Date of Award
Spring 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Experimental Pathology
First Advisor
Qyang, Yibing
Abstract
The leading cause of death in the United States and worldwide is heart disease, a category that includes a variety of cardiovascular diseases both inherited and acquired. Because of the urgency and complexity of the problem, copious research has been conducted on the causes and progression of these diseases with an eye towards therapeutic solutions to their manifestations. For diseases that require tissue replacement, such as vessel implantation in coronary artery blockage or single ventricle disease, cardiovascular tissue engineering (CVTE) has emerged as an attractive way to provide high quality implantable therapeutics. CVTE is a field using chemistry, biology, and engineering principles to develop three-dimensional tissues that can replace or repair diseased cardiovascular tissues, made more plausible with the invention of induced pluripotent stem cells (iPSCs), cells that are capable of both self-renewal and differentiation into any cell type. While many exciting new tissues have been developed for a variety of treatments through CVTE, only very few of these therapeutics have moved into clinical trials, and almost all of these are exclusively decellularized tissues. This asymmetry is due in part to the lack of sufficient tools for preclinical modeling of cellularized therapeutics in large animals, which is necessary to test their safety and efficacy given the invasiveness of the intervention and their unique challenges. For stem cell-based CVTE constructs, which make up a large proportion of potential cellularized therapeutics, there is a particularly high bar for their clinical application, given their potential for forming tumors or transdifferentiate. Pigs have long been considered the ideal preclinical large animal model for cardiovascular therapeutics because of the similarities of their cardiovascular system to humans in both size and function, but they have been unable to be used effectively to test cellularized CVTE constructs due to the inability to create pig iPSCs of comparable quality to human iPSCs combined with the difficulty in using xenogeneic human cellularized CVTE therapeutics in pigs without massive immunosuppression that limits the safety and efficacy information obtained in these tests. This latter problem has only been exacerbated by the advent of hypoimmunogenic iPSCs, which has opened up the possibility of allogeneic cellularized CVTE constructs that would require preclinical large animal modeling in a completely immunocompetent animal. Therefore, there is an urgent need for the development of pig iPSC technology to allow for the seamless testing of a variety of stem cell-based CVTE therapeutics in this preclinical large animal model. Herein, I report the creation and validation of the first transgene-free piPSCs, their differentiation into vascular smooth muscle (SMCs) and endothelial cells (ECs), and the application of these piPSC-derived vascular cells in proof-of-principle, small animal CVTE studies, along with the derivation of hypoimmunogenic piPSCs for future use. I accomplished this by using a modified form of pig expanded potential stem cell medium (pEPSCM) leveraged with Sendai virus delivery of human Yamanaka reprogramming factors. Once validated through canonical stem cell verification tests, I differentiated these cells into SMCs using the embryoid body (EB) differentiation protocol developed in our lab and applied the piPSC-SMCs in various screens to allow for the future creation of tissue engineered vascular grafts (TEVGs). Additionally, I differentiated the piPSCs into ECs and demonstrated that they were equally functional as primary pig ECs. As a proof-of-principle of the use of these cells in modeling a human CVTE therapeutic, I created tissue engineered vascular conduits (TEVCs) by endothelializing decellularized human umbilical cord arteries (dHUA) with piPSC-ECs and training them in a flow bioreactor to mature them for implantation. These pig TEVCs showed similar effectiveness to human TEVCs created using native human ECs, and they were capable of preventing thrombosis as an interposition inferior vena cava (IVC) graft in contrast to dHUA alone. Finally, I have made strides in the creation of hypoimmunogenic piPSCs by using gene-editing to ablate expression of pig major histocompatibility complexes I and II. Together, this demonstrates the creation of piPSCs capable of producing differentiated cells suitable for performing CVTE applications, setting the stage for future use of these cells as a vital preclinical model for allogeneic cell-based CVTE therapeutics in farm pigs.
Recommended Citation
Batty, Luke Daniel, "Pig Induced Pluripotent Stem Cells as a Preclinical Model for Cardiovascular Tissue Engineering" (2023). Yale Graduate School of Arts and Sciences Dissertations. 953.
https://elischolar.library.yale.edu/gsas_dissertations/953
