Keratin 8 enhances the efficacy of mesenchymal stem cells in preventing acute graft-versus-host disease

Abstract Background: Acute graft-versus-host disease (aGVHD) remains a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The development of methods that preserve graft-versus-leukemia (GVL) activity while preventing aGVHD remains a long-sought goal. Recent studies suggest that mesenchymal stem cells (MSCs), particularly human umbilical cord-derived MSCs (HUC-MSCs), are effective at mitigating aGVHD, yet their varied efficacy remains poorly understood. Methods: We performed a comprehensive analysis of MSCs heterogeneity through single-cell transcriptomic mapping across nine human tissue sources and identified the keratin 8 high-expression (KRT8 + ) subpopulation of HUC-MSCs with enhanced functional properties. We further validated the functional roles of the KRT8 + subpopulation through lentivirus-mediated KRT8 overexpression. Bulk RNA sequencing was used to analyze the genomic impact of KRT8 overexpression. Additionally, we examined the effects of KRT8 overexpression on cellular senescence via Western blotting and β-galactosidase (SA-β-gal) staining. The immunosuppressive function of HUC-MSCs was evaluated by coculturing them with CD3 + T Cells and liver organoids. Cell migration ability was assessed through Transwell assays and PKH26 dye labeling in vivo . An MHC-mismatched GVHD mice model (C57BL/6J to BALB/c) was established to test the therapeutic effect of HUC-MSCs. Clinical signs of aGVHD were monitored, and pathological changes were analyzed using hematoxylin and eosin staining. The GVL effect was evaluated through the inoculation of luciferase-transfected A20 cells and bioluminescence imaging. Results: KRT8 was expressed predominantly in HUC-MSCs. Compared with the KRT8 low-expression (KRT8 lo ) subpopulation of HUC-MSCs, the KRT8 + subpopulation presented increased stemness and reduced immunogenicity. KRT8 overexpression in HUC-MSCs significantly ameliorated aGVHD in vivo. Importantly, increased KRT8 expression in HUC-MSC did not impaire GVL activity in A20 lymphoma cells. Functional experiments further confirmed that HUC-MSCs with increased KRT8 expression significantly reduced cellular senescence by increasing enhancer of zeste homolog 2 ( EZH2 )-mediated histone h3 lysine 27 trimethylation (H3K27me3). This upregulation resulted in increased secretion of hepatocyte growth factor (HGF). This, in turn, suppressed T-cell proliferation and activation, significantly reducing liver organoid damage in vitro . Conclusions: Our results suggest that the infusion of KRT8 + HUC-MSCs enhances the efficacy of preventing aGVHD but does not interfere with the GVL effect. This strategy represents a promising therapeutic advancement in the setting of allo-HSCT.