Dual Preconditioned Mesenchymal Stem Cells-Derived Culture Conditioned Media Augment Immunomodulation and Drive Metabolic Reprogramming in Acute Graft-versus-Host Disease
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Background
The toxicity associated with conventional conditioning regimens limits treatment outcomes in autoimmune disorders such as acute graft-versus-host disease (aGVHD). Developing effective adjunctive interventions to restore immune balance is therefore essential. This study investigated the immunomodulatory potential of mesenchymal stem cells (MSCs)-derived culture-conditioned media (CCM) from naïve and preconditioned MSCs-hypoxic (MSCs HYP ), apoptotic (MSCs APO ), and dual preconditioned (MSCs HYP+APO ) in aGVHD.
Methods
Human MSCs isolated from bone marrow and Wharton’s Jelly were preconditioned under hypoxia (1% O₂), apoptosis (1 µM staurosporine, 24 h), or both. The immunoregulatory and antioxidant properties of their CCM were evaluated through T-cell proliferation assays, Treg induction, macrophage polarization, mitochondrial function, and T-cell bioenergetics. Comparative proteomic profiling of CCM from WJ-MSCs and WJ-MSCs HYP+APO co-cultured with aGVHD patient-derived activated PBMNCs was performed using LC-MS/MS, alongside in vivo validation in a chemotherapy-induced aGVHD murine model.
Results
WJ-MSCs HYP+APO -CCM exhibited superior immunomodulatory efficacy, suppressing T-cell proliferation, promoting Treg and Th2/Th9 differentiation, and driving M2 macrophage polarization. It reduced mitochondrial ROS, enhanced mitochondrial polarization, and shifted T-cell metabolism from glycolysis toward oxidative phosphorylation. Proteomic analysis revealed modulation of IL-12, IL-17, and JAK–STAT pathways, along with regulation of complement, coagulation, and metabolic cascades. Interaction with immune cells further enhanced its antioxidant and tissue-reparative properties via extracellular matrix remodeling.
Conclusion
Dual preconditioning under hypoxia and apoptosis amplifies the immunomodulatory, antioxidant, and reparative efficacy of WJ-MSC-CCM, offering a potent non-cellular therapeutic strategy for aGVHD management.
GRAPHICAL ABSTRACT
Immunomodulatory and immune metabolic reprogramming potential of naïve and preconditioned MSCs (MSCs, MSCs HYP , MSCs APO , MSCs HYP+APO ) derived CCM in Acute GVHD (created using Biorender.com )
