Oculodentodigital dysplasia, or ODDD, is an ultra-rare autosomal dominant disorder caused by germline variants in GJA1. Patients typically show a pleiotropic constellation of features affecting the eyes, teeth, digits, and sometimes the central nervous system, with severity varying dramatically even within the same family. Connexin 43 is the most ubiquitously expressed connexin in the human body, forming gap junction channels that allow direct cytoplasmic exchange between adjacent cells. But the protein’s long C-terminal tail also does channel-independent work, physically associating with signaling partners such as β-catenin and various kinases. It is this second, scaffolding role that the new research places at the center of the disease mechanism.

The team, led by researchers at Peking Union Medical College Hospital and Children’s Hospital of Soochow University, recruited two families with classic ODDD presentations. Proband 1, a young girl, displayed short palpebral fissures, hypoplastic alae nasi, sparse hair, bilateral syndactyly of the fourth and fifth fingers, and dental anomalies including microdontia and enamel hypoplasia. Her affected younger brother carried the same craniofacial and hand phenotype, while their mother showed only mild bilateral brachydactyly of the fifth fingers—a striking example of intra-familial variability. Proband 2 presented similar features along with syndactyly of the toes, and whole-genome sequencing revealed his variant arose de novo, absent from both clinically healthy parents. Sanger sequencing confirmed heterozygous missense variants, c.253G>C in Family 1 and c.605G>A in Family 2, both segregating perfectly with disease status.

What makes the study unusual is its reliance on primary patient-derived cells rather than engineered model systems. The researchers cultured skin fibroblasts from both patients and mesenchymal stromal cells, or MSCs, harvested from discarded surgical bone and skin tissue collected during syndactyly release and corrective osteotomy procedures. Controls came from age- and sex-matched pediatric patients undergoing hand trauma surgery at the same anatomical site, with whole-genome sequencing used to exclude any pathogenic variants in known disease genes. Flow cytometry confirmed the MSC identity, with cells positive for CD90, CD105, and CD73 and negative for CD45, meeting the International Society for Cellular Therapy minimal criteria.

RNA sequencing on patient fibroblasts and MSCs revealed a transcriptional landscape split cleanly in two. Downregulated gene sets were dominated by Wnt signaling, extracellular matrix–receptor interaction, osteoblast differentiation, DNA replication, and cell cycle regulation. Upregulated sets lit up with inflammatory response genes, cytokine-mediated signaling, JAK-STAT signaling, tumor necrosis factor response, and ECM structural constituents. The team then validated these patterns at the protein level using RT-qPCR, Western blotting, immunofluorescence, and ELISA. In both fibroblasts and MSCs, mRNA levels of canonical Wnt targets—AXIN2, CCND1, PCNA, and c-MYC—fell sharply. Protein levels of Cx43, β-catenin, and IκBα dropped in parallel, while the ratio of phosphorylated p65 to total p65 rose, a signature of NF-κB pathway activation.

The inflammatory profile that emerged is the study’s most surprising finding. Interleukin-6 expression and secretion surged in patient cells, yet CXCL8—better known as IL-8—was paradoxically and significantly reduced, despite NF-κB being switched on. Interferon-stimulated genes MX1 and IFIT1 were simultaneously elevated. The researchers traced this uncoupling to c-MYC, the Wnt-dependent transcription factor whose loss appears to lift normal repression on MMP3 while simultaneously starving CXCL8 of a required activator. MMP3, or stromelysin-1, is a matrix metalloproteinase that degrades components of the extracellular matrix, and its abnormal upregulation points to excessive ECM remodeling—a process that could plausibly underlie the tissue fusion seen in syndactyly, though the authors caution that this connection remains speculative and requires in vivo validation.

Functional assays confirmed the molecular story has real cellular consequences. Transwell migration assays showed patient fibroblasts and MSCs crossed basement membranes at markedly reduced rates, while EdU incorporation revealed suppressed proliferation. These defects align with the coordinated downregulation of CCND1, PCNA, and c-MYC, key regulators of the G1/S cell cycle transition, suggesting GJA1 deficiency stalls cells before they enter DNA synthesis. Differentiation assays added another layer: patient MSCs showed compromised osteogenic capacity, with reduced alkaline phosphatase activity and diminished Alizarin Red S mineralization, alongside aberrantly enhanced adipogenic potential measured by Oil Red O staining. This lineage shift mirrors the classical biology of Wnt signaling, which normally maintains MSC stemness, promotes bone formation, and suppresses fat cell differentiation—offering a cellular explanation for the bone hypoplasia seen in ODDD patients.

To establish causality rather than mere correlation, the team ran bidirectional genetic interventions. Knocking down GJA1 with siRNA in normal control cells, reducing expression to roughly the level seen in patient cells, faithfully reproduced the entire patient phenotype: Wnt suppression, the IL-6-high/CXCL8-low inflammatory bias, elevated MMP3, impaired migration, and skewed differentiation. Conversely, overexpressing wild-type connexin 43 in patient cells substantially reversed these defects, restoring IκBα levels, reducing p65 phosphorylation, suppressing MMP3, and rescuing migration. A second round of experiments isolated c-MYC’s role directly: silencing c-MYC in normal MSCs reproduced the CXCL8 collapse and MMP3 surge, while overexpressing c-MYC in patient MSCs rescued both, establishing c-MYC as a necessary and sufficient regulator of these two genes downstream of the GJA1/Wnt axis.

Pharmacological experiments then mapped the hierarchy of the pathway. Treating patient cells with CHIR99021, a selective GSK-3β inhibitor that activates canonical Wnt signaling, restored c-MYC expression, corrected the CXCL8 deficit, suppressed MMP3, reduced IL-6 secretion, and improved both migration and osteogenic differentiation. In contrast, a neutralizing antibody against IL-6 acted only on downstream readouts: it dampened the interferon response but failed to restore c-MYC, could not rescue CXCL8, and left MMP3 overexpression untouched. The combination of both agents produced the strongest overall rescue, simultaneously targeting the core upstream defect and the downstream inflammatory overactivation. The comparison makes a clear translational argument: blocking a single cytokine treats symptoms, but reactivating the Wnt/c-MYC axis addresses the disease’s root signaling failure.

The authors are candid about the study’s limits. Fibroblasts came from only two patients and MSCs from one, so transcriptomic findings are explicitly framed as exploratory, and technical replicates from a single donor cannot substitute for independent biological replicates. The researchers also could not perform promoter-reporter or chromatin immunoprecipitation experiments to demonstrate direct c-MYC binding at the CXCL8 or MMP3 promoters, again because of the scarcity of pediatric patient cells. Connexin 43’s broad expression in heart, brain, and skin means long-term neurological and cardiac follow-up of the two families is warranted, even though neither child currently shows overt deficits in those systems. Still, the work delivers a coherent new pathogenic framework for ODDD—one in which a broken gap junction protein silently rewires Wnt, NF-κB, and matrix remodeling all at once—and it nominates Wnt activation, rather than isolated cytokine blockade, as the therapeutic strategy most worth pursuing in this rare and poorly understood disorder.

Subject of Research: Molecular mechanisms of GJA1-related oculodentodigital dysplasia in patient-derived fibroblasts and mesenchymal stromal cells

Article Title: GJA1 dysfunction suppresses Wnt/c-MYC signaling and promotes inflammatory imbalance and MMP3-associated ECM remodeling in fibroblasts and mesenchymal stromal cells from patients with ODDD

Article References: Tao, S., Zhao, B., Wang, X., Shen, X., & Zhao, X. (2026). GJA1 dysfunction suppresses Wnt/c-MYC signaling and promotes inflammatory imbalance and MMP3-associated ECM remodeling in fibroblasts and mesenchymal stromal cells from patients with ODDD. Frontiers in Cell and Developmental Biology, 14, Article 1959966. https://doi.org/10.3389/fcell.2026.1959966

Image Credits: AI Generated

DOI: 10.3389/fcell.2026.1959966

Keywords: oculodentodigital dysplasia, GJA1, connexin 43, Wnt signaling, c-MYC, NF-κB, MMP3, mesenchymal stromal cells, fibroblasts, rare disease, osteogenic differentiation, CHIR99021

The research team, working at the Korea Research Institute of Bioscience and Bioscience-affiliated laboratories, took an approach that sidesteps a long-standing obstacle in the field. Bone marrow from children with Legg-Calvé-Perthes disease is scarce and difficult to study, and even when mesenchymal stromal cells, the body’s principal skeletal progenitors, are isolated from osteonecrotic bone, they carry the imprint of a diseased, inflamed, poorly perfused tissue environment. Any abnormality observed in such cells could reflect that hostile microenvironment rather than an intrinsic flaw. To separate the two, the researchers reprogrammed skin fibroblasts from two patients with the disease, and from a healthy donor, into induced pluripotent stem cells using a non-integrating Sendai virus system that delivers the reprogramming factors OCT4, SOX2, KLF4 and c-MYC. Because skin cells can be reset to an embryonic-like state and then steered into mesenchymal stem cells under identical, standardized conditions, any difference that emerges between patient-derived and healthy cells must be written into the cells themselves rather than imposed by the tissue around them.

The quality control on this cellular reset was rigorous. The resulting induced pluripotent stem cell lines formed compact colonies with the characteristic morphology of embryonic stem cells, stained strongly for alkaline phosphatase, and expressed the canonical pluripotency markers OCT4, NANOG, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4 at levels comparable across all three lines. Quantitative PCR confirmed expression of OCT4 and REX1 in the undifferentiated state, and when the cells were allowed to spontaneously differentiate into embryoid bodies, they activated ectodermal markers such as SOX1 and PAX6, mesodermal markers including HAND1 and PDGFRA, and endodermal markers SOX7 and AFP. Immunostaining for TUJ1, NESTIN, DESMIN, alpha-SMA, FOXA2 and SOX17 confirmed protein-level differentiation into all three germ layers, and teratoma assays in immunodeficient mice produced tissues representing ectoderm, mesoderm and endoderm. G-band karyotyping showed normal diploid chromosomes in every line, ensuring that any phenotype found later was not an artefact of culture stress or genomic instability.

With validated stem cell lines in hand, the team differentiated them into induced mesenchymal stem cells using a defined, xeno-free protocol, first patterning the cells toward the mesodermal lineage for four days and then maturing them for a further seventeen days on fibronectin-coated plates. Flow cytometry confirmed that the resulting cells expressed the canonical mesenchymal markers CD73 and CD105 and lacked the haematopoietic markers CD34 and CD45, consistent with bona fide MSC identity. One subtlety stood out: the two patient-derived lines expressed CD90, a surface glycoprotein involved in adhesion and MSC immunophenotype, at only about sixty to seventy percent of the level seen in healthy donor cells, which reached roughly ninety-five percent. Proliferation rates, however, were indistinguishable between the groups, meaning the patient cells grew normally even as they showed this subtle phenotypic deviation, hinting that the real differences lay deeper, in what the cells could become rather than how fast they could multiply.

What the cells could become turned out to be the central finding. Under osteogenic induction, healthy donor cells produced abundant mineralized matrix, revealed by intense Alizarin Red S staining, and strongly upregulated the osteoblast genes RUNX2 and BGLAP. Both patient-derived lines formed dramatically fewer mineral deposits and mounted only weak induction of the same genes. Adipogenesis fared no better: healthy cells accumulated plentiful lipid droplets stained by Oil Red O and induced the adipogenic regulators PPARG and ADIPOQ, while patient cells showed markedly reduced lipid accumulation and gene induction. Chondrogenesis, assessed by Alcian Blue staining of sulfated glycosaminoglycans and by expression of SOX9 and COL2A1, was also reduced but comparatively better preserved. Crucially, this pattern of a simultaneous, broad suppression of all three mesenchymal lineages argues against the classic idea of a simple lineage shift, in which bone-forming cells are diverted into fat cells. Instead, it points to a deeper loss of overall differentiation competence within the patient-derived progenitors themselves.

The molecular culprit emerged when the researchers examined the Wnt/beta-catenin pathway, the master regulator of mesenchymal lineage commitment. Beta-catenin, the transcriptional co-activator at the heart of canonical Wnt signalling, promotes osteogenesis and chondrogenesis while restraining adipogenesis, and its intracellular abundance is normally kept in check by glycogen synthase kinase-3, which phosphorylates beta-catenin and tags it for proteasomal destruction. In the patient-derived cells, the team found a striking double abnormality. Transcripts for CTNNB1, the gene encoding beta-catenin, were significantly reduced, while GSK3B mRNA was markedly elevated. At the protein level, phosphorylation of GSK3alpha at Tyr279 and GSK3beta at Tyr216, the modifications associated with enhanced enzymatic activity, was substantially increased, reaching roughly 1.8- to 2.2-fold above healthy donor levels in the more severely affected patient line. Total beta-catenin protein and intracellular beta-catenin fluorescence were correspondingly diminished, painting a coherent picture of a kinase pushed into overdrive and a key transcriptional regulator destroyed faster than it should be.

The disease signature extended into inflammation, a domain long suspected to matter in this condition because children with active disease show elevated IL-6 and HMGB1 in synovial fluid. Even without any inflammatory stimulus, the patient-derived mesenchymal cells expressed higher baseline levels of IL6, PTGS2 and CCL2, indicating a primed inflammatory state. When the cells were challenged with IL-1beta, the patient cells responded with larger fold increases in these cytokines than healthy cells, and the Wnt target genes AXIN2 and LEF1, already slightly reduced at baseline, were driven down even further. Western blotting showed that IL-1beta pushed GSK3 phosphorylation higher in patient cells than in controls while beta-catenin, already low, declined still more. These exaggerated responses persisted after osteogenic differentiation, where patient-derived osteoblasts displayed stronger NF-kappaB phosphorylation and greater degradation of its inhibitor I-kappa-B-alpha upon inflammatory stimulation. Transcriptomic sequencing reinforced the theme, revealing enriched alterations in inflammatory signalling, extracellular matrix organization and Wnt-related regulatory pathways in both patient fibroblasts and patient-derived mesenchymal cells, with upregulation of multiple extracellular Wnt antagonists including DKK1, SFRP1, SFRP2, WIF1 and SOST.

The most striking result, and the one with the clearest translational implication, came next. When the researchers treated the patient-derived cells with lithium chloride, a long-established inhibitor of GSK3, beta-catenin fluorescence recovered to levels comparable to untreated healthy donor cells, with the rescue being strongest precisely where the deficit had been greatest. More importantly, the functional defect yielded to the same treatment. Lithium chloride substantially increased mineralized nodule formation and Oil Red O staining in both patient lines during osteogenic and adipogenic induction, and quantitative PCR confirmed that either lithium chloride or the more selective inhibitor CHIR99021 significantly boosted expression of BGLAP, RUNX2, ADIPOQ and PPARG. The differentiation programs had not been deleted; they had merely been silenced by an overactive kinase, and silencing the kinase switched them back on. This established that the mesenchymal failure in Legg-Calvé-Perthes disease reflects a reversible, kinase-dependent signalling imbalance rather than irreversible cellular damage.

The study’s authors are careful to place their findings in context. Observations in patients, such as elevated circulating leptin and increased adipose tissue within the affected femoral head, reflect systemic and tissue-level changes that arise from ischemia, inflammation and altered marrow mechanics, and the in vitro reduction in adipogenesis seen here does not contradict that clinical picture. Rather, the platform isolates the intrinsic cellular component of the disease, complementing animal models in which ischemia was surgically induced in piglets and rodents. Those models have shown that ischemia activates hypoxia-inducible factor 1-alpha and drives inflammatory and angiogenic responses, and the new human data now connect those tissue-level events to a specific, druggable intracellular node inside the progenitors that must rebuild the bone. The authors propose a reciprocal feedback loop in which inflammatory activation sustains GSK3 activity, further destabilizing beta-catenin and progressively compromising skeletal regeneration.

For a disease whose current treatments range from conservative containment to major reconstructive surgery, the prospect of a pharmacological strategy that restores the bone-building capacity of a child’s own progenitor cells is genuinely exciting. Lithium is an old drug with a well-characterized safety profile in other contexts, and the iPSC-based model developed here provides a patient-specific testing ground for optimizing GSK3-targeted regenerative therapies before they reach the clinic. Much work remains, including validating the findings in larger patient cohorts and determining how a GSK3 inhibitor could be delivered safely to the developing femoral head. But the central message stands: the cellular failure underlying a devastating childhood hip disease is not a life sentence written into the genome, but a reversible regulatory state, and with the right key, the machinery of skeletal repair can be switched back on.

Subject of Research: Reversible GSK3/beta-catenin dysregulation in patient-derived iPSC-mesenchymal stem cells from children with Legg-Calvé-Perthes disease

Article Title: Patient‐Derived iPSC‐MSC Modelling Reveals Reversible GSK3‐Mediated Suppression of Mesenchymal Lineage Differentiation in Legg‐Calvé‐Perthes Disease

Article References: Seol, B., Lim, H. J., Song, C. L., Lee, J., & Cho, Y. S. (2026). Patient‐Derived iPSC ‐ MSC Modelling Reveals Reversible GSK3 ‐Mediated Suppression of Mesenchymal Lineage Differentiation in Legg‐Calvé‐Perthes Disease. Journal of Cellular and Molecular Medicine, 30(17), Article e71350. https://doi.org/10.1111/jcmm.71350

Image Credits: AI Generated

DOI: 10.1111/jcmm.71350

Keywords: Legg-Calvé-Perthes disease, induced pluripotent stem cells, mesenchymal stem cells, GSK3, beta-catenin, Wnt signalling, osteonecrosis, osteogenesis, inflammation, lithium chloride, CHIR99021, paediatric orthopaedics