Background:
Although studies have feasibility of
in vivo
cardiac transplantation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in animal experiments, 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. However, with a new genetically encoded probe, the monomeric 28-kDa peroxidase reporter 2 (APEX2), which withstands strong EM fixation, this problem can now be done. We have now been able to test whether APEX2 can identify iPSC-CMs in host heart after long-term engrafting, and evaluate the engrafted iPSC-CMs in post-myocardial infarction using 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. One and 3 months after transplantation, we mapped engraft iPS-CMs using high resolution micro-CT and evaluated their ultrastructure by EM.
Results:
APEX2 was stably expressed and labeled histone H2B in iPSCs before and after
in vitro
differentiation into CM. Graft efficiency of APEX2 iPSC-CMs in NOG heart was excellent and APEX2 expression sustained over 3 months
in vivo
. APEX2 reaction observed in EM clearly identified engrafted APEX2 iPSC-CMs in niches surrounded by host CMs and their physical interaction was visualized. EM also revealed a progression in the maturation of sarcomeric structure and mitochondria in engrafted iPSC-CMs, by comparing data at 1 and 3 months after transplantation.
Conclusion:
We demonstrate that APEX2 is a versatile genetic reporter to trace cell fates in living animals over many months. Using APEX2-based staining, we were able to identify and characterize the maturation process of iPSC-CMs, and determine how they distribute within myocardial niches, as well as their interaction with host CMs. This method should be useful to many studies of stem cell-based cell replacement therapy, as it allows both tracking of cells and the ultrastructural characterization of engrafted cell and graft-host interactions.
In vitro ubiquitination and degradation of bacteria-purified human histone H2B by the nucleoli fractions
