HUMAN AND MOUSE KERATOCYTE MORPHOLOGY AND EXPRESSION OF KERATOCAN AND CD 34 ARE MAINTAINED DUING EXPANSION BY SERUM CONTAINING MEDIA ON AMNIOTIC MEMBRANE STROMA

Abstract

Abstract: : Purpose:To expand human and murine keratocytes in vitro while maintaining their dendritic morphology and expression of cornea specific keratocan and CD34 glycoprotein. Methods:Collagenase–released keratocytes from human and murine corneal stroma were expanded on plastic or human amniotic membrane (AM) stroma in DMEM with ITS (insulin, transferrin, and sodium selenite) or 10% FBS. After confluence, cells were continuously passed at 1:2 split on plastic or AM. Cells continuously cultured on plastic in 10% FBS were switched to DMEM with ITS, and cell cultured on plastic in DMEM with ITS were challenged with 10, 100, 1000 pg/ml TGF–ß1. Cell morphology and cell–cell networks were assessed by phase contrast microscopy and Live and Dead® assay. Proteins and mRNA were evaluated for the expression of keratocan, CD34 and α–smooth muscle actin (α–SMA). Results:In 10% FBS, human and murine cells cultured on plastic adopted a spindle–shape morphology and lost CD34 protein expression and keratocan mRNA and protein at passage 1 (P1) and thereafter. In contrast, human and murine cells continuously maintained three–dimensional dendritic morphology and expression of keratocan mRNA and protein for up to 6 and 8 passages, respectively, when cultured on AM. Keratocan expressed by AM cultures retained keratan sulfate glycosylation similar to in vivo state but to a lesser extent. Expression of CD34 protein was maintained on AM in 10% FBS for up to P3 and in ITS despite addition of 1 ng/ml TGF–ß1 without the expression of α–SMA. In contrast, expression of CD34 protein was lost on plastic with concomitant expression of α–SMA when as low as 10 pg/ml TGF–ß1 was added. Cells that were serum withdrawn on plastic at P2 dramatically reverted to a dendritic shape, but still did not express keratocan or CD34 protein. Conclusions:Human and murine keratocyte phenotype, characterized by dendritic morphology and expression of keratocan and CD34, can be maintained on AM while expanded in a serum–containing medium, and effectively withstand the challenge of exogenous TGF–ß1. The loss of such a phenotype on plastic cultures cannot be completely reversed by serum withdrawal.

Keywords: cornea: stroma and keratocytes • extracellular matrix • protein structure/function