Fig 2: BCAM is a downstream target of the transcription factor p63(A) Representative immunostaining analysis of p63 (green) and BCAM (red) expression in the limbus and central cornea. Nuclei stained with Hoechst 33342 (blue). n = 3. Scale bar: 20 µm.(B) Bar graph depicts BCAM expression in control and TP63 siRNA-treated limbal epithelial cells (mean ± SD; n = 8; ****p < 0.0001). Data were analyzed using a Dunnett’s multiple comparisons test. KD, knockdown.(C) Bar graph illustrates relative percentage of BCAM-positive cells in control and TP63 siRNA-treated limbal epithelial cultures analyzed by flow cytometry (mean ± SD; n = 6; ***p < 0.001). Data were analyzed using a Dunnett’s multiple comparisons test.(D) The graph illustrates p63 binding sites within 2-kb up- and downstream of the BCAM transcription start site (TSS) of BCAM identified by p63 ChIP-seq in human skin (Kouwenhoven et al., 2010). P63 ChIP-seq data were downloaded from GEO: GSE17611 and visualized using the UCSC genome browser.(E) Bar graph depicts enrichment of the BCAM promoter/enhancer and the BCAM first intron sequences in anti-p63 antibody immunoprecipitates compared with the isotype control immunoprecipitates as determined by ChIP-qPCR analyses. Myoglobin exon 2 sequence was used as a negative control (mean ± SD; n = 6).(F) Bar graph represents the fold change in luciferase activity in ?Np63a-overexpressing and control 293T cells co-transfected with luciferase reporter vector containing the promoter region 2-kb upstream of BCAM TSS (mean ± SD; n = 4; *p < 0.05). Data were analyzed using a paired t test. OE, overexpression.

Fig 3: BCAM maintains corneal epithelial stratification through regulation of cell adhesion and migration(A) Left, representative H&E-stained images of the cell sheets generated by limbal epithelial cells subjected to siRNA-induced BCAM blockade. Right, bar graph depicts the comparative thickness analyses of the cell sheets generated by control and BCAM siRNA-treated cells (mean ± SD; n = 7; *p < 0.05, **p < 0.01). Data were analyzed using a Dunnett’s multiple comparisons test. KD, knockdown.(B) Left, representative macroscopic images of the cell colonies generated by control and BCAM siRNA-treated cells. Right, bar graph depicts comparative analyses of colony-forming efficiency (mean ± SD; n = 8; **p < 0.01). Data were analyzed using a Dunnett’s multiple comparisons test.(C) Left, line graph represents the quantitative analyses of wound closure in control and BCAM siRNA-treated cells (mean ± SEM; n = 7; p = 0.0475). Data were analyzed using a two-way ANOVA. A representative phase-contrast image is shown on the right.

Fig 4: scRNA-seq analysis of ABCB5-positive LSCs identifies stem cell heterogeneity(A) Left, illustration of ABCB5-positive LSC isolation from human limbus. Right, flow cytometric analyses of ABCB5 expression in human donor limbal tissues used for scRNA-seq analyses (n = 3).(B) Uniform manifold approximation and projection (UMAP) plot of 3,417 limbal epithelial cells isolated from three human donors (left). Feature plot (middle) and violin plot (right) depicting the expression of BCAM.(C) Feature plots (left) and violin plots (right) depicting the expression of the eye transcription factor PAX6 and the LSC markers KRT15 and TP63.(D) Feature plots (left) and violin plots (right) illustrating expression of MYC, p15, and Cyclin D2.

Fig 5: 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.