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Mechanisms of Dedifferentiation of Adult Human Retinal Pigment Epithelial Cells in vitro. Morphological and Molecular Genetic Analysis

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Abstract

Changes in morphology and molecular genetic profile of human retinal pigment epithelial (RPE) cells in vitro were studied when exposed to the basic fibroblast growth factor (bFGF). The cell morphology was estimated by cell area and perimeter, spreading and polarization coefficients. It was demonstrated that the number of elongated (fibroblast-like) cells and the number of cells, which size was less than in the control increased in 48 h after adding the factor to the culture. At the same time, the cell proliferative activity decreased (according to MTT test). Immunocytochemical analysis demonstrated a decrease in the staining for connexin Cx43 and an increase in the intensity of staining for protein the Otx2 neuroepithelium protein. Simultaneously, the number of nestin-positive cells and βIII-tubulin positive cells increased. Using quantitative real-time PCR method, an increase in mRNA expression of the KLF4, OCT4, NANOG, OTX2, and NES and decrease in mRNA expression of the MITF and KRT18 were detected in RPE cells treated with bFGF; this indicates enhancement of cell dedifferentiation. These data are confirmed by a decrease in the COL1A1 mRNA expression indicating a decrease in synthetic cell activity. The results indicate that single (short-time) effect by bFGF is sufficient to activate the mechanism, which decreases the cell differentiation level toward neuroepithelium.

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ACKNOWLEDGMENTS

This work was performed within part of a state order of the Koltzov Institute of Developmental Biology, Russian Academy of Sciences, on topic no. 0108-2018-0004 using the equipment of the Center for Collective Use of the Koltzov Institute of Developmental Biology, Russian Academy of Sciences.

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  1. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 119334, Moscow, Russia

    A. V. Kuznetsova, A. M. Kurinov, L. A. Rzhanova & M. A. Aleksandrova

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  1. A. V. Kuznetsova
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Statement of compliance with standards of research involving humans as subjects. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants involved in the study.

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Translated by A. Barkhash

Abbreviations: ICC—immunocytochemistry, IPSC—induced pluripotent stem cells, qPCR—quantitative real-time polymerase chain reaction, bFGF—basic fibroblast growth factor, RPE—retinal pigment epithelium.

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Kuznetsova, A.V., Kurinov, A.M., Rzhanova, L.A. et al. Mechanisms of Dedifferentiation of Adult Human Retinal Pigment Epithelial Cells in vitro. Morphological and Molecular Genetic Analysis. Cell Tiss. Biol. 13, 107–119 (2019). https://doi.org/10.1134/S1990519X19020068

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