0027: Consistent Method to Generate Hyaline Cartilage from Human Induced Pluripotent Stem Cell-Derived Multi-Tissue Organoids

Background/Purpose: Existing methods to produce hyaline cartilage from human induced pluripotent stem cells (hiPSCs) face significant limitations, such as complex culture conditions, instability of the cartilage phenotype, and variability across batches. Our objective was to develop a robust, consistent, and animal component-free (xeno-free) method to generate hyaline cartilage through self-organized multi-tissue organoids (MTOs).

Methods: We developed a xeno-free culture method for differentiating hiPSCs to MTOs in which hyaline cartilage differentiation emerged by 8 weeks in culture. MTOs were cultured under defined conditions for up to 15 weeks, with evaluations conducted at multiple time points. Differentiation into cartilage was characterized by histological analysis for cartilage-specific matrix components. Independent bulk RNA sequencing (bulk-seq) at weeks 8, 11, and 15 was performed to profile gene expression changes over time, while single-cell RNA sequencing (scRNA-seq) was independently employed to elucidate cellular heterogeneity and specific differentiation trajectories within the organoids. Both sequencing approaches were essential for thoroughly characterizing hiPSC-derived cartilage.

Results: Histological analysis confirmed robust cartilage formation at 15 weeks, evidenced by intense immunohistochemical staining for collagen type II and aggrecan, indicative of characteristic cartilage extracellular matrix deposition (Fig 1). Bulk-seq analyses comparing weeks 8, 11, and 15 showed significant maturation of cartilage phenotype, with notable upregulation of cartilage-specific genes, including COL2A1 and ACAN (p< 0.01), and downregulation of pluripotency markers (p< 0.001) by week 15. Further bulk-seq analysis revealed that the gene expression profile of week-15 hiPSC-derived cartilage closely resembled that of developing human cartilage (Fig 2), and was distinct from adult cartilage, suggesting a developmental progression in vitro. Single-cell RNA sequencing identified minimal off-target differentiation, with chondrocytes constituting the majority ( >85%) of cells and minimal residual pluripotent cell population ( > 3%) (Fig 3). Additional staining exhibited limited pluripotent marker expression across batches, with 6.94 ± 1.65% Oct4+ cells and 17.54 ± 4.68% SSEA4+ cells (Fig 3).

Conclusion: We present a reproducible, xeno-free method for generating high-quality hyaline cartilage from hiPSC-derived MTOs. The integrated use of bulk-seq and scRNA-seq validated the chondrogenic differentiation process, demonstrating stable cartilage phenotype, minimal off-target differentiation, and developmental progression, thus making this approach highly promising for cartilage tissue engineering and the basis for potential clinical applications.