Agrin promoted the outgrowth and increased the numbers of cultured limbal stem cells in vitro. (A1) Representative images of limbal stem cell explants treated with different concentrations of Agrin (0, 10 and 100 ng/mL) at day 3, scale bar = 80 µm. (A2) Quantification of the outgrowth area of limbal stem cell explants treated with different concentrations of Agrin (0, 10, and 100 ng/mL) at day 3, n = 6 explants for each group, * P < 0.05 vs. control. (B1) Representative images of expanded limbal stem-like cells with prominent nuclei and high nucleus/cytoplasm ratio in outgrowth areas at day 5, scale bar = 20 µm. (B2) Quantification of limbal stem-like cells numbers at day 5. n = 5 explants for each group (8 microscopic fields for 1 explant), * P < 0.05 vs. control. (C) Quantification of limbal stem cells proliferation indirectly determined by Cell Count Kit-8 after treatment with Agrin (0, 10, and 100 ng/mL) for 24, 36, and 48 hours, the absorbance at 450 nm was measured, n = 5 samples for each group. * P < 0.05 vs. control.