CellSight Ocular Stem Cell and Regeneration Research Program, Department of Ophthalmology, Sue Anschutz-Rodgers Eye Center, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
Methods Mol Biol. 2025;2848:197-214. doi: 10.1007/978-1-0716-4087-6_13.
Retinal pigment epithelium (RPE) cells derived from induced pluripotent stem cells (iPSCs) serve multiple roles, including among others, modeling RPE development in normal and pathological conditions, investigating mechanisms of RPE physiology, modeling retinal diseases involving the RPE, and developing strategies for regenerative therapies. We have developed a simple and efficient protocol to generate RPE tissue from human iPSCs-derived retinal organoids. The RPE tissue present in the retinal organoids is analogous to the native human RPE in differentiation timeline, histological organization, and key features of functional maturation. Building upon this system, we established a method to generate functionally mature, polarized RPE monolayers comparable to human primary RPE. This comprehensive protocol outlines the steps for isolating and culturing RPE tissue using retinal organoids. The outcome is a pure population of cells expressing mature RPE signatures and organized in a characteristic cobblestone monolayer featuring robust ultrastructural polarization. These RPE monolayers also exhibit the functional hallmarks of bona fide mature RPE cells, providing a suitable system to mimic the biology and function of the native human RPE.
诱导多能干细胞（iPSC）衍生的视网膜色素上皮（RPE）细胞具有多种功能，包括模拟正常和病理条件下的 RPE 发育、研究 RPE 生理学机制、模拟涉及 RPE 的视网膜疾病，以及开发再生治疗策略。我们已经开发出一种简单高效的方案，可从人诱导多能干细胞衍生的视网膜类器官中生成 RPE 组织。这些视网膜类器官中的 RPE 组织在分化时间、组织学结构和功能成熟的关键特征上与天然人 RPE 相似。在此基础上，我们建立了一种生成功能成熟、极化的 RPE 单层的方法，与人类原代 RPE 相当。本综合方案概述了使用视网膜类器官分离和培养 RPE 组织的步骤。结果是获得了表达成熟 RPE 特征的纯细胞群体，并以特征性的鹅卵石状单层排列，具有稳健的超微结构极化。这些 RPE 单层还表现出真正成熟 RPE 细胞的功能特征，为模拟天然人 RPE 的生物学和功能提供了合适的系统。
