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Carolina B Del Debbio, Dânia E Hamassaki; Small Molecules and Adult Ciliary Epithelium Cells Reprogramming. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1363.
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© ARVO (1962-2015); The Authors (2016-present)
The Ciliary Epithelial cells (CE) of adult mammalian eyes are a quiescent population of cells able to proliferate and generate neurospheres with retinal progenitor profile. Despite the potential of CE to generate retinal progenitors, the efficiency is still low, probably due to the inefficient reprogramming and the presence of reminiscent epithelial parental properties. It is known that small molecules have been effective in stem and iPS cells reprogramming and, for that reason, we evaluate the effect of two small molecules on CE reprogramming efficiency,the Compound C (CC), inhibitor of AMP-activated protein kinase and bone morphogenetic protein, and NSC23766, a Rac1 GTPase inhibitor
After 4 consecutive intraocular injections of the small molecules (CC =10ng/eye, NSC = 200ng/eye) in adult Wistar rats, we dissected the CE, sectioned in cryostat or extracted the RNA and synthesized the cDNA. Later, the expression of the pluripotent genes c-Myc, Oct4 and Sox2 was analyzed. For in vitro experiments, CE cells were dissected and cultured in the presence of growth factors (FGF and EGF), to form neurospheres, and small molecules. Proliferation rates and progenitor profile were analyzed
Our data indicated that CC injections increased the expression of c-Myc and Oct4 (6 and 14 folds change (fc), respectively), in comparison to controls (PBS), but no effect was observed in Sox2 expression. Rac1 inhibition increased the expression of c-Myc and Oct4 (5 and 9 fc), however, Sox2 expression was decreased (4 fc). Furthermore, Rac1 inhibition increased CE cells proliferation rates observed through Ki67 immunostaining (32.8±0.20, control = 5.10±0.87), and quantitative PCR (5 fc). Methyltransferase transcripts analysis indicated that both molecules decreased the expression of DNMT1 and DNMT3b (CC = 1.5 and 1.2 folds; NSC = 1.6 and 2 folds), indicating the regulation of epigenetic factors. Neurospheres treated in the presence of small molecules showed an apparently increased expression of proliferation transcripts (ki67 and cyclins), and decreased epithelial properties genes (palmdelphin and Rab27), in comparison to controls
Our results suggest that the use of small molecules can be efficient for CE reprogramming, and the information gleaned from this study may provide valuable insight into the cellular and molecular events that underlie the reprogramming response of CE cells and the mechanism of retinal recovery
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