Detection of neurone-specific enolase in long-term cultures of human corneal endothelium

BACKGROUND: Human corneal endothelial cells cultivated in monolayer culture for protracted periods undergo morphological dedifferentiation, whereby they assume a more fibroblast-like appearance. These cultures may also become overgrown with contaminating stromal fibroblasts and/or with keratocytes, when non-selective media are employed, thus rendering identification of actual endothelial cells difficult on a strictly morphological basis. METHODS: The endothelium of the human cornea stains for neurone-specific enolase (NSE) in situ, and we therefore wished to study the expression of this marker in primary and long-term monolayer cultures of these cells. Ten such cultures were established, six being stained for NSE at the primary and first-passage stage, the other four for 6, 8, 10 and 12 months. The NSE-staining pattern manifested in co-cultures of corneal endothelium and fibroblasts or keratocytes (first to fifth passage cultures) was also investigated, and co-cultures established from each of the latter two cell types served as controls. RESULTS: In monolayers of corneal endothelium which had retained their cobblestone-like morphology, NSE could be demonstrated even after more than 20 passages, which amounted to 1 year in culture. Dedifferentiated or degenerating endothelial cells stained poorly and inhomogeneously. Control cultures of fibroblasts or keratocytes were consistently NSE-negative, and when each of these cell types was co-cultured separately with corneal endothelium, only the latter expressed the marker protein. CONCLUSION: Since antibodies against NSE are commercially available, practical use may be made of this marker protein for confirming corneal endothelial status in long-term cultures.