Pretreatment With a Gamma-Secretase Inhibitor Prevents Tumor-Like Overgrowth In Human iPSC-Derived Transplants for Spinal Cord Injury

Toshiki Okubo 1,2, Narihito Nagoshi 1, Jun Kohyama 2, Go Itakura 2, Morio Matsumoto 1, Hideyuki Okano2, Masaya Nakamura 1

1Department of Orthopaedic Surgery, Keio University School of Medicine, Shinjyuku-ku, Tokyo, Japan 2Department of Physiology, Keio University School of Medicine, Shinjyuku-ku, Tokyo, Japan

Disclosures: Toshiki Okubo (N), Narihito Nagoshi (N), Jun Kohyama (N), Go Itakura (N), Morio Matsumoto (N), Hideyuki Okano (N), Masaya Nakamura (N) Introduction: We previously reported that transplantation of certain neural stem/progenitor cells derived from human iPS cells (hiPSC-NS/PCs) into the injured spinal cord resulted in tumor-like overgrowth and subsequent deterioration of motor function. Control of tumor-like overgrowth is critical in clinical applications of iPSC-based transplantation therapy for spinal cord injury (SCI). Remnant immature NS/PCs must be removed or induced into more mature cell types, which may avoid tumor-like overgrowth following transplantation. Meanwhile, Notch signaling controls the neuronal differentiation of NS/PCs [1], and inhibition of this signaling with gamma-secretase inhibitor (GSI) induces NS/PCs to differentiate into more mature neuronal cells with limited proliferation. The purpose of the present study was to elucidate the effects of GSI on tumorigenic hiPSC-NS/PCs. Methods: hiPSC-NS/PCs, a potentially tumor-like overgrowth cell line [2], were cultured with GSI. Immunocytochemistry (ICC) was performed to compare their characterizations. Cell cycle/apoptosis analyses were also performed using flow cytometry, and global analyses of the gene expression profiles were performed by DNA microarray and RT-PCRs. Next, we induced contusive SCI in mice and transplanted hiPSC-NS/PCs with or without GSI pretreatment nine days after SCI. The growth/survival and histological analyses of transplanted cells were examined with bioluminescence imaging (BLI) and immunohistochemistry. Behavioral analyses were also performed by BMS score, rota-rod test and DigiGait system. Results: The number of cells and the size of neurospheres significantly decreased in the GSI pretreatment (GSI+) group compared with the non-treatment (control) group (p=0.0082). Consistently, the proportion of S phase in cell cycle analyses significantly reduced (p=0.0064), and the proportion of Ki67+ proliferative and Nestin+ immature cells in ICC analyses also significantly decreased (p=0.0071), whereas βIII-tubulin+ mature neuronal cells significantly increased in the GSI+ group compared with the control group (p=0.0078). The gene expression profiles using DNA microarray and RT-PCR showed decreases in pluripotency and self-renewal marker-related genes, such as Sox2, Nanog, Pax6 and Oct3/4. BLI analysis revealed that the photon counts of transplanted cells increased more than 10-fold from their initial values, suggesting tumor-like overgrowth in the control group, in accordance with the motor functional deterioration. However, in the GSI+ group, the photon counts of grafted cells reached a plateau around 4 weeks after transplantation, which resulted in no tumor-like overgrowth through inhibition of cell proliferation and long-lasting motor functional recovery (Figure 1 and 2). In the GSI+ group, the proportion of Hu+ mature neuron increased significantly (p=0.023), which were further integrated with the reconstruction of the host neuronal network and form synapses, whereas the proportion of Ki67+ and Nestin+ cells significantly decreased compared with the control group (p=0.036). Discussion: We confirm that GSI pretreatment of hiPSC-NS/PCs reduce the proportion of dividing cells and increase neuronal maturation in vitro, and prevent tumor-like overgrowth of transplanted cells by inhibiting cell proliferation, resulting in safe and long-lasting functional recovery in vivo. Pretreatment of hiPSC-NS/PCs with GSI prior to transplantation can improve their safety in clinical setting. Significance: This study revealed that GSI induces undifferentiated hiPSC-NS/PCs to more neuronal maturation with limited proliferation. Therefore, inhibition of Notch signaling can prevent a tumor formation and promote motor functional recovery in human iPSC-grafted therapy of SCI. Reference: 1. Nelson, B.R., Hartman, B.H., Georgi, S.A., Lan, M.S. and Reh, T.A. (2007). Transient inactivation of Notch signaling synchronizes differentiation of neural progenitor cells. Dev Biol 304, 479–498. 2. Nori, S., Okada, Y., Toyama, Y., Nakamura, M., Okano, H., (2015). Long-term safety issues of iPSC-based cell therapy in a spinal cord injury model: oncogenic transformation with epithelial-mesenchymal transition. Stem Cell Reports 4, 360-373 Figures:

Figure 1: Long-term observation after transplantation of hiPSC-NS/PCs

Pretreated with GSI

Figure 2: Motor function analyses after transplantation of hiPSC-NS/PCs

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ORS 2017 Annual Meeting Poster No.0137