Single-cell dissection of urine-derived stem cell hierarchies reveals robust chondrogenesis and clinical scalability

Development of models of human cartilage and bone growth is essential for the study of growth disorders and pave the way for personalized therapeutic interventions. The majority of in vitro strategies depend on the use of invasively obtained mesenchymal stem cells (MSCs) or the generation of intricate induced pluripotent stem cells (iPSCs). We have established urine-derived stem cells (USCs) as a non-invasive stem cell source capable of osteogenic and robust chondrogenic differentiation in 3D. Single-cell RNA sequencing of USCs revealed a hierarchy originating from parietal epithelial cells of the kidney, with a proliferative TOP2A⁺ subpopulation governed by MYC and E2F4 regulatory networks. Integration of chondrogenic USCs with a human cartilage single-cell reference has led to the identification of a TIMP3⁺-expressing chondrocyte-like subpopulation that exhibits cartilage-specific gene ontologies. Pseudotime analysis uncovers an ALDH1A2⁺ intermediate state and alternative chondrogenic differentiation trajectories. In conclusion, a streamlined, xeno-free culture and differentiation protocol has been developed, thereby establishing a foundational framework for clinical-grade expansion and cartilage matrix formation. This positions USCs as a powerful model for cartilage biology and a platform for regenerative therapies.