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Ya-Ping Lin, Steven W. Wang; RGC-potent retinal stem cells generated by replacing Atoh7 with Hes1. Invest. Ophthalmol. Vis. Sci. 2012;53(14):420.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of this project is to create retinal stem cells that are potent to give rise to retinal ganglion cells (RGCs) using a novel genetic engineering strategy. The proneural factor Atoh7 is required for cell cycle exit and essential for retinal ganglion cell (RGC) formation. Contrarily, the inhibitor type bHLH factor Hes1 prevents neurogenesis by promoting cell cycle entry and inhibiting Atoh7 activation. We hypothesize that replacing Atoh7 with Hes1 would lead to incessant cell cycle reentry in retinal progenitor cells, thus creating retinal stem cells (RSCs). We also hypothesize that reactivate Atoh7 in these RSCs can restore their RGC forming potential. The current report provides experimental results for testing these two hypotheses.
A mouse line (Atoh7Hes1/Hes1) carrying a genetic element of loxP-Hes1-IRES-dsRed-loxP in place of Atoh7 coding sequence was generated. Excision of the floxed element would allow an Atoh7-IRES-hrGFP cassette to resume Atoh7’s genomic position. The final mouse line (Atoh7Hes1/Hes1;CreERTM) was generated by crossing the Atoh7Hes1/Hes1 mice with a Cre-ERTM line that Cre activity could be induced by administrating Tamoxifen. Dissociated retinal cells from E13.5 Atoh7Hes1/Hes1;CreERTM retinas were cultured to form neurospheres. Generated neurospheres were treated with 4OH-Tamoxifen to restore Atoh7 and cells were allowed for differentiation. Resulting cells were tested for their RGC properties.
The Atoh7 promoter remained active in the neurospherical cells after prolonged culture. This result indicated that Atoh7 could be restored after removal of the inserted Hes1 element. Immunolabeling with a combination of RGC markers showed that at least 90% of the neurosphere derived cells are positive for all tested RGC markers after Atoh7 restoration. Co-culture of these cells with cells isolated from RGC-depleted retinas showed that at least 50% of neurosphere derived Atoh7 reactivated cells exhibited RGC morphology.
Neurospheres were produced due to incessant cell cycle reentry when a Hes1 cDNA was used to replace the Atoh7 coding sequence. A large portion of neurosphere derived cells acquired RGC fate after Atoh7 restoration. We present here a novel strategy for generating stem cells for specific neural cell type.
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