טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentZwi-Dantsis Limor
SubjectGeneration and Characterization of Functional Cardiomyocytes
from Induced Pluripotent Stem (iPS) Cells -
Possible Applications for The Treatment
of Heart Diseases
DepartmentDepartment of Biotechnology
Supervisor Professor Lior Gepstein
Full Thesis textFull thesis text - English Version


Abstract

Myocardial cell replacement therapies are emerging as novel therapeutic paradigms for heart failure, but are hampered by the lack of sources for human cardiomyocytes, by the need for production of cardiomyocytes in a scalable manner, and by the anticipated immune rejection of allogeneic cell-sources. A potential solution to these challenges maybe the groundbreaking induced pluripotent stem cells (iPSCs) technology. This approach allows the reprogramming of adult somatic cells into pluripotent stem cells by the introduction of a set of transcription factors. The generated human iPSCs (hiPSCs) can then be coaxed to differentiate into a variety of cell types. In the current work we aimed to evaluate the potential of the hiPSCs technology in the emerging field of cardiovascular regenerative medicine.

We first aimed to establish, a reproducible and well-characterized cardiomyocyte differentiation system from hiPSCs. Using the embryoid-body differentiation system, hiPSCs were successfully differentiated into spontaneously-beating cardiomyocytes. The generated myocytes displayed molecular, structural, and functional properties of early-stage human cardiomyocytes, generated a functional syncytium, and responded appropriately to adrenergic and cholinergic signaling. Proof-of-concept pharmacological studies demonstrated the potential of the hiPSCs-derived cardiac-tissue to identify drug-induced conduction and repolarization abnormalities.

Next, we aimed to establish a bioprocess for scaling-up of the differentiation process into cardiomyocytes. We utilized a scalable stirred-suspension bioreactor (spinner flasks) for direct embryoid-bodies formation of murine iPSCs. Our findings showed that the differentiation system can be efficiently scaled-up using this bioreactor technique; and that the differentiation efficacy, gene expression profile, and electrophysiological properties of the iPSC-derived cardiomyocytes were comparable between the static and stirred systems.

Finally, we aimed to evaluate the potential of the hiPSCs technology for   personalized myocardial regenerative medicine applications by the derivation of patient-specific hiPSCs. We demonstrated the ability to reprogram skin fibroblasts derived from patients with advanced heart failure into hiPSCs. The heart-failure hiPSCs were then coaxed to differentiate into the cardiac-lineage. Importantly, the molecular, structural, and functional properties of the heart-failure hiPSCs-derived cardiomyocytes (hiPSCs-CMs) did not differ from hiPSCs-CMs derived from healthy individuals. Functional integration and synchronized electrical activities were then demonstrated between the heart failure-hiPSC-CMs and neonatal rat myocytes in co-culture studies. Finally, in-vivo transplantation studies in the rat heart revealed the ability of the generated cardiomyocytes to engraft, survive, and structurally integrate with host cardiac tissue.

In conclusion, our study demonstrates the potential of the hiPSCs technology for future autologous cardiovascular cell-replacement therapies and for individualized drug testing and drug development.