Denuded Descemet’s membrane supports human embryonic stem cell-derived retinal pigment epithelial cell culture

Abstract

Recent clinical studies suggest that retinal pigment epithelial (RPE) cell replacement therapy may preserve vision in retinal degenerative diseases. Scaffold-based methods are being tested in ongoing clinical trials for delivering pluripotent-derived RPE cells to the back of the eye. The aim of this study was to investigate human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells survival and behaviour on a decellularized Descemet’s Membrane (DM), which may be of clinical relevance in retinal transplantation. DMs were isolated from human donor corneas and treated with thermolysin. The DM surface topology and the efficiency of the denudation method were evaluated by atomic force microscope, scanning electron microscopy and histology. hESC-RPE cells were seeded onto the endothelial-side surface of decellularized DM in order to determine the potential of the membrane to support hESC-RPE cell culture, alongside maintaining their viability. Integrity of the hESC-RPE monolayer was assessed by measuring transepithelial resistance. RPE-specific gene expression and growth factors secretion were assessed to confirm maturation and functionality of the cells over the new substrate. Thermolysin treatment did not affect the integrity of the tissue, thus ensuring a reliable method to standardize the preparation of decellularized DM. 24 hours post-seeding, hESC-RPE cell attachment and initial proliferation rate over the denuded DM were higher than hESC-RPE cells cultured on tissue culture inserts. On the new matrix, hESC-RPE cells succeeded in forming an intact monolayer with mature tight junctions. The resulting cell culture showed characteristic RPE cell morphology and proper protein localization. Gene expression analysis and VEGF secretion demonstrate DM provides supportive scaffolding and inductive properties to enhance hESC-RPE cells maturation. Decellularized DM was shown to be capable of sustaining hESC-RPE cells culture, thus confirming to be potentially a suitable candidate for retinal cell therapy.

Advances in the study of tissue-engineered retinal pigment epithelial cell sheets

“… [ 37 ] Corneal Membrane Obtained by dissecting corneas of cattle/pigs Autologous RPE and IPE Monolayer cell patch was formed on Descement membrane Both cell types form a complete monolayer and microvilli structure on the membrane [ 38 ] Isolated from the donor human cornea and treated with thermolysin to remove the cells hESC-RPE hESC-RPE forms an intact monolayer on decellularized Descemet's membrane hESC-RPE cells successfully formed intact monolayers with mature tight junctions on decellularized Descemet membranes, displaying characteristic RPE cell morphology and protein localization, and gene expression analyses and VEGF secretion indicated that DM provided supportive scaffolding and inducible properties. [ 39 ] Lens Capsule The lens capsule was obtained by removal during cataract surgery, immersed in PBS at 37 ℃, UV-sterilized for 3 h, and subsequently digested with trypsin to remove cells. ARPE19 The cells are inoculated onto the capsular membrane of the lens to form a monolayer ARPE19 cells cultured on the lens capsule exhibited a monolayer with the presence of actin bands and tight junctions.…”

“…Elena Daniele et al. [ 39 ] demonstrated that hESC-RPE could also attach and form a complete monolayer structure on Descemet's membrane isolated from human donor corneas and decellularized. The cell morphology, protein localization, VEGF secretion, and gene expression patterns were comparable to those of mature RPE.…”

Biomaterials used for tissue engineering of barrier-forming cell monolayers in the eye

“…Descemet’s membrane is another natural substrate with features resembling Bruch’s membrane characteristics concerning the abundant presence of type IV collagen, laminin and fibronectin. Daniele et al (2023) used this membrane to produce a polarized monolayer of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells in vitro .…”