br Results br Discussion Our studies highlight the potential

Results
Discussion Our studies highlight the potential clinical significance of the iC9 safeguard system. Recent studies introducing suicide gene-based safeguard systems into mouse or rhesus iPSCs report that teratoma formation was significantly delayed or inhibited only when treatment started before implantation or soon after iPSC injection (Chen et al., 2013; Lim et al., 2013; Wu et al., 2014; Zhong et al., 2011). In our study, this was not so for CID treatment; when started 30 days after iC9-iPSC implantation, it caused dramatic teratoma regression. These results indicate that our lentiviral iC9/CID safeguard system may be useful practically. Looking toward the introduction of the iC9/CID safeguard system to clinical use, we applied this system to iPSC-derived (rejuvenated) CTL therapy to show how this system could actually work. Our results indicated that introduction of iC9 into iPSCs does not interfere with their pluripotency and differentiation ability. iC9-iPSCs could efficiently differentiate into virus-specific CTLs that exhibited continuously high iC9/mCherry expression. These iC9-iPSC-derived CTLs could be eliminated by CID treatment in vivo. EF1α-iC9 GSK503 in rhesus iPSCs reportedly is downregulated during in vitro and in vivo differentiation due to DNA methylation within the promoter (Wu et al., 2014). We initially compared the percentages of apoptosis between Ubc-iC9-iPSCs and EF1α-iC9-iPSCs 24 hr after CID treatment. CID could induce apoptosis more strongly in Ubc-iC9-iPSCs than in EF1α-iC9-iPSCs. Ubc-iC9 expression levels were still high during and after differentiation into virus-specific CTLs, and CID also induced robust apoptosis of Ubc-iC9-iPSC-derived CTLs. These data suggest that the Ubc promoter provides stable expression of the iC9 transgene after iC9/mCherry sorting and is suitable for iPSC-derived cell therapy. To increase the efficacy of this iC9 system further, iC9-transduced iPSCs could be cloned and selected for maximum sensitivity for CID and a minimum number of mutations in the genome. If increased efficiency of this iC9/CID system is required, the proteasome inhibitor bortezomib can be used. Bortezomib and iC9/CID are strongly synergistic, enhancing apoptosis in lung cancer cells by blocking the degradation of active caspase-3 and caspase-9 (Ando et al., 2014). Of interest in this regard is that the E3 ubiquitin ligase XIAP, an inhibitor of apoptosis, binds to caspase-3 and targets it for proteasomal degradation (Albeck et al., 2008; Huang et al., 2001; Suzuki et al., 2001). Knockdown of XIAP enhances iC9-induced apoptosis, suggesting that bortezomib maximizes iC9-induced apoptosis (Ando et al., 2014). Given that bortezomib downregulates nuclear factor κB (Hideshima et al., 2009), which is a transcriptional target of XIAP (Stehlik et al., 1998), bortezomib likely downregulates XIAP expression. As far as we know, tumor formation in a clinical study using lentivirus vectors has not been reported. However, in clinical studies, to examine integration sites of lentivirus-iC9 and choose clones most likely to be safe appears prudent. An alternative approach may be to insert the iC9 system into a “safe harbor” site, such as AAVS1 or ROSA26, through homologous recombination using highly efficient new genetic manipulation technologies involving zinc-finger nucleases (Hockemeyer et al., 2009), transcription activator-like effector nucleases (Boch and Bonas, 2010), or clustered regularly interspaced short palindromic repeats (Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013). Our concept of the iC9/CID safeguard system for iPSC-derived cell therapy can be presented as a schema (Figure 5E). This safeguard system can eliminate contaminating iPSCs, debulk tumors originated from iPSCs, stop cytokine release syndrome associated with iPSC-derived CTL therapy, and control “on-target, off-tumor toxicities”. It should be applicable to other cell therapies using iPSC-derived cells. The iC9 safeguard system can be an efficient and reliable approach to provide safety for future regenerative therapy and first-in-human cell therapy.