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S. Singhal, B. Bhatia, J. S. Ellis, P. T. Khaw, G. A. Limb; Inhibition of Notch Signalling Pushes Müller Stem Cells From the Adult Human Retina Towards a Retinal Ganglion Cell Fate in vitro. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5034.
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Notch signalling keeps retinal progenitors in an undifferentiated state during development. Retinal stem cells/progenitors must escape notch signalling in order to begin differentiating towards a retinal ganglion cell (RGC) fate. MIO cells (Müller stem cell lines) derived from the adult human retina demonstrate retinal stem cell properties in vitro and have the potential for retinal regeneration. This study examined the role of notch signalling in regulating the differentiation of these cells towards the retinal ganglion cell (RGC) fate.
MIO cells were cultured in vitro under baseline and differentiating culture conditions. Cells were either fixed with 4% PFA for immunocytochemistry or pellets isolated for RNA and protein extraction. Levels of notch activity as well as markers of RGC development- ATOH7 (RGC competence) and Brn3b (RGC commitment) were determined under baseline and differentiating conditions. Notch activity of MIO cells was then inhibited using 50uM DAPT/ml. Expression of RGC markers was reassessed following notch inhibition.
Notch activity was detected in MIO cells and found to be down regulated when cells were exposed to differentiating conditions, as seen in embryonic retinal progenitors. MIO cells also showed low levels of ATOH7 and Brn3b expression which was up regulated upon culture on matrix substrates (matrigel) in the presence of fibroblast growth factor b (FGFb). Upon inhibition of Notch activity with DAPT, the expression of Brn3b was further up regulated almost 3 fold. This was also accompanied by a change in cell morphology towards a neural phenotype (elongated cell processes with secondary and tertiary branching).
MIO cells contain RGC precursors which are regulated by Notch signalling. The RGC precursor population of MIO cells can be amplified by the use of matrix substrates and growth factors. Like embryonic retinal progenitors, MIO cells maintain their progenicity in the presence of Notch activity. Inhibition of Notch signalling by DAPT pushes these cells to differentiate down a retinal ganglion cell (RGC) fate. These observations suggest that in conjunction with notch inhibition, MIO cells might constitute a useful source of RGC precursors to replace damaged RGC in vivo.
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