PROJECT SUMMARY/ABSTRACT Recapitulation of normal lung function following a severe acute injury implies an inherent regenerative ability of the lung. However, sources and relative regenerative capacities of lung epithelial stem/progenitor cells remain unclear, especially in the human lung. Depending on the injury type and severity, several distinct progenitors are activated and respond by proliferating and differentiating to aid in near complete recovery. Both airway and alveolar stem/progenitor cells are activated and contribute to alveolar repair following severe injuries such as influenza or bleomycin. To this end, recent studies from our lab have uncovered an airway epithelial progenitor cell marked by elevated levels of Major Histocompatibility Complex (MHC) Class I protein, H2-K1. Despite having a transcriptome highly similar to the mature club cells, the H2-K1high progenitors, unlike mature club cells, selectively proliferate post injury and aid in improved oxygenation in injured mice after orthotopic transplantation. However, mechanisms underlying early and selective activation of these progenitors remain unknown. In addition, there are several more aspects of distal lung regeneration that are yet unclear. The chief among them is whether the distal epithelial progenitor hierarchy that we observe in mouse lungs is maintained in distal human lungs. The distal human lung airways have a higher proportion of basal cells and have more heterogeneous secretory cell populations than the mouse airway epithelium. Furthermore, the human type 2 alveolar epithelial cells (AEC2s) have remarkable in vitro proliferative and regenerative capacity. Therefore, there is an unmet need to understand the identity and regulation of distal human airway and alveolar progenitors. To this end, our preliminary data show that a distal human secretory subpopulation that is analogous to the mouse H2-K1high club-like progenitors can give rise to AEC2s in vitro. Conversely, we have uncovered a novel and unexpected ability of mature human AEC2s to differentiate towards airway lineages in vitro and in vivo, suggesting at least two sources of distal epithelial regeneration in the human. Therefore, it is critical to clarify the identity and characteristics of both distal mouse and human epithelial progenitors primed for alveolar repair. This proposal seeks to answer these questions through three aims: 1) Determine the mechanisms underlying activation of H2-K1high progenitors in alveolar repair. 2) To identify distal secretory cells as a source of alveolar cells post injury in the distal human lung. 3) Determine whether a subpopulation of mature human AEC2s has reversible bi-directional potential to differentiate into alveolar basal cells. These aims will utilize single cell mRNA and open chromatin sequencing, in vitro and in vivo manipulation of proposed signaling pathways, and orthotopic transplantation of progenitor cells to clarify the epithelial stem/progenitor cell hierar...