274P Generation of cardiac organoids from DuchenneMuscularDystrophy patient-derived induced pluripotent stem cells: a novel approach to understanding cardiomyopathy

Abstract

Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by mutations in the dystrophin gene. Cardiomyopathy is the leading cause of death in DMD patients, but the exact mechanisms underlying it remain unclear. Due to the limitations of animal models, patient-derived human models need to be developed to investigate cardiac pathophysiological mechanisms better. Hence, 3D human cardiac organoids (hCOs) are designed to more closely resemble the structural and functional properties of the human heart than traditional 2D cell cultures or animal models. For this purpose, we generated DMD patient-derived induced pluripotent stem cells (hiPSC) and corrected DMD mutation with CRISPR/Cas9 gene editing, establishing isogenic control lines. Healthy controls and CRISPR/Cas9 edited cells with introduced DMD gene exon 50 deletion were also sources for these cell types. Under specified culture conditions, these stem cells differentiate into various cardiac cell types, including cardiomyocytes (CM), endothelial cells (EC), and cardiac fibroblasts (CF). We affirmed the presence of specific markers (troponin T for CM, DDR2 for CF, and CD31 and VE-cadherin for EC), indicating successful differentiation. Self-aggregation of CM, CF, and EC at controlled ratios (CM: 75%, CF: 15%, EC: 15%) resulted in the formation of 3D cardiac organoids. Overall, we successfully generated cardiac organoids from hiPSCs derived from DMD patients, healthy donors, and isogenic control samples, which will be used to understand the mechanisms of DMD cardiomyopathy further. Grants support: National Science Centre: MAESTRO 2018/30/A/NZ3/00412.