May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Localization of P–63 to Fluorescent GFP Expressing Cells in a Corneal Epithelial Cell Culture Derived from Cornea–scleral Rim Explants of GFP Transgenic Pig
Author Affiliations & Notes
  • N. Shamie
    Ophthalmology, University of California, Irvine, Irvine, CA
  • T.I. Almeda
    Ophthalmology, University of California, Irvine, Irvine, CA
  • P.H. Schwartz
    National Human Neural Stem Cell Resource,
    Childrens Hospital Orange County, Orange, CA
  • H.J. Klassen
    Stem Cell Research,
    Childrens Hospital Orange County, Orange, CA
  • M.C. Kenney
    Ophthalmology, University of California, Irvine, Irvine, CA
  • Footnotes
    Commercial Relationships  N. Shamie, None; T.I. Almeda, None; P.H. Schwartz, None; H.J. Klassen, None; M.C. Kenney, None.
  • Footnotes
    Support  none
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 1440. doi:
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      N. Shamie, T.I. Almeda, P.H. Schwartz, H.J. Klassen, M.C. Kenney; Localization of P–63 to Fluorescent GFP Expressing Cells in a Corneal Epithelial Cell Culture Derived from Cornea–scleral Rim Explants of GFP Transgenic Pig . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1440.

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

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Abstract

Abstract: : Purpose: To demonstrate colocalization of a putative stem cell marker (p–63) to green–fluorescent protein (GFP)–expressing cells cultured from cornea–scleral rims of a transgenic GFP–expressing pig model. This cell line can then be used as a donor for corneal limbal transplantation so that the innate GFP fluorescence can be used to follow the outcome of the graft. Methods: Cornea–scleral rims from midgestational transgenic GFP–expressing pigs were harvested. The cornea–scleral rims were cultured on collagen type IV coated cell culture dishes with serum–free keratinocyte medium supplemented with recombinant EGF, L–glutamine, and bovine pituitary extract. Cells were passaged with 0.2% trypsin at 3 days. Immunohistochemistry of the cultured monolayer of cells was performed using a monoclonal antibody to p–63, a presumed marker for corneal epithelial stem cells, and a rhodamine–conjugated secondary antibody. Negative controls were also carried out using only the secondary antibody. The cells were then veiwed using a Leica fluorescent microscope with a digital camera. The double labeled patterns of fluorescent/GFP and rhodamine/p–63 were examined. Results: A confluent monolayer of epithelial cells budding from the explant rim was obtained within three days in the above culture conditions. In the third passage of the cultured cells, every cell expressing the GFP had co–localization of the p–63 antibody. Conclusions: The innately fluorescent GFP–expressing, limbal–derived corneal epithelial cells cultured from transgenic pigs were also found to be positive for expression of p–63, a presumed marker for corneal epithelial stem cells. This model for co–expression of both fluorescent/GFP and rhodamine/p–63 can be used to track the fate of the epithelial stem cells after surgical implantation onto a limbal stem deficient cornea.

Keywords: cornea: basic science • cornea: epithelium 
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