May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Effect of Crx Expression on Differentiation of Human Adult Iris and Retinal Stem Cells
Author Affiliations & Notes
  • C. Jomary
    Retinitis Pigmentosa Research Unit, King's College London, London, United Kingdom
  • H. Thomson
    Vision Research, Clinical Neurosciences, University of Southampton, Southampton, United Kingdom
  • A. Lotery
    Vision Research, Clinical Neurosciences, University of Southampton, Southampton, United Kingdom
  • S. Jones
    Retinitis Pigmentosa Research Unit, King's College London, London, United Kingdom
  • Footnotes
    Commercial Relationships  C. Jomary, None; H. Thomson, None; A. Lotery, None; S. Jones, None.
  • Footnotes
    Support  British Retinitis Pigmentosa Society
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 5780. doi:
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      C. Jomary, H. Thomson, A. Lotery, S. Jones; Effect of Crx Expression on Differentiation of Human Adult Iris and Retinal Stem Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5780.

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

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Abstract

Purpose: : To assess the effect of exogenous expression of the transcription factor Crx on the differentiation of human adult iris and retinal stem cells into cells with a functional photoreceptor phenotype exhibiting light-sensitive properties.

Methods: : Stem cells isolated from fresh trabeculectomy iris tissue and from post-mortem retinal ciliary body were maintained in serum-free culture, and genetically modified by electroporation to express exogenous epitope-tagged murine Crx. Changes in the expression of stem cell markers (homeodomain transcription factor Pax6, POU transcription factor Oct3/4, proliferating cell nuclear antigen), neuronal markers (nestin, neuron-specific class III β-tubulin and neurofilament), and photoreceptor-specific markers (rhodopsin, cyclic nucleotide-gated cation channel-3, blue-cone opsin, cyclic (c)GMP phosphodiesterase) expression were evaluated by immunocytochemistry and western blot analysis. Photo-transduction cascade activity was assessed by measuring light-induced hydrolysis of cGMP levels using a cGMP enzyme-linked immunoassay.

Results: : Expression of stem cell markers of proliferation and pluripotency was significantly decreased by exogenous Crx expression. Notably, the homeodomain transcription factor Pax-6, which is expressed developmentally in embryonic retina, showed decreased expression in Crx-electroporated stem cells in comparison with controls. Concomitantly, Crx induced expression of the analysed neuronal and photoreceptor-specific markers. Light-induced cGMP hydrolysis was increased in stem cells expressing exogenous Crx, and inhibition of PDE resulted in elevated cGMP levels.

Conclusions: : Our previous investigations (Jomary & Jones, IOVS in press) into exogenous Crx expression in murine retinal stem cells demonstrated the potential for this factor to promote differentiation of these cells into cells expressing photoreceptor-specific markers and displaying light-induced sensitivity characteristic of an activatable visual phototransduction cascade. The present study extends these findings to the effect of exogenous Crx expression in human iris- and retina-derived stem cells and is consistent with the notion that Crx has a broadly-applicable ability to promote differentiation of such cells into photoreceptor phenotypes.

Keywords: differentiation • gene/expression • photoreceptors 
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