Intersectional genetic tracing reveals the origin of alveolar stem cells during lung repair and regeneration

Alveolar type 2 (AT2) cells are stem cells of the alveolar epithelia. Previous genetic lineage tracing studies reported multiple cellular origins for AT2 cells after injury. However, conventional lineage tracing based on Cre-loxP has the limitation of non-specific labeling. Thus, the exact contribution of various epithelial cells to the pools of AT2 cells under different conditions remains unclear. Here, we used dual recombinases-mediated intersectional genetic lineage tracing to investigate the cellular origins of AT2 cells during lung homeostasis, injury and repair. In contrast to previous studies, we found AT1 cells were terminally differentiated cells and did not contribute to AT2 cells after lung injury and repair. Distinctive, but simultaneous, labeling of club cells, bronchioalveolar stem cells (BASCs), and AT2 cells revealed the exact contribution of each to AT2 cells after lung injury. Moreover, we found that club cells have the potential to rebuild virtually all alveoli in some severely injured lung regions. Mechanistically, Notch signaling promotes a BASCs-to-AT2 cell transition, but it inhibits club cell-to-AT2 cell conversion during lung repair. This intersectional genetic lineage tracing strategy with enhanced precision allowed us to elucidate the physiological role of AT1, club, BASCs, and AT2 cells to alveolar regeneration after injury. tdT+, ZsG+, tdT+ZsG+ lung epithelial cell of the Scgb1a1-CreER;Sftpc-DreER;R26-TLR mice at 4 weeks after bleomycin injury were isolated by Fluorescence-activated cell sorting (FACS) and analyzed using scRNAseq.