Isolation of adult progenitor cells with neuronal potential from rabbit corneal epithelial cells in serum- and feeder layer-free culture conditions

PURPOSE: To isolate progenitor cells from rabbit corneal epithelial cells (CEC) in serum- and feeder layer-free culture conditions and to compare the self-renewal capacity of corneal epithelial progenitor cells obtained from the central and limbal regions of the cornea. METHODS: Tissue samples of New Zealand white rabbit corneas were dissected from the limbal and central regions to obtain CEC for sphere-forming culture, in which the cells formed spheres in serum-free medium containing growth factors. The number of primary and secondary sphere colonies and the size of the primary spheres were compared between the limbal and central regions. To promote differentiation, isolated sphere colonies were plated in dishes coated with poly-L-lysine (PLL)/laminin. The expression of epithelial, neural, and mesenchymal mRNAs was examined in the sphere colonies and their progeny by immunocytochemistry and/or the reverse transcription–polymerase chain reaction (RT–PCR). Adherent differentiated cells from the sphere colonies were also examined morphologically. RESULTS: Primary spheres were isolated from both the limbal and central regions of the cornea. The rate of primary sphere formation by CEC from the limbal region (55.6±10.6/10,000 cells) was significantly higher than that by cells from the central cornea (43.1±7.2/10,000 cells, p=0.0028), but there was no significant difference in the size of primary spheres derived from both regions. The self-renewal capacity of cells from the limbal region was higher than that of cells from the central region, as evidenced by the significantly higher secondary sphere formation rate of limbal cells (38.7±8.5/10,000 cells) in comparison with that for central cells (31.3±5.7/10,000 cells, p=0.013). The primary sphere colonies expressed bromodeoxyuridine (BrdU), a 63-kDa protein (p63), p75 neurotrophin receptor (p75(NTR)), and nestin, whereas their progeny expressed cytokeratin 3, cytokeratin 12, vimentin, α-smooth muscle actin, microtubule-associated protein 2, and neuron-specific enolase on immunocytochemical analysis. These markers were confirmed by RT–PCR. CONCLUSIONS: Our findings indicate that limbal CEC contain more progenitor cells with a stronger self-renewal capacity than cells from the central region. These progenitor cells differentiate into the epithelial lineage, and can also produce neuronal protein.