Role of the AhR pathway in the enhanced platelet production with MSCs. (A) Inhibition of CYP1B1 expression in CD34 cells cultured with MSC and its prevention with an AhR agonist. CD34⁺ cells were cultured as in Figure 1 for 10 days under control conditions or in the presence of SR1 (1 µM), MSCs, or MSCs + the AhR agonist FICZ (0.2 µM). mRNA was extracted, and the level of CYP1B1 transcript, a downstream effector of the AhR, was quantified by real-time polymerase chain reaction. The level of transcript, normalized in reference to control cultures (dimethyl sulfoxide samples that were normalized such that their median value for CYP1B1 mRNA level was 1), represented a ratio of 0.04 ± 0.04, 0.09 ± 0.04, and 6.5 ± 1 in SR1, MSC, and MSC + FICZ conditions, respectively (mean ± SEM in 3 experiments; 2-way ANOVA and a Bonferroni posttest, **P < .01, ***P < .001). (B) An AhR agonist prevents the positive effect of MSCs on proplatelet and platelet formation. CD34⁺ cells were cultured for 14 days under control conditions or with MSCs or with MSCs + FICZ (0.2 µM). Incubation with FICZ reversed the MSC-dependent increase in proplatelet formation (left: representative DIC microscopy photographs of culture wells) and platelet release (right: 8.3 ± 1.9 × 10⁵ platelets per well for MSCs vs 0.7 ± 0.5 × 10⁵ platelets per well for MSCs + FICZ; mean ± SEM in 3 experiments; 2-way ANOVA and a Bonferroni posttest, *P < .1; and P > .5, not significant [n.s.]).