Gene expression analysis of basal cells expanded with UK-5099 or dual SMAD inhibitors
Dual SMAD inhibitors (TGF-beta inhibitor A-8301 and BMP inhibitor DMH-1) were previously reported to promote long-term expansion of basal cells. Since UK-5099 showed a comparable effect to dual SMAD inhibitors in promoting basal cells expansion, we performed RNA-seq  to determine if cells expanded by these two methods exhibit a similar transcriptional profile. The Pierson correlation test indicated that cells expanded by UK5099 displayed a transcriptional profile similar to those expanded with dual-SMAD inhibitors. The expression levels of canonical basal cell marker genes, such as Krt5 and p63, were comparable in cells expanded using both methods, as well as genes related to the TCA cycle and glycolysis. Interestingly, despite the similarity in the expression of canonical basal cell markers, cells expanded via UK-5099 exhibited higher activity in differentiation-related signaling pathways, such as the Hippo and TGF-ß signaling pathways.This observation suggests that mitochondrial pyruvate transport is a critical metabolic checkpoint for basal cell differentiation. Overall design: Primary mouse airway basal cells were isolated and then cultured with UK-5099 and dual SMAD inhibitors for 4 days