Abstract
Purpose: :
Corneal stroma has a unique extracellular matrix essential for tissue integrity and transparency. Cells for corneal tissue engineering must have the ability to secret such a cornea–specific matrix. Multipotent adult progenitor cells have recently been identified in a number of tissues including human corneal stroma (Du et al., Stem Cells 23:1266 2005). These cells appear to be good candidates for corneal cellular therapy and tissue engineering. In this study, we investigated conditions in which cultured human corneal stromal derived stem cells (CSSC) might adopt a keratocyte phenotype secreting extracellular matrix resembling that of the corneal stroma.
Methods: :
Human CSSCs were isolated by cell sorting using Hoechst 33342 dye exclusion. Corneal stromal fibroblasts cultured in fetal bovine serum served as negative controls. CSSCs and fibroblasts were cultured as substratum–free pellets 2–4 weeks in media containing fibroblast growth factor and insulin. Cultures were stained by Hematoxylin–Eosin, immunostained for keratocan, keratan sulfate, collagen V and collagen VI, or fixed and examined by electron microscopy. mRNA abundance was determined by real–time RT–PCR. Keratocan and keratan sulfate secreted in media by the cells were detected by immunoblotting.
Results: :
After 3–weeks, CSSCs cultured as pellets secreted abundant connective tissue containing keratocan, keratan sulfate, collagen V and collagen VI, whereas fibroblasts did not express the same markers. H&E and electron microscopic analysis showed that in outer regions of pellet cultures, cells were flattened between layers of matrix and exhibited extended processes. Extracellular matrix contained fibrillar collagen with regions of high organization with aligned parallel collagen fibrils decorated with crosslinking proteoglycans. In these cultures expression of genes characteristic of stem cells was reduced and keratocyte–specific genes expression was increased.
Conclusions: :
Stem cells from human corneal stroma cultured as scaffolding–free pellets, differentiate into cells which express a gene profile similar to that of human keratocytes and which deposit a tissue–like extracellular matrix with a composition and structure similar to that of the cornea stroma. These studies show that stromal stem cells cultured under appropriate conditions may represent the starting material for development of bioengineered corneal bioprostheses.
Keywords: cornea: stroma and keratocytes • extracellular matrix • gene/expression