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.


CONCLUSIONS
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.
Department of Ophthalmology, University of Bern, Switzerland.
M
M
Böhnke
K
K
Vogelberg
K
K
Engelmann
D015415:Biomarkers; D002454:Cell Differentiation; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000201:enzymology / Q000648:ultrastructure; D004728:Endothelium, Corneal / Q000201:enzymology* / Q000648:ultrastructure; D005347:Fibroblasts / Q000201:enzymology / Q000648:ultrastructure; D005500:Follow-Up Studies; D006801:Humans; D007124:Immunoenzyme Techniques; D008855:Microscopy, Electron, Scanning; D008858:Microscopy, Phase-Contrast; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*
D015415:Biomarkers; D010751:Phosphopyruvate Hydratase