Ultrastructural Maturation of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes in a Long-Term Culture

Abstract

Background:In the short-to mid-term, cardiomyocytes generated from human-induced pluripotent stem cells (hiPSC-CMs) have been reported to be less mature than those of adult hearts. However, the maturation process in a long-term culture remains unknown. Methods and Results:A hiPSC clone generated from a healthy control was differentiated into CMs through embryoid body (EB) formation. The ultrastructural characteristics and gene expressions of spontaneously contracting EBs were analyzed through 1-year of culture after cardiac differentiation was initiated. The 14-day-old EBs contained a low number of myofibrils, which lacked alignment, and immature high-density Z-bands lacking A-, H-, I-, and Mbands. Through the long-term culture up to 180 days, the myofibrils became more tightly packed and formed parallel arrays accompanied by the appearance of mature Z-, A-, H-, and I-bands, but not M-bands. Notably, M-bands were finally detected in 360-day-old EBs. The expression levels of the M-band-specific genes in hiPSC-CMs remained lower in comparison with those in the adult heart. Immunocytochemistry indicated increasing number of MLC2v-positive/MLC2a-negative cells with decreasing number of MLC2v/MLC2a double-positive cells, indicating maturing of ventricular-type CMs. Conclusions:The structural maturation process of hiPSC-CMs through 1-year of culture revealed ultrastructural sarcomeric changes accompanied by delayed formation of M-bands. Our study provides new insight into the maturation process of hiPSC-CMs. (Circ J 2013; 77: 1307 -1314

Induced pluripotent stem-cell-derived cardiomyocytes: cardiac applications, opportunities, and challenges

“…Once differentiated, hiPSC-CMs can be kept in culture for several months (Bedada et al 2014;Kamakura et al 2013). During differentiation, hiPSC-CMs rapidly express contractile proteins (Fig.…”

“…The β-adrenergic stimulation pathway also undergoes major modifications during the differentiation process (Jung et al 2016). Longer culture times are known to greatly improve the morphology and function of hiPSC-CMs (Kamakura et al 2013;Lundy et al 2013). Lundy et al reported that hiPSC-CM circularity decreases with aging and is accompanied by an increase in myofibril density and organization (Lundy et al 2013).…”

Stem Cells and Stem Cell-derived Tissues and Their Use in Safety Assessment

“…Alternatively, hPSC-derived cardiomyocytes at the current maturity could be studied and the cardiac channels deconstructed such that a mature phenotype could be generated via computer-based modeling (31). A less optimal approach to maturity is to culture for extended periods of time, as 1 year in culture led to myosin and band changes indicative of adult cardiomyocytes (32,33). Thus, there are a multitude of avenues to explore maturing cardiomyocytes should the scientific need arise.…”

Differentiation of Human Atrial Myocytes From Endothelial Progenitor Cell-Derived Induced Pluripotent Stem Cells

“…Embryoid bodies are three-dimensional aggregates of pluripotent stem cells that undergo differentiation and cell specification into all the three-germ layers including CM in a manner similar to the early embryo [216][217][218][219]. In this technique, small clumps of pluripotent stem cell are dispersed in a suspension culture containing serum media to form spherical aggregates.…”

“…Over the years that followed, a series of optimization studies examined the capacity of first generation iPSC lines to differentiate into CM lineages [214,215]. These approaches include Embryoid Body formation (EB) [216][217][218][219] co-culture with stromal layer [220] and direct cytokine-guided differentiation [221].…”