title: Gene Signature-Based Development of ELISA Assays for Reproducible Qualification of Cultured Human Corneal Endothelial Cells.
abstract: To develop a method to qualify the function of cultured human corneal endothelial cells (cHCECs) applicable for clinical settings.
  
  
  
  The diversified gene and microRNA (miRNA) signatures in HCECs from a variety of tissue donors were confirmed by three-dimensional (3D) gene human miRNA profiling. These were compared with those of more than 20 cHCECs distinct in their cell morphology or culture lots. Candidate genes were selected after quantitative (q)RT-PCR validation, and gene products were assayed by ELISA. After three additional screening steps, final candidate cytokines for qualification were selected.
  
  
  
  Gene and miRNA signatures among distinct cHCEC lots were greatly diversified compared with those among fresh tissues from different age donors. By comparing more than 20 lots of cultures, 32 candidate genes were assigned to be seemingly linked to distinct cHCEC morphologic features. The validation of candidate genes by qRT-PCR revealed the genes, either upregulated or downregulated, corresponding to morphologic variances in cHCECs (e.g., epithelial-mesenchymal transition or cell senescence). Further adding the ELISA results by Bio-Plex Human Cytokine 27-Plex Panel, 11 candidate cytokines suitable to qualify cHCEC function were selected. In consideration of the presence of these cytokines in the anterior chamber, IL-8, tissue inhibitors of metalloproteinases 1 (TIMP-1), monocyte chemotactic protein-1 (MCP-1), and platelet-derived growth factor-BB (PDGF-BB) were ultimately selected and applied in practice for the qualification of cHCECs actually used in our clinical cell-injection studies.
  
  
  
  The specified cytokines properly discriminating the functional features of cHCECs indicates a correlation between profiling signatures and cell morphology.
authors.affiliation: Department of Ophthalmology Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Morio
authors.initials: M
authors.lastname: Ueno
authors.affiliation: Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Kazuko
authors.initials: K
authors.lastname: Asada
authors.affiliation: Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Munetoyo
authors.initials: M
authors.lastname: Toda
authors.affiliation: Department of Ophthalmology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Ursula
authors.initials: U
authors.lastname: Schlötzer-Schrehardt
authors.affiliation: Toray, Inc., Kamakura, Japan.
authors.forename: Kazue
authors.initials: K
authors.lastname: Nagata
authors.affiliation: SightLife, Inc., Seattle, Washington, United States.
authors.forename: Monty
authors.initials: M
authors.lastname: Montoya
authors.affiliation: Department of Ophthalmology Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Chie
authors.initials: C
authors.lastname: Sotozono
authors.affiliation: Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kinoshita
authors.affiliation: Department of Ophthalmology Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Junji
authors.initials: J
authors.lastname: Hamuro
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002478:Cells, Cultured; D002648:Child; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics*; D004728:Endothelium, Corneal / Q000166:cytology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation; D006801:Humans; D021621:Imaging, Three-Dimensional; D008297:Male; D008858:Microscopy, Phase-Contrast; D008875:Middle Aged; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D055815:Young Adult
chemical_list: D016207:Cytokines; D012333:RNA, Messenger