Cultivation of Corneal Endothelial Cells on a Pericellular Matrix Prepared from Human Decidua-Derived Mesenchymal Cells

Ryohei Numata,Daisuke Kanematsu,Yonehiro Kanemura,Naoki Okumura,Morio Ueno,Makiko Nakahara,Shigeru Kinoshita,Yoshiki Sasai,Noriko Koizumi,Che John Connon

doi:10.1371/journal.pone.0088169

Abstract

The barrier and pump functions of the corneal endothelium are essential for the maintenance of corneal transparency. Although corneal transplantation is the only current therapy for treating corneal endothelial dysfunction, the potential of tissue-engineering techniques to provide highly efficient and less invasive therapy in comparison to corneal transplantation has been highly anticipated. However, culturing human corneal endothelial cells (HCECs) is technically difficult, and there is no established culture protocol. The aim of this study was to investigate the feasibility of using a pericellular matrix prepared from human decidua-derived mesenchymal cells (PCM-DM) as an animal-free substrate for HCEC culture for future clinical applications. PCM-DM enhanced the adhesion of monkey CECs (MCECs) via integrin, promoted cell proliferation, and suppressed apoptosis. The HCECs cultured on the PCM-DM showed a hexagonal morphology and a staining profile characteristic of Na+/K+-ATPase and ZO-1 at the plasma membrane in vivo, whereas the control HCECs showed a fibroblastic phenotype. The cell density of the cultured HCECs on the PCM-DM was significantly higher than that of the control cells. These results indicate that PCM-DM provides a feasible xeno-free matrix substrate and that it offers a viable in vitro expansion protocol for HCECs while maintaining cellular functions for use as a subsequent clinical intervention for tissue-engineered based therapy of corneal endothelial dysfunction.

Highlights

The corneal endothelium is the inner layer of the cornea, and it plays an essential role in the maintenance of corneal transparency via its barrier and pump functions [1]
The monkey CECs (MCECs) cultured on the pericellular matrix of decidua-derived mesenchymal cells (PCM-DM) showed lamellipodia and stress fibers, whereas the control MCECs cultured on the noncoated dish exhibited less formation of lamellipodia and stress fibers (Fig. 2A)
We demonstrated that PCM-DM provides a xeno-free culture substrate for efficient culture of human corneal endothelial cells (HCECs)

Summary

Introduction

The corneal endothelium is the inner layer of the cornea, and it plays an essential role in the maintenance of corneal transparency via its barrier and pump functions [1]. A distinct feature of human corneal endothelial cells (HCECs) in the clinical setting is that they are essentially nonregenerative in vivo [2]. Severe damage of CECs due to Fuchs’ corneal endothelial dystrophy, trauma, or surgical intervention causes corneal blindness associated with decompensation of the barrier and pump functions of the corneal endothelium [2]. Researchers have used tissue engineering-based techniques to transplant cultured CECs in animal corneal endothelial dysfunction models and to resolve corneal transparency [8,9,10,11,12,13]. Coincident to other organs, such as heart [14], pancreas [15], cartilage [16], and corneal epithelium [17], regenerative therapy for corneal endothelium is expected to be introduced in clinical settings

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