July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Using cell reprogramming technology to convert Müller glia into photoreceptors
Author Affiliations & Notes
  • Raymond Ching Bong Wong
    Centre for Eye Research Australia , East Melbourne, Victoria, Australia
    Ophthalmology, Surgery, University of Melbourne, Melbourne, Victoria, Australia
  • Tu Nguyen
    Centre for Eye Research Australia , East Melbourne, Victoria, Australia
    Ophthalmology, Surgery, University of Melbourne, Melbourne, Victoria, Australia
  • Lucy Lyujie Fang
    Centre for Eye Research Australia , East Melbourne, Victoria, Australia
  • Camden Lo
    Monash University, Melbourne, Victoria, Australia
  • Jafar Jabbari
    Australia Genome Research Facility, Melbourne, Victoria, Australia
  • Sandy Hung
    Centre for Eye Research Australia , East Melbourne, Victoria, Australia
  • Rick Guei-Sheung Liu
    Centre for Eye Research Australia , East Melbourne, Victoria, Australia
    University of Tasmania, Hobart, Tasmania, Australia
  • Chi D Luu
    Centre for Eye Research Australia , East Melbourne, Victoria, Australia
    Ophthalmology, Surgery, University of Melbourne, Melbourne, Victoria, Australia
  • Mark C Gillies
    Save Sight Institute, Sydney, New South Wales, Australia
  • Alex Hewitt
    Centre for Eye Research Australia , East Melbourne, Victoria, Australia
    University of Tasmania, Hobart, Tasmania, Australia
  • Footnotes
    Commercial Relationships   Raymond Ching Bong Wong, None; Tu Nguyen, None; Lucy Lyujie Fang, None; Camden Lo, None; Jafar Jabbari, None; Sandy Hung, None; Rick Liu, None; Chi Luu, None; Mark Gillies, None; Alex Hewitt, None
  • Footnotes
    Support  Raymond Wong was supported by grants from the National Health and Medical Research Council (1084256), the University of Melbourne (Louisa Jean De Bretteville Bequest) and the Kel and Rosie Day Foundation. The Centre for Eye Research Australia receives operational infrastructure support from the Victorian Government.
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 4578. doi:
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      Raymond Ching Bong Wong, Tu Nguyen, Lucy Lyujie Fang, Camden Lo, Jafar Jabbari, Sandy Hung, Rick Guei-Sheung Liu, Chi D Luu, Mark C Gillies, Alex Hewitt; Using cell reprogramming technology to convert Müller glia into photoreceptors. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4578.

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

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Abstract

Purpose : The loss of photoreceptors in many degenerative eye diseases is a leading cause of blindness. Since there are no effective treatments to restore vision once photoreceptors are lost, photoreceptor regeneration has the very real prospect of alleviating blindness in these eyes. Direct reprogramming using master transcription factors can be used to convert one somatic cell type directly into another, without passing through an intermediate stem cell state. Previous studies have highlighted the feasibility to reprogram glial cells into different types of neurons both in vitro, and in vivo in the brain, spinal cord and retina. This study aims to develop an reprogramming technology to convert Müller glia into photoreceptors (induced photoreceptors, iPHs) as an innovative approach for photoreceptor generation.

Methods : We have adapted the CRISPR activation (CRISPRa) system to activate multiple endogenous genes, which allow us to activate up to 9 reprogramming factors simultaneously. This allows us to efficiently perform in vitro screening in human Müller glia cell line (MIO-M1) to identify the optimal cocktail of reprogramming factors for generation of iPHs. Photoreceptor marker analysis was performed in iPHs by qPCR, immunocytochemistry and single cell transcriptome profiling using the 10x Chromium system.

Results : Using the CRISPRa platform, we have screened and identified cocktails of reprogramming factors that allow reprogramming of human Müller glia into iPHs in vitro. qPCR and Immunocytochemical analysis demonstrated that the reprogrammed iPHs expressed the rod markers RHO, PDE6B and GNAT. To comprehensively analyse the reprogrammed iPHs, we utilised perform single cell transcriptome profiling of ~10000 iPH cells. Transcriptome analysis demonstrated the transition of glial to neuron through reprogramming, the activation of photoreceptor markers in iPHs and the presence of different populations of reprogrammed cells.

Conclusions : Our study has demonstrated the use of transcription factors to promote reprogramming of Müller glia into photoreceptors. Development of cell reprogramming technology represents a potential strategy to generate photoreceptors and future application for in vivo reprogramming provides an exciting regenerative approach to replace photoreceptor losses in a range of retinal diseases.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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