Induced Pluripotent Stem Cell–Based Treatment of Acquired Heart Block: The Battle for Tomorrow Has Begun!

Electronic pacing devices are designed to maintain a normal heart rate and have been used for >50 years to correct bradycardias caused by atrioventricular node block or sinus node dysfunction. These electronic devices work very well but are limited by their high cost and limited battery life, as well as risks for generator/lead failure, infection, hemorrhage, and cardiac/pulmonary collapse.¹ Biological pacing is an attractive concept because it avoids these limitations while providing hardware-free cardiac pacing. Early studies used virus-mediated somatic gene transfer to modify the electrophysiologic properties of quiescent atrial or ventricular cells with the intent of achieving spontaneous electric firing to maintain chronotropic competency. Of these, the 2 best known approaches focus on (1) inhibiting the chamber-specific inward rectifier K⁺ current, which normally keeps the resting membrane potential at a strongly hyperpolarized voltage to electrically stabilize membrane potentials² or (2) introducing a pacemaker-specific hyperpolarization-activated cyclic nucleotide–gated channel that mediates the funny current (I_f), which is activated by hyperpolarization and mediates an inward current leading to diastolic depolarization and pacemaker activity.³ While these approaches successfully conferred pacemaker activity, genetically modified cells lack many other features of the native sinoatrial node (such as a short action potential and heterogenous cell-to-cell conductance coupling), which limits the ability of these approaches to mimic the natural control of heart rhythm and respond appropriately to cardiovascular stressors.

With the advent of stem cell and cellular reprogramming techniques,⁴ sinoatrial node–like pacemaker cells have been generated by reprogramming ventricular myocytes^5,6 or directing the differentiation of embryonic stem cells.^7–10 In the former, sinoatrial node pacemaker cells were obtained by reprogramming rat or guinea pig ventricular myocytes via overexpression of Tbx18 (a transcription factor critical for embryonic development of the sinoatrial node tissue).⁵ Successful in vivo reprogramming of pig ventricular myocytes into pacemaker cells has since shown that this Tbx18-reprogrogramming strategy can be translated to large animals.⁶ In contrast, overexpression of Tbx3 has been shown to promote the differentiation of mouse embryonic stem cells into pacemaker cells such that >80% of reprogrammed cells attain a pacemaker phenotype.⁷ Altering other critical regulators of embryonic sinoatrial node development in murine embryonic stem cells (such as Shox2) increase pacemaker cell generation and have been shown to correct bradycardia in small animal models of acquired heart block.⁸ Most recently, pacemaker cells have been derived from human embryonic stem cells using inductive media conditions to prevent the development of Nkx2.5+ cardiomyocytes, resulting in cells that exhibit properties of sinoatrial node cells and function as biological pacemakers in small animal models of heart block.⁹

In this issue of Circulation: Arrhythmia and Electrophysiology, Chauveau et al¹¹ report the generation of pacemaker-like myocytes from human induced pluripotent stem cells (iPSCs), which were derived from human hair follicle keratinocytes donated by healthy volunteers. iPSCs were differentiated using the serum-driven embryoid body formation protocol, and spontaneously beating areas within the culture system were mechanically isolated and collected for downstream studies. GeneChip expression analysis showed robust expression of cardiac-specific genes, whereas immunostaining documented protein expression of cardiac-specific markers (ie, troponin T). Patch-clamp recordings demonstrated automaticity (spontaneous firings of action potentials) and a prominent characteristic sinoatrial node I_f current, which was sensitive to ivabradine (an I_f blocker). Continuous ambulatory electrocardiographic monitoring revealed that 1 to 4 weeks after transplant of human iPSC-derived spontaneously beating cardiomyocyte aggregates into the left ventricular free wall of immunosuppressed dogs with atrioventricular block, 60% to 80% of the heartbeats originated at regions consistent with the site of cell injection. These ectopic beats were also catecholamine sensitive as ventricular ectopy from the site of injection increased during adrenergic receptor stimulation using epinephrine infusion. Finally, the authors labeled iPSC cardiomyocytes with the lipophilic membrane stain DiI before transplantation, and, 9 days after intramyocardial injection, histology revealed that DiI+ cells were found near the injection site. Most notably, these DiI+ cells also expressed connexin 43, suggesting effective electric coupling to existing myocardium.

Thus, this study is the first to test a human iPSC-based biological pacemaker in a large animal model of heart block.¹¹ As iPSCs can be made for individual patients, it provides important proof of concept that it may be possible to generate patient-specific cardiac pacemaker cells—an important step toward autologous cell therapy for bradycardia. This study also showed that iPSC-based pacemaker cells have I_f-based automaticity, pace atrioventricular node blocked hearts at physiologic rates, and demonstrate rate responsiveness to adrenergic challenges (the latter being an obvious advantage compared with electronic devices). The large animal model used in this study allowed testing the iPSC-based pacemaker cells in a preclinical setting, facilitating future translation of this strategy to patients. Interesting although these findings might be, there are several critical limitations that need to be addressed before iPSC-based pacing can hope to usurp electronic pacemakers, including (1) clear confirmation that these iPSC-derived cells represent true pacemaker cells that express sinoatrial node–specific markers (eg, Tbx18, Tbx3, Shox2, Hcn4, and Cx45) rather than immature electrically unstable chamber myocytes that exhibit automaticity and I_f, (2) clear evidence supporting transplanted cell survival and functional integration (ie, electric coupling) within the host myocardium at time points much later than 9 days after injection, (3) detailed analysis of iPSC-derived pacemaker cells maturation status as pacemaker cell size is critical to overcome source–sink mismatch although stable ongoing electric depolarization is essential to enable long-term function.⁴ Nevertheless, this study is an important step forward in the journey to enable development of biological therapies for the pacemaker patients of tomorrow.