PB1987 SELECTIVE ACTIVATION OF TOLEROGENIC CELL DEATH PATHWAYS IN MESENCHYMAL STROMAL CELLS

Abstract

Background:Mesenchymal stromal cells (MSCs) have been successfully employed for their tolerogenic and immunosuppressive capabilities in different conditions. We have demonstrated that, following infusion, MSCs undergo apoptosis and that this apoptosis results in an immunosuppressive activity. However, it is unclear how MSCs respond to different death inducing stimuli and whether MSCs from different sources respond similarly when following analogous patterns.Aims:To characterize the molecular response of MSCs, obtained from different sources, to distinct cell death stimuli.Methods:MSCs were isolated from human bone marrow (BM‐MSCs) or umbilical cord (UC‐MSCs) of healthy donors and exposed to recombinant human Tumor necrosis factor‐a(TNF‐a) and Interferon‐g(IFN‐g) in combination or individually, anti‐FAS (activator), TNF‐related apoptosis‐inducing ligand (TRAIL), phytohemagglutinin (PHA) activated Peripheral Blood Mononuclear Cells (PHA‐aPBMCs), hydrogen peroxide (H2O2) with or without the presence of the pan‐caspase inhibitor Z‐VAD. After 24 h, the cells were harvested and stained for annexin V/7‐AAD and analyzed by flow‐cytometry. The cancer cell line MCF‐7 was used as a positive control.Results:BM‐MSCs showed high sensitivity to apoptosis induction when treated with FAS activator (11% of AnnV+/7‐AAD‐ and 72% AnnV+/7‐AAD+) and PHA‐aPBMCs (7% of AnnV+/7‐AAD‐ and 78% AnnV+/7‐AAD). Conversely, UC‐MSCs were more resistant to apoptosis mediated by FAS, since the percentages of AnnV+/7‐AAD‐ and AnnV+/7‐AAD+ were 63% and 9%. Both BM‐MSCs or UC‐MSCs were only marginally sensitive to TRAIL (35% of apoptosis). In all cases, apoptosis induction was caspase dependent, as demonstrated by the significant reduction (p < 0.001) of the proportion of apoptotic cells in both BM‐MSCs and UC‐MSCs in the presence of the pan‐caspase inhibitor Z‐VAD. Conversely, neither BM‐MSCs or UC‐MSCs were induced to undergo apoptosis when treated with TNF‐aalso at very high concentrations or in combination with IFN‐g. The efficacy of the recombinant TNF‐ain inducing cell death was confirmed by using the cell line MCF‐7 as sensitive controls. Finally, all MSCs were sensitive to H2O2.Summary/Conclusion:MSCs exhibit varying susceptibility to different apoptotic stimuli. Interestingly, although this property seems to be maintained across different tissues of MSC origin, we could find variable degrees of sensitivity to specific stimuli, thus suggesting that different MSCs may play distinct roles according to the specific environment.