Clonogenic analysis of ciliary epithelial derived retinal progenitor cells in rabbits

In lower vertebrates, multipotential retinal stem cells reside in a far peripheral retinal zone known as the ciliary marginal zone while more fate-restricted progenitor cells are located immediately adjacent to this retinal margin. To determine whether mammalian ciliary epithelium contains heterogenous stem cell and progenitor cell populations similar to lower vertebrates, we investigated the heterogeneity of the retinal progenitor (or stem) cells isolated from adult rabbit eyes, which are large and suited for precise identification of the region. Under clonogenic culture conditions for a neurosphere assay, spheres were generated from approximately 0.1% of the ciliary epithelial cells and expressed retinal selective markers when plated under conditions producing differentiation. Subsequently, we found that 13.7% of the primary spheres gave rise to a secondary sphere and that 77% of the primary sphere colonies were unipotential progenitors that generated either neurons or glial cells exclusively, while 23% were at least bipotential that could give rise to both cell types using double-labelled immunocytochemistry. Taken together, our results demonstrate that the ciliary epithelium of adult rabbits contains both (at least) bipotential retinal progenitor (or stem) cells and unipotential fate-restricted progenitor cells, similar to lower vertebrates.