Abstract
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.
Highlights
Because of the worldwide shortage of graftable corneas that are to be used to restore visual impairments, alternatives, such as the production of a functional human cornea by tissue engineering, have emerged
In order to ensure these biological processes, the engineered epithelium must harbor and sustain a sub-population of corneal stem cells in the basal cell layer. To preserve these epithelial cell’s properties, the culture conditions must allow for a good proliferation of the human corneal epithelial cells by delaying their terminal differentiation while ensuring long-term regeneration of the epithelium once grafted on a patient
In order to evaluate the impact of the feeder layer type on the morphology and growth characteristics of corneal epithelial cells, human corneal epithelial cells (hCECs) were cultured either alone or in the presence of a murine (i3T3) or human feeder layer (Figure 1)
Summary
Because of the worldwide shortage of graftable corneas that are to be used to restore visual impairments, alternatives, such as the production of a functional human cornea by tissue engineering, have emerged. In order to ensure these biological processes, the engineered epithelium must harbor and sustain a sub-population of corneal stem cells in the basal cell layer To preserve these epithelial cell’s properties, the culture conditions must allow for a good proliferation of the human corneal epithelial cells (hCECs) by delaying their terminal differentiation while ensuring long-term regeneration of the epithelium once grafted on a patient. 3T3 cells have an endogenous retrovirus containing a 3600-bp region of xenotropic murine leukemia virus-related virus (XMRV) which are associated with human prostate cancer and chronic fatigue syndrome [10] Because of all these evidences, the need for producing engineered tissues that use human rather that murine feeder cells emerged, especially when such tissues are to be used for grafting purposes. Our goal is to characterize the impact of different feeder layers on the proliferative properties of hCECs in monolayer cultures
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