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(Investigative Ophthalmology and Visual Science. 2005;46:1626-1631.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-1263
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Human Corneal Endothelial Cell Precursors Isolated by Sphere-Forming Assay

Seiichi Yokoo,1 Satoru Yamagami,1 Yasuo Yanagi,2 Saiko Uchida,2 Tatsuya Mimura,2 Tomohiko Usui,2 and Shiro Amano2

1From the Departments of Corneal Tissue Regeneration and 2Ophthalmology, Tokyo University Graduate School of Medicine, Tokyo, Japan.

PURPOSE. To isolate precursors of human corneal endothelial cells (HCECs) in vitro.

METHODS. HCECs were subjected to a sphere-forming assay in which spheres floated in serum-free medium containing growth factors. To promote differentiation, the isolated sphere colonies were plated in dishes coated with poly-L-lysine (PLL)/laminin or fetal bovine endothelium extracellular matrix. Marker expression of neural and mesenchymal cells was examined in the sphere colonies and their progenies by immunocytochemistry and/or reverse transcription–polymerase chain reaction (RT-PCR). Adherent differentiated cells from the sphere colonies were evaluated morphologically and functionally.

RESULTS. HCECs formed primary and secondary spherical colonies, as shown by sphere-forming assay in vitro. The colonies expressed nestin, ß3-tublin, glial fibrillary acidic protein, and {alpha}-smooth muscle actin on immunocytochemistry. The progeny, proliferating on extracellular matrix derived from bovine corneal endothelium, but not on PLL/laminin-coated and noncoated dishes, expressed nestin and ß3-tublin. These markers were confirmed by RT-PCR. Adherent differentiated cells from the sphere colonies had an HCEC-like hexagonal shape and satisfactory transport activity that is essential in HCECs.

CONCLUSIONS. These findings indicate that the HCEC contains precursor cells with a propensity to differentiate into HCECs and that these cells can also produce neuronal and mesenchymal cell proteins.




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