Abstract
Purpose: :
Sox2 expression is associated with uncommitted dividing stem and progenitor cells of the developing nervous system and is also expressed in adult neural stem cells. In this study we investigated the role of the transcription factor Sox2 in maintaining the progenitor characteristics of Müller stem cells from the adult human retina.
Methods: :
RNA interference was used to investigate the consequences of Sox2 downregulation on the progenitor characteristics of MIO cells. MIO cell lines are spontaneously immortalized Müller cells from the adult human retina that exhibit characteristics of retinal stem cells. Knockdown of Sox2 was achieved using two Sox2 shRNA constructs and one scrambled construct ligated into the pGSU6-GFP vector. Analysis of transfected MIO cells was carried out by RT-PCR, Western blotting and confocal analysis of immunostained cells.
Results: :
After knockdown of Sox2 with the shRNA constructs, Müller stem cells adopted a neural morphology and extended long axons with varicosities characteristic of neural cells. Cells transfected with the silencing constructs upregulated markers of differentiating retinal neurons including HuD, Brn3b, Rhodopsin and S-Opsin whilst downregulating neural stem cell markers such as Notch1. Transfection with one of the constructs markedly reduced the proliferating ability of these cells in culture.
Conclusions: :
The present observations strongly indicate that Sox2 may play a very important role in maintaining the neural stem cell characteristics of MIO cells. These results also suggest that Sox2 may confer Müller stem cells the potential to regenerate retinal neurons in the adult human eye.
Keywords: Muller cells • differentiation • gene/expression