Single Cell Profiling and In Vivo Cellular Interrogation of Alveolar Stem Cells

 描述(由申请人提供)：尽管肺泡功能对整体健康至关重要，而且导致婴儿和成人急性和慢性呼吸衰竭的疾病的发病率和死亡率很高，但关于肺泡如何在一生中维持和修复自己，人们知之甚少，或至少达成了共识。我们最近使用克隆分析绘制了体内单个AT2细胞的整个生命周期图，并确定了一个罕见的亚群 表现出干细胞特性，包括多潜能，自我更新和整个过程中的活性 生命的长短。在这里，我们建议在这项基础性工作的基础上，回答有关肺泡维持和干细胞调节的细胞和分子机制的重要悬而未决的问题。我们的方法采用了一些最新颖和最尖端的遗传和单细胞基因组技术来实现这些雄心勃勃的目标。具体目的是：a)通过aa)量化AT2细胞的核标记保留和ab)测量老化过程中at2细胞克隆命运的动态，来确定体内生理性肺泡上皮细胞更新的程度和细胞机制，b)经验性地表征AT2细胞群体中的分子异质性，并识别区分at2干细胞的标记bb)使用AT2细胞的单细胞RNA测序和bb)通过标记稀释和标记保留的AT2细胞的批量RNA测序(在目标aa中确定)，C)直接测试体内EGFR信号是否调节AT2干细胞的功能，以及重复消融EGFR缺失的肺上皮细胞是否会导致肺泡扩大或肺纤维化。通过使用尖端技术和方法了解肺的气体交换表面如何在一生中保持健康，以及如何制造新细胞来取代因损伤而失去的细胞，我们正在为开发医疗或基于细胞的治疗铺平道路，以治疗毁灭性和致命的肺部疾病，如肺气肿和婴儿呼吸窘迫综合征。

 DESCRIPTION (provided by applicant): Despite the critical importance of alveolar function for overall health and the significant morbidity and mortality attributable to diseases causing acute and chronic respiratory failure in infants and adults, little is known, or at least agreed upon, about how alveoli maintain and repair themselves throughout life. We recently used clonal analysis to map the entire life cycle of individual AT2 cells in vivo, and identified a rare subset that exhibited stem cell properties, including multi-potency, self-renewal, and activity throughout the lifespan. Here, we propose to build upon this foundational work to answer important outstanding questions about the cellular and molecular mechanisms of alveolar maintenance and stem cell regulation. Our approach employs some of the most novel and cutting-edge genetic and single cell genomic technology to accomplish the ambitious aims. Specific Aims are: A) to determine the extent and cellular mechanism of physiologic alveolar epithelial renewal in vivo by Aa) quantifying nuclear label retention by AT2 cells and Ab) measuring AT2 cell clonal fate dynamics during ageing, B) to empirically characterize molecular heterogeneity within the AT2 cell population and identify markers that distinguish AT2 stem cells Ba) using single cell RNA-sequencing of AT2 cells and Bb) by bulk cell RNA-sequencing of label-diluting versus label-retaining AT2 cells (identified in Aim Aa), C) to directly test if signaling via EGFR regulats AT2 stem cell function in vivo and whether repetitive ablation of EGFR-deleted lung epithelial cells results in alveolar enlargement or pulmonary fibrosis. By employing cutting edge technology and approaches to understand how the gas exchange surface of the lungs remain healthy throughout life, and how new cells are made to replace those that are lost from injury, we are paving the way towards developing medical or cell-based treatments to treat devastating and fatal lung diseases like emphysema and infant respiratory distress syndrome.