A Cartilaginous Organoid System Derived From Human Expanded Pluripotent Stem Cells (hEPSCs).

The development of human organotypic models of cartilage provides essential insights into chondrogenesis and chondrocyte hypertrophy while enabling advanced applications in drug discovery, gene editing, and tissue regeneration. Here, we present a robust and efficient protocol for differentiating human expanded pluripotent stem cells (hEPSCs) into hypertrophic chondrocytes through a sclerotome intermediate. The protocol involves initial sclerotome induction, followed by 3D chondrogenic culture and subsequent hypertrophic maturation induced by bone morphogenetic protein-4 (BMP4), thyroid hormone (T3), and β-glycerophosphate. This protocol also allows for sensitive testing of the effects of various compounds on hypertrophic differentiation during the maturation process. Furthermore, we identify an α-adrenergic receptor antagonist, phentolamine, as an inhibitor of hypertrophic differentiation. This organoid system provides a practical platform for exploring cartilage hypertrophy mechanisms and testing therapeutic strategies for cartilage regeneration. Key features • This differentiation protocol generates hypertrophic chondrocytes from hEPSCs through a sclerotome intermediate. • This protocol facilitates sensitive testing of compounds during the hypertrophic maturation stage, enabling the study of molecular mechanisms and therapeutic interventions for cartilage hypertrophy. • This protocol identifies the α-adrenergic receptor antagonist phentolamine as a modulator of hypertrophic differentiation.