Recently several clinical trials of stem cell

Recently, several clinical trials of stem-cell-based therapy for SCI using either human NS/PCs (Cummings et al., 2005; Salazar et al., 2010) or human ESC-derived OPCs (Strauss, 2010) have been initiated. Compared with these stem cells, iPSCs raise fewer ethical concerns in certain countries (Nori et al., 2011; Tsuji et al., 2010). On the other hand, the use of iPSC-derived cells risks tumorigenesis (Miura et al., 2009; Tsuji et al., 2010). The present study demonstrates that even unsafe iPSC-NSs can confer therapeutic benefits against SCI, at least in the short term. However, long-term observation is required to assess the safety of iPSC-NSs, because slow-growing tumors could cause motor function to deteriorate over longer periods of time. In the present study, we used retrovirally generated iPSCs (Nakagawa et al., 2008; Takahashi et al., 2007) and showed that activation of OCT4- and KLF4-Tg might be related to tumor formation. Thus, from a clinical-applications perspective, NS/PCs derived from integration-free iPSCs (Okita et al., 2008, 2011) should be chosen to avoid Tg-induced tumorigenesis. Recently, a pilot clinical study of integration-free iPSC-based therapy for age-related macular degeneration was approved following review by the Japanese government (Garber, 2013; Kamao et al., 2014). As a step toward clinical applications in the SCI field, we have already initiated integration-free iPSC-NS transplantation in the NOD-SCID mouse SCI model. At the same time, transplantation into immune-deficient animals, accompanied by subsequent long-term observation, should be used to determine the safety and effectiveness of these cells (Okano et al., 2013).
Experimental Procedures Additional details regarding several of the protocols used in this work are provided in Supplemental Experimental Procedures.
Acknowledgments We thank A. Iwanami, S. Kaneko, K. Fujiyoshi, O. Tsuji, A. Yasuda, Y. Takahashi, S. Kawabata, Y. Nishiyama, T. Iida, S. Shibata, T. Harada, S. Miyao, and H.J. Okano for technical assistance and scientific discussions, and H. Saya, M. Ko, M. Jakt, and K. Horiuchi for critical readings of the manuscript. We also thank S. Yamanaka and M. Nakagawa for the human iPSC clones (253G1 and 201B7). The p-STAT3, p-ERK1/2, and p-AKT gsk3 inhibitor were kindly provided by N. Onishi. This work was supported by grants from the JST-CIRM collaborative program; Grants-in-Aid for Scientific Research from JSPS and the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT); Research Center Network for Realization of Regenerative Medicine from by the Japan Science and Technology Agency (JST); the Kanrinmaru Project (Keio University); Research Fellowships for Young Scientists from the Japan Society for the Promotion of Science; Keio Gijuku Academic Development Funds; and a Grant-in-Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network) from the MEXT.
Introduction Embryonic stem cells (ESCs) are derived from the inner cell mass of the blastocyst, during a stage of development defined by rapid cell division rates. Mouse and human ESCs grown in culture retain the rapid proliferation observed in early embryonic cells, exhibiting an accelerated cell-cycle program characterized by a shortened G1 phase and differentially regulated cell-cycle checkpoints (Orford and Scadden, 2008). When ESCs differentiate, their cell-cycle structure changes to incorporate a longer G1 phase and slower proliferation rates. Whether their unique cell-cycle program alters ESC dependency on cell-cycle regulatory proteins has not been previously established. Cell-cycle adaptations that account for the altered ESC cell-cycle structure were first identified in mouse ESCs (mESCs) (Ballabeni et al., 2011; Orford and Scadden, 2008). Cyclin/CDK complexes represent the key enzymes that regulate orderly progression through the mammalian cell cycle. In somatic cells, cyclin abundance fluctuates throughout the cell cycle, in part due to degradation by the anaphase-promoting complex/cyclosome (APC/C) at the end of mitosis (reviewed in Morgan, 2007). In mESCs, however, APC/C activity is attenuated due to high levels of EMI1 (early mitotic inhibitor 1), resulting in reduced fluctuation of cyclin expression (Ballabeni et al., 2011). Additionally, mESCs express higher levels of cyclins E, A, and B compared to somatic cells (Stead et al., 2002) and do not appreciably express the endogenous CDK inhibitors, including INK family members (p15, p16, and p19) and CIP/KIP family members (p21 and p27) (Sabapathy et al., 1997).