Dual Preconditioned Mesenchymal Stem Cells-Derived Culture Conditioned Media Augment Immunomodulation and Drive Metabolic Reprogramming in Acute Graft-versus-Host Disease

BackgroundThe 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 naive and preconditioned MSCs-hypoxic (MSCsHYP), apoptotic (MSCsAPO), and dual preconditioned (MSCsHYP+APO) in aGVHD. MethodsHuman MSCs isolated from bone marrow and Whartons Jelly were preconditioned under hypoxia (1% O2), apoptosis (1 {micro}M 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-MSCsHYP+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. ResultsWJ-MSCsHYP+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. ConclusionDual 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 ABSTRACTImmunomodulatory and immune metabolic reprogramming potential of naive and preconditioned MSCs (MSCs, MSCsHYP, MSCsAPO, MSCsHYP+APO) derived CCM in Acute GVHD (created using Biorender.com) O_FIG O_LINKSMALLFIG WIDTH=95 HEIGHT=200 SRC="FIGDIR/small/686182v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@f7ed73org.highwire.dtl.DTLVardef@1063015org.highwire.dtl.DTLVardef@13ed827org.highwire.dtl.DTLVardef@e103c0_HPS_FORMAT_FIGEXP M_FIG C_FIG