Effective Keratocyte Culture Using Amniotic Membrane Matrix and Differentiation of Mesenchymal Stem Cells

ABSTRACT

PURPOSE: To investigate the characteristics of cultured rabbit corneal keratocytes in vitro and evaluate the possibility of differentiation of mesenchymal stem cells to keratocytes using the keratocyte conditioned medium (KCM). METHODS: Isolated keratocytes were seeded on the stromal side of amniotic membranes (AM) or plastic dishes, and morphologic changes were evaluated. Rabbit mesenchymal stem cells were cultured on AM with alpha-MEM (minimum essential medium alpha) and KCM. The gene expression patterns of specific keratocyte markers (keratocan, lumican, and aldehyde dehydrogenase family, member A1 (ALDH1A1)) of cultured cells were evaluated by RT-PCR. RESULTS: Keratocytes on AM showed dendritic morphology with slow proliferation in contrast, cells on dishes were stellate in shape with fast proliferation. Cultured keratocytes on AM maintained the expression of keratocan, lumican and ALDH1A1 while keratocytes on plastic dishes steadily lost their keratocyte marker gene expression. Additionally, mesenchymal stem cells cultured with KCM on AM induced expression of keratocan and ALDH1A1. CONCLUSIONS: Keratocytes cultured on AM stromal matrix maintained their characteristic morphology and marker gene expression. Morphology changes and marker gene expressions of mesenchymal stem cells suggest an ability to differentiate into keratocytes when grown on AM with KCM.