Mesenchymal Transition of Ex-vivo Expanded Human Corneal Endothelial Cells is reversible

Abstract

Purpose: Cultivation of pure human corneal endothelial cells (HCEC) is important for development of tissue engineered corneal endothelial layer for transplantation purposes. Studies have shown that ex vivo expansion of human corneal endothelial cells may undergo endothelial mesenchymal transition. The purpose of this study is to evaluate mesenchymal transition in ex vivo expanded human corneal endothelial cells under different conditions, developed at our department, and evaluate whether mesenchymal transition is reversible.

Methods: Descemet’s membrane with the attached endothelium was carefully dissected from human corneas in small strips. HCECs were expanded on plastic in our culture medium (CM) for 3 weeks at 37°C with 5% CO2 in a humidified atmosphere. Thereafter, HCECs were cultured in expansion medium (EM) for further 3 weeks. After ex vivo expansion of HCECs, cells were transferred on a biological carrier and incubated first in EM and then in stabilizing medium (SM) for 3 weeks in each step. HCECs were harvested at different stages and analyzed by light microscopy, qRT-PCR, immunohistochemistry, transmission and scanning electron microscopy (TEM and SEM).

Results: HCECs are mitotically stimulated in CM and show characteristic corneal endothelial cell morphology with low expansion potential. In EM, the cells proliferate fast and loose their typical endothelial morphology and to some extent show fibroblastic shape. After transferring of the expanded HCECs onto a biological carrier and further incubation in EM and SM, proliferation potential of cells were extremely reduced and typical corneal endothelial cell morphology recovered. qRT-PCR analysis of MKI67, CDH2, NES, SNAIL1, SNAIL2 , VADC2, VADC3, ZO1, GJA1 (Connexin 43), ATPase, CDK1, CDK2 and CDK5PAR genes, Immunohistochemistry of related mesenchymal transition proteins, TEM and SEM electron micrographs confirm this observation.

Conclusions: The preliminary results show that mesenchymal transition in ex vivo expanded human corneal endothelial cells is reversible.

Keywords: 481 cornea: endothelium • 480 cornea: basic science