Specimens were fixed in 2.5% glutaraldehyde in PBS, washed three times in PBS, and post fixed for 1 h in 2% aqueous osmium tetroxide. 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 (4), 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. Introduction Cultivated autologous epithelial sheets are used to treat limbal AM 114 stem cell deficiency (LSCD) in unilateral cases. Autologous transplantation of limbal tissue or cultured limbal epithelial cells from the contralateral eye can result in the reestablishment of a long-term repopulating cell population in the recipient eye [1-5]. However, in bilateral cases only AM 114 allogeneic limbal epithelial cells are available for homotypic transplantation, bringing about the attendant systemic adverse effects associated with postoperative immunosuppression. Furthermore, the success rate of allogenic transplantation shows great variability [6, 7] suggesting that either the intrinsic incompatibilities of the allogeneic transplantation and/ or the effects of the postoperative immunosuppressive drugs interfere with long-term engraftment. Accordingly, in preceding years, there has been strong interest in the use of alternative autologous AM 114 ectodermal lineages to replace the limbal epithelium as a source tissue for ocular surface reconstruction in blind patients having bilateral LSCD. Pioneer studies were performed with oral mucosa [8-11]. The conjunctival epithelial (CjE) lineage, which shares with the limbal-corneal epithelium a common embryological origin and a somatic expression of the PAX6 morphogen , is particularly attractive as a heterotypic autologous source, provided that two phenotypic characteristics of conjunctival epithelium, vascular compatibility and mucinous cell generation, are spontaneously lost during the processes of in vivo cell expansion and reengraftement in the avascular cornea or are purposely eliminated by genetic manipulation. Ang et al. AM 114 previously demonstrated that conjunctival epithelial cells grown on human amniotic membrane in a fetal bovine serum (FBS)-complemented medium with 3T3 mouse feeder cell support can modify their phenotype toward the corneal phenotype when grown in vivo on the corneal surface in a short-term human-to-rabbit xenograft transplantation model . Recently, explant cultures from conjunctival biopsies made over human amniotic membrane in the presence of a FBS-complemented medium were used to attain corneal surface regeneration in patients having LSCD . While the results of the cellular phenotype obtained under these conditions are highly encouraging for the purpose of regenerative procedures in patients, it will be desirable to eliminate the involvement of xenogeneic components, in particular the co-culture with animal cells and sera. We have previously demonstrated that the same culture approach used to generate conjunctival epithelial equivalents following a large Col13a1 in vivo expansion AM 114 of the epithelial precursor populations  can be used to establish a CjE equivalent that recapitulates critical features of the source human CjE, including in selected cases, generation of mucinous, Goblet-like cells. The culture approach, derived from the original Boyce and Ham low-calcium press strategy for the development of epidermal precursors , avoids reliance on mouse feeder cells and fetal calf serum. Our study demonstrates when these human being CjE equivalents are transplanted into the de-epithelialized ocular surface of rabbits having a carrier-free approach, after two weeks in the in vivo corneal environment, the human being cells establish a five- to six-layer stratified epithelium that a) presents a defect-free outer surface; b) establishes a profusion of intercellular desmosomes and well developed substratum-anchoring hemidesmosomes; c) includes a coating of basal cells that is rich in nuclear p63, a marker of extended epithelial cells proliferative potential;.