Each day, about 2000 U.S. workers have a job-related eye injury requiring medical treatment. Corneal diseases are the fifth cause of blindness worldwide. Most of these diseases can be cured using one form or another of corneal transplantation, which is the most successful transplantation in humans. In 2012, it was estimated that 12.7 million people were waiting for a corneal transplantation worldwide. Unfortunately, only 1 in 70 patients received a corneal graft that same year. In order to provide alternatives to the shortage of graftable corneas, considerable progress has been achieved in the development of living corneal substitutes produced by tissue engineering and designed to mimic their in vivo counterpart in terms of cell phenotype and tissue architecture. Most of these substitutes use synthetic biomaterials combined with immortalized cells, which makes them dissimilar from the native cornea. However, studies have emerged that describe the production of tridimensional (3D) tissue-engineered corneas using untransformed human corneal epithelial cells grown on a totally natural stroma synthesized by living corneal fibroblasts, that also show appropriate histology and expression of both extracellular matrix (ECM) components and integrins. This review highlights contributions from laboratories working on the production of human tissue-engineered corneas (hTECs) as future substitutes for grafting purposes. It overviews alternative models to the grafting of cadaveric corneas where cell organization is provided by the substrate, and then focuses on their 3D counterparts that are closer to the native human corneal architecture because of their tissue development and cell arrangement properties. These completely biological hTECs are therefore very promising as models that may help understand many aspects of the molecular and cellular mechanistic response of the cornea toward different types of diseases or wounds, as well as assist in the development of novel drugs that might be promising for therapeutic purposes.

Purpose In this study, we evaluated the feasibility of recovering the corneal surface integrity in a patient suffering from unilateral LSCD through the transplantation of cultured autologous corneal epithelial cells. Methods Human corneal epithelial cells (HCECs) were isolated from a limbal biopsy of the contralateral eye of a patient with unilateral LSCD and cultured in monolayer in the presence of an irradiated human fibroblasts feeder layer (iHFL). To produce a cultured autologous corneal epithelium (CACE), HCECs were seeded on a fibrin substrate and maintained in culture until confluence. The in vitro obtained CACE was then used to treat the affected eye of the patient. Two years later, a successful penetrating keratoplasty was performed. Results Efficient restoration of the corneal epithelium was achieved following transplantation of CACE indicating probable re-colonization of the cornea by stem cells. Corneal transparency was restored after removing the scarred stroma by performing a penetrating keratoplasty. Conclusion CACE produced in vitro was shown to restore a normal corneal surface capable of sustaining a viable and clear penetrating keratoplasty and reestablished a near normal vision in a unilateral LSCD patient.

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.

Damage to limbal epithelial stem cells can lead to limbal stem cell deficiency (LSCD). Current autologous treatment procedures for unilateral LSCD bear a significant risk of inducing LSCD in the donor eye. This complication can be avoided by grafting a stem cell containing cultured autologous corneal epithelium (CACE). The primary objective of this study was to demonstrate the safety of CACE grafted on eyes with LSCD. The secondary objective was to assess the efficacy of a CACE graft in restoring a self-renewing corneal surface with adequate anatomic structures, as well as improving the best corrected visual acuity (BCVA). Fifteen patients were grafted with a CACE on a fibrin gel produced from a 3 mm2 limbal biopsy harvested from the donor eye. Data were collected at baseline and after grafting. Follow-ups from 1 to 5 years were conducted. No major adverse events related to the CACE graft were observed. For every visit, an anatomic score based on corneal opacity as well as central vascularization and a functional score based on BCVA were determined. Safety was demonstrated by the low occurrence of complications. Anatomical (93%) and functional (47%) results are promising for improving vision in LSCD patients. Combined functional success and partial success rates with inclusion of BCVA were 53% [CI95: 27–79%] one year after CACE grafting. At the last follow-up, 87% [CI95: 60–98%] of the patients had attained corneal clarity. The outcomes demonstrate the safety of our technique and are promising regarding the efficacy of CACE in these patients.