Serial Passaging Drives Inflammatory–Metabolic Reprogramming and Replicative Senescence in Human Umbilical Cord Mesenchymal Stem Cells

expansion alters their molecular and functional properties. Understanding replicative senescence trajectories is critical for optimizing cell manufacturing. We performed longitudinal RNA sequencing across passages P3, P6, P9, P12, and P15, using P6 as a clinical reference. Transcriptomic dynamics were bioinformatically analyzed and validated by targeted quantitative real-time PCR. Functional assays assessed senescence-associated β-galactosidase (SA-β-gal) activity, mitochondrial membrane potential, iron accumulation, and glucose uptake. Transcriptomic profiling revealed progressive, passage-dependent divergence with an inflection point after P9. Late-passage cells (P12, P15) exhibited coordinated upregulation of proinflammatory pathways (TNFα, IL-17, and MAPK) and senescence programs, alongside downregulation of oxidative phosphorylation and autophagy. Functional validations confirmed that P12 cells acquired a senescent phenotype: enhanced SA-β-gal, mitochondrial depolarization, iron overload, and a glycolytic shift. Molecular analyses corroborated increased expression of senescence-associated secretory phenotype factors (IL6, CCL2) and glucose transporters (SLC2A1), with suppression of mitochondrial genes (MT-CO2, MT-ND4). P6 cells maintained stable profiles and robust integrity. Serial passaging drives hUC-MSCs toward senescence through bidirectional reprogramming of inflammation and metabolism. Our findings identify P6-P9 as a safe manufacturing window and propose metabolic and iron-related markers for quality control of clinical-grade MSCs, ensuring therapeutic potency.