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Analysis of mechanisms regulating self-renewal and neural differentiation of neural stem cells derived from primate embryonic stem cells

灵长类胚胎干细胞来源的神经干细胞自我更新和神经分化调节机制分析

基本信息

批准号：
17500256
负责人：
INOUE Nobuo
金额：
$ 2.24万
依托单位：
Tokyo Metropolitan University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17500256/
关键词：
Embryonic stem cell ES cell Neural differentiation Neural stem cell Neuron Astrocyte

项目摘要

We have reported that neural differentiation of mouse and monkey embryonic stem (ES) cell can be efficiently induced by Neural Stem Sphere (NSS) method. In the first stage of the method, the NSS was formed from a colony of ES cells by culture in astrocyte-conditioned medium under free-floating conditions. Here we analyzed changes in gene expression during the formation of NSSs from mouse and monkey ES cell colonies by quantitative real-time RT-PCR and found that expression of ES cell markers was down-regulated by the culture. In contrast, expression of an epiblast marker, a neuroectodermal marker, neural stem cell markers and neuronal markers became marked sequentially during the culture. However, expression of an early mesoderm marker, a primitive endodermal marker and an epidermal marker was low and did not significantly increase throughout the culture. In the second stage of the method, culturing the spheres on an adhesive substrate in ACM promoted neurogenesis. These results suggest that mouse and primate ES cells can unidirectionally differentiate into neurons via neural stem cells by the NSS method. We applied the NSS method to human ES cells which were supplied from Kyoto University and demonstrated that human ES cells could be efficiently differentiated into neurons by the method. We examined gene expression and protein profiling during differentiation of mouse embryonic stem (ES) cells into neurons via neural stem cells by microarray-based hybridization and proteomic analysis, and we could identified some genes and proteins with known and unknown biological functions. Neural stem cells prepared from the NSS were demonstrated to be induced to differentiate into astrocytes by withdrawing fibroblast growth factor-2 from the medium without any additional instruction.

本文报道了用神经干细胞球（NSS）法可有效地诱导小鼠和猴胚胎干细胞向神经细胞分化。在该方法的第一阶段中，NSS通过在自由漂浮条件下在星形胶质细胞条件培养基中培养从ES细胞集落形成。在这里，我们通过定量实时RT-PCR分析了小鼠和猴ES细胞集落形成NSS过程中基因表达的变化，发现ES细胞标志物的表达被培养物下调。相反，在培养过程中，上胚层标记物、神经外胚层标记物、神经干细胞标记物和神经元标记物的表达依次变得显著。然而，早期中胚层标记物、原始内胚层标记物和表皮标记物的表达较低，并且在整个培养过程中没有显著增加。在该方法的第二阶段，在ACM中的粘性基质上培养球体促进神经发生。这些结果表明，小鼠和灵长类动物ES细胞可以通过NSS方法经由神经干细胞单向分化为神经元。我们将NSS方法应用于由京都大学提供的人ES细胞，并证明人ES细胞可以通过该方法有效地分化为神经元。本研究通过基因芯片杂交和蛋白质组学分析，研究了小鼠胚胎干细胞经神经干细胞分化为神经元过程中的基因表达和蛋白质谱，并鉴定了一些具有已知和未知生物学功能的基因和蛋白质。从NSS制备的神经干细胞被证明通过从培养基中取出成纤维细胞生长因子-2而被诱导分化成星形胶质细胞，而无需任何额外的指导。