Although NPCs derived from iPSCs exhibited beneficial effects if safety-tested lines were selected, the risk of tumorigenicity after transplantation remains a major concern. Even if iPSCs were considered safe when established, they occasionally present tumor-like growth or differentiation resistance when induced into NPCs and subsequently transplanted into the injured spinal cord . To establish a safe cell therapy, it is necessary to introduce a process of removing tumorigenic cells before or even after transplantation, regardless of the types of cell lines used. Hence, we performed several prophylactic studies against tumorigenicity.
Safety issue for molecular level
The optimal way to prevent tumor formation in the spinal cord is to select the safe and validated iPSC-NPC lines before transplantation. To identify factors that were associated with tumorigenicity, we evaluated and compared gene expression profiles of tumorigenic and non-tumorigenic iPSC-NPCs by comprehensive DNA methylation analyses . The results revealed that several tumor suppressor genes were methylated in the tumorigenic NPC lines and this epigenomic status could promote the tumor formation after the cell transplantation. Interestingly, tumorigenicity was enhanced when the number of passages increased even for the initially safety-validated iPSC-NPCs. The underlying mechanism for this abnormality was the progression of hypermethylation for the tumor suppressor genes. Altogether, the methylation profiles could be included in the criteria to choose safe iPSC-NPCs in an actual clinical setting, and passage numbers should be limited not to generate aberrational transplanting cells.
Suppression of tumorigenic cells using a notch signal inhibitor
Ideally, in cell therapy using iPSC-NPCs, tumorigenic cells were eliminated in vitro before transplantation. In this regard, we investigated a γ-secretase inhibitor (GSI), which inhibits Notch signaling, which controls the status of undifferentiated NPCs. Inhibition of this signaling promotes NPCs for additional maturation and neuronal differentiation . In our study, tumorigenic human iPSC-NPCs were treated with GSI in vitro, for only 1 day and already, the cells exhibited neuronal differentiation, a reduction in cell proliferation, and suppression of tumor-related gene expression. Upon transplant into the SCI model of NOD/SCID mice, the iPSC-NPCs generated mature neurons around the injury site and did not form tumors at 89 days after transplantation. On the other hand, in non-GSI-treated NPCs, tumor formation was observed with declining motor function. Thus, pretreatment with GSI can eliminate tumor-initiating cells in human iPSC-NPCs or promote differentiation of the cells into mature neuronal cells. These beneficial mechanisms potentially mitigated the safety issue related to tumorigenicity after cell transplantation.
Detection of tumor-like proliferating cells using positron emission tomography
In some cell lines, iPSC-NPCs with differentiation-resistant properties caused abnormal cell growth after transplantation [14, 16], which makes immediate identification of these unsafe cell lines critical during cell proliferation in the grafted tissues. The remnant undifferentiated NPCs showed a high expression of the 18-kDa translocator protein (TSPO), also known as the peripheral-type benzodiazepine receptor. Because the TSPO-selective radioligand [18F] FEDAC is available as a clinically relevant nuclide, we proposed that the remaining NPCs after transplantation could be detected using positron emission tomography (PET) imaging . When non-tumorigenic iPSC-NPCs were transplanted into central nervous system (CNS) tissue, the cells did not show uptake of the [18F] FEDAC in PET imaging. However, the NPCs which had proliferative properties presented nuclide uptake 4 weeks after transplantation. Thus, PET imaging with a NPC-specific nuclide is an effective modality in identifying the remnant undifferentiated cells and ensuring safety in future clinical treatments using cell transplantation.
Transplantation of tumorigenic iPSC-NPCs with integrated suicide genes
Although pretreating NPCs with GSI prior to transplantation and/or utilizing oligogenic cells may reduce the risk of tumorigenesis, completely eradicating the risk in certain iPSC lines is difficult. We have proposed several possible methods for ablating the transplanted cells retrospectively using suicide gene systems [18, 19]. First, induced caspase-9 was transduced into known tumorigenic iPSC-NPCs following transplantation . These cells were transplanted into the spinal cord of mice and observed for several weeks to allow for tumor growth. When the apoptosis inducer (dimerizer) was systemically injected into these mice, the transplanted cells were successfully ablated with only an insignificant loss of motor function. However, this system killed all the transplanted cells, including fully functional neural cells that may have contributed to functional recovery. In order to spare fully differentiated neural cells from ablation, we tried transducing the herpes simplex virus type I thymidine kinase (HSVtk) gene into the same line of tumorigenic cells . HSVtk is known to phosphorylate its prodrug ganciclovir (GCV) which is toxic to immature/proliferative cells such as tumor cells. A similar experiment with mice transplanted with HSVtk-induced iPSC-NPCs resulted in the selective eradication of tumorigenic cells whilst sparing the mature, differentiated neural cells. Improved motor function was also promoted, affirming the role of transplanted cells within the injured host spinal cord. Because the HSVtk/GCV system has already been applied in some clinical trials without any safety problems , this technique can, theoretically, be used in the clinical application of our iPSC project for SCI patients.