Secretome of hypoxia-preconditioned apoptotic wharton's jelly mesenchymal stem cells: A superior non-cellular approach for managing acute graft-versus-host disease

Abstract

Background: The therapeutic potential of MSCs in treating aGVHD is often questioned due to their undetectable presence post-administration, highlighting importance of their secretome. Our study explores immunomodulatory effects of WJ-MSCs (viable/hypoxia preconditioned apoptotic) and their culture-conditioned media (CCM) in aGVHD. Methods: Human MSCs were isolated from Wharton's Jelly (WJ) and preconditioned under hypoxia (1% O₂) with 1μM staurosporine (STS) for 24 hours to generate WJ-MSCs^HYP+APO. CCM were collected after 48 hours from WJ-MSCs and WJ-MSCs^HYP+APO. Their immunomodulatory effects were evaluated in vitro by assessing T-cell proliferation, Tregs induction, macrophage polarization, Th1/2/17 subtypes, mitochondrial ROS, and membrane potential. A comparative analysis of CCM from WJ-MSCs, WJ-MSCs^HYP+APO, and their co-culture with aGvHD patients-derived aPBMNCs was performed using LC-MS/MS, alongside in vivo testing in a chemotherapy-based aGVHD murine model. Results and Conclusion: WJ-MSCs were more effective than WJ-MSCs^HYP+APO in inhibiting T-cell proliferation (77.23% vs 93.71%; p ≤0.0001). In contrast, WJ-MSCs^HYP+APO outperformed in enhancing Tregs (10.08% vs 16.99%; p ≤0.01) and promoting M2 macrophage polarization (19.26% vs 72.89%; p ≤0.0001). WJ-MSCs^HYP+APO-CCM was superior to WJ-MSCs-CCM in suppressing T-cell proliferation (21.03% vs 51.25%; p ≤0.001), increasing Tregs (24.73% vs 11.67%; p ≤0.0001), and promoting M2 macrophages (92.48% vs 42.85%; p ≤0.0001) while reducing mitochondrial ROS (12.638% vs 62.638%; p: ≤0.0001) and polarizing mitochondria (9.172 vs 0.872; p: ≤0.0001) effectively. It also significantly decreased the Th1/Th2 ratio (1.21 vs 12.84; p ≤0.0001). Bioinformatic analysis revealed 336 and 369 differentially expressed proteins in secretome of WJ-MSCs and WJ-MSCs^HYP+APO, respectively, when co-cultured with aPBMNCs from aGvHD patients. WJ-MSCs^HYP+APO-CCM enriched in proteins that regulate immune responses and maintain homeostasis through complement (100.0%), and carbohydrate metabolism (50.0%) while promoting M2 macrophage polarization, Tregs generation, and reducing oxidative stress, evidenced by downregulation of proteins involved in aerobic glycolysis (66.67%), gluconeogenesis (66.67%), antigen processing and activation (16.67%). It also protects against secondary infections by upregulating antimicrobial peptides (50.0%) and fine-tuned inflammation by modulating coagulation (73.08%). Both MSCs and their CCM demonstrated efficacy in alleviating GVHD in vivo model, as evidenced by increased weight, reduction in skin ruffling, hunching, eyelid closure, and diarrhea. Notably, the WJ-MSCs^HYP+APO-CCM produced a more pronounced therapeutic effect than MSCs and might be used as a non-cellular therapeutic approach for managing aGVHD.