Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells.
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.
Budak
Murat T
MT
Department of Ophthalmology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.
Alpdogan
Onder S
OS
Zhou
Mingyuan
M
Lavker
Robert M
RM
Akinci
M A Murat
MA
Wolosin
J Mario
JM
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001562:Benzimidazoles; D015415:Biomarkers; D002454:Cell Differentiation / Q000502:physiology*; D019070:Cell Lineage / Q000502:physiology; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D003228:Conjunctiva / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D016850:Limbus Corneae / Q000166:cytology / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010641:Phenotype; D011817:Rabbits; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D001562:Benzimidazoles; D015415:Biomarkers; D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; C017807:bisbenzimide ethoxide trihydrochloride