Figure 3

Characterization of cultured CD117⁺ cells for melanocyte characteristics. (A) Quantitative real-time polymerase chain reaction (qRT-PCR) primer assays confirm the differential expression of established melanocyte markers (KIT, MLANA, TYRP1) in cultured LEPC, LMSC, and LMs. Data are expressed as means (2^−ΔCT) ± SEM (n = 5). *p < 0.05; Mann–Whitney U test. (B) Immunocytochemical analysis of cultured CD117⁺ cells showing expression of melanocyte markers Melan-A, Sox-10, TRP1, and HMB45 (red); nuclear counterstaining with 4′,6‐diamidino‐2‐phenylindole (blue) (C) CD117 and Melan-A protein expression in CD117⁺ cells were confirmed by western blot analysis. Reprobing with an anti-GAPDH antibody served as a control. Uncropped versions of Western blot are shown in Supplementary Figure 1. (D) Flow cytometry analysis for double staining for CD117 and Melan-A markers in cells expanded. Data are expressed as a percentage as means ± SEM. LEPC, limbal epithelial progenitor cells; LMSC, limbal mesenchymal stromal cells; LM, limbal melanocytes; KIT, CD117; MLANA, Melan-A; SOX10, sex-related HMG box 10; TYRP1 or TRP1, tyrosinase-related protein 1; HMB45, human melanoma black-45; GAPDH; Glyceraldehyde 3-phosphate dehydrogenase).