title: Concomitant Evaluation of a Panel of Exosome Proteins and MiRs for Qualification of Cultured Human Corneal Endothelial Cells.
abstract: 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.
authors: Department of Ophthalmology Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors: Morio
authors: M
authors: Ueno
authors: Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors: Kazuko
authors: K
authors: Asada
authors: Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors: Munetoyo
authors: M
authors: Toda
authors: Toray Industries, Inc., Tokyo, Japan.
authors: Kazue
authors: K
authors: Nagata
authors: Department of Ophthalmology Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors: Chie
authors: C
authors: Sotozono
authors: Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan.
authors: Nobuyoshi
authors: N
authors: Kosaka
authors: Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan.
authors: Takahiro
authors: T
authors: Ochiya
authors: Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors: Shigeru
authors: S
authors: Kinoshita
authors: Department of Ophthalmology Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors: Junji
authors: J
authors: Hamuro
mesh_terms: D002478:Cells, Cultured; D004728:Endothelium, Corneal / Q000166:cytology / Q000378:metabolism*; D055354:Exosomes / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics*; D008858:Microscopy, Phase-Contrast; D010641:Phenotype; D012313:RNA / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D035683:MicroRNAs; D012313:RNA