Abstract 18307: T-tubules and Dyad Formation in Engrafted iPSC-Derived Cardiomyocytes With Stable APEX2 Expression
Background: Although studies have shown the potential of in vivo cardiac transplantation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to treat cardiac diseases in animal experiments, immaturity of iPSC-CMs remains as a critical obstruction. 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 reduced coupling gains and arrhythmogenic risks after cardiac transplantation. With a newly adapted genetically encoded probe, the monomeric 28-kDa peroxidase reporter 2 (APEX2), which withstands strong electron microscopy (EM) fixation, cells and molecules are identified in EM and their nano-structures are confirmed both in vitro and in vivo. We evaluated the engrafted iPSC-CMs with nuclear APEX2 expression in post-myocardial infarction heart in EM.
Methods: We established human iPSC lines which stably expressed histone H2B-APEX2 (APEX2 iPSCs). After differentiating APEX2 iPSCs into CM in vitro, purified cells were transplanted into NOG mouse hearts with myocardial infarction by direct injections into the myocardium. Six months after transplantation, we evaluated the ultrastructure of engrafted iPSC-CMs using EM.
Results: APEX2 did not give significant influences on cardiac differentiation, and stably expressed in iPSC-CMs over 6 months in vivo. APEX2 reaction clearly identified engrafted APEX2 iPSC-CMs in EM 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. This is the first report unequivocally to demonstrate 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.
Author Disclosures: T. Hatani: None. S. Funakoshi: None. T.J. Deerinck: None. E.A. Bushong: None. T. Kimura: None. S. Yamanaka: Consultant/Advisory Board; Modest; iPS Academia Japan. M.H. Ellisman: None. M. Hoshijima: None. Y. Yoshida: Ownership Interest; Modest; iPS Portal.
- © 2016 by American Heart Association, Inc.