Background
Mesenchymal stem cells (MSCs) are immunomodulators investigated in allotransplantation, yet their mechanism of action is poorly defined We hypothesized that MSCs package anti-inflammatory cytokines into secreted vesicles, exosomes (EXO), which execute the immunomodulatory effects of MSCs Additionally, we postulated that differential EXO packaging may be driven by oxygen tension and inflammatory milieu Our aims were:(1)Determine whether MSCs secrete EXOs;(2)Characterize MSC-EXO cargo under differential conditions;(3)Investigate MSC-EXOs in preventing rejection in allotransplantation.
Methods
Lewis rat-derived MSCs were cultured in normoxia(21% O2), hypoxia(5% O2), or with 100U IFNÎ3 MSC-EXOs were isolated by ultracentrifugation and confirmed by TEM, immunoblot, and flow cytometry for EXO markers MSC-EXO cytokine and transcript cargo were characterized by immunoblot, flow cytometry, and quantitative real time PCR Functional biologic MSC-EXO activity was assessed in an in vitro syngeneic Treg induction model and in an in vivo allogeneic rat hindlimb allotransplantation model.
Results
MSC-EXOs from normoxic, hypoxic, and IFNÎ3-primed MSCs expressed EXO markers Cd63 and Cd81, and hypoxic cells produced a 16-fold greater quantity of EXOs than normoxic cells(p<005) MSC-EXOs packaged and were enriched for the anti-inflammatory cytokines IDO, TGFÎ21, and iNOS when compared to total cellular lysates as imaged by immunoblot Transcriptional analysis revealed that MSC-EXOs package over 1,000-fold more of the anti-inflammatory transcript IL10 than any other transcript examined, whereas the most highly expressed cellular anti-inflammatory transcript was TGFÎ2 Additionally, EXO transcript packaging was dependent on environmental stimuli, as MSC-EXOs derived under inflammatory conditions packaged over 10 million-fold more IDO transcript than under non-inflammatory conditions Normoxic MSC-EXOs induced a 6-fold increase in a CD4+FoxP3+Treg population(p < 0005) versus negative control; a 2-fold greater induction than normoxic MSCs(p < 00001) In an allotransplantation model, ex vivo seeded DiI–labeled MSC-EXOs localized to the perivascular space following re-implantation.
Conclusions
MSC-EXOs package the anti-inflammatory cytokines IDO, iNOS, and TGFÎ21 and the IL10 transcript, and are able to stimulate Treg production Furthermore, MSC-EXOs localize to perivascular cells in an allotransplantation model This suggests that MSC-EXOs package cytokines and transcripts for intercellular delivery and gene transfer, which may explain the mechanism of action of MSCs and their unique immunomodulatory properties.