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C.-Y. Chang, C. R. Green, C. N. J. McGhee, T. Sherwin; Is the Limbus Really the Niche for Human Corneal Stem Cells?. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1766.
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The limbus of the cornea is said to be the niche for limbal stem cells (LSCs) and the primary source of corneal epithelial maintenance. In this model, adult corneal epithelial cells are maintained by LSCs that cycle slowly and give rise to transient amplifying (TA) cells. These migrate centripetally, differentiate outwards to the surface, and are then lost by desquamation. However, we have shown that central human epithelial cells are capable of corneal regeneration after wounding (IOVS. 2008 May 30 [Epub ahead of print] PMID: 18515566 ) andit has been reported that oligopotent stem cells are distributed across the entire animal cornea (Nature. 2008 Nov 13;456(7219):250-4.). In this study, we investigated whether central epithelial cells in human corneas have limbal stem cell properties.
Human corneal epithelial cells were separated from the central cornea and the limbus by scraping in conjunction with three consecutive enzyme treatments (dispase II 40min, dispase II 20min, collagenase type II and hyaluronidase 4h at 37°C). Cells were collected between each step and cultured to form holoclones. Hoechst 33342 dye was used for FACS sorting and side population (SP) cells isolated based upon cell size and Hoeschst dye efflux ability via ABCG2 transporters.
Human limbal epithelial cells and central epithelial cells were both capable of forming holoclones although fewer were derived from central epithelium overall (C:L ratio =2:5). In FACS, central SP and limbal SP cells showed no significant difference based upon size and dye efflux.
Both limbal and central epithelial cells are capable of holoclone formation in culture. Central and limbal epithelial cells cannot be differentiated using FACS. Thus, on the basis of putative stem cell identifiers, FACS SP separation and propensity to form holoclones in culture, both central and limbal human corneal epithelium contain stem cells.
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