Single-cell atlas of human urine-derived stem cell chondrogenesis enables a non-invasive, xeno-free platform for translational cartilage and skeletal disease research

Background Clinically compatible, non-invasively harvested stem cell sources are needed to model human skeletal disorders and advance regenerative strategies. Human urine-derived stem cells (USCs) offer patient-specific accessibility, yet their developmental hierarchy and chondrogenic mechanisms remain poorly defined. Methods USCs were isolated, expanded and profiled in detail using single-cell RNA sequencing with lineage reconstruction to map progenitor states and differentiation trajectories. Chondrogenesis was induced in two-dimensional (2D) and three-dimensional (3D) systems. To support translational use, we established a fully xeno-free expansion and differentiation workflow using autologous human serum and benchmarked it against conventional serum-based conditions. Results Single-cell analysis resolved a structured USC hierarchy originating from MYC/E2F4-regulated TOP2A⁺ proliferative progenitors, progressing through an ALDH1A2⁺ retinoic-acid–responsive intermediate, and culminating in TIMP3⁺ chondrocyte-like cells exhibiting high transcriptional similarity to native cartilage. This trajectory featured coordinated activation of canonical chondrogenic regulators (SOX9, SOX5, SOX6) and enrichment of extracellular matrix programs associated with cartilage formation. Under xeno-free autologous serum conditions, USCs preserved proliferative capacity, enhanced mesenchymal condensation, and generated matrix‑rich cartilage-like constructs in 2D and 3D with superior maturation signatures compared with standard culture conditions. Conclusions We provide a mechanistic single-cell atlas of human USC chondrogenesis and establish USCs as a non-invasive, patient-compatible, and fully xeno-free stem cell platform for translational cartilage research, skeletal disease modelling, and personalized regenerative medicine applications.