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Bo Lu, Wei Hu, Xinxin Zhang, Na Yang, Neil Bressler, Jun Kong; Epithelial-Mesenchymal Transition of Retinal Pigment Epithelium from Different Sources in Vitro. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3009.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate features associated with initiation of mesenchymal changes when retinal pigment epithelium (RPE) cells are subcultivated serially in vitro and to compare how to change peculiarities of fetal RPE and RPE differentiated from human embryonic stem cells (hESCs-RPE) after they experience epithelial-mesenchymal transition (EMT).
fRPE and hESC-RPE cells are respectively subcultured in vitro and passaged by trypsin-EDTA in 20min or 10min. The changes in cell configuration are observed by inverted phase contrast microscope and hematoxylin-eosin staining. Real time quantification PCR (qPCR) western blot and immunofluorescent assays were employed to analyze changes in biomarkers (epithelial markers: RPE65, E-cadherin, ZO-1; mesenchymal markers are: alpha smooth muscle actin(α-SMA), N-cadherin, Fibronectin) of fetal RPE and hESCs9-RPE.
1. Normal fRPE and hESC-RPE cells presented a cobblestone appearance with pigment surrounding the nuclei. However, when RPE cells had abundant tonofilament, pigment sharply decreased or disappeared, which suggested mesenchymal changes. The proliferating ability of cells reduced when continuously passaged in vitro, especially in EMT transdifferentiated cells. 2. hESCs-RPE and fRPE passed with a concentration of 6x105 cells/ml and complete dissociation during passagingmight show EMT characteristics and express mesenchymal markers as early as in the fourth passage, while RPE cells passed with a concentration of 3x105 cells/ml or insufficient trypsinization time might transdifferentiate as early as in the first passage. 3. Results of qRT-PCR analysis showed increased a-SMA, N-cadherin, fibronectin mRNA expression and decreased RPE65, E-cadherin and ZO-1 mRNA expression (p < 0.05) after RPE experienced EMT. Western blot and immunoblotting confirmed the increased expression of EMT markers and decreased expression of epithelial markers at protein level. However, hESCs-RPE behaved similarly with EMT changes except E-cadherin expression increased.
These findings suggest that EMT change can occur when RPE from aborted fetuses and human embryonic stem cells are cultured in vitro. There are also differences in protein expression between fetal RPE and hESCs9-RPE. These data also suggest in future clinical RPE transplantation, one should consider the condition of RPE cells as well as their different sources.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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