title: Characterisation of human limbal side population cells isolated using an optimised protocol from an immortalised epithelial cell line and primary limbal cultures.
abstract: The challenges in limbal stem cell biology largely remain in the process of identification, isolation and expansion of these adult corneal epithelial stem cells of the eye. Due to the absence of specific limbal stem cell markers, identification and isolation of putative limbal stem cells is a complicated task. The side population assay is an isolation method that utilises the ability of stem cells to efflux the DNA-binding dye Hoechst 33342 (or other vital dyes) combined with dual wavelength flow cytometry and is a valuable strategy to enrich for limbal stem cells. This assay has been used to successfully identify stem/ progenitor cell populations in a variety of tissues and cell lines. Here we optimise this assay to identify SP cell populations in both primary human limbal epithelial cultures and in an established human corneal epithelial cell line. The limbal SP fraction showed higher expression of ATP-binding cassette sub-family G member 2 (ABCG2), ΔNp63--a common limbal stem cell marker and the stem cell marker Sox2 compared to non-SP cells (NSP).
authors.affiliation: Institute of Genetic Medicine, International Centre for Life, Newcastle University, Newcastle Upon Tyne, NE1 3BZ, UK, b.shaharuddin@ncl.ac.uk.
authors.forename: Bakiah
authors.initials: B
authors.lastname: Shaharuddin
authors.forename: Ian
authors.initials: I
authors.lastname: Harvey
authors.forename: Sajjad
authors.initials: S
authors.lastname: Ahmad
authors.forename: Simi
authors.initials: S
authors.lastname: Ali
authors.forename: Annette
authors.initials: A
authors.lastname: Meeson
mesh_terms: D002461:Cell Line, Transformed; D002469:Cell Separation; D004847:Epithelial Cells / Q000502:physiology; D019573:Epithelium, Corneal / Q000166:cytology; D005434:Flow Cytometry; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D016850:Limbus Corneae / Q000166:cytology*; D010641:Phenotype; D061251:Primary Cell Culture; D058985:Side-Population Cells / Q000187:drug effects / Q000502:physiology*; D014700:Verapamil / Q000494:pharmacology
chemical_list: D007211:Indoles; D014700:Verapamil; C010522:tryptoquivaline