Phenotypic alterations and PI3K-AKT pathway regulation in senescence of human tonsil mesenchymal stem cells

Tonsil mesenchymal stem cells (TMSCs) are a promising regenerative medicine source but require continuous subculturing for expansion. Long-term expansion in vitro induces cellular senescence, impairing their function. This study aimed to elucidate senescence-related phenotypic alterations and regulatory mechanisms in human tonsil-derived mesenchymal stem cells.

Human-derived TMSCs were isolated from palatine tonsils, cultured under standard conditions, and characterized for mesenchymal markers. Senescence-associated changes were evaluated across early (P1-P5) and late passages (beyond P10). Proliferation capacity was assessed via CCK-8 assays, while senescence-associated β-galactosidase (SA-β-gal) activity and protein levels of p16, p53, and p21 were quantified. RNA sequencing identified differentially expressed genes (DEGs) between young and senescent TMSCs, followed by KEGG pathway enrichment analysis. Key findings were validated by measuring the p-Akt/Akt ratio via Western blot.

TMSCs showed a progressive decline in proliferative capacity with increasing passages. SA-β-gal staining revealed a significantly higher percentage of positive cells in late-passage TMSCs compared to early-passage cells. Expression levels of P16, P53, and P21 proteins were markedly upregulated in aged TMSCs. KEGG analysis of DEGs indicated significant enrichment in the PI3K-Akt signaling pathway, ECM-receptor interaction, and calcium signaling. Consistent with this, Western blot confirmed a significantly increased p-Akt/Akt ratio in senescent TMSCs.

Our research proved that replicative senescence in TMSCs is associated with PI3K-Akt pathway activation, which likely orchestrates senescence via p16 and p53-p21 cascades. These findings provide new insights into the mechanisms of stem cell aging and suggest potential molecular targets for developing strategies to delay senescence in TMSCs for regenerative medicine.