Engineering human iPS cells into an airway epithelium capable of ion transport

描述(申请人提供)：人类的呼吸道由高度专业化的假复层上皮构成，它是抵御吸入病原体的第一道防线。这种组织包含祖细胞和分化的细胞类型，有助于粘液纤毛清除和损伤反应中的上皮修复。上皮的损伤可能导致慢性炎症、重塑和显著损害防御功能，这些都是许多大呼吸道疾病的特征，包括囊性纤维化(CF)、慢性阻塞性肺疾病(COPD)、哮喘和癌症。尽管进行了几十年的研究，但这些呼吸道疾病的适当临床解决方案仍然难以找到。这项研究的长期目标是从人诱导的多能干细胞(IPS)中开发出一种生理反应和临床适用的组织工程化气道。该项目将利用最近发表的从人类干细胞衍生出肺上皮的技术来生产一种假复层上皮，可以在临床相关的基质上生长，用作外科组织移植。本申请提供的初步数据显示，我们已经能够将人iPS细胞分化为表达p63/CK5的气道基底细胞样祖细胞。我们建议使用额外的分化过程来产生完全分化的呼吸道上皮细胞，能够正常的离子转运功能和自分泌信号分子的调节。为了实现这一目标，提出了以下目标：目的1)将人诱导性多能干细胞分化为可用于在去细胞的呼吸道基质上生成假复层上皮的上皮祖细胞；目的2)鉴定参与粘液纤毛清除的关键离子转运蛋白的表达和亚细胞定位，并评估已分化的假复层上皮的跨皮转运特性；目的3)鉴定已分化的上皮细胞表达的关键受体介导的信号通路，这些信号通路对于正常调节气道上皮的离子转运功能是必不可少的。这些研究的结果将为从人iPS细胞中开发临床适用的组织工程化呼吸道移植物提供重要的见解。此外，这些目标的成功完成不仅将对肺组织工程产生影响，还将对其他器官系统产生影响，在这些系统中，上皮细胞的分化和运输功能对临床应用至关重要。

DESCRIPTION (provided by applicant): The human airways are lined by a highly specialized pseudostratified epithelium, which acts as the first line of defense against inhaled pathogens. This tissue contains both progenitor and differentiated cell types that facilitate mucociliary clearance and epithelial repair in response to injury. Damage to the epithelium may lead to chronic inflammation, remodeling and markedly compromised defense function that are hallmarks of many large airway diseases including cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), asthma and cancer. Despite decades of research, an adequate clinical solution to these airway diseases remains elusive. The long term goal of the research described in this proposal is to develop a physiologically responsive and clinically applicable tissue-engineered airway from human induced pluripotent stem (iPS) cells. This project will utilize recently published techniques on derivation of pulmonary epithelium from human stem cells to produce a pseudostratified epithelium that can be grown on clinically relevant substrates for use as a surgical tissue graft. Preliminary data presented in this application shows that we have been able to differentiate human iPS cells into p63+/CK5+ expressing airway basal cell-like progenitor cells. We propose to employ additional differentiation procedures to produce a fully differentiated airway epithelium capable of normal ion transport function and regulation by autocrine signaling molecules. To accomplish this goal, the following aims are proposed: Aim 1) Differentiate human iPS cells into airway epithelial progenitor cells that can be used for generating a pseudostratified epithelium on decellularized airway substrates; Aim 2) Characterize the expression and subcellular localization of key ion transport proteins involved in mucociliary clearance and assess the transepithelial transport properties of the differentiated pseudostratified epithelium; Aim 3) Identify key receptor-mediated signaling pathways expressed by differentiated epithelial cells that are known to be essential for normal regulation of ion transport function of the airway epithelium. The results of these studies will provide important insight regarding the development of a clinically applicable tissue- engineered airway graft from human iPS cells. Moreover, successful completion of the aims will have an impact not only on pulmonary tissue-engineering but also on other organ systems where epithelial cell differentiation and transport function are essential for clinical applications.