Characterization of cytoskeleton features and maturation status of cultured human iPSC-derived cardiomyocytes

Abstract

Recent innovations in stem cell technologies and the availability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have opened new possibilities for studies and drug testing on human cardiomyocytes in vitro. Still, there are concerns about the precise nature of such ‘reprogrammed’ cells. We have performed an investigation using immunocytochemistry and confocal microscopy on several cellular features using commercially available hiPSC-CMs. For some selected developmentally regulated or cardiac chamber-specific proteins, we have compared the results from hiPSC-derived cardiomyocytes with freshly isolated, ventricular cardiomyocytes from adult rats. The results show that all typical cardiac proteins are expressed in these hiPSC-CMs. Furthermore, intercalated disclike structures, calcium cycling proteins, and myofibrils are present. However, some of these proteins are only known from early developmental stages of the ventricular myocardium or the diseased adult heart. A heterogeneous expression pattern in the cell population was noted for some muscle proteins, such as for myosin light chains, or incomplete organization in sarcomeres, such as for telethonin. These observations indicate that hiPSC-CMs can be considered genuine human cardiomyocytes of an early developmental state. The here described marker proteins of maturation may become instrumental in future studies attempting the improvement of cardiomyocyte in vitro models.

Quantitative Evaluation of the Sarcomere Network of Human hiPSC-Derived Cardiomyocytes Using Single-Molecule Localization Microscopy

“…This is in agreement with numerous studies showing that the differentiation status of CMs generated from stem cells resemble an embryonic or neonatal state [4,21,22]. The average sarcomere length in iPSC-derived CM is about 1.84 µm, which corresponds to former reports showing that the distance between two adjacent sarcomeres is~1.7-2.0 µm (Figure 2) [14,16,23]. This large variety in sarcomere length, observed in several studies, might be attributed to different imaging techniques used, causing varying extents of artefacts [24,25].…”

“…To evaluate the structural maturation of iPSC CMs, cells are usually subjected to microscopic approaches that allow the quantification of sarcomere organization and alignment [14][15][16]. While electron microscopy provides high spatial resolution, sample preparation and image acquisition is complex and extensive user skills are required.…”

3D Co-culture of hiPSC-Derived Cardiomyocytes With Cardiac Fibroblasts Improves Tissue-Like Features of Cardiac Spheroids

“…The intermediate filament protein vimentin was used as marker for cardiac fibroblasts because it is expressed in both quiescent and activated cardiac fibroblasts in vitro and in vivo (Ali et al, 2014;Kofron et al, 2017). Vimentin is not found in freshly isolated adult rat cardiomyocytes, but it is expressed to some extent in 2D cultured hiPSC-CMs as we have previously shown (Zuppinger et al, 2017). Using cryosections of adult murine left ventricle we examined the distribution and morphology of cardiac fibroblasts in normal murine ventricular tissue (Figure 2A).…”

“…For the identification of hiPSC-CMs in 2D-and 3D cultures we used sarcomeric proteins that are well-organized at this stage in hiPSC-CM in 2D culture and in cardiac spheroids (Beauchamp et al, 2015;Zuppinger et al, 2017). The intermediate filament protein vimentin was used as marker for cardiac fibroblasts because it is expressed in both quiescent and activated cardiac fibroblasts in vitro and in vivo (Ali et al, 2014;Kofron et al, 2017).…”

Disruption of histamine/H1R signaling pathway represses cardiac differentiation and maturation of human induced pluripotent stem cells

“…Myofibrils and sarcomeres are mature indicators related to the contraction force of cardiomyocytes [35,36]. Ultrastructural characterization by TEM demonstrated that histamine treatment showed better-organized cross-striated myofilaments and more mitochondria in hiPSC-CMs on day 18 compared with those in the control group (Fig.…”