Exosomes from Mesenchymal Stem Cells are Anti-Inflammatory Mediators in VCA
Jonathan P. Massie, BS, Catherine C. Motosko, BS, Trevor J. Ellison, BS, Sophia G. Hameedi, BA, Bukhtawar Waqas, BA, J. Rodrigo Diaz-Siso, MD, Daniel J. Ceradini, MD.
NYU Langone Medical Center, New York, NY, USA.
Mesenchymal stem cells(MSCs) are immunomodulators investigated in vascularized composite allotransplantation(VCA), yet their mechanism is poorly defined. We hypothesized that MSCs package anti-inflammatory cytokines into exosomes(EXO) which execute the immunomodulatory effects of MSCs, and differential EXO packaging could 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 VCA.
Lewis rat-derived MSCs were cultured in normoxia(21% O2), hypoxia(5% O2), or with 100U IFNγ. MSC-EXOs were isolated by ultracentrifugation and confirmed by TEM, immunoblot, and flow cytometry for EXO markers. MSC-EXO cytokine cargo were characterized by flow cytometry, and assessed for syngeneic Treg induction, and in an allogeneic rat hindlimb VCA model.
MSC-EXOs from normoxic, hypoxic, and IFNγ-primed MSCs expressed EXO markers Cd63 and Cd81. Hypoxic MSC-EXOs packaged significantly greater quantities of IDO(6.4-fold,p<0.0001), IL-10(1.4-fold,p<0.05), iNOS(2.1-fold,p<0.0001), PGE2(1.7-fold,p<0.05) and TGFβ(1.4-fold,p<0.005) versus an isotype control. IFNγ-primed MSCs packaged greater quantities of iNOS and TGFβ compared to normoxic and hypoxic MSC-EXOs(p<0.05 for all). Normoxic MSC-EXOs induced a 6-fold increase in a CD4+FoxP3+ Treg population(p<0.005) versus negative control; a 2-fold greater induction than normoxic MSCs(p<0.0001).In a VCA model, ex vivo seeded DiI-labeled MSC-EXOs localized to the perivascular space following re-implantation.
MSC-EXOs package all known mediators of MSC immunomodulation and stimulate Treg production. Preliminary VCA studies suggest that MSC-EXOs localize to sites of immunologic importance similar to MSCs. MSC-EXOs are a promising explanation for MSC immunomodulatory properties, and may offer a novel therapeutic approach in VCA.
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