Production of Engineered Cardiac Tissue from Encapsulated hiPSCs for Scale-up Studies and Drug-Testing

Abstract

Cardiovascular disease is the leading cause of death worldwide, causing a global health and financial burden. The first chapter of this dissertation highlights the needs for scalable, clinically relevant production of human pluripotent stem cell-derived cardiac tissue and provides an introduction to cardiac tissue engineering. The first project (Chapter 2) presents a rapid, scalable, and single-cell handling approach for the production of 3D functional cardiac tissue microspheres directly differentiated from encapsulated hiPSCs. Encapsulation occurred using a custom microfluidic system, which provides tight control over size and shape of the microspheres both between and within batches. The microspheres supported efficient cardiac differentiation, and the resulting CMs had appropriate temporal changes in gene expression and response to pharmacological and electrical stimuli. This microsphere direct differentiation platform using microfluidic encapsulation of hiPSCs was expanded in Chapter 3 to demonstrate ECT microsphere production with a variety of sizes and in chemically defined conditions. Chapter 4 highlights the flexibility of the microfluidic encapsulation system to produce ECT microspheroids with varying sizes and axial ratios (AR) with initial diameters ranging from 400–1000 microns with ARs from 1–9, and the impact of these parameters along with initial cell and PEG-fibrinogen concentrations on cardiac differentiation outcomes was assessed. Furthermore, initial scale-up studies were performed showing that microspheroids can be cultured and differentiated in shaker flasks, producing over 40 million cells per batch with consistent cardiac differentiation efficiencies. Photocrosslinking of PEG-fibrinogen for ECT production using the photoinitiator, LAP, which is commonly used in bioprinting, is demonstrated in Chapter 5. Two light sources were used for photocrosslinking of LAP, which was compared with the established Eosin Y crosslinking system. There were no differences in cardiac differentiation efficiency or cell numbers from the resulting ECTs photocrosslinked with the three light sources. Photocrosslinking with LAP allowed for non-destructive monitoring of action potentials using a genetically encoded voltage indicator cell line. Finally, the thalidomide induced changes to cardiac tissue formation, differentiation, and function were investigated in Chapter 6, showing that drug-induced changes during cardiac differentiation from hiPSCs could be detected. Overall, the results here demonstrate advancements in production of ECTs directly differentiated from encapsulated hiPSCs towards scalable, clinically relevant production with potential for use in high-throughput drug screening, bioprinting, and regenerative medicine