Sorbitol causes preferential selection of Muller glial precursorsfrom late retinal progenitor cells in vitro

[摘要] Purpose: The replacement of glucose by sorbitol in growth mediumcauses selection of astroglial cells from heterogeneous primary culturesderived from the brains of newborn mice. The present study wasundertaken to investigate the effects of sorbitol on in vitro selectionof Müller glial precursors from expanded late retinal progenitor cells(RPCs).Methods: RPCs used in these studies were isolated from the neuralretina of postnatal day one green fluorescent protein (GFP) transgenicmice. The resulting GFP positive neurospheres were dissociated into asingle cell suspension and grown on poly-D-lysine/laminin coated tissueculture flasks or slides to generate adherent RPCs. These adherent cellswere treated with glucose free medium containing 25 mM sorbitol for 7days and the expression of retinal-specific cell markers was determinedby immunocytochemistry, reverse transcriptase polymerase chain reaction(RT-PCR) and immunoblot analysisResults: In vitro studies showed that sorbitol treatment of lateRPCs altered cellular morphology. Immunocytochemical studies showed anincrease in the proportion of cells expressing glial cell markers, mostof which co-expressed CRALBP, GFAP, and vimentin. An increase in theproportion of cells expressing PKCα, a bipolar cell marker, wasalso observed. RT-PCR analysis showed down-regulation of nestintranscripts with a concomitant increase in CRALBP, GFAP, vimentin andPKCα. These findings were confirmed by immunoblot analysis, wheredown-regulation of nestin expression with simultaneous up-regulation ofCRALBP, GFAP and PKCα was observed.Conclusions: Sorbitol treatment of multipotent late RPCs, in theabsence of glucose, results in the preferential selection of Müllerglial precursors and their subsequent differentiation into cells thatmorphologically resemble Müller cells and co-express multiple glialmarkers.

[学科分类] 生物化学/生物物理