Pluripotent stem cell-derived cardiac tissue patch with advanced structure and function

Abstract

Recent advances in pluripotent stem cell research have provided investigators with potent sources of cardiogenic cells. However, tissue engineering methodologies to assemble cardiac progenitors into aligned, 3-dimensional (3D) myocardial tissues capable of physiologically relevant electrical conduction and force generation are lacking. In this study, we introduced 3D cell alignment cues in a fibrin-based hydrogel matrix to engineer highly functional cardiac tissues from genetically purified mouse embryonic stem cell derived cardiomyocytes (CMs) and cardiovascular progenitors (CVPs). Procedures for CM and CVP derivation, purification, and functional differentiation in monolayer cultures were first optimized to yield robust intercellular coupling and maximize velocity of action potential propagation. A versatile soft-lithography technique was then applied to reproducibly fabricate engineered cardiac tissues with controllable size and 3D architecture. While purified CMs assembled into a functional 3D syncytium only when supplemented with supporting non-myocytes, purified CVPs differentiated into cardiomyocytes, smooth muscle, and endothelial cells, and autonomously supported the formation of functional cardiac tissues. After a total culture time similar to period of mouse embryonic development (21 days), the engineered cardiac tissues exhibited unprecedented levels of 3D organization and functional differentiation characteristic of native neonatal myocardium, including: 1) dense, uniformly aligned, highly differentiated and electromechanically coupled cardiomyocytes, 2) rapid action potential conduction with velocities between 22 and 25 cm/s, and 3) significant contractile forces of up to 2 mN. These results represent an important advancement in stem cell-based cardiac tissue engineering and provide the foundation for exploiting the exciting progress in pluripotent stem cell research in the future tissue engineering therapies for heart disease.

Physiological aspects of cardiac tissue engineering

“…Cardiomyocytes, similar to skeletal muscle cells, increase active force development in response to increased preload (Frank-Starling mechanism). Several studies have shown the same to be true in engineered cardiac constructs [ Table 1 and (4,26,50,94,108,110)] and reported increases between 1.9 -10-fold. The substantial variation is likely in part due to practical difficulties in defining baseline values for force development.…”

“…The heart consists of fibroblasts, endothelial cells, and cardiomyocytes, raising the question as to whether a certain ratio of these three cell populations supports constitution of cardiac tissues in vitro. A few reports have addressed this question systematically and most support the hypothesis that endothelial cells and fibroblasts support tissue development (12,60,71,94), but one also reported inferior outcomes in the presence of fibroblasts (50), indicating that more work is required to answer this question.…”

Biomimetic engineered muscle with capacity for vascular integration and functional maturation in vivo

“…Engineered muscle constructs were loaded into a custom-made force-measurement setup containing an optical force transducer and a linear actuator (ThorLabs), as previously described (25,26,42). Samples were stimulated (10 ms, 3 V/mm pulses) at different frequencies (1-40 Hz), and isometric passive and active (contractile) forces were measured at different muscle lengths.…”

Cardiac Tissue Engineering

“…74 Cardiac 3D constructs from chickens, rats, mice, and humans show a positive inotropic and chronotropic response to β-adrenergic stimulation, which is sensitive to the muscarinic agonist carbachol. 74 Furthermore, they increase force with ; in rat, 76 murine, 22 and mouse EHTs, 77 it was negative. Collectively, this demonstrates that 3D engineered tissues function qualitatively like native muscle.…”