Mechanisms of Development and Regeneration of Mammalian Central Nervous System

首先，我们开发了一种系统，通过胚胎样体（EB）形成诱导分化和随后的称为神经球培养的浮动培养，选择性扩增神经干/祖细胞（NSPCs），从小鼠胚胎干细胞中获得包括干细胞在内的神经祖细胞。这些祖细胞实际上只能分化为主要是胆碱能或gaba能的神经元。然后，将这些es来源的神经祖细胞通过中隔胆碱能损伤移植到有记忆缺陷的小鼠海马中，可以改善这些神经祖细胞的缺陷，并向包括胆碱能神经元在内的神经元分化。另一方面，我们成功地从PA-6基质细胞饲养层分化的转基因小鼠胚胎干细胞中纯化了表达EGFP的神经干细胞和多巴胺能神经元。将这些经FACS纯化的多巴胺能神经元移植到帕金森模型大鼠纹状体中，可部分改善症状。我们还开发了一种衍生NSPCs的系统，该系统可以通过EB形成小鼠ES细胞，有效地产生表达HB9转录因子的体细胞运动神经元和表达PhoxB转录因子的后脑内脏运动神经元。此外，我们发现胚胎干细胞的各种类型的运动神经元的产生可以通过暴露于不同浓度的视黄酸来控制。最后，我们成功地开发了一种从食蟹猴和人类胚胎干细胞中衍生出NSPCs和神经元的系统，该系统在培养条件和培养周期上进行了轻微的修改。

Firstly, we have developed a system to obtain neural progenitor cells including stem cells from mouse ES cells through the induction of differentiation by embryoid body (EB) formation and a subsequent floating culture called neurosphere culture which selectively amplifies neural stem/progenitor cells (NSPCs). These progenitor cells differentiate into virtually only neurons that are mainly cholinergic or GABAergic. Then, transplantation of these ES-derived neural progenitors into hippocampus of mice that had memory deficit by cholinergic lesion in their septum resulted amelioration of the deficit and differentiation of the progenitors into neurons including cholinergic neurons.On the other hand, we have succeeded to purify EGFP expressing neural stem cells and dopaminergic neurons derived from genetically modified mouse ES cells differentiated on the PA-6 stromal cell feeder layer by FACS. Transplantation of these FACS purified dopaminergic neurons into striatum of Parkinsonian model rats resulted partial amelioration of the symptoms.We also have developed a system to derivate NSPCs which can efficiently generate somatic motoneurons expressing HB9 transcription factor and hindbrain visceral motoneurons expressing PhoxB transcription factor from mouse ES cells through EB formation. Moreover, we have found that generation of various types of motoneurons from ES cells can be controlled by the exposure EBs to various concentration retinoic acid.Finally, we have successfully developed a system to derivate NSPCs and neurons from crab-eating monkey and human ES cells by applying the systems developed for mouse ES cells with slight modifications in the culture conditions and periods.