="width:119px;">BP2944-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">5ml Pipettes</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td>1367811D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">10ml Pipettes</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td>1367811E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">25ml Pipettes</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td>1367811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">9&#34; Pasteur Pipettes</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td style="width:119px;">13-678-20D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">NIH 3T3 Cells</td>
			<td style="width:172px;">ATCC</td>
			<td style="width:119px;">CRL1658</td>
			<td></td>
		</tr>
	</tbody>
</table>

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?...

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 ...

Research

JoVE Journal

Developmental Biology

Live Imaging of Mouse Secondary Palate Fusion

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 ...

科研

发育生物学

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

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

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

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

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

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

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

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a NaÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¯ve-like State with Improved Multilineage Differentiation Potency

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

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

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 ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...