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Steven W. Wang; Genetic engineering for enriched retinal ganglion cell production from primary retinal culture. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1368.
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© ARVO (1962-2015); The Authors (2016-present)
Stem cell research pushed in vitro neuronal production to new heights. However, stem cells for retinal ganglion cell (RGC) production, or any other projection neurons, remain lacking amid countless efforts and claims. The purpose of this study, distinct from the conventional induction formulation, is to explore genetic methods for enriched RGC production.
During retinogenesis, Hes1 and Atoh7 (Math5) play opposing roles in keeping cells in a proliferative state and permitting neuronal differentiation. I exploited these countering traits by conditionally replacing the Atoh7 coding sequence with a floxed Hes1 and placing Atoh7 downstream of the floxed Hes1 to allow later Atoh7 reactivation – Atoh7flHes1/flHes1. To remove Hes1, restore Atoh7, and trace the produced cells, a stable Math5flHes1/flHes1;Ai9;CreER line was generated. Neurospheres were cultured from dissociated Atoh7flHes1/flHes1;Ai9;CreER retinas collected between embryonic day 13 (E13) and E15. Neurospheres were dissociated, treated with 4OH-Tamoxifen, and cultured on varies substrates in RGC promoting media.
Dissociated retinal cells from the Atoh7flHes1/flHes1 retinas produce at least 6 times more neurospheres than their wildtype counterparts indicating the employed genetic strategy pomotes cell proliferation. Dissociated neurosphere cells did not continue to proliferate after removal of flHes1 and restoration of Atoh7. Greater than 95% of these cells are positive of all 5 RGC markers tested. When cultured on laminin-coated substrate, the cells do not resemble RGCs. They exhibited a flattened cytoplasm closely attached to the substrate. Most notably, when cultured on a monolayer of retinal cells derived from Atoh7-null mice, most of the cells extended long axons and complex neurite structures representing typical RGC morphologies.
Using a novel genetic engineering approach, I report here the first time, a readily accessible way to produce large numbers of RGCs in vitro. This will now afford us the opportunity to study in more detail the molecular features of neurosphere-derived RGCs, and their in vitro properties. In addition, these in vitro-produced RGCs will allow us to devise new strategies for transplanting RGCs into RGC-deficient retinas from a host of useful mouse models.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
RGC-like morphologies can be seen in live cells (tdTomato) dissociated from neurospheres and allowed to differentiated in vitro.
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