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To characterise the origin, ultrastructure and functional properties of corneal limbal neurospheres (LNS).Limbal cells were isolated from the corneal limbus of adult mice and cultured in a serum-free sphere forming culture system. LNS were characterised by immunocytochemistry, Reverse-transcription-PCR and electron microscopy. LNS cells were also cocultured with neonatal mouse retinal cells. Phenotype and function were then assessed by immunofluorescence and a calcium influx/efflux assay.LNS cells displayed clonal growth and self-renewal, and expressed a wide range of stem cell and neural lineage markers. The acquisition of neural properties was concordant with expression of neural crest markers including CD34, Sca1, Sox9, Twist1, but not CD45. LNS exhibited similar morphology and microstructure to neurospheres derived from the central nervous system. Following culture in a conducive environment, the derived cells displayed mature neural markers and exhibited electrical excitability.Corneal limbal stromal progenitor cells are a potential and convenient autologous cell source to generate functional neurons.