Elucidating the corneal endothelial cell proliferation capacity through an interspecies transcriptome comparison

The regenerative capacity of corneal endothelial cells (CECs) differs between species; in bigger mammals such as humans and sheep, CECs are arrested in a non-proliferative state. Damage to these cells can compromise its function leading to corneal opacity and impaired vision. Corneal transplantation is the current treatment for the recovery of a clear eyesight, but the donor tissue demand is higher than the offer and there is the need to develop novel treatment modalities. In contrast, rabbit CECs retain a high proliferative profile and have the capacity to repopulate the endothelium upon injury. There is currently a lack of fundamental knowledge to explain the difference in the proliferation capacity of these cells across species. Gaining information on the transcriptomic differences across species could allow the identification of crucial proliferation drivers of CEC proliferation to develop novel regenerative medicine therapies. In this study we have analyzed at the transcriptomic level corneal endothelial samples originating from human, sheep, and rabbit. To understand the differences across the CECs proliferation capacity in each species, we have generated an automated pipeline for the analysis of pathways with different activity. Our results revealed that 52 pathways had commonly different activity when comparing species with non-proliferative endothelium (human and sheep) to species with proliferative endothelium (rabbit). Overall design: This study presents an interspecies transcriptome comparison of CECs originating from species with proliferative (rabbit) and quiescent endothelium (sheep and human). This study was performed in compliance with the tenets of the Declaration of Helsinki. All human research-grade corneal tissue (n=5) was obtained from the ETB-BISLIFE Multi-Tissue Center (Beverwijk, the Netherlands), with informed consent from the next of kin. All human corneas were stored up to 22 days in organ culture media at 31 °C and had a minimum endothelial cell density of 2300 cells/mm2. Organ culture media comprised the following: minimum essential medium (MEM) supplemented with 20 mM HEPES, 26 mM sodium bicarbonate, 2% (v/v) newborn calf serum (Thermo Fisher Scientific), 10 IU/mL penicillin, 0.1 mg/mL streptomycin and 0.25 µg/mL amphotericin. All eyes from rabbits (n=5) and sheep (n=5) were enucleated from the animals used for other experimental procedures approved by the regulatory authorities of Maastricht University, Utrecht University, and MSD and performed in accordance with Dutch national and European guidelines. All animal ocular tissue was used within 4 h after sacrifice. All donor and animal tissue information is specified in Table 1.