Reversible immortalization: towards an unlimited supply of primary human corneal endothelial cells to cure a blinding disease.

The corneal endothelium is the most inner layer of the human cornea, the window of the human eye. This cell layer is of utmost importance, since it functions to maintain corneal transparency. Upon damage to the endothelium, corneal oedema ensues and subsequently, visual impairment. The only solution for these patients is to transplant a new, functional corneal endothelium sourced from a donor eye. However, on a worldwide scale there are not enough donor corneas to meet for the patients in need.Not only does this shortage result in long waiting lists, but this donor scarcity also hinders corneal endothelial research. The limited tissue supply make research time-consuming, but also the corneal endothelial cells are very difficult to bring in culture. That is why until this very day the pathophysiology of corneal endothelial diseases remains to be elucidated. In this project I propose the reversible immortalization of corneal endothelial cells to overcome both the worldwide donor shortage and the intricate ex vivo growth. The process of reversible immortalization consists of four different steps: 1) insertion of removable genes to stimulate proliferation, (2) positive selection of transfected cells, (3) gene excision and (4) an additional negative selection to eliminate any residual engineered cells. In this small research project, I will investigate to establish the first three steps using either a lentiviral vector or a transposon system. The final aim is to effectively use the transposon system, while lentiviruses are part of this application as a positive control, since protocols and results are found throughout the literature, but are scarce for PiggyBac. However, the PiggyBac displays the intrinsic advantage such as a finger-print free excision of a transgene.Upon success, we can easily create vast amounts of corneal endothelial cells in our laboratory. These cells can will feed into our established projects of developing a corneal endothelial cell therapy or the development of a so-called cornea-on-a-chip.

Keywords:TRANSFECTION, OPHTHALMOLOGY, REGENERATION

Disciplines:Ophthalmology