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Haibin Tian, Jing-Ying Xu, Caixia Jin, Furong Gao, Juan Wang, Jieping Zhang, Jingfa Zhang, Weiye Li, Lixia Lu, Guo-Tong Xu; A cell culture condition that induces the mesenchymal-epithelial transition of dedifferentiated porcine retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3093.
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© ARVO (1962-2015); The Authors (2016-present)
The pathological change of retinal pigment epithelial (RPE) cells is one of the main reasons for the development of retinal degenerative diseases (RD). Thus, cultured RPE cells are proper cell model to study the etiology of RD in vitro. However, such cultured RPE cells easily undergo epithelial-mesenchymal transition (EMT) that results in changes of morphological characteristics and functions of the cells. Thus a new cell culture condition was created to restore and maintain mesenchymal-epithelial transition (MET) of the cultured RPE cells
We cultivated dedifferentiated porcine RPE (pRPE) cells and compared their behaviors in four conditions: 1) in cell culture dish with DMEM/F12 containing FBS (CC dish-FBS), 2) in petri dish with DMEM/F12 containing FBS (Petri dish-FBS), 3) in cell culture dish with DMEM/F12 containing N2 and B27 supplements (CC dish-N2B27), and 4) in petri dish with DMEM/F12 containing N2 and B27 (Petri dish-N2B27). In addition to observe the cell morphology and behavior, RPE specific markers, as well as EMT related genes and proteins, were examined by immunostaining, Quantitative real-time PCR and Western blot.
The results showed that dedifferentiated pRPE cells maintained EMT in CC dish-FBS, Petri dish-FBS and CC dish-N2B27 groups, whereas MET was induced only when the dedifferentiated pRPE cells were cultured in Petri dish-N2B27. Such induced pRPE cells showed polygonal morphology with increased expression of RPE specific markers and decreased EMT associated markers. Furthermore, during the re-differentiation of those dedifferentiated pRPE cells, Petri dish-N2B27 induced F-actin rearrangement, which promoted nuclear exclusion of transcriptional co-activator with PDZ-binding motif (TAZ), an EMT inducing factor. As a result, the expression of TAZ target molecule zinc finger E-box binding protein (ZEB-1), another EMT inducing factor, was reduced.
This study provides a simple and reliable method to reverse dedifferentiated pRPE cells into epithelialized phenotype, which is more appropriate for studying RD in vitro, and suggests that MET of other cell types might be induced by a similar approach.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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