Spatiotemporal coordination of stem cell behavior following alveolar injury

Abstract

SUMMARYTissue repair requires a highly coordinated cellular response to injury. In the lung, alveolar type 2 (AT2) cells act as stem cells and can replace both themselves and alveolar type 1 cells (AT1); however, the complex orchestration of AT2 stem cell activity following lung injury is poorly understood owing to the inability of tracking individual stem cells and their dynamic behavior over time. Here, we apply live time lapse imaging to ex vivo mouse precision cut lung slice (PCLS) culture and in vivo mouse lung to track individual GFP-labeled AT2 cells following induction of alveolar injury by bleomycin. We observe highly dynamic movement of AT2 cells, including migration within and between alveoli. To map the dynamic evolution of AT2 cell behavior, we introduce Live Cell Encoder (LCE-PHATE), a novel method for converting static snapshots from time lapse imaging into single points representative of entire, dynamic cellular trajectories. Applying LCE-PHATE, we observe the emergence of at least three distinct morphokinetic AT2 cell states associated with AT2 stem cell injury response. Finally, small molecule-based inhibition of Rho-associated protein kinase (ROCK) pathway significantly reduced motility of AT2 stem cells following injury and reduced expression of Krt8, a marker of intermediate progenitor cells. Together, our results uncover motility of alveolar stem cells as a new injury response mechanism in the lung and reveal properties of stem cell motility at high cellular resolution.