Purchase this article with an account.
Djida Ghoubay, Otman Asndali, Pablo L. Goldschmidt, Christine Chaumeil, Laurent Laroche, vincent Borderie; Kinetic Assessment Of Limbal Epithelial Progenitor Cultures Explanted From Human Donors. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3500.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To assess growth, maintenance and survival of potential stem limbal epithelial progenitor cultures obtained from human corneal explants.
Limbal epithelial cells were cultured from 2-mm2 human limbal explants (1/explant per dish) in cholera-toxin-free medium with and without murine cell feeders. Growth kinetics was followed by phase contrast microscopy after 9, 11, 14, 18, and 21 days. The differentiation marker expression (CK3, CK19, and vimentin) and the progenitor ("stem like") markers (p63, ABCG2) were detected by immunofluorescence. RNA messengers were identified and quantified by reverse transcription followed by polymerase chain reaction. The ability of murine 3T3 feeders (reference) and human corneal stromal feeders to produce clones of progenitors (colony forming efficiency) was compared.
The mean number of cultured cells obtained after 7, 14, and 21 days was, respectively, 500, 60489, and 195949. Broad spectrum cytokeratins, CK3, CK19, vimentin, p63, and ABCG-2 were expressed at all culture times, both by immunofluorescence and RT-PCR. When limbal epithelial cells were cultured with murine 3T3 irradiated feeders the number of colonies was significantly higher, with a peak at 11 days. Colonies were observed at 9, 11, 14 and 18 of culture but not at 21 days, both with murine and human feeders.
Compared to murine 3T3 fibroblasts, the human corneal stromal cells was less effective to promote limbal epithelial progenitor cell expansion. Moreover, the results obtained in the present cell-growth kinetic follow-up show that the viability and replication capacities of the epithelial progenitors after 2 weeks of culture are higher than after 3 weeks.
This PDF is available to Subscribers Only