Human corneal endothelial cell precursors isolated by sphere-forming assay.
OBJECTIVE
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, beta3-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 beta3-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.
Yokoo
Seiichi
S
Department of Corneal Tissue Regeneration, Tokyo University Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
Yamagami
Satoru
S
Yanagi
Yasuo
Y
Uchida
Saiko
S
Mimura
Tatsuya
T
Usui
Tomohiko
T
Amano
Shiro
S
D000199:Actins / Q000378:metabolism; D000328:Adult; D000368:Aged; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007381:Intermediate Filament Proteins / Q000378:metabolism; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018874:Spheroids, Cellular / Q000166:cytology / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
D000199:Actins; D016895:Culture Media, Serum-Free; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; C577484:NES protein, human; D009419:Nerve Tissue Proteins; D064231:Nestin; D014404:Tubulin