Heterogeneity and bias of lineage negative progenitors in lung epithelial repair

 描述(由申请人提供)：项目摘要/摘要远端肺上皮发挥着气体交换的关键生物学功能，是阻止有害的空气传播物质进入人体的屏障。为了维持这些功能，肺有广泛的能力对损伤作出反应，并再生丢失或受损的细胞。目前的模型认为，上皮修复可以归因于细胞表达成熟的谱系标记。相反，我们最近确定了存在于正常肺远端的罕见的谱系阴性上皮干/祖细胞(LNEP)的再生作用。LNN原位移植显示出显著的直接分化为2型肺泡细胞和远端呼吸道细胞的能力。然而，在严重的流感损伤后，LNN激活了ΔNp63和细胞角蛋白5(KRT5)重塑程序，随后细胞增殖并向严重损伤的肺泡区迁移。这一途径在很大程度上导致了异常的上皮屏障，而不是II型细胞分化。发育不良的肺泡上皮囊肿是持续过度的Notch信号的一部分，使人联想到纤维化的人肺中的“蜂窝状”囊肿。有趣的是，移植全部LNEP群体显示出细支气管性和肺泡性分化，而表达低水平Krt5的细胞严重偏向于呼吸道样囊性扩张。此外，单细胞转录本的主成分分析表明细胞有多个亚群。这些观察表明，LNEP种群内的异质性是血统启动的亚种群倾向于特定命运的表现。这项建议旨在解决这种异质性的生物学意义，具体目的如下：1)测试小鼠LNN是否由无偏见和启动的干细胞/祖细胞群体组成。2)确定LNEP的人类等价物(S)，并探讨其在人类肺部疾病中的作用。3)定义调节无偏LNEP亚群和启动LNEP亚群的呼吸道和肺泡再生反应的不同信号线索。这些目标将利用单细胞RNA-Seq、谱系追踪、器官培养和原位细胞移植来询问细胞异质性和高度可驯化的再生动力学。这些研究将为开发肺上皮损伤反应的定量和预测模型奠定基础。这样的模型将为未来操纵再生机制以实现更好的疾病结果的努力提供信息。

 DESCRIPTION (provided by applicant): Project Summary/Abstract The epithelium of the distal lung performs the critical biological function of gas exchange and serves as a barrier to prevent access of deleterious airborne agents into the body. In order to maintain these functions the lung has an extensive ability to respond to injury and regenerate lost or damaged cells. Current models posit that epithelial repair can be attributed to cells expressing mature lineage markers. By contrast, we have recently defined the regenerative role of rare lineage-negative epithelial stem/progenitor (LNEP) cells present within normal distal lung. Orthotopic transplantation of LNEPs reveals a striking capacity for direct differentiation into type 2 pneumocytes and distal airway cells. However, after severe influenza injury, LNEPs activate a ΔNp63 and cytokeratin 5 (Krt5) remodeling program whereupon cells proliferate and migrate toward heavily injured alveolar areas. This pathway largely results in an abnormal epithelial barrier rather than type II cell differentiation. The dysplastic alveolar epithelial cysts result i part from ongoing excessive Notch signaling and are reminiscent of "honeycombing" cysts in fibrotic human lungs. Interestingly, transplantation of the total LNEP population demonstrates both bronchiolar and alveolar differentiation, whereas cells expressing low levels of Krt5 are heavily biased toward airway-like cystic expansion. Furthermore, principle component analysis of single cell transcriptomes indicates multiple subpopulations of cells. These observations suggest that heterogeneity within the LNEP population is a manifestation of lineage-primed subpopulations predisposed towards particular fates. This proposal seeks to address the biological significance of this heterogeneity with the following specific aims: 1) Test whether murine LNEPs consist of both unbiased and primed stem/progenitor cell populations. 2) Identify the human equivalent(s) of LNEPs and explore their role in human lung disease. 3) Define distinct signaling cues that modulate airway and alveolar regenerative responses of unbiased and primed LNEP subpopulations. These aims will utilize single cell RNA-Seq, lineage tracing, organoid culture, and orthotopic cell transplantation to interrogate cellular heterogeneity and regenerative dynamics with a high degree of tractability. These studies will lay the foundation for development of a quantitative and predictive model of lung epithelial injury responses. Such a model will inform future efforts to manipulate regenerative mechanisms in order to achieve better disease outcomes.