Derivation and characterization of a patient-derived hiPSC line carrying the HCN2 p.G460D variant with neuronal and cardiac differentiation potential

Background Pathogenic variants in HCN2 , encoding the hyperpolarization-activated cyclic nucleotide-gated channel 2, have recently emerged as a cause of developmental and epileptic encephalopathies (DEEs). Because HCN2 is expressed in both the central nervous and cardiovascular systems, disease mechanisms may extend beyond neuronal dysfunction and involve both neuronal and cardiac tissues. However, the lack of patient-specific human models has limited the investigation of tissue-specific and shared consequences of HCN2 dysfunction and the development of targeted therapeutic approaches. Methods A human induced pluripotent stem cell (hiPSC) line was generated via non-integrating Sendai viral vectors from dermal fibroblasts of a patient with severe drug-resistant DEE carrying the HCN2 c.1379G>A (p.G460D) variant. Following validation of genomic integrity, vector clearance, pluripotency, trilineage potential, and retention of the disease-causing variant, hiPSCs were subjected to directed differentiation toward cortical neurons and cardiomyocytes. Neuronal and cardiac lineage specification, subtype marker profiles, and endogenous HCN2 retention were assessed by RT-qPCR and immunofluorescence. In cardiomyocyte monolayers, functional beating kinetics, rhythm stability, and spatial electromechanical coupling were quantified using video-based motion tracking. Results Reprogramming established a patient hiPSC line carrying the heterozygous HCN2 p. G460D variant with a stable 46,XX karyotype, vector clearance, robust pluripotency, and verified trilineage potential. Directed differentiation yielded deep-layer glutamatergic cortical neurons expressing FOXG1 , MAP2 , BCL11B , and SLC17A7 , forming extensive networks positive for TUBB3, MAP2, and NeuN. In parallel, cardiac differentiation produced functional cardiomyocytes expressing TNNT2 , M YH6 , and MYH7 with organized cTnT, ACTN2, and RYR2 sarcomeric structures. Both lineages retained endogenous HCN2 expression. Video kinematic profiling demonstrated progressive functional maturation of the beating cardiomyocytes, showing a 59.1% reduction in rhythm variability and enhanced spatial electromechanical coupling over time. Conclusion We established the first patient-derived hiPSC platform carrying a pathogenic HCN2 variant associated with DEE. The ability to generate both neuronal and cardiac lineages from the same genetic background provides a valuable translational resource future mechanistic studies, cell-type-specific functional comparisons, and exploratory evaluation of potential therapeutic approaches in HCN2 -related channelopathies.