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

 描述（由申请人提供）：尽管肺泡功能对于整体健康至关重要，并且婴儿和成人中引起急性和慢性呼吸衰竭的疾病具有显著的发病率和死亡率，但对于肺泡在整个生命过程中如何维持和修复自身知之甚少，或至少达成一致。我们最近使用克隆分析来绘制个体AT 2细胞在体内的整个生命周期，并鉴定了一个罕见的亚群 表现出干细胞特性，包括多能性、自我更新和整个过程中的活性。 寿命在这里，我们建议建立在这个基础工作，回答重要的悬而未决的问题，肺泡维护和干细胞调节的细胞和分子机制。我们的方法采用了一些最新颖和最前沿的遗传和单细胞基因组技术来实现雄心勃勃的目标。具体目标是：A）通过Aa）定量AT 2细胞的核标记保留和Ab）测量老化过程中AT 2细胞克隆命运动力学来确定体内生理性肺泡上皮更新的程度和细胞机制，B）凭经验表征AT 2细胞群内的分子异质性并鉴定区分AT 2干细胞的标志物Ba）使用AT 2细胞的单细胞RNA测序和B B）通过大量细胞RNA测序，对标记稀释的与标记保留的AT 2细胞（在目的Aa中鉴定）进行测序，C）以直接测试经由EGFR的信号传导是否在体内调节AT 2干细胞功能，以及EGFR缺失的肺上皮细胞的重复消融是否导致肺泡扩大或肺纤维化。 通过采用尖端技术和方法来了解肺部的气体交换表面如何在整个生命过程中保持健康，以及如何制造新的细胞来取代那些因受伤而丢失的细胞，我们正在为开发医疗或基于细胞的治疗方法铺平道路。

 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.