Figure 4.

MSCs (Alofisel/darvadstrocel) undergo apoptosis when in contact with CD patients’ PBMCs and secrete PGE2 in a caspase-dependent manner

(A) 5 × 10⁴ adipose tissue-derived MSCs (Alofisel/darvadstrocel) were cultured for 4 h with 1 × 10⁶ PBMCs and apoptosis was evaluated by assessing Annexin-V/7AAD staining by flow cytometry. MSC alone and MSC co-cultured with PBMCs from healthy donors were used as controls. (B) 5 × 10⁴ MSCs were cultured for 4 h with 1 × 10⁶ PBMCs either in direct contact or in transwell and apoptosis was evaluated by assessing Annexin-V/7AAD staining by flow cytometry. (C) Levels of PGE2 were detected in the supernatant by ELISA. MSCs alone (n = 8), PBMCs alone (n = 3), co-culture of MSC:PBMC obtained from healthy controls (HC, n = 4) and Crohn’s disease patients (CD, n = 24). (D) PGE2 levels detected by ELISA after 4 h MSC:PBMC co-culture either in straight contact or in a transwell system. (E) Expression levels of PGE2 were measured in the co-culture supernatant by ELISA after treatment with the pan-caspase inhibitor Z-VAD-FMK (50 μM) and compared with the untreated control. Data are expressed as mean ± SD. Unpaired t test was used to analyze mean differences between the samples in (A) and (B). Nonparametric Wilcoxon signed rank test and Kruskal-Wallis test followed by Dunn’s post-test were used to compare the differences between two or more groups in (C), (D), and (E) (∗p values <0.05, ∗∗p values <0.01, ∗∗∗p values <0.001, ∗∗∗∗p values <0.0001, n.s, not significant).