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W. Mao, S.-Z. Wang; Exploring Rpe as a Source of Retinal Neurons: A Physiological Study of Ash1-Induced Rpe Transdifferentiation Towards Amacrine Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2934.
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© ARVO (1962-2015); The Authors (2016-present)
RPE cells, unlike retinal neurons, can be readily cultured and amplified. Our laboratory is interested in coaxing cultured RPE cells to give rise to retinal neurons. This study tests whether proneural gene Ash1 is able to induce RPE cells to transdifferentiate towards amacrine cells with physiological properties.
Replication-competent retrovirus RCAS was used to drive the expression of chick ash1 (ash1) or GFP (as a control) in RPE cell cultures derived from day 5 - 6 chick embryos. Neuronal properties of transdifferentiating cells were examined by calcium imaging. Intracellular calcium concentration was monitored by Fluo-4 AM, a calcium indicator, and its fluorescence emission was monitored by a digital camera mounted on a microscope. Fluorescent images were taken sequentially with the exposure time of 400ms or 700ms before and after application of 10µl 1mM glutamate. Images were analyzed by Image J software. The relative change of fluorescence was calculated by using the formula: ΔF/Fo. ΔF = F-Fo. Fo = the averaged integrated optic density (IOD) of the images before glutamate application. F = IOD of an image.
In RPE cultures infected with RCAS-ash1,,cells displayed morphological properties that were similar to those of retinal neurons. Some of the transdifferentiating cells, especially those highly resembling amacrine cells, showed responses to exogenous glutamate at a concentration of 1mM. We observed that after glutamate application, fluorescence intensity increased in the cell body and in processes. This indicated a fast calcium influx,through calcium channels upon activation of functional glutamate receptors. . After reaching its peak, the fluorescence intensity decreased quickly and soon returned to the base level.
Our previous study has shown that ash1 induced cultured RPE cells to acquire thin, long processes and large rounded cell bodies. The morphological changes highly resembled those of amacrine cells. In addition, there was de novo expression of neural markers, especially amacrine cell specific markers, in RPE cell cultures infected with RCAS-ash1. Both morphological and immunochemical findings suggested ash1 is able to drive RPE cells toward amacrine cells. The present calcium imaging data showed that these cells were also able to respond to glutamate, like neurons. Our study suggests that ash1 is capable of inducing chick embryonic RPE cells to transdifferentiate into amacrine-like cells with morphological, molecular biological and physiological similarities.
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