April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Epigenetic and Transcriptional Regulation of RAX in Retinal Fate Determination using Human Induced Pluripotent Stem Cells
Author Affiliations & Notes
  • Akshayalakshmi Sridhar
    Biology, Indiana Univ Purdue Univ Indianapolis, Indianapolis, IN
  • Sarah Ohlemacher
    Biology, Indiana Univ Purdue Univ Indianapolis, Indianapolis, IN
  • Jason S Meyer
    Biology, Indiana Univ Purdue Univ Indianapolis, Indianapolis, IN
    Stark Neurosciences, Indianapolis, IN
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 1366. doi:
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      Akshayalakshmi Sridhar, Sarah Ohlemacher, Jason S Meyer; Epigenetic and Transcriptional Regulation of RAX in Retinal Fate Determination using Human Induced Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1366.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Human pluripotent stem cells (hPSCs) allow for the unprecedented ability to study the earliest events in human retinal fate determination in vitro. Previous studies have been successful in the derivation of retinal cell types from hPSCs, however the mechanism of retinal specification from an unspecified pluripotent population remains to be clearly defined. Thus, efforts were undertaken to better elucidate the transcriptional and epigenetic mechanisms underlying retinal fate specification from hPSCs.

Methods: Following the differentiation of hPSCs to neural and retinal lineages, expression patterns of candidate transcription factors were characterized using immunocytochemistry and qRT-PCR. Based on these studies, the transcription factor RAX (retinal and anterior neural fold homeobox) was identified as influential in retinal fate due to its early widespread expression followed by restricted expression in retinal progenitor cells. To further investigate the role of RAX, gene overexpression and shRNA mediated knockdown approaches were undertaken. Furthermore, methylation analyses of CpG islands in the RAX promoter region were investigated at various stages of retinal development to better elucidate epigenetic modifications associated with early retinal fate determination.

Results: Analyses by immunocytochemistry and qRT-PCR demonstrated that the expression of RAX peaked early and then became restricted to a subpopulation of cells, specifically retinal progenitor populations identified by subsequent transcriptional analysis. Additionally, the expression of RAX was subsequently lost from non-retinal neural populations. Methylation analysis of the RAX promoter region suggested that epigenetic mechanisms could be regulating the expression of RAX in the establishment of a retinal fate. The ability of RAX to regulate the adoption of a retinal fate was subsequently tested through lentiviral-mediated overexpression and shRNA experiments.

Conclusions: Overall, the results of this study help to elucidate the role of RAX in the establishment of a retinal fate. These studies will assist in the establishment of more efficient methods to generate retinal cells from hPSCs for translational purposes, and serve to further establish hPSCs as an important in vitro model system for studies of the earliest stages of human retinal development.

Keywords: 721 stem cells • 698 retinal development • 497 development  
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