The aim of the present study

The aim of the present study was to explore the induction of apoptosis in tumors formed from hiPSC-derived products. Our results demonstrate that apoptosis can be induced by CID in tumors grown from transplanted gap 26 that have undergone differentiation after engraftment and have been in close interaction with host tissues independent of the underlying mechanism of tumor formation and differentiation. Our results also showed that injected CID is able to reach transplanted cells and induce their apoptosis even in the spinal cord, which is segregated from the bloodstream by the BBB, just as in subcutaneous tissues and circulating blood. We also confirmed that the administered CID inhibited the growth of tumors formed after cell transplantation, and quickly and completely ablated the transplanted cells. In the future, it may become feasible to specifically conserve differentiated neural cells by integrating a suicide gene under a tumorigenesis-specific promoter using a factor such as the HSV-TK gene rather than by means of total ablation. This will allow for a more selective ablation of tumor-initiating cells and proliferative cells. Under the present conditions, whereby the conceivable mechanisms of tumorigenic transformation are diverse and other risks involved in the transplantation of cells remain unclear, methods for completely ablating or “undoing” all possible adverse events must be an option. In the present study, the iCaspase9 system was shown to be able to induce apoptosis in a broad range of cell and tissue types, including hiPSCs, differentiated hiPSC-NS/PCs, neurons, teratomas formed from hiPSC-derived products, and differentiated neural tumors. We also found no evidence of remnant cells on long-term follow-up, suggesting the ablation, rather than the involution, of tumors by the iCaspase9 system. Furthermore, iCaspase9 gene transfection into good clones may lead to elucidation of the neural regeneration mechanism of transplanted cells. We recognize that the transduction of the iCaspase9 system using a lentiviral vector represents a potential shortcoming of the present study. There are still a number of problems that need to be resolved, and at present it is still not suitable for clinical application. The use of a non-integrating vector (Uno et al., 2015) or gene introduction into a particular gene locus, e.g., a safety harbor, would help to minimize the risks associated with gene introduction by transfection. Furthermore, the potential effects of iCaspase9 gene on the differentiation and characteristics of the transplanted cells cannot be ruled out; hence, long-term follow-up is necessary. Once these issues are resolved, the iCaspase9 system may become a useful method for reducing risk in clinical applications of hiPSC-derived products.
Experimental Procedures
Author Contributions
Acknowledgments We appreciate the help of Dr. S. Shibata, Dr. F. Renault-Mihara, Dr. M. Shinozaki, Dr. S. Tashiro, Dr. K. Matsubayashi, Dr. K. Ito, Dr. Y. Tanimoto, Dr. Y. Hoshino, Dr. Y. Fukushima, and Ms. M. Isoda who are all members of the spinal cord research team at the Department of Physiology, Orthopedic Surgery and Rehabilitation Medicine, Keio University School of Medicine, Tokyo, Japan. We also thank Prof. Douglass Sipp (Keio University) for invaluable comments on the manuscript and Ms. T. Harada for assistance with animal care. We also thank Dr. S. Yamanaka and M. Ando for the human iPSC clones (253G1 and TKDA3-4). This work was supported by the Research Center Network for Realization of Regenerative Medicine by the Japan Science and Technology Agency (JST) and the Japan Agency for Medical Research and Development (AMED) (grant no. 16bm0204001h0004 to H.O. and M.N.), a Grant-in-Aid for Scientific Research by Japan Society for the Promotion of Science (grant no. 15K10422 to A.I.), and a grant by The General Insurance Association of Japan (grant no. 15-2B-13 to A.I.). H.O. is a founding scientist and a paid SAB of San Bio, Co., Ltd.