April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Generation of purified human lens epithelial cells and identification of a novel lens transcription factor using pluripotent stem cells.
Author Affiliations & Notes
  • Michael O'Connor
    School of Medicine, University of Western Sydney, Penrith, NSW, Australia
    Molecular Medicine Research Group, University of Western Sydney, Campbelltown, NSW, Australia
  • Patricia Murphy
    School of Medicine, University of Western Sydney, Penrith, NSW, Australia
    Molecular Medicine Research Group, University of Western Sydney, Campbelltown, NSW, Australia
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5042. doi:
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      Michael O'Connor, Patricia Murphy; Generation of purified human lens epithelial cells and identification of a novel lens transcription factor using pluripotent stem cells.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5042.

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

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Abstract

Purpose: An inability to access large numbers of human lens cells has obstructed development of accommodation-retaining treatments for presbyopia and primary cataract, as well as high-throughput drug screening for primary and secondary cataract. We hypothesized that human pluripotent stem cells could be used to produce large numbers of purified human lens cells for developmental biology, drug discovery, and clinical applications.

Methods: We developed a novel culture method to produce purified human lens epithelial cells from pluripotent stem cells. Passaging of the lens cells produced sufficient numbers for a proof-of-principle anti-secondary cataract assay in multi-well format. We also investigated lens signaling via bioinformatics, PCR and Western blotting.

Results: Using a targeted culture approach we differentiated human pluripotent stem cells into purified lens epithelial cells that express typical lens cell markers and could be differentiated into lens fibre cells. Expansion of these epithelial cells allowed seeding in 96 well-plates, and exposure to control or H2O2 led to rapid and total cell death with H2O2 but not control treatment. Bioinformatics analysis predicted novel transcription factors to be involved in lens epithelial cell maintenance, with PCR and Western blotting confirming expression of at least one of these in human lens cells.

Conclusions: Previous work from our group (O’Connor and McAvoy, 2007) showed that 3-dimensional, focusing lenses can be regenerated in vitro from rat lens explants. These in vitro lenses ultimately developed a cataract similar to age-related human cataract. Together, this work suggested in vitro lens regeneration might be used to develop novel cataract treatments or anti-cataract drug screens. To humanize our rat lens regeneration system we have developed a method for large-scale production of purified human lens epithelial cells from pluripotent stem cells. These new human lens cells express key lens epithelial cell markers, can be differentiated into fibre cells, and enable anti-secondary cataract drug screening. These new human lens cells are now being used by our group to further investigate in vitro human lens regeneration, secondary cataract drug screening, the role of the novel lens transcription factor we identified, and potential accommodation-retaining treatments for primary cataract.

Keywords: 721 stem cells • 687 regeneration • 739 transcription factors  
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