Morio Ueno1, Kazuko Asada2, Munetoyo Toda2, Kazue Nagata3, Chie Sotozono1, Nobuyoshi Kosaka4, Takahiro Ochiya4, Shigeru Kinoshita2, Junji Hamuro1. 1. Department of Ophthalmology Kyoto Prefectural University of Medicine, Kyoto, Japan. 2. Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan. 3. Toray Industries, Inc., Tokyo, Japan. 4. Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan.
Abstract
PURPOSE: We elucidate a method to use secreted miRNA profiles to qualify cultured human corneal endothelial cells (cHCECs) adaptable for cell-injection therapy. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
PURPOSE: We elucidate a method to use secreted miRNA profiles to qualify cultured human corneal endothelial cells (cHCECs) adaptable for cell-injection therapy. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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: Ricardo F Frausto; Vinay S Swamy; Gary S L Peh; Payton M Boere; E Maryam Hanser; Doug D Chung; Benjamin L George; Marco Morselli; Liyo Kao; Rustam Azimov; Jessica Wu; Matteo Pellegrini; Ira Kurtz; Jodhbir S Mehta; Anthony J Aldave Journal: Sci Rep Date: 2020-05-04 Impact factor: 4.379