| Co-Investigator(Kenkyū-buntansha) |
TOYAMA Yoshiaki Keio University, School of Medicine, Professor, 医学部, 教授 (40129549)
ISHII Ken Keio University, School of Medicine, Instructor, 医学部, 助手 (00276289)
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| Budget Amount *help |
¥15,200,000 (Direct Cost: ¥15,200,000)
Fiscal Year 2006: ¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 2005: ¥8,700,000 (Direct Cost: ¥8,700,000)
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| Research Abstract |
(1)Recently, we have shown that the transplantation of neural stem/progenitor cells (NSPCs) can contribute to the repair of injured spinal cord in adult rats and non-human primates. However, in some cases, most of the transplanted cells adhered to the cavity wall and failed to migrate and integrate into the host spinal cord. In this study, we focused on chondroitin sulfate proteoglycan (CSPG) as a putative inhibitor on NSPC migration in vivo that is known as a constituent of glial scar strongly express after spinal cord injury. First, in vitro study revealed that the migration of NSPCs was inhibited by CSPG, and this inhibitory effect of CSPG on cell migration was attenuated by C-ABC pretreatment. Consistently, in vivo study of C-ABC treatment combined with NSPC transplantation into the injured spinal cord revealed that C-ABC pretreatment promoted the migration of grafted NSPCs, whereas CSPG immunopositive scar tissue around the lesion cavity prevented their migration into host spinal
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cord without the C-ABC pretreatment. Furthermore, this combined treatments significantly induced the appearance of a greater number of GAP-43-positive nerve fibers at the lesion epicenter compared with the single treatment of NSPC transplantation. These findings suggested that the application of C-ABC enhanced the benefits of NSPCs transplantation for spinal cord injury due to overcoming the inhibitory effects of the glial scar, and that the combined treatment of NSPC transplantation and C-ABC application may be a promising strategy for the regeneration of injured spinal cord.
(2)Understanding the survival time of grafted NSPCs and determining the extent of migration away from transplantation sites is essential for optimizing treatment regimens. Here, we used in vivo bioluminescence imaging to non-invasively assess the survival and residence time of transplanted NSPCs at injury sites in living animals and histologic analyses to assess cell morphology. Third-generation lentiviral vectors enabled efficient transduction and stable expression of both luciferase and a variant of green fluorescent protein in primary cultured NSPCs. Signals from these cells were detectable for up to 6 months or more after transplantation into the injured spinal cords of mice and cell survival depended on the time of transplantation relative to injury. Histological and functional data supported the imaging data. Optimization of cell therapies can be greatly accelerated and refined by imaging, and we demonstrate that the timing of NSPC transplantation relative to time of injury may be a key determinant of the fates and function of integrated cells as indicated by morphology and functional recovery.
(3)Recent reports have described the presence of neural crest-derived stem cells (NCSCs) that are multipotent and can self-renew in various tissue even in adults. Here we identify NCSCs in the bone marrow (BM), dorsal root ganglia, and skin and prospectively isolated them in adult transgenic mice encoding neural crest-specific Protein 0 promoter-Cre/Floxed-EGFP. Cultured EGFP-positive cells from each tissue form neurosphere-like structures that express NCSC genes and can differentiate into neurons, glial cells, and myofibroblasts. However, comparison of these NCSCs in three tissue sources revealed distinct differences. Interestingly, we observed NCSCs in the aorta-gonad-mesonephros region at E11.0, suggesting the migration of NCSCs through the blood circulation to the BM, providing an explanation for the generation of neural cells from the BM. The identification of NCSCs in accessible adult tissue provides a new source of multipotent cells for autologous cell therapy. Less
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