FOXO1-mediated cell cycle arrest enhances osteogenic differentiation of spheroid-cultured human periodontal ligament–derived multipotent mesenchymal stromal cells

Introduction: Periodontitis is a chronic inflammatory disease characterized by progressive alveolar bone loss. Although spheroid culture enhances the osteogenic and regenerative potential of human periodontal ligament-derived multipotent mesenchymal stromal cells (hPDLMSCs), the underlying molecular mechanisms remain unclear. This study aimed to investigate the transcriptional features of spheroid-cultured hPDLMSCs, with a particular focus on cell cycle regulation and Forkhead Box O (FOXO) transcription factors. Methods: Monolayer- and spheroid-cultured hPDLMSCs were subjected to transcriptome analysis using RNA sequencing. Differentially expressed genes and enriched signaling pathways were identified, followed by validation through qRT-PCR and Western blotting. Flow cytometric analysis was then performed to compare cell cycle characteristics between monolayer- and spheroid-cultured hPDLMSCs. The functional roles of FOXO1 and FOXO4 were examined using siRNA-mediated knockdown combined with cell cycle and osteogenic differentiation analyses. Results: Transcriptome analysis revealed significant alterations in cell cycle-related genes and FOXO signaling in spheroid-cultured hPDLMSCs, including downregulation of cyclin family genes associated with cell cycle progression. Spheroid culture induced cell cycle arrest, characterized by an increased G0/G1 phase population and elevated expression of FOXO1, FOXO4, and cyclin-dependent kinase inhibitors (CDKN1A, CDKN1B, and CDKN1C). FOXO1 knockdown promoted cell cycle progression and markedly reduced stemness and osteogenic marker expression as well as alkaline phosphatase activity. Similarly, FOXO4 knockdown decreased stemness-related gene expression but had a limited effect on osteogenic differentiation. Conclusions: FOXO1-mediated cell cycle regulation supports the maintenance of stemness and osteogenic potential in spheroid-cultured hPDLMSCs, suggesting that FOXO1 may serve as a promising target for periodontal tissue regeneration.