Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, 606-8507, Japan; Center for iPS Cell Research and Application, Kyoto University, Kyoto, 606-8507, Japan.
Center for iPS Cell Research and Application, Kyoto University, Kyoto, 606-8507, Japan.
Biochem Biophys Res Commun. 2018 Nov 10;505(4):1251-1256. doi: 10.1016/j.bbrc.2018.10.020. Epub 2018 Oct 15.
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.
许多研究已经在动物实验中证明了将人诱导多能干细胞衍生的心肌细胞（iPSC-CMs）进行体内心脏移植的可行性。然而，尚未通过电子显微镜（EM）等纳米结构确认成功整合了移植的 iPSC-CMs，部分原因是在 EM 中鉴定移植物细胞被证明很困难。我们使用 APEX2，一种经过工程改造的抗坏血酸过氧化物酶成像标签，成功地定位和分析了在梗死小鼠心脏中移植 6 个月后的 iPSC-CMs 的肌节和兴奋收缩机制的精细结构。APEX2 使 iPSC-CMs 在多种成像模式下可见，包括荧光显微镜、X 射线显微镜断层扫描、透射电子显微镜和扫描电子显微镜。EM 断层扫描允许评估 APEX2 阳性 iPSC-CMs 的分化状态，并分析肌节的精细结构，包括 T 小管和二联体。
