Induction Of Alveolar Epithelial Type II-Like Cells By Direct Reprogramming

Alveolar type II cells (AT2s) are essential for the lung’s regenerative capacity following injury, and AT2 disorders are associated with multiple lung diseases. These cells are complex to maintain in culture, and the lack of a model to study AT2s remains a major roadblock in understanding their self-renewal and identity. Direct reprogramming by transcription factor (TF) overexpression allows for the fast
generation of specific cell types without transitioning through a pluripotent state. There are, however, no reports of pulmonary cell induction by cell reprogramming in the respiratory system, keeping this lineage largely unexplored despite its therapeutic potential. Therefore, we hypothesized that overexpression of AT2-specific TFs would allow for the direct conversion of fibroblasts to AT2-like cells.
In this study, we validated a direct reprogramming approach to screen TFs on mouse embryonic fibroblasts harboring the AT2-specific surfactant protein C (SPC) reporter. We identified 28 TFs based on gene enrichment, loss-of-function studies, and bioinformatic analysis as candidates to induce AT2 cell fate. To mimic the distal lung environment, we isolated AT2s from murine lungs and established alveolospheres capable of proliferating and maintaining of surface marker expression. In both 3D spheroids and 2D in vitro cultures, combined overexpression of the 28 TFs was sufficient to activate the SPC reporter. We then refined the combination to 5 AT2-restricted TFs, FOXA1, FOXA2, CEBPA, NKX2.1, and ETV5, and confirmed their capacity to activate the SPC reporter. Removal of FOXA1,
FOXA2 or CEBPA from the combination reduced reporter activation suggesting their requirement to induce AT2s. We suggest a network where FOXA1 and FOXA2 open the chromatin for CEBPA which recruits NKX2.1, promoting AT2 identity. Generating AT2-like cells by direct reprogramming not only gives insight into the transcriptional regulators of their identity, but also the constant supply of AT2s is an attractive potential strategy for clinical therapies.