The Effect of DNA Demethylation and IFN‐γ Licensing on HLA‐G Expression in Wharton's Jelly–Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) play a central role in regenerative medicine and immunotherapy owing to their multilineage differentiation capacity and potent immunomodulatory properties. Among MSC sources, Wharton's jelly-derived MSCs (WJ-MSCs) are particularly attractive due to their non-invasive procurement, high proliferative potential, and low immunogenicity. Accumulating evidence indicates that human leukocyte antigen G (HLA-G), a non-classical major histocompatibility complex (MHC) class I molecule, is a key mediator of MSC-induced immune tolerance. However, the molecular mechanisms regulating HLA-G expression in MSCs remain incompletely understood. Epigenetic regulation, particularly DNA methylation within the HLA-G promoter region, has emerged as a critical determinant of HLA-G transcriptional activity. In addition, interferon-gamma (IFN-γ) is widely recognised as a licensing factor that enhances the immunomodulatory functions of MSCs. In this study, we investigated the effects of DNA demethylation followed by IFN-γ licensing, applied either sequentially or concurrently, on HLA-G expression in WJ-MSCs at early and later culture passages. Our findings demonstrate that DNA demethylation significantly enhances HLA-G expression in early-passage WJ-MSCs, indicating preserved epigenetic plasticity at this stage. In contrast, IFN-γ licensing did not consistently increase HLA-G levels unless applied in an epigenetically permissive context. Notably, concurrently applied DNA demethylation and IFN-γ licensing resulted in elevated HLA-G protein expression, highlighting the importance of epigenetic priming for effective cytokine responsiveness. In later-passage WJ-MSCs, however, responsiveness to both epigenetic modulation and IFN-γ licensing was reduced and highly variable. Collectively, these results indicate that HLA-G expression in WJ-MSCs is regulated by a dynamic interplay between epigenetic status and cytokine licensing, with passage-dependent epigenetic remodelling critically influencing immunomodulatory capacity. These findings underscore the importance of epigenetic context and passage selection when optimising MSC-based immunoregulatory strategies for clinical applications.