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Developmental Biology

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Published: August 20th, 2016

DOI:

10.3791/54329

1Department of Ophthalmology, Far Eastern Memorial Hospital, 2Institute of Clinical Medicine, National Taiwan University, 3Department of Ophthalmology, National Taiwan University Hospital, 4College of Medicine, National Taiwan University

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of β-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

Corneal endothelial cells (CECs) play a vital role in maintaining corneal clarity and thus visual acuity by regulating the hydration status of the corneal stroma through active pumping1. Because of the limited proliferative potential of human CECs, the cell number decreases with age, and the repair of corneal endothelial wounds following injury is usually achieved through cell enlargement and migration, rather than cell mitosis2. When the CEC count decreases below a threshold of approximately 500 cells/mm2, the dehydration status of the corneal stroma cannot be maintained, leading to bullous keratopathy and vision impairment3,4

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All the procedures followed in this study accorded with the Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee of National Taiwan University Hospital.

1. Isolation, Primary Culture Preparation, and Immunostaining of Bovine CECs

  1. Acquire fresh bovine eyes from a local abattoir.
  2. Disinfect the eyes in a 10% w/v povidone-iodine solution for 3 min. Wash them with pho.......

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After the isolation of bovine CECs, the cells were cultured in vitro. Figure 1 presents the phase contrast images of the bovine CECs. The hexagonal shape of the cells at confluence indicated that the cells were not contaminated by corneal stromal fibroblast during cell isolation. Figure 2 depicts the immunostaining that was performed using antibodies against ABC, snail, and slug at an indicated time point. Apart from phenotypic changes in the

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CECs are known for their propensity to undergo EnMT during cell proliferation. To develop strategies for suppressing the EnMT process for therapeutic purposes, a thorough understanding of the EnMT mechanism is necessary. We described 2 models to investigate EnMT, namely the bovine CEC in vitro culture model and rat corneal endothelium cryoinjury model. Our results demonstrated the EnMT process in both models. Furthermore, the EnMT-suppressing effect of marimastat was reproduced in both models, suggesting that th.......

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We thank the staff of the Second Core Lab, Department of Medical Research, National Taiwan University Hospital for their technical support.

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Name Company Catalog Number Comments
trypsin ThermoFisher Scientific 12604-013
Dulbecco’s modified Eagle medium and Ham's F12 medium ThermoFisher Scientific 11330
fetal bovine serum ThermoFisher Scientific 26140-079
dimethyl sulfoxide Sigma D2650
human epidermal growth factor ThermoFisher Scientific PHG0311
insulin, transferrin, selenium  ThermoFisher Scientific 41400-045
cholera toxin Sigma C8052-1MG
gentamicin ThermoFisher Scientific 15750-060
amphotericin B ThermoFisher Scientific 15290-026
paraformaldehyde Electron Microscopy Sciences 111219
Triton X-100 Sigma T8787 
bovine serum albumin Sigma A7906
marimastat Sigma M2699-25MG
anti-active beta-catenin antibody Millpore 05-665
anti-snail antibody Santa cruz sc28199
anti-slug antibody Santa cruz sc15391
goat anti-mouse IgG (H+L) secondary antibody ThermoFisher Scientific A-11001 for staining of ABC of bovine CECs
goat anti-mouse IgG (H+L) secondary antibody ThermoFisher Scientific A-11003 for staining of ABC of rat corneal endothelium
goat anti-rabbit IgG (H+L) secondary antibody ThermoFisher Scientific A-11008 for staining of snail and slug of bovine CECs
antibody diluent Genemed Biotechnologies 10-0001
4',6-diamidino-2-phenylindole ThermoFisher Scientific D1306
mounting medium Vector Laboratories H-1000
laser scanning confocal microscope ZEISS LSM510
xylazine  Bayer N/A
tiletamine plus zolazepam Virbac N/A veterinary drug
proparacaine hydrochloride ophthalmic solution Alcon N/A veterinary drug
0.1% atropine Wu-Fu Laboratories Co., Ltd N/A clinical drug 
0.3% gentamicin sulfate Sinphar Group N/A clinical drug 
basic fibroblast growth factor ThermoFisher Scientific PHG0024 clinical drug 

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