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Seiichi Yokoo, Satoru Yamagami, Yasuo Yanagi, Saiko Uchida, Tatsuya Mimura, Tomohiko Usui, Shiro Amano; Human Corneal Endothelial Cell Precursors Isolated by Sphere-Forming Assay. Invest. Ophthalmol. Vis. Sci. 2005;46(5):1626-1631. doi: 10.1167/iovs.04-1263.
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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, β3-tublin, glial fibrillary acidic protein, and α-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 β3-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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