SummaryIn this study we dissected retinal organoid morphogenesis in human embryonic stem cell (hESC)-derived cultures and established a convenient method for isolating large quantities of retinal organoids for modeling human retinal development and disease. Epithelialized cysts were generated via floating culture of clumps of Matrigel/hESCs. Upon spontaneous attachment and spreading of the cysts, patterned retinal monolayers with tight junctions formed. Dispase-mediated detachment of the monolayers and subsequent floating culture led to self-formation of retinal organoids comprising patterned neuroretina, ciliary margin, and retinal pigment epithelium. Intercellular adhesion-dependent cell survival and ROCK-regulated actomyosin-driven forces are required for the self-organization. Our data supports a hypothesis that newly specified neuroretina progenitors form characteristic structures in equilibrium through minimization of cell surface tension. In long-term culture, the retinal organoids autonomously generated stratified retinal tissues, including photoreceptors with ultrastructure of outer segments. Our system requires minimal manual manipulation, has been validated in two lines of human pluripotent stem cells, and provides insight into optic cup invagination in vivo.
During embryonic development, the fibroblast growth factor (FGF) 3 signal transduction pathway regulates a range of cellular processes including cell proliferation, survival, migration, and differentiation (1). The mammalian FGF signaling is mediated by the interaction of specific secreted FGFs (i.e. FGF1 to FGF10) that work in conjunction with a specialized class of transmembrane receptor tyrosine kinases, the FGF receptors (FGFR1 to FGFR4). Formation of a complex between the dimeric FGFR and its FGF ligand dimer triggers a cascade of intracellular processes relayed by mitogen-activated kinases (MAPKs) such as Erk1 (official gene name: Mapk3) and Erk2 (Mapk1), PI-3/Akt kinase system, and other kinases. Upon entering the nucleus, Erk1/2 kinases elicit transcription of specific DNA-binding transcription factors and/or their post-translational modifications. While the majority of FGF signaling output includes activation of cell proliferation, survival, and motility, FGF signaling also regulates lens, myoblast, and osteogenic terminal differentiation (1, 2).The ocular lens has served as an advantageous model for studies of FGF signaling over many years (2). Primary rodent lens cell culture experiments showed that addition of a "high" concentration of bFGF/FGF2 (40 ng/ml) alone induced lens fiber cell terminal differentiation while "low" (0.15 ng/ml) and moderate (3 ng/ml) concentrations control cell survival and migration, respectively (3-5). FGF signaling is also modulated by the lens capsule, an extracellular matrix serving as an interface between the lens, aqueous and vitreous humor (6, 7). Subsequent genetic studies of FGF receptors (8, 9), components of the Frs2␣/Ras/MAPK signaling arm (10 -13), and the cooperating heparan sulfate biosynthesis pathway (14, 15) demonstrated in vivo roles of FGF signaling in mouse lens fiber cell survival and differentiation, and identified a set of lens regulatory genes, including c-Maf, Prox1, Etv1 (ER81), and Etv5 (ERM), whose expression was attenuated following genetic disruption of the FGF signaling pathway (9,14,15).Among these factors, Etv1 and Etv5 are well-established nuclear components of FGF signaling during neural development (16). The bZIP nuclear oncogene c-Maf encodes an important DNA-binding transcription factor that controls lens fiber cell differentiation through crystallin target genes (17). In addition to the lens, c-Maf regulates T-cell (18) and chondrocyte differentiation (19). Up-regulation of MAF was found in multiple myeloma cells and is a potential therapeutic target to treat this cancer (20). Therefore, a thorough understanding of c-Maf transcriptional control relates not only to the basic question of embryonic development but also for dysregulated gene expression during oncogenesis.
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