Abstract
Purpose: :
To compare the retinal progenitor/stem cell characteristics of cells isolated from the neural retina and ciliary body of the adult human eye and to identify markers which could potentially discriminate between these two cell populations.
Methods: :
Neural retina and non-pigmented and pigmented ciliary epithelium (CE) were separated under a dissecting microscope. Cells were cultured in suspension as spheres and also as monolayers following isolation. Expression of neural progenitor and epithelial cell markers was examined by RT-PCR, western blotting and immunostaining.
Results: :
Cells from the CE were able to form neurospheres in primary culture. Following dissociation of these spheres, non-pigmented cells were able to expand as a monolayer and exhibited an epithelial morphology. Dissociated pigmented cells were able to attach but acquired a flattened morphology and did not proliferate. In contrast, cells isolated from the neural retina did not form spheres in suspension culture and only proliferated if they were cultured as a monolayer after isolation. Upon attachment, these cells started to proliferate after 2-3 weeks in culture, exhibited a characteristic glial morphology and expressed markers of Müller stem cells. RT-PCR analysis revealed that CE and Müller stem cells expressed markers of retinal progenitors such as Sox2, Pax6, Chx10 and Notch1, and markers of retinal pigmented epithelium including RPE65, ZO-1, MERTK and MITF-A. However, non-pigmented CE cells expressed higher levels of mRNA coding for epithelial markers compared to Müller stem cells. Müller stem cells stained for CD44, nestin, vimentin and Sox2 whereas CE cells with progenitor characteristics did not stain for these markers and only a sub-population were positive for Sox2. Unlike Müller stem cells, CE cells showed staining for cytokeratins, further confirming their epithelial nature.
Conclusions: :
These results showed that Müller and CE stem cells with neural stem/progenitor characteristics constitute two distinct populations in the eye, and suggest that stem cells from the neural retina have Müller glial characteristics whereas CE stem cells are epithelial in character. Proteins such as CD44, vimentin and nestin were able to discriminate between these two cell types. These results show that both populations of stem/progenitor cell have the potential to differentiate into retinal neurons and may have applications in the development of stem cell therapies.
Keywords: retinal culture • Muller cells • transcription factors