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

Abstract

Background: 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. Methods: 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. Results: 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. Conclusion: 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.

1. Introduction

Mesenchymal stem cells (MSCs) possess self-renewal capacity, rapid proliferation, and multi-lineage differentiation potential. They exhibit low immunogenicity and immune regulatory functions, contributing to tissue repair, anti-inflammatory, anti-aging, and therapeutic interventions for autoimmune diseases [1–3]. Usually, these cells are expanded before clinical application. However, like any normal somatic cells, MSCs have a limited lifespan when cultured in vitro. Several studies have shown that long-term culture of MSCs leads to persistent changes in the cells, including reduced proliferation rates, decreased stemness, and altered differentiation potential [4, 5].

Tonsil mesenchymal stem cells (TMSCs), as a new source of MSCs, exhibit unique advantages such as minimally invasive isolation via routine tonsillectomy, derivation from young pediatric donors, and minimal ethical constraints. Thus, they have garnered significant attention in the therapeutic applications for various diseases in recent years [6–8]. For instance, prior studies have demonstrated that TMSCs encapsulated within reactive oxygen species (ROS)-releasing hydrogels significantly enhance osteogenic differentiation and promote bone regeneration, highlighting their potential in orthopedic repair [9]. Additionally, Byeongmoon Jeong and colleagues reported that tonsil-derived stem cells are capable of differentiating into functional hepatocyte-like cells, which have been further utilized for the repair of liver tissue damage [10]. Despite these promising findings, MSCs senescence remains a critical barrier to their clinical translation, as it markedly impairs their regenerative and reparative capacities. The molecular mechanisms underlying TMSCs senescence also remain unclear. Given this, our study aims to systematically investigate the biological properties and senescence-associated mechanisms of TMSCs. By doing so, we hope to provide novel mechanistic insights that can inform the development of strategies to mitigate MSC senescence, thereby enhancing their therapeutic efficacy for MSC-based disease interventions.