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

Abstract

Background Urine-derived stem cells (USCs) represent an accessible and non-invasive cell source with reported chondrogenic differentiation potential. However, the cellular heterogeneity and transcriptional dynamics underlying USC differentiation remain incompletely understood, limiting their translational interpretation. Methods We combined functional differentiation assays with single-cell RNA sequencing to characterize USC differentiation at both phenotypic and transcriptional levels. Chondrogenic and osteogenic differentiation were assessed using histological staining, quantitative PCR, and three-dimensional spheroid cultures. Single-cell transcriptomic analysis was performed on integrated datasets of undifferentiated and differentiated USCs, followed by pseudotime trajectory inference and mapping to a human cartilage reference atlas. Results Chondrogenic induction resulted in reproducible acquisition of cartilage-associated features, including glycosaminoglycan-rich extracellular matrix deposition, increased expression of SOX9, and formation of aggrecan-positive spheroids. In this donor, single-cell analysis mapped an inferred differentiation trajectory from proliferative states towards differentiated populations, although the fine-grained pseudotemporal ordering was sensitive to analytical choices and is therefore interpreted qualitatively. Along this inferred trajectory, we identified a candidate transient transcriptional state associated with elevated CDH1 expression and epithelial-like aggregation features. Probabilistic mapping to a human cartilage reference atlas indicated that overall mapping confidence was low (median prediction score 0.34) and that only a minority of cells showed confident transcriptional similarity (prediction score ≥ 0.5) to mature/articular cartilage-associated reference states (7.8% of all cells and 17.6% of chondrogenically induced cells). This confident similarity was concentrated in a few clusters at the differentiated end of the trajectory rather than representing the bulk of the culture, and label-transfer confidence was not equated with chondrocyte identity. Despite this enrichment, differentiated populations exhibited transcriptional heterogeneity, including subsets

1. Introduction

Understanding human cartilage development and its dysregulation in skeletal disorders remains a major challenge, largely due to the limited accessibility of primary human cartilage tissue [1–3]. Functional studies of chondrogenesis often rely on mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs), which require invasive sampling or complex reprogramming procedures and may not fully recapitulate physiological differentiation processes [4–7].

Urine-derived stem cells (USCs) have emerged as an attractive cell source due to their non-invasive accessibility and robust expansion capacity [8–10]. Previous studies have demonstrated multilineage differentiation potential in USCs, including chondrogenic differentiation [11, 12]. However, the cellular composition of cultured USCs and the transcriptional programs underlying their differentiation remain incompletely understood [11, 13, 14]. In particular, it remains unclear whether USC-derived cells follow structured differentiation trajectories and to what extent they resemble defined cartilage-associated transcriptional states. Moreover, inter-individual variability in USC composition and differentiation potential has been reported, highlighting the need for careful interpretation of findings derived from individual samples [13–15].

Recent advances in single-cell RNA sequencing (scRNA-seq) enable high-resolution dissection of cellular heterogeneity and lineage progression [16, 17]. These approaches provide the opportunity to map differentiation processes at the level of individual cells and to identify transitional states and regulatory programs [18]. However, to our knowledge, such analyses have not been systematically applied to USC chondrogenesis. These approaches are particularly relevant for systems such as USC differentiation, where cellular heterogeneity and intermediate states are not resolved by bulk analyses. In addition, the translational potential of USCs depends on clinically compatible culture conditions.