br Results br Discussion We

Results
Discussion We successfully achieved quick induction of apoptosis in approximately 95% of the iPSCs in vitro, as in a previous report (Ando et al., 2015). This apoptosis induction rate was essentially comparable with the data reported from experiments carried out using the HSV-TK system (Chen et al., 2013). We observed iCaspase9 gene expression in all cell types that were examined, including the hiPSC-NS/PCs and their terminally differentiated products, namely, neurons. Furthermore, we found that there was no detectable influence of the iCaspase9 gene system on the efficiency of terminal differentiation of hiPSC-NS/PCs into neurons and astrocytes. No significant differences were observed between the EF-iC9-iPSCs and UbC-iC9-iPSCs or between the EF-iC9-iPS-NS/PCs and UbC-iC9-iPS-NS/PCs, although the rate of apoptosis induction was somewhat higher in the UbC-iC9-iPS-NS/PCs. Reports of studies of iCaspase9 gene transduction under the EF1α promoter revealed that DNA methylation occurs during the process of differentiation, which results in downregulation of the iCaspase9 gene expression (Wu et al., 2014) or iCaspase9 gene silencing (Pfaff et al., 2013). Contaminant non-iCaspase9-expressing cyclobenzaprine hydrochloride may have had a lower rate of apoptosis induction in the EF-iC9 iPSC and hiPSC-NS/PC populations in the present study via the same mechanism. We performed further observation by serial subculturing after exposure to CID, whereby no viable cells were observed. However, this may not represent complete killing, because annexin V/7-AAD-positive cells failed to reach 100%. Therefore, new genetic manipulation technology that ensures gene insertion and enhances the effectiveness of the iCaspase9 system is required to be widely used. We also evaluated the apoptosis-inducing effect of iCaspase9 in vivo. Tumorous proliferation of diverse histological types was observed following grafting of the hiPSC-NS/PCs. In all of the EF-iC9-TKDA3-4 iPS-NS/PCs, EF-iC9-253G1 hiPSC-NS/PCs, and UbC-iC9-253G1 hiPSC-NS/PCs grafted groups in this study, non-teratomatous neural tumor formation occurred, consistent with previous reports (Itakura et al., 2015; Nori et al., 2015). Although a larger portion of the tumors exhibited solid growth of Nestin-positive cells that had not terminally differentiated, some of the transplanted cells had differentiated into neurons, astrocytes, or oligodendrocytes. In the UbC-iC9-TKDA3-4 hiPSC-NS/PCs grafted group, however, the graft rapidly increased in size, growing into a teratoma containing embryonic elements of all three primary germ cell layers. In the apoptosis-induced groups, administration of CID was followed by quick disappearance of all the transplanted cells, regardless of the type of tumor formed, and there was neither gross evidence of graft re-enlargement nor histological evidence of remnant cells, even at long-term follow-up. It had previously been unclear whether systemically administered CID is distributed into the cerebrospinal fluid; however, in the present study we found that intraperitoneally injected CID rapidly reached cells engrafted in the CNS and induced apoptosis, similar to reports of previous studies of subcutaneous tissue and blood. This suggests that CID is an effective inducer even in the CNS protected by the BBB. In the present study, nevertheless, the CID treatment was undertaken after tumor formation. Thus, it is likely that pronounced neovascularization was present in the peritumoral region at the point of CID treatment, which would result in efficient induction of apoptosis in the presence of the undeveloped newly formed BBB. Further discussion of CID permeability through the BBB is needed in the future. The EF-iC9-TKDA3-4 hiPSC-NS/PCs, EF-iC9-253G1 hiPSC-NS/PCs, and UbC-iC9-253G1 hiPSC-NS/PCs grafted mice exhibited improved hindlimb motor function until week 4 after transplantation, with a subsequent gradual decrease of function. In the CID-treated groups there was a slight decline in function followed by a plateau. In the UbC-iC9-TKDA3-4 iPS-NS/PC-grafted mice, compared with hindlimb function at the time of the transplantation, hindlimb function declined due to enlargement of the teratoma to the extent that the ankle joint was slightly movable but barely capable of bearing weight. In the CID-treated groups, on the other hand, no such deterioration due to tumor enlargement was noted after the disappearance of the transplanted cell-derived tumors in response to CID injection, and there was a slight recovery of function compared with function at the time of transplantation. However, this was comparable with the hindlimb motor function observed in the PBS-grafted groups reported in previous studies and may represent spontaneous progression post spinal cord injury in NOD/SCID mice (Nori et al., 2011). According to previous studies in which tumorigenic transformation was not observed post transplantation, transplanted cells appeared to contribute to the improvement of motor function (Fujimoto et al., 2012; Nori et al., 2011). When these cells that contributed to the functional recovery were ablated, motor function declined, but there was a slight recovery in the final motor function compared with the PBS-transplanted groups (Abematsu et al., 2010). Owing to the intact motor function in the mice of the transplanted cell ablation group among the present iC9-TKDA3-4 hiPSC-NS/PCs grafted groups, it did not appear that the iC9-TKDA3-4 hiPSC-NS/PCs were able to improve hindlimb motor function through mechanisms such as reconstruction of neural circuits in the injured spinal cord after the transplantation. Furthermore, it has been reported that hiPSC-NS/PCs liberate a variety of humoral factors (Kobayashi et al., 2012). Our data showed no evidence of functional recovery attributable to humoral factors after transplantation of the UbC-iC9-TKDA3-4 hiPSC-NS/PCs that formed teratomas. Eventually, a gradual depression of hindlimb function due to a mass effect associated with tumor enlargement was observed. Subsequently, we noted that ablation of the transplanted cells prevented the depression of hindlimb function, leading to a restoration to spontaneous progression after spinal cord injury. For the iC9-253G1 hiPSC-NS/PCs grafted groups, on the other hand, a slight decrease in hindlimb function was observed in the CID-treated group, which suggests that the treatment may contribute to functional recovery even in the presence of abnormal proliferation of undifferentiated neural cells. Furthermore, changes in motor function associated with the rapidly growing tumors, such as teratomas, were reversible provided that the duration of tissue displacement by the tumor was <3 weeks, and full rescue from tumor-related adverse events by administration of CID seems achievable. Previous assessments have shown that, in the case of slowly growing tumors in the spinal cord, hindlimb motor function begins to decline due to tumor enlargement and progresses to complete paralysis. However, in our study, recovery from depressed hindlimb motor function was evident even after tumor ablation on day 100 post transplantation (Itakura et al., 2015). We cannot exclude the possibility, however, that additional functional depression caused by a rapidly growing tumor may appear during longer-term follow-up.