September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Increasing the population of retinal ganglion-like cells among retinal cells differentiated from mouse ES cells using 3D culture method
Author Affiliations & Notes
  • Satoshi Iraha
    Retinal Regeneration, RIKEN Centor for Developmental Biology, Kobe, Japan
    Application biology and regenerative medicine , Graduate School of Medicine Kyoto University, Kyoto, Japan
  • Michiko Mandai
    Retinal Regeneration, RIKEN Centor for Developmental Biology, Kobe, Japan
  • Hidenobu Tanihara
    Ophthalmology, Kumamoto university, Kumamoto, Japan
  • Masayo Takahashi
    Retinal Regeneration, RIKEN Centor for Developmental Biology, Kobe, Japan
    Application biology and regenerative medicine , Graduate School of Medicine Kyoto University, Kyoto, Japan
  • Footnotes
    Commercial Relationships   Satoshi Iraha, None; Michiko Mandai, None; Hidenobu Tanihara, None; Masayo Takahashi, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 6073. doi:
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    • Get Citation

      Satoshi Iraha, Michiko Mandai, Hidenobu Tanihara, Masayo Takahashi; Increasing the population of retinal ganglion-like cells among retinal cells differentiated from mouse ES cells using 3D culture method. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6073.

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

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Abstract

Purpose : Diseases such as central retinal artery occlusion (CRAO) or angle closure glaucoma are ophthalmic emergencies leading to the irreversible damage to retinal ganglion cells (RGCs) with sever and abrupt loss of visual function. Presently, there are no guideline-endorsed therapies for acute RGC loss. Cell replacement therapy emerged as one possibility for a therapeutic option in these diseases since the introduction of 3D retinal differentiation method that practically enables us to obtain retinal cells of any desired developmental stage. The aim of this study was to increase the proportion of RGCs among differentiated retinal cells for the purpose of RGC transplantation.

Methods : The Rax gene is expressed in neural retinal progenitor cells. Retinal cells were differentiated from the Rax-GFP mouse ES cell (mESC) line using 3D culture method, and GFP-positive optic vesicle-like structures were cut from cultured aggregates to purify retinal sheets as previously described (Assawachananont et al., Stem Cell Reports, 201). We first added DAPT (N-[N-(3,5-Difluorophenacetyl)- L -alanyl]-S-phenylglycine t-butyl ester: Notch signaling inhibitor) at different concentrations (0, 2, 10, 50 μM) on differentiation day (DD) 10, BrdU was added on DD13, and the samples were fixed on DD 14 for immuno-histochemical evaluation. Next, the effective dose of DAPT was added at different timing and/or period (2, 3, 4 days) and retinal tissue was attached to the plate on cessation of DAPT treatment and fixed on DD 17. Immuno-histochemical analysis for Brn3 (RGC marker) and BrdU was performed. Neurite extension was also monitored during the adhesion culture.

Results : The percentages of Brn3+ RGCs increased dose-dependently with 4-day DAPT treatment from 12.6% (0 μM) to 14.4 % (2 μM), 25.5 % (10 μM) and 25.1% (50 μM). There were few BrdU positive cells after 10 or 50 μM DAPT treatment. DAPT treatment of different periods all increased the Brn3 positive cell population and neurite extension.

Conclusions : We could increase RGC population in retinal tissue by adding DAPT. We plan to transplant RGC rich retinal cells into CRAO model mice.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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