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.

Figure 1. Histology of multi-tissue organoids (MTOs) at 8, 12, and 30 weeks. a. MTOs at 8 weeks show a developing cartilage nodule with diffuse Alcian blue staining (blue) of the early cartilaginous matrix, diffuse labeling for aggrecan (ACAN, brown) in cells and matrix, and type II collagen labeling (COL2A1, brown) in the cartilaginous matrix. Size bars = 1000 µm, 200 µm, 50 µm. Arrows indicate areas magnified in the second and third rows. b. MTOs at 12 weeks show maturing cartilage with increased Alcian blue positive cartilaginous matrix separating chondrocytes, moderate diffuse staining for type II collagen, and diffuse aggrecan labeling. Arrows indicate areas magnified in the second and third rows. Size bars = 1000 µm, 200 µm, 50 µm. c. MTOs after 30 weeks in culture show further maturation to hyaline cartilage morphology with chondrocytes surrounded by an abundant matrix with diffuse Alcian blue and type II collagen staining, and aggrecan staining apparent in the periphery. Arrows indicate areas magnified in the second and third rows. Size bars = 1000 µm, 200 µm, 50 µm.

Figure 2. Transcription signatures in MTOs compared to human lower limb bud cartilage at 6 weeks of development (hindlimb bud week-6), fetal weeks 14-17, adolescent knee cartilage (kao), and adult knee and costal cartilage (kau). a. Principal component analysis (PCA) on 325 chondrocyte-specific genes shows closest matching for wk15 MTOs and human lower limb bud cartilage (green ellipse). b. Pearson correlation plot of 325 chondrocyte-specific genes showing closest match for wk15 MTOs and human lower limb bud cartilage, but with overall close matching with all stages of human cartilage. c. Representative comparisons of marker transcripts (COL2A1, COL9A1, COL6A2, COL11A1, COL10A1, MMP13, ACAN, CD44, PRG4) in MTOs and human cartilage at each growth stage. Wk15 MTOs showed closest matches to all stages of human cartilage. MTOs – multi-tissue organoids; GPC – growth plate chondrocytes.

Figure 3. Single-cell RNA-sequencing data showing uniformity of cell populations within and across MTOs. a. Uniform Manifold Approximation Projection (UMAP) and b. Split-bar graph generated using scRNA-seq data of cells derived from 3 batches of MTOs. Results show homogeneity at the cluster levels across the MTO batches, with clusters 0, 1, 2, and 4 representing cells within the chondrogenic lineage, cluster 3 corresponding to neurogenic lineage cells, cluster 5 indicative of non-specific proliferating cells, and cluster 6 containing cells consistent with a mesenchyme phenotype. c. SingleR algorithmic labeling of the unique cell populations found across MTOs at the single cell level in comparison to undifferentiated hiPSC control based on the Human Primary Cell Atlas (HPCA) reference. d. Dot plot depicting enriched biological process terms related to chondrogenesis in the top 300 upregulated marker genes across chondrogenic-lineage clusters. e. Feature plots of positive and negative chondrogenic markers, as well as positive neurogenic markers. f. Immunocytochemistry images of singularized cells of MTOs verifying the expression of chondrogenic markers, type VI collagen (Col VI) and Aggrecan (ACAN) at the protein level, co-stained with DAPI. Scale bar = 100 µm g. Quantification of ICC images of COL VI, ACAN, and pluripotent markers OCT4 and SSEA4. Data represent mean ± SD from each MTO biological replicate (n=8) in comparison to respective positive control (n=3). Statistical significance was assessed by Tukey’s HSD test. (* = p < 0.05).

« Back to ACR Convergence 2025

ACR Meeting Abstracts - https://acrabstracts.org/abstract/consistent-method-to-generate-hyaline-cartilage-from-human-induced-pluripotent-stem-cell-derived-multi-tissue-organoids/