Molecular profiles of functionally effective MSC-Derived extracellular vesicles in immunomodulation

Abstract

Background & Aim Accumulating evidence indicates that extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have immunomodulatory effects. Although the molecular mechanisms underlying the EV-mediated immunomodulation have not been fully understood, several studies demonstrated that MSC-derived EVs (MSC-EVs) carry numerous numbers of mRNAs, microRNAs, and proteins and these factors are responsible for the therapeutic effect of MSC-EVs. However, the contents of MSC-EVs can change as their parent cell changes since EVs carry a subset of proteins and RNAs of their parent cell, suggesting that the therapeutic efficacy of MSC-EVs can be also varied depending on donors and culture conditions of MSCs. Therefore, the aim of our study is to establish a molecular profile of functionally effective MSC-EVs in immunomodulation. Methods, Results & Conclusion MSCs lose many of their biological properties as they are expanded in culture, we therefore compared immunomodulatory effects of EVs derived from early passage (passage 2: P2) MSCs and late passage (P7) MSCs using splenocyte cultures and mice with ocular Sjogren's syndrome. In parallel, we performed proteomics and RNA sequencing with both of the EVs. We found that the P2 EVs more effectively suppressed the activation of splenocytes in vitro and improved inflammatory dry eye in mice with ocular Sjogren's syndrome. Proteomics showed that about 40% of proteins enriched in both P2 and P7 EVs were binding proteins, but the top ranked binding proteins of the P2 EVs were classified as extracellular region or matrix proteins while P7 EV binding proteins were classified as chromatin or nucleic acid binding proteins. In addition, the well-known immunomodulators, TGFβ1 and PTX3 (Pentraxin 3) were enriched in the P2 EVs and inhibition or overexpression of TGFβ1 or PTX3 in MSCs directly affected the immunomodulatory potency of their EVs. Moreover, RNA sequencing revealed that about 80 miRNAs were differentially expressed between P2 and P7 EVs and the well-known immunomodulators, let-7b-5p and miR-146b were also enriched in P2 EVs. In summary, our comparative analyses between EVs from early and late passage MSCs provided the distinctive molecular profile of functionally effective MSC-EVs and also helped understand the molecular mechanism underlying the EV-mediated immunomodulation. Furthermore, our data suggest that the enriched factors in functionally effective EVs can be used as biomarkers to evaluate their regulatory function in immunomodulation.