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Akshayalakshmi Sridhar, Sarah Ohlemacher, Jason S Meyer; In vitro modeling of human retinogenesis with pluripotent stem cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3575.
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© ARVO (1962-2015); The Authors (2016-present)
Human pluripotent stem cells (hPSCs) provide a unique ability to study some of the earliest events of human development, particularly some of the earliest events in human retinogenesis such as the establishment of a definitive retinal fate from a more primitive neural progenitor source. In this role, hPSCs may provide an in vitro model for understanding the complex interplay of transcription factors involved in the acquisition of a retinal fate from an unspecified pluripotent cell population.
hPSCs were differentiated as previously described and samples were collected every two days, starting from the undifferentiated state through when cells acquired either retinal or non-retinal forebrain identities. Immunocytochemistry and qRT-PCR approaches were undertaken to identify candidate transcription involved in retinal fate establishment. Lentiviral-mediated overexpression and shRNA knockdown approaches assessed the role of candidate transcription factors in the specification of a retinal fate apart from other non-retinal neural fates. Furthermore, epigenetic approaches assessed the role of DNA methylation in retinal and forebrain fate determination.
Candidate transcription factors were identified underlying the establishment of a retinal fate apart from other neural lineages. Neural transcription factors including PAX6 and OTX2 were expressed early while retinal-associated transcription factors such as SIX6 were expressed at slightly later timepoints. Upon establishment of an anterior neural identity, expression patterns of certain transcription factors such as RAX became more restricted to subpopulations of cells, indicating the emergence of retinal and forebrain populations from the same primitive anterior neural population. Gene overexpression and knockdown experiments investigated the mechanism of action of these candidate transcription factors. Furthermore, epigenetic analysis demonstrated that DNA methylation could potentially account for differential gene expression in the establishment of retinal phenotypes apart from alternate neural lineages.
Preliminary results begin to elucidate the complex interplay of transcription factors involved in the specification of a retinal fate from differentiating hPSCs. Overall, these results will help to better establish hPSCs as a valuable in vitro system with which to study some of the earliest events of human retinogenesis.
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