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H. Qi, D.-Q. Li, Z. Chen, H. D. Shine, S. C. Pflugfelder, D. B. Jones; Identification of Neurotrophic Factors NGF, GDNF and Their Corresponding Receptors TrkA, GFR-1 as Potential New Markers for Human Corneal Epithelial Stem Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):481.
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Neurotrophic factors play a vital role in survival and self renewal of neuron and non-neuron stem cells. This study was to explore their expression and function in human cornea and limbus to evaluate whether nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF) and their corresponding receptors TrkA , GFRα-1 could serve as potential markers for human corneal epithelial stem cells.
Expression of NGF, GDNF, TrkA and GFRα-1 was evaluated by immunofluorescent staining with laser scanning confocal microscopy and real-time PCR using cryosections and total RNA prepared from fresh human corneoscleral tissues and limbal epithelial cultures. The co-localization between these 4 molecules and stem cell associated markers were evaluated by double staining. Colony forming efficiency and in vitro wound-healing assays were performed with or without recombinant human NGF and GDNF.
NGF and GDNF were exclusively and sparsely localized in the basal layer of limbal epithelium where stem cells reside, but they were not detected in whole corneal and suprabasal limbal epithelium and stroma. TrkA and GFRα-1 shared a similar pattern to their ligands, with extended expression to a few suprabasal limbal cells and scattered basal cells of peripheral cornea. The immunoreactivity of these 4 proteins was co-localized with stem cell associated markers ABCG2, p63, and EGFR, but not Keratin 3. The expression of these neurotrophic mRNAs was significantly higher by limbal than corneal epithelium. In primary limbal epithelial culture, the antibodies to these 4 molecules stained cytoplasm and membrane of small cells co-expressing ABCG2, p63 and EGFR. Multi-fold more abundant transcripts encoding these 4 genes were detected in cells at an early growth stage that co-expressed higher levels of ABCG2, p63 and EGFR mRNA, than confluent cells. Colony forming efficiency and in vitro wound-healing were significantly promoted by addition of recombinant human NGF and GDNF to media of limbal epithelial cultures.
These findings revealed that neurotrophic factors NGF, GDNF and their corresponding receptors TrkA , GFRα-1 were exclusively expressed by a subpopulation of basal limbal epithelial cells, and may serve as new markers for human corneal epithelial stem cells.
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