Development and Characterization of Isogenic Cardiac Organoids Derived from Human Pluripotent Stem Cells

Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide, causing nearly 25% of deaths in the United States. Despite the efforts to create in vitro models for the study and treatment of CVDs, these are still limited in their recapitulation of the heart tissue. Thus, the engineering of accurate cardiac models is imperative to gain more understanding and improve the outcome of CVDs. This Ph.D. dissertation focuses on the development and characterization of isogenic cardiac organoids derived from human induced pluripotent stem cells (hiPSCs). Additionally, the integration of chemical and biological cues for enriching their microenvironment and promoting their maturation state and function were studied. First, hiPSC-derived cardiac cells were utilized for the fabrication of multicellular spherical microtissues, namely isogenic cardiac organoids. The cellular composition and culture time of the engineered tissues were optimized to induce cellular aggregation and the formation of cell-cell interactions. Also, ribbon-like gold nanoparticles, namely gold nanoribbons (AuNRs), were synthesized, characterized, and biofunctionalized for their integration into the isogenic cardiac organoids. In-depth biological evaluation of the organoids showed enhanced cardiac maturation markers. Furthermore, a supplement-free cell culture regime was designed and evaluated for fabricating isogenic cardiac organoids. Mechanistic, cellular, and molecular-level studies demonstrated that the presence of hiPSC-derived cardiac fibroblasts (hiPSC-CFs) significantly improves the morphology and gene expression profile of the organoids. Electrophysiological-relevant features of the organoids, such as conduction velocity (CV), were further investigated utilizing a microelectrode array (MEA) platform. It was shown that MEA offers a simple, yet powerful approach to assessing electrophysiological responses of the tissues, where a trend in decreased CV was found due to the presence of hiPSC-CFs. Overall, this dissertation has a broad impact casting light on the development strategy and biological mechanisms that govern the formation and function of isogenic cardiac organoids. Moreover, this study presents two unique approaches to promote maturation of stem cell-derived cardiac organoids: 1) through the integration of novel biofunctionalized nanomaterials, and 2) through a cell culture regime, leading to enhanced function of the organoids. The proposed micro-engineered organoids have broad applications as physiologically relevant tissues for drug discovery, CVDs modeling, and regenerative medicine.