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
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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), PEanti 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 Ecadherin, 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 Ecadherin. (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.
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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
1
Kim 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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