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cpr817-sup-0001-Fig1.tifimage/tif1924KFig. S1 Effect of pre-hypoxia exposure on the osteogenic differentiation of hAT-MSCs. Von Kossa's staining in monolayer cultures. (a) Experimental plan. hAT-MSCs were cultured in monolayer under hypoxia in complete growth control medium (GM) for 7 days and transferred to normoxia in the presence of GM (b) or the osteogenic differentiation cocktail medium (DM) (c) for a period of 34 days. Alternatively, hAT-MSCs were cultured in normoxia and then exposed for the same time either to GM (d) or DM (e). Von Kossa's staining was performed to quantify mineralization. Von Kossa's staining controls: (f) negative and (g) positive (mouse E18.5) are shown illustrating embryonic bone. Bar = 100 μm.
cpr817-sup-0002-Fig2.tifimage/tif1907KFig. S2 Effect of pre-hypoxia exposure on the osteogenic differentiation of hAT-MSCs. Alizarin red S staining in monolayer cultures. (a) Experimental plan. hAT-MSCs were cultured in monolayer under hypoxia in complete growth control medium (GM) for 7 days and transferred to normoxia in the presence of GM (b) or the osteogenic differentiation cocktail medium (DM) (c) for a period of 34 days. As controls, hAT-MSCs were cultured in normoxia and then exposed for the same time either to GM (d) or DM (e). Alizarin red S staining was performed to quantify calcium depositions. Alizarin red S staining controls: (f) negative and (g) positive (mouse E 18.5) are shown illustrating embryonic bone. Bars = 100 μm.
cpr817-sup-0003-Fig3.tifimage/tif224KFig. S3 Effect of hypoxia on the DNAdistribution. (a, b) Cell cycle distribution and percent (c) of PI-labelled hAT-MSCs in different phases of the cell cycle after 7 days culture either in normoxia or in hypoxia (n = 3). The percentage of sub-G1 cells (apoptotic bodies) was lower in hypoxia compared to the normoxic counterpart.
cpr817-sup-0004-Fig4.tifimage/tif1746KFig. S4 Effect of pre-hypoxia exposure on chondrogenic differentiation of hAT-MSCs: Alcian blue staining in monolayer cultures. (a) Experimental plan. hAT-MSCs were pre-cultured in monolayer under hypoxia in control growth medium (GM) for 7 days and transferred to normoxia in the presence of GM (b) or the chondrogenic differentiation cocktail medium (DM) (c) for a period of 19 days. As controls, hAT-MSCs were cultured in normoxia and then exposed for the same time either to GM (d) or DM (e). Chondrogenic differentiation was assessed with Alcian blue staining to visualize sulphated proteoglycan production. Alcian blue control staining: (f) positive control (adult mouse large intestine illustrating positive goblet cells) is shown. Bars = 100 μm.
cpr817-sup-0005-Fig5.tifimage/tif1192KFig. S5 Effect of pre-hypoxia exposure on chondrogenic differentiation of hAT-MSCs: Toluidine blue staining in pellet cultures. Toluidine blue staining was performed to detect the presence of cartilage-typical glycosaminoglycans. (a) Experimental plan. hAT-MSCs were pre-cultured in monolayer under hypoxia in control growth medium (GM) for 7 days and transferred to normoxia, in pellet cultures, in the presence of GM (b) or the chondrogenic differentiation cocktail medium (DM) (c) for a period of 27 days. As controls, hAT-MSCs were cultured in normoxia and then exposed for the same time either to GM (d) or DM (e). A high magnification view (400×) of c and e in the insets, are shown. Toluidine positive staining control (mouse adult lung) is shown illustrating bronchial cartilage. Bars = 100 μm.
cpr817-sup-0006-Fig6.tifimage/tif1419KFig. S6 Effect of pre-hypoxia exposure on chondrogenic differentiation of hAT-MSC: Safranin O staining in pellet cultures. Safranin O staining was performed to detect cartilage. (a) Experimental plan. hAT-MSCs were pre-cultured in monolayer under hypoxia in control growth medium (GM) for 7 days and transferred to normoxia, in pellet cultures, in the presence of GM (b) or the chondrogenic differentiation cocktail medium (DM) (c) for a period of 27 days. As controls, hAT-MSCs were cultured in normoxia and then exposed for the same time either to GM (d) or DM (e). A high magnification view (400×) of c and e in the insets, are shown. Safranin O positive staining control (mouse E18.5) is shown illustrating embryonic cartilage. Bars = 100 μm.
cpr817-sup-0007-Fig7.tifimage/tif1609KFig. S7 Effect of pre-hypoxia exposure on chondrogenic differentiation of hAT-MSC: Masson's Trichrome staining in pellet cultures. Masson's Trichrome staining was performed to visualise collagen fibres. (a) Experimental plan. hAT-MSCs were pre-cultured in monolayer under hypoxia in control growth medium (GM) for 7 days and transferred to normoxia, in pellet cultures, in the presence of GM (b) or the chondrogenic differentiation cocktail medium (DM) (c) for a period of 27 days. As controls, hAT-MSCs were cultured in normoxia and then exposed for the same time either to GM (d) or DM (e). A high magnification view (400×) of c and e in the insets, are shown. Human tongue was used as a Masson's Trichrome positive staining control for collagen. Bars = 100 μm.

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