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  • Previous studies have reported that

    2018-10-24

    Previous studies have reported that Notch signaling controls the induction of neural stem cystic fibrosis chloride channel in vitro and in vivo during differentiation (Crawford and Roelink, 2007; Nelson et al., 2007). As established by Ogura et al. (2013), inhibition of this signaling pathway promotes the neuronal differentiation of neural progenitor cells derived from human iPSCs. Our in vitro experiments confirmed that by inhibiting Notch signaling with the GSI, a greater number of NS/PCs derived from two lines of human iPSCs with tumorigenesis-/overgrowth-prone characters (253G1 and 836B3) are induced to undergo neuronal differentiation and maturation, although they present limited proliferation. Nelson et al. (2007) showed treatments with a GSI (DAPT) lasting at least 6 hr commits NS/PCs to neuronal differentiation, although HES5 expression is reduced after 3 hr. In the present study, significant differences were not observed between the GSI-1d and GSI-4d group in hiPSC-NS/PC proliferation and differentiation, suggesting that inhibition of Notch signaling with a GSI promotes the neuronal differentiation and maturation of hiPSC-NS/PCs regardless of the culture period or cell lines. Nori et al. (2011) showed that the 201B7 hiPSC-NS/PC-transplanted group exhibited significantly better functional recovery than the PBS-injected group more than 21 days after SCI as measured by the BMS score. In this study, the GSI+ group of 253G1 hiPSC-NS/PC transplants (“bad” clone) exhibited confirmed functional recovery more than 21 days after SCI (BMS score: PBS group 2.5 ± 0.3 at 12 days post transplantation and 3.0 ± 0.1 at 89 days post transplantation; GSI+ group 3.8 ± 0.7 at 12 days post transplantation and 4.8 ± 0.4 at 89 days post transplantation). Borghese et al. (2010) showed that the neural stem cell derived from human embryonic cells pretreated with DAPT were strongly positive for human synaptophysin, and exhibited strong human doublecortin staining compared with untreated cells after transplantation. In this study, the GSI+ group of 201B7 hiPSC-NS/PC transplants (“good” clone) exhibited a significantly greater tendency to enhance the axonal regrowth in the mouse injured spinal cord, which are likely to have resulted in improved motor function compared with the control group at 26 days after transplantation. The improved motor function was maintained after this point in the GSI+ group (BMS score: control group 3.8 ± 0.2 at 26 days post transplantation and 4.4 ± 0.5 at 89 days post transplantation; GSI+ group 4.3 ± 0.5 at 26 days post transplantation and 4.9 ± 0.3 at 89 days post transplantation). It is possible that pretreatment with GSI might have other mechanisms besides axonal regrowth, and could have a clinically meaningful effect and improve human iPSC-based transplantation for SCI. Improved cell quality and safety, particularly with respect to the risk of tumor-like overgrowth, will be crucially important for any clinical use of hiPSC-NS/PCs. Okada et al. (2005) revealed that the photon counts measured by BLI analyses were significantly proportional to the number of the transplanted cells in vivo. Nori et al. (2015) indicated that some mice transplanted with 253G1 hiPSC-NS/PCs showed temporary motor function recovery for up to 47 days after transplantation; however, the photon counts of the transplanted cells increased more than 10-fold from its initial value, and these mice also developed tumor-like overgrowth and deterioration of motor function at 103 days after transplantation. Therefore, the photon counts measured by BLI analyses could be useful for the diagnosis for detecting tumor-like overgrowth in the mouse injured spinal cords. In the present study, the control group (i.e., the group without GSI pretreatment) showed a rapid increase in the photon count of the transplanted cells and improvements in hindlimb motor function, which subsequently deteriorated gradually upon long-term observations. However, in the GSI+ groups, the photon counts increased more slowly and reached a plateau after transplantation. Greater functional recovery was also observed, and was maintained at significantly higher levels compared with the control group. In addition, we previously reported that the size of the tumors formed by the transplanted hiPSC-NS/PCs in the injured spinal cord correlated with the proportion of Nestin+ cells in the graft (Nori et al., 2015). The proportion of transplanted Nestin+ cells decreased from 10.7% ± 2.2% at 47 days to 7.5% ± 1.0% at 103 days after transplanting 201B7 hiPSC-NS/PCs, resulting in no tumor-like overgrowth. The proportion of Nestin+ cells increased from 19.6% ± 0.5% at 47 days to 33.1% ± 7.4% at 103 days after transplanting 253G1 hiPSC-NS/PCs. Therefore, we suggest that differentiation-resistant Nestin+ cells proliferated over time and formed tumors. However, the present study showed that the proportion of transplanted Nestin+ cells in the control group increased to 30.3% ± 1.6% at 89 days after transplantation and resulted in tumor-like overgrowth, whereas the proportion of Nestin+ cells in the GSI+ group decreased to 5.3% ± 0.8% at 89 days after transplantation and did not show evidence of tumor-like overgrowth. The proportion of Ki-67+ cells in the control group was significantly increased to 17.9% ± 0.9% at 89 days after transplantation compared with the GSI+ group. Furthermore, the proportion of pan-ELAVL (Hu)+ neuronal cells in the GSI+ groups significantly increased to 51.0% ± 1.8% compared with the control group at 89 days after transplantation, although significant differences were not observed in glial differentiation. Most of the GSI-pretreated, transplanted βIII-tubulin+/HNA+ cell-derived neurons were co-localized with Bsn+ synaptic boutons of the host neurons, and hSyn+ boutons were apposed to βIII-tubulin+/HNA− host mouse neurons. These findings indicate that the proportion of proliferating/undifferentiating cells increases over time and induces tumor-like overgrowth in the vertebral canal of the mice. Therefore, the motor functional recovery observed in the control group subsequently deteriorated gradually upon long-term observations. However, pretreatment with GSI differentiated nearly all of these cells into mature neurons, which were further integrated into the reconstructed host neuronal network, where they formed synapses and prevented tumor-like overgrowth, even in mice transplanted with the “bad” clone, after observations for long periods. Therefore, significantly greater motor functional recovery was maintained compared with the control group.