Abstract
The in vitro generation of human cardiomyocytes derived from induced pluripotent stem cells (iPSC) is of great importance for cardiac disease modeling, drug-testing applications and for regenerative medicine. Despite the development of various cultivation strategies, a sufficiently high degree of maturation is still a decisive limiting factor for the successful application of these cardiac cells. The maturation process includes, among others, the proper formation of sarcomere structures, mediating the contraction of cardiomyocytes. To precisely monitor the maturation of the contractile machinery, we have established an imaging-based strategy that allows quantitative evaluation of important parameters, defining the quality of the sarcomere network. iPSC-derived cardiomyocytes were subjected to different culture conditions to improve sarcomere formation, including prolonged cultivation time and micro patterned surfaces. Fluorescent images of α-actinin were acquired using super-resolution microscopy. Subsequently, we determined cell morphology, sarcomere density, filament alignment, z-Disc thickness and sarcomere length of iPSC-derived cardiomyocytes. Cells from adult and neonatal heart tissue served as control. Our image analysis revealed a profound effect on sarcomere content and filament orientation when iPSC-derived cardiomyocytes were cultured on structured, line-shaped surfaces. Similarly, prolonged cultivation time had a beneficial effect on the structural maturation, leading to a more adult-like phenotype. Automatic evaluation of the sarcomere filaments by machine learning validated our data. Moreover, we successfully transferred this approach to skeletal muscle cells, showing an improved sarcomere formation cells over different differentiation periods. Overall, our image-based workflow can be used as a straight-forward tool to quantitatively estimate the structural maturation of contractile cells. As such, it can support the establishment of novel differentiation protocols to enhance sarcomere formation and maturity.
Highlights
The generation of induced pluripotent stem cell was a hallmark in stem cell research, enabling the generation of almost any cell type from individual donors
We showed that sarcomere maturation can be profoundly improved when induced pluripotent stem cells (iPSC)-CMs are gown on structured surfaces that facilitate the development of adult-like cell morphology
Large progress has been made in the development of iPSC-derived CMs, limited maturation is still a major obstacle that impedes their use in basic research and for clinical applications
Summary
The generation of induced pluripotent stem cell (iPSCs) was a hallmark in stem cell research, enabling the generation of almost any cell type from individual donors Since their derivation does not require the use of human embryos ethical problems are avoided [1, 2]. Cardiomyocytes (CM) derived from iPSCs are commonly used for human disease modeling, therapeutic applications and drug-testing approaches [3, 4]. The immature phenotype of these CMs is a major limitation for their pre-clinical and clinical use [6, 7] This cardiogenic maturation involves multiple developmental changes of the cellular physiology, including morphological alterations, electrophysiological changes, metabolic maturation, and establishment of a proper contraction machinery [7]. Splicing modifications have been found for titin and the regulatory light chain of myosin [6, 10]
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