Influence of electrical stimulation on cardiac differentiation of human induced pluripotent stem cells

© 2016 Dr. Héctor Damián Hernández de SantiagoThe possibility of remodelling human cardiac tissue raises hopes that in the future we may be able to fully restore cardiac function after myocardial infarction. In addition, the ability of human induced pluripotent stem cells (iPSCs) to differentiate into bona fide cardiomyocytes provides a platform for cardiac disease modelling, drug discovery and pharmacological safety testing of new drugs. One of the major limitations for the use of cardiomyocytes derived from iPSCs is that they resemble foetal cardiomyocytes and are immature, both morphologically and functionally. Considering that the developing heart grows in an electric field, I hypothesised that electrical stimulation might affect cardiogenesis of human iPSCs. The overall aim of this PhD thesis is to investigate whether appropriate electrical stimulation can promote cardiac differentiation and maturation of cardiomyocytes derived from human iPSCs. A custom made electrical stimulator was developed to investigate the effect of brief electrical stimulation on cardiac differentiation of iPSCs. Embryoid bodies (EBs) generated from human iPSCs were electrically stimulated for a few minutes with an electric field of 65 or 200 mV/mm at 1 Hz frequency. Brief electrical stimulation for 5 minutes significantly increased the percentage of beating EBs when compared to the non- electrically stimulated contros after 14 days after re-plating them (11.7 ± 2.3% vs. 4.9 ± 1.4% in control, p<0.05). This cardiogenic effect was not observed in EBs electrically stimulated for longer (10-15 minutes) or shorter (1.5 minutes) durations, suggesting 5 minutes of electrical stimulation is not only sufficient to promote cardiac differentiation, but also optimal. In addition, electrically stimulated EBs displayed increased gene expression of cardiac transcription factors (TBX4 and NKX2.5) and cardiac contractile proteins (α-actinin, cardiac troponin-T, myosin heavy chain beta isoform and myosin light chain atrial isoform), compared to the non-electrically stimulated group. Furthermore, only beating EBs from the electrically stimulated group were responsive to the chronotopic agents, carbachol and isoprenaline. In a separate study, brief electrical stimulation did not further enhance the cardiogenic effect of growth factors and small molecules (a combination of trichostatin A, activin A and BMP4 treatment during EB formation). To test the effect of long-term electrical stimulation on cardiomyocytes derived from human iPSCs, immature cardiomyocytes at day 27 post-differentiation were subjected to continuous electrical stimulation at 200 mV/mm for 7 days. The long-term electrical stimulation significantly increased the percentage of cardiomyocytes with organized sarcomeres (39 ± 8% vs. 23 ± 11% in control, p<0.05). Electrical stimulation also promoted alignment of cardiomyocytes in parallel to the electric field (10 ± 1% vs. 6 ± 2% in control, p<0.05) and decreased the circularity index (0.69 ± 0.02 vs. 0.74 ± 0.02 in control, p