Irradiated Human Fibroblasts as a Substitute Feeder Layer to Irradiated Mouse 3T3 for the Culture of Human Corneal Epithelial Cells: Impact on the Stability of the Transcription Factors Sp1 and NFI
| Autor: | Sergio Cortez Ghio, Louis-Philippe Guérin, Lucie Germain, Sylvain L. Guérin, Francis Bisson, Gaëtan Le-Bel |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Cellular differentiation human corneal epithelial cells Cycloheximide NFI Catalysis Article Sp1 Inorganic Chemistry 03 medical and health sciences chemistry.chemical_compound Mice 0302 clinical medicine Feeder Layer Tissue engineering Cornea medicine Animals Humans Physical and Theoretical Chemistry Molecular Biology Spectroscopy Corneal epithelium Cell Proliferation Tissue Engineering feeder layer Stem Cells Organic Chemistry Epithelium Corneal Feeder Cells Cell Differentiation Epithelial Cells General Medicine 3T3 Cells Fibroblasts Epithelium eye diseases Coculture Techniques Computer Science Applications Cell biology 030104 developmental biology medicine.anatomical_structure chemistry gene profiling 030221 ophthalmology & optometry sense organs Stem cell |
| Zdroj: | International Journal of Molecular Sciences Volume 20 Issue 24 |
| ISSN: | 1422-0067 |
| Popis: | Because of the worldwide shortage of graftable corneas, alternatives to restore visual impairments, 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. In this study, we investigated the impact of replacing irradiated 3T3 (i3T3) murine fibroblast feeder cells by irradiated human corneal fibroblasts (iHFL) on the expression of Sp1 and NFI and evaluated their contribution to the proliferative properties of human corneal epithelial cells (hCECs) in both monolayer cultures and human tissue engineered corneas (hTECs). hCECs co-cultured with iHFL could be maintained for up to two more passages than when they were grown with i3T3. Western Blot and electrophoretic mobility shift assays (EMSAs) revealed no significant difference in the feeder-layer dependent increase in Sp1 at both the protein and DNA binding level, respectively, between HCECs grown with either i3T3 or iHFL. On the other hand, a significant increase in the expression and DNA binding of NFI was observed at each subsequent passage when hCECs were co-cultured along with i3T3. These changes were found to result from an increased expression of the NFIA and NFIB isoforms in hCECs grown with i3T3. Exposure of hCECs to cycloheximide revealed an increased stability of NFIB that likely resulted from post-translational glycosylation of this protein when these cells were co-cultured with i3T3. In addition, iHFL were as efficient as i3T3 at preserving corneal, slow-cycling, epithelial stem cells in the basal epithelium of the reconstructed hTECs. Furthermore, we observed an increased expression of genes whose encoded products promote hCECs differentiation along several passages in hCECs co-cultured with either type of feeder layer. Therefore, the iHFL feeder layer appears to be the most effective at maintaining the proliferative properties of hCECs in culture most likely by preserving high levels of Sp1 and low levels of NFIB, which is known for its gene repressor and cell differentiation properties. |
| Databáze: | OpenAIRE |
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