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

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 If 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 If 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 If (n=7 cells) illustrating the dose-dependent If attenuation by ivabradine. BaCl2 (500 μmol/L) was included in the extracellular solution to inhibit IK1. 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.