Maturation, Scale-up and Vascularization of Engineered Cardiac Muscle from Human Pluripotent Stem Cells

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2018

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Abstract

Over recent decades, the continued clinical burden of ischemic heart disease has created a need for alternative therapies to improve the function of diseased myocardium. While the existing clinical interventions are often aimed at restoring blood supply to infarcted myocardium, they have been unable to effectively replace damaged muscle with healthy new tissue. Transplantation of a pre-formed, tissue-engineered cardiac muscle has been proposed as a potential strategy to remuscularize the infarcted heart and prevent adverse remodeling leading to heart failure. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are currently the only robust cell source for engineering of functional cardiac tissues. Despite more than 15 years of research with hPSC-CMs, human engineered cardiac tissues still fall short of replicating the mature electrical and contractile properties of adult human myocardium important for safety and efficacy of the future therapies. Furthermore, most of engineered cardiac tissues are currently engineered to be avascular and are too small for use in clinical applications. As such, the primary goals of this thesis were to: 1) generate mature engineered cardiac tissues from human pluripotent stem cells, 2) scale-up engineered tissue size to clinically relevant dimensions without loss of function, and 3) pre-vascularize engineered cardiac tissues in vitro and test their survival and ability to remain functional in vivo.

To address these goals, we first utilized our established hydrogel-based method to engineer 3D human cardiac tissues (“Cardiopatches”) and determine the effects of cardiomyocyte purity and culture conditions on the tissue structure and function. After 2wks of culture, hPSC-CMs in 3D cardiopatches exhibited faster conduction velocity (CV), longer sarcomeres, and increased expression of genes involved in cardiac contractile function compared to hPSC-CMs cultured in age- and purity-matched 2D monolayers. Furthermore, higher hPSC-CM fraction in cardiopatches yielded faster CV, while maximum forces of contraction were achieved for a particular range of hPSC-CM purities (60-80%). Furthermore, engineered cardiopatches demonstrated a positive inotropic response to β-adrenergic stimulation and generated contractile stresses in excess of 10mN/mm2. These functional results were reproducibly achieved using 4 independent hPSC lines.

Through further optimization, we identified low seeding density and transition from serum-free to serum-containing media as critical factors for accelerated maturation in vitro. These modifications yielded cardiopatches with improved electrical and mechanical function, with average contractile stresses (>20 mN/mm2) and CVs (>28 cm/s) approaching values in adult myocardium. Ultrastructural analysis of cardiopatches revealed highly organized sarcomeric structures, characterized by consistent H-zone and I-bands, frequent intercalated discs, abundant mitochondria, and occasional appearance of T-tubules and central M-bands. Continual increase in functional output during 3-week cardiopatch culture was associated with significant upregulation of molecular maturation markers that, in many instances, reached near-adult levels of expression. Under these optimized conditions, we successfully scaled up engineered cardiopatches to clinically relevant dimensions (4x4cm), while preserving high CVs and contractile strength (with absolute forces exceeding 20 mN) and lacking spontaneous or pacing-induced arrhythmias.

Finally, we explored the ability of cardiopatches to survive and undergo vascularization in vivo using a dorsal skinfold window chamber model in immunocompromised mice and following epicardial implantation onto healthy rat hearts. Within 2 weeks post-implantation into window chambers, initially avascular cardiopatches underwent robust vascularization in vivo and maintained ability to fire Ca2+ transients, albeit at an expense of declined electrical function. In an attempt to improve the vascularization and function in vivo, we developed methods to pre-vascularize cardiopatches with highly branched vascular networks made of hPSC-derived endothelial cells (hPSC-ECs). Upon implantation in window chambers, hPSC-ECs in cardiopatches co-localized with host vasculature and formed hybrid microvessels, indicative of host-donor vascular integration. To further establish their translational potential, we implanted cardiopatches into the mechanically active environment of healthy rat hearts, and demonstrated their survival, vascular integration, and higher levels of electrical function relative to those in dorsal window chambers. Lastly, as a proof-of-concept study, we implanted cardiopatches into a porcine model of myocardial infarction and demonstrated evidence of hiPSC-CM survival, thus providing a foundation for future large animal studies aimed at clinical translation.

In summary, this thesis describes novel methodologies to engineer human cardiac tissues with near-adult levels of electrical and mechanical function, capacity for pre-vascularization, scalability to clinical dimensions, and ability to survive, vascularize, and remain functional in vivo. To the best of our knowledge, the reported functional parameters of cardiopatches are the highest in the field, and our scale-up and pre-vascularization of cardiopatches without loss of function are the first reported in the field. As such, this thesis represents a significant advancement in human heart tissue engineering research that will enable development of next generation cell-based therapies for cardiac repair.

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Shadrin, Ilya (2018). Maturation, Scale-up and Vascularization of Engineered Cardiac Muscle from Human Pluripotent Stem Cells. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/16776.

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