Gene Editing Human Lung Cells to Better Understand Distal Lung Progenitors

The human lung contains two major types of basal cells: large airway basal cells (LABCs) and distal airway basal cells (DABCs). LABS are found in proximal airways of the lung, such as the trachea or large bronchi, while DABCs are found in the periphery, alveolar regions of the lung. Using known single cell data, the TP63 transcription factor is expressed at different levels in LABCs and DABCs. TP63 transcription factor is important for lung and epithelial cell development. Data shows that LABCs express high levels of TP63 and DABCs express low levels of TP63. Using an ORF gene editing plasmid with red fluorescence, TP63 levels were overexpressed in DABCs. Cells that contained mCherry, red fluorescent protein, were sorted using Fluorescence-Activated cell sorting (FACS). FACS allows us to observe the success of reprogramming distal airway cells as they become large by isolating successfully infected cells, which emit a red fluorescent color. Red fluorescent cells are then differentiated via ALI to create basal cell progeny as a second checkpoint of a successful reprogramming. Future steps include obtaining three samples to successfully differentiate and analyze the modified cells by staining them for known large airway cell markers.