Patient-Derived iPSC-MSC Modelling Reveals Reversible GSK3-Mediated Suppression of Mesenchymal Lineage Differentiation in Legg-Calvé-Perthes Disease

Abstract

Legg-Calvé-Perthes disease (LCPD) is an idiopathic paediatric osteonecrosis characterized by avascular injury to the femoral head, resulting in structural collapse and early osteoarthritis. Although mesenchymal stem cell (MSC) dysfunction has been implicated, the underlying mechanisms remain unclear due to the absence of human disease models. Here, we generated patient-specific induced pluripotent stem cells (iPSCs) from dermal fibroblasts of LCPD patients and differentiated them into mesenchymal stem cells (iMSCs) to establish an in vitro platform for mechanistic interrogation. LCPD-iMSCs exhibited hallmark disease-associated phenotypes, including concurrent suppression of osteogenic, adipogenic and chondrogenic differentiation, accompanied by enhanced phosphorylation of GSK3α/β, reduced β-catenin abundance and attenuated canonical Wnt signalling. Transcriptomic analyses revealed alterations in extracellular matrix organization, adhesion pathways and PI3K-Akt/MAPK signalling, consistent with a cellular environment predisposed to impaired mesenchymal function. Pharmacological inhibition of GSK3 using lithium chloride restored β-catenin levels, improved osteogenic and adipogenic marker expression, and enhanced matrix deposition, demonstrating functional reversibility of the LCPD phenotype. These findings identify GSK3-mediated β-catenin destabilization as a central regulator of MSC lineage impairment in LCPD and establish a patient-derived iPSC-MSC model as a valuable platform for mechanistic and therapeutic exploration in paediatric osteonecrosis.