A MULTI-CELLULAR APPROACH TO ENGINEER VASCULARIZED CARDIAC GRAFTS FOR MYOCARDIAL REGENERATION

Due to the limited endogenous regenerative potential of the myocardium, therapeutic interventions are necessary to restore function following the occurrence of myocardial infarction. Cardiac tissue engineering offers the promise of restoring lost functionality. With the highly complex make-up of the myocardium, a multi-component/multi-stage approach to regeneration is essential. Cardiomyocytes are highly metabolic and require continuous amounts of oxygen and nutrients following transplantation onto the native myocardium. To address this, several groups have attempted to create pre-vascularized grafts, in which the engineered vasculature would be able to anastomose with the host blood vessels to promote survival of transplanted cells. Hence, the culture of cardiomyocytes has been coupled with endothelial cell types. To stabilize endothelial cell-derived vascular networks, vascular mural cells are necessary. In an effort to improve vascular development, we investigate the use of an abundant clinically relevant cell source, the human adipose derived stem/stromal cells. The objective of this thesis is to identify the cell-cell interactions of human adipose derived stem/stromal cells (hASCs) when cultured with cardiomyocytes and endothelial cells and to also develop a biomaterial platform for engineering aligned, contractile, vascularized myocardium that can be used to regenerate a fibrotic heart. This objective was achieved utilizing a multi-step approach which included first investigating the electrophysiological effects of co-culturing hASCs with cardiomyocytes (CMs). After determining the concentration of hASCs that would not statistically impede the electrophysiological properties of CMs [NRVCM:hASC:HUVEC ~ 500:100:0 and 500:50:0 cells], we used these concentrations of hASCs with endothelial cells (ECs) to promote stabilized vasculature. After exploring the ratios of each co-culture, we combined the optimized ratios to develop tri-cultures for a vascularized cardiac system. The ultimate ratio of 250,000:25,000:12,500 cells/cm2 resulted in dense vessel network development with electrophysiological properties with an average conduction velocity of 20 ± 2 cm/s, APD80 and APD30 of 122 ± 5 ms and 59 ± 4 ms, respectively, and maximum captured rate of 7.4 ± 0.6 Hz. Following this, novel fibrin microfiber sheets were used in conjunction with these cell types to develop vascularized cardiac patches. Initially, the fibrin microfibers were characterized for mechanical properties. NRVCMs were seeded onto fibrin microfibers at a concentration of 1.5 106 cells/cm2. NRVCMs were cultured for up until 56 days and achieved electrical conduction velocities of 22 2.1 cm/s. Using a tri-culture system, we engineered contractile cardiac patches with vascular network structures that were capable of being implanted into the ischemic myocardium. Ultimately cardiomyocytes were seeded for 14 days prior to adding hASCs and HUVECs. Upon the addition of the supporting cell types, we found that the electrophysiological properties at the ratio of 1500:37.5:150 were conduction velocity of 14 ± 0.6 cm/s, APD80 and APD30 of 152 ± 11 ms and 71 ± 6 ms, respectively, and maximum capture rate of 3.9 ± 0.7 Hz. The values obtained at this ratio were not statistically significant different from the NRVCM only control and appeared to match the closest compared to other tri-cultures. Lastly, we investigated the human interactions of development of a tri-culture system with completely translatable cell types which include human induced pluripotent stem cells differentiated towards the cardiomyocyte lineage and the endothelial cell lineage. This work will ultimately lead to the development of clinically translatable vascularized cardiac patches

Understanding Arrhythmogenic Cardiomyopathy: Advances through the Use of Human Pluripotent Stem Cell Models

Cardiomyopathies (CMPs) represent a significant healthcare burden and are a major cause of heart failure leading to premature death. Several CMPs are now recognized to have a strong genetic basis, including arrhythmogenic cardiomyopathy (ACM), which predisposes patients to arrhythmic episodes. Variants in one of the five genes (PKP2, JUP, DSC2, DSG2, and DSP) encoding proteins of the desmosome are known to cause a subset of ACM, which we classify as desmosome-related ACM (dACM). Phenotypically, this disease may lead to sudden cardiac death in young athletes and, during late stages, is often accompanied by myocardial fibrofatty infiltrates. While the pathogenicity of the desmosome genes has been well established through animal studies and limited supplies of primary human cells, these systems have drawbacks that limit their utility and relevance to understanding human disease. Human induced pluripotent stem cells (hiPSCs) have emerged as a powerful tool for modeling ACM in vitro that can overcome these challenges, as they represent a reproducible and scalable source of cardiomyocytes (CMs) that recapitulate patient phenotypes. In this review, we provide an overview of dACM, summarize findings in other model systems linking desmosome proteins with this disease, and provide an up-to-date summary of the work that has been conducted in hiPSC-cardiomyocyte (hiPSC-CM) models of dACM. In the context of the hiPSC-CM model system, we highlight novel findings that have contributed to our understanding of disease and enumerate the limitations, prospects, and directions for research to consider towards future progress

Pharmacological, Physiochemical, and Drug-Relevant Biological Properties of Short Chain Fatty Acid Hexosamine Analogues Used in Metabolic Glycoengineering

In this study, we catalog structure activity relationships (SAR) of several short chain fatty acid (SCFA)-modified hexosamine analogues used in metabolic glycoengineering (MGE) by comparing <i>in silico</i> and experimental measurements of physiochemical properties important in drug design. We then describe the impact of these compounds on selected biological parameters that influence the pharmacological properties and safety of drug candidates by monitoring P-glycoprotein (Pgp) efflux, inhibition of cytochrome P450 3A4 (CYP3A4), hERG channel inhibition, and cardiomyocyte cytotoxicity. These parameters are influenced by length of the SCFAs (e.g., acetate vs n-butyrate), which are added to MGE analogues to increase the efficiency of cellular uptake, the regioisomeric arrangement of the SCFAs on the core sugar, the structure of the core sugar itself, and by the type of <i>N</i>-acyl modification (e.g., <i>N</i>-acetyl vs <i>N</i>-azido). By cataloging the influence of these SAR on pharmacological properties of MGE analogues, this study outlines design considerations for tuning the pharmacological, physiochemical, and the toxicological parameters of this emerging class of small molecule drug candidates