Regeneration of distal lung epithelium via epithelial-mesenchymal co-culture and modulation of Wnt signaling

项目总结/摘要 终末期肺病的唯一治愈性治疗选择是移植，然而，这种疗法是不可行的。 受局限性的困扰：那些有幸接受捐赠肺的人将面临终身的 免疫抑制，十年生存率低于30%。然而，证据正在积累， 肺中许多上皮细胞和间充质细胞类型促进修复的先天潜力， 损伤后的再生，甚至在整个组织水平上。本项目建议使用全肺工程 研究上皮-间充质相互作用的作用的策略，沿着关键的发育 信号通路，促进肺泡上皮细胞分化和肺泡单位形成。具体而言是 这项计划的首要目标是研究肺泡上皮II型细胞-成纤维细胞相互作用是否 和典型的Wnt-β-连环蛋白可溶性效应物可以在体外以三维（3-D）仿生 用改进的表面活性剂产生和上皮屏障工程化远端肺上皮的大鼠肺模型 功能本项目的第一个目的是重点描述典型的Wnt信号转导模式， 天然肺以及分离的II型细胞（此处为RTII+细胞，通过大鼠II型标记物RTII-70选择） 和成纤维细胞（FB）。在肺损伤的晚期阶段， 发育，在此期间成熟的II型细胞产生表面活性剂，分化成I型细胞后代，和 成为功能性肺泡的一部分，可能会告知肺泡修复过程。第二个目标是 评价典型Wnt可溶性效应物对RTII+细胞的表型和分化的影响， 有和没有FB共培养，使用标准的2-D和3-D类器官培养系统。它试图确定 根据目标1中确定的时间尺度，这些效应器的时间调制是否可能 用于促进体外肺泡上皮成熟。最后，第三个目标着眼于增量 脱细胞肺细胞外基质对RTII+细胞成熟的影响，有或没有FB共培养。 在这最后一个目的中，RTII+细胞和FB将被接种到无细胞大鼠肺支架中，并安装到 仿生生物反应器该系统允许在整个组织水平上评估RTII+细胞功能， 肺顺应性和上皮屏障功能。总的来说，该项目具有重要意义， 了解促进能够产气的成熟肺泡上皮形成的因素 交换，以及发展促进患者内源性肺再生的策略 患有慢性肺病。

PROJECT SUMMARY/ABSTRACT The only curative treatment option for end-stage lung disease is transplantation, however, this therapy is plagued by limitations: those who are fortunate enough to receive a donor lung face a lifetime of immunosuppression and a ten-year survival rate of less than 30%. Nevertheless, evidence is accumulating for the innate potential of numerous epithelial and mesenchymal cell types in the lung to promote repair and regeneration after injury, even at the whole tissue level. This project proposes to use whole lung engineering strategies to investigate the role of epithelial-mesenchymal interactions, along with a key developmental signaling pathway, in promoting alveolar epithelial differentiation and alveolar unit formation. Specifically, the overarching goal of this proposal is to investigate whether alveolar epithelial type II cell-fibroblast interactions and canonical Wnt-β-catenin soluble effectors can be modulated in vitro in a 3-dimensional (3-D) biomimetic rat lung model to engineer a distal lung epithelium with improved surfactant production and epithelial barrier function. The first Aim of this project focuses on characterizing the pattern of canonical Wnt signaling in the native lung, as well as in isolated type II cells (here RTII+ cells, selected for by the rat type II marker RTII-70) and fibroblasts (FBs), during alveologenesis. Patterns of Wnt signaling during this late stage of lung development, during which mature type II cells produce surfactant, differentiate into type I cell progeny, and become part of the functional alveolus, will likely inform alveolar repair processes. The second Aim focuses on evaluating the effects of canonical Wnt soluble effectors on the phenotype and differentiation of RTII+ cells, with and without FB co-culture, using standard 2-D and 3-D organoid culture systems. It seeks to determine whether temporal modulation of these effectors, according to a timescale determined in Aim 1, might be utilized to promote maturation of alveolar epithelium in vitro. Finally, the third aim looks at the incremental effect of decellularized lung extracellular matrix on the maturation of RTII+ cells, with or without FB co-culture. In this last Aim, RTII+ cells and FBs will be seeded into an acellular rat lung scaffold and mounted into a biomimetic bioreactor. This system allows for assessment of RTII+ cell function at the whole tissue level, in terms of lung compliance and epithelial barrier function. Overall, this project has important implications, both for understanding the factors that promote the formation of a mature alveolar epithelium capable of gas exchange, as well as for the development of strategies to promote lung regeneration endogenously in patients suffering from chronic lung disease.