Ph.D Thesis


Ph.D StudentAbboud Yousef
SubjectMethods to Improve Cell Engraftment for Myocardial Repair
and Disease Modeling
DepartmentDepartment of Medicine
Supervisors PROF. Lior Gepstein
ASSISTANT PROFESSOR Anat Aharon


Abstract

The adult heart has limited regenerative capacity; therefore, any significant cell loss may result in the development of progressive heart failure. Cardiomyocyte replacement therapy has become an option for myocardial repair but has been hampered by the significant donor cell loss after cell/tissue engraftment. Here, we examine the ability of human-villous-trophoblast-secreted extracellular vesicles (HVT-EVs) and a unique tissue engineering approach (decellularization/recellularization) to improve cell-graft survival and therefor myocardial function. In addition, we created the first in-vivo model for a human genetic disease: Pompe’s disease.

HVT-EVs were isolated then characterized by a nanoparticle tracking analyzer. Their pro-survival and pro-angiogenic effects were initially evaluated by in-vitro assays that confirmed their ability to improve cardiomyocyte survival and endothelial migration and sprouting. Myocardial infarction (MI) was then induced through permanent LAD coronary ligation in rats. Animals were randomized 7-10 days later to be transplanted with either human induced pluripotent stem cell derived cardiomyocytes (hIPSC-CMs), HVT-EVs, both, or PBS as control. Cell engraftment and survival was evaluated by detailed histological evaluation. Myocardial function was evaluated through serial echocardiograms up to 30 days after transplantation, which showed a significant advantage in functional outcome for the combined therapy. Graft-host coupling was evident using optical mapping in the Langendorf isolated heart model by tracking the activity of a genetically-encode voltage indicator (Arclight) expressed in the hIPSC-CMs. The positive effects achieved by HVT-EVs could stem from their potential role in creating more mature blood vessels, as they bare a load of pro-angiogenic and pro-survival proteins. However, the functional benefit observed seemed to partially diminish with time.

To address the latter shortcoming, we used a decellularized rat heart tissue as a cardiac patch scaffold and transplanted rats after MI induction with the patch alone, or patches seeded with either hiPSC-CMs, HVT-EVs or both. HVT-EVs in the patch were slowly released, improving heart contractility and function. Hence, when combined with hIPSC-CMs they had an additive effect that further improve fractional shortening. In contrast to the first set of experiments, cardiac function did not show any deterioration with time.

In the final stage of the project, we established an in-vivo model for Pompe’s disease by transplanting Pompe-hIPSC-CMs to mouse hearts. Pompe-CMs engrafted, survived, were perfused, and demonstrated the typical severe pathological feature of the disease: enlarged glycogen-filled lysosomal compartment. Pompe-CMs were successfully treated with α-glucosidase enzyme replacement therapy (ERT) which reversed the pathological features. Interestingly, beyond restoring the ultrastructural properties, ERT also restored the ability of the Pompe-CMs to restore ventricular function following transplantation in the infarct model.

In summary, this thesis presented two main uses of hIPSC technology:
1. Cell therapy, where we show that HVT-EVs transplantation can improve cardiac function after MI either alone or even better if coupled with hIPSC-CMs transplantation. This is probably achieved by attenuating cell death, improving tissue vascularization, and cultivating damaged tissues resulting in better reception of the cell-graft.
2. Disease modeling, where we created the first in-vivo model of Pompe’s disease and demonstrated that it can be useful for studying disease pathophysiology and therapy in a patient specific manner.