Gene expression analysis during mouse airway basal cell differentiation
To investigate the dynamic gene expression change of tracheal epithelial cells during differentiation, we performed RNA-seq analysis on cells from day 1, day 3, and day 5 of ALI culture, corresponding to the stages of basal cell proliferation, epithelial cell stratification, and early luminal cell specification in vivo. Gene expression and Go analysis revealed that metabolic pathways, such as ATP biosynthesis, cellular respiration, and TCA cycle activity, were highly activated in cells from day 3 of culture compared to those from day 1 of culture.Consistently, the expression of TCA cycle related genes was significantly upregulated during epithelium regeneration, such as Sdhd and Idh2, which encode key enzymes of tricarboxylic acid cycle (TCA cycle); Mpc2 and Pdgb, which function in the mitochondrial pyruvate oxidation process. The activity of these metabolic pathways was further elevated  in cells from day 5 of culture. Overall design: For investigating the dynamic changes in gene expression during tracheal epithelial cell differentiation, we isolated primary airway basal stem cells and cultured them using ALI (air-liquid interface) culture. We collected cells at three time points during the ALI culture: day 1, day 3, and day 5, corresponding to the stages of basal cell proliferation, epithelial cell stratification, and luminal cell specification, respectively, mirroring the in vivo differentiation process.