Mixed lymphocyte reaction-conditioned MSC-derived extracellular vesicles enhance graft survival via miR-638-mediated immunoregulation

  • Stem Cells Transl Med. 2025 Apr 22;14(4):szaf009. doi: 10.1093/stcltm/szaf009.
Yue Ding  1 ,  Jiyuan Wang  1 ,  Xueyang Zheng  1 ,  Yu Chen  1 ,  Fanyuan Zhu  1 ,  Fang Lin  2 ,  Kexin Ma  3  2  4 ,  Xiaoting Liang  3  2 ,  Shu Han  1
Affiliations
  • 1. Department of Organ Transplantation, Shanghai Changzheng Hospital (Second Affiliated Hospital of Naval Medical University), 200003 Shanghai, People's Republic of China.
  • 2. Shanghai Heart Failure Research Center, Shanghai East Hospital, Tongji University School of Medicine, 200120 Shanghai, People's Republic of China.
  • 3. Translational Medical Center for Stem Cell Therapy and Institute for Regenerative Medicine, Shanghai East Hospital, Tongji University School of Medicine, 200120 Shanghai, People's Republic of China.
  • 4. Shanghai East Hospital, Jinzhou Medical University, 121001 Jinzhou, People's Republic of China.
PMID: 40261200 DOI: 10.1093/stcltm/szaf009
Abstract

Background: Mesenchymal stem cells (MSCs) require priming by proinflammatory stimuli for optimal immunosuppressive effects. Our previous work identified mixed lymphocyte reaction-conditioned medium (MLR-CdM) as a potent enhancer of MSC immunosuppressive properties. This study evaluates the immunomodulatory potential of MSC-derived extracellular vesicles preconditioned with MLR-CdM (MSC-EVMLR) compared to IFN-γ (MSC-EVIFN), focusing on key miRNAs and mechanisms involved.

Methods: We assessed the ability of MSC-EVMLR and MSC-EVIFN to modulate lymphocyte proliferation and cytokine expression in vitro. To identify potential effector molecules within MSC-EVMLR, we performed miRNA array analysis combined with dose-response experiments using MLR-CdM under varying stimulation conditions. We used a murine allogeneic heterotopic heart transplantation model to investigate the impact of MSC-EVMLR on graft survival and its immunomodulatory effects.

Results: MSC-EVMLR outperformed MSC-EVIFN in suppressing lymphocyte proliferation and steering cytokine expression toward an anti-inflammatory profile in vitro. Through miRNA array analysis and dose-response experiments with MLR-CdM, miR-638 was identified as a potential effector molecule in MSC-EVMLR. In vivo study demonstrated that MSC-EVMLR significantly prolonged graft survival, which was associated with a marked decreased proinflammatory cytokines IL6 and IFN-γ and increase in regulatory T cells (Tregs) and within the transplanted heart tissue. These effect was significantly reduced upon miR-638 knockdown. Additionally, the miR-638/Fosb axis was identified as a key pathway that promoted Treg differentiation and induced immune tolerance.

Conclusions: Preconditioning MSCs with MLR-CdM, a blend of inflammatory stimuli, potentiates the immunoregulatory capacity of MSC-EV beyond the effects of IFN-γ stimulation alone. This study advances the understanding of MSC-EV-based therapies in transplantation.

Keywords
Treg; graft survival; mesenchymal stem cell-derived extracellular vesicles; miR-638; mixed lymphocyte reaction-conditioned medium.
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