Abstract
The maturation of iPSC-derived cardiomyocytes is still a critical point for their application in cardiovascular research as well as for their clinical use. Although multiple differentiation protocols have been established, researchers failed to generate fully mature cardiomyocytes in vitro possessing identical phenotype-related and functional properties as their native adult counterparts. Besides electrophysiological and metabolic changes, the establishment of a well structured sarcomere network is important for the development of a mature cardiac phenotype. Here, we present a super resolution-based approach to quantitatively evaluate the structural maturation of iPSC-derived cardiomyocytes. Fluorescence labelling of the α-actinin cytoskeleton and subsequent visualization by photoactivated localization microscopy allows the acquisition of highly resolved images for measuring sarcomere length and z-disc thickness. Our image analysis revealed that iPSC and neonatal cardiomyocyte share high similarity with respect to their sarcomere organization, however, contraction capacity was inferior in iPSC-derived cardiac cells, indicating an early maturation level. Moreover, we demonstrate that this imaging approach can be used as a tool to monitor cardiomyocyte integrity, helping to optimize iPSC differentiation as well as somatic cell direct-reprogramming strategies.
Highlights
In recent years, induced pluripotent stem cell technology has become a major technology in cardiovascular research as it enables efficient in vitro generation of functional cardiomyocytes (CM) without any ethical concerns
To quantitatively evaluate the structural maturation of induced pluripotent stem cell (iPSC)-derived CM, the sarcomere network was visualized by labelling α-actinin filaments and samples were subjected to photoactivated localization microscopy (PALM) imaging (Figure 1)
Localization uncertainty is reduced, which provokes a decrease of the lateral resolution (Figure 3C). These data highlight the importance of appropriate imaging conditions when evaluating sarcomere organization in cardiac cells using single-molecule localization microscopy
Summary
In recent years, induced pluripotent stem cell (iPSC) technology has become a major technology in cardiovascular research as it enables efficient in vitro generation of functional cardiomyocytes (CM) without any ethical concerns. The proper maturation is a common problem as iPSC-CM do not display the same morphological and functional features as their adult counterparts, rather resembling fetal CM [4,5] In this regard, it has been found that the low level of maturation can be critical in drug development since immature CM seem to be more susceptible to HERG blockers than mature CM [6]. While adult CM are characterized by a well aligned, highly organized sarcomere network, iPSC-derived CM commonly demonstrate disarranged sarcomeric filaments, possessing a lower contraction capability which is more comparable to fetal, immature cardiac cells [12,13]. Correlation with functional data showed that the contraction capacity related to cell size is superior in neonatal cells if compared to iPSC-derived CM, which confirms their immature structural phenotype
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