Isolation and Differentiation of Lung Stem Cells

Primary lung stem cell (LSC) culture was performed as previously described [14 (link)]. LSCs were isolated from neonatal CD-1 mice by FACS sorting using phycoerythrin- (PE-) conjugated anti-CD157 (BioLegend, CA, USA), fluorescein isothiocyanate- (FITC-) conjugated anti-CD54 (BD Biosciences, CA, USA), and allophycocyanin- (APC-) conjugated anti-CD45 (eBioscience, CA, USA) antibodies. Isolated CD45⁻CD54⁺CD157⁺ cells or irradiated cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS, 1% insulin–transferrin–selenium (ITS), and 1 ng/ml epidermal growth factors (EGF) (all obtained from Thermo Fisher Scientific, CA, USA) through several passages in a collagen I-coated plate. To conduct differentiation studies, the attached LSCs were incubated in MCDB-201 medium (Sigma-Aldrich, MO, USA) supplemented with 1% FBS, 1% ITS, and 10 ng/ml EGF for 7 or 14 days to induce AECII or AECI cells.
To determine the fibrogenic effect of transforming growth factor beta (TGF-β) and connective tissue growth factor (CTGF) (PeproTech, NJ, USA), LSCs (2.5 × 10⁴ cells/cm²) in 12-well culture plates were treated with TGF-β (5 ng/ml) or CTGF (50 ng/ml) for 3 days.