Inhibition of TGF-β Signaling Enables Human Corneal Endothelial Cell Expansion In Vitro for Use in Regenerative Medicine

Naoki Okumura,Eunduck P Kay,Makiko Nakahara,Noriko Koizumi,Shigeru Kinoshita,Junji Hamuro,Che John Connon

doi:10.1371/journal.pone.0058000

Naoki Okumura, Eunduck P Kay + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0058000

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Abstract

Corneal endothelial dysfunctions occurring in patients with Fuchs' endothelial corneal dystrophy, pseudoexfoliation syndrome, corneal endotheliitis, and surgically induced corneal endothelial damage cause blindness due to the loss of endothelial function that maintains corneal transparency. Transplantation of cultivated corneal endothelial cells (CECs) has been researched to repair endothelial dysfunction in animal models, though the in vitro expansion of human CECs (HCECs) is a pivotal practical issue. In this study we established an optimum condition for the cultivation of HCECs. When exposed to culture conditions, both primate and human CECs showed two distinct phenotypes: contact-inhibited polygonal monolayer and fibroblastic phenotypes. The use of SB431542, a selective inhibitor of the transforming growth factor-beta (TGF-β) receptor, counteracted the fibroblastic phenotypes to the normal contact-inhibited monolayer, and these polygonal cells maintained endothelial physiological functions. Expression of ZO-1 and Na+/K+-ATPase maintained their subcellular localization at the plasma membrane. Furthermore, expression of type I collagen and fibronectin was greatly reduced. This present study may prove to be the substantial protocol to provide the efficient in vitro expansion of HCECs with an inhibitor to the TGF-β receptor, and may ultimately provide clinicians with a new therapeutic modality in regenerative medicine for the treatment of corneal endothelial dysfunctions.

Highlights

Corneal endothelial dysfunction is a major cause of severe visual impairment leading to blindness due to the loss of endothelial function that maintains corneal transparency
In our effort to overcome those substrate-related problems, we previously demonstrated that the transplantation of cultivated corneal endothelial cells (CECs) in combination with a Rho kinase (ROCK) inhibitor enhanced the adhesion of injected cells onto the recipient corneal tissue without the use of a substrate and successfully achieved the recovery of corneal transparency in two cornealendothelial-dysfunction animal models [10,11]
Though corneal transplantation is widely performed for corneal endothelial dysfunction, researchers are currently seeking alternative methods to restore healthy corneal endothelium

Summary

Introduction

Corneal endothelial dysfunction is a major cause of severe visual impairment leading to blindness due to the loss of endothelial function that maintains corneal transparency. Since corneal endothelium is composed of a monolayer and is a structurally flexible cell sheet, corneal endothelial cells (CECs) have been cultured on substrates including collagen sheets, amniotic membrane, or human corneal stroma. The cultured CECs are transplanted as a cell sheet These techniques require the use of an artificial or biological substrate that may introduce several problems such as substrate transparency, detachment of the cell sheet from the cornea, and technical difficulty of transplantation into the anterior chamber. In our effort to overcome those substrate-related problems, we previously demonstrated that the transplantation of cultivated CECs in combination with a Rho kinase (ROCK) inhibitor enhanced the adhesion of injected cells onto the recipient corneal tissue without the use of a substrate and successfully achieved the recovery of corneal transparency in two cornealendothelial-dysfunction animal models (rabbit and primate) [10,11]

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