Cell surface markers of functional phenotypic corneal endothelial cells.
OBJECTIVE
Cultured human corneal endothelial cells (HCECs) are anticipated to serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction. However, corneal endothelial cells (CECs) tend to exhibit fibroblastic transformation, thereby losing their functional phenotype when cultured. The purpose of this study was to investigate the usefulness of surface markers of CECs displaying fibroblastic phenotypes as a means of cell characterization.


METHODS
The expression levels of 242 cell surface antigens were screened in cultured human and monkey CECs using flow cytometry. An expression intensity ratio of nonfibroblastic/fibroblastic CECs > 2 and of fibroblastic/nonfibroblastic CECs > 2 were selected as indicating nonfibroblastic and fibroblastic markers, respectively. Nonfibroblastic and fibroblastic CECs were mixed, and CD73-positive and -negative cells were sorted using flow cytometry and further cultured. The functional phenotype of the sorted cells was evaluated according to morphology and the expression of function-related (Na(+)/K(+)-ATPase and ZO-1) and fibroblastic (type I collagen and fibronectin) markers.


RESULTS
Flow cytometry analysis demonstrated that CD98, CD166, and CD340 are elevated in HCECs of nonfibroblastic phenotype, while CD9, CD49e, CD44, and CD73 are markers of fibroblastic phenotype HCECs. The CECs that sorted as CD73-negative exhibited normal hexagonal morphology and expressed functional markers, whereas CECs that sorted as CD73-positive exhibited the fibroblastic phenotype.


CONCLUSIONS
These markers will be useful for quality control to characterize the phenotype of cells destined for tissue engineering-based therapy. In addition, this selection protocol will provide a novel method for purification of functional cells.
Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Naoki
N
Okumura
Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
Hiroatsu
H
Hirano
Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
Ryohei
R
Numata
Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
Makiko
M
Nakahara
Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Morio
M
Ueno
Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Junji
J
Hamuro
Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Shigeru
S
Kinoshita
Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
Noriko
N
Koizumi
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D003316:Corneal Diseases / Q000473:pathology / Q000601:surgery*; D016039:Corneal Transplantation / Q000379:methods*; D004728:Endothelium, Corneal / Q000166:cytology / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D000882:Haplorhini; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D012313:RNA / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D023822:Tissue Engineering / Q000379:methods*
D015415:Biomarkers; D008565:Membrane Proteins; D012313:RNA
cell surface marker; corneal endothelial cells; regenerative medicine; tissue engineering