Clusters of Corneal Epithelial Cells Reside Ectopically in Human Conjunctival Epithelium

Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kioto, Kyōto, Japan
Investigative Ophthalmology &amp Visual Science (Impact Factor: 3.4). 05/2006; 47(4):1359-67. DOI: 10.1167/iovs.05-1084
Source: PubMed


The ocular surface is covered by two biologically distinct epithelia: corneal and conjunctival. The expression of keratin12 (K12) is currently considered a hallmark of cornea-type differentiation. In the current study, the biological features of K12-positive cells in human bulbar conjunctival epithelium were examined.
Human conjunctival tissues were subjected to investigate the K12-positive cells in conjunctiva by immunostaining, in situ hybridization, Western blot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR), and fluorescence-activated cell sorting (FACS). Gene expression profiling of these cells was performed with introduced amplified-fragment length polymorphism (iAFLP). To determine the presence of stem- or progenitor cells, immunostaining and colony-forming assays were performed.
Western blot analysis, RT-PCR revealed that K12 was expressed in conjunctival epithelium. Immunostaining analysis showed that K12-positive cells reside mainly in clusters in conjunctival epithelium. FACS analysis showed that 0.2% to 1.7% of conjunctival epithelial cells collected from the inferior bulbar conjunctiva were K12 positive. iAFLP analysis revealed that the gene expression patterns of these cells were highly similar to that of corneal epithelial cells. p63 and ABCG2 were expressed beneath the K12-positive cells. Some colony-forming cells expressed K12.
The K12-positive cells appear to be ectopically residing, self-maintaining corneal epithelial cells in the conjunctival epithelium.

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    • "This phenomenon is similar to Kawasaki et al.’s finding that keratin 12-positive corneal epithelial cells can ectopically reside in the conjunctival epithelium. The researchers found that between 0.2% and 1.7% of the conjunctival epithelial cells collected from the inferior bulbar conjunctiva were keratin 12 positive [34]. Although these keratin 12-positive cells may directly extend from the limbus, they may be maintained by their own stem or progenitor cells in the conjunctiva. "
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    ABSTRACT: To understand whether the epithelial phenotype in total sclerocornea is corneal or conjunctival in origin. Four cases of total sclerocornea (male:female = 1:3; mean age = 5.4±4.3; 1-11 years old) who received penetrating keratoplasty (PKP) at our hospital between 2008 and 2011 were included. Corneal buttons obtained during PKP were used for transmission electron microscopy (TEM) as well as immunoconfocal microscopy for cytokeratins 3, 12, and 13, goblet cell mucin MUC5AC, connexin 43, stem cell markers p63 and ABCG2, laminin-5, and fibronectin. After a mean follow-up period of 38.8±14.0 (12-54) months, the grafts remained clear in half of the patients. TEM examination revealed a markedly attenuated Bowman's layer in the scleralized corneas, with irregular and variably thinned collagen lamellar layers, and disorganization and random distribution of collagen fibrils, which were much larger in diameter compared with a normal cornea. Immunoconfocal microscopy showed that keratin 3 was expressed in all four patients, while p63, ABCG2, and MUC5AC were all absent. Cornea-specific keratin 12 was universally expressed in Patients 1 to 3, while mucosa (including conjunctiva)-specific keratin 13 was negative in these patients. Interestingly, keratin 12 and 13 were expressed in Patient 4 in a mutually exclusive manner. Linear expression of laminin-5 in the basement membrane zone and similar expression of fibronectin were observed. The epithelia in total sclerocornea are essentially corneal in phenotype, but in the event of massive corneal angiogenesis, invasion by the conjunctival epithelium is possible.
    Molecular vision 04/2014; 20:468-xxx. · 1.99 Impact Factor
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    • "Last but not least, as previously shown by Ang et al. [13], the human CjE cells have acquired the tonofilament pattern that characterizes the corneal epithelial cells, the expression of CK3 and CK12. With respect to CK12, gene expression comparisons by microarray [19] and histology have shown that certain areas of the human CjE the epithelium may spontaneously express a substantial level of the CK12 mRNA [20]. However, no such observation has been made in regard to CK3. "
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    ABSTRACT: 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. 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. 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. 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.
    Molecular vision 12/2013; 19:2542-50. · 1.99 Impact Factor
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    • "These cytokeratins are the hallmark of corneal differentiation and are rarely reported in conjunctival cells. The presence of clusters of corneal progenitor cells in conjunctiva has been reported, and it is possible that the expression of K3 and K12 observed in this study could be due to the proliferation of corneal stem cells in conjunctiva [34]. It is also possible that these differences are due to the different niches that the culture conditions used here provide and thereby lead to changes in the expression profile. "
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    ABSTRACT: To culture oral mucosal epithelial cells on deepithelialized human amniotic membrane without the use of feeder cells and to compare the characteristics of cultured oral cells with cultured limbal and conjunctival epithelial cells for use in ocular surface reconstruction. Oral biopsies were obtained from healthy volunteers after informed consent and were cultured on deepithelialized amniotic membrane for three to four weeks. Confluent cultures of limbal, oral, and conjunctival cells were subjected to characterization of markers of stem cells and of epithelial differentiation by reverse-transcription polymerase chain reaction (RT-PCR) and by immunohistochemistry. Ultrastructural studies were also performed using electron microscopy. A sheet of healthy, stratified oral epithelial cells was obtained within three to four weeks of culture. Electron microscopy demonstrated that the cells formed gap junctions and desmosomes. RT-PCR analysis showed that cultured oral epithelial cells expressed markers of epithelial differentiation such as cytokeratin K3, K4, K13, K15 and connexin 43. The cells also expressed stem cell markers of epithelial cells such as DeltaN isoforms of p63 as well as p75, a marker for stem cells of oral epithelium. The cells did not express cytokeratin K12 or Pax-6, an eye-specific transcription factor. Oral epithelial cells can be cultured as explants on deepithelialized amniotic membrane without using feeder cells. Characterization showed that these cells maintain the phenotypic characteristics of oral epithelial cells and that the culture is a heterogeneous population of differentiated cells and stem cells. We find the cultured oral epithelial cells usable for ocular surface reconstruction in patients having bilateral ocular surface diseases.
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