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purpose. To demonstrate the presence of corneal stromal precursors that express neural markers in vitro.

methods. To isolate sphere-forming cells, human corneal stromal cells were subjected to a reaggregation-free neurosphere assay in medium containing methylcellulose gel matrix. To promote differentiation, the isolated sphere colonies were plated in wells with medium containing fetal bovine serum. Expression of nestin, vimentin, keratocan, α-smooth muscle actin (αSMA), β-III tubulin, neurofilament M (NFM), and glial fibrillary acidic protein (GFAP) was examined in the sphere colonies and their progeny (ie, cells migrated from sphere colonies), by immunocytochemistry and/or reverse transcription–polymerase chain reaction (RT-PCR).

results. Human corneal stromal cells formed sphere colonies that had no self-renewal capability. The frequency of sphere-forming cells was 1.5%±0.1%(range, 1.3%–1.6%). Most of the cells within these colonies expressed nestin and vimentin, whereas some expressed β-III tubulin, NFM, GFAP, and αSMA by immunocytochemistry. Ninety-one percent and 89% of the progeny expressed vimentin and αSMA, respectively, whereas nestin was undetectable. β-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 increased in the progeny.