N at 3 months/6 months)Graft failureDNTUDNTBLTNSPC transplantation, as well as safety issues and possibilities for

N at 3 months/6 months)Graft failureDNTUDNTBLTNSPC transplantation, as well as safety issues and possibilities for clinical applications.Discussion The risk of teratoma formation by transplanted iPSCderivatives is widely recognized, and many attempts have been made to minimize such risks [26, 27]. Although the transformation of iPSC-derived products has not been studied as extensively, a number of reports have showed the transformation of iPSC-derived intermediate progenitor cells as the result of genetic modification [14, 28, 29], transgene activation [30], or epigenetic events [31]. We used integration-free iPSCs to minimize the risk of genetic modification and/or transgene re-activation, which were observed in a previous report by our group [14]. However, some of the NSPCs from the three integration-free iPSCs used in this study retained proliferative characteristics invitro and in vivo, which appeared to be attributable to karyotype abnormalities or de novo CNVs that occurred during differentiation and culture processes. As the three iPSC-lines utilized in the present study were induced from blood samples from a single donor, the different genomic abnormalities observed in our iPSC lines may have occurred either during the reprogramming process or in cell culture. This PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26024392 suggests that even when integration-free iPSCs are used, it is not possible to completely eliminate the risk of genetic instability during NSPC production. This also indicates that the neural induction protocols that we used cannot eliminate all cells with genetic instability, and that genetic differences that emerged during the reprogramming process from the original iPSCs cannot be fully standardized. To control the proliferation of the derivatives in vivo, we thus should use iPSC lines without karyotype abnormalities or genomic instabilities.Sugai et al. Molecular Brain (2016) 9:Page 11 ofCulture duration is also important in controlling the genomic and epigenomic character of cultured cells. Young iPSCs may be immature and unstable, but it has also been reported that culture-induced genomic and epigenomic aberrations can occur at any stage, as the undifferentiated state of iPSCs is inherently unstable and sensitive [32]. We decided to use iPSCs as early as 11 passages for neural induction from feeder-free cultured iPSCs (1210B2 and 1231A3), in order to lower the risk of genetic abnormality associated with long-term culture process. Induced pluripotent stem cells established using the same method have been reported to express pluripotency-markers by passage 5 [33]. We also confirmed expression of pluripotent markers in our cells by passage 11. Additionally, we needed to cultivate the cells for this length to obtain a sufficient number of cells for further use. With respect to on-feeder iPSCs (1201C1), we needed to culture these until passage 19 in order to obtain sufficient numbers of cells. The adequate passage numbers of iPSCs or NSPCs may thus differ depending on the culture method and the purpose of usage of cells, a condition that should be evaluated for each case. It has been reported that iPSCs tend to acquire epigenetic and genetic modifications, including CNVs, during the reprogramming process and PD-148515 structure subsequent cell culture [34?6], and that these genetic alterations may occur in genomic sites related to cancer development. It is impossible to eliminate the risk of genetic abnormality; however, our results with the 1210B2 EB- and NRNSPCs indicate that the prol.