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

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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