Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells

Murat T. Budak, Onder S. Alpdogan, Mingyuan Zhou, Robert M. Lavker, M. A.Murat Akinci, J. Mario Wolosin*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

186 Scopus citations

Abstract

When cell populations are incubated with the DNA-binding dye Hoechst 33342 and subjected to flow cytometry analysis for Hoechst 33342 emissions, active efflux of the dye by the ABCG2/BCRP1 transporter causes certain cells to appear as a segregated cohort, known as a side population (SP). Stem cells from several tissues have been shown to possess the SP phenotype. As the lack of specific surface markers has hindered the isolation and subsequent biochemical characterization of epithelial stem cells this study sought to determine the existence of SP cells and expression of ABCG2 in the epithelia of the ocular surface and evaluate whether such SP cells had features associated with epithelial stem cells. Human and rabbit limbal-corneal and conjunctival epithelial cells were incubated with Hoechst 33342, and analyzed and sorted by flow cytometry. Sorted cells were subjected to several tests to determine whether the isolated SP cells displayed features consistent with the stem cell phenotype. Side populations amounting to <1 % of total cells, which were sensitive to the ABCG2-inhibitor fumitremorgin C, were found in the conjunctival and limbal epithelia, but were absent from the stem cell-free corneal epithelium. Immunohistochemistry was used to establish the spatial expression pattern of ABCG2. The antigen was detected in clusters of conjunctival and limbal epithelia basal cells but was not present in the corneal epithelium. SP cells were characterized by extremely low light side scattering and contained a high percentage of cells that: showed slow cycling prior to tissue collection; exhibited an initial delay in proliferation after culturing; and displayed clonogenic capacity and resistance to phorbol-induced differentiation; all features that are consistent with a stem cell phenotype.

Original languageEnglish (US)
Pages (from-to)1715-1724
Number of pages10
JournalJournal of cell science
Volume118
Issue number8
DOIs
StatePublished - Apr 15 2005

Keywords

  • Conjunctiva
  • Cornea
  • Epithelia
  • Limbus
  • Side population
  • Stem cells

ASJC Scopus subject areas

  • Cell Biology

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