SUPPLEMENTARY MATERIALS AND METHODS
MSCs differentiation assays
MSCs at passage 3 were cultured in a 6-well plate (2 × 104 cell/cm2) to differentiate into
adipocytes, osteoblasts and chondrocytes in corresponding induction medium. An osteogenic
medium consisting of Dulbecco’s modified Eagle’s medium/F-12 supplemented with 10% fetal
bovine serum (Hyclone), 10nM dexamethasone, 5 mg/mL sodium ascorbate-2 phosphate, 10 mM
b-glycerol-phosphate, and 100U penicillin/100 mg streptomycin/0.25 mg Fungizone was used.
Cultures were fed with a fresh medium every 3 days. After 21 days, the medium was removed.
Cells were washed with 0.09% NaCl and fixed in 70% ethanol. Afterward, cells were incubated
in von Kossa stain (1% wt/vol silver nitrate solution; Sigma Aldrich) and developed under direct
light for 60 min.
After 3 weeks of culture with adipogenic induction medium containing 1026 M
dexamethasone, 10 mg/mL insulin, and 100 mg/mL 3-isobutyl-L-methylxantine (Sigma), MSCs
were stained with Oil Red-O to detect lipid.
For chondrocyte differentiation, MSCs were cultured in DMEM (high glucose)
containing 1027 M dexamethasone, 50 mg/ml ascorbate-2-phosphate, 100 mg/ml pyruvate
(Sigma Aldrich), 10 ng/ml TGF-b1 (R&D Systems) and 50 mg/ml ITS Premix (BD Biosciences,
6.25 mg/ml insulin, 6.25 mg/ml transferrin, 6.25 ng/ml selenious acid, 1.25 mg/ml bovine serum
albumin, and 5.35 mg/ml linoleic acid). Cultures at 3 weeks were fixed and stained with Safranin
O (Sigma Aldrich).
SUPPLEMENTARY FIGURE LEGEND
FIGURE 1. A diagram shows the Transwell-Clear system filled with a 1:1 mixture of airway
epithelial cell growth and MesenCult MSC basal media. MSCs (2 × 104 cells/cm2) were cultured
in the upper Transwell chamber (i.e., on the polyester membrane) and lung cells (6 ×104
cell/cm2) in the lower chamber.
FIGURE 2. MSCs differentiation assays into osteoblasts, adipocytes and chondrocytes were
used to confirm the use of pure MSCs in this study. Osteoblasts are stained with von Kossa stain
(Figure 2A); Adipocytes are stained with Oil Red-O (Figure 2B); Chondrocytes are stained with
Safranin O (Figure 2C). Scale bar: 34 µm at 400x.
FIGURE 3. Mean of fold change in genes expression ± SD after 5 days of coculture; *P < 0.05;
**P < 0.01; ***P < 0.001. In figure 3A; lung cultures obtained from normal non-irradiated rats
were used as a reference sample; in figure 3B; lung cultures obtained from 16 Gy-irradiated rats
were used as a reference sample; in figure 3C; lung cultures obtained from 20 Gy-irradiated rats
were used as a reference sample. Cocultured MSCs expressed surfactant B gene (type II
pneumocytes) that was significantly up-regulated in normal and 16 Gy irradiated cocultures
(19.5 & 15.5 fold change in 3A & 3C, respectively) but down regulated in 20 Gy irradiated
cocultures. Control MSCs did not express this gene along the experiment. Cocultured Lung cells
showed significant fold changes in most genes compared to controls.
FIGURE 4. Mean of fold change in genes expression ± SD after 5 days of coculture; *P < 0.05;
**P < 0.01; ***P < 0.001. In figure 4A; lung cultures obtained from normal non-irradiated rats
were used as a reference sample; in figure 4B; lung cultures obtained from 16 Gy-irradiated rats
were used as a reference sample; in figure 4C; lung cultures obtained from 20 Gy-irradiated rats
were used as a reference sample. Cocultured MSCs expressed surfactant B gene (type II
pneumocytes) that was significantly up-regulated (7 &16 & 20 fold change in 4A & 4B & 4C,
respectively). Control MSCs did not express this gene along the experiment. Cocultured Lung
cells showed significant fold changes in most genes compared to controls.