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

Abstract

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, alpha-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.

Figure 1

Sphere-forming culture of rabbit corneal epithelium. A: Anterior view of a rabbit cornea and a diagram of the corneal epithelium. B, C: Rabbit corneal epithelial cells from the limbus or central cornea formed spheres. Growth of a representative single sphere is shown until 7 day.

Figure 2

Comparison of primary sphere formation by rabbit CEC from the periphery and center of the cornea. A: The number of spheres obtained from limbal CEC (n=10) was significantly higher than that from central CEC (n=10) after 7 days of culture. The experiment was repeated twice and representative data are shown. The asterisk indicates a p=0.0028 (unpaired t-test). B: The mean sphere size exceeded 250 μm on day 7.

Figure 3

Formation of secondary spheres. A: Secondary spheres were generated from dissociated primary limbal or central spheres. B: The replating efficiency for formation of secondary spheres was higher when the cells were derived from the limbal cornea than from the central cornea (p=0.013, unpaired t-test).

Figure 4

Analysis of spheres and their progeny. A: Immunocytochemical analysis of sphere colonies on day 7. Bright-field images and immunostaining of spheres for p63 (an epidermal stem/progenitor cell marker), p75^NTR (an epidermal basal progenitor cell marker), cytokeratins 3 and 12 (differentiated epithelial cell markers, nestin (a neural stem cell marker), microtubule-associated protein 2 (MAP2: a differentiated neuronal cell marker), and neuron-specific enolase (NSE: a differentiated neuronal cell marker). Each colony has been labeled by BrdU. As a control, IgG was used instead of the primary antibody. Scale bar=100 µm. B: Double immunocytochemical staining of a sphere colony. The spheres are double immunostained by nestin and cytokeratin 12 or by p63 and alpha smooth muscle actin (αSMA). Scale bar=100 µm. C: RT–PCR of corneal epithelial tissues, spheres, and their progeny. Genes for P63, keratin 3, keratin 12, and nestin are present in corneal epithelial tissues, spheres, and their progeny derived from the limbal or central regions, but not in total RNA processed without reverse-transcription. D: Immunocytochemical analysis of differentiated cells obtained from spheres. Cells migrating out of the spheres express both cytokeratin 3 and cytokeratin 12 (differentiated epithelial cell markers). These cells are also positive for MAP2, and NSE. Scale bar=100 µm.