MSC immunomodulation is mediated by paracrine molecules.
(a) Schematic representation of Transwell® system with MSCs in the bottom well and IECs in the top well. A 0.4 μm-porous membrane was used to prevent cell-cell interaction and permit soluble molecule exchange. Sorted-IECs (T, B and NK cells) were stimulated with specific stimuli and cultured alone or in the presence of resting or primed allogeneic MSCs. At the end of co-culture, IEC proliferation was assessed using carboxyfluorescein succinimidyl ester (CFSE) dilution method, as described in Materials and Methods section. CFSE fluorescence was analyzed after 6 days for T (at 10:1 T/MSC ratio) and NK (at 1:1 NK/MSC ratio) cells (b,d, respectively), while for B cells (c) the fluorescence was detected after 4 days of co-culture (at 1:1 B/MSC ratio). The same IEC:MSC ratios were maintained to assess the effect of MSC paracrine molecules on sorted-T, -B and -NK cells (b–d, respectively) proliferation by use of Transwell® 24 system. The results are expressed as relative proliferation percentage of IECs, normalized to IEC cultured alone (100%). Error bars represented mean ± SEM of twelve independent experiments for standard immunological assays and four independent experiments for Transwell® assays. ***P < 0.001.