June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Pluripotency marker TRA-1-60 is expressed in stromal, perivascular and intraepithelial cells at the human limbus
Author Affiliations & Notes
  • Friedrich Kruse
    Department of Ophthalmology, University of Erlangen Nurnberg, Erlangen, Germany
  • Naresh Polisetti
    Department of Ophthalmology, University of Erlangen Nurnberg, Erlangen, Germany
  • Johannes Menzel-Severing
    Department of Ophthalmology, University of Erlangen Nurnberg, Erlangen, Germany
  • Ursula Schlotzer-Schrehardt
    Department of Ophthalmology, University of Erlangen Nurnberg, Erlangen, Germany
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3225. doi:
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      Friedrich Kruse, Naresh Polisetti, Johannes Menzel-Severing, Ursula Schlotzer-Schrehardt; Pluripotency marker TRA-1-60 is expressed in stromal, perivascular and intraepithelial cells at the human limbus. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3225.

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

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Abstract

Purpose: The proteoglycans TRA-1-60 and TRA-1-81, which are expressed on the surface of human embryonic stem cells, are widely used in human stem cell research as positive indicators of true pluripotency. To evaluate the presence of pluripotent cells at the human limbus, we mapped the expression of TRA-1-60 in corneo-limbal tissue specimens in situ.

Methods: Expression of the TRA-1-60 antigen was investigated in corneo-limbal specimens from 10 human donor eyes using light and electron microscopic immunohistochemistry. TRA-1-60 mRNA expression was analyzed after laser capture microdissection of basal limbal epithelial cell clusters and limbal stromal cells, respectively, and pre-amplification of RNA by real-time PCR. In addition, antibodies against putative stem/progenitor markers, including ABCG2, p63alpha, cytokeratin 15, N-Cadherin, Sox-9, and Oct-4, as well as against laminin-1, Melan A, CD11c, and CD45 were used in double labelling experiments.

Results: The TRA-1-60 epitope was abundantly expressed on the surface of a subset of mesenchymal cells in the anterior limbal stroma displaying a well-defined demarcation to the peripheral corneal stroma. The TRA-1-60 positive cells cumulated adjacent to limbal basal epithelial cell clusters expressing putative stem/progenitor markers and established intimate contact with epithelial cells by numerous cell processes. In addition, few TRA-1-60 positive cells were found to be integrated within the basal epithelial layer overlying the epithelial basement membrane. Dual staining showed that all TRA-1-60 positive cells co-expressed vimentin, but were negative for Melan A, CD11c, and CD45 excluding their nature as intraepithelial melanocytes, dendritic cells or lymphocytes. Moreover, TRA-1-60 staining was also associated with perivascular cells of anteriormost limbal blood vessels. Real-time PCR confirmed differential expression of TRA-1-60 in limbal stromal cells compared to corneal stromal cells.

Conclusions: The unequivocal presence of pluripotent cells within subepithelial stroma, blood vessel walls, and basal epithelium at the human limbus provides new insights into the constitution of the limbal niche and its interaction with limbal epithelial stem/progenitor cells. These cells may have a great potential in corneal regeneration and wound healing.

Keywords: 480 cornea: basic science • 482 cornea: epithelium • 721 stem cells  
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