Gene Signature-Based Development of ELISA Assays for Reproducible Qualification of Cultured Human Corneal Endothelial Cells

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Details zur Publikation

Autor(en): Ueno M, Asada K, Toda M, Schlötzer-Schrehardt U, Nagata K, Montoya M, Sotozono C, Kinoshita S, Hamuro J
Zeitschrift: Investigative Ophthalmology & Visual Science
Jahr der Veröffentlichung: 2016
Band: 57
Heftnummer: 10
Seitenbereich: 4295-305
ISSN: 0146-0404


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.



FAU-Autoren / FAU-Herausgeber

Schlötzer-Schrehardt, Ursula apl. Prof. Dr.
Medizinische Fakultät


Zusätzliche Organisationseinheit(en)
Augenklinik


Autor(en) der externen Einrichtung(en)
Kyoto Prefectural University of Medicine / 京都府立医科大学
SightLife Inc.
Toray Industries Inc


Zitierweisen

APA:
Ueno, M., Asada, K., Toda, M., Schlötzer-Schrehardt, U., Nagata, K., Montoya, M.,... Hamuro, J. (2016). Gene Signature-Based Development of ELISA Assays for Reproducible Qualification of Cultured Human Corneal Endothelial Cells. Investigative Ophthalmology & Visual Science, 57(10), 4295-305. https://dx.doi.org/10.1167/iovs.16-19806

MLA:
Ueno, Morio, et al. "Gene Signature-Based Development of ELISA Assays for Reproducible Qualification of Cultured Human Corneal Endothelial Cells." Investigative Ophthalmology & Visual Science 57.10 (2016): 4295-305.

BibTeX: 

Zuletzt aktualisiert 2018-07-10 um 02:16