Towards safer iPSC-CM transplantation: steroid-sparing immunosuppression and arrhythmia prevention

This editorial refers to ‘Immune regulation following allogeneic iPSC-derived cardiomyocyte transplantation in non-human primates’, by S. Chino et al., https://doi.org/10.1093/cvr/cvaf249. Despite tremendous progress in heart failure treatment, truly regenerative therapies are still sorely lacking. Heart transplantation remains the gold standard for end-stage disease, but the number of patients in need far exceeds the available donor organs.1 Remuscularization of the heart with induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) has been proposed as a potential solution, with a theoretically unlimited source of differentiated cells enabling repair of the failing heart. After more than two decades of work, cell-based approaches to remuscularize damaged myocardium have moved from concept to reality, and multiple clinical trials are now ongoing.2 However, several barriers still impede the maturation of this promising technology into standardized therapies. Broadly, these barriers can be divided into three interconnected categories: (ⅰ) graft immunogenicity, whereby allogeneic iPSC-CMs are recognized and rejected by the host immune system; (ⅱ) graft survival, as its implantation into the hostile environment expose the cells to severe stress and early loss; and (ⅲ) arrhythmogenicity, as multiple large-animal studies have shown that transplanted iPSC-CMs can trigger ventricular arrhythmias3–5. In this issue of Cardiovascular Research, Chino et al.6 report a series of experiments in cynomolgus monkeys in which 2 × 108 allogeneic iPSC-CMs were injected intramyocardially 2 weeks after ischaemia-reperfusion injury (Figure 1). In an initial cohort, the authors sought to define the minimal immunosuppressive regimen necessary to prevent graft rejection. While receiving triple therapy with a calcineurin inhibitor (CNI), mycophenolate mofetil (MMF), and methylprednisolone (MPL), histology at 3–4 weeks after implantation showed no signs of acute rejection. However, withdrawal of MPL at 4 weeks while maintaining MMF and CNI resulted in rejection at 8 weeks. In a second cohort, replacing MMF with abatacept (ABT) permitted withdrawal of MPL at 4 weeks without rejection at 8 weeks, suggesting that costimulation blockade may allow for steroid-sparing regimens. The investigators also transplanted hypoimmunogenic β2-microglobulin knockout and CD47 overexpression iPSC-CMs to assess whether genetic modification could minimize the need for intensive immunosuppression. In these animals, withdrawal of MPL after 4 weeks of triple therapy did not lead to graft rejection, consistent with reduced allorecognition. Nonetheless, the grafts exhibited marked central necrosis, indicating poor engraftment and survival of the transplanted cells. Follow-up in vitro experiments implicated serum-dependent toxicity by showing that exposure to bovine serum induced apoptosis in the engineered iPSC-CMs but not in the wild-type cells. Cynomolgus iPSC-CM transplantation experiments and interacting translational challenges. (A) Schematic of the cynomolgus monkey myocardial infarction (MI) model and subsequent experiments. After ischaemia-reperfusion MI, animals underwent intramyocardial injection of allogeneic iPSC-CMs. Post-transplant, three experimental interventions are explored: (i) minimization of the immunosuppression protocol starting with triple therapy: cyclosporine A or tacrolimus (CyA/TAC) in combination with MMF and MPL, then minimizing triple therapy to CyA/TAC with or without MMF, and then treating with TAC plus ABT; (ii) transplantation of hypoimmune iPSC-CMs, generated by β2-microglobulin knockout and CD47 overexpression (B2M KO/CD47 OE); and (iii) antiarrhythmic treatment with amiodarone and ivabradine to mitigate post-transplantation ventricular arrhythmias. (B) Conceptual framework highlighting key translational hurdles for iPSC-CM therapy and potential approaches. Immunogenicity can be addressed by immunosuppressive regimens, autologous iPSCs or hypoimmunogenic iPSCs; graft survival may be enhanced by improving vascularization, genetically engineering the cells for superior survival, refining delivery methods and developing interventions that inhibit systemic and local stress reactions; and arrhythmogenicity may be reduced by antiarrhythmic drugs, genetic engineering to modify ion-channel expression, optimizing electrical coupling between the host myocardium and the graft, and promoting iPSC-CM maturation. Importantly, the early engraftment period was plagued by ventricular arrhythmias. By week 2, six of the seven monkeys in the initial cohort exhibited near-persistent ventricular tachycardia (VT), and three (43%) died suddenly between weeks 3 and 7. As the severity of post-transplantation arrhythmia was inconsistent with the authors’ previous work, they compared electrophysiological and transcriptomic characteristics between the iPSC-CMs used herein and those used previously. Their results indicated that the cells transplanted in this study were more prone to arrhythmia, as they had shorter action potential duration, an increased spontaneous beating rate, and a transcriptional profile consistent with a more excitable phenotype. The second cohort was treated with a combination of amiodarone and ivabradine. This treatment resulted in a substantial reduction in arrhythmia burden and mortality. After withdrawal of antiarrhythmic therapy, ventricular arrhythmia burden increased immediately. These results directly address two of the major hurdles described above: immunogenicity, and arrhythmogenicity. Though it is agreed upon that immunosuppressive therapy can effectively protect the transplanted graft from rejection, the question of which immunosuppressive drugs to use, and for how long, is far more complicated. Triple therapy, the standard regimen used following heart transplantation, is a reasonable starting point for iPSC-CM grafts, which should not be more immunogenic than whole hearts. As long-term steroid use and intense immunosuppression are associated with significant morbidity, characterizing the minimal immunosuppressive protocol necessary for iPSC-CM transplantation is of great importance. High-quality large-animal studies are especially critical in this endeavour because the ability to monitor immune rejection via intramyocardial biopsy is limited or impossible with iPSC-CM grafts. Hypoimmunogenic iPSC-CMs are also an appealing approach to this problem.7 If the implanted cells can be engineered to limit immune recognition and activation, it may be possible to minimize immunosuppression even further or, in the future, forgo it entirely. The results of the present study underscore the importance of testing hypoimmunogenic cells in-vivo and in a species-specific context, as improvements in immunogenicity may result in graft loss through alternative mechanisms that may otherwise go unnoticed. This study further emphasizes that arrhythmogenicity may be a significant barrier for clinical translation of iPSC-CM transplantation. The substantial arrhythmia burden noted is in-line with previous reports describing development of VTs following iPSC-CM engraftment in swine and primate models3–5. These graft-related VTs likely reflect a combination of automaticity/triggered activity and imperfect host-graft coupling in an immature, heterogeneous cardiomyocyte product. Subtype heterogeneity (including nodal-like cells) and immature ventricular-like cells with increased If and reduced Ik1 can drive automaticity, and recent mapping and pacing/entrainment studies suggest a focal VT source rather than macroreentry.8 The fact that transplanting engineered cells modified to be ‘quiescent-yet-excitable’ reduces arrhythmias further supports abnormal automaticity as a dominant underlying mechanism.9 Even so, triggered activity (DADs/EADs) and re-entry may also contribute, considering the immature Ca2+ handling, reduced repolarization reserve, slow conduction, and patchy coupling. Efforts to reduce pro-arrhythmia aim to shift the phenotype of the iPSC-CM cell-graft from a pro-arrhythmic, immature, ectopic source into a well-coupled, adult-like myocardial graft. Strategies include graft maturation, cardiomyocyte subtype purification, improved structural/electrical integration, inflammation control, and engineered safety mechanisms such as suicide genes or chemogenetic ion channels that can induce electrical silencing.10 Coupling these approaches with short-term anti-arrhythmic therapy and backup ICD device implantation may be sufficient to overcome the arrhythmogenic risks inherent to iPSC-CMs. The reported use of amiodarone and ivabradine supports this approach. Ivabradine is an If current blocker, further supporting the role of abnormal automaticity in pro-arrhythmia. Nevertheless, the If current is not the only pacemaking mechanism, with the ‘calcium-clock’ mechanism being an alternative pathway. Accordingly, genetically reducing the HCN-4 derived If current alone was not sufficient to prevent in-vivo anti-arrhythmic activity of iPSC-CMs11 and achieving this effect required knockout of three other currents and overexpression of Ik1.9 Although this study addresses two issues that are critical for the translation of iPSC-CM transplantation, important questions remain. Small animal numbers, a multitude of regimens, short follow-up, and disparate endpoints limit the strength of any single comparison. As such, definitive conclusions about immunosuppressive or antiarrhythmic regimens cannot yet be drawn. Still, these data are important as they outline first steps towards tackling these important questions and justify future studies with longer follow-ups, a randomized head-to-head design, and a more comprehensive set of immunosuppressive regimens. Because the field of iPSC-CM transplantation is still in its infancy, there remain major differences in graft production and transplantation methodologies. It is important to interpret the results of this study within the framework of its experimental protocol and in the context of intramyocardially injected iPSC-CMs. Epicardial engineered heart patches12 or intramyocardial cardiac spheroids5 will likely elicit different immune responses, confer distinct patterns of graft survival, and carry varying degrees of arrhythmogenic propensity, underscoring that as the field and its methods mature, larger studies with more clinically oriented protocols will be required. In conclusion, the study by Chino et al.9 highlights immunogenic and arrhythmogenic factors that are crucial to the clinical translation of iPSC-CM transplantation. Their findings point towards promising strategies for more minimal immunosuppression regimens and effective periprocedural antiarrhythmic therapy. The Authors' work was supported by the Israel Science Foundation MAVRI - Biomedical Research Grant (3589/25). No new data were generated or analysed in support of this research.