Interferon-mediated in vitro and in vivo Regulation of Airway Progenitor Cells in Regeneration of Functional Alveoli

项目总结 严重急性损伤后正常肺功能的重现意味着肺固有的能力 再生，但肺上皮干/祖细胞的来源和相对再生能力仍然存在 不清楚。尽管几乎所有分化的上皮细胞都可以重新进入细胞周期，但越来越多的证据表明 表明远端呼吸道细胞亚群在其扩张、迁移、 并重建肺泡屏障。我们实验室最近的研究发现了Sox2pos/p63neg上皮细胞 具有再生潜能和向肺泡命运分化能力的祖细胞(EP)。此外，还有许多 其他研究已经描述了一部分Scgb1a1标记的细胞在体外和体内也具有再生能力 容量。然而，他们的确切身份、地点和监管仍不清楚。为了进一步定义环境保护计划，我们 对富含EPs的肺上皮细胞进行了大规模的单细胞测序，发现 静止的EP人口。这些细胞在转录水平上与胚胎Sox9pos肺芽细胞相似，具有 干扰素(干扰素)调节的基因表达增加。有趣的是，这些细胞还表达一些 与细胞周期相关的基因和关键的周期蛋白抑制剂CDKN1A(P21)和CDKN1c(P57)，提示它们 在有利的条件下增殖的能力。事实上，IFNpos细胞代表了 不同体外实验条件下的呼吸道上皮细胞(包括Scgb1a1标记细胞)。重要的是，这些 细胞也有向基底层(以细胞角蛋白5的表达为标志)或肺泡分化的能力 (以表面活性蛋白C表达为标志)细胞。因此，我的初步数据已经确定了再生的 呼吸道上皮的一部分。然而，它们在体内的调节和它们在功能上的贡献 肺上皮损伤后的再生仍不清楚。为此，我们假设低水平的 结构性干扰素信号调节和维持动员再生的稀有呼吸道EPs池 损伤后的肺泡上皮。我们将在以下两个目标中检验这一假设：1)确定 干扰素信号在维持未损伤小鼠肺组织静止期p63neg EPs中的作用2)确定 严重创伤后p63neg EPs体内反应中的干扰素信号。我们将利用鼠标模型来 阻断损伤小鼠活跃的干扰素信号，研究其在维持EPs中的作用。同样，条件性呼吸道 损伤后STAT1上皮细胞的缺失将决定干扰素/STAT1信号在损伤反应中的作用。 最后，将外源性扩增的EPs移植到受伤的肺中，并测量其功能恢复情况。 通过氧合和肺功能。这个项目有很大的潜力来阐明信号通路。 调节静息状态下肺上皮祖细胞的反应并确定它们的潜力 上皮祖细胞在帮助损伤后肺再生中的作用。此外，小说研究涉及到 移植外源性扩增的祖细胞将为其整体再生潜力奠定基础 作为未来细胞疗法的治疗剂。

PROJECT SUMMARY Recapitulation of normal lung function following a severe acute injury implies an inherent capability of lung to regenerate but the sources and relative regenerative capacities of lung epithelial stem/progenitor cells remain unclear. Although virtually all differentiated epithelial lineages can re-enter the cell cycle, increasing evidence indicates that subpopulations of distal airway cells may be particularly robust in their capacity to expand, migrate, and reconstitute alveolar barriers. Recent studies from our lab have uncovered Sox2pos/p63neg epithelial progenitors (EPs) with regenerative potential and ability to differentiate towards alveolar fate. Further, numerous other studies have described a fraction of Scgb1a1-labeled cells to also have in vitro and in vivo regenerative capacity. However, their exact identities, location, and regulation remains unclear. To further define the EPs, we performed large scale single cell sequencing of flow-sorted lung epithelial cells enriched in EPs, which identified a quiescent EP population. These cells, with a transcriptomic similarity to embryonic Sox9pos lung bud cells, have increased expression of interferon (IFN)-regulated genes. Interestingly, these cells also express a number of genes associated with cell cycle and key cyclin inhibitors Cdkn1a (p21) and Cdkn1c (p57), suggestive of their ability to proliferate under favorable conditions. Indeed, IFNpos cells represent the regenerative fraction of the airway epithelium (including that of Scgb1a1-labeled cells) in various in vitro conditions tested. Importantly, these cells also have the ability to differentiate towards either basal (marked by Cytokeratin 5 expression) or alveolar (marked by surfactant protein C expression) cells. Thus, my preliminary data have identified the regenerative fraction of the airway epithelium. However, their regulation in vivo and their ability to functionally contribute to lung epithelial regeneration post injury remain unknown. To this end, we hypothesize that low levels of constitutive IFN signaling regulates and maintains a pool of rare airway EPs that mobilize to regenerate alveolar epithelium following injury. We will test this hypothesis in following two aims: 1) To determine the role of IFN signaling in maintenance of quiescent p63neg EPs in uninjured mouse lung. 2) Determine the role of IFN signaling in in vivo responses of p63neg EPs following major injury. We will leverage mouse models to abrogate active IFN signaling in injured mice to study its role in maintenance of EPs. Likewise, conditional airway epithelial deletion of Stat1 followed by injury will determine the role of IFN/Stat1 signaling in injury response. Finally, exogenously expanded EPs will be transplanted in injured lungs and functional recovery will be measured by oxygenation and lung function. This project has a significant potential to clarify the signaling pathways regulating lung epithelial progenitor cell response during quiescence and determine the potential of these epithelial progenitors in aiding lung regeneration following an injury. Further, the novel studies involving transplantation of exogenously expanded progenitors will lay foundation for their overall regenerative potential as a therapeutic agent for future cell based therapies.