title: A simple and scalable process for the differentiation of retinal pigment epithelium from human pluripotent stem cells.
abstract: Age-related macular degeneration (AMD), the leading cause of irreversible vision loss and blindness among the elderly in industrialized countries, is associated with the dysfunction and death of the retinal pigment epithelial (RPE) cells. As a result, there has been significant interest in developing RPE culture systems both to study AMD disease mechanisms and to provide substrate for possible cell-based therapies. Because of their indefinite self-renewal, human pluripotent stem cells (hPSCs) have the potential to provide an unlimited supply of RPE-like cells. However, most protocols developed to date for deriving RPE cells from hPSCs involve time- and labor-consuming manual steps, which hinder their use in biomedical applications requiring large amounts of differentiated cells. Here, we describe a simple and scalable protocol for the generation of RPE cells from hPSCs that is less labor-intensive. After amplification by clonal propagation using a myosin inhibitor, differentiation was induced in monolayers of hPSCs, and the resulting RPE cells were purified by two rounds of whole-dish single-cell passage. This approach yields highly pure populations of functional hPSC-derived RPE cells that display many characteristics of native RPE cells, including proper pigmentation and morphology, cell type-specific marker expression, polarized membrane and vascular endothelial growth factor secretion, and phagocytic activity. This work represents a step toward mass production of RPE cells from hPSCs.
authors.affiliation: Wilmer Eye Institute.
authors.forename: Julien
authors.initials: J
authors.lastname: Maruotti
authors.forename: Karl
authors.initials: K
authors.lastname: Wahlin
authors.forename: David
authors.initials: D
authors.lastname: Gorrell
authors.forename: Imran
authors.initials: I
authors.lastname: Bhutto
authors.forename: Gerard
authors.initials: G
authors.lastname: Lutty
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Zack
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D016764:Cell Polarity / Q000235:genetics; D002469:Cell Separation; D048430:Cell Shape; D002470:Cell Survival; D002999:Clone Cells; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D007621:Karyotyping; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D010587:Phagocytosis; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000378:metabolism; D033581:Stem Cell Transplantation; D013997:Time Factors; D014158:Transcription, Genetic; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D015415:Biomarkers; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A
keywords: Defined; Pluripotent stem cells; Retinal pigment epithelium; Serum-free