Alu methylation level, morphological, and senescence changes during in vitro aging of human dental pulp stem cells

Introduction: Human dental pulp stem cells (DPSCs) are pivotal in tissue engineering and cell-based therapies due to their significant differentiation potential and accessibility. A major challenge in in vitro cell expansion is their replicative senescence, which impacts their regeneration and differentiation capabilities. While genetic factors influence these processes, epigenetic regulations such as Alu methylation also play crucial roles. Changes in Alu methylation have been associated with human aging and age-related diseases, contributing to cellular dysfunction and stem cell senescence. Despite this, the implications of Alu methylation alterations in stem cell senescence remain underexplored. This study focuses on examining Alu methylation during the replicative senescence of DPSCs. Methods: The methylation status of Alu elements in serially passaged, long-term cultured human DPSCs was assessed using combined bisulfite restriction analysis. Morphological changes and indicators of replicative senescence were also evaluated. DPSCs were divided into three passage groups for analysis: early, middle, and late. Methylation levels across these groups were compared to identify trends correlating with passage number. Results: Significant morphological changes and markers of replicative senescence were observed predominantly in the late-passage DPSCs. These cells exhibited notably lower levels of Alu methylation and higher proportions of hypomethylated Alu CpG sites compared to those in early passages. Conclusion: The study confirmed that alterations in Alu methylation are evident in the replicative senescence of human DPSCs, suggesting that epigenetic modifications could influence the aging process of these cells and potentially impact their therapeutic efficacy.