Substrate Stiffness, Cell Anisotropy, and Cell-Cell Contact Contribute to Enhanced Structural and Calcium Handling Properties of Human Embryonic Stem Cell-Derived Cardiomyocytes.

ABSTRACT

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) can be utilized to understand the mechanisms underlying the development and progression of heart disease, as well as to develop better interventions and treatments for this disease. However, these cells are structurally and functionally immature, which undermines some of their adequacy in modeling adult heart tissue. Previous studies with immature cardiomyocytes have shown that altering substrate stiffness, cell anisotropy, and/or cell-cell contact can enhance the contractile and structural maturation of hPSC-CMs. In this study, the structural and calcium handling properties of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were enhanced by exposure to a downselected combination of these three maturation stimuli. First, hESC-CMs were seeded onto substrates composed of two commercial formulations of polydimethylsiloxane (PDMS), Sylgard 184 and Sylgard 527, whose stiffness ranged from 5 kPa to 101 kPa. Upon analyzing the morphological and calcium transient properties of these cells, it was concluded that a 21 kPa substrate yielded cells with the highest degree of maturation. Next, these PDMS substrates were microcontact-printed with laminin to force the cultured cells into rod-shaped geometries using line patterns that were 12, 18, or 24 µm in width. We found that cells on the 18 and 24 µm pattern widths had structural and functional properties that were superior to those on the 12 µm pattern. The hESC-CMs were then seeded onto these line-stamped surfaces at a density of 500 000 cells per 25-mm-diameter substrate, to enable the formation of cell-cell contacts at their distal ends. We discovered that this combination of culture conditions resulted in cells that were more structurally and functionally mature than those that were only exposed to one or two stimuli. Our results suggest that downselecting a combination of mechanobiological stimuli could prove to be an effective means of maturing hPSC-CMs in vitro.