Identification of single senescent cells from precision cut lung slices ex vivo

Introduction: Chronic lung diseases (CLD) are linked to aging and are characterized by different hallmarks of aging, such as cellular senescence. Within CLD, several cell types have been shown to become senescent, however, it is unknown whether these are disease-specific, and moreover, potential disease- and/or cell-specific senescence inducers, remain unexplored. WNT/β-catenin is an oncogenic pathway increased in pulmonary fibrosis and has been reported to induce senescence. Here, we investigate which cells are affected by WNT/β-catenin induced senescence in the lung. Methods: Precision-cut lung slices (PCLS) were treated with WNT stimulators for up to 7 days. PCLS were characterized by qPCR, Immunofluorescence and Flow Cytometry directly after slicing or after 24h, 5 days or 7 days of culture. Results: WNT-stimulation in PCLS led to significant induction of WNT target genes, such as Axin2. After 7 days of treatment, cellular senescence was assessed by p16 and p21 gene expression and Immunofluorescence. In particular, we found strong p21 staining in lung epithelial cells in PCLS. In order to characterize single cells from PCLS, we established a protocol to isolate single cells from PCLS for subsequent flow cytometry and sorting. Single cell suspensions generated from PCLS showed similar proportions of epithelial (EpCam+), inflammatory (CD45+), endothelial (CD31+) and mesenchymal (CD90+) cells as compared to the native lung. Conclusions and Outlook: Cellular senescence can be induced in PCLS ex vivo and PCLS further allow mechanistic analysis of single cell-specific senescence. Ongoing analysis of single cell sequencing data from human aging and CLDs will be utilized to validate cell- / disease-specific senescence.