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

• Published in: Circulation Journal 2013, Vol.77(5), pp.1307-1314
• Main Authors: Kamakura, Tsukasa, Makiyama, Takeru, Sasaki, Kenichi, Yoshida, Yoshinori, Wuriyanghai, Yimin, Chen, Jiarong, Hattori, Tetsuhisa, Ohno, Seiko, Kita, Toru, Horie, Minoru, Yamanaka, Shinya, Kimura, Takeshi
• Format: Article
• Language: English
• Subjects: Biomarkers - metabolism; Cardiomyocytes; Cell Differentiation; Cell Line; Cell Lineage; Gene Expression Regulation, Developmental; Humans; Immunohistochemistry; Induced pluripotent stem cells; Induced Pluripotent Stem Cells - metabolism; Induced Pluripotent Stem Cells - ultrastructure; Microscopy, Electron, Transmission; Muscle Proteins - genetics; Muscle Proteins - metabolism; Myocardial Contraction; Myocytes, Cardiac - metabolism; Myocytes, Cardiac - ultrastructure; Sarcomeres - diagnostic imaging; Sarcomeres - metabolism; Time Factors; Ultrasonography; Ultrastructure
• ISSN: 1346-9843; 1347-4820
• DOI: 10.1253/circj.CJ-12-0987

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 M-bands. 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)