Clinical And Basic Researches Of Limbal Stem Cells

Partâ… Staging System for Limbal Stem Cells DeficiencyUsing in Vivo Confocal MicroscopyObjective To describe the corneal and limbal epithelial changes in patients with imbalstem cells deficiency (LSCD) using in vivo laser scanning confocal microscopy (LSCM),and to classify the stages of LSCD at the cellular level.Method This is a prospective, noncomparative clinical study. Patients were selectedaccording to their clinical presentations. Twenty four eyes of 18 subjects with LSCD wereclassified into 4 stages groups initially according to their cornea status. In vivo imagings ofthe corneal and limbal epithelium in all the 24 eyes were obtained with HRT 3 Rostockcornea module LSCM. The LSCM images were reviewed.Results Five useful parameters were found to evaluate all the LSCM images: theepithelium layers; epithelial cells size and activity; invading cells; epithelial andsubepithelial nerve, intraepithelial vessels. Both the central cornea and limbus showed 4different stages changes which correlated with the initial clinical 4 stages group. Thestaging system for LSCD at the cellular level was established.Conclusion The morphological and structural changes of corneal and limbal epithelium at the cellular level correlated with clinical presentations at different stages in LSCD.Confocal microscopy might be a useful tool to detect very early stage of LSCD and couldaid in the staging of LSCD.Partâ…¡Expression of Human Embryonic Stem Cells Markers in theLimbal and Corneal Epithelial CellsObjective To investigate the expression of five human embryonic stem cells (hESCs)markers, Alkaline phosphatase (ALP), Stage-specific embryonic antigen (SSEA)-1,SSEA-4, Nanog and Oct-4, in the limbal and corneal epithelial cells.Method Frozen sections were obtained from human corneoscleral tissues.Immunohistochemistry study for five hESCs markers, ALP, SSEA-1, SSEA-4, Nanog andOct-4, was performed to evaluate their expression at the protein level in the human limbusand cornea. The mRNA expression of SSEA-4, Nanog and Oct-4 was further confirmed byreverse transcription-PCR (RT-PCR).Results Immunohistochemistry study showed that SSEA-4 was present in all layers ofthe limbal and corneal epithelial cells and ALP was detected in a small subgroup of stromalcells in the anterior limbus. The expression of SSEA-4 was confirmed by RT-PCR. SSEA-1and Oct-4 were not detected at the protein nor the mRNA level in the limbus and cornea.Nanog mRNA transcript was detected in the cornea and limbus, but Nanog protein was notdetected using immunostaining.Conclusion The expression of SSEA-4 in mature corneal epithelial cells indicates thatit is not a marker for corneal epithelial progenitor/stem cells. Whether Nanog is expressedon the ocular surface needs to be further confirmed at the protein level. Partâ…¢Using an Inducible Transgenic Mice Model toStudy Limbal Stem CellsObjective To establish an inducible GFP transgenic mice model to study the limbalstem cells.Method An inducible GFP transgenic mice (ROSA26-rtTA-Col1a1-tetOP-H2BGFP)model was selected and the expression of GFP was turned on for 21 days starting atdifferent periods and followed by 1 month and 4 months, which was counted from the datethe GFP was turned on. After the mice were euthanized, the left eyes were frozen with OCTand immunohistochemistry study for GFP expression pattern was performed on the frozensections. The sclerocornea tissues from the right eyes were isolated, mounted and observedunder confocal and fluorescence microscopy. Images were processed.Results Expression of GFP was present on the whole ocular surface in both 1 monthand 4 months mice and stronger GFP expression band was seen in the limbus. In 4 monthsmice, the expression of GFP was weaker than 1 month and parts of higher GFP expressionlimbal cells were organized in a radial stripe fashion towards the center of the corneaforming a vortex pattern. Three-dimensional reconstructions of the vortex pattern confocalimages revealed that the stripe cells were seen in the basal and suprabasal corneal epitheliallayers and different from the other cells in the peripheral and central cornea.Conclusion The epithelial cells in the limbus retained the higher GFP expression in 4months after the GFP was turned on indicating that they are the slowest cycling cells. Theradial vortex pattern suggests that these epithelial cells might migrate from the limbustowards the center of the cornea. This mouse model is a useful tool to study the in vivohomeostasis of corneal epithelial cells.