PURPOSE

The replacement of glucose by sorbitol in growth medium causes selection of astroglial cells from heterogeneous primary cultures derived from the brains of newborn mice. The present study was undertaken to investigate the effects of sorbitol on in vitro selection of Müller glial precursors from expanded late retinal progenitor cells (RPCs).

METHODS

RPCs used in these studies were isolated from the neural retina of postnatal day one green fluorescent protein (GFP) transgenic mice. The resulting GFP positive neurospheres were dissociated into a single cell suspension and grown on poly-D-lysine/laminin coated tissue culture flasks or slides to generate adherent RPCs. These adherent cells were treated with glucose free medium containing 25 mM sorbitol for 7 days and the expression of retinal-specific cell markers was determined by immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblot analysis

RESULTS

In vitro studies showed that sorbitol treatment of late RPCs altered cellular morphology. Immunocytochemical studies showed an increase in the proportion of cells expressing glial cell markers, most of which co-expressed CRALBP, GFAP, and vimentin. An increase in the proportion of cells expressing PKCalpha, a bipolar cell marker, was also observed. RT-PCR analysis showed down-regulation of nestin transcripts with a concomitant increase in CRALBP, GFAP, vimentin and PKCalpha. These findings were confirmed by immunoblot analysis, where down-regulation of nestin expression with simultaneous up-regulation of CRALBP, GFAP and PKCalpha was observed.

CONCLUSIONS

Sorbitol treatment of multipotent late RPCs, in the absence of glucose, results in the preferential selection of Müller glial precursors and their subsequent differentiation into cells that morphologically resemble Müller cells and co-express multiple glial markers.