Transcriptional profiling of CD44hi AT2 cells under homeostasis and after lung injury [I]

The epithelium of alveoli is composed of alveoli type I cells (AT1) that cover ~95% of the surface area and alveoli type II cells (AT2) that secret surfactant as well as function as adult stem cells. To characterize a subgroup of AT2 that express higher levels of hyaluronan receptor CD44 (the CD44high, or CD44hi AT2) and behave as alveoli epithelial stem cells during steady-state homeostasis, we used RNA-sequencing to compare the expression profiles of these cells with the bulk CD44low (or CD44lo ) AT2. CD44hi AT2 showed gene signatures that indicate partial de-differentiation and a higher potential to proliferate compared with CD44lo AT2. At steady-state conditions, CD44hi AT2 also showed higher expression of genes activated by NF?B mediated inflammatory responses and transcriptome signatures that resemble early-stage Kras-induced adenocarcinoma. Furthermore, these cells were more responsive to KrasG12D induction both in 3D tumor organoid culture and in mouse lung cancer cell transplantation models. In summary, CD44hi AT2 plays an essential role in the homeostatic maintenance of the alveolar epithelium and tumor initiation. Overall design: The goal of this study was to compare the gene expression profiles of CD44hi alveolar type 2 (AT2) cells versus CD44lo AT2 cells. CD44hi and CD44lo AT2 cells were sorted using fluorescence activated cell sorter (FACS) from 8 female SpC-creER/Rosa-mTmG mice at 6-8 weeks of age. Total RNA profiles were generated by deep sequencing using Illumina HiSeq4000. Three biological replicates were included for each cell type.