<p>Supplementary information</p><p>Isolation of alveolar epithelial type II progenitor cells from adult human lungs.</p><p>Naoya Fujino1, Hiroshi Kubo1, Takaya Suzuki2, Chiharu Ota 1, Ahmed E. Hegab1, Mei He1, Satoshi </p><p>Suzuki3, Takashi Suzuki4, Mitsuhiro Yamada5, Takashi Kondo2, Hidemasa Kato6, Mutsuo Yamaya1.</p><p>Materials and Methods</p><p>Cell culture</p><p>A549 (human lung adenocaricnoma cell line) was cultured with Dulbecco's Modified Eagle </p><p>Medium (DMEM; Invitrogen) containing 10% FBS (Invitrogen), 100 units/mL penicillin and 100 </p><p>μg/mL streptomycin (antibiotics; Sigma-Aldrich). HMC-1 (human mast cell line) was cultured with</p><p>Iscove's Modified Dulbecco's Medium (IMDM; Invitrogen) containing 10% FBS (Invitrogen) and the antibiotics (Sigma-Aldrich). Human umbilical vein endothelial cells (HUVEC) was cultured with HuMedia-EG2 (Kurabo, Osaka, Japan). </p><p>Separation of human lung cells and cell culture</p><p>Human lung cells were isolated as previously described with some modifications (1). We separated distal lung tissues within 3 cm from pleura. After pleura were separated bluntly, lung specimens were cut into approximately 1x1x1 cm pieces. The samples were inflated with dispase II </p><p>(Roche Applied Science, Mannheim, Germany; final concentration, 2.0 U/mL) and placed in conical tubes containing dispase II, collagenase/dispase (Roche Applied Science; final concentration, 1 mg/mL) and DNase I (Sigma-Aldrich, St. Luis, MO; final concentration, 0.1 mg/mL), and incubated for 90-120 minutes at 37℃ with shaking. Enzymatically digested samples were minced with scissors, passed through needles, and filtered through a 100 μ m mesh (BD </p><p>1 Biosciences, San Jose, CA). After treatment with red blood cell lysis buffer (Roche Applied </p><p>Science), the cells were filtered through a 40 μ m mesh (BD Biosciences) and resuspended in </p><p>DMEM (Invitrogen, Carlsbad, CA) containing 10% FBS (Invitrogen), 1% amino acids solution </p><p>(Invitrogen), 100 units/mL penicillin, 100 μ g/mL streptomycin (antibiotics; Sigma-Aldrich), and 2.5</p><p>μg/mL amphotericin B.</p><p>Hematopoietic CD45+ cells and non-hematopoietic CD45- lung cells were collected using the autoMACS Separator with anti-human CD45 antibody-coated micro-beads according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany).</p><p>Flow cytometry</p><p>Cell-surface antigens were examined by flow cytometry with the FACSCalibur flow cytometer (BD Biosciences). The data was analyzed by CellQuest Pro (BD Biosciences). We used the following antibodies: FITC-anti human CD45, phycoerythrin (PE)-anti human E-cadherin </p><p>(Biolegend, San Diego, CA), FITC-anti human CD31, PE-anti human c-kit (BD Biosciences), PE- anti human CD133/1 (Miltenyi Biotec), FITC-anti human CD34 (IMMUNOTECH) and PE-anti human vascular endothelial growth factor receptor 2 (VEGFR2; BD Biosciences). To stain E- cadherin, cultured cells were harvested using 1 mM EDTA. Isotype-matched antibodies were used as negative controls.</p><p>2 Figure legends</p><p>Supplementary figure1: Positive controls for flow cytometric analyses. (A) A549 expressed E- cadherin. (B) HMC-1 expressed c-kit. (C) Primary CD45- lung cells contained CD133-expressing cells. (D) Hematopoietic CD45+ cells isolated from human lung tissues binded to anti-CD45 antibody derived from a different clone. (E) Primary CD45- lung cells contained CD34-expressing endothelial cells. (F, G) HUVEC expressed CD31 and VEGFR2. </p><p>Supplementary figure2: Immunofluorescence staining for pro SP-C (green) and CD90 (red) in clonally expanded cells. The staining pattern of these cells was similar to that of original cells </p><p>(Figure 1C). Inset, isotype control. Scale bar, 50 μ m.</p><p>3 References</p><p>1 Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I, Vogel S, et al. Identification of</p><p> bronchioalveolar stem cells in normal lung and lung cancer. Cell 2005; 121(6):823-835.</p><p>4</p>