Abstract: Abstract: Objective To evaluate the effect of PF-4708671, an inhibitor of mTOR downstream element ribosomal protein S6 kinase 1 (S6K1), on the proliferation and differentiation of mouse airway stem/progenitor cells. Methods A total of ten C57BL/6 mice were included in the present study. Mouse airway stem cells (vClub) and progenitor cells (Club) were isolated from lungs by fluorescent activated cell sorting. Organoid culture model was used to culture vClub cells, Club cells and supportive fibroblast MLg cells in transwells respectively. Stem/progenitor cells were maintained with PF-4708671 (0, 4, 20 and 100 nmol / L). Organoid cultures were imaged with inverted microscopy at day 8 after seeding. Stem cell-derived colonies were counted. Real-time PCR was conducted to analyze mRNA expression of S6K1 kinase gene Rps6kb1, Club cell marker cytochrome P450 family 2 subfamily, polypeptide 2 (Cyp2f2), ciliated cell marker acetylated tubulin (Act) and Forkhead box J1 (Foxj1), goblet cell marker Chloride channel accessory 3 (Clca3) and Forkhead box A3 (Foxa3) at day 10 after seeding. Results The number of colonies generated by vClub cells showed no difference between the PF-4708671 treatment groups (P＞0.05). Also, numbers of colonies generated by Club cells were significantly decreased in PF-4708671 treatment groups (4, 20 and 100 nmol/L) compared with those of 0 nmol/L group (P＜0.05). Different concentrations of PF- 4708671 showed no significant effects on the differentiation of vClub into Club cells. There was no significant difference in mRNA expression level between characteristic molecular Cyp2f2. PF-4708671 showed no significant effect on the differentiation ability of Club cells into ciliated cells or goblet cells. There were no significant differences in expression levels of ciliated cells Act, Fox j1, Clca3 and Foxa3 between the four groups (P＞0.05). Conclusion mTOR / S6K1 signaling promotes the proliferation of mouse airway stem/progenitor cells, and has little effect on the differentiation.

Key words: mTOR/S6K1, airway stem/progenitor cells, cell proliferation, cell differentiation