Identification of Prdm genes in human corneal endothelium

Corneal endothelial cells (CECs) are essential for maintaining corneal stromal hydration and ensuring its transparency, which is necessary for normal vision. Dysfunction of CECs leads to stromal decompensation, loss of transparency and corneal blindness. Corneal endothelium has low proliferative potential compared to surface epithelial cells leading to poor regeneration of CEC following injury. Additionally, the tissue exhibits age related decline in endothelial cell density with re-organisation of the cell layer, but no regeneration. The mechanisms which control proliferation and differentiation of neural crest derived CEC progenitors are yet to be clearly elucidated. Prdm (Positive regulatory domain) family of transcriptional regulators and chromatin modifiers are important for driving differentiation of a variety of cellular types. Many Prdm proteins are expressed in specific precursor cell populations and are necessary for their progression to a fully differentiated phenotype. In the present work, we sought to identify members of the Prdm gene family which are specifically expressed in human (h) CECs with a view to begin addressing their potential roles in CEC biology, focussing especially on Prdm 4 and 5 genes. By performing semi-quantitative reverse transcription coupled to PCR amplification we found that in addition to Prdm4 and Prdm5, Prdm2 and Prdm10 genes are expressed in hCECs. We further found that cultured primary hCECs or immortalised HCEC-12 cells express all of the Prdm genes found in CECs, but also express additional Prdm transcripts. This difference is most pronounced between Prdm gene expression patterns of CECs isolated from healthy human corneas and immortalised HCEC-12 cells. We further investigated Prdm 4 and Prdm 5 protein expression in cultured primary hCECs and HCEC-12 cells as well as in a human cadaveric whole cornea. Both Prdm 4 and Prdm 5 are expressed in human corneal endothelium, primary hCECs and in HCECs- 12 cells, characterised by expression of the Naþ/Kþ-ATPase. We observed that both proteins exhibit cytosolic (intracellular, but non-nuclear and distinct from extracellular fluid) as well as nuclear localisation within the endothelial layer, with Prdm 5 being more concentrated in the nuclei of the endothelial cells than Prdm 4. Thus, our work identifies novel Prdm genes specifically expressed in corneal endothelial cells which may be important in the control of CEC differentiation and proliferation

Evaluation of corneal endothelial cell therapy using an in vitro human corneal model

Aim: To establish an in vitro human corneal decompensation model and to use it for the evaluation of a cell-therapy approach for treating corneal endothelial (CE) disorders and to test the expression profile of positive regulatory domain proteins (PRDMs) as potential markers for corneal endothelial cells (CECs). Materials and Methods: Human cadaveric corneas were obtained from Bristol and Manchester Eye Banks, UK. A CE decompensation model was established by removal of the Descemet’s membrane (DM)/Endothelium complex from donor corneas and placing them in air-interface organ culture. The corneal thickness was used as a surrogate measure of CE function and was measured using Optical Coherence Tomography (OCT). Decompensated corneas were subjected to cultured endothelial cell therapy using immortalized HCEC -12 cells (group 1), primary human corneal endothelial cells (hCECs) at 0 passage (group 2) and hCECs at passage 2 (group 3) with defined seeding cell density. The effect on stromal de-swelling in cell therapy treated corneas was assessed 3, 7 and 10 days post-transplantation followed by histological evaluation. In addition, expression of PRDM genes in the corneal endothelium was undertaken using reverse transcriptase polymerase chain reaction (RT-PCR), immunocytochemistry and immunohistochemistry. Results: Organ culture of human cadaveric corneas in air-interface following the selective removal of the DM/Endothelium complex resulted in stromal thickness of 903.6 ± 86.51 μm, whereas normal corneas maintained a physiological thickness of 557.51 ± 72.64 μm. When transplanted directly onto the posterior corneal stroma the human CECs were able to attach and achieved physiological corneal thickness of 458.91 ± 90.07 μm, 489.65 ± 94.62 μm and 613.7 ± 94.62 μm for cell therapy groups -1, -2 and -3 respectively. The study identified PRDMs 1, 2, 4, 5 and 10 in the human CE and revealed a differential expression between normal CE and cultured hCECs. Conclusion: Removal of the DM/Endothelium complex from cadaveric human corneas held in air interface organ culture resulted in corneal endothelial decompensation. Direct transplantation of cultured primary hCECs to bare posterior corneal stroma devoid of DM resulted in the formation of an endothelial monolayer and restoration of stromal hydration to physiological thickness, substantiating the role of cell therapy to treat corneal endothelial disorders. The identification of PRDM proteins in the human corneal endothelium paves the way for future studies to understand their role in hCEC proliferation control

Feasibility Study of Human Corneal Endothelial Cell Transplantation Using an In Vitro Human Corneal Model

Purpose: To test the feasibility of a cell-therapy approach to treat corneal endothelial (CE) disorders using an in vitro model of human corneal decompensation. Materials and Methods: A CE decompensation model was established by removal of the DM/Endothelium complex from human cadaveric corneas in an air-interface organ culture system (Group 2) and compared to normal corneas (Group 1). The posterior stroma of decompensated corneas was seeded with immortalized human corneal endothelial cells (HCEC-12) in Group 3 and passage 0 primary human corneal endothelial cells (hCECs) in Group 4 corneas. Functional effects on stromal thickness were undertaken with histological analysis 3-10 days post-cell therapy treatment. Results: Removal of the DM/Endothelium complex in Group 2 corneas resulted in a stromal thickness of 903 ± 86 μm at 12 hours in comparison to 557 ± 72 μm in Group 1 corneas. The stromal thickness reduced from 1218 ± 153 μm to 458 ± 90 μm (63 ± 6 %, p=0.001) post cell transplantation in Group 3) and from 1100 ± 86 μm to 489 ± 94 μm (55 ± 7 %, p=0.00004 in Group 4 respectively. Post-transplantation histology demonstrated the formation of a monolayer of corneal endothelium attached to the posterior stromal surface. Conclusion: Direct transplantation of cultured hCECs and immortalized HCEC-12 to bare posterior corneal stroma resulted in the formation of an endothelial monolayer and restoration of stromal hydration to physiological thickness, demonstrating the feasibility of cell therapy in the treatment of corneal endothelial decompensation in a human in vitro model

Experimental models of corneal endothelial cell therapy and translational challenges to clinical practice

The human corneal endothelium (CE) is a post-mitotic monolayer of endothelial cells, thought to be incapable of in vivo regeneration. Dysfunction of the CE is a commonly cited indication for corneal transplantation, with corneal blindness being the fifth most common cause of blindness globally. In 2012 alone 184,576 corneal transplants were performed in 116 countries (Gain et al., 2016). Presently, outcomes following human corneal transplantation have been reported to have over 97% success rate in restoring the recipient's vision (Patel et al., 2019). However, the continuing demand for cadaveric human corneas has driven research into alternative sources of CE and with the advent of protocols to produce cultured hCECs there is now the potential for cell therapy to regenerate the damaged CE. This review aims to examine the merits and limitations of different types of human and animal models used so far to test the concept of CE cell therapy