Fig 1.
Phase contrast photographs of human limbal explant outgrowth on plastic dishes without or with 10uM Y-27632 at the end of third week in culture.
(A). Magnification: 200×. Many pyknotic, fibroblastic-like and irregularly shaped cells were found in cultures without Y-27632 as indicated by arrow heads. A distinct population of cells with prominent nuclei and high nucleus/cytoplasm ratio as indicated by arrows were noted in the presence of 10 μM Y-27632. Quantitative comparison of the outgrowth area between control and Y-27632 group was performed once every two or three days and shown in (B). Statistical analyses were performed using a paired test, and *p<0.05 was considered significant.
Fig 2.
Y-27632 promoted the proliferation of human limbal epithelial cells.
Phase contrast photographs of human limbal epithelial cells expanded on plastic dishes with or without adding various concentrations of Y-27632 in culture medium (A). Magnification: 200×. Cells with prominent nuclei and high nucleus/cytoplasm ratio as indicated by arrows were noted in dishes with Y-27632. There was a positive dose-dependent correlation between the concentrations of Y-27632 and proliferation of expanded limbal epithelial cells (B). Statistical analyses were performed using a paired test, and *p<0.05 was considered significant.
Fig 3.
Y-27632 enhanced the colony forming efficiency of human limbal epithelial cells.
Human limbal epithelial cells at low cell density were expanded on plastic dishes with or without adding various concentrations of Y-27632 in culture medium for one week. (A) Photographs were taken after fixation and nuclear hematoxylin staining. (B) Numbers of hematoxylin-stained nuclei were counted and statistical analyses were performed using a paired test, and *p<0.05 was considered significant. There was a positive dose-dependent correlation between the concentrations of Y-27632 and colony forming efficiency of human limbal epithelial cells.
Fig 4.
Y-27632 increased expression of Ki67, a proliferation marker, in human limbal epithelial cells.
Human limbal epithelial cells were expanded on cover slides in plastic dishes with or without various concentrations of Y-27632 in culture medium for 30 hours. (A) After fixation, immunofluorescent staining was performed with anti-ki67 antibody. The nuclei were counterstained by DAPI (blue). Scale bar, 50 μm. Magnification: 630×. (B) The percentage of Ki67-positive limbal epithelial cells (pink fluorescence in nuclei) under at least 5 high power fields in each slide was counted and statistical analyses were performed using a paired test, and *p<0.05 was considered significant. There was a dose-dependent correlation between the concentrations of Y-27632 and percentage of ki67-positive limbal epithelial cells. (C) Whole cell protein extracts were analyzed by western blot using anti-ki67 antibody with tubulin as an internal control.
Fig 5.
Y-27632 increased expression of p63, a presumed limbal stem cell marker, in human limbal epithelial cells.
Human limbal epithelial cells were expanded on cover slides in plastic dishes with or without various concentrations of Y-27632 in culture medium for 30 hours. (A) After fixation, immunofluorescent staining was performed with anti-p63 antibody. The nuclei were counterstained by DAPI (blue). Scale bar, 50 μm. Magnification: 630×. (B) The percentage of p63-positive limbal epithelial cells (pink fluorescence in nuclei) under at least 5 high power fields in each slide was counted and statistical analyses were performed using a paired test, and *p<0.05 was considered significant. There was a dose-dependent correlation between the concentrations of Y-27632 and percentage of p63-positive limbal epithelial cells. (C) Nuclear protein extracts were analyzed by western blot using anti-p63 antibody with lamin B as an internal control.
Fig 6.
Effects of Y-27632 on the cell cycle distribution and apoptosis of human limbal epithelial cells.
Human limbal epithelial cells were expanded on plastic dishes with or without 20uM Y-27632 in culture medium at 37°C for 30 hours. After trypsinization, cells were fixed, stained with PI and analyzed by flow cytometry. Statistical analyses were performed using a paired test, and *p<0.05 was considered significant. The percentage of apoptotic cells (subG1 phase) was reduced from 2.9±0.9% (control) to 1.7±0.5% in the presence of Y-27632 (p = 0.24, n = 5). On the other hand, the percentage of proliferating cells (S-phase) was increased from 5.1±1.3% (control) to 7.8±1.9% in the presence of Y-27632 (p = 0.0295, n = 5).
Fig 7.
Effects of Y-27632 on the expression of Keratin 12, a corneal epithelial differentiation marker, in limbal epithelial cells.
Human limbal epithelial cells were expanded on cover slides in plastic dishes with or without 20μm Y-27632 in culture medium for 30 hours. (A) Total protein extracts were analyzed by western blot using anti-keratin 12 antibody with GAPDH as an internal control. (B) After fixation, immunofluorescent staining was performed with anti-keratin 12 antibody. The nuclei were counterstained by DAPI (blue). There was no significant difference in the expression of keratin 12 between Y-27632 and control groups. Statistical analyses were performed using a paired test, and *p<0.05 was considered significant. Scale bar, 1000 μm. Magnification: 40×.
Fig 8.
Y-27632 promoted in vivo corneal epithelial wound healing.
Corneal epithelial wounds were created in both eyes of Sprague Dawley rats. Y-27632 or PBS was applied topically five times a day to the left or right eye, respectively. (A) After wounding, the area of epithelial defect in each cornea was revealed by staining with 1% fluorescein, and photographed every 24 hours until healed. Representative photographs of corneal healing in five different experiemnets were shown here. The corneal epithelial wounds were rapidly healed in Y-27632-treated eyes. (B) The area of the photographed corneal epithelial defect was measured by a computer-assisted digitizer and statistical analyses were performed using a paired test, and *p<0.05 was considered significant (n = 5, p = 0.02 and 0.002 at day 2 and day 3, respectively). (C). Healed corneas were fixed and stained with anti-Ki67 antibody. Ki67 was more abundantly expressed in the Y-27632-treated corneas, confirming that proliferation was actually increased by Y-27632. The nuclei were counterstained by DAPI (blue). Scale bar, 100 μm. Magnification: 200×.