AClinical Neurosciences Division, School of Medicine, BInfection, Inflammation and Immunity Division, School of Medicine, 1University of Southampton, Southampton, United Kingdom
Commercial Relationships: X. Chen, None; H. Thomson, None; P. Hossain, None; A. Lotery, None.
Support: Foresight, NERC and BRPS
Purpose:Cells from the adult rodent corneal limbal epithelium basal layer have been demonstrated to display neuronal potential and differentiate towards functional neurons in vitro. A sphere forming culture system has also been used to expand stem cells of a neural crest origin from mouse corneal stroma. The aim of this study was to investigate whether progenitors with neuronal potential could be cultured from adult murine limbus to form neuronal characteristics.
Methods:Cells from adult (8 weeks) murine corneal limbus were isolated and cultured in a serum-free sphere-forming system in the presence of mitogens. Cells were maintained in the presence or absence of the bone morphogenetic protein 4 (BMP4) inhibitor (Noggin). Sphere derived cells and their progeny were characterized using immunocytochemistry and/or reverse transcription-polymerase chain reaction (RT-PCR).
Results:Adult mouse limbal cells formed sphere colonies with the ability to self renew. The frequency of sphere-forming cells was 0.45-1.0% subject to culture conditions. Noggin did not have an effect on the efficiency of sphere formation. In the presence and absence of Noggin, sphere cells expressed ABCG2, Sox2, Nestin and beta-III tubulin (early differentiated neuron) but not P63 (epithelial stem cell marker) as shown by immunocytochemistry. Expression of neuronal stem/progenitor cells markers (nestin, Sox2, Musashi, beta-III tubulin) and neural crest markers (Twist1 and Slug) increased with time in culture and passage, concomitant with a decrease in expression of the epithelium lineage markers P63 and cytokeratin12, as shown by RT-PCR.
Conclusions:The adult mouse corneal limbus contains stem / precursor cells which express neural stem cell markers in vitro. These stem cells/ progenitor cells appear to be neural crest & are likely to be derived from the corneal limbal stroma.
Keywords: cornea: basic science cornea: stroma and keratocytes cornea: epithelium
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