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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)
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.
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.
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.
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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