To isolate precursor cells from HCEC, we used the sphere-forming assay. Cells were isolated without contamination by corneal epithelial cells, as demonstrated by RT-PCR analysis of corneal epithelial markers (K3 and K12 genes), as well as the characteristic hexagonal shape of cells in primary culture (data not shown). There was almost complete disaggregation into single cells, since counting the percentage of single, double, and triple cells showed that >99% of all cells were single
(Fig. 1A) . To determine the appropriate culture conditions, we prepared dishes at a density of 1, 10, 30, and 50 viable cells/μL and stained the cells with the fluorescent cell tracker CM-DiI to investigate the possibility of spheres being formed by reaggregation. No spheres were generated in the cultures with 1 viable cell/μL, but a significant number of spheres were formed at 30 and 50 cells/μL, with some spheres arising from reaggregation as evidenced by DiI-staining. The spheres were divided into DiI-positive and DiI-negative when culturing was performed at 10 cells/μL
(Fig. 1B) , indicating that the spheres were derived from proliferation, not from reaggregation of the dissociated cells. After 5 days of culturing, small floating spheres formed, and these spheres grew larger after 10 days, while the nonproliferating cells died and were eliminated
(Fig. 1C) . To verify that the increase in colony size was actually due to proliferation, we added the thymidine analogue BrdU at 24 hours before cell fixation. BrdU labeled most of the cells within each sphere on day 10
(Fig. 1D) , indicating that the colonies contained proliferating cells. These results suggest that the sphere colonies arose from single isolated HCECs and that the sphere-forming cells possessed proliferative capacity. When the number of spheres obtained was counted after 10 days of culture, showing that 257 ± 83 spheres (mean ± SD,
n = 8) were generated per dish (50,000 cells). In a typical case, 2.5 × 10
4 cells were isolated from a 10-mm piece of corneal tissue and then generated approximately 130 spheres after 10 days. These spheres were 88.3 ± 15.9 μm in diameter (mean ± SD,
n = 35). The replating efficiency decreased dramatically between the primary sphere colonies and secondary colonies. When the primary colonies were trypsinized and incubated in serum-free floating culture, secondary colonies were generated
(Fig. 1E) , at approximately 15 ± 1 (
n = 3) per dish (10,000 cells). This suggested that HCECs have the capacity for self-renewal of sphere colonies, but this capacity is limited.