Characterization of the corneal surface in limbal stem cell deficiency and after transplantation of cultivated limbal epithelium.
OBJECTIVE
Transplantation of in vitro-cultivated limbal epithelium (TCLE) recently was developed to treat limbal stem cell deficiency (LSCD). The objective of this study was to characterize changes in the cornea during LSCD and on the corneal surface after TCLE.


METHODS
Experimental study.


METHODS
The pannus tissue excised from the corneas of 17 LSCD patients was analyzed to characterize the changes in the cornea during LSCD. Five corneal buttons obtained during perforating keratoplasty (pKP) from patients who had undergone TCLE at least 6 months before pKP were examined to assess the effect of TCLE. Six samples of healthy central cornea and 6 of bulbar conjunctiva served as control tissue.


METHODS
The expression of epithelial lineage markers (keratin [K] 3, K12, K19, and mucin 5AC) and inflammatory markers (interleukin-1alpha [IL-1alpha], IL-1beta, intercellular adhesion molecule 1 [ICAM-1], vascular cell adhesion molecule 1 [VCAM-1], and vascular endothelial growth factor [VEGF]) were analyzed using real-time polymerase chain reaction, Western blotting, and immunofluorescence in the tissue samples.


METHODS
Comparison of the markers' expression patterns.


RESULTS
The expression of all markers differed in healthy cornea and conjunctiva. Expression of lineage markers was similar in pannus to conjunctiva, but not to cornea. Interleukin-1beta, ICAM-1, VCAM-1, and VEGF were increased significantly in pannus compared with the levels in healthy cornea. Interleukin-1alpha, IL-1beta, and ICAM-1 were increased compared with healthy conjunctiva. The TCLE improved vision and reduced inflammation, vascularization, and discomfort. After TCLE, the lineage markers in the excised corneal buttons showed a corneal phenotype and a significant reduction in inflammatory markers in 4 of 5 cases.


CONCLUSIONS
Limbal stem cell deficiency is characterized by ingrowth of abnormal inflamed tissue with a conjunctival phenotype. Transplantation of limbal epithelium cultivated in vitro on intact amniotic membrane restored a noninflamed ocular surface and a corneal phenotype.


BACKGROUND
The author(s) have no proprietary or commercial interest in any materials discussed in this article.
Pauklin
Mikk
M
Department of Ophthalmology, University of Duisburg-Essen, Essen, Germany.
Steuhl
Klaus-P
KP
Meller
Daniel
D
D000328:Adult; D000368:Aged; D000650:Amnion / Q000166:cytology; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D019070:Cell Lineage; D017690:Cell Transplantation; D002478:Cells, Cultured; D002648:Child; D003316:Corneal Diseases / Q000378:metabolism / Q000473:pathology / Q000601:surgery*; D004591:Electrophoresis, Polyacrylamide Gel; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism; D007378:Interleukins / Q000235:genetics / Q000378:metabolism; D007633:Keratins / Q000235:genetics / Q000378:metabolism; D016850:Limbus Corneae / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D055271:Mucin 5AC / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000473:pathology*; D054457:Tissue Scaffolds; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D055815:Young Adult
D015415:Biomarkers; D007378:Interleukins; D055271:Mucin 5AC; C467484:VEGFA protein, human; D019010:Vascular Cell Adhesion Molecule-1; D042461:Vascular Endothelial Growth Factor A; D018799:Intercellular Adhesion Molecule-1; D007633:Keratins