Fig 2: Central cornea after four and 14 days of culture. Panels (A), (B), and (C) represent three different sections from the cornea cultured for four days. (A) Compared to limbal or peri-limbal region, eGFP expression is less pronounced in central cornea (arrowheads). The lack of p63a and KRT19 expression suggests the differentiated nature of this area. (B) eGFP expression can be seen in basal layers of central epithelium that co-localizes with KRT3 expression (arrowheads). (C) Non-primary control confirms the specificity of primary antibody expression. DAPI stained nuclei are shown in gray. (D) H&E image demonstrates where the confocal images were taken. Panels (E), (F), and (G) represent three different sections from the cornea that was cultured for 14 days. (E) eGFP + cells can be seen in predominantly superficial layers of the epithelium (arrowheads). (F) Co-expression of KRT3 in eGFP + cells suggests their differentiated state (arrowheads). (G) Non-primary control confirms the specificity of primary antibody expression. (H) H&E image demonstrates where the confocal images were taken. Scale bar indicates 50 µm. All images were taken in 20× magnification.

Fig 3: Peri-limbal region after four and 14 days of culture. Panels (A), (B) and (C) represent three different sections from the cornea that was cultured for four days. (A) Strong eGFP signal can be seen in peri-limbal corneal epithelium that co-localizes with stem cell markers p63a and KRT19 (arrowheads). eGFP expression can also be observed in stromal cells underneath the limbus (asterisk) resulting from sub-limbal injection. (B) eGFP + /p63a + cells are markedly visible in basal layers of peri-limbal epithelium (arrowheads), while differentiation marker KRT3 signal becomes stronger in superficial epithelial layers towards the central cornea (arrows). (C) Non-primary control confirms the specificity of primary antibody expression. DAPI stained nuclei are shown in gray. (D) H&E image demonstrates where the confocal images were taken. Panels (E), (F) and (G) represent three different sections from the cornea cultured for 14 days. (E) Stronger eGFP signal is visible in superficial epithelial layers (arrowheads). (F) eGFP + cells co-express KRT3 (arrowheads), suggesting their differentiated nature. (G) Non-primary control confirms the specificity of primary antibody expression. (H) H&E image demonstrates where the confocal images were taken. All images were captured in 20× magnification, and the scale bar indicates 50 µm.

Fig 4: Corneal limbus after four and 14 days of culture show eGFP + cells in situ. Panels (A), (B) and (C) represent three different sections from the cornea that was cultured for four days. (A) Stem cell markers p63a (red) and KRT19 (magenta) co-localize with eGFP signal (green) in cells with successful transduction (arrowheads). (B) Scant expression of differentiation marker KRT3 (magenta) in eGFP + /p63a + cells (arrowheads). (C) Non-primary control confirms the specificity of primary antibody expression. DAPI stained nuclei are shown in gray. (D) H&E image demonstrates where the confocal images were taken. Panels (E), (F) and (G) represent three different sections from the cornea cultured for 14 days. (E) After 14 days, the eGFP signal is stronger in upper limbal layers (arrowheads) where it no longer co-localizes with stem cell markers p63a and KRT19. (F) The eGFP + cells in upper limbal layers co-express differentiation marker KRT3 (arrowheads). (G) Non-primary control confirms the specificity of primary antibody expression. (H) H&E image demonstrates where the confocal images were taken. All images were captured in 20× magnification and the scale bar indicates 50 µm.