Analysis of p63 and cytokeratin expression in a cultivated limbal autograft used in the treatment of limbal stem cell deficiency.
(1/22)
AIM: To investigate the expression of p63 and cytokeratins throughout the course of producing a cultivated autograft of limbal epithelial cells. METHODS: A 75 year old male with a severe alkali burn to his right eye received two cultivated autografts of limbal epithelial cells on amniotic membrane followed by a corneal allograft. Immunostaining for p63 and cytokeratins was performed during ex vivo expansion with 3T3 fibroblasts, following subcultivation on amniotic membrane, and on the excised corneal button. RESULTS: Cultures grown in the presence of 3T3 fibroblasts or on amniotic membrane displayed positive staining for keratins 14 and 19, and p63, but poor staining for keratin 3 (K3). The excised corneal button possessed a stratified epithelium of K3 positive cells residing on amniotic membrane. CONCLUSIONS: Our results document for the first time the co-expression of cytokeratins 14 and 19 with p63 in a cultivated limbal graft. These data support the conclusion that cultivated grafts of limbal epithelium contain predominantly undifferentiated cells with the potential to regenerate a normal corneal epithelium. (+info)
Characterization of corneal pannus removed from patients with total limbal stem cell deficiency.
(2/22)
PURPOSE: To determine the epithelial lineage of origin in corneal pannus tissue surgically removed from patients with total limbal stem cell (SC) deficiency. METHODS: The lineage of origin of the entire conjunctivalized pannus removed from eight corneas with a diagnosis of total limbal SC deficiency was characterized by anti-keratin (K)-3 and anti-K19 monoclonal antibodies. The protein and mRNA of epithelial outgrowth from segments of five such pannus specimens were analyzed by Western blot and reverse transcription-polymerase chain reaction, respectively. RESULTS: Cross sections of all eight specimens showed a stratified epithelium with goblet cells expressing mucin (MUC)-5AC, and a stroma showing blood vessels and inflammatory cell infiltrates. Immunostaining showed full-thickness expression of K19 in the entire pannus of all eight specimens. Expression of K3 was negative in seven patients, but was sporadically positive in a patient with Stevens-Johnson syndrome. In culture, all five pannus specimens generated a compact, small epithelial cell outgrowth, and except for one, reached confluence in 2 to 3 weeks. The K3/K12 pair was expressed by extracts of cell outgrowth from the control limbal epithelial explant, but not in all five pannus specimens. A 60-kDa band of DeltaNp63 was expressed in the control specimen and in all five pannus specimens. Cell outgrowth expressed K3 transcript in three, but none showed K12 transcript. CONCLUSIONS: The resultant epithelial phenotype of the pannus tissue was not corneal, as evidenced by the negative staining to cornea-specific K12 mRNA and protein, but was conjunctival, as evidenced by the presence of goblet cells, the weak expression of K3, and the strong expression of K19. The abundant expression of DeltaNp63 in such conjunctiva-derived epithelium in eyes with total limbal SC deficiency raises doubts as to its validity as a limbal SC marker. (+info)
Ultrastructural analysis of in vivo expanded corneal epithelium on amniotic membrane.
(3/22)
The purpose of this study is to characterize and compare the ultrastructural changes occurring during the in vivo cultivation of corneal epithelium on amniotic membrane (AM) at several different time points. Corneal burn patients (n=7) with a corneal epithelial defect and severe limbal damage were selected. Initially, AM transplantation with limbal autograft was performed at the acute stage of corneal burn to reconstruct the damaged ocular surface. One to six (mean interval; 3.3+/-1.2) months later, the central part of AM containing an in vivo expanded corneal epithelium was excised and retransplanted in adjacent lesions. The excised epithelium with AM was examined by electron microscopy and immunohistochemical study. By electron microscopy, one and two months after expansion, cultivated epithelium on AM showed an undifferentiated epithelium and an incomplete basement membrane (BM). But, after three months, the cultivated epithelium began to differentiate into a multilayered epithelium with a continuous BM with increased hemidesmosomes. These findings were further confirmed by immunohistochemical study, that cytokeratin K3 was expressed in the cultivated corneal epithelium and newly formed BM was partially positive of collagen IV at three months. At least 3 months may be needed for the proliferation and differentiation of in vivo cultivated corneal epithelium on AM. (+info)
Proliferation and differentiation of transplantable rabbit epithelial sheets engineered with or without an amniotic membrane carrier.
(4/22)
PURPOSE: To report a novel method of engineering transplantable, carrier-free corneal epithelial sheets by using a biodegradable fibrin sealant and to compare its characteristics with epithelial sheets cultivated on denuded amniotic membrane carriers. METHODS: Stratified corneal epithelial sheets were prepared in culture dishes coated with biodegradable fibrin glue. Amniotic membrane (AM) carriers served as the control. The quality of cultivated sheets was compared by immunohistochemistry for cytokeratin (K)3, K12, K14, p63, occludin, and integrin beta1; electron microscopy; and colony-forming assays. K3 protein expression was compared by Western blot analysis. In a limbal-deficient rabbit transplantation model, postoperative adaptation and proliferation of BrdU-labeled cell sheets were examined by histology and anti-Ki67 staining. RESULTS: Epithelial sheets were successfully engineered by using a biodegradable fibrin sealant. Cell sheets in both groups were multilayered, expressed K3, K12, and K14, and had functioning occludin(+) apical tight junctions as well as p63 and integrin beta1 staining in basal cells. The carrier-free sheets appeared to be more differentiated than the AM sheets, which was also demonstrated by the higher levels of K3 in the Western blots. The colony-forming efficiency of dissociated cells was similar in both groups, although larger colonies were observed on the AM sheets. AM sheets retained higher levels of BrdU-labeled cells and fewer Ki67(+) cells compared with carrier-free sheets after transplantation. CONCLUSIONS: Tissue engineering with a commercially available fibrin sealant was an effective means of creating a carrier-free, transplantable corneal epithelial sheet. Carrier-free sheets were more differentiated compared with AM sheets, while retaining similar levels of colony-forming progenitor cells. (+info)
Human corneal epithelial equivalents for ocular surface reconstruction in a complete serum-free culture system without unknown factors.
(5/22)
Genetics of Meesmann corneal dystrophy: a novel mutation in the keratin 3 gene in an asymptomatic family suggests genotype-phenotype correlation.
(6/22)
PURPOSE: Juvenile epithelial corneal dystrophy of Meesmann (MCD, OMIM 122100) is a dominantly inherited disorder characterized by fragility of the anterior corneal epithelium and intraepithelial microcyst formation. Although the disease is generally mild and affected individuals are often asymptomatic, some suffer from recurrent erosions leading to lacrimation, photophobia, and deterioration in visual acuity. MCD is caused by mutations in keratin 3 (KRT3) or keratin 12 (KRT12) genes, which encode cornea-specific cytoskeletal proteins. Seventeen mutations in KRT12 and two in KRT3 have been described so far. The purpose of this study was to investigate the genetic background of MCD in a Polish family. METHODS: We report on a three-generation family with MCD. Epithelial lesions characteristic for MCD were visualized with slit-lamp examination and confirmed by in vivo confocal microscopy. Using genomic DNA as a template, all coding regions of KRT3 and KRT12 were amplified and sequenced. Presence of the mutation was verified with restriction endonuclease digestion. RESULTS: In the proband, direct sequencing of the polymerase chain reaction (PCR) product from amplified coding regions of KRT3 and KRT12 revealed a novel 1493A>T heterozygous missense mutation in exon 7 of KRT3, which predicts the substitution of glutamic acid for valine at codon 498 (E498V). Using PCR-Restriction Fragment Length Polymorphism (RFLP) analysis, the mutation was demonstrated to segregate with the disease (four affected members, three non-affected) and to be absent in 100 controls from the Polish population, indicating that it is not a common polymorphism. CONCLUSIONS: Location of the E498V mutation emphasizes the functional relevance of the highly conserved boundary motifs at the COOH-terminus of the alpha-helical rod domain in keratin 3 (K3). (+info)
The use of human mesenchymal stem cell-derived feeder cells for the cultivation of transplantable epithelial sheets.
(7/22)