Int. J. Dev. Biol. 65: 537 - 543 (2021)

https://doi.org/10.1387/ijdb.210115fs

Vol 65, Issue 10-11-12

Human C2a and C6a iPSC lines and H9 ESC line have less efficient cardiomyogenesis than H1 ESC line: Beating enhances cardiac differentiation

Original Article | Published: 13 December 2021

Ana Sepac¹, Zeljko J. Bosnjak², Sven Seiwerth^1,3, Suncana Sikiric¹, Tihana Regovic Dzombeta^1,4, Ana Kulic⁵, Jelena Marunica Karsaj⁶ and Filip Sedlic*^,7

¹Department of Pathology, University of Zagreb School of Medicine, Zagreb, Croatia, ²Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, USA, ³Department of Pathology and Cytology, University Hospital Center Zagreb, Zagreb, Croatia, ⁴Department of Pathology, University Hospital Center Sestre milosrdnice, Zagreb, Croatia, ⁵Department of Oncology, Clinical Unit of Pathophysiology and Experimental Oncology, University Hospital Center Zagreb, Zagreb, Croatia, ⁶University Hospital Center Sestre milosrdnice, Zagreb, Croatia and ⁷Department of Pathophysiology, University of Zagreb School of Medicine, Zagreb, Croatia

Abstract

Background: Human induced pluripotent stem cells (hiPSCs) need to be thoroughly characterized to exploit their potential advantages in various aspects of biomedicine. The aim of this study was to compare the efficiency of cardiomyogenesis of two hiPSCs and two human embryonic stem cell (hESC) lines by genetic living cardiomyocyte labeling. We also analyzed the influence of spontaneous beating on cardiac differentiation. Methods: H1 and H9 hESC lines and C2a and C6a hiPSC lines were induced into in vitro directed cardiac differentiation. Cardiomyogenesis was evaluated by the analysis of cell cluster beating, cardiac protein expression by immunocytochemistry, ability of cells to generate calcium transients, and cardiomyocyte quantification by the myosin light chain 2v-enhanced green fluorescent protein gene construct delivered with a lentiviral vector. Results: Differentiation of all cell lines yielded spontaneously beating cell clusters, indicating the presence of functional cardiomyocytes. After the cell dissociation, H1-hESC-derived cardiomyocytes exhibited spontaneous calcium transients, corresponding to autonomous electrical activity and displayed ability to transmit them between the cells. Differentiated hESC and hiPSC cells exhibited striated sarcomeres and expressed cardiac proteins sarcomeric α-actinin and cardiac troponin T. Cardiomyocytes were the most abundant in differentiated H1 hESC line (20% more than in other tested lines). In all stem cell lines, cardiomyocyte enrichment was greater in beating than in non-beating cell clusters, irrespective of cardiomyogenesis efficiency. Conclusion: Although C2a and C6a hiPSC and H9 hESC lines exhibited efficient cardiomyogenesis, H1 hESC line yielded the greatest cardiomyocyte enrichment of all tested lines. Beating of cell clusters promotes cardiomyogenesis in tested hESCs and hiPSCs.

Keywords

cardiomyogenesis, hiPSC, hESC, beating, calcium transients