A novel experimental in vitro model of Wilson’s disease based on definitive endoderm cells

BACKGROUND: Wilson’s disease is a rare autosomal recessive disorder caused by mutations in the ATP7B gene, which encodes a copper-transporting ATPase. Dysfunction of this protein disrupts copper excretion through bile, leading to its pathological accumulation in hepatocytes. Currently, the range of available cell models for studying the molecular basis of this pathology and exploring new therapeutic approaches remains significantly limited. AIM: Development of an in vitro model using human induced pluripotent stem cells (iPSCs) to study molecular mechanisms of Wilson’s disease and screen therapeutic strategies. METHODS: We developed a 2D cellular model based on definitive endoderm cells from a healthy donor using validated induced pluripotent cells, which were subsequently differentiated using the differentiation inducers Activin A and CHIR99021. Copper overload, characteristic of the pathogenesis of Wilson’s disease, was mimicked by introducing exogenous copper into the growth medium. Alamar Blue dye was used to assess relative cell viability. RESULTS: The used iPSC line exhibits a normal karyotype; morphology characteristic of embryonic stem cells; expresses pluripotency markers SOX2, OCT4, TRA-1-60, and SSEA-4; and forms tissues of all three germ layers upon spontaneous differentiation in embryoid bodies. Differentiation of these iPSCs using the proposed method results in the generation of definitive endoderm cells with corresponding morphology, which express markers SOX17, FOXA2, and ATP7B. The obtained model demonstrates sensitivity to exogenous copper overload with an IC₅₀ of 197 μM. CONCLUSION: The platform based on iPSC-derived human definitive endoderm cells enables the modeling of copper overload in vitro, mimicking the disruption of cellular metabolism in Wilson’s disease. Thus, the proposed model can be used for both fundamental research and the development of new therapeutic approaches.