P3 mice were anesthetized, the abdominal cavity was opened and ventricular vein and artery were cut. The chest was opened and the right atrium nicked. Lungs were perfused through the right ventricle of the heart with 1 ml ice-cold 16PBS using a 26G syringe, cut in small pieces using a razor blade and digested with the Enzyme mix [Elastase (Sigma-Aldrich, 45124): 3 U/ml, Collagenase: 0.1 U/ml, Dispase: 0.8 U/ml (Roche Applied Science, 11097113001) and DnaseI 50 mg/ml (Sigma-Aldrich, DN25] in PBS, at 37uC for 1 hour with rotation. Equal volume of HBSS ++ [HBSS 16 (Gibco, 14170) , supplemented with 2% FCS (Gibco, 10500), 0.1 M HEPES (Sigma-Aldrich, H0887) and 15% Cell Dissociation Buffer (Gibco, 1315-016) or EGTA 2 mM] was added. Cells were gently agitated several times, passed through a 40 mm cell strainer (BD Biosciences, 352340) and centrifuged 10 minutes at 1200 rpm, at 4uC. The supernatant was discarded and cells were re-suspended in HBSS ++ and counted with a hemocytometer. Specific subpopulations of lung cells were isolated using a BD FACS-Aria IIu cell sorter by staining the isolated lung cells as previously described by Teisanu et al. [38] with biotinylated anti-CD31 (BD Biosciences, 553371), anti-CD45 (Biolegend, 103103) and anti-CD34 (eBioscience, 13-0341-85) combined with Streptavidin APCCy7 (Biolegend, 405208), with anti-Sca1-Alexa Fluor647 (Biolegend, 108118) andanti-EpCam-PECy7 (Biolegend, 118216). DAPI was included for dead cell exclusion. The EpCam pos -CD31 neg -CD45 neg -CD34 neg cells were separated into Sca1 neg and Sca1 low and each of these two populations were separated by sorting into eGFP neg , eGFP low and eGFP high . Isolated cells were used either for further in-vitro cultivation, RNA expression or Western Blot analysis.