Figure 6.

MSCs interact with primary AML cells to modify their ROS levels, p38MAPK activation, and GPX3 expression. Analysis was performed in primary AML cells cultured alone (blue), with MSC-CM (green) or with MSCs (red). (A) ROS levels were analyzed in primary AML cells by flow cytometry after CM-H₂DCFDA labelling (n = 13 with MSC-CM; n = 26 with MSC-contact). Primary BM-blasts from 20 AML patients were studied by coculture experiments on normal BM-MSCs from five donors (26 coculture experiments were performed due to various combinations). Seventeen AML patients (among these 20 patients) presented a decrease in ROS levels upon contact with MSCs. Blasts were used no more than three times. As expected, when AML patient blasts were studied on different MSCs, the results were not different, ruling out any MSC batch effect. Whenever possible (sufficient number of blasts), the effect of CM-MSCs was evaluated (n = 13), and in all cases, CM-MSCs did not induce a decrease in ROS levels in AML blasts. All statistics were performed comparing different groups using the Kruskal–Wallis test comparing coculture condition (n = 26), CM-MSCs condition (n = 13) and control condition (blasts alone). (B) p38MAPK expression and activation were studied by flow cytometry after labelling with anti-p38 or anti-phospho (T180/Y182)-p38, respectively (n = 7); (C) GPX3 expression was studied by real-time PCR. Results are presented as percentage of increased or decreased relative GPX3 quantity (RQ = 2^−ΔΔCt) in MSC-CM or MSCs cocultured AML cells vs. AML cells cultured alone (n = 6). * p < 0.05.