Development of stem (neural stem-, embryonic stem-, medullary stem) cells to dopaminergic neurons and their application to reconstruction medicine.

神经干细胞具有自我更新和多向分化潜能，一旦定向分化为神经元，是神经移植的理想供体细胞。在本研究中，从神经干细胞分化DA能神经元和他们的应用重建医学进行了调查。神经干细胞在含血清的普通培养体系中主要发育为星形胶质细胞。然而，大多数（超过80%）的ES细胞来源的神经干细胞可以开发成神经元，使用5步培养方案与细胞因子和营养因子。微阵列分析显示，许多基因的上调或下调与神经干细胞分化为神经元。其中，在NSCs中高表达的多效生长因子对DA能神经元和祖细胞具有较强的营养和存活作用。在胚胎脑中，同源异型因子ATBF 1在位于分化区的细胞中表达，与β-微管蛋白和MAP 2共表达。在神经上皮细胞、Neuro 2A细胞和P19细胞的表达研究中发现，当ATBF 1在胞浆中表达时，细胞持续增殖，当ATBF 1在核细胞中表达时，细胞离开细胞周期并分化为神经元。ATBF 1的核内转运依赖于PI（3）K-激酶的活性，细胞核（胞浆）定位依赖于CRM 1。因此，同源异型因子ATBF 1与神经元分化、脑发育、脑结构的形成等有关，其研究结果将在神经分化、重建医学、脑发育和教育等领域得到应用。

Neural stem cells (NSCs) are self-renewing and multi-potent, thus are reasonable candidates of donor cells for neural transplantation once they steadily directed to neurons. In the present study, differentiation from NSCs to DAergic neurons and their application to reconstruction medicine were investigated. NSCs developed mostly to astrocytes in an ordinary culture system with serum. However, majority (over 80%) of ES cells-derived NSCs could be developed to neurons using 5 steps culture protocol with cytokines and trophic factors. Micro-array analysis revealed many genes were up-regulated or down-regulated in association with differentiation to neurons from NSCs. Among them, pleiotrophin highly expressed in NSCs showed a strong trophic and survival effects on DAergic neurons and progenitors. In embryonic brains, a homeotic factor ATBF1 was expressed in cells located in differentiation zone coexpressing with β- tubulin and MAP2. In expression study of neuroepithelial cells, Neuro 2A cells and P19 cells, it was found that when ATBF1 was expressed in cytosole cells proliferated continuously, and when ATBF1 was expressed inside nucleus cells left cell cycle and differentiated to neurons. Intra-nuclear transport of ATBF1 was dependent on the activity of PI(3) K-kinase and extranuclear (cytosol) localization was CRM1dependent. Thus, a homeotic factor ATBF1 was found be involved in neuronal differentiation, brain development, formation of brain architecture and so on. Results of the present study would be applied to various fields such as neural differentiation, reconstruction medicine, brain development and education.