MSCs preconditioned by an MLR-milieu exhibit decreased HO-1 levels. (A) After 5 days of a priming MLR (pMLR) and removal of the PBLs (MSC purity ≥ 90%), (i-ii) HO-1 levels (MFI) were determined in the MSCs as shown in the representative histogram for MSC113 cells and compared with nonprimed (native) MSCs (n = 5). (B) Relative gene expression (mRNA) levels of HO-1 in the MSCs were determined after 2 and 5 days of MLR, respectively (n = 5). Equivalent experiments were carried out in regular or trans-wells (TW). (C) After 5 days of initial pMLR, the suppressive activity of the primed MSCs was evaluated compared with nonprimed (native) cells in a subsequent MLR (n = 5). (D) After 5 days of MLR, MSCs were evaluated with regard to apoptosis and necrosis by flow cytometry. As shown (i) in the representative dot plot analysis (ii) for 6 independent experiments, viable cells were defined as 7-AAD⁻/annexin V⁻. (E) Relative gene expression levels (mRNA) of Nrf2 in MSCs were determined after 2 and 5 days of MLR, respectively (n = 5). All experimental settings were conducted with MSCs derived from at least 3 individual donors, and n refers to the number of repeated experiments. Bars represent SD. *P ≤ .05. **P ≤ .01. ***P ≤ .001.