Differentiation of Retinal Pigment Epithelium (RPE) and Retinal Organoids from Human iPSCs

In recent decades, human-induced pluripotent stem cell (iPSC) technology has revolutionized in vitro disease modeling and personalized medicine, enabling the generation of patient-specific "disease-in-a-dish" systems across a wide range of tissues, including retinal pigment epithelium (RPE) and retinal organoids. Numerous protocols have been developed for iPSC-RPE and retinal organoid differentiation, typically involving prolonged culture durations, low overall efficiency, and reliance on multiple extrinsic signaling factors. Here we describe an RPE differentiation protocol over 6 weeks, incorporating three additional factors, followed by pigment isolation to isolate differentiated RPE cells for subsequent maturation. After 4 weeks of maturation, the resulting monolayer is well polarized, exhibiting hexagonal cobblestone morphology, expressing canonical RPE markers. We also describe an effective of retinal organoid differentiation protocol, using nicotinamide and a stepwise reduction of KnockOut™ Serum Replacement (KOSR) concentration over the first 9 days, that facilitates the initiation of retinal differentiation in challenging iPSC lines.