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Identification of mouse retinal stem cell and cell lineages by gene expression patterns

通过基因表达模式鉴定小鼠视网膜干细胞和细胞谱系

基本信息

批准号：
17390075
负责人：
WATANABE Sumiko
金额：
$ 9.34万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
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/grant/KAKENHI-PROJECT-17390075/
关键词：
Retina gene expression transgenic mouse retinal progenitor cells cell sorter cell surface marker 網膜発生分化増殖シグナル伝達トランスジェニックマウス転写因子 siRNA BAC ゼブラフィッシュ

项目摘要

The vertebrate neural retina is organized into a laminar structure comprising types of neurons and 1 type of glial cells.. During retinogenesis, these various cell types are derived from a common population of multipotent retinal progenitor cells in relatively fixed chronological sequence. It has been shown that intrinsic cues and extrinsic signals play critical roles for defining the type of cells generated from common retinal progenitor cells. In the course of the development process, retinal progenitor cells are believed to change their intrinsic properties during their environmental transition, so that they can respond to extrinsic signals and generate appropriate types of retinal cells. This retinal competence model suggests that retinal progenitors are not a temporally homogeneous population of cells. However, the nature of the heterogeneous intrinsic properties is still elusive. This is in part due to the lack of markers identifying distinct stages of retinal progenitor cells. A … More lthough combinational expression of transcriptional factors and cell cycle regulators may represent intrinsic properties of the cells, these molecules are intracellular, limiting their usefulness for stem cell enrichment and suggesting the importance of defining surface markers to identify progenitor cells of the retina. Surface antigens make it possible to isolate specific subset of progenitor populations from cell mixture without damaging the cells, thus making it possible to characterize their nature and identify molecules that regulate their proliferation and differentiation. However, the definition of retinal progenitor cells in terms of their expression of surface antigen has not been investigated. Therefore, we sought to identify markers of retinal stem or progenitor cells by using the technique of flow cytometry/cell sorting on retinal cells in culture. We screened retinal cells from mice at various developmental stages for their reactivity with a panel of antibodies against cell-surface antigens and obtained unique expression patterns of more than 30 antigens in the developing retina. Among them, we focused on CD15, SSEA-1, which was reported to be expressed in CNS stem cells, as a candidate to be a marker of retinal progenitor cells in an early immature stage. SSEA-1 antigen, Lewis X carbohydrate, was strongly expressed in the retinal marginal region at embryonic stage. As retinal development proceeds to perinatal stage, the expression of the SSEA-1 became weaker but remained significantly in the marginal region. Immunohistochemical and flow cytometric approaches revealed that SSEA-1 marks immature subset of retinal progenitor cells in early stage. Furthermore, we found that c-kit was expressed in a defined stage of progenitor cells and that analysis of c-kit and SSEA-1 expression patterns enabled us to characterize centrally and peripherally located retinal progenitor cells. We found that these subsets of retinal progenitor cells were differently regulated and possess distinct proliferative and differentiation potentials. Furthermore, we found that prolonged c-kit activation induced the proliferation and accumulation of nestin positive retinal cells. These effects were in part mediated by a MAPK signaling pathway. Using amplifying cDNA from purified SSEA-1 or c-kit positive cells, we made DNAchip analysis and obtained gene expression patterns of these subset of retinal progenitor cells. This information may serve important molecular basis for stem cell studies. Together, our results provide a combinational marker that define temporally and spatially distinct retinal progenitor cells. Less

脊椎动物的神经视网膜被组织成层状结构，包括多种类型的神经元和1种类型的神经胶质细胞。在视网膜发生期间，这些不同的细胞类型以相对固定的时间顺序来源于多能视网膜祖细胞的共同群体。已经表明，内在线索和外在信号在确定由普通视网膜祖细胞产生的细胞类型方面起着关键作用。在发育过程中，视网膜祖细胞被认为在其环境转变期间改变其内在特性，使得它们能够响应外部信号并产生适当类型的视网膜细胞。这种视网膜能力模型表明，视网膜祖细胞不是一个时间上同质的细胞群体。然而，异质性的本质仍然是难以捉摸的。这部分是由于缺乏识别视网膜祖细胞不同阶段的标志物。一 ...更多信息尽管转录因子和细胞周期调节因子的组合表达可能代表细胞的内在特性，但这些分子是细胞内的，限制了它们用于干细胞富集的有用性，并提示了定义表面标记以鉴定视网膜祖细胞的重要性。表面抗原使得有可能从细胞混合物中分离祖细胞群体的特定亚群而不损伤细胞，从而使得有可能表征其性质并鉴定调节其增殖和分化的分子。然而，视网膜祖细胞在其表面抗原的表达方面的定义尚未研究。因此，我们试图通过对培养的视网膜细胞使用流式细胞术/细胞分选技术来鉴定视网膜干细胞或祖细胞的标志物。我们筛选了不同发育阶段的小鼠视网膜细胞与细胞表面抗原抗体的反应性，并在发育中的视网膜中获得了30多种抗原的独特表达模式。其中，我们重点研究了CD 15，SSEA-1，据报道其在CNS干细胞中表达，作为早期未成熟阶段视网膜祖细胞的候选标记物。SSEA-1抗原，即刘易斯X糖，在胚胎期视网膜边缘区强表达。随着视网膜发育进入围产期，SSEA-1的表达逐渐减弱，但在边缘区仍有显著表达。免疫组化和流式细胞术显示，SSEA-1标志着视网膜前体细胞的早期未成熟亚群。此外，我们发现c-kit在祖细胞的确定阶段中表达，并且c-kit和SSEA-1表达模式的分析使我们能够表征位于中心和外周的视网膜祖细胞。我们发现这些视网膜祖细胞亚群受到不同的调节，并具有不同的增殖和分化潜力。此外，我们还发现c-kit激活时间延长可诱导nestin阳性视网膜细胞的增殖和聚集。这些作用部分由MAPK信号通路介导。从纯化的SSEA-1或c-kit阳性细胞中扩增cDNA，进行DNA芯片分析，获得这些视网膜前体细胞亚群的基因表达谱。这些信息可能为干细胞研究提供重要的分子基础。总之，我们的研究结果提供了一个组合标记，定义时间和空间上不同的视网膜祖细胞。少