Ex vivo models of human ocular surface regeneration indicate that conjunctiva possess the capacity of restoring corneal epithelium

AbstractPurposeWe recently showed that corneas stored in the corneal bioreactor (BR) that restores IOP and storage medium renewal had increased endothelial viability and enzymatic pump expression, less stromal edema and posterior folds and more stratified and differentiated epithelium than paired corneas stored passively in the same medium.AimTo use this model to analyze the respective contribution of corneal(C), limbal(L) and conjunctival(Conj) epithelium in regeneration of corneal epithelium.MethodsFive pairs of fresh human corneas (body donation to Sciences, death to retrieval time <20 hr) were used. During retrieval, a rim of conjunctiva was left intact. Five models were then built by totally mechanically removing 1, 2 or 3 epithelial population(s): C‐L+Conj+, C‐L‐Conj+, C‐L+Conj‐, C+L‐Conj‐, C‐L‐Conj‐ (control). Corneas were then stored for 1 month in the BR (21 mmHg, 2.5 µl/min of CorneaMax, Eurobio) to allow epithelial regrowth. The epithelia were then examined by immunostaining of flat mounted whole cornea using cytokeratine CK12 (corneal epithelium), CK15 (limbal epithelium) and Hoechst (all cells nuclei). Transparency was assessed by a contrast transmission analyzer.ResultsNo epithelium regenerated in the controls. In the 4 other models, the cornea was covered by a multilayered corneal epithelium, even in the C‐L‐Conj+, characterized by expression of K3/K12 and a transparent tissue.ConclusionsThe 3 ocular surface epithelia (comprising the conjunctival one) contain stem‐cells or progenitors able to migrate onto the whole cornea and regenerate corneal epithelium independently of each other. In this ex vivo model of corneas deprived of blood supply and innervation, the neovascularization cannot develop. This suggests that the corneal conjunctivalization characteristic of limbal stem cell deficiency is not due to the absence of a progenitor capable of transdifferentiating into corneal epithelium but mainly to the absence of an anti‐angiogenic barrier.