Fig 2: Histologic cross sections (hematoxylin, eosin, and saffron) and immunostaining on corneal cross sections or on flat-mounted corneas after 14 days’ storage in organ culture, on agar, on Agar-AL, or in the ASM. K3–K12 (green), E-cadherin (green), laminin 5 (red), ZO-1 (red), and MUC16 (green). Nuclei were stained with TO-PRO-3 Iodide (blue). MUC16 staining pictures presented are the en face (upper image) and the orthogonal (lower image) views. No staining was observed in negative controls (data not shown).

Fig 3: Corneal differentiation potential of limbal BCAM-positive cells(A) Left, representative macroscopic images of the cell colonies generated by limbal BCAM-positive and BCAM-negative cells. Individual colonies are stained with Rhodamine B (pink). Right, bar graph depicts comparative analyses of colony-forming efficiency (mean ± SD; n= 12; ****p < 0.0001). Data were analyzed using a paired t test.(B) Bar graph illustrates comparative analyses of limbal BCAM-positive and -negative cell holoclone-forming efficiency (mean ± SD; n = 7; **p < 0.01). Data were analyzed using a paired t test.(C) Left, representative macroscopic images of the cell colonies generated by central corneal BCAM-positive and -negative cells. Right, bar graph depicts comparative analyses of colony-forming efficiency (mean ± SD; n = 7; *p < 0.05). Data were analyzed using a paired t test.(D) Left, representative H&E-stained images of the cell sheets generated by BCAM-positive and -negative cells isolated from the limbus and central cornea. Right, bar graph depicts the comparative thickness analyses of the cell sheets generated by BCAM-positive and -negative cells isolated from the limbus and central cornea (mean ± SD; n = 7; ****p < 0.0001). Data were analyzed using a Tukey’s multiple comparisons test.(E) Representative immunostaining analyses of MUC16 (green) and KRT12 (red) expression in the cell sheet derived from limbal BCAM-positive cells. Nuclei stained with Hoechst 33342 (blue), n = 4. Scale bar, 20 mm.

Fig 4: BCAM marks limbal corneal epithelial progenitors(A) Schematic illustration of the ocular surface anatomy.(B) Schematic illustration of the experimental design in (C)–(F).(C) Representative flow cytometric analyses of ABCB5 and BCAM expression in freshly isolated limbal epithelial cells. FSC, forward scatter; A, area. n = 3.(D) Representative flow cytometric analyses of ABCB5 and BCAM expression in in-vitro-expanded limbal epithelial cell cultures. FSC, forward scatter; A, area. n = 5.(E) Bar graphs represent the relative percentages of ABCB5-positive/BCAM-positive, ABCB5-positive/BCAM-negative, ABCB5-negative/BCAM-positive, and ABCB5-negative/BCAM-negative subpopulations in freshly isolated limbal epithelial cells and in-vitro-expanded limbal epithelial cultures (mean ± SD; **p < 0.01, ****p < 0.0001). Data were analyzed using a t test.(F) Top, representative flow cytometric analyses of ABCB5 and BCAM expression in in-vitro-expanded ABCB5-positive/BCAM-positive limbal epithelial cells. Bottom left, bar graph illustrates the relative percentages of ABCB5-positive/BCAM-negative, ABCB5-positive/BCAM-positive, ABCB5-negative/BCAM-positive, and ABCB5-negative/BCAM-positive subpopulations (mean ± SD; n = 3; ***p < 0.001, ****p < 0.0001). Data were analyzed using a Tukey’s multiple comparisons test. Bottom right, schematic illustration of the ABCB5-positive/BCAM-positive cell fate. FSC, forward scatter; A, area.(G) Representative immunostaining analyses of integrin ß4 (green) and BCAM (red) expression in the limbus and central cornea. Nuclei stained with Hoechst 33342 (blue), n = 3. Scale bar, 20 µm.(H) Representative immunostaining analyses of KRT12 (green) and BCAM (red) co-expression in the limbus and central cornea. Nuclei stained with Hoechst 33342 (blue), n = 3. Scale bar, 20 µm.(I) Representative immunostaining analyses of NUMB (green) and BCAM (red) co-expression in the limbus and central cornea. Nuclei stained with Hoechst 33342 (blue), n = 3. Scale bar, 20 µm.