Electrical Stimulation Promotes Cardiac Differentiation of Human Induced Pluripotent Stem Cells

Abstract

Background. Human induced pluripotent stem cells (iPSCs) are an attractive source of cardiomyocytes for cardiac repair and regeneration. In this study, we aim to determine whether acute electrical stimulation of human iPSCs can promote their differentiation to cardiomyocytes. Methods. Human iPSCs were differentiated to cardiac cells by forming embryoid bodies (EBs) for 5 days. EBs were then subjected to brief electrical stimulation and plated down for 14 days. Results. In iPS(Foreskin)-2 cell line, brief electrical stimulation at 65 mV/mm or 200 mV/mm for 5 min significantly increased the percentage of beating EBs present by day 14 after plating. Acute electrical stimulation also significantly increased the cardiac gene expression of ACTC1, TNNT2, MYH7, and MYL7. However, the cardiogenic effect of electrical stimulation was not reproducible in another iPS cell line, CERA007c6. Beating EBs from control and electrically stimulated groups expressed various cardiac-specific transcription factors and contractile muscle markers. Beating EBs were also shown to cycle calcium and were responsive to the chronotropic agents, isoproterenol and carbamylcholine, in a concentration-dependent manner. Conclusions. Our results demonstrate that brief electrical stimulation can promote cardiac differentiation of human iPS cells. The cardiogenic effect of brief electrical stimulation is dependent on the cell line used.

Figure 1

Characterisation of human iPSC line CERA007c6. Representative images showing expression of pluripotency markers (a) OCT4 and (b) TRA1-60 in the undifferentiated cells. CERA007c6 can be differentiated into all three lineages: (c) endoderm (alpha-fetoprotein, AFP), (d) mesoderm (smooth muscle actin, SMA), and ectoderm (Nestin). (f) An IgG negative control. Scale bar = 100 μm.

Figure 2

Custom made electrical stimulator. (a) A panel of 16 platinum-coated gold electrodes that fit into an 8-well chamber slide. Each well contained a pair of electrodes to stimulate the EBs in suspension. (b) Representative oscilloscope diagram of a pair of electrodes (at 200 mV/mm of the electric field) showing a charge-balanced biphasic current pulse (red) and the corresponding electrode voltage (yellow). Each current pulse was 1 ms/phase and was stimulated at 1 Hz. EB: embryoid body.

Figure 3

Acute electrical stimulation enhances cardiac differentiation of iPS(Foreskin)-2 cell line. (a-b) The effect of acute electrical stimulation at 65 mV/mm (a) or 200 mV/mm (b) on cardiac differentiation of human iPS(Foreskin)-2 cell line (n = 5–15 independent experiments). (c-d) Quantitative RT-PCR analysis for the expression of cardiac transcription factors (c) and cardiac contractile muscle proteins (d) in electrically stimulated and control EBs at days 0, 1, and 7 after electrical stimulation (n = 7 independent experiments). Data are expressed as mean ± SEM. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 by one-way (a-b) or two-way (c-d) ANOVA with the Bonferroni post hoc test.

Figure 4

Acute electrical stimulation did not promote cardiac differentiation of CERA007c6 iPSCs. (a) The effect of acute electrical stimulation at 200 mV/mm for 5 min on cardiac differentiation of CERA007c6 iPSCs at day 14 after plating (n = 3 independent experiments). (b-c) Quantitative RT-PCR analysis for the expression of cardiac transcription factors (b) and cardiac contractile muscle proteins (c) in electrically stimulated and control EBs at days 0, 1, and 7 after electrical stimulation (n = 3 independent experiments). Data are expressed as mean ± SEM.

Figure 5

Expression of cardiac-specific markers in cardiomyocytes derived from iPS(Foreskin)-2 cell line. (a) Beating EBs expressed cardiac contractile proteins, cardiac troponin T, sarcomeric α-actinin, myosin heavy chain, and gap junctional protein connexin 43 (Cx43, white arrow) (scale bar = 50 μm). Quantitative RT-PCR analysis for the expression of cardiac transcription factors (b) and cardiac contractile muscle proteins (c) in electrically stimulated and control beating EBs at day 14 after plating (n = 5–7 independent experiments). (d) MYL2/MYL7 gene expression ratio. (e) Percentage of cardiac troponin T-positive cells in each beating EB at day 14 after plating (n = 5-6 independent experiments). Data are expressed as mean ± SEM. ES: electrically stimulated and undiff: undifferentiated iPSCs.

Figure 6

Electrophysiological properties of cardiomyocytes derived from iPS(Foreskin)-2 cell line. (a) Beating EBs at day 14 after plating were cycling calcium. Intensity of fluorescence has been normalized with background. (b) A representative image of a microelectrode array with beating pieces and the extracellular field potential recorded. (c) The percentage change in beating rate and (d) the cFPD of cardiomyocytes from control and electrically stimulated (ES, 200 mV/mm, 5 min) groups treated with isoproterenol hydrochloride (isoprenaline, 1–1000 nM) or carbamylcholine (carbachol, 1–1000 nM) (n = 3 independent experiments). (e) Quantitative RT-PCR analysis for the expression of muscarinic receptor 2 (CHM2), β-adrenergic receptor-1 (ADRB1), and β-adrenergic receptor-2 (ADRB2) in control and electrically stimulated beating EBs (n = 4–7 independent experiments). ES: electrically stimulated and undiff: undifferentiated iPSCs. Data are expressed as mean ± SEM. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 by one-way ANOVA with the Bonferroni post hoc test.