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J. S. Meyer, E. E. Capowski, K. A. Wallace, A. D. Verhoeven, A. V. Sloman, J. M. Martin, L. S. Wright, R. Stewart, J. A. Thomson, D. M. Gamm; Isolation, Differentiation and Developmental Modeling of Early Retinal Progenitor Cell Populations From Human Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1230.
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© ARVO (1962-2015); The Authors (2016-present)
Established methods for deriving early retinal progenitors from human pluripotent stem cells (hPSCs) typically yield a heterogeneous population of cells, which complicates studies of retinal development. Therefore, we sought to develop a simple method of isolating a highly enriched population of optic vesicle (OV) stage, multipotent retinal progenitor cells from human ES and iPS cells.
hPSCs were differentiated toward a retinal lineage using a previously described protocol. Highly enriched populations of OV stage retinal progenitors were manually separated from forebrain progenitor populations and allowed to differentiate for up to 120 days. Differences in gene expression between retinal and forebrain progenitor populations were determined via PCR and microarray analyses. Differentiating OV populations were then examined by qPCR and ICC to ascertain their ability to generate retinal cell types. Subsequently, selected inducing factors were added for discrete periods to study effects on cell fate choice.
Upon initial isolation, >90% of all OV stage hPSC populations expressed the definitive neural retinal progenitor marker Vsx2 (Chx10). Comparison of retinal and forebrain progenitor populations revealed key differences in the expression of numerous transcription factors, including Rx, Six6, Dlx1 and Nkx2.1. In vitro maturation of OV populations produced all major classes of retinal cell types in a manner reminiscent of normal development. Furthermore, treatment of hPSC-derived, multipotent early progenitors with factors known to influence retinal development affected cell fate choice in a predictable fashion.
Results from this study demonstrate that highly enriched populations of OV stage, multipotent retinal progenitors can be isolated from hPSCs. This capability will facilitate future studies of mechanisms of human retinogenesis and disease as well as efforts to develop hPSC-based therapies.
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