The activation of Notch has

The activation of Notch1 has been found to contribute to the invasion and metastasis of cancer via the EMT. In addition, Saad et al. (2010) showed that the overexpression of NOTCH1 induces the expression of SNAIL. In the present study, after GSI treatment the expression of NOTCH1, SNAIL1, and SNAIL2 was downregulated in vitro compared with the control group. Furthermore, the proportion of Notch1+, activated Notch1+, and Snail+ harpagoside was also significantly decreased after transplantation compared with the control group, which indicated that the presence of activated Notch1 on the transplanted cells reflected the γ-secretase-dependent activation of Notch1 in vivo. Therefore, the GSI pretreatment could reduce the acquisition of EMT and inhibit the activation of Notch1 and Snail, which induced the transplanted cells to undergo neuronal maturation without tumor-like overgrowth upon transplantation of hiPSC-NS/PCs for SCI. However, Kopan and Ilagan (2004) showed that other signaling molecules act as targets for GSI. Thus, we cannot rule out the possibility that the GSI-induced inhibition of other signaling pathways, such as the expression of EMT-related genes, contributed to these results.
Experimental Procedures
Author Contributions
Acknowledgments We appreciate the assistance of Drs. N. Nagoshi, K. Kojima, and S. Ito, who are all members of the spinal cord research team in the Department of Orthopedic Surgery, Keio University School of Medicine, Tokyo, Japan. We also thank Ms. T. Harada, Ms. R. Kashiwagi, and Ms. K. Yasutake for their assistance with the experiments and animal care. We thank Prof. Douglass Sipp (Keio University) for providing invaluable comments on the manuscript. This work was supported by Research Center Network for Realization of Regenerative Medicine from the Japan Science and Technology Agency (JST), the Japan Agency for Medical Research and Development (AMED) (grant no. 15bm0204001h0003 to H.O. and M.N.), and in part by a medical research grant on traffic accidents from The General Insurance Association of Japan (grant no. 15-1-42). H.O. is a compensated scientific consultant of San Bio, Co., Ltd.
Introduction Parkinson\'s disease (PD) is the second most common neurodegenerative disorder and is characterized by the selective loss of midbrain dopamine (mDA) neurons in the substantia nigra (SN). While the majority of PD cases are sporadic, there has been considerable progress in the identification of genes related to familial forms of PD. The study of such rare mutations may illuminate novel strategies to predict, understand, and potentially treat PD. A number of PD animal models have been established, but most of these have failed to faithfully reproduce the human disease (Dawson et al., 2010). Recent advances in the derivation and differentiation of human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs) present new opportunities for disease modeling (Bellin et al., 2012). The advent of iPSC technology has enabled PD modeling directly in patient-specific and disease-relevant human cells such as mDA neurons, the cell type preferentially lost in the disorder. To study mechanisms of mDA neuron loss in vitro, it is essential to select an optimized in vitro protocol to obtain the appropriate cell type. Many previous PD modeling studies in patient-specific iPSC lines were conducted using early generation differentiation protocols, which resulted in variable dopamine (DA) neuron populations in both quality and quantity (Cooper et al., 2012; Devine et al., 2011; Jiang et al., 2012; Nguyen et al., 2011). Therefore, the iPSC-derived DA neuron populations in those studies may have been heterogeneous and possibly distinct from the actual cells affected in the PD brain. We previously reported a successful protocol for deriving mDA neurons from human ESCs and iPSCs (Kriks et al., 2011), which facilitates robust induction of more “authentic” midbrain neurons that express key mDA markers including the transcription factors FOXA2 and LMX1A.