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Abstract Many studies have shown the feasibility of in vivo cardiac transplantation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in animal experiments. However, nano-structural confirmation of the successful incorporation of the engrafted iPSC-CMs including electron microscopy (EM) has not been accomplished, partly because identification of graft cells in EM has proven to be difficult. Using APEX2, an engineered ascorbate peroxidase imaging tag, we successfully localized and analyzed the fine structure of sarcomeres and the excitation contraction machinery of iPSC-CMs 6 months after their engraftment in infarcted mouse hearts. APEX2 made iPSC-CMs visible in multiple imaging modalities including light microscopy, X-ray microscopic tomography, transmission EM, and scanning EM. EM tomography allowed assessment of the differentiation state of APEX2-positive iPSC-CMs and analysis of the fine structure of the sarcomeres including T-tubules and dyads. [ABSTRACT FROM AUTHOR]

Background: Although many studies have shown the feasibility of in vivo cardiac transplantation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in animal experiments, nano-structural confirmation of the engrafted iPSC-CMs including electron microscopy (EM) has not been accomplished, partly because identification of engrafted cells in EM has proven to be difficult. However, with a new genetically encoded probe, the monomeric 28-kDa peroxidase reporter 2 (APEX2), which withstands strong EM fixation can resolve this problem. Moreover, immaturity of iPSC-CMs is a critical problem. Especially, excitation-contraction coupling is one of the fundamental properties of cardiomyocytes, the absence of dyad formed between T-tubule and junctional portion of the sarcoplasmic reticulum is one of the major reasons of arrhythmogenic risks after transplantation. Using the APEX2 system, we evaluated morphological alteration of the engrafted iPSC-CMs in EM in a mouse model of myocardial infarction. Methods: We established human iPSC lines which stably expressed histone H2B-APEX2 (APEX2 iPSCs). After differentiating APEX2 iPSCs into CMs in vitro , purified cells were transplanted into NOG mouse hearts with myocardial infarction by direct injections into the myocardium. We evaluated the ultrastructure of engrafted iPSC-CMs using EM at 3 and 6 months after transplantation. Results: APEX2 did not give significant influences on cardiac differentiation in vitro and stably expressed in iPSC-CMs over 6 months in vivo. APEX2 reaction observed in EM clearly identified engrafted APEX2 iPSC-CMs surrounded by host CMs. The maturation of sarcomeric structure and mitochondria were evident, and T-tubules and dyads started to emerge in engrafted iPSC-CMs at 6 months after transplantation. Conclusions: We demonstrated that APEX2 is a versatile genetic reporter to trace cell fates in living animals over many months. We unequivocally demonstrated that T-tubules and dyads can be formed in iPSC-CMs after a substantially long period of engraftment. This method should be useful to many studies of stem cell-based cell replacement therapy, as it allows direct nano-scale structural characterization of engrafted cells in EM. [ABSTRACT FROM AUTHOR]