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Florence Pecha, Djida Ghoubay, Laurent Laroche, Vincent Borderie; Cholera Toxin And Isoproterenol Effects On Limbal Epithelial Cells Culture. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5129.
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Limbal stem cells (LSCs) are responsible for maintaining the corneal epithelium throughout life. The LSCs deficiency (LSCD) is a disease characterized by the loss or dysfunction of LSCs. Ex vivo expansion of LSCs is an increasingly well recognized treatment modality for LSCD. LSCs are usually cultured with cholera toxin-supplemented basal medium.The aim of this project was to study the effect of cholera toxin and isoproterenol on limbal epithelial cell culture to select progenitor cells.
Human donor corneas discarded for transplantation due to low endothelial cell counts or corneoscleral rims obtained in the operating after 8-mm trephinations of donor corneas were used. Kinetics of growth, morphometry and the phenotype of cells in superficial limbal explants cultivated in cholera toxin -supplemented basal medium and isoproterenol-supplemented basal medium were study. The expression of epithelial and progenitor cell associated markers CK3, MNF116, CK19, delatNp63, and vimentin were carried out with Immunocytochemistry, Flow cytometry, and RT PCR.
Cell count number was higher with isoproterenol at 0,5 (p=0,049), 1 (p=0,005) and 2 µg/ml (p=0,008) than with cholera toxin supplementation. Analysis of cell morphology realized at day 14 showed smaller cells with isoproterenol at 0,5 (p=0,02), 1 (p=0,02), 2 (p=0,04) and 4µg/ml (p= 0,01). Cultured cells expressed cytokeratins which confirmed their epithelial phenotype. Positive immunostaining for vimentin, CK19, and deltaNp63 confirmed presence of progenitor cells.
Isoproterenol act on epithelial limbal cell primary culture and on cellular size at day 14 without alter the race of progenitor cells and differenciated cells. Isoproterenol supplementation enhances growth of epithelial limbal cells more efficiently than cholera toxin and has no xenobiotic risk.
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