Determining the Mechanism of Exosome Release from Mesenchymal Stem Cells and their Immunomodulatory Effects for Use in Vascularized Composite Allotransplantation
Salma A. Abdou, BA, William J. Rifkin, BA, Joshua A. David, BS, Jennifer Kwong, BA, Darren L. Sultan, BA, Jonathan P. Massie, MD, Rohini Kadle, MD, Piul S. Rabbani, PhD, Daniel J. Ceradini, MD.
Hansjörg Wyss Department of Plastic Surgery, New York University School of Medicine, New York, NY, USA.
PURPOSE: Accumulating evidence indicates that the powerful immunomodulatory effects of mesenchymal stem cells (MSCs) are largely mediated by the release of exosomes. A better understanding of the cellular mechanisms involved in exosome release as well as their immunomodulatory effects is an important step in facilitating their use as a non-cellular therapy for use in VCA.
METHODS: CD4+ T-cells were added to a co-culture of MSCs and endothelial cells and T-regulatory cell differentiation was determined. To specifically determine exosomes’ effects on T-cell differentiation, splenocytes were cultured with MSCs and with MSC derived exosomes alone. MSCs were transfected with Rab27a siRNA and knockdown was confirmed with PCR. Exosomes harvested from Rab27a knockdown MSCs were quantified and analyzed on Western Blot.
RESULTS: In this model, the endothelial cells acted as antigen presenting cells in an allograft. CD4+ cells cultured alone showed 8% T regulatory differentiation as compared to 28% in T cells co-cultured with MSCs and endothelial cells. In the in vitro co-culture of naïve CD4- and CD4+ T cells, exosomes were biologically active and able to induce a 6-fold increase in a population of CD4+FoxP3+ T regulatory cells versus a negative control. Exosomes alone induced 14.3% T regulatory formation as compared to 6.22% by MSCs. PCR confirmed 57% (p =0.007) Rab27a knockdown of BM-MSCs transfected at 50 picomoles of siRNA/500,000 cells. The concentration of exosomes harvested from wild type MSCs was 86.86ug/mL as compared to 36.2ug/mL from the Rab27a knockdown MSCs. This represents a 58.3% decrease in exosome release with 57% Rab27a knockdown. On immunoblot analysis, the relative expression of CD81, a distinct marker of exosomes, was decreased in the exosomes harvested from transfected MSCs.
CONCLUSION: Exosomes are essential to the immunomodulatory properties of MSCs. They have functional biologic activity and demonstrate superior T regulatory cell induction potential than cells. Rab27a is critical for the release of exosomes from MSCs. Exosomes derived from MSCs are a promising non-cellular therapy to decrease risk of rejection in VCA.
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