Redefining Transplants: Bioengineering the Corneal Endothelium using Human Stem Cells & a Hydrogel Film.

Abstract

Purpose : In response to the global deficit in tissue for corneal transplantation, we intended to bioengineer the corneal endothelium. The challenge was sourcing human corneal endothelial cells (hCECs) due to their scarcity and developing a suitable biodegradable material to carry them into the eye.

Aiming to produce a reliable source of cells, we developed a protocol to generate induced pluripotent stem cells (iPSCs)-derived corneal endothelial-like cells (iCECs) within 10 days. We further passaged them onto different biomaterials to study their maturation and to create an artificial graft.

Methods : We cultured iPSCs (P12-15(n=4)) and iCECs (n=4) on tissue culture plastic (TCP) plates coated with either vitronectin-N (VTN) or laminin-511 (L511), RGD-functionalized PEG-based hydrogel films (PHFs) and a PET-permeable membrane (PPM). We employed E8-Flex, basal induction and human endothelium (M5) media. These were supplemented with small molecules at different stages of development. Cultures were characterized by immunocytochemistry and RT-qPCR using a selected range of pluripotency, neural and hCEC markers. iPSCs before seeding and hCECs from 3 donors were used as controls. Statistical analysis was performed using nested ANOVA in biological and technical triplicates (ns=p>0.05).

Results : By day 10, characterization with OCT4 and NANOG showed reduced pluripotency. Areas lacking Nestin were noted and the gene expression of NES and SOX10, neural markers, were comparable to hCECs in both types of coating (p>0.6). The expression of ZO1 was consistently present, assisting in tracking morphological shifts. L511-coated cultures exhibited organoid characteristics and smaller polygonal cells. Corneal markers CDH2 and GRIP1 showed similar expression to hCECs in VTN-coated cultures (p>0.07).

Passaging iCECs onto TCP further reduced the expression of OCT4, NES and SOX10, comparable to hCECs (p>0.2); the opposite was found in PHFs and PPMs (p<0.001). Finally, integrating iCECs onto PHFs, thus bioengineering a corneal endothelium, exhibited similar gene expression profiles (PAX6, PITX2, COL8A1, AQP1 and MRGPRX3) to hCECs (p>0.05).

Conclusions : In light of the global shortage of corneal tissue, our induced corneal endothelial cells present a promising alternative for artificial grafts and cell injection therapies, potentially revolutionizing treatments to restore vision in patients with corneal endothelial disease.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.