Reconstruction of Corneal Stroma With Gelatin and Multipotent Precursor Cells From Human Corneal Stroma

Abstract

Abstract: : Purpose: To isolate multipotent precursor cells from human corneal stroma and reconstruct corneal stroma with the precursor cells and gelatin. Methods: To isolate sphere–forming cells, human corneal stromal cells were subjected to a reaggregation–free neurosphere assay using medium containing methylcellulose gel matrix. To promote differentiation, the isolated sphere colonies were plated in wells with medium containing fetal bovine serum. Gene expression was examined in the sphere colonies and their progeny by immunocytochemistry and/or the reverse–transcriptase polymerase chain reaction (RT–PCR). Corneal stroma was reconstructed by cultivating the precursor cells in porous gelatin for 1 week. This reconstructed corneal stromal sheet was transplanted in a pocket of 6 rabbits’ corneal stroma (sphere/gelatin group). A sheet of gelatin on which corneal keratocytes were cultivated for 1 week was transplanted in other 6 eyes (keratocyte/gelatin group). Only a sheet of gelatin was transplanted in other 6 eyes (gelatin group). Gene expression and extracellular matrix production were examined immunohistochemically in each group 4 weeks after the surgery. Results: Human corneal stromal cells formed sphere colonies. The frequency of sphere forming cells was 1.5 ± 0.1%. Most of the cells within these colonies expressed nestin and vimentin, while some cells expressed ß–III tubulin, NFM, GFAP, and αSMA by immunocytochemistry. Ninety one and 89 percent of the progeny expressed vimentin and αSMA, respectively, whereas expression of nestin was undetectable. ß–III tubulin–, NFM–, and GFAP– positive cells were detected in the progeny at the frequency of 7.2, 0.9, and 0.5%, respectively. Semiquantitative RT–PCR showed that nestin, NFM, GFAP, and keratocan gene expression was higher in the sphere colonies, whereas vimentin and αSMA expression was increased in the progeny. In the gelatin sheet of sphere/gelatin group, more cells and more intense expressions of collagen 1, 4, laminin, vimentin, CD34, αSMA, and nestin were observed than in the other two groups. Conclusions: Adult human corneal stroma contains multipotent precursor cells. They have a strong propensity to differentiate into mesenchymal fibroblasts, but these cells can also differentiate into the neuronal lineage. The precursor cells from human corneal stroma may supply more extracellular matrix than keratocytes.

Keywords: cornea: stroma and keratocytes