Isolation of Alveolar Epithelial Type II Progenitor Cells from Adult Human Lungs
Supplementary information
Isolation of alveolar epithelial type II progenitor cells from adult human lungs.
Naoya Fujino1, Hiroshi Kubo1, Takaya Suzuki2, Chiharu Ota1, Ahmed E. Hegab1, Mei He1, Satoshi Suzuki3, Takashi Suzuki4, Mitsuhiro Yamada5, Takashi Kondo2, Hidemasa Kato6, Mutsuo Yamaya1.
Materials and Methods
Cell culture
A549 (human lung adenocaricnoma cell line) was cultured with Dulbecco's Modified Eagle Medium (DMEM; Invitrogen) containing 10% FBS (Invitrogen), 100 units/mL penicillin and 100 μg/mL streptomycin (antibiotics; Sigma-Aldrich). HMC-1 (human mast cell line) was cultured with 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).
Separation of human lung cells and cell culture
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 (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 μm mesh (BD Biosciences, San Jose, CA). After treatment with red blood cell lysis buffer (Roche Applied Science), the cells were filtered through a 40 μm mesh (BD Biosciences) and resuspended in DMEM (Invitrogen, Carlsbad, CA) containing 10% FBS (Invitrogen), 1% amino acids solution (Invitrogen), 100 units/mL penicillin, 100 μg/mL streptomycin (antibiotics; Sigma-Aldrich), and 2.5 μg/mL amphotericin B.
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).
Flow cytometry
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 (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.
Figure legends
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.
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 (Figure 1C). Inset, isotype control. Scale bar, 50 μm.
References
1Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I, Vogel S, et al. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 2005; 121(6):823-835.
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