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

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.

Stem cells of the adult cornea: From cytometric markers to therapeutic applications

“…2A), and $10% of the limbal epithelial cells are stained. On the other hand, only about 3% of the cells appear ABCG2 positive when measured by flow cytometry after isolation of single limbal epithelial cells by dispase II-trypsin digestion of corneas (76,77). Both numbers are higher than the proportion of LESCs, which is estimated to be less than 1% based on the fraction of the side population.…”

“…Both numbers are higher than the proportion of LESCs, which is estimated to be less than 1% based on the fraction of the side population. These differences may be explained by cytoplasmic (nonfunctional) expression of ABCG2 in some limbal epithelial cells (77) and indicate that ABCG2 labeling with antibodies not only marks LESCs but possibly also some transient amplifying cells.…”

Niche regulation of corneal epithelial stem cells at the limbus

“…Interestingly, using the clonal culture system based on 3T3 fibroblast feeder layers, SP cells generate much less colonies than non-SP cells in freshly isolated limbal epithelial cells from both human [24] and rabbit [24,81,82]. These results prompt one to suspect whether expression of ABCG2 is a salient feature of limbal SCs as discussed above.…”

“…Using this method, SP cells have been isolated from human [23][24][25], rat [80], and rabbit [24,81,82] limbal tissues. The frequency of SP cells from the freshly isolated limbal epithelium varied from 0.2% to 0.64% in humans and from 0.4% to 1.21% in rabbits, while no SP cells were detected in human and rabbit central corneas.…”

“…In contrast, the SC-containing limbal epithelium has a high proliferative potential in different cultures [12][13][14][15], and their in vitro proliferation is resistant to the inhibition by tumor-promoting phorbol esters [13,16,17]. Furthermore, limbal basal epithelial cells express cytokeratin 19 [18], and integrin a9 [10,19], and preferentially express such progenitor markers as p63 [20], especially its ∆Np63a isoform [21,22], Bcrp1/ABCG2 [10,[23][24][25], and N-cadherin [26].…”