Engineering 3D Cardiac Microtissues from Human Pluripotent Stem Cells

There has been limited progress toward the development of in vitro cardiac tissue models and cellular cardiac therapies due to the lack of an easily obtainable and expandable cell source. Human pluripotent stem cells (hPSCs) represent a potentially unlimited source of cardiomyocytes for the study of cardiovascular development, disease, and tissue homeostasis, as well as regenerative cardiac therapies. An enhanced understanding of interactions between cardiomyocytes and their surrounding microenvironment is required in order to generate appropriate in vitro models of cardiac diseases. In this study, we model cardiac microenvironments by generating 3D cardiospheres of hPSC-derived cardiomyocytes using controlled formation and culture conditions to modulate cardiomyocyte phenotype and physiological function.

Differentiation of cardiomyocytes from hPSCs was achieved within 14 days with both growth factor- and small molecule-directed protocols on 2D monolayer cultures using a serum-free medium. Following 2D differentiation, cultures were trypsinized and seeded into 400 µm diameter microwells (Aggrewell™) at a density of 500-1500 cells/microwell. After 24 hours of formation, 3D cardiospheres were transferred to suspension culture and maintained for 7 days. Spheroid size and the percentage of beating spheroids were monitored over time and cardiac marker expression was analyzed after 7 additional days.

The differentiation protocols yielded weakly beating cells by day 10 of differentiation and strongly beating regions across the cultures by day 14. Regardless of the initial cardiomyocyte efficiency, cells aggregated into 3D cardiospheres with nearly 100% exhibiting spontaneous contractile beating, synchronous intracellular calcium transients, and expression of cardiac markers α-actinin and connexin 43. Within a given experiment, cardiospheres maintained their size for the duration of culture, however, the relative sizes of cardiospheres was modulated by controlling the initial cell seeding density (500-1500 cells/sphere). The cell population within the 3D aggregates expressed higher levels of cardiac specific genes, and cells dissociated from cardiospheres exhibited an enrichment of cardiomyocytes from a starting population of 10-40% to a final population of 80-99% cardiomyocytes when cultured in 3D cardiospheres as opposed to the purity of parallel 2D monolayer cultures, which remained similar to the starting population.

These studies demonstrate that the combination of forced aggregation and suspension culture enables reliable generation of 3D cardiospheres with nearly 100% spontaneously beating constructs independent of the starting purity, providing a straight-forward method for enrichment of cardiomyocytes. Additionally, microparticles can be incorporated within both hPSC aggregates and cardiospheres, enabling the delivery of factors to direct differentiation or promote cardiomyocyte maturation and survival within the 3D constructs. Ongoing studies aim to understand the complex physical and biochemical elements of 3D cardiac environments and how aggregated cardiospheres can be utilized both as in vitro models to study cardiac health and disease, as well as serve as transplantable constructs for regenerative cardiac therapies.