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.

我们报告说，小鼠和猴子胚胎（ES）细胞的神经分化可以通过神经茎球（NSS）方法有效诱导。在该方法的第一阶段，在自由浮动条件下，由星形胶质细胞调节培养基中的培养物培养在ES细胞的群落中形成NSS。在这里，我们通过定量的实时RT-PCR分析了NSS从小鼠和猴子ES细胞菌落形成过程中基因表达的变化，发现ES细胞标记物的表达被培养物下调。相比之下，在培养过程中，层状标记，神经外皮标记，神经干细胞标记和神经元标记的表达被依次标记。然而，早期中胚层标记，原始内皮标记和表皮标记的表达较低，并且在整个培养物中没有显着增加。在该方法的第二阶段，在ACM中培养球体上的球体促进了神经发生。这些结果表明，小鼠和灵长类动物ES细胞可以通过NSS方法通过神经干细胞单向分化为神经元。我们将NSS方法应用于京都大学提供的人类ES细胞，并证明可以通过该方法将人ES细胞有效地分化为神经元。我们通过基于微阵列的杂交和蛋白质组学分析在小鼠胚胎干细胞（ES）细胞分化过程中研究了基因表达和蛋白质分析，我们可以鉴定出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是通过从培养基中撤回成纤维细胞生长因子-2而没有任何额外指导的。