Figure 1.

mASCs expanded in hypoxia maintain their immunosuppressive activities. (a) Proliferative response of mASCs expanded in normoxia (21% O₂) or in hypoxia (5% O₂). mASCs were seeded at 5000 cells/cm² in T25 flasks and counted at different time points when reaching 70–80% of confluence. Data are shown as mean (SD) of one representative experiment out of three. ^∗p < 0.05 versus 21% O₂. ((b) and (c)) Immune phenotypic characterization of mASCs. mASCs were expanded under hypoxic conditions and then cultured with medium (unstimulated) or stimulated with LPS (1 μg/mL) or TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) for different time points (24 hours for NO₂ determination). Gene expression of different immune markers was analyzed using semiquantitative PCR. iNOS activity was measured in TNF-α/IFN-γ-stimulated mASCs by the detection of NO₂ in culture supernatants using Griess assay. COX-1/2 activity was determined by measuring the PGE₂ levels in culture supernatants from unstimulated mASCs. (d) Arginase activity is induced in mASC: splenocyte cocultures. Splenocytes were cultured with or without mASCs and stimulated with ConA for 3 days. Defined quantities of cell lysates were then subjected to an in vitro arginase activity assay. Data are shown as mean (SEM) of 3 independent experiments. ^∗p < 0.05 versus splenocytes + ConA. ((e), (f), and (g)) mASCs suppress T cell proliferation in vitro via iNOS and COX-1/2 but not arginase activity. Splenocytes (10⁶ cells/mL) were stimulated with ConA (2.5 μg/mL) in the presence of different numbers of mitomycin C-treated mASCs (ratio 20 : 1 for IFN-γ and CXCL10 determination). ((e) and (f)) The iNOS inhibitor L-NAME (1 mM), the COX-1/2 inhibitor indomethacin (20 μM), and the arginase inhibitor L-norvaline (10 mM) were added to cultures when indicated. After 3 days, cell proliferation was measured by [³H]-thymidine incorporation and (g) IFN-γ and CXCL10 contents in the supernatants were determined by ELISA. Data are shown as mean (SEM) of 3 independent experiments. ^∗p < 0.05 versus mASC in (f) and ^∗p < 0.05 versus ConA in (g). (h) iNOS and COX-2 activities are increased in mASC: splenocyte cocultures. ConA-stimulated splenocytes (10⁶ cells/mL) were cultured with mitomycin C-treated mASCs (ratio 20 : 1) in the presence of L-NAME or indomethacin for 3 days and the PGE₂ and NO₂ contents measured in the culture supernatants. Results are shown as mean (SEM) of 3 separate experiments. ^∗p < 0.05 versus ConA; ^∧p < 0.05 versus mASC + ConA.