title: Defined Medium Conditions for the Induction and Expansion of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium.
abstract: We demonstrate that a combination of Noggin, Dickkopf-1, Insulin Growth Factor 1 and basic Fibroblast Growth Factor, promotes the differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells. We describe an efficient one-step approach that allows the generation of RPE cells from both human embryonic stem cells and human induced pluripotent stem cells within 40-60 days without the need for manual excision, floating aggregates or imbedded cysts. Compared to methods that rely on spontaneous differentiation, our protocol results in faster differentiation into RPE cells. This pro-retinal culture medium promotes the growth of functional RPE cells that exhibit key characteristics of the RPE including pigmentation, polygonal morphology, expression of mature RPE markers, electrophysiological membrane potential and the ability to phagocytose photoreceptor outer segments. This protocol can be adapted for feeder, feeder-free and serum-free conditions. This method thereby provides a rapid and simplified production of RPE cells for downstream applications such as disease modelling and drug screening.
authors.affiliation: Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia.
authors.forename: Grace E
authors.initials: GE
authors.lastname: Lidgerwood
authors.affiliation: O'Brien Institute Department, St Vincent's Institute of Medical Research, Fitzroy, VIC, Australia.
authors.forename: Shiang Y
authors.initials: SY
authors.lastname: Lim
authors.affiliation: Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia.
authors.forename: Duncan E
authors.initials: DE
authors.lastname: Crombie
authors.affiliation: School of Medicine, Menzies Institute for Medical Research, University of Tasmania, TAS, Australia.
authors.forename: Ray
authors.initials: R
authors.lastname: Ali
authors.affiliation: Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia.
authors.forename: Katherine P
authors.initials: KP
authors.lastname: Gill
authors.affiliation: O'Brien Institute Department, St Vincent's Institute of Medical Research, Fitzroy, VIC, Australia.
authors.forename: Damián
authors.initials: D
authors.lastname: Hernández
authors.affiliation: Department of Anatomy and Neuroscience, University of Melbourne, Parkville, VIC, Australia.
authors.forename: Josh
authors.initials: J
authors.lastname: Kie
authors.affiliation: Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia.
authors.forename: Alison
authors.initials: A
authors.lastname: Conquest
authors.affiliation: Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia.
authors.forename: Hayley S
authors.initials: HS
authors.lastname: Waugh
authors.affiliation: Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia.
authors.forename: Raymond C B
authors.initials: RC
authors.lastname: Wong
authors.affiliation: Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia.
authors.forename: Helena H
authors.initials: HH
authors.lastname: Liang
authors.affiliation: Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia.|School of Medicine, Menzies Institute for Medical Research, University of Tasmania, TAS, Australia.
authors.forename: Alex W
authors.initials: AW
authors.lastname: Hewitt
authors.affiliation: Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia.|Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia.
authors.forename: Kathryn C
authors.initials: KC
authors.lastname: Davidson
authors.affiliation: Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia. apebay@unimelb.edu.au.
authors.forename: Alice
authors.identifier: 0000-0002-7408-9453
authors.initials: A
authors.lastname: Pébay
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D010587:Phagocytosis / Q000502:physiology; D010786:Photoreceptor Cells / Q000166:cytology / Q000378:metabolism; D010858:Pigmentation / Q000502:physiology; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D012160:Retina / Q000166:cytology* / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000378:metabolism
chemical_list: D017077:Culture Media, Conditioned; D016222:Fibroblast Growth Factor 2
keywords: Differentiation; Electrophysiology; Human embryonic stem cells; Human induced pluripotent stem cells; RNA sequencing; Retinal pigment epithelium