Wnt pathway activation unlocks disease-neutral proliferative potential in human iPSC-derived cardiomyocytes: A comparative study across healthy and inherited cardiac disease models

The therapeutic potential of Wnt/β-catenin signaling to enhance proliferation in differentiated cardiomyocytes remains underexplored, particularly in genetically diverse disease models. Here, we systematically evaluated whether pharmacological Wnt activation overrides genetic constraints to drive expansion of induced pluripotent stem cell-derived cardiomyocytes (iCMs) from healthy donors and inherited cardiomyopathy models (GAA-Pompe disease, RYR2-catecholaminergic polymorphic ventricular tachycardia, and KCNQ1-long QT syndrome type 1). Using a component-defined GiWi protocol, functionally mature iCMs were generated from a high-quality iPSC line with validated trilineage differentiation capacity. Longitudinal analysis of CHIR-induced Wnt/β-catenin activation demonstrated dose-dependent proliferative amplification, with CHIR-treated iCMs achieving > 400-fold monolayer expansion by passage 4 versus ∼8-fold in controls. Immunofluorescence quantification revealed significantly elevated Ki67⁺ /cTnT⁺ double-positive cardiomyocytes under CHIR treatment (∼20 % vs. ∼9 % in controls at passage 3). Strikingly, proliferative responses showed genetic neutrality: healthy iCMs exhibited ∼432-fold expansion compared to ∼406-fold in disease models (p = 0.72), with comparable Ki67⁺/cTnT⁺ ratios by passage 4 (healthy: ∼8.9 %; disease: ∼8.3 %). These findings demonstrate that timed Wnt activation overrides genetic lesions to enable disease-agnostic proliferation in differentiated iCMs. This genetic neutrality supports standardized regenerative strategies for genetically heterogeneous cardiomyopathies and arrhythmias, addressing a critical challenge in developing personalized cardiac therapies.