Expression of PAX6 and Keratocyte-Characteristic Markers in Human Limbal Stromal Cells of Congenital Aniridia and Healthy Subjects, <i>In Vitro</i>

Our aim was to examine the expression of PAX6 and keratocyte-specific markers in human limbal stromal cells (LSCs) in congenital aniridia (AN) and in healthy corneas, <i>in vitro</i>. Primary human LSCs were extracted from individuals with aniridia (AN-LSCs) (<i>n</i> = 8) and from healthy corneas (LSCs) (<i>n</i> = 8). The cells were cultured in either normal-glucose serum-containing cell culture medium (NGSC-medium) or low-glucose serum-free cell culture medium (LGSF-medium). Analysis of PAX6 and keratocyte-specific markers was conducted using qPCR and Western blotting. The keratocyte-specific markers included Collagen I (COL1A1), Collagen III (COL3A1), Collagen V (COL5A1), α-smooth muscle actin (ACTA2), Aldehyde Dehydrogenase 3 Family, Member A1 (ALDH3A1), Keratocan (KER), Lumican (LUM), and CD34. <i>PAX6</i> mRNA expression exhibited a significant decrease in AN-LSCs compared to LSCs in both NGSC- and LGSF-medium (<i>p</i> = 0.04; <i>p</i> = 0.014). There was a marked reduction in <i>COL5A1</i> mRNA expression (<i>p</i> = 0.011), accompanied by notably higher <i>ALDH3A1</i> and <i>KER</i> mRNA levels (<i>p</i> = 0.007; <i>p</i> = 0.013) in AN-LSCs compared to LSCs when using NGSC-medium. In LGSF-medium, AN-LSCs showed a significant increase in <i>COL1A1</i> and <i>COL5A1</i> mRNA expression compared to LSCs (<i>p</i> = 0.048; <i>p</i> = 0.002). Moreover, COL1A1 and α-SMA protein expression were significantly elevated in AN-LSCs compared to LSCs in LGSF-medium (<i>p</i> = 0.048, <i>p</i> = 0.008). Our investigation affirms the altered expression of PAX6 and keratocyte-specific markers in AN-LSCs relative to healthy controls. Both NGSC- and LGSF-medium exerted distinct effects on both LSCs and AN-LSCs. The observed variations in PAX6 and keratocyte-specific marker expression in AN-LSCs may play a pivotal role in the development and progression of aniridia-associated keratopathy.