Hypoxia combined with vitamin C and N-acetylcysteine attenuates telomere shortening and replicative senescence in human umbilical cord mesenchymal stem cells

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are promising candidates for regenerative medicine; however, prolonged in vitro expansion promotes oxidative stress, replicative senescence, functional deterioration, and telomere attrition. This study evaluated whether integrating physiological hypoxia with vitamin C (VC) and N-acetylcysteine (NAC) could maintain the quality of multiple dimensions of hUC-MSCs during long-term expansion. hUC-MSCs derived from three independent umbilical cord donors were cultured from passage 3 (P3) to passage 10 (P10) under normoxic (21% O₂) or hypoxic (5% O₂) conditions with VC and/or NAC supplementation. Cell proliferation, senescence, intracellular ROS-associated fluorescence, glutathione redox status, and multilineage differentiation potential were assessed using CCK-8, SA-β-gal staining, H2DCFDA-based flow cytometry, GSH/GSSG measurements, and lineage-specific differentiation assays, respectively. Relative telomere length and telomere signal-free chromatid ends were evaluated by Q-FISH, and telomerase activity was measured using the TRAP assay. RNA-seq and functional enrichment analyses were complemented by RT‒qPCR, Western blotting, and siRNA-mediated RTEL1 knockdown. The hypoxia + VC + NAC condition produced the most consistent overall protective profile during prolonged expansion. Treated hUC-MSCs for 10 passages maintained their proliferative capacity; exhibited reduced SA-β-gal positivity and intracellular ROS-associated fluorescence; showed an increased GSH/GSSG ratio; and retained adipogenic, chondrogenic, and osteogenic differentiation potential. Q-FISH demonstrated attenuation of the passage-associated decline in relative telomere fluorescence and a reduced frequency of signal-free chromatid ends. These changes were not accompanied by a detectable increase in telomerase activity. Transcriptomic analyses revealed enrichment of HIF-1, AMPK, glutathione metabolism, DNA repair, and telomere maintenance-associated programs, together with reduced inflammatory and senescence-related signalling. Independent RT‒qPCR confirmed increased RTEL1, CTC1, and WRN expression, and Western blotting validated increased RTEL1 protein expression. RTEL1 depletion increased p21 expression and SA-β-gal activity, partially reversing the anti-senescence phenotype induced by the combined culture conditions. Hypoxic culture combined with VC and NAC provides an effective, non-genetic strategy for reducing oxidative stress, delaying replicative senescence, retaining multilineage differentiation potential, and attenuating passage-associated telomere shortening during prolonged hUC-MSC expansion. RTEL1 contributes, at least in part, to the anti-senescent phenotype induced by the combined culture conditions.