Cellular Heterogeneity of Pluripotent Stem Cell Derived Cardiomyocyte Grafts is Mechanistically Linked to Treatable Arrhythmias [Spatial Transcriptomics]

Exciting pre-clinical data have confirmed that human pluripotent stem cell derived cardiomyocytes (PSC-CMs) can remuscularise the injured or diseased heart, with several clinical trials now in planning or recruitment stages worldwide. However, ventricular arrhythmias are a predictable complication following engraftment of intramyocardially injected PSC-CMs. Therefore, there is an urgent unmet need to gain mechanistic insights and treatment strategies to control or prevent these engraftment arrhythmias (EAs). We used a porcine model of myocardial infarction and PSC-CM transplantation to investigate efficacy of pharmacologic and catheter based anti-arrhythmic strategies in mitigating EAs. Furthermore, cell doses were robustly phenotyped using single cell ribonucleic acid sequencing and high parameter flow cytometry to identify cellular characteristics predictive of arrhythmogenesis. Combination therapy with amiodarone and ivabradine significantly reduced EA rate and burden following PSC-CM transplantation. Catheter ablation was also a feasible and effective treatment strategy which could be considered in the case of pharmacologically refractory arrhythmias. In addition, we show that EAs are mechanistically linked to cellular heterogeneity in the input PSC-CM and resultant graft. Specifically, we identify atrial and pacemaker-like cardiomyocytes as culprit arrhythmogenic subpopulations. We further describe two unique surface marker signatures, SIRPA+/CD90-/CD200+ and SIRPA+/CD90-/CD200-, which identify arrhythmogenic and non-arrhythmogenic cardiomyocytes respectively. Our data deepens mechanistic understanding of EAs and suggests that modifications to current PSC-CM production and/or selection protocols could ameliorate this problem. We further show that current clinical pharmacologic and interventional anti-arrhythmic strategies can control and potentially abolish these arrhythmias, an important safety consideration given several impending clinical trials. Overall design: Spatial transcriptomics analysis of engrafted PSC-CMs. Using a tissue microarray punch, the tissue samples of 3mm diameter were collected from regions confirmed to contain PSC-CM grafts.

临床前研究的突破性数据已证实，人多能干细胞来源心肌细胞（human pluripotent stem cell derived cardiomyocytes, PSC-CMs）可实现受损或病变心脏的心肌重建，目前全球已有多项临床试验处于规划或招募阶段。然而，心肌内注射PSC-CMs移植后，室性心律失常是可预见的并发症。因此，亟需阐明其潜在机制并开发调控或预防此类移植相关心律失常（engraftment arrhythmias, EAs）的治疗策略。本研究采用心肌梗死联合PSC-CM移植的猪模型，探究药物及导管消融类抗心律失常策略对移植相关心律失常的缓解效果。此外，本研究通过单细胞核糖核酸测序（single cell ribonucleic acid sequencing）与高参数流式细胞术对细胞制剂进行了严格的表型鉴定，以识别可预测致心律失常性的细胞特征。胺碘酮与伊伐布雷定联合给药可显著降低PSC-CM移植后的移植相关心律失常发生率与负荷量。对于药物难治性心律失常，导管消融亦是一种可行且有效的治疗方案。此外，本研究证实移植相关心律失常的发生机制与输入性PSC-CM及最终移植物的细胞异质性密切相关。具体而言，本研究明确心房样心肌细胞与起搏样心肌细胞为致病的致心律失常亚群。本研究进一步定义了两种独特的表面标志物特征：SIRPA+/CD90-/CD200+与SIRPA+/CD90-/CD200-，可分别识别致心律失常性与非致心律失常性心肌细胞。本研究数据加深了对移植相关心律失常的机制理解，并提示对现有PSC-CM制备及分选方案进行优化，可缓解该问题。此外，本研究证实当前临床应用的药物与介入性抗心律失常策略可有效控制甚至消除此类心律失常，鉴于多项即将开展的临床试验，该结果具有重要的安全参考价值。研究整体设计：对移植的PSC-CMs进行空间转录组学分析。本研究通过组织芯片穿刺术，从已确认存在PSC-CM移植物的区域中采集直径3mm的组织样本。