Abstract
We report directed differentiaion of retinal precursors in vitro from mouse ES cells. Six3+ rostral brain progenitors are generated by culturing ES cells under serum-free suspension conditions (SFEB culture) in the presence of Wnt and Nodal antagonists (Dkk1 and LeftyA), and subsequently steered to differentiate into Rx+ cells (16%) by treatment with activin and serum. Consistent with the characteristics of early neural retinal precursors, the induced Rx+ cells coexpress Pax6 and the mitotic marker Ki67, but not Nestin. The ES cell-derived precursors efficiently generate cells with the photoreceptor phenotype (rhodopsin+, recoverin+) when cocultured with embryonic retinal cells. Furthermore, organotypic culture studies demonstrate the selective integration and survival of ES cell-derived cells with the photoreceptor phenotype (marker expression and morphology) in the outer nuclear layer of the retina. Taken together, ES cells treated with SFEB/Dkk1/LeftyA/serum/activin generate neural retinal precursors, which have the competence of photoreceptor differentiation.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Activins / pharmacology
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Animals
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Cell Differentiation / drug effects*
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Cells, Cultured
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Culture Media, Serum-Free / pharmacology
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Eye Proteins / metabolism*
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Genetic Vectors
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Homeobox Protein SIX3
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Homeodomain Proteins / metabolism*
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Immunohistochemistry
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In Situ Hybridization
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Intercellular Signaling Peptides and Proteins / pharmacology
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Left-Right Determination Factors
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Lentivirus
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Mice
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Nerve Tissue Proteins / metabolism
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PAX6 Transcription Factor
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Paired Box Transcription Factors / metabolism*
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Repressor Proteins / metabolism*
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Retina / cytology*
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Retina / metabolism
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Rhodopsin / metabolism
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Serum
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Stem Cells / cytology*
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Stem Cells / drug effects
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Stem Cells / metabolism
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Transforming Growth Factor beta / pharmacology
Substances
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Culture Media, Serum-Free
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Dkk1 protein, mouse
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Eye Proteins
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Homeodomain Proteins
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Intercellular Signaling Peptides and Proteins
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Left-Right Determination Factors
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Nerve Tissue Proteins
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PAX6 Transcription Factor
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Paired Box Transcription Factors
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Pax6 protein, mouse
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Repressor Proteins
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Rx protein, mouse
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Transforming Growth Factor beta
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Activins
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Rhodopsin