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Gaëtan Le-Bel, Karine Zaniolo, Francis Bisson, Sylvain Guérin, Lucie Germain; Preservation of the stem cell population in tissue-engineered human corneas. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4342.
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In order to maintain a good transparency, the stratified epithelium of the tissue-engineered cornea yielded by the limbic stem cells must renew itself on a regular basis. It has been shown that monitoring the expression of ubiquitous transcription factors such as Sp1 and NFI that play critical functions in cell proliferation and migration, may be of a considerable interest in order to assess the quality of the epithelial stem cells population that are to be selected for the production of tissue engineered corneas. In the present study, we compared the efficiency of different feeder layers (murine or human) to maintain the proliferative properties of primary cultures of human corneal epithelial stem cells in vitro.
A human corneal stem cells population (HCSCs; n = 1) was grown as a monolayer in the presence of irradiated fibroblasts of either mouse (i3T3) or human (irradiated Human Feeder Layer or iHFL) origin. The cells were amplified through several passages until they reached a high rate of replicative senescence. The morphological and growth rate characteristics were evaluated at each cell passage. Both the expression and DNA binding properties of Sp1 and NFI were assessed using Western blot and electrophoretic mobility shift assays, respectively. Gene expression of Sp1 and NFI was also monitored by RT-qPCR.
HCSCs grown in the presence of iHFL as a feeder layer could be maintained until passage 8 before they reached terminal differentiation whereas they stopped proliferating at passage 6 when grown in the presence of i3T3. No difference could be observed in the amount and DNA binding activity of Sp1 between the different conditions selected. On the other hand, HCSCs have an increased NFI binding activity and also express an additional band for NFI in Western blot when they were grown in the presence of i3T3. RT-qPCR analyses revealed a strong increase in the mRNA level of the NFIX isoform when HCSCs are cultured with an i3T3 but not an iHFL feeder layer.
The feeder layer made from irradiated human fibroblasts appears to be the most effective for the maintenance of the HCSCs proliferative capacity. This might be related to the fact that expression of one of the NFI isoforms is considerably reduced in cells grown in the presence of iHFL as this transcription factor is well known to function as efficiently as a repressor than an activator of gene transcription.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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