Core binding factor β preserves early chondrogenic identity and prevents hypertrophic transition in cartilage organoids formation

Human-induced pluripotent stem cells (hiPSCs) represent a promising cell source for cartilage regeneration because of their self-renewal capacity and chondrogenic potential. However, the propensity of hiPSC-derived chondrocytes to undergo hypertrophic maturation remains a major obstacle to generating stable articular cartilage. Here, we identified core binding factor β (CBFβ) as a critical regulator of early chondrogenic identity and a suppressor of hypertrophic transition during hiPSC-derived cartilage organoid formation. CBFβ expression was markedly diminished in degenerative articular cartilage from both human osteoarthritis (OA) specimens and mouse OA models, and cartilage-specific ablation of Cbfβ accelerated cartilage structural deterioration and matrix loss. Notably, CBFβ was secreted by non-mineralizing cells, including chondrocytes and vascular smooth muscle cells, suggesting an autocrine/paracrine regulatory role. Pharmacological inhibition with Brefeldin A reduced extracellular CBFβ levels, whereas blockade of exosome release by GW4869 had minimal effect, indicating a secretion-associated mechanism independent of exosomes. Recombinant human CBFβ (rhCBFβ) treatment enhanced the chondrocyte phenotype by upregulating early chondrogenic markers (SOX9, COL2A1) while suppressing hypertrophic and catabolic markers (RUNX2, MMP13). In hiPSC-derived cartilage organoids, rhCBFβ enhanced matrix deposition and increased COL2A1 and SOX9 expression. Transcriptomic profiling and qRT-PCR validation further demonstrated that rhCBFβ activated cartilage matrix-associated and anti-hypertrophic transcriptional programs, including upregulation of PTHRP, HIF1α, HDAC4, MGP, CILP, and ALK5, together with suppression of RUNX2. Collectively, these findings establish CBFβ as a key regulator of articular cartilage homeostasis and highlights its therapeutic potential for cartilage regeneration in OA. The ability of rhCBFβ to preserve early chondrogenic identity while preventing hypertrophic maturation offers a promising strategy for cartilage tissue engineering. Further preclinical studies are warranted to evaluate its efficacy and accelerate clinical translation for OA therapy.