Cell Homogeneity Indispensable for Regenerative Medicine by Cultured Human Corneal Endothelial Cells.
To identify the subpopulation (SP) among heterogeneous cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition applicable for cell-based therapy.



Subpopulation presence in cHCECs was confirmed via surface CD-marker expression level by flow cytometry. CD markers effective for distinguishing distinct SPs were selected by analyzing those on established cHCECs with a small cell area and high cell density. Contrasting features among three typical cHCEC SPs was confirmed by PCR array for extracellular matrix (ECM). Combined analysis of CD markers was performed to identify the SP (effector cells) applicable for therapy. ZO-1 and Na+/K+ ATPase, CD200, and HLA expression were compared among heterogeneous SPs.



Flow cytometry analysis identified the effector cell expressing CD166+CD105-CD44-∼+/-CD26-CD24-, but CD200-, and the presence of other SPs with CD166+ CD105-CD44+++ (CD26 and CD24, either + or -) was confirmed. PCR array revealed three distinct ECM expression profiles. Some SPs expressed ZO-1 and Na+/K+ ATPase at comparable levels with effector cells, while only one SP expressed CD200, but not on effector cells. Human leukocyte antigen expression was most reduced in the effector SP. The proportion of effector cells (E-ratio) inversely paralleled donor age and decreased during prolonged culture passages. The presence of Rho-associated protein kinase (ROCK) inhibitor increased the E-ratio in cHCECs. The average area of effector cells was approximately 200∼220 μm2, and the density of cHCECs exceeded 2500 cells/mm2.



A specified cultured effector cell population sharing the surface phenotypes with mature HCECs in corneal tissues may serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction.
Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Junji
J
Hamuro
Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Munetoyo
M
Toda
Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Kazuko
K
Asada
Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Asako
A
Hiraga
Department of Ophthalmology, University Hospital Erlangen, Erlangen, Germany.
Ursula
U
Schlötzer-Schrehardt
SightLife, Inc., Seattle, Washington, United States.
Monty
M
Montoya
Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Chie
C
Sotozono
Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Morio
M
Ueno
Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Shigeru
S
Kinoshita
D000293:Adolescent; D000328:Adult; D015703:Antigens, CD / Q000378:metabolism; D002452:Cell Count; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D055954:Corneal Endothelial Cell Loss / Q000378:metabolism / Q000473:pathology / Q000601:surgery*; D016039:Corneal Transplantation / Q000379:methods*; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D007223:Infant; D007231:Infant, Newborn; D008858:Microscopy, Phase-Contrast; D008875:Middle Aged; D044968:Regenerative Medicine / Q000379:methods*; D014019:Tissue Donors; D055815:Young Adult
D015703:Antigens, CD