Date of Award

Spring 3-31-2023

Embargo Period

5-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Biopathology

College

College of Graduate Studies

First Advisor

Ying Mei

Second Advisor

Amy Bradshaw

Third Advisor

Daniel Judge

Fourth Advisor

Donald Menick

Fifth Advisor

Robin Muise-Helmericks

Sixth Advisor

Hai Yao

Abstract

Cardiovascular disease is the constant leading cause of death worldwide. While substantial efforts have been undertaken to improve disease outcomes, the lack of adequate human cardiac tissue models exacerbates research and development costs and clinical trial failures, hampering novel therapeutic discovery. To address this, human engineered cardiac microtissues composed of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) show potential in recapitulating hallmark qualities of natural human myocardium for novel therapeutic testing, disease modeling and cardiotoxicity screening. However, as these models are engineered in vitro, they are limited in their capacity to fully recapitulate myocardium and are vulnerable to influences often associated with in vitro techniques. To investigate the capability of engineered cardiac microtissues to model natural human myocardium, we performed an RNA-sequencing analysis of our previously established cardiac organoid model to compare their transcriptomic similarity to human myocardial samples. We identified that the inclusion of primary supporting cell types commonly found in natural myocardium, such as cardiac fibroblasts (CF), improves engineered cardiac microtissues’ recapitulation of human myocardium. Yet, it was clear that the lack of immune cells within engineered cardiac tissues prevented full recapitulation of the myocardium. However, our engineered cardiac organoid model is composed of genetically mismatched cell types and thus is incapable of predicting patient specificity for disease modeling, therapeutic discovery, and cardiotoxicity. To address this, we sought to develop an isogenic cardiac organoid model replacing the cell types found in our established model with hiPSC-derived cell types. Unfortunately, transcriptomic analysis of hiPSC-cardiac fibroblasts (hiPSC-CF) revealed their similarity to activated cardiac fibroblasts associated with numerous cardiomyopathies. We reasoned that the in vitro cell culture substrates of Matrigel and tissue culture plastic (TCP) utilized during hiPSC-CF differentiation induce their activated pathogenic phenotype. Proteomic analyses identified that Matrigel contains SPARC, a known regulator of fibroblast activation and often associated with cardiomyopathy. Further, universal TCP is known to induce the activation of fibroblasts through mechanical stimulation. We hypothesized that the activation of hiPSC-CF could be alleviated by incorporating cardiac-specific biomimetic substrates for the differentiation and expansion of hiPSC-CF. To do so, we utilized decellularized porcine heart extracellular matrix (HEM) as a TCP surface coating for hiPSC-CF differentiation and expansion. We identified that HEM reduces activated fibroblast characteristics yet does not impact hiPSC-CF differentiation. Further, we show that the alleviation in activation translates into 3D cell culture conditions and even improves cardiac organoid function in an isogenic cardiac organoid model. The results of this thesis provide an understanding of limitations and considerations in current hiPSC differentiation techniques and provide a novel solution for improving engineered cardiac tissues using biomimetic substrates.

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Copyright is held by the author. All rights reserved.

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