To identify the LSC population in different culture methods, we next examined the phenotype of cultivated LECs using several putative corneal epithelial stem cells markers, including ATP-binding cassette family G2 (ABCG2), ΔNp63, N-cadherin, and K14.
31–36 We used K12 as a marker of mature cornea epithelial cells,
1 whereas Ki67 was used as a marker for active cell proliferation. When compared to the control, we observed a steady increase in expression of ABCG2 in the CC-LMCs (1.3-fold,
P = 0.314) and 3D CC-LMC (2.0-fold,
P = 0.251) cultures compared to the control (
Fig. 3). Likewise, we saw a similar increase in ΔNp63 expression (20%,
P = 0.01) in 3D CC-LMC culture, but a 20% decrease was observed in CC-LMC culture (
P = 0.04). The N-cadherin was expressed at high levels in the CC-LMC (27.6-fold,
P = 0.02) and 3D CC-LMC (5.4-fold,
P = 0.01) cultures. Interestingly, we did observe an almost identical decrease in K14 mRNA expression in the CC-LMC (2.3-fold,
P = 0.001) and 3D CC-LMC (2.1-fold;
P = 0.011) cultures compared to that in the control. Conversely, K12 mRNA expression was significantly lower in CC-LMC (1.4-fold decrease,
P = 0.045) and 3D CC-LMC (3.5-fold decrease,
P = 0.005) cultures. Finally, Ki67 expression from all culture methods was consistent with the cell count numbers quantitated. The Ki67 expression was the highest in the control (0.34-fold decrease in CC-LMC,
P = 0.015 and 0.55-fold decrease in 3D CC-LMC,
P = 0.003).