Induced Pluripotent Stem Cell-Derived Cardiomyocytes Provide In Vivo Biological Pacemaker Function

Abstract

Background: Although multiple approaches have been used to create biological pacemakers in animal models, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have not been investigated for this purpose. We now report pacemaker function of iPSC-CMs in a canine model.

Methods and results: Embryoid bodies were derived from human keratinocytes, their action potential characteristics determined, and their gene expression profiles and markers of differentiation identified. Atrioventricular blocked dogs were immunosuppressed, instrumented with VVI pacemakers, and injected subepicardially into the anterobasal left ventricle with 40 to 75 rhythmically contracting embryoid bodies (totaling 1.3-2×10⁶ cells). ECG and 24-hour Holter monitoring were performed biweekly. After 4 to 13 weeks, epinephrine (1 μg kg^-1 min^-1) was infused, and the heart removed for histological or electrophysiological study. iPSC-CMs largely lost the markers of pluripotency, became positive for cardiac-specific markers. and manifested I_f-dependent automaticity. Epicardial pacing of the injection site identified matching beats arising from that site by week 1 after implantation. By week 4, 20% of beats were electronically paced, 60% to 80% of beats were matching, and mean and maximal biological pacemaker rates were 45 and 75 beats per minute. Maximum night and day rates of matching beats were 53±6.9 and 69±10.4 beats per minute, respectively, at 4 weeks. Epinephrine increased rate of matching beats from 35±4.3 to 65±4.0 beats per minute. Incubation of embryoid bodies with the vital dye, Dil, revealed the persistence of injected cells at the site of administration.

Conclusions: iPSC-CMs can integrate into host myocardium and create a biological pacemaker. Although this is a promising development, rate and rhythm of the iPSC-CMs pacemakers remain to be optimized.

Keywords: action potentials; atrioventricular block; dogs; embryoid bodies; myocardium.

Figure 1.

SOX2 (brown) and troponin T (blue) immunostaining identify the presence of multipotent cells and differentiated cardiac cells, respectively. Arrows indicate cells with a dual positive immunostaining suggestive of cells in a differentiating or dedifferentiating state.

Figure 2.

The effect of ivabradine on automaticity of induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs). A, Representative experiment showing action potentials recorded in absence (control panel) and presence of 0.1, 1, 3, and 10 μmol/L ivabradine. B, Summary of mean beat rate in absence and presence of 0.1, 1, 3, and 10 µmol/L ivabradine. Ivabradine induced a dose-dependent decrease in the mean beat rate (n=12 cells). In B, One-way ANOVA was performed followed by the Holm–Sidak method. *P<0.05 vs control. P <0.05 per Holm–Sidak method was considered statistically significant. All experiments were performed at 36°C.

Figure 3.

The effect of ivabradine on I_f in induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs). Combined current- and voltage-clamp recordings were performed in the same cell. A, Spontaneous action potentials recorded in control Tyrode solution (left). Ivabradine at 10 μmol/L blocked automaticity (right). B, A representative experiment illustrating the dose-dependent attenuation of I_f by ivabradine. C, In the voltage-clamp protocol, the membrane was clamped from a holding potential of −40 to −120 mV in 10 mV steps for 2 s pulse duration. Interpulse interval: 15 s. D, Current-voltage relations of I_f (n=7 cells) illustrating the dose-dependent I_f attenuation by ivabradine. BaCl₂ (500 μmol/L) was included in the extracellular solution to inhibit I_K1. All experiments were performed at 36°C.

Figure 4.

A, Percentage of beats matched to the pacing site in 10 dogs during 4- to 5-wk follow-up period. B, Percentage of electronically paced beats in 8 dogs during 4- to 5-wk follow-up period. Two dogs with longer follow-up periods shown in (A) and (C) were not included because they had no electronic pacemaker. C, Mean and maximum pace-mapped beating rates during 4- to 5-wk follow-up period. Number of dogs that displayed matching rhythm was 9 at wk 1 and 4 to 5, and 10 at wk 2 to 3. *P<0.05 vs wk 1 (ANOVA and Bonferroni correction). In addition, (A) and (C) are plotted individual data of the 2 animals that were followed through wk 8 to 9 and 12 to 13, respectively.

Figure 5.

A, Representative ECGs recorded during pace mapping at time of implant and 5 and 19 d after embryoid bodies (EBs) implantation. B, Time course of the percent and maximum rate of matching beats recorded from the same dog during 4 wk of follow-up. C, Automatic rhythm recorded from a ventricular slab removed from the site of EB injection on the day of the terminal study.

Figure 6.

Induced pluripotent stem cells (iPSCs) were stained with the vital dye Dil, and serial sections were stained as indicated in Methods. Upper, from left to right, Dil, Cx43, and DAPI staining. The merged image (lower) is from an area containing Dil-stained cells intercalated between myocytes. Green=Cx43 (Connexin 43) staining, Blue=DAPI, Red–Orange=Dil. Arrows indicate Cx43 staining within the region occupied by Dil-stained cells. Red background is because of leaching of the Dil from the delivered iPSCs.