טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLesman Ayelet
SubjectTissue Engineering of Vascularized Cardiac Muscle from
Human Embryonic Stem Cells
DepartmentDepartment of Biomedical Engineering
Supervisor Professor Shulamit Levenberg
Full Thesis textFull thesis text - English Version


Abstract

Myocardial cell-replacement and tissue-engineering strategies are hampered by the paucity of sources for human cardiomyocytes and by the scarce vasculature in the ischemic area limiting the engraftment and survival of the transplanted muscle. As a possible solution to these obstacles, we assessed the ability to construct a three-dimensional tissue-engineered human, vascularized, cardiac-muscle and subsequently to engraft it in the in-vivo rat heart and to promote functional vascularization.

Human embryonic stem cell-derived cardiomyocytes were cultured alone or together with endothelial cells and embryonic fibroblasts within highly-porous, biodegradable, polymeric scaffolds. Our in-vitro studies show that a three-dimensional beating human cardiac muscle-construct containing a dense vessel-like network was successfully created. Functional properties of the generated cardiac tissue-construct with synchronous activity mediated by action potential propagation through gap junctions were demonstrated. The presence of endothelial capillaries was found to be crucial for tissue development and proliferation. Furthermore, the presence of embryonic fibroblasts improved the stability of the generated vessels, to decrease endothelial cell death, and to increase endothelial proliferation.

Medium flow imposed on the cellular scaffolds during in-vitro cultivation was found to trigger endothelial cells organization and to enhance vessel network depth. Thus, allowing optimization of tissue vascularity in-vitro.

Transplantation of the engineered cardiac tissue in the rat heart resulted in formation of stable grafts. The human cardiomyocytes were located within the transplanted scaffolds. Some of them have undergone structural maturation demonstrating elongated pattern, organized striations and the expression of gap-junctions.

Intense vascularization was evidenced throughout the engrafted scaffolds. Significant portion of the generated vessels were of human origin. The presence of the preexisting human vessels was found to increase tissue vascularity and resulted in the generation of functional human vessels that became integrated with host vascular network and contribute to perfusion of the tissue-construct.

Our study highlights the important role of pre-vascularization of the engineered tissue in-vitro and following implantation in-vivo. This tissue-construct could contribute significantly to the emerging discipline of cardiovascular regenerative medicine, stem cell research as well as the ability to study the interactions between different cell types in a complex biological process such as functional tissue vascularization.