A single-donor proof-of-concept single-cell analysis maps heterogeneous differentiation trajectories toward cartilage-like states in human urine-derived stem cells

• Single-cell RNA sequencing of urine-derived stem cells (USCs) reveals heterogeneous differentiation trajectories toward cartilage-like states, with only a minority of cells achieving confident transcriptional similarity to mature cartilage. • Chondrogenic induction of USCs leads to reproducible cartilage-associated features, including glycosaminoglycan deposition, SOX9 upregulation, and aggrecan-positive spheroid formation. • A candidate transient transcriptional state characterized by elevated CDH1 expression and epithelial-like aggregation features is identified along the inferred differentiation trajectory. • The study is a single-donor proof-of-concept, emphasizing the need for multi-donor validation before translational conclusions can be drawn for cartilage regeneration. • Xeno-free culture conditions using human serum were evaluated, but their impact on differentiation trajectories remains insufficiently characterized.