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Abstract
Age-related macular degeneration (AMD) is a highly prevalent form of blindness caused by loss death of cells of the retinal pigment epithelium (RPE). Transplantation of pluripotent stem cell (PSC)-derived RPE cells is considered a promising therapy to regenerate cell function and vision.ObjectiveThe objective of this study is to develop a rapid directed differentiation method for production of RPE cells from PSC which is rapid, efficient, and fully defined and produces cells suitable for clinical use.DesignA protocol for cell growth and differentiation from hESCs was developed to induce differentiation through screening small molecules which regulated a primary stage of differentiation to the eyefield progenitor, and then, a subsequent set of molecules to drive differentiation to RPE cells. Methods for cell plating and maintenance have been optimized to give a homogeneous population of cells in a short 14-day period, followed by a procedure to support maturation of cell function.ResultsWe show here the efficient production of RPE cells from human embryonic stem cells (hESCs) using small molecules in a feeder-free system using xeno-free/defined medium. Flow cytometry at day 14 showed ~ 90% of cells expressed the RPE markers MITF and PMEL17. Temporal gene analysis confirmed differentiation through defined cell intermediates. Mature hESC-RPE cell monolayers exhibited key morphological, molecular, and functional characteristics of the endogenous RPE.ConclusionThis study identifies a novel cell differentiation process for rapid and efficient production of retinal RPE cells directly from hESCs. The described protocol has utility for clinical-grade cell production for human therapy to treat AMD.
Highlights
Age-related macular degeneration (AMD) is the most common cause of blindness in people aged over 60 years and affects approximately 200 million people worldwide [1]
Differentiation of Human embryonic stem cells (hESC) to hESC-retinal pigment epithelium (RPE) cells Initially, RPE cell differentiation from the MEL-1 hESC line was confirmed using protocols adapted from previous studies
These data indicate that the MEL-1 line is not defective for RPE differentiation using established methods [31] and that differentiation can be enhanced by early exposure to NIC [25]
Summary
Age-related macular degeneration (AMD) is the most common cause of blindness in people aged over 60 years and affects approximately 200 million people worldwide [1]. In advanced stages of AMD, death and/or dysfunction of RPE cells in the macula trigger photoreceptor degeneration, resulting in loss of central vision [5]. Early-stage AMD presents as drusen deposits underneath the RPE, which advances to one of two major forms: wet and dry AMD [6]. Wet AMD accounts for Limnios et al Stem Cell Research & Therapy (2021) 12:248. Anti-VEGF therapy can delay the advancement of wet AMD, 10–15% of patients is non-responsive or develops tachyphylaxis [9]. Dry AMD accounts for 90% of AMD cases and is associated with geographic atrophy of the RPE and retinal degeneration in the macula [10]. There is currently no effective treatment for dry AMD; disease onset can be delayed by dietary supplementation [11]
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