|Ph.D Student||Riba Roitblat Inbar|
|Subject||Induced Pluripotent Stem Cell Model of Familial|
|Department||Department of Medicine||Supervisor||Professor Lior Gepstein|
|Full Thesis text|
Pompe and Danon diseases are inherited skeletal and cardiac myopathies caused by deficiency of the glycogen-degrading lysosomal enzyme acid alpha-glucosidase (GAA) and Lysosomal-Associated Membrane Protein 2, respectively. We aimed to establish patient-specific human induced pluripotent stem cells (hiPSCs) models of Pompe and Danon diseases; to provide insights into the pathophysiological mechanisms underlying these disorders; to evaluate the role of abnormal autophagy in these cardiomyopathies; to characterize the resulting functional abnormalities; and to evaluate potential therapies. To this end, patient-specific hiPSCs were generated from an infantile Pompe patient and from an adolescent Danon patient, coaxed to differentiate into cardiomyocytes (hiPSC-CMs) and compared to healthy-control hiPSC-CMs. Electron microscopy, immunostainings and protein electrophoresis revealed significant lysosome and autophagosome accumulation in both types of diseased cardiomyocytes. The Pompe hiPSC-CMs presented with more progressive lysosomal pathology and intracellular damage compared to Danon cells. Treatment with recombinant human GAA reversed the pathological lysosomal glycogen storage in Pompe hiPSC-CMs but failed to clear the abnormal autophagic buildup. Protein electrophoresis of cells treated with autophagy pathway modulators suggested the abnormalities in the autophagy process and autophagic buildup identified in the Danon and Pompe hiPSC-CMs was primarily due to reduced autophagosome clearance as a result of abnormities in autophagosome-lysosome fusion. Cellular hypertrophy evaluation using immunostaining analysis demonstrated larger cell area of Danon hiPSC-CMs relative to control. Functional characterization included calcium imaging and contractile properties studies. Calcium imaging displayed calcium-handling irregularities in Pompe hiPSC-CMs and faster calcium transient kinetics in both diseased hiPSC-CMs. Finally, contractile analysis of 3D engineered heart tissues revealed a trend for diminished force in the Pompe specimens relative to controls, whereas Danon tissues produced similar forces as controls. In conclusion, patient-specific hiPSC-CMs recapitulated the phenotype of Pompe and Danon diseases in both single cell and 3D tissue in-vitro models. These displayed the underlying lysosomal and autophagosomal pathology, suggested that the cause for the autophagic abnormalities in both diseases was reduced clearance and provided further insights into cardiac functional properties of Pompe and Danon cardiomyopathies. Enzyme replacement therapy may clear the glycogen accumulation in lysosomes in Pompe cardiomyocytes but failed to prevent the abnormal autophagy process, potentially explaining the treatment failure in some patients.