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

脊椎动物的神经视网膜被组织成层状结构，由不同类型的神经元和1种神经胶质细胞组成。在视网膜形成过程中，这些不同的细胞类型是从一个相对固定的时间顺序的多潜能视网膜前体细胞共同群体中衍生出来的。已有研究表明，内源性信号和外源性信号在确定由普通视网膜前体细胞产生的细胞类型方面起着关键作用。在发育过程中，视网膜前体细胞被认为会在环境转变过程中改变其固有特性，从而对外部信号做出反应，并产生适当类型的视网膜细胞。这一视网膜能力模型表明，视网膜前体细胞在时间上不是同质的细胞群体。然而，异质内在属性的本质仍然难以捉摸。这在一定程度上是由于缺乏识别视网膜前体细胞不同阶段的标记。A…虽然转录因子和细胞周期调节因子的组合表达可能更能代表细胞的内在特性，但这些分子是在细胞内的，限制了它们对干细胞浓缩的有用性，并表明定义表面标记来识别视网膜前体细胞的重要性。表面抗原使从细胞混合物中分离出特定亚群的祖细胞群体成为可能，而不损害细胞，从而使表征它们的性质和识别调节其增殖和分化的分子成为可能。然而，根据视网膜祖细胞表面抗原的表达来定义视网膜祖细胞还没有被研究过。因此，我们试图通过对培养的视网膜细胞进行流式细胞术/细胞分选技术来鉴定视网膜干/祖细胞的标志物。我们从不同发育阶段的小鼠的视网膜细胞中筛选出它们与一组针对细胞表面抗原的抗体的反应性，并获得了30多种抗原在发育中的视网膜中的独特表达模式。其中，CD15、SSEA-1被报道在CNS干细胞中表达，被认为是视网膜祖细胞早期未成熟阶段的标志。SSEA-1抗原Lewis X碳水化合物在胚胎期视网膜边缘区域有强表达。随着视网膜发育到围产期，SSEA-1的表达减弱，但在边缘区仍有明显表达。免疫组织化学和流式细胞仪检测显示SSEA-1标记早期未成熟的视网膜祖细胞亚群。此外，我们发现c-kit在特定阶段的祖细胞中表达，对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围的视网膜前体细胞。我们发现，这些视网膜前体细胞亚群受到不同的调控，并具有明显的增殖和分化潜力。此外，我们还发现，长时间的c-kit激活诱导了Nestin阳性视网膜细胞的增殖和积聚。这些效应部分是通过MAPK信号通路来实现的。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，我们的结果提供了一个组合标记，定义了时间和空间上不同的视网膜前体细胞。较少

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