Abstract

Introduction Urine-derived stem cells (USCs) are a promising stem-cell source because they can be obtained non-invasively and are readily available. However, their biological characteristics under hypoxic conditions remain insufficiently characterized. This study evaluated the effects of hypoxia on USCs in vitro . Methods USCs isolated from six healthy male donors were cultured under normoxic (21% O2) or hypoxic (3% O2) conditions. Colony formation, viability, proliferation, cell cycle, apoptosis, migration, surface markers, stemness-related gene expression, multilineage differentiation, cytokine secretion, and transcriptomic changes were assessed. Results Hypoxia enhanced colony formation, proliferation, migration, stemness-related gene expression, and osteogenic, adipogenic, and chondrogenic differentiation, while maintaining cell viability and not significantly affecting apoptosis or surface marker expression. VEGF secretion increased, whereas other assessed cytokines showed no significant differences. RNA sequencing identified 1,267 differentially expressed genes, including 748 upregulated and 519 downregulated genes. Hypoxia upregulated oxygen-sensing, metabolic, and HIF-1 pathways and downregulated Hippo signaling. Discussion Hypoxia at 3% O2 enhanced multiple biological characteristics of USCs without compromising viability. These findings provide a foundation for further investigation of hypoxia-conditioned USCs in regenerative medicine.