טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentEliyahu Sivan
SubjectExperimental Model of Long Q-T Syndrome in Human Embryonic
Stem Cells-Derived Cardiomyocytes
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Ofer Binah
Full Thesis textFull thesis text - English Version


Abstract

The successful production of hiPSC-CM is suggested as the most promising candidate for cardiovascular, regenerative and personalized medicine. hiPSC-CM were generated through the infection of human skin fibroblasts (HDF-iPSC-CM) and human hair follicle karatinocyte cells (HFKT-iPSC-CM), with retroviruses containing the four human genes:  OCT4, Sox2, Klf4 and C-Myc. In the current study we tested the hypothesis that HFKT-iPSC-CM and HDF-iPSC-CM are similar in their functional properties with regard to Ca2 handling and the role of the sarcoplasmic reticulum (SR) in the contraction process. For this purpose, we investigated the responsiveness of these cells to β-adrenergic stimulation (isoproterenol), ryanodine receptor (RyR) blocker and caffeine. Furthermore, we evaluated their response in mechanical tests such as force-frequency relations (FFR) and post-rest potentiation (PRP) by measurements of [Ca2]i transients and contractions. In addition, we exposed HFKT-iPSC-CM to the L-type Ca2 channel blocker nifedipine and the Na channel blocker, tetrodotoxin (TTX) and investigated their response by measurements of extracellular electrograms. Our major findings are; (1) both HFKT-iPSC-CM and HDF-iPSC-CM displayed negative FFR and lack of PRP. (2) The RyR blocker and caffeine affected [Ca2]i transients and contractions in both cultures, indicating functional SR Ca2 release. (3) There was a positive inotropic response in both hiPSC-CM to β-adrenergic stimulation (isoproterenol). (4) HFKT-iPSC-CM responded to nifedipine by shortening the QT intervals and increasing the beat rate. Moreover, HFKT-iPSC-CM responded to TTX by slowing the beat rate, reducing the maximal voltage change of the QRS (dV/dtmax) and reducing the QRS amplitude. In conclusion, HFKT-iPSC-CM and HDF-iPSC-CM exhibit similar behavior with regard to Ca2 handling. The major difference in hiPSC-CM is associated with age progression; the older the culture, the more pronounced the response to the drugs that were tested.