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G. Zhan, I. Ahmad; Differentiation of Muller Cells Into Retinal Ganglion Cells in vitro and in vivo. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3565.
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© ARVO (1962-2015); The Authors (2016-present)
Muller cells, the sole glia generated by the retinal stem cells/progenitors, have recently been identified to possess latent stem cell properties and potential (Das et al., 2006, Dev. Biol. 299:283). When removed from their niche and cultured in the presence of growth factors or activated in vivo by neurotoxins they display stem cell properties and demonstrate their neurogenic ability to give rise to retinal neurons. Here, we have examined their potential to differentiate into retinal ganglion cells, the cell type vulnerable in glaucoma.
Muller cells were isolated from four weeks old rats and cultured in the presence of EGF and FGF2 to generate neurospheres. Stem cell properties of cells in neurospheres were determined by their ability to proliferate and express pan neural and retinal progenitor markers. To induce their differentiation along RGC sub lineage Muller cell-derived neurospheres were co-cultured in the presence of embryonic day 3 (E3) chick retinal cells that are known to elaborate RGC promoting factors (Hegde et al., 2007, Exp. Eye Res. 84:577). Determination of RGC differentiation included immunocytochemical and RT-PCR analyses of RGC-specific markers. Muller cells displaying stem cell properties were transplanted into normal rats or those carrying ischemia-reperfusion injury intravitreally and the transplantation outcomes were examined two weeks post-transplantation.
A subset of Muller cells in the presence of growth factors generated neurospheres that contained proliferating (BrdU+) cells expressing immunoreactivities and transcripts corresponding to progenitor markers (e.g., Nestin, Sox2, Rx). A subset of cells in neurospheres, cultured in the presence of E3 chick retinal cells or conditioned medium, expressed immunoreactivities corresponding to RGC regulators and markers, Brn3b and RPF1. Results obtained by immunocytochemical analysis were confirmed by the detection of Brn3b, RPF1 and Ath5 transcripts by RT-PCR. Muller cells transplanted in the normal rats eyes were observed incorporated in different lamina of the retina and those integrated in the RGC layers expressed RGC-specific markers Brn3b and RPF1.
Muller cells, induced to activate their stem cell properties by growth factors, have the potential to differentiate into RGCs in vitro and in vivo, based on biochemical and molecular criteria.
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