Mechanisms of Development and Regeneration of Mammalian Central Nervous System

首先，我们开发了一种系统，通过类胚体(EB)诱导分化和随后的漂浮培养选择性地扩增神经干细胞/祖细胞(NSPC)，从小鼠ES细胞中获得包括干细胞在内的神经前体细胞。这些祖细胞实际上只分化为主要是胆碱能或GABA能的神经元。然后，将这些ES来源的神经前体细胞移植到隔区胆碱能损伤所致记忆障碍小鼠的海马区，改善了这些神经前体细胞的缺陷，并向包括胆碱能神经元在内的神经元分化。另一方面，我们成功地通过FACS纯化了表达EGFP的神经干细胞和从PA-6基质细胞饲养层分化的转基因小鼠ES细胞来源的多巴胺能神经元。将纯化的多巴胺能神经元移植到帕金森病模型大鼠的纹状体内，部分缓解了帕金森病模型大鼠的症状。我们还开发了一种衍生NSPC的系统，该系统可以通过EB形成从小鼠ES细胞中高效地产生表达HB9转录因子的躯体运动神经元和表达PhoxB转录因子的后脑内脏运动神经元。此外，我们还发现EBS在不同浓度的维甲酸中可以控制ES细胞产生不同类型的运动神经元。最后，我们应用为小鼠ES细胞开发的培养条件和培养周期的轻微改变，成功地开发了一种从食蟹猴和人ES细胞中衍生NSPC和神经元的系统。

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.