Replicative senescence in amniotic fluid-derived mesenchymal stem cells and its impact on their immunomodulatory properties.

The expansion of mesenchymal stem cells (MSCs) for clinical applications is often limited by replicative senescence, a growth arrest induced by various stresses during in vitro culture, yet its impact on the immunomodulatory properties of MSCs remains unclear. This study derived MSCs from the amniotic fluid (AF-MSCs) of seven first-trimester pregnancies, characterized them through flow cytometry, and evaluated their osteogenic differentiation potential before expanding the cells to compare immunoregulatory gene expression in proliferative and senescent states. Additionally, an assessment of the adipogenic differentiation potential of AF-MSCs from three samples was conducted following their recovery from approximately 9 months of cryopreservation, with results showing that these recovered cells retain the capacity for adipogenic differentiation. Molecular analysis revealed no significant differences in the expression of key immunoregulatory genes, such as TGFβ, IL-10, IDO, and VCAM-1, between proliferative and senescent cells, although senescent cells showed downregulation of FASL and upregulation of IL-6, COX1, and HLA-G. Markers of cell proliferation, including FOXM1 and B-MYB, were significantly downregulated in senescent cells, confirming the progression of replicative senescence. Despite expectations, the results indicated that some immunomodulatory markers remained stable or were even enhanced in senescent AF-MSCs. These findings highlight the resilience of AF-MSC immunomodulatory properties during prolonged in vitro expansion, supporting their potential for therapeutic applications despite the challenges posed by replicative senescence.