Single-cell dissection of cell hierarchies in urine-derived stem cells – application to chondrogenesis and general scalability

Developing of models of human cartilage and bone growth is essential for the study of growth disorders and to advance towards personalized therapeutic interventions. The majority of in vitro strategies depend on the use of invasively obtained mesenchymal stem cells (MSCs) or the laborious generation of induced pluripotent stem cells (iPSCs). We have now established urine-derived stem cells (USCs) as a non-invasive stem cell source capable of osteogenic and robust chondrogenic spheroid differentiation. 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. Pseudo-time analysis of chondrogenic USCs uncovered alternative chondrogenic differentiation trajectories with an ALDH1A2 ⁺ intermediate state and a TIMP3 ⁺-expressing chondrocyte-like subpopulation as the major endpoint, exhibiting cartilage-specific gene ontologies. In conclusion, a streamlined, xeno-free culture and differentiation protocol was developed, thereby establishing the basis for clinical-grade cell expansion and cartilage matrix formation. This positions USCs as a powerful tool for studying cartilage biology and a potential platform for development and use in regenerative therapies.