Morphological and functional characterization of retinal pigment epithelium cells differentiated from stem cells for retinal therapeutic applications

Age-related macular degeneration (AMD) is a complex progressive retinal disease considered the leading cause of blindness among people over 65 years. It is characterized by the degeneration of retinal pigment epithelium (RPE) cells, resulting in photoreceptor loss and severe visual impairment. Two types of AMD have been identified: the first one is the dry-atrophic form, characterized by drusen and geographic atrophy, and the second is the wet-neovascular type, characterized by choroidal neovascularization. While some therapies have been developed for wet AMD, an efficient treatment is still missing for the more severe dry AMD. Since the RPE has limited regenerative capacity, there is an urgent need to develop new and innovative therapeutic strategies able to restore retina functionality. Stem cell-based therapy has emerged as a potential solution in overcoming the limitations of current treatments. Human embryonic stem cells (hESCs) could be differentiated into RPE cells and thus substitute the damaged cells of the patients. The following study aimed at obtaining RPE cells from hESC (hESC-RPE) and compare their phenotype with donor derived RPE cells using both marker characterization and functional assays. In details, the hESC WA09 cell line was used to differentiate RPE cells with a direct triphasic differentiation protocol, inducing in the first phases the neuroectoderm and RPE progenitor markers and finally seeding cells on appropriate substrates to enable their complete differentiation and the formation of a polarized RPE layer. Given the limited availability of comprehensive data in the literature, a protocol for the isolation of RPE cells from donor eyeballs was set up. Several approaches were tested, including both enzymatic and mechanical methods. Various seeding coatings were also evaluated, such as Matrigel and Synthemax together with the introduction of a sucrose gradient to isolate the cell clusters from single cells. To confirm the morphological, structural and functional features of hESC-RPE cells after differentiation, key RPE markers including bestrophin 1 (BEST1), retinoid isomerohydrolase 65 (RPE65), premelanosome protein (PMEL) and zonula occludens-1 (ZO-1) were tested by immunofluorescence, revealing the obtainment of fully differentiated RPE cells which also developed full pigmentation. In addition, transepithelial electrical resistance (TEER) was measured during hESC-RPE culture to evaluate tight junction formation and the integrity of the epithelium. Finally, the secretion of two growth factors was assessed by enzyme-linked immunosorbent assay (ELISA): vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), both critical for maintaining retinal homeostasis. The increase in TEER values above 400 Ω·cm², which represents the minimum TEER value to identify a functional epithelium, showed that hESC-derived RPE cells formed a polarized monolayer with intact tight junctions as early as the day 61 post seeding. The secretion of VEGF and PEDF further confirms the functional integrity of RPE, as these factors play essential roles in maintaining the retinal microenvironment, and their adequate secretion is critical for the long-term health of retinal tissues. In conclusion, this comparative study with donor derived primary RPE established the efficacy of stem cell differentiation to obtain RPE cells emulating native functions, thus opening new perspectives in RPE reconstruction as treatment strategy for AMD.