Growth Factor Changes in Ex Vivo Expansion of Human Limbal Epithelial Cells on Human Amniotic Membrane

Abstract

Human limbal epithelial cells cultured on human amniotic membrane have been used for transplantation to treat corneal surface injuries. We determined whether the amniotic basement membrane affects the growth of human limbal epithelial cells through the production of growth factors. The epithelial cells grown out from limbal basal epithelium were placed on conventional culture plastic or on the epithelial side of denuded amniotic membrane under serum-free conditions. Culture supernatant was assayed for growth factor release at 24, 48, and 96 hours. The cells grown on both substrata produced similar levels of epidermal growth factor (EGF). Cells grown on amniotic membrane showed enhanced secretion of tissue inhibitor of metalloproteinase type 1 (TIMP1) and reduced production of transforming growth factor beta1 and beta2. Depletion of EGF and TIMPI in cell culture slowed down cell growth and reduced EGF receptor expression, respectively. Increased TIMPI influences the proteolytic system in the cell and extracellular matrix interaction, and decreased transforming growth factor beta1 and beta2 may stimulate corneal cell proliferation. We show that the amniotic membrane leads to differential expression of cytokines of limbal epithelial cells cultured on its surface. Such effects may be favorable to the growth and differentiation of the cells when used for ocular surface reconstruction.