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sijia ding, Chao Jiang, Chen Zhao; The mechanism of mTOR signaling pathway in the regulation of differentiation of iPS into retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3583.
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© ARVO (1962-2015); The Authors (2016-present)
To explore the role of mTOR signaling pathway in the regulation of differentiation of iPS into retinal pigment epithelial (RPE) cells.
After embryoid bodies were formed by cultured iPS in suspension condition, they were induced to differentiate into RPE cells. The expressions of RPE specific proteins (RPE65, LRAT, ZO-1) in iPS-RPE cells during differentiation were detected by immunocytochemistry. Q-PCR and Western Blotting were carried out to analyze RPE specific genes, proteins and mTOR activity in iPS-RPE at different time points of differentiation (after one month, two months, three months). Finally, iPS-RPE were treated with mTOR inhibitor rapamycin and RPE specific protein expression was evaluated.
The RPE specific proteins (RPE65, LRAT, zo-1) of iPS-RPE cells were observed after one month of differentiation by fluorescence microscope. Compared with the control iPS, Q-PCR results showed that iPS-RPE exhibited significant higher level of RPE specific genes (RPE65, Best1, MerTK, CK18) after three months of differentiation (P <0.01). Western Blotting also showed that the expressions of RPE specific proteins BEST1, catenin and MerTK significantly increased in iPS-RPE cells as differentiation process went on. The activity of the mTOR was inhibited in this process. In addition, rapamycin treated cells exhibited higher expression of catenin, but expression of BEST1, MerTK and CK18 was not changed.
We established an efficient method to obtain iPS-RPE cells. And mTOR signaling pathway is gradually suppressed during the process of iPS differentiation into RPE cells in vitro.
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