June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Exploring mouse retinal ganglion cell diversity within iPSC derived optic cups
Author Affiliations & Notes
  • Julia Oswald
    affiliate of Harvard Medical School, Schepens Eye Research Institute, Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Petr Baranov
    affiliate of Harvard Medical School, Schepens Eye Research Institute, Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Julia Oswald, None; Petr Baranov, None
  • Footnotes
    Support  Shore/Alice Adler fellowship
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1361. doi:
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      Julia Oswald, Petr Baranov; Exploring mouse retinal ganglion cell diversity within iPSC derived optic cups. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1361.

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

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Abstract

Purpose : In glaucoma and other optic neuropathies, transplantation of retinal ganglion cells (RGCs) has been proposed as a universal approach to achieve neuroprotection and cell replacement. Experiments addressing axon regeneration furthermore demonstrated a correlation between functional recovery and specific RGC subtypes, including direction selective RGCs (DSGCs) and α-RGCs. Hence, to address the possibility of subtype-specific RGC transplantation we have studied the diversity of RGCs in retinas, derived from differentiated optic cups, originating from mouse embryonic (mES) and induced pluripotent stem cells.

Methods : In this study we compared retinal ganglion cell populations amongst a mouse derived, Thy1-GFP iPS cell line and a mouse ES cell line (both on C57Bl/6 background). Using both cell lines, optic cups were generated in-vitro using a three-dimensional approach, aided by Matrigel. After optic cup maturation, Thy1+ cells were isolated by either magnetic micro-beads targeting Thy1 or fluorescence activated cell sorting (FACS) for Thy1-GFP at day 16 or day 21 of culture. Subsequently, the isolated Thy1+ cell populations were characterized by RT-PCR and Flow-Cytometry for expression of subtype specific RGC markers. Following re-plating, Thy1+ cells were characterized by immunocytochemistry and calcium imaging.

Results : Following the generation of defined optic cup structures from both, mouse ES and iPS cells, Thy1+ cells isolated by magnetic micro-beads and FACS were shown to express a diverse set of retinal ganglion subtype specific markers. Theses included HoxD10, Fstl4 (for ON DSGCs), Spp1, Kcng4 (ON/OFF α-RGCs) as well as CART, Cdh6, Mmp17 (ON-OFF DSGCs) and JAM2 for OFF J-RGCs. While J-RGCs and α-RGCs were most abundant, both DSGCs and melanopsin (Opn4)-positive RGCs could also be detected. After re-plating Thy1+ RGCs developed characteristic morphology and functionality in-vitro. Notably, ES- and iPS-derived optic cups displayed comparable RGC diversity across experiments.

Conclusions : Retinal tissue, derived from in-vitro differentiated optic cups is a reliable source of various RGC subclasses. RGCs isolated from those may be used for transplantation studies addressing the subtype-specific effect of RGCs towards neuroprotection and cell replacement.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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