In vitro expansion and characterization of corneal stem cells isolated from an eye with malignant melanoma

The objective of this study was the identification, characterization and in vitro replication of the human corneal stem cells, taking into consideration the difficulties in obtaining sufficient corneal material from living donors. The study explored a variety of stem cell markers, usually found in embryonic or adult mesenchymal stem cells. Culture medium and replication substrates had to be identified, with no data available on this subject in our country (there are no other reports on corneal stem cells in Romania, to our knowledge).

Corneal epithelial limbus was harvested from an enucleated eye, containing also a choroid malignant melanoma. Stem cells from the limbus were isolated and cultivated in vitro. Expression of specific stem cell markers was evaluated with immunocytochemistry.

Corneal stem cell expansion in primary culture was slow, achieving 70-80% confluence after 28 days. Stem cells were easily isolated in standard medium, showed fibroblastoid morphology and were positive for certain stem cell specific markers in immunocytochemical staining: Oct3÷4, SOX2, Nanog, SSEA4, CD44, CD90, CD133, and CD34. They also expressed pan-cytokeratin. Donor age (72 years) and the presence of a malignant tumor close to limbal stem niche could have had an impact on the proliferation rate and the characteristics of the corneal stem cells.

Isolated limbal cells were adult type stem cells with an epithelial orientation. The characterization of these cells with immunocytochemistry allowed us to observe surface markers that other stem cells also express.