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Morio Ueno, Kazuko Asada, Munetoyo Toda, Kazue Nagata, Chie Sotozono, Nobuyoshi Kosaka, Takahiro Ochiya, Shigeru Kinoshita, Junji Hamuro; Concomitant Evaluation of a Panel of Exosome Proteins and MiRs for Qualification of Cultured Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(10):4393-4402. doi: 10.1167/iovs.16-19805.
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We elucidate a method to use secreted miRNA profiles to qualify cultured human corneal endothelial cells (cHCECs) adaptable for cell-injection therapy.
The variations of cHCECs in their composites of heterogeneous subpopulations (SPs) were verified in relation to their surface cluster-of-differentiation (CD) markers. Integrated analysis of micro RNA (miRNA) profiles in culture supernatants (CS) were investigated by 3D-Gene Human microRNA Chips. To validate 3D-Gene results, quantitative real-time PCR was done from numerous cultures with distinct morphology and SP composition. Exosomes and miRNAs in CS also were analyzed.
Secreted miRNA profiles among morphologically-diverse cHCEC SPs proved useful for individual distinction. Candidate miRNAs to discriminate CD44− SPs from those with CD44++ ∼ CD44+++ phenotypes were miRs 221-3p, 1246, 1915-3p, and 4732-5p. The levels of the latter-three miRs decreased dramatically in cHCEC CS without cell-state transition (CST) compared to those of control medium, whereas those from cHCECs with senescence-like CST showed an increase. MicroR184 decreased inversely in parallel with the upregulation of CD44 on cHCECs. CD9+ exosomes were more elevated in cHCEC CS with senescence-like CST than those without CST, indicating the possible import of these extracellular vesicles (EVs) into cHCECs without CST.
Cultured HCECs sharing a CD44− phenotype of matured HCECs may be discriminated by measuring the amount of miRNAs or exosome in CS. Thus, miRNA in CS may serve as a tool to qualify cHCECs. Future detailed analysis of cell-to-cell communication via these EVs might open novel pathways for a better understanding of CST in HCEC cultures.
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