Regeneration of the corneal epithelium with conjunctival epithelial equivalents generated in serum- and feeder-cell-free media

Abstract

Purpose: An alternative autologous tissue for ocular surface reconstruction is a potential treatment for the patients with bilateral limbal stem cell deficiency. For the purpose of regenerative procedures in patients, it is desirable to eliminate the involvement of xenogeneic components, such as nonhuman sera and feeder cells. In the present study, we examined the behavior and phenotypic features of cultured conjunctival epithelial sheets generated in serum- and 3T3-free culture conditions when transplanted into the de-epithelialized limbal corneal surface.

Methods: Epithelial cells from normal conjunctiva obtained by neutral protease digestion were expanded by culture in a serum-free low-calcium medium and set in an air-liquid interface culture for 14 days. The resulting multilayered epithelial sheets were grafted onto rabbit ocular surfaces made epithelial-free by alkali treatment. Pre-grafted and post-grafted epithelia were analyzed by electron microscopy and immunohistochemistry.

Results: At graft time the cultured epithelial sheet consisted of 6-8 layers of properly stratified epithelium that displayed a CK19(+)/MUC5AC(+)/ CK3 (-)/CK12(-) phenotype, consistent with the conjunctival epithelial lineage. Two weeks after xeno-grafting the in vivo epithelium consisted of 5-6 well compacted layers expressing the precursor cell-related protein p63, the proliferation marker Ki67, desmosomes, hemidesmosomes and its integrin (β4), and the corneal specific cytokeratins CK3, and CK12. Conjunctival goblet cell mucin (MUC5AC) was not visible. The engrafted epithelium stained positively for the anti-human nuclei antibody, confirming that the epithelial cells on the rabbit corneas were of human origin.

Conclusions: Our results suggest that conjunctival epithelial sheets generated in serum- and 3T3-free culture conditions can acquire the corneal epithelial phenotype when transferred to the in vivo corneal stromal environment.

Figure 1

Immunohistochemical examinations of the cultivated human conjunctival epithelia (CjE) equivalent. The cultivated human CjE equivalent was stained green for MUC5AC (A), CK19 (B), CK3 (C), CK12 (D), red for p63 (E) by indirect immunofluorescence as described in Methods. Nuclei have been counterstained with DAPI. A: The arrow points to positive cell for MUC5AC. Representative images of three independent cultures are shown.

Figure 2

Micrographs of the rabbit ocular surface two weeks after transplantation of human conjunctival epithelia (CjE) sheets onto alkali-treated ocular surfaces. A, C: In these eyes with transplants, the ocular surfaces are smooth and show no fluorescein penetration. B, D: The eyes were treated exactly as A and C but without human CjE sheet application. Note the incipient corneal neovascularization in B and the extensive corneal epithelial defect in D. Representative images of eight grafted corneas are shown.

Figure 3

Hematoxylin and Eosin (H and E), and transmission electron microscopy (TEM) of the engrafted human conjunctival epithelia (CjE). A, B: H and E stain of corneas with or without transplanted epithelial sheets two weeks after transplantation. C, D: TEM of the grafted tissue showing hemidesmosomal profiles at the basal cell-central corneal stromal interface (C, arrows) and desmosomes between wing shaped cells (D, arrowheads). Representative images shown of four grafted corneas.

Figure 4

Immunohistochemistry of the engrafted human conjunctival epithelia (CjE). The engrafted human CjE over the rabbit cornea surfaces were stained red for human nuclei (A), p63 (B), MUC1 (E), CK3 (G), and CK12 (H) by indirect immunofluorescence as described in Methods. The engrafted human CjE was stained green for Ki67 (C) and Integrin β4 (D). MUC5AC expression was not detected (F). Nuclei have been counterstained with DAPI except sections stained with anti-human nuclei antibody. Represented images of eight engrafted human CjEs over rabbit cornea surfaces.