Chondrocyte dedifferentiation during monolayer expansion is a major barrier to producing clinically reliable cells for cartilage regeneration. The upstream regulators governing this phenotypic instability are not fully defined. This study identifies WNT inhibitory factor 1 (WIF1) as a potential key molecular determinant of chondrocyte stability. Human auricular chondrocytes (HACs) were expanded to early (P2) and late (P7) passages. Transcriptomic profiling, qPCR, GAG quantification, and histology were performed in 2D monolayer and 3D atelocollagen pellet cultures. WIF1 function was interrogated using lentiviral knockdown and overexpression. β-catenin, p -SMAD2/3, p -JNK, COL1A1, COL2A1, and WIF1 were evaluated by immunohistochemistry. Late-passage HACs showed marked dedifferentiation, including reduced WIF1, COL2A1, ACAN, and SOX9 with concomitant increases in COL1A1, ACTA2, and MMP9. WIF1 knockdown in early-passage cells reproduced this phenotype, inducing β-catenin nuclear accumulation, increased SMAD2/3 and JNK activation, diminished GAG synthesis, and loss of cartilage-specific ECM architecture. Conversely, WIF1 overexpression in P7 chondrocytes restored chondrogenic markers, enhanced proteoglycan-rich ECM formation, and suppressed fibroblastic gene expression. WIF1 overexpression re-established a signaling profile resembling early-passage cells, characterized by low β-catenin, reduced p -SMAD2/3, and attenuated p -JNK. WIF1 functions as a critical upstream regulator that maintains chondrocyte identity by simultaneously suppressing WNT/β-catenin, TGF-β/SMAD, and JNK stress signaling. Its loss is a hallmark of dedifferentiation, while its restoration reverses transcriptional drift and rescues ECM-producing capacity. These findings highlight WIF1 as a promising therapeutic target to improve the stability of expanded chondrocytes and enhance the quality of regenerative cartilage constructs. • WIF1 suppresses canonical and non-canonical WNT pathways in human chondrocytes. • WIF1 overexpression maintains chondrogenic gene expression through late passages. • Downregulation of WIF1 activates β-catenin, TGF-β/SMAD, and JNK signaling. • WIF1 serves as a key multi-pathway regulator to prevent chondrocyte dedifferentiation.
Chu et al. (Sun,) studied this question.