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Akshayalakshmi Sridhar, Casey A Miller, Kimberly Ho-A-Lim, Jessica A Cooke, Sarah Ohlemacher, Jason S Meyer; Elucidating the role of RAX in human retinogenesis with pluripotent stem cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1135.
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© ARVO (1962-2015); The Authors (2016-present)
Human pluripotent stem cells (hPSCs) allow for the unprecedented ability to recapitulate the earliest stages of human retinogenesis, including the establishment of a definitive retinal fate from a more primitive neural progenitor source. Recent studies have clearly illustrated this ability, with hPSCs possessing the capacity to give rise to three-dimensional optic vesicle-like structures in vitro. In this role, hPSCs provide an attractive in vitro model for elucidating complex mechanisms of human retinogenesis in an easily accessible in vitro system.
hPSCs were directed to a retinal lineage as via previously described, resulting in three-dimensional optic vesicle-like structures. Initially, the expression patterns of critical eye field transcription factors were analyzed over a timecourse of differentiation via immunocytochemistry and quantitative RT-PCR. Lentiviral-mediated overexpression and shRNA knockdown approaches were also utilized to evaluate the role of critical transcription factors in retinal differentiation. Additionally, DNA methylation analyses were performed to identify epigenetic modifications associated with early retinal fate determination.
At early stages of differentiation, RAX was observed to be expressed broadly throughout most cells, followed by its restriction to a subpopulation of cells shortly thereafter. This restriction was closely associated with the onset of more definitive retinal transcription factors such as SIX6 and CHX10. Interestingly, the expression of RAX was also absent from any cells adopting a non-retinal forebrain phenotype, suggesting a critical role for RAX in retinal specification. Methylation analysis of the RAX promoter suggested that epigenetic mechanisms could potentially account for the restricted expression of RAX in early retinal differentiation. Overexpression and knockdown of RAX further demonstrated its requirement in the establishment of a retinal fate, as well as the specification of particular retinal cell types.
Overall, the results of this study help to demonstrate the suitability of hPSCs as an effective in vitro model of human retinogenesis, with RAX demonstrated to be critical for the initial establishment of a retinal fate as well as the specification of particular retinal cell types. Such results help to establish hPSCs as a valuable in vitro model system for studies of human retinogenesis.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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