טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentAmitai-Lange Aya
SubjectNeuronal Networks Derived from Healthy and Diseased Human
Pluripotent Stem Cells
DepartmentDepartment of Medicine
Supervisor Professor Lior Gepstein
Full Thesis textFull thesis text - English Version


Abstract

Human pluripotent stem cells (hPSCs), namely human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold great promise for basic and translational neuroscience. Yet, to fulfill their unique potential, their neuronal derivatives must display the relevant electrical properties at both the single-cell and network levels. Here we aimed to test the hypothesis that hPSCs-derived neurons can form an electrically-active neuronal-network.

Both hESCs and hiPSCs were coaxed to differentiate into neurons. Immunostaining studies revealed the presence of glial cells, excitatory (Glutamatergic) neurons, inhibitory (GABAergic) neurons, and development of neuronal synapses. Extracellular microelectrode array recordings revealed the development of spontaneous synchronized bursts within the hPSCs-derived neuronal-networks. Similarly, laser-confocal calcium imaging demonstrated synchronized intracellular calcium transients within the networks. Network activity was suppressed by the sodium channel blocker tetradotoxin and by the glutamate receptor antagonists CNQX and AP-5 in both hESCs and hiPSCs neuronal-cultures. In contrast, application of the GABA-A receptor antagonist picrotoxin resulted in a paradoxical decrease in network activity in hESCs- but not in hiPSCs-derived neuronal-cultures, suggesting an earlier maturation stage of the former.

We next aimed to test the hypothesis that patient/disease-specific hiPSCs-derived neuronal tissue can be used to model inherited disorders. To this end we created patient-specific hiPSCs from Pompe and Rett syndromes patients. Pompe glyocogen-storage disease is a devastating skeletal and cardiac myopathy caused by deficiency of the glycogen-degrading lysosomal enzyme Acid Alpha-Glucosidase (GAA). Here, we aimed to test whether neuronal tissue is also affected by the disease process. Live cell-imaging, lysosomal immunostaining, and transmission electron microscopy of the Pompe hiPSCs-derived neuronal tissue revealed enlarged glyocogen-containing lysosomes in both neurons and glial cells that significantly distorted cell morphology. Treatment with labeled recombinant human GAA resulted in its intracellular uptake and lysosomal trafficking; eventually reversing glycogen storage in these cells.

We next established an in-vitro hiPSCs model of Rett syndrome and demonstrated its role in studying disease mechanisms in both neurons and cardiomyocytes. Initial evidence suggested the presence of abnormal network activity in the Rett-hiPSCs derived neuronal cultures. Similarly, repolarization abnormalities and increased arrhythmogenicity were noted in the Rett-hiPSCs-derived cardiomyocytes. The latter finding suggests that the cardiac phenotype of Rett syndrome is independent of the nervous system pathology.

In summary, our analysis of structural and electrical properties of neuronal-cultures derived from healthy and diseased hPSCs demonstrates the unique potential of these experimental models for developmental biology, physiology, drug discovery, disease modeling, and tissue replacement therapy.