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X. Deng, D. Zhu, C. Spee, S. J. Ryan, D. R. Hinton; Photoreceptor-Like Differentiation of RPCInduced by RPE Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4284.
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The emerging field of retinal cell replacement therapy provides a promising new approach for the treatment of retinal degeneration. The current study was focused on evaluating conditions for in vitro induction of photoreceptor-like differentiation of retinal progenitor cells (RPCs).
1). RPC isolation: retinas were dissected from human fetal eyes (18-20 gestational weeks) and digested with trypsin. The isolated RPCs were maintained in RPC medium supplemented with 10% knockout replacement serum, N2 and B-27 supplement , EGF, bFGF, and IGF on fibronectin pre-coated culture dishes. 2). Induction of RPC differentiation: Fetal retinal pigment epithelium (fRPE) and mouse embryonic fibroblast (MEF) cells were treated with Mitomycin C (10 ug/ml), seeded on cell culture dishes (8x105 cells/cm2), and incubated overnight. The human RPCs were then seeded either on top of fRPE or MEF cells, or on fibronectin coated dishes, cultured up to 7days, with culture medium changed every other day. 3). Immunofluorescent labeling: RPCs on chamber slides were immuno-stained with anti-Nestin, Pax6 , Chx10 and Rhodopsin antibody. The stained cells were observed and digitally imaged under fluorescence microscopy.
RPCs isolated from human fetal retina exhibited positive immunofluorescent staining for three RPC markers; Nestin, Pax6 and Chx10. RPCs could temporally grow on top of fRPE, MEF cells or on fibronectin coated dishes. However, after 7 days of culture, only the RPCs on fRPE cells remained viable and healthy; the RPCs grown on fibronectin coating showed degenerative changes; while most of the RPCs grown on MEF cells died. There were more rhodopsin positive cells in the RPCs cultured on RPE cells, than in cultures in which RPCs were grown on fibronectin.
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