Abstract Background Mesenchymal stem cells (MSCs) hold great potential for regenerative medicine and tissue engineering. However, their clinical use is limited by low in vivo availability and rapid senescence during ex vivo expansion, posing a major challenge for scaled production. Growing evidence indicates that microenvironmental oxygen tension critically regulates MSCs fate. This study aims to systematically evaluate how different oxygen tensions modulate MSCs aging and function, providing evidence to optimize MSCs expansion protocols for clinical applications. Methods Human umbilical cord derived-mesenchymal stem cells (hUC-MSCs) were maintained under normoxic (20%O 2 ) and hypoxic (5%O 2 ) conditions through continuous passaging until senescence occurred. In vitro analysis systematically compared proliferation, cell cycle distribution, apoptosis, and senescence biomarkers across serially expanded populations from both oxygen concentrations. For in vivo evaluations, a type 2 diabetic nephropathy mouse model was used to investigate therapeutic efficacy. Mice received systemic transplantation of hUC-MSCs from each group, and renal functional recovery along with histopathological changes were assessed. Results Cells cultured under both 20%O 2 (normoxia) and 5%O 2 (hypoxia) stably expressed MSC-specific surface markers throughout serial passages. Hypoxia-cultured cells retained enhanced adipogenic and osteogenic differentiation potential, along with superior proliferative capacity and growth factor secretion, through at least 16 passages, consistently outperforming normoxic counterparts. Hypoxic conditions also attenuated replicative senescence, suppressed DNA damage, and reduced secretion of SASP-associated pro-inflammatory factors (including IL-6, IL-8, and TGF-β), while decreasing apoptosis. In a diabetic nephropathy mouse model, P16 MSCs cultured under hypoxia still significantly improved renal function and attenuated fibrosis post-transplantation, as demonstrated by reduced 24-h urinary protein, serum creatinine, and blood urea nitrogen levels compared to the normoxic MSCs group. Conclusion We provide the first in vivo evidence that hypoxic culture conditions enables high-passage hUC-MSCs(P16) to retain therapeutic potency in diabetic nephropathy. Our findings demonstrate that hypoxia effectively mitigates replicative senescence through suppression of oxidative damage and SASP secretion, thereby preserving the proliferative capacity and paracrine repair functions of long-term cultured cells.
Jia et al. (Tue,) studied this question.