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.

Polyethylene Terephthalate Textiles Enhance the Structural Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

“…Previous studies analyzing the effect of CM orientation and anisotropy on calcium handling of hiPSC CMs have reported inconsistent results [16,18,21,38,43,44]. According to our data, CMs cultured on PET textiles had slightly altered calcium handling properties, but no significant changes were observed.…”

“…By providing a three-dimensional (3D) environment with proper topographical cues and an extracellular matrix, cells can create more cell–ECM connections, which potentially affect cell proliferation and even differentiation and maturation [20]. Stiffness of a flat, 2D culture substrate affects cell properties, proliferation, and differentiation [21]. To increase the cardiac functionality and maturity, hiPS-CMs have been cultured with, for example, endothelial cells, and this has shown to enhance cardiomyocyte proliferation and functionality [22].…”

Striated myocyte structural integrity: Automated analysis of sarcomeric z-discs

“…Using improved z-line extraction and a biologically motivated approach to segmenting off-target staining, we were able to automatically and accurately isolate z-lines. We also designed and implemented an algorithm to measure the length of continuous z-lines, which is physically related to z-line registration [32,34,41,68,69]. Improved z-line extraction coupled with a variety of metrics allowed ZlineDetection to capture differences in z-line architecture among standard engineered cardiac tissues (Fig 4).…”

“…In addition to the orientational order, the relative spatial location of z-lines in neighboring myofibrils (e.g. continuity or registration of z-lines) has been used to evaluate striated myocytes [34,41,68,69]. In this work, we created and automated an algorithm to measure continuous z-lines by grouping z-line pixels based on relative orientation and location.…”

Next generation of heart regenerative therapies: progress and promise of cardiac tissue engineering

“…Promoting this level of cellular organization can be achieved using grooved patterned substrates where hPSC-CMs already showed elongation along the line patterns, electromechanical coupling with neighboring CMs, and electrophysiological improvements 68 , 102 . Of note, substrate stiffness, groove width, and cell density seem to have an impact on the structural maturation of hPSC-CMs 103 . In fact, the stiffness of a substrate can by itself modulate the mechanical load experienced by hPSC-CMs, affecting the cytoskeleton organization 104 and contractility 102 .…”