Gene expression changes associated with endothelial cell loss during corneal organ culture

Abstract

Abstract Purpose: Prior to transplantation corneas are stored in organ culture for up 4 weeks. However, up to 30% of corneas are discarded, mainly due to a poor endothelial cell count. In this study we have investigated the genes expressed during the process using micro array technology. Methods: Human corneas where stored in organ culture and the endothelial cell numbers where assessed after 10 days. RNA was extracted from 3 corneas with a low and 3 with a high cell count. The RNA samples were converted to cDNA using a Poly A PCR amplification protocol. These were used to probe Affymetrix whole genome micro array chips. Results: Results showed over 1000 genes are either up or down regulated by the endothelial cells of corneas with a low cell count compared to corneas with high counts. As expected there are genes involved in apoptosis, cell signalling, cell cycle, extracellular matrix, cell adhesion and cytoskeleton genes. However, of more interest are the genes involved in water channels and transport. For example we have identified several aquaporin genes (AQP) which are integral membrane proteins that serve as channels in the transfer of water across the membrane. AQP1 is increased and AQP7 is decreased in the corneas with low cell counts. It is likely that the changes in expression of these genes further increase the swelling of the corneas in culture and cause damage to the endothelium leading to higher levels of cell death in the affected corneas. Conclusions: These results add to our understanding of the effects of cell culture conditions on the corneal endothelium. A better understanding of endothelial cell loss in culture would lead to improved culture protocols and increase the number of corneas available for transplantation.