Improvement of limbal and corneal phenotype after transplantation of human adipose tissue-derived mesenchymal stem cells in an in vivo model of ocular surface failure

Abstract

Purpose: In order to restore the ocular surface in cases of ocular surface failure due to limbal stem cell deficiency (LSCD), an extraocular source of stem cells is needed to avoid dependence on limbal stem cells which are difficult to obtain, isolate, and culture. Recent evidence indicates that mesenchymal stem cells (MSC) can play a beneficial role in the restoration of the ocular surface phenotype in LSCD. The aim of this work was to test the role of human adipose tissue-derived MSC (hAT-MSC) to restore corneal epithelial phenotype in ocular surface failure due to LSCD in rabbits.

Methods: After corneal epithelial debridement, a surgical 3600 limbal peritomy was performed in 12 rabbits. Three weeks later, 2.5x10⁵ hAT-MSC, labeled with bromodeoxyuridine (BrdU) and seeded onto amniotic membrane, were transplanted onto the ocular surface of 6 rabbits. The remaining 6 rabbits were left untreated as controls. Histopathology analyses at the end of the follow-up (11 weeks) evaluated the presence of inflammation and goblet cells (as a sign of conjunctival in-growth) in the limbus and central cornea. Immunofluorescence studies were done to locate hAT-MSC (BrdU) and to analyze the expression of corneal (CK3, E-cadherin) and limbal (CK15, p63) epithelial cell specific markers.

Results: Goblet cells were observed in the limbal epithelium of both non-transplanted and transplanted eyes. There was less presence of inflammatory cells in transplanted eyes in comparison with non-transplanted eyes. Corneal stroma showed a more disorganized structure in the non-transplanted group. hAT-MSC labeled with BrdU were located in inflamed areas of the limbal stroma. The expression of CK3, E-cadherin, CK15 and p63 markers was lost in untreated rabbits, whereas CK3 and E-cadherin expression increased in the limbal and corneal epithelium of hAT-MSC transplanted group. Additionally, the expression of CK15 and p63 was partially restored in the basal layers of the limbal epithelium after the hAT-MSC transplantation.

Conclusions: These results demonstrate that hAT-MSC can migrate to inflamed areas of the ocular surface, and partially restore limbal and corneal epithelial phenotype in a rabbit model of ocular surface failure due to total LSCD.