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Akshayalakshmi Sridhar, Melissa Steward, Manav Gupta, Jason Meyer; Identification of transcriptional regulators underlying retinal fate determination from human induced pluripotent stem cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2209. doi: https://doi.org/.
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The establishment of a definitive retinal fate from a more primitive neural progenitor source occurs early in development and thus, efforts to study the initial stages of human retinal development have been largely limited. Human induced pluripotent stem cells (hiPSCs) allow for the unique ability to study the earliest events in human retinogenesis in vitro. To examine this potential, studies were undertaken to investigate the roles of critical transcription factors in the specification of a definitive retinal fate.
hiPSCs were directed to differentiate to a neural and retinal lineage following previously established methods and expression patterns of genes essential to retinal development were characterized using immunocytochemistry and qRT-PCR. Fluorescence-activated cell sorting (FACS) was utilized to quantify the percentage of cells expressing candidate transcription factors. The roles of these transcription factors in retinal specification were further investigated through up- and down-regulation of gene expression.
Immunocytochemistry and qRT-PCR experiments revealed differential patterns of gene expression among retinal and non-retinal cell populations. While many transcription factors maintained high levels of expression over the first 20 days of differentiation, others displayed expression patterns that suggested a more influential role in retinal fate determination. RAX and SIX6 were identified as candidate transcription factors to promote retinal differentiation, as the expression of RAX peaked early and then became restricted to a subpopulation, as determined by qRT-PCR and FACS. Additionally, the expression of SIX6 was not apparent until later and was exclusively restricted to a subpopulation of cells. The necessity of these genes was then tested through up- and down-regulation of gene expression.
The results of this study help to elucidate transcriptional mechanisms of the earliest stages of retinal fate determination, identifying RAX and SIX6 as candidate transcription factors underlying definitive retinal fate determination. Such information will assist in the establishment of more efficient methods to generate retinal cells from hiPSCs for translational purposes, as well as establish hiPSCs as a unique in vitro model system for studies of the earliest stages of human development.
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