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Isolation, Cultivation and Characterization of Cells from the Ciliary Margin of Adult Mice

1Ader M., 2Linke S., 1Meininghaus N., 2Richard G., 3Schachner M., 3Bartsch U.,
1Zentrum für Molekulare Neurobiologie Hamburg (Hamburg)
2Universitätsklinikum Hamburg-Eppendorf, Klinik und Poliklinik für Augenheilkunde (Hamburg)
3Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Institut für Biosynthese neuraler Strukturen (Hamburg)

Purpose: Transplantation of stem cells might provide a tool to replace degenerated cell types in dystrophic retinas. With the final aim to use retinal stem cells for such transplantations, we have isolated, cultivated and characterized cells from the ciliary margin of adult mice.
Method: Cells from the ciliary margin of adult wild-type and EGFP-transgenic mice were isolated, cultivated and passaged as described (Tropepe et al., 2000). In brief, the ciliary margin was isolated, enzymatically dissociated, and cells were maintained in a medium supplemented with B27 and fibroblast growth factor-2 (FGF-2). Cultures were passaged once every week by enzymatic dissociation of spheres. In some experiments, pigment epithelium of the retina and the iris was isolated in addition to the ciliary margin, and cultivated under identical conditions. Spheres were incubated with antibodies to nestin, a marker for undifferentiated cells. To induce differentiation of cells, spheres were plated onto a laminin substrate and fetal calf serum was added to the medium.
Results: Cells isolated from the ciliary margin of adult mice formed free-floating spheres in the presence of FGF-2. Cultures could be passaged for up to six times (further passaging was not attempted). Formation of spheres was observed in cultures prepared from the ciliary margin, but not in cultures prepared from the pigment epithelium of the retina or the iris. A large fraction of cells within the spheres expressed nestin. Plating of spheres onto a laminin substrate and addition of serum to the medium induced differentiation into cells with a epithelial or neuronal morphology.
Conclusions: Cells from the ciliary margin of the adult mouse proliferate in response to FGF-2, form free-floating spheres, can be passaged, and express nestin. Thus, cells display characteristics of retinal stem cells. However, their differentiation potential in vitro and after transplantation into dystrophic retinas in vivo remains to be investigated. 
The project is supported by the Bundesministerium für Bildung und Forschung (01GN0126 to U.B.).