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

ABSTRACT: 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.

ORGANISM(S): Sus scrofa Homo sapiens

PROVIDER: GSE248949 | GEO | 2023/12/14

REPOSITORIES: GEO

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Project description: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.

Project description:Isolation of specific cell types, including pluripotent stem cell (PSC)-derived populations, is frequently accomplished using cell surface antigens expressed by the cells of interest. However, specific antigens for many cell types have not been identified, making their isolation difficult. Here, we describe an efficient method for purifying cells based on endogenous miRNA activity. We designed synthetic mRNAs encoding a fluorescent protein tagged with sequences targeted by miRNAs expressed by the cells of interest. These miRNA switches control their own translation levels by sensing miRNA activities. Several miRNA switches (miR-1-, miR-208a-, and miR-499a-5p-switches) efficiently purified cardiomyocytes differentiated from human PSCs, and switches encoding the apoptosis inducer Bim enriched for cardiomyocytes without cell sorting. This approach is generally applicable, as miR-126-, miR-122-5p- and miR-375-switches purified endothelial cells, hepatocytes and INSULIN-producing cells differentiated from hPSCs, respectively. Thus, miRNA switches can purify cell populations for which other isolation strategies are unavailable. MYH6-EIP4 day8 differentiated cells (EGFP+) for miRNA, N=1 MYH6-EIP4 day8 differentiated cells (EGFP-) for miRNA, N=1 MYH6-EIP4 day20 differentiated cells (EGFP+) for miRNA, N=1 MYH6-EIP4 day20 differentiated cells (EGFP-) for miRNA, N=1 HUVEC for miRNA, N=2 AoSMC for miRNA, N=2 Hepatocytes for miRNA, N=2 NHDF for miRNA, N=2 MYH6-EIP4 iPSCs for mRNA, N=3 MYH6-EIP4 day18 cardiomyocytes (before transfection) for mRNA, N=3 MYH6-EIP4 day19 cardiomyocytes (miR-1-switch day1) for mRNA, N=3 MYH6-EIP4 day19 cardiomyocytes (without transfection) for mRNA, N=3 MYH6-EIP4 day25 cardiomyocytes (miR-1-switch day7) for mRNA, N=3 MYH6-EIP4 day25 cardiomyocytes (without transfection) for mRNA, N=3 201B7 d22 miR-208a-BFP sorted cells for mRNA, N=1 201B7 d22 miR-208a-BFP negative fraction sorted cells for mRNA, N=1 201B7 d22 SIRPA+LIN- sorted cells for mRNA, N=1 201B7 d22 SIRPA+LIN- negative fraction sorted cells for mRNA, N=1 201B7 d22 VCAM1+ sorted cells for mRNA, N=1 201B7 d22 VCAM1+negative fraction sorted cells for mRNA, N=1 MYH6-EIP4 day19 cardiomyocytes (miR-208a-Bim-switch day1) for mRNA, N=1 MYH6-EIP4 day19 cardiomyocytes (without transfection) for mRNA, N=1 MYH6-EIP4 day21 cardiomyocytes (miR-208a-Bim-switch day7) for mRNA, N=1 MYH6-EIP4 day21 cardiomyocytes (without transfection) for mRNA, N=1 MYH6-EIP4 day25 cardiomyocytes (miR-208a-Bim-switch day7) for mRNA, N=1 MYH6-EIP4 day25 cardiomyocytes (without transfection) for mRNA, N=1

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

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