Timing and cell specificity of senescence drives postnatal lung development and injury

Senescence causes age-related diseases and stress-related injury. Paradoxically, it is also essential for organismal development. Whether senescence contributes to lung development or injury in early life remains unclear. This work defines the ontogeny of lung senescence and provides an optimal therapeutic window for mitigating neonatal hyperoxic lung injury by clearing senescence. Newborn C57BL/6J (<12 h old) along with their mothers were exposed to room air or hyperoxia (>95% O2) for 3 days in an A-chamber. The dams were switched between exposed litters and room air control litters every 24 h in 3 days exposed animals to avoid maternal injury. For time points beyond pnd3, the pups were allowed to recover in room air until postnatal da 7 (pnd7). All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Brown University. At pnd7, lung from hyperoxia-exposed mice was dissociated into single cell, and suspensions from three mouse lungs were pooled. The FACS was utilized to sort C12FDG positive cells from the lung suspensions. Single cell encapsulation was performed using the Chromium Single Cell Chip G kit on the 10× Genomics Chromium Controller, and single cell cDNA was sequenced by Genewiz on the Illumina Hiseq (2×150 bp paired-end runs). We also revisited and mined our published scRNA-seq dataset from hyperoxia- or air-exposed mice at pnd7 as controls (PMID: 34417156). Computational analysis on expression of genes involved in senescence and SASP (senescence-associated secretory phenotype) was analyzed using the R package Seurat v 3.2.1.