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Gaëtan Le-Bel, Francis Bisson, Sylvain Guérin, Lucie Germain; Contribution of the feeder layer type to the preservation of stem cells in cultured human corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3869. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Because of the worldwide shortage of graftable corneas that are to be used to restore visual impairments, alternative solutions, such as the production of a functional human cornea by tissue-engineering, have emerged. Self-renewal of the corneal epithelium through the maintenance of a sub-population of corneal stem cells is required to maintain the functionality of such a reconstructed cornea. We previously reported an association between stem cell differentiation and the level to which they express the transcription factors Sp1 and NFI. Our goal is to characterize the impact of different feeder layers on the proliferative properties of human cornea epithelial cells (HCECs) in monolayer cultures.
HCECs were isolated from the corneal limbus of human donor’s eyes and grown in monolayer alone (negative control), or with either irradiated murine (i3T3) or human (iHFL) fibroblasts as feeder layers. The cells were amplified on several passages until they reached replicative senescence. Growth rate analyses were performed at each passage. The expression and DNA binding properties of Sp1 and NFI were determined by microarray, Western Blot and gel shift analyses. Influences of feeder layers on Sp1 and NFI half-life were analysed by immunoprecipitation and by treatment of HCECs with cycloheximide. The impact of the feeder layers on the pattern of genes expressed by HCECs cultured at different passages (P3, P5 and P7) was also assessed by microarrays.
HCECs co-cultured with iHFL were maintained for two additional passages in culture relative to HCECs grown with i3T3. No difference was observed in the amount and DNA binding activity of Sp1 between the different conditions. Expression and DNA binding of NFI were increased at each subsequent passages when co-cultured along with i3T3. These changes correlated with increased expression of the NFIA and NFIB isoforms that are less important in HCECs grown with iHFL. Furthermore, we observed an increased expression of genes whose encoded promote HCECs differentiation along several passages in HCECs co-cultured with i3T3.
Our results suggest that the iHFL feeder layer is the most effective in maintaining the proliferative abilities of HCECs in culture. This could be explained by the decreased expression of NFIB, which is known for its gene repressor properties.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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