Restoration of the epithelial stem cell population by the grafting of a tissue-engineered human corneal epithelium: a case report

Abstract

Purpose : Limbal stem cell deficiency (LSCD) leads to conjunctivalization and opacification of the corneal surface. Replacement of the epithelium on the eye of a LSCD patient by an autologous human corneal epithelium produced in vitro by tissue-engineering represents an interesting option to prevent the loss of vision consecutive to LSCD. We tested the hypothesis that limbal stem cells are present in the basal layer of the tissue-engineered epithelium and can recolonize an empty limbus following grafting to a patient with LSCD.

Methods : Human corneal epithelial cells (HCECs) were grown as a monolayer in the presence of irradiated fibroblasts of either mouse (i3T3) or human (iHFL) origin and then seeded on tissue-engineered human corneal stromas to allow regeneration of the corneal epithelium. The reconstructed corneas were then exposed to bromodeoxyuridin (BrdU) followed by a chase period of 1, 7 and 21 days. Localization of stem cells was then assessed by indirect immunofluorescence targeting BrdU and Keratine 19 (K19). In addition, an autologous corneal epithelial graft was reconstructed using HCECs cultivated on iHFL from a biopsy of the contralateral eye of a patient with unilateral LSCD resulting from a post-shingles neurotrophic keratitis. The corneal graft was used to treat the affected eye.

Results : Twenty-one days after the BrdU pulse, highly positive BrdU cells expressing K19 were observed in the basal epithelial layer of the tissue-engineered epithelium indicating the presence of HCECs stem cells. The corneal epithelium rebuilt from HCECs grown with iHFL has a more important stem cell population relative to that produced using HCECs co-cultured with i3T3. Moreover, restoration of the corneal epithelium was attained following transplantation of the autologous corneal epithelial graft, and relieved the LSCD by recolonizing the stem cell niche (limbus) although a central penetrating keratoplasty had to be performed in order to replace the damaged stroma and to recover corneal transparency.

Conclusions : The tissue-engineered human corneal epithelium possesses a subpopulation of stem cells located in its basal layer that can, once grafted to an eye with LSCD, recolonize the limbus, thus providing permanent restoration of a self-renewing corneal epithelium and reestablishing a clearer vision to the patient.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.