June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Feline Müller glia with stem cell characteristics differentiate towards a retinal ganglion cell phenotype in vitro
Author Affiliations & Notes
  • Karen Eastlake
    Institute of Ophthalmology, UCL, London, United Kingdom
  • Silke Becker
    Institute of Ophthalmology, UCL, London, United Kingdom
  • Hari Jayaram
    Institute of Ophthalmology, UCL, London, United Kingdom
  • Phillippa Cottrill
    Institute of Ophthalmology, UCL, London, United Kingdom
  • Megan Jones
    Institute of Ophthalmology, UCL, London, United Kingdom
  • G Astrid Limb
    Institute of Ophthalmology, UCL, London, United Kingdom
  • Footnotes
    Commercial Relationships Karen Eastlake, None; Silke Becker, None; Hari Jayaram, None; Phillippa Cottrill, None; Megan Jones, None; G Astrid Limb, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2234. doi:
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      Karen Eastlake, Silke Becker, Hari Jayaram, Phillippa Cottrill, Megan Jones, G Astrid Limb; Feline Müller glia with stem cell characteristics differentiate towards a retinal ganglion cell phenotype in vitro. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2234.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: A strategy to combat retinal ganglion cell (RGC) loss that may be explored for potential therapeutic application is cell replacement by transplantation of stem cell derived RGCs. Since Müller glia with stem cell characteristics can be found in higher mammals including humans, and these cells have the potential for cell based regenerative therapies, we investigated whether these cells were present in the feline eye, and whether they have similar characteristics of Muller stem cells isolated from the adult human retina.

Methods: Feline Müller glia was isolated using standard protocols, following by culture on fibronectin in the presence of EGF. We characterized a feline cell preparation (FM9) that became spontaneously immortalized (a characteristic of stem cells) for their phenotypic and gene expression characteristics of Müller stem cells, including CRALBP, Notch1, Sox2 and Wnt2. We also investigated their ability to differentiate towards a retinal ganglion cell (RGC) phenotype following culture on matrigel in the presence of the γ-secretase inhibitor DAPT (a Notch inhibitor). RGC phenotype was examined by gene (RT-PCR) and protein (western blotting/immunostaining) expression of Hes1, Vimentin, Thy1, Isl-1 and Brn3b.

Results: FM9 cells were shown to express markers of Müller stem cells, including CRALBP, Notch1, Sox2 and Wnt2. Following culture in the presence of the Notch inhibitor DAPT they acquired morphological characteristics of neural cells, compatible with changes towards a RGC phenotype. These observations were supported by up-regulation of mRNA coding for the RGC marker Brn3b and down-regulation of Vimentin and Hes1 following Notch inhibition. Protein expression studies showed that Notch down-regulation in FM9 cells induced a decrease in the expression of the Müller glia markers Vimentin and CRALBP, accompanied by an increase in the RGC markers Isl-1 and Thy1.

Conclusions: The results indicate that a population of Müller cells with stem cell characteristics is present in the feline retina as that seen in higher mammals including humans. These cells can be induced to differentiate into a RGC phenotype in vitro, providing an important tool for future transplantation studies to restore retinal ganglion function in this species.

Keywords: 603 Muller cells • 721 stem cells • 531 ganglion cells  
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