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A2459 - Integrated Analysis of Alveolar Epithelial Progenitor Cells Identifies Extensive Occult Diversity During Alveolar Regeneration
Author Block: W. Zacharias1, M. Morley2, J. Zepp2, E. Cantu3, E. E. Morrisey2; 1Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Pennsylvania, Philadelphia, PA, United States, 2Department of Medicine, Cardiovascular Institute, University of Pennsylvania, Philadelphia, PA, United States, 3Department of Surgery, University of Pennsylvania, Philadelphia, PA, United States.
RATIONALE: The alveolar region of the mammalian lung is a complex, precisely structured tissue required for gas exchange and provision of oxygen for cellular metabolism. In human disease, the alveolar region is damaged as a result of many insults, including viral infection, pneumonia, and ARDS. During recovery from illness, alveoli must regenerate in order to restore gas exchange. We have recently reported the identification of a novel subset of the mouse and human alveolar type 2 (AT2) cell, the alveolar epithelial progenitor cell (AEP), which is Wnt responsive and regenerates substantial portions of injured alveolar epithelium after influenza injury. A key open question is the major pathways and factor which define the activity of the AEP during regeneration. Here, we describe the results of in depth bioinformatic analysis of this cellular population using epigenomic, transcriptomic, and single cell RNA-seq analysis. METHODS: Mouse AEPs and AT2s were isolated using FACS sorting. Cells were analyzed by deep RNA sequencing, chromatin architecture was analyzed via ATAC-seq, and differential expression and chromatin architecture were identified using standard pipelines. Next, the AEP population was assessed for subsets using single cell RNA sequencing on a 10x genomics platform. Finally, these studies were repeated at multiple time points following influenza injury to identify key pathways driving the regenerative response of AEPs after acute lung injury. Finally, the mouse AEP genetic signature was compared to human AEPs to identify common pathways. RESULTS: The AEP population is distinct at the transcriptomic and epigenomic levels from other AT2 cells despite identical morphological characteristics. Integrated analysis demonstrates a number of AEP-specific primed genes which are differentially activated after influenza-mediated injury. Network and transcription factor binding analysis demonstrate that differential transcriptional pathways activate in AEP vs AT2 cells after injury. Finally, single cell analysis demonstrates that the AEP subpopulation contains additional diversity, with differential expression of progenitor cell and cell surface markers. CONCLUSIONS: Alveolar epithelial progenitor cells are characterized by distinct gene expression and chromatin architecture which prime AEPs for differential activation of progenitor cell genes and pathways following acute lung injury. Single cell analysis suggests that further diversity may lie within the AEP population. These findings demonstrate that detailed analysis of defined cellular populations in the lung can identify unexpected diversity. Understanding this diversity will be a key step in developing an integrated understanding of lung regeneration.